INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 by "Catering Guide dergisi"

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

PROCEEEDINGS OF INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS NOVEMBER 7-8, 2019

LVIV, UKRAIN

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

PROCEEEDINGS OF INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 7-8 NOVEMBER, 2019, LVIV, UKRAIN In Lviv, Ukrain Organized by Trakya University ISBN # : 978-975-374-249-8 Trakya University Publisher No: 220

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

WELCOME NOTES You are welcome to our BIALIC Congress that is organized by Trakya University. The aim of our conference is to present scientific subjects of a broad interest to the scientific community, by providing an opportunity to present their work as oral or poster presentations that can be of great value for global science arena. Our goal is to bring three communities, namely science, research and private investment together in a friendly environment of Lviv, Ukraine in order to share their interests and ideas and to benefit from the interaction with each other. In November 2019, it will be held the first edition of the BIALIC Congress, with ambition of the organizers to make it a periodical event. We are proud to announce that in the BIALIC 2019 will take part more than 300 scientists and researchers from all over the world. There were submitted 376 scientific papers, of which 226 will be presented as oral talks and 150 as poster presentations. The full author list of all submitted papers comprises 936. Our conference is a premier international science, technology and business forum focusing on Agriculture, Biology and Life Science. The technical sessions highlight invited and volunteer speakers. We love our nature and care about the environment. We wanted to make our conference as much greener as possible, using less paper. The participantsâ&#x20AC;&#x2122; posters were submitted via conference web page and will be presented on electronic poster screens, developed particularly for this purpose. Abstract book is published in electronic version in bluetooth in the web which will be provided on each participant. Conference Topics: Agriculture, Forestry, Life Sciences, Agricultural Engineering, Aquaculture and Biosystems, Animal Science, Biomedical science, Biochemistry and Molecular Biology, Biology, Bioengineering, Biomaterials, Biomechanics, Biophysics, Bioscience, Biotechnology, Botany, Chemistry, Chemical Engineering, Earth Sciences, Environmental Science, Food Science, Genetics and Human Genetics, Medical Science, Machinery, Pharmaceutical Sciences, Physics, Soil Science. Lviv is not only a very nice, lovely and historical city at the edge of Europe, but located just at the heart of Eastern Europe region. We are much pleased to host all of you in Lviv, Ukraine. We would like to thank all of you for joining this conference and we would like to give also special thanks to our sponsors and collaborators for giving us a big support to organize this event. We wish you nice stay in Lviv! Prof Dr Yalcin KAYA Head of the Organizing Committee

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

ORGANIZING COMMITTEE Prof. Dr. Yalcin KAYA

Trakya University, Turkey

Chair

Assoc. Prof. Dr. Semra HASANCEBI

Trakya University, Turkey

Co-Chair

Asst. Prof. Dr. Necmi BESER Trakya University, Turkey

Co-Chair

Prof. Dr. Yaroslav BLUME

Director of Institute of Food Biotechnology and Genomics, National Academy of Sciences of Ukraine

Member

Prof. Dr. Loannis TOKATLIDIS

Trakia Democritus University, Greece

Member

Dr. Leanordo VELASCO

CSIC, Sevilla, Spain

Member

Prof. Dr. Maria DUCA

University of National Academy of Sci. Moldova Member

Assoc.Prof.Dr. Nooduan MUANGSAN Assoc. Prof. Dr. Gokhan KACAR

Suranaree University of Technology, Thailand

Member

Trakya University, Turkey

Member

Assoc. Prof. Dr. Guzin Tunca

Trakya University, Turkey

Member

Asst. Prof. Dr. Mehmet YABAS

Trakya University, Turkey

Member

Assoc Prof. Dr. Suleyman KOK Trakya University, Turkey

Member

Asst.Prof.Dr. Orhan Onur ASKIN

KÄ±rklareli University, Turkey

Member

Emrah AKPINAR

Trakya University, Turkey

Secreatary

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

SCIENTIFIC COMMITTEE NAME

INSTITUTION-COUNTRY

Acad. Prof. Dr. Atanas ATANASSOV

Joint Genomic Center- Sofia Bulgaria

Prof. Dr. Teodora POPOVA

Institute of Animal Science- Kostinbrod Bulgaria

Prof. Dr. Viliana VASSILEVA

Institute of Pleven Bulgaria

Prof. Dr Zhao JUN

Inner Mongolia Agricultural University China

Prof. Dr. Ioannis TOKATLIDIS

Trakia Democritus University Greece

Prof. Dr. Rishi K BEHL

CCS Haryana Agricultural University India

Prof. Dr Mohamed RAMDANI

University of Mohamed V Agdal Maroc

Prof. Dr Maria DUCA

University of National Academy of Sci Moldova

Prof Dr Velibor SPALEVIC

University of Montenegro Montenegro

Prof. Dr. Saeed RAUF

Muhammad Nawaz Sharief Agric Univ Pakistan

Prof. Dr. Dejana PANKOVIC

Educon University Serbia

Prof. Dr. Bulent UZUN

Akdeniz University Turkey

Prof. Dr. Nedim MUTLU

Akdeniz University Turkey

Prof. Dr. Ahmet ULUDAG

Onsekizmart University Turkey

Prof. Dr. Hulya ILBI

Ege University Turkey

Prof. Dr. Doganay TOLUNAY

Forestry Faculty, Istanbul University Turkey

Prof. Dr. Ahu ALTINKUT UNCUOGLU

Marmara University Turkey

Prof. Dr. Metin TUNA

Namık Kemal Universit Turkey

Prof. Dr. Sezen ARAT

Namık Kemal Universit Turkey

Prof. Dr. Mehmet Emin CALISKAN

Omer Halisdemir University Turkey

Prof. Dr. Murat YURTCAN

Trakya University Turkey

Prof. Dr. Fatma GUNES

Trakya University Turkey

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

Assoc.Prof.Dr. Natiga Nabiyeva

Genetic Resources Institute of The National Academy of Sciences Azerbaijan

Prof. Dr. Ismail CAKMAK

Sabanci University Turkey

Prof. Dr. Mustafa CULHA

Yeditepe University Turkey

Prof. Dr. Yaroslav BLUME

National Academy of Sciences Ukraine

Prof. Dr. Nurhan T. DUNFORD

Oklahoma State University USA

Assoc. Prof. Dr. Nooduan MUANGSAN

Suranaree University of Technology Thailand

Assoc. Prof. Dr. Ina ZIVATKAUSKIENE

University of Applied Sciences Lithuania

Asst. Prof. Dr. Buket ASKIN

KÄ±rklareli University Turkey

Asst. Prof. Dr. Hayati ARDA

Trakya University Turkey

EDITORS OF THE PROCEEDING BOOK Prof Dr Yalcin KAYA

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

Content LETHAL EFFECT OF TURKISH DIATOMACEOUS EARTH (K14) AGAINST ADULTS OF AMERICAN COCKROACHES (PERIPLANETA AMERICANA L.) ........................................... 11 DETERMINATION OF TOXICITY OF GASEOUS OZONE AGAINST NYMPH STAGES OF GERMAN COCKROACH (BLATELLA GERMANICA L.) ........................................................... 15 BIOLOGICAL EFFICIENCY OF TREATMENT OF OZONE GAS AGAINST PLODIA INTERPUNCTELLA (HÃ&#x153;BNER) (LEPIDOPTERA: PYRALIDAE) (INDIAN MEAL MOTH) IN HAZELNUT ........................................................................................................................................ 19 INSECTICIDAL ACTIVITY OF LOCAL DIATOMACEOUS EARTH IN COMBINATIONS WITH ENTOMOPATHOGENIC FUNGUS, BEAUVERIA BASSIANA (BALS.) VUILL. AGAINST RHYZOPERTHA DOMINICA (F.).................................................................................. 25 MICROWAVE RADIATION TREATMENT FOR CONTROLLING COWPEA WEEVIL (CALLOSOBRUCHUS MACULATUS (FABRICIUS)) ON STORED CHICKPEA ..................... 34 MORTALITY OF SPODOPTERA LITTORALIS LARVAE CAUSED BY EICOSANOID BIOSYNTHESIS INHIBITORS AND TWO NATIVE ISOLATES OF METARHIZIUM ANISOPLIAE ....................................................................................................................................... 41 EFFECT OF FEEDING BEHAVIOR OF RHYZOPERTHA DOMINICA ON ITS SENSITIVITY TO BEAUVERIA BASSIANA INFECTIONS ................................................................................... 45 EFFECT OF HOST POPULATION ON THE EFFICACY OF BEAUVERIA BASSIANA AGAINST SITOPHILUS ORYZAE ................................................................................................... 49 VARIATION IN THE EFFECT OF BEAUVERIA BASSIANA ISOLATES AGAINST THREE COLEOPTERAN STORED-PRODUCT PESTS: CONCENTRATION-MORTALITY RELATION.......................................................................................................................................... 54 ENTOMOLOGICAL RISKS AND MANAGEMENT STRATEGIES IN APRICOT ORCHARDS (DROSOPHILA SUZUKII, CERATITIS CAPITATA, LYRISTES PLEBEJUS) ..... 59 HOW SEED BUGS SPREAD (HETEROPTERA: LYGAEIDAE) FROM CEREAL CROPS TO FRUITS ................................................................................................................................................ 63 INVESTIGATION OF SOME AGRICULTURAL TRAITS AND TOLERANCE TO VERTICILLIUM WILT ON COTTON ............................................................................................. 66 INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) IN POULTRY .................................................. 71 DEFOLIANT APPLICATIONS AND EFFECTS FOR COTTON HARVESTER ...................... 76 THE EFFECT OF TOP CUTTING HEIGHT OF COTTON STALK AND CHEMICAL APPLICATION ON COTTON YIELD ............................................................................................ 84 THE EFFECT OF DIETARY SUPPLEMENTATION OF DIFFERENT MULTI-ENZYMES ON PRODUCTION PERFORMANCE AND EGG QUALITY CHARACTERISTICS IN LAYING HENS ................................................................................................................................... 89 IMPROVED NUTRITIONAL QUALITIES OF SUNFLOWER MEAL BY FERMENTATION ............................................................................................................................................................... 98 SYNTHESIS OF CARVACROL DERIVATIVES AS ANTIOXIDANT AGENTS ................... 106 PLANT TASTE AND THERAPY IN HAUSA TRADITIONAL MEDICINE ........................... 112 EFFECT OF IMAZAMOX USAGE ON PHYTOHORMONE LEVELS IN SUNFLOWER (HELIANTHUS ANNUUS L.) CULTIVATION ............................................................................. 126

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 OBSERVATIONS FOR THE DETERMINATION OF GERMINATION RATE OF JUNIPERUS EXCELSA SUBSP. POLYCARPOS (TURKESTAN JUNIPER) SEEDS ................ 135 A RESEARCH ON ELECTRONIC PROPERTIES OF TEMPERATURE SENSORS USED IN ENGINEERING ................................................................................................................................ 137 NANOTECHNOLOGICAL STRATEGIES TO IMPROVE STABILITY AND BIOAVAILABILITY OF ASTAXANTHIN ................................................................................... 145 FOOD-GRADE EMULSIFIERS: SOURCE, PROPERTIES AND APPLICATIONS .............. 150 MARINE PHOSPHOLIPIDS: IMPORTANCE, CHARACTERIZATION AND APPLICATIONS IN FOOD INDUSTRY ....................................................................................... 155 ANTIOXIDANT ACTIVITY OF TOTAL ANTHOCYANINS EXTRACTED FROM SWEET CHERRY CULTIVARS ................................................................................................................... 159 COLOR CHANGES IN DIFFERENT OILS USED IN A CANNED EEL .................................. 163 THE DETERMINATION OF NUTRITIVE VALUE OF ALFALFA AND CORN SILAGES AT DIFFERENT RATES........................................................................................................................ 171 OXIDATIVE STRESS RELATED KIDNEY TOXICITY OF A GLYPHOSATE-BASED HERBICIDE ...................................................................................................................................... 176 THE EFFECTS OF DIFFERENT NITROGEN DOSES ON SOME AGRICULTURAL CHARACTERISTICS OF PHASELIA (PHACELIA TANACETIFOLIA BENTHAM) ............ 182 TRADITIONAL CHEESE DESSERTS AND KÃ&#x153;NEFE............................................................... 186 ACUTE TOXICITY OF LOW DOSE DIAZINON PUPA STAGE OF ROSE TORTRIX ARCHIPS ROSANA (LINNAEUS, 1758) (LEPIDOPTERA: TORTRICIDAE) ............................ 202 FACTORS INFLUENCING CONSUMPTION OF MEAT IN TURKEY .................................. 207 AMINO ACID PROFILES OF WHITE WINES FROM THREE AUTOCHTHONOUS GALICIAN VARIETIES.................................................................................................................. 217 BACTERIOPHAGES AS COMPLEMENTARY AGENTS IN THE MANAGEMENT STRATEGIES OF FIRE BLIGHT DISEASE................................................................................ 224 SCREENING OF RESISTANCE GENES AND SOME REACTIVE OXYGEN SPECIFIC ENZYMES AGAINST XANTHOMONAS AXONOPODIS PV. PHASEOLI AND PSEUDOMONAS SAVASTANOI PV. PHASEOLICOLA IN BEAN VARIETIES ...................... 230 RESOLVING OF CERTAIN CONSERVED MIRNA IN OLEA EUROPAEA........................... 239 GENERATION OF RNASEQ DATA FOR CORYLUS EVELLANA L. ...................................... 249 PRODUCTION AND CHARACTERIZATION OF BIODIESEL FROM SCENEDESMUS QUADRICAUDA (TURPIN) ISOLATED FROM KANYE WATER RESERVOIR IN KANO STATE, NIGERIA ............................................................................................................................ 255 INVESTIGATION OF THE POTENTIAL PROTECTIVE EFFECT AGAINST DNA DAMAGE OF SOUR CHERRY PEELS WASTES FROM FRUIT JUICE PRODUCTION ... 264 INDUCTION MUTATION IN BREAD WHEAT (TRITICUM AESTIVUM L.) GENOTYPES FOR IMPROVE YIELD COMPONENT AND QUALITY PARAMETERS ............................. 269 IRRIGATION SCHEDULING OF COOL AND WARM SEASON TURFGRASSES IRRIGATED WITH SPRINKLER METHOD* ............................................................................ 281 EFFECT OF ALOE VERA AND MAP TREATMENTS ON SENSORY EVALUATION OF JUJUBE FRUITS DURING COLD STORAGE AND SHELF LIFE .......................................... 296

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 IN-SILICO SHOTGUN METAGENOMICS DATA COMPARISON EXPANDS THE NUMBER OF POSSIBLE CANDIDATES TO BE USED FOR BIOCONTROL OF FIRE BLIGHT DISEASE ........................................................................................................................... 301 ORGANIC DAIRY FARMING AND ANIMAL HOUSINGS ...................................................... 305 EFFECTS OF DIFFERENT APPLICATIONS AND TEMPERATURE LEVELS TO CUTTINGS ROOTING IN KIWIFRUIT (ACTINIDIA DELICIOSA A. CHEV.) ..................... 309 ADAPTATION OF THE SELECTED CHERRY LAUREL GENOTYPES IN ORDU (TURKEY) ......................................................................................................................................... 317 EVALUATION OF ERWINIA AMYLOVORA STRAINS BY CRISPR TECHNOLOGY ........ 324 CYTOTOXICITY OF NATURAL AND SYTNHETIC COLOURANTS USED IN TRADITIONAL MARDIN BLUE ALMOND TOFFEE............................................................... 331 YIELD AND YIELD COMPONENTS OF DIFFERENT SWEET POTATO (IPOMOEA BATATAS) GENOTYPES ................................................................................................................ 337 ROLE OF PROCESSING ON FLAVOR DEVELOPMENT IN MEAT ..................................... 341 THE EFFECT OF USING SPICES ON PHYSICAL, SENSORIAL PROPERTIES AND VITAMIN C CONTENT OF RED BEET CHIPS ......................................................................... 358 DETERMINATION OF QUALITY CHARACTERISTICS OF DIFFERENT SWEET POTATO GENOTYPES .................................................................................................................. 364 DETERMINING THE PERFORMANCE OF SOME NEW POTATO CULTIVARS AND CANDIDATES IN CENTRAL BLACK SEA REGION ............................................................... 368 EMPATHY AND PRO-SOCIAL BEHAVIOR IN RATS ............................................................. 372 ORGANIC DAIRY GOAT PRODUCTION MODEL FOR THE SOUTHEASTERN REGION IN TURKEY ...................................................................................................................................... 375 CONSERVATIONAL NOTES ON THE ACANTHOLIMON AVANOSICUM DOĞAN & AKAYDIN (AVANOS: NEVŞEHIR) IN TURKEY ....................................................................... 378 DETERMINATION OF CONSUMERS’ BEHAVIORS TOWARDS IRRADIATED FOODS 383 CALCULATION OF IBMR SCORE VALUES BY USING PYTHON PROGRAMMING LANGUAGE ...................................................................................................................................... 390 DEVELOPING A SOFTWARE BASED ON LEAFPACS2 CALCULATIONS BY USING PYTHON PROGRAMMING LANGUAGE ................................................................................... 394 ASSESSMENT OF ECOLOGICAL QUALITY BY USING AQUATIC MACROPHYTES IN LAKE AVLAN .................................................................................................................................. 398 IS SKIN PRICK TEST EFFECTIVE TO DETERMINE AEROALLERGENS? ...................... 407 ASSESSMENT OF GLOBAL DNA METHYLATION IN LUNG TISSUE OF RABBITS THAT EXPOSED ROCURONIUM DURING ANESTHESIA ................................................................ 410 EFFECT OF THE USE OF AUTOCHTHONOUS STARTER CULTURES ON THE COLOUR CHANGES THAT TAKE PLACE THROUGHOUT THE DRYING-RIPENING PROCESS OF GALICIAN CHORIZO SAUSAGE ................................................................................................ 414 CARBON NANOMATERIALS PRODUCTION FROM THE GAS PRODUCT OF COMPOSITE PACKAGING WASTE PYROLYSIS.................................................................... 421 LIFE CYCLE ASSESSMENT OF MULTI WALLED CARBON NANOTUBES SYNTHESIS VIA CHEMICAL VAPOR DEPOSITION ..................................................................................... 426

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 EFFECT OF THE ADDITION OF DATES ON THE PHYSICOCHEMICAL AND SENSORY CHARACTERISTICS IN RAW-CURED BEEF SAUSAGES ..................................................... 434 EFFECTS OF PROCESSING TECHNIQUES AND STORAGE ON OXIDATIVE STABILITY OF DIFFERENT OILS IN A CANNED EEL ................................................................................ 441 MACROPHYTE BIODIVERSITY OF THE NORTH AEGEAN BASIN IN TURKEY ........... 446 ADAPTATION OF NEW APRICOT VARIETIES FOR TURKEY ........................................... 460 OUR SPONSORS .......................................................................................................................... 467 Supporting Associations................................................................................................................ 473

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

LETHAL EFFECT OF TURKISH DIATOMACEOUS EARTH (K14) AGAINST ADULTS OF AMERICAN COCKROACHES (PERIPLANETA AMERICANA L.) Necati AL1, Hasan TUNAZ1*, Ali Arda IŞIKBER1, Mehmet Kubilay ER1 1

Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Plant Protection Department, Avşar Campus, 46100 Kahramanmaraş, Turkey * Corresponding author’s e-mail htunaz@ksu.edu.tr ABSTRACT

In this study, mortality effects of K14 which is local diatomaceous earths, were investigated against adults of American cockroach (Periplaneta americana (L.)) on concrete, ceramic floor tile and laminate flooring. On these three different surfaces, P. americana adults were exposed to K14 diatomaceous earth at the doses of 2.5, 5, 10, 20, 40, 80 and 100 g/m2 along 11 days. In all surface applications of K14 diatomaceous earth, exposure time and dose caused significant effect on mortality rates of P. americana adults. It was determined that K14 coded Turkish diatomaceous earth has the lowest mortality effect on all application surfaces at the dose of 2.5 g/m2 after 11 days. Starting from the 40 g/m2 dose of the K14 local diatomaceous earth, all doses have reached 100% mortality at the end of the eleventh day on all application surfaces. In general, the mortality activity of K14 diatomites against P. americana adults was found to be similar on all three surfaces at the end of the seventh to eleventh days. At the end of this study, local diatomaceous earth coded K14 was found to be good alternatives for controlling P. americana which is a medical pest insect. Keywords: Turkish diatomaceous earth, P. americana, Surface application INTRODUCTION The American cockroach is commonly found living area with people and scattered all over the world. It is also major carriers of pathogens and main source of allergens. Therefore, it is an important primary medical and economical insect pest. The cockroach is mainly controlled by synthetic insecticides (Rust et al., 1993). However, this cockroach widely developed resistance to these insecticides (Jialin et al., 2007). Hence, the development of new types of selective cockroach-control alternatives are needed. Diatomaceous earth (DE) is a component of organic origin and is a precipitate formed from the fossilized siliceous shells of algae that have lived in all aquatic ecosystems. The cell walls of the algae are amorphous cystite (SiO2 + H2O). Recent studies have shown that DE has a significant impact on warehouse pests (Waksh and Shabbir, 2005; Athanassiou et al., 2007). In addition to the insecticidal effect of DE, it can be used as filtration, absorbant, filling material in the industry, silicon reinforcing in humans, and as a moisture retainer in the packaging of nutrients (Durmuşkaya, 2009). Diatomaceous earth is probably the most effective of the natural powders that can be used as insecticides. The insecticidal effect of diatomaceous earth is regarded as a method of physical struggle because it does not have a chemical effect on insects. This physical struggle; DE has an effect on the insect cuticle and results in death of the insect's water loss (Ebeling, 1971). DE, besides its water absorber feature, can also abrade the oil quite well. For this reason it is also very effective on the protective waxy layer present on the insect cuticle. As a result, death in insects occurs due to water loss and drying (Cloarec et al., 1992). Furthermore, the killing effect of insects may vary significantly depending on the test conditions used, the diathermy (marine or freshwater diatoms), the geographical area taken, the 11

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

formulation process, the oil absorption capacity and the chemical / mechanical modification of the diatomaceous earths (Faulde ve ark., 2006). Therefore, in this study, mortality effects of K14 which is local diatomaceous earths, were investigated against adults of American cockroach (P. americana (L.)) on concrete, ceramic floor tile and laminate flooring. MATERIAL AND METHODS Insect Colonies of P. americana were reared in plastic containers (60 liter) and maintained at room temperature. The cockroaches were provided with water in glass tubes with cotton stoppers and dry dog food. Each container was provided with paper egg cartons as shelter. The adult cockroaches (5-10 days old) were tested for each bioassays at 25 ( 2) oC and 50 (5) % relatıve humidity. Local diatoms used in biological tests In this study, K14 coded local diatoms from Kayseri/Turkey province were used. At least 5 kg samples were taken from the diatom reserve. The samples were mixed in the gutters and brought to the laboratory. The diatom specimen brought in rock form is prepared in natural form. For natural (natural) preparation of the diatom specimen, it was dried at 100 ± 10 ° C for 2 hours, until it had a 3-5% moisture content in a controllable ventilated oven. After drying, the small pieces were grinded in a laboratory mill for 10 seconds at the highest speed. All samples were then sieved through a 100 mesh (149 μm) standard sieve and the damp, soft small pieces left under the sieve were dried in a ventilated oven at 40 ° C for 24 hours. Thus, natural powdered diatomaceous earth of a particle size of 149 microns or less is obtained. Surfaces used in biological tests During the test, concrete, ceramic, parquet surfaces were prepared in plastic boxes (100x100x60 mm) and ventilation holes were opened with the help of a needle to the cover parts of plastic boxes in order to provide air in and out during the established tests. Concrete Surface: The mortar obtained by using 200 g + 50 ml water was poured into plastic boxes (100x100x60 mm) and the mortar was obtained by drying. Ceramic Substrate: The ceramic surfaces used during this work were produced from a mixture of clay, kaolin, quartz, feldspar and limestone in sizes of 150x150x5.5 mm according to TS202 standards. The ceramic surfaces produced in TS202 standards are reduced in dimensions of 100x100 mm and working dimensions are obtained. Parquet Substrate: Laminated parcels which are manufactured according to the standards of High Density Fiberboard (HDF) and 717 E-1 are 8x195x1200 mm in size and 100x100 mm in size and working dimensions are obtained. Biological tests and test method Biological tests were carried out in the climate chamber with 25 ± 1 ° C and 65 ± 5% relative humidity. Water and feed were not given to insects during the experiment and insects were exposed to 2.5, 5, 10, 20, 40, 80 and 100 g/m2 doses of diatomaceous earth. Diatomaceous earth weighed with the aid of precision scales for dosing experiments is placed on concrete, ceramic and parquet surfaces. After the diatomaceous earth was distributed on the surface, ten newborn individuals from P. americana, which we cultured in the laboratory environment, were left. The experiments were carried out in four replicates, with 5 individuals each time. The control unit was also set up in four replications and no diathermy was applied, such as water and feed. 12

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

Dose experiments were taken into the climate chamber as soon as the establishment was established and dead-live insects were counted for eleven days. RESULTS AND CONCLUSIONS In this study, the mortality effect of seven different dosages of Turkish diatomaceous earth coded K14 against Periplaneta americana adults on three different surfaces (concrete, parquet, ceramic) was determined in laboratory conditions. In all surface applications of K14 diatomaceous earth, exposure time and dose caused significant effect on mortality rates of P. americana adults. It was determined that K14 coded Turkish diatomaceous earth has the lowest mortality effect on all application surfaces at the dose of 2.5 g/m 2. However, as the dose rate and exposure time increased, the efficacy of the local diatoma coded K14 increased. According to these determinations, it was determined that the effectiveness of K14 coded local diatom soil at 2.5 g / m2 dose on concrete, parquet and ceramics surfaces was low, but its effectiveness increased as the exposure time increased. It was determined that the effectiveness of K14 coded local diatom soil at the dose of 100 g / m2 on concrete, parquet and cercamic surfaces was high at the end of the eleventh day and had a 100% mortality rate at the end of the eleventh day and it was determined as the most effective dose value. At the end of the eleventh day on the ceramic surface of K14 coded local diatom, the efficacy of 2.5, 5, 10 and 20 g / m2 doses was low while the efficacy of all doses was high and the mortality rate reached to 100% from 40 g / m2 dose. All these results demonstrate that K14 encoded native diatomaceous earth has potential for use in the P. americana adult struggle and may be an alternative to synthetic instecticides with a broad-spectrum spectrum used in the struggle for this bug. However, diatom earth species should be demonstrated in a comprehensive study of the applicability of the American cockroach under natural habitat conditions and the determination of its interaction with other living factors outside cockroaches when applied in natural conditions. REFERENCES Athanassiou, C.G., N.G., Kavallieratos, C.M. Meletsis (2007). Insecticidal effect of three diatomaceous earth formulations, applied alone or in combination, against three storedproduct beetle species on wheat and maize. Journal of Stored Products Research. 43, 330334. Cloarec, A., C. Rivauit, F. Fontaine, A. Le Guyander (1992). Cockroaches as caries of bacteria in multifamily dwellings. Epidemiololgy and Infection 109, 483-490. Durmuşkaya, C. (2009). Nano teknoloji uzmanı, Diyatomeler. Bilim Teknik Dergisi (Ocak): 5659. Ebeling W., 1971. Sorptive dusts for pest control. Annual Review of Entomology, 16, 123-158. Faulde, M. K., J.J. Scharninghausen, S. Cavaljuga (2006). Toxic and behavioural effects of different modified diatomaceous earths on the German cockroach, Blattella germanica (L.)(Orthoptera: Blattellidae) Under Simulated Field Conditions. Journal of Stored products research, 42(3), 253-263. Rust, M.K., D.A. Reierson, B.C. Ziechner (1993). Relationship between insecticide resistance and performance in choice tests of field collected German cockroaches (Dictyoptera: Blattellidae). J.Econ. Entomol. 86, 1124–1130. JiaLin, Z., W. MingSheng, C. JianMing (2007). Resistance investigation of Blattella germanica to six insecticides and control strategy in Hefei city. Chinese. J. Vector Bio. Cont. 18, 9899.

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Wakil W., A. Shabbir 2005. Evaluation of diatomaceous earth admixed with rice to control Sitophilus oryzae (L.) (Coleoptera: Curcilionidae). Pakistan Entomologist, 27, 15-18.

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

DETERMINATION OF TOXICITY OF GASEOUS OZONE AGAINST NYMPH STAGES OF GERMAN COCKROACH (BLATELLA GERMANICA L.) Uğur GUZ1, Hasan TUNAZ1*, Mehmet Kubilay ER1, Ali Arda IŞIKBER1, İnanc Safak DOĞANAY1 1

Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Plant Protection Department, Avşar Campus, 46100 Kahramanmaraş, Turkey * Corresponding author’s e-mail: htunaz@ksu.edu.tr

ABSTRACT In this study under, the effects of two different concentrations of ozone gas (16.7 and 33.3 mg / L) against Blatella germanica nymphs at different exposure times (10, 20, 30, 40 and 50 minutes) were investigated laboratory conditions. It was determined that the ozone gas had important effect on mortality of B.germanica nymphs. In general, ozone gas caused higher paralyisis-mortality rates of B.germanica nymphs than mortality rates of B.germanica nymphs at both concentrations and all exposure times. A concentration of 33.3 mg / L of ozone gas with 40 and 50-minute exposure times killed all cockroach nymphs after 24 hours. On the other hand, only 16.7 mg / L concentration of ozone gas with 50 minute exposure time killed 100% of the B. germanica nymphs after 24 hours. In terms of the exposure time of the ozone gas to nymphs of B. germanica, the concentration of 33.3 mg / L. with 10-minute exposure times resulted 83% mortality, with 20-minute exposure times resulted 90 % mortality and with 3050-minute exposure times resulted 100 % mortality after 24 hours. 16.7 mg / L of ozone gas, the nymphs resulted in 73% mortality with 10-minute exposure times, 83% mortality with 2030-minute exposure times, and 100% mortality with 40-50-minute exposure times after 24 hours. All these results show that the ozone gas (33.3 mg / L) with 40-50-minute exposure times can successfully control B. germanica nymphs. Keywords: Ozone gas, Blatella germanica, Mortality, Biological efficacy

INTRODUCTION Cockroaches are an insect species that have remained unchanged since ancient times (Appel, 1995). There are approximately 3,500 species of cockroach in the world (Atkinson et al, 1991). Most types of cockroaches are insect species that live in outdoor environments. However, a few cockroach species are found in the living areas of insects. One of the cockraoch species found in people's habitats is the German cockroach, Blatella germanica (L.), which is one of the most common cockroach species all over the world. It is easily distributed when indoor and outdoor temperature and humidity are suitable for this species. In addition to the psychological harm to humans, the most important damage is a medical harm. cockraoches are vectors for many diseases such as bacteria, fungi, helminths, protozoa, viruses that cause disease in humans (Mullen et al., 2002). Cockracohes cause allergies, which are harmful to humans as well as diseases and spread to the world. Cockraoches also cause asthma in many people with allergies (Roberts, 1996). That is why, in addition to psychological harm, cockroaches are potential vectors of important diseases in humans and struggle must be done seriously. In the world, the control of German cockraoches is traditionally made with inorganic and synthetic organic insecticides (Rust et al., 1993). Due to all these adverse effects of the use of intensive chemical insectides in control of the German cockroach, 15

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an alternative control method which is not harmful to the environment, human and animal is needed. Ozone is a form of three atomic oxygen (O3) molecule. Ozone is produced as a bluish or colorless gas characterized by fresh clean odor in the air following the thunder storm. Ozone is an unbalanced gas and quickly converts oxygen to temperatures above 35 ° C. Therefore, it must be produced during use and can not be stored after it is produced. There is a striking characteristic odor that many people in the ozone can notice even at very low concentrations (0.02 ppm by volume) (Kim et al., 2003). Commercially, mostly ozone is produced with pure oxygen or airborne corona current generators (Kim et al., 2003). The control of insects with ozone gas has started with stored product pests. Isikber and Öztekin (2009) tested the ozone gas on some stored product pests. These are the application of ozone gas to the insects with products and without product environment. The toxicity data obtained from fumigating studies with ozone gas in unfilled environment (empty volume) revealed significant differences between ozone gas sensitivities of developmental periods of E. cautella and P. interpunctella. Ozone application in unfilled environment (empty volume) caused 100% death of E. cautella and P. interpunctella in all periods except egg period. The toxicity results obtained in study showed that T. confusum is generally more resistant to ozone gas than E. kuehniella. There is no study of the deadly effect of ozone gas on the German cockroaches until the date, so the possible use is not known. For this reason, in this study, it was aimed to determine the optimum ozone concentration and ozonation duration by using the osmotic gas in the nymph control of German cockroach. MATERIAL AND METHODS Insect Colonies of B. germanica were reared in plastic containers (60 liter) and maintained at room temperature. The cockroaches were provided with water in glass tubes with cotton stoppers and dry dog food. Each container was provided with paper egg cartons as shelter. The nymphal cockroaches were tested for each bioassays at 25 ( 2) oC and 50 (5) % relatıve humidity. Ozone gas fumigation chamber In empty volume applications, 3 liters of glass jars with a metal cover of 9 cm in diameter were used in all of the biological tests. These hatches have 2 entry pipes 0.5 cm in diameter and 3 cm in length. A 5 cm long silicone hose is connected to the end of each record, one of the hoses is connected to the vacuum pump and the other to the ozone generator. One of the hoses was provided with ozone gas from the other hole while the air in the glass jar where the biological tests were conducted was discharged. Thus, the ozone gas was periodically circulated in the glass jar. The adjustment of the ozone gas concentration is adjusted according to the flow rate of the pure oxygen gas. The flow rate of the oxygen gas is controlled by the flowmeter placed between the oxygen tube and the ozone generator. Biological tests and Empty volume applications For empty volume applications, biological tests were carried out in 3 l metal-lined glass jars (fumigation chamber) at a temperature of 26 ± 1 and 65 ± 5% relative humidity. In all tests, B. germenica 1-3. term nymphs were used. The nymphs used in the biological tests were placed in 3 l jars where ozone fumigation was carried out and 2-3 dogs were added to the bottles. The solution was prepared by adding 10 ml of purified water to 100 g of MgNO2 (Magnesium 16

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Nitrate) to keep the media noodle in the jars constant at 65 Âą 5%. This solution was soaked in a jar until wetted to a drying paper size of 5 x 2 cm. As the individuals exposed to ozone gas were placed in glass jars, the air in the jars used in the tests was evacuated to 760 mm Hg by low pressure pump (KNF, Germany). As the air in the jars is evacuated, the hoses on the covers of the jars are closed with the help of plastic clips to prevent gas in and out of the jars. After taking the air in the jars used in the biological tests, ozone gas is delivered to the ozone generator of the oxygen gas with the help of the flowmeter and ozone gas is produced. In order to produce ozone gas at different concentrations, the oxygen gas flow was set at 5 and 10 l / h and the flow rate was monitored from the flowmeter screen. When the desired flow rate is reached, the clips in the lids of the jars are opened and the produced ozone gas is directed to the fumigation chamber and the pressure inside the jar is filled with ozone gas until reaching normal pressure conditions. Since ozone gas is not a stable gas, it quickly transforms into oxygen form due to the effect of temperature and relative huminity. Therefore, ozone gas application has been completely applied to biological tests. In empty volume applications, application times were determined as 10, 20, 30, 40 and 50 minutes and the air in the jars where the tests were carried out once every 30 minutes in 40 and 50 minute applications was evacuated by vacuum pump and ozone gas was applied again. Experiments were carried out in 3 replicates of 10 individuals each time, leaving 3 controls for each trial. Upon completion of the application period, the ozone gas applied jars were ventilated and the insects were removed from the jars. The nymphs exposed to ozone gas were placed in 1 l glass jars and 2-3 dogs that were not exposed to ozone gas were added to the bottles. Dead and alive individuals were counted 1 hour, 6 hours and 24 hours after the opening of the experiments. RESULTS AND CONCLUSIONS In this study, the toxicity of ozone gas against nymphs of Blatella germanica was demonstrated at two different application concentrations and different application times. In this study, as a result of the biological tests, ozone gas was generally observed in both the ozone gas concentration and the paralysis-mortality rate in all the application periods in the nymphs of B. germanica; was higher than the death rate. This has shown that the ozone gas is a knockdown feature on this insect and that it died shortly after death. When the concentration of 33.3 mg / L of ozone gas was applied to nymphs of B. germanica with 40 and 50 minutes, respectively, it killed all cockroaches after 24 hours of application. On the other hand, when a concentration of 16.7 mg / L of ozone gas was applied to B. germanica nymphs for 50 minutes, it killed 90% of the nymphs of B. germanica after 24 hours of application. When the ozone gas is evaluated in terms of exposure time to B. germanica nymphs, the concentration of 33.3 mg / L of ozone gas with 10-20 minute exposure times caused 65 % the nymphs mortality, with 30 minute exposure time caused 90% nymphs mortality and with 50 minute exposure times caused 100 % nymphs mortality after 24 hours. At a concentration of 16.7 mg / L of ozone gas, as the exposure times increased, the nymph mortalities gradually increased after 24 hours and the nymph mortality reached 90% with 50 minute exposure times. At a concentration of 16.7 mg / L of ozone gas, as the exposure times increased, the nymph mortalities gradually increased after 24 hours and the nymph mortality reached 90% with 50 minute exposure times. All the results have shown that ozone gas has potential for controlling of B. germanica and may be an alternative to the synthetic chemicals used in the control of this insect. However, a comprehensive study of the ozone gas's applicability in the natural habitat of the German cockroach and the determination of its effect on live and non-cockroach factors when applied in natural conditions is necessary.

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REFERENCES Appel, A.G. (1995). Blattella and related species, pp. 1-19. In M. K. Rust, J. M.Owens, and D. A. Reierson [eds.], Understanding and controlling the German cockroach. Oxford University Press, New York. Atkinson, T.H., P.G. Koehler, R.S. Patterson (1991). Catalog and atlas o f the cockroaches (Dictyoptera) o f the North America north o f Mexico. Entomol. Soc. Am. Miscell. Publication. 78,1-86. Isikber, A.A., S. Oztekin (2009). Comparison of susceptibility of two stored product insects, Ephestia kuehniella Zeller and Tribolium confusum du Val to gaseous ozone. Journal of Stored Products Research 45, 159-164. Kim, J.G., A.E. Yousef, M.A. Khadre (2003). Ozone and its current and future exposure in the food industry. Advances in Food and utrition Research 45: 167-218. Mullen, G., D. Lance, C. Cameron, P. Daniel, L. Lynsey, G. Michael, E. Rebecca (2002). Medical and Veterinary Entomology. Academic Press. 0-12-510451-0. ss. sf.32. Amsterdam. Roberts, J. (1996). Cockroaches linked with asthma. Br Med J. 312 (7047), 1630. Rust, M.K., D.A. Reierson, B.C. Ziechner (1993). Relationship between insecticide resistance and performance in choice tests of field collected German cockroaches (Dictyoptera: Blattellidae). J.Econ. Entomol. 86, 1124â&#x20AC;&#x201C;1130.

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BIOLOGICAL EFFICIENCY OF TREATMENT OF OZONE GAS AGAINST PLODIA INTERPUNCTELLA (HÜBNER) (LEPIDOPTERA: PYRALIDAE) (INDIAN MEAL MOTH) IN HAZELNUT Ali Arda IŞIKBER1*, Hasan TUNAZ1, Mehmet Kubilay ER1 1

Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Plant Protection Department, Kahramanmaraş, Turkey *

Corresponding author: aaisikber@gmail.com

ABSTRACT In biological tests conducted in presence of hazelnuts, ozone gas at different concentrations (16.7, 33.3 and 66.6 mg/L) were exposed to all biological stages (egg, larva, pupa and adult) of Plodia interpunctella placed at top and bottom of the commodity for various exposure periods (2, 4 and 6 hours). In biological tests conducted in presence of hazelnut, 100% mortalities of all biological stages of P. interpunctella placed at top of the commodity were obtained at tested ozone concentrations and exposure periods. Generally, the mortalities of all life stages of P. interpunctella placed at bottom of the commodity for ozone treatments were lower than those placed at top of the commodity. It was easy to kill the pupae and adults of P. interpunctella placed at bottom of the commodity while the ozone treatments resulted in low mortalities of the eggs and larvae placed at bottom of the commodity. Just as 100% mortalities of the larvae and adults were not obtained even at the highest ozone concentration for the longest exposure period. In conclusion, in this study, it was observed that ozone gas only at high concentrations can control all biological stages of P. interpunctella in hazelnut and therefore could have an alternative potential for methyl bromide in quarantine applications in short application period. Keywords: Plodia interpunctella, Ozone gas, Hazelnut, Fumigation

INTRODUCTION

Hazelnut production is an important agricultural activity in Turkey. Hazelnut cultivation is mainly performed on steeplands in Black Sea region of Turkey and it has been an important source of income for a large number of family farms (Dikmen, 1999). Turkey produces 73% of world production and exports 84% of its production, which accounts for around 20% of total agricultural exports from Turkey (Fiskobirlik, 2003). Storage pests infesting hazelnut especially during drying and storage period may cause significant problems in hazelnut sector. The fig moth (Ephestia cautella (Walker) and Indianmeal moth (Plodia intepunctella (Hubner) reduce fruit quality by feeding and damaging the fruit and contaminating by leaving its excretions and other residues as silky net weaves (Damarlı et al., 1997). Large populations can develop before being detected and severe damage may occur rapidly (Jarratt, 2001). Moreover, from a phytosanitary point of view, during export, the presence of insects, or their fragments, has cost inestimable losses, due to cargo returns. Ozone is a triatomic form of oxygen (O3) and is referred to as activated oxygen, allotropic oxygen or pure air. It is an unstable gas and its life span lasts about 20 minute, depending on the temperature. Electrical generation of ozone eliminates the handling, storage, and disposal problems of conventionally used post-harvest pesticides. Attractive aspect of ozone is that it 19

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decomposes rapidly (half-life of 20-50 min) to molecular oxygen without leaving a residue. These attributes make ozone an attractive candidate for controlling insects and fungi in stored products. Ozone in its gaseous form has been also considered to have potential to kill insect pests in commodities and was subjected of several research studies (Erdman, 1980; Mason et al., 1997; Kells et al., 2001). High mortality was achieved for adults of the maize weevil, Sitophilus zeamais (Mostsch.), and the red flour beetle Tribolium confusum (Jacqueline du Val), and the larval stage of the Indian meal moth, P. interpunctella exposed to lower ozone concentrations ranging from to 5 to 45 ppm (Erdman, 1980; Kells et al., 2001). Methyl bromide has frequently used as a fumigant for disinfestations of other stored agricultural commodities such as nuts, cereals and fruits since it kills the insects rapidly, has a wide spectrum of activity and relatively low-cost (Fields and White, 2002). However it was banned according to the Montreal Protocol on Substances that Deplete Ozone Layer (Schneider et al., 2003) except quarantine, laboratory and pre-shipment purposes. As a consequence, there is a critical need to develop new fumigants for quarantine purposes, where rapid insect mortality is required (exposure time less than 1 day). Objective of present study was to determine toxicity of ozone at high concentrations and short exposure time against all life stages of P. interpunctella in hazelnut under laboratory conditions. MATERIALS AND METHOD Test Insects Biological tests were carried out on all life stages (adult, larva, pupa and egg) of Plodia interpunctella. Plodia interpunctella was reared yeast at 26  1C and 65  5% r.h. on diet of a 10:2:1 mixture of wheat flour: wheat germ: dried brewers. Eggs aged 1-2 days in 9 cm Petri dishes were placed in 3 litter jars and then were exposed to the treatments. Larvae were removed from culture jars and exposed to the treatments 21 days after oviposition. Two days old pupae were obtained by daily separation from culture jars and were exposed to the treatments. Newly emerged, aged 0-1 day, adults were placed in culture jars and then were exposed to the treatments. Commodity In-shell hazelnuts with m.c. of 10.5%0.5 were used in the tests. In order to minimize the reaction of microbial loads in the commodity with ozone the hazelnuts used in the tests were sterilized under high pressurized stem. Fumigation Chambers Test chambers consisted of 3-liter glass jar, each capped with a metal stopper equipped with entry and exit tubing. Two pieces of rubber tubing, 5 cm long, 6.2 mm ID, were attached to the tubing and sealed with pinch-clamps. The desiccators were sealed with silicone vacuum grease. Ozone Fumigation Procedures Ozone generator in laboratory scale was provided from the company Ozomax Inc., Ozone gas was generated using a laboratory corona discharge ozone generator (Model OZO-1VTT) from purified extra dry oxygen feed gas. Ozone was introduced as gaseous into the exposure jars using a ozone generator. Pressure in each jar was measured using a 0 to 800 mm Hg vacuum digital gauge. The 100 mm Hg measure referred to herein is absolute pressure, with 760 mm Hg considered as atmospheric pressure. Prior to each test, twenty larvae, pupae or adults were 20

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confined, separately, inside 3 cm diameter by 8 cm long wire-mesh cages. For eggs, fifty eggs placed in opened Petri dishes were used per fumigation. For intermittently repeated ozone treatment in presence of commodity, each desiccators was loaded separately with 1.3 kg of in-shell hazelnut, and then 50 eggs, 25 pupae, adults and larvae confined inside the wire-mesh cages were inserted into top and bottom position of the commodity the desiccators were briefly evacuated to 760 mm Hg. Afterwards ozone gaseous at concentrations of 16.7, 33.3 and 66.6 mg/L was flushed into exposure jar until reaching atmospheric pressure and it was repeated every half an hour for 2, 4 and 6 hours. Untreated control insects were exposed to atmospheric conditions. Each test was replicated at three times. For all ozone fumigations, temperature and relative humidity were maintained at 261C and 655% at atmospheric pressure, respectively. Data Processing and Analysis After each treatment, larvae, pupae, and adults were transferred to 250-mL jars containing standard diets and were held at 26  1C and 65  5% r.h. until examined for mortality. The eggs in their Perspex slides were held under the same conditions until the oviposition sites were examined for egg hatch. Mortality data was subjected to Arcsin transformation and then, were analyzed using one-way analysis of variance (ANOVA). The means were separated using the LSD method at 5% level. RESULTS AND DISCUSSION In biological tests conducted in presence of hazelnuts, 100 % mortalities of all biological stages of P. interpunctella placed at top of the commodity were obtained at tested ozone concentrations for 6-h exposure time. Generally, it was easier to kill the adults and pupae than the larvae and eggs (Table 1, 2 3 and 4). While it was possible to kill 100 % of the adults and pupae placed at bottom of the commodity at tested ozone concentrations and exposure periods, 100 % mortality of the larvae and eggs were not obtained at any of the ozone treatments. There was significant difference in susceptibility of biological stages of P. interpunctella to ozone treatments and the mortalities of the adults and pupae had higher than those of the larvae and eggs. Table 1. Mortality (%) of Plodia interpunctella adults placed at top and bottom position after 2, 4 and 6-h exposure intermittently ozone gaseous treatment at different concentrations in presence of 1.3 kg of in-shell hazelnut Ozone concentration

Insect Position

66.6 mg/L

Top Bottom

33.3 mg/L 16.7 mg/L Control

Top

Mortality rate (%) ± S. Error Exposure time (h) 2h 4h 6h 100±0 A 100±0 A 100±0 A 91.7±3.3 BC 100±0 A 100±0 A 100±0 A 73.3±3.3 CD

100±0 A 96.7±1.6 A

100±0 A 100±0 A

93.3±4.4 AB

100±0 A

Bottom

48.3±1.6 D

83.3±1.6 B

98.3±1.6 A

Top Bottom

0±0

Bottom Top

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F and P value

F5,12= 32.66 P<0.0001

F5,12= 28.28 P<0.0001

F5,12= 0.458 P=1

Table 2. Mortality (%) of Plodia interpunctella larvae placed at top and bottom position after 2, 4 and 6-h exposure intermittently ozone gaseous treatment at different concentrations in presence of 1.3 kg of in-shell hazelnut Ozone concentration 66.6 mg/L 33.3 mg/L 16.7 mg/L Control F and P value

Insect Position

Mortality rate (%) ± S. Error Exposure time (h) 4h 6h 88.3±3.3 A 100±0 A 56.6±.0 ABC 94.9±2.9 AB 73.3±8.8 AB 100±0 A 40±5.7 BC 47.5±6.1 C

Top Bottom Top Bottom

2h 15±7.6 A 8.3±1.6 A 11.6±.2 A 6.6±1.6 A

Top

6.6±4.4 A

51.7±10.1 BC

81.0±6.9 B

Bottom

1.6±1.6 A

33.3±8.8 C

40.7±8.9 C

Top Bottom -

1.6±1.6 6.6±1.6 F5,12=1.10 P=0.4099

1.6±1.6 6.6±1.6 F5,12=7.67 P<0.01

1.6±1.6 6.6±1.6 F5,12= 28.69 P<0.0001

Generally, the mortalities of all life stages of P. interpunctella placed at bottom of the commodity for ozone treatments were lower than those placed at top of the commodity. It was easy to kill the pupae and adults of P. interpunctella placed at bottom of the commodity while the ozone treatments resulted in low mortalities of the eggs and larvae placed at bottom of the commodity. Just as 100 % mortalities of the larvae placed at bottom of the commodity were not obtained even at the highest ozone concentration for the longest exposure period. Table 3. Mortality (%) of Plodia interpunctella pupae placed at top and bottom position after 2, 4 and 6-h exposure intermittently ozone gaseous treatment at different concentrations in presence of 1.3 kg of in-shell hazelnut Ozone concentration

66.6 mg/L 33.3 mg/L 16.7 mg/L

Insect Position 2h

Mortality rate (%) ± S. Error Exposure time (h) 4h

Top

90±5 A

100±0 A

Bottom

78.3±6.0 AB

100±0 A

Top

80±7.6 AB

100±0 A

Bottom

73.3±8.3 AB

93.3±3.3 A

94.9±2.9 AB

Top

60±0 BC

96.6±3.3 A

100±0 A

Bottom

45±5 C

58.3±8.8 B

81.3±7.3 B

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Control F and P value

Top

0±0

Bottom

1.6±1.6

F5,12=4.84

F5,12=14.17

F5,12=9.08

P<0.05

P<0.0001

P<0.001

Table 4. Mortality (%) of Plodia interpunctella eggs placed at top and bottom position after 2, 4 and 6-h exposure intermittently ozone gaseous treatment at different concentrations in presence of 1.3 kg of in-shell hazelnut Ozone concentration

66.6 mg/L 33.3 mg/L 16.7 mg/L Control F and P value

Insect Position Top Bottom Top Bottom Top Bottom Top Bottom -

Mortality rate (%) ± S. error Exposure time (h) 2h 90±5 A 78.3±6.0 AB 80±7.6 AB 73.3±8.3 AB 60±0 BC 45±5 C 0±0 1.6±1.6 F5,12=4.84 P<0.05

4h 100±0 A 100±0 A 100±0 A 93.3±3.3 A 96.6±3.3 A 58.3±8.8 B 0±0 1.6±1.6 F5,12=14.17 P<0.0001

6 hour 100±0 A 100±0 A 100±0 A 94.9±2.9 AB 100±0 A 81.3±7.3 B 0±0 1.6±1.6 F5,12=9.08 P<0.001

Conclusion In this study, it was observed that ozone gas only at high concentrations can control all biological stages of P. interpunctella in hazelnut and therefore could have an alternative potential for methyl bromide in quarantine applications in short application period. Acknowledgments: This study was a part of a project granted by The Scientific and Technological Research Council of Turkey (TÜBİTAK) with project number 109O802. REFERENCES Damarlı, E., Gün H., Özay G., Bülbül S., Oechsle P., 1997. An alternative method instead of methyl bromide for insect disinfestations on dried figs: controlled atmosphere, Acta Hort., 480: 209-215. Dikmen, N., 1999. The Importance of Hazelnut in Black Sea Coast and Turkish Economy; Problems and Recommendations. Agricultural Production and Marketing Symposium in the Black Sea Region, Directorate of Black Sea Agricultural Research Institute, Samsun, Turkey pp. 289-297 (in Turksih). Erdman, H.E. 1980. Ozone toxicity during ontogeny of two species of flour beetles, Tribolium confusum and T. castaneum. Environmental Entomology, 9: 16-17.

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Fields, P.G., White N.D.G., 2002. Alternatives to methyl bromide treatments for stored-product and quarantine insects, Annual Review of Entomology, 47: 331-359. Fiskobirlik, 2003. World Hazelnut Production and World Hazelnut Export. Webpage of Agricultural Sales Cooperative Association (Fiskobirlik) Statistics. Http://www.fiskobirlik.org.tr/istatis.htm. (In Turkish). Jarratt, J.M., 2001. Pest management principles: Industrial, institutional and structural pest control, http://msucares.com/publications/p2247ch7.pdf. Kells, S.A., Mason L.J., Maier D.E., Woloshuk C.P., 2001. Efficacy and fumigation characteristics of ozone in stored maize, Journal of Stored Products Research, 37: 371382. Mason, L.J., Woloshuk C.P., Maier D.E., 1997. Efficacy of ozone to control insects, moulds and mycotoxins. In: Donahaye, E.J., Navarro, S., Varnava, A. (Eds.), Proceedings of the International Conference on Controlled Atmosphere and Fumigation in Stored Products. Nicosia, Cyprus Printer Ltd., Nicosia,. pp. 665-670. Schneider, S.M., Rosskopf E.N., Leesch J.G., Chellemi D.O., Bull C.T., Mazzola M., 2003. Research on alternatives to methyl bromide: pre-plant and post-harvest. Pest Management Science, 59: 814-826.

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INSECTICIDAL ACTIVITY OF LOCAL DIATOMACEOUS EARTH IN COMBINATIONS WITH ENTOMOPATHOGENIC FUNGUS, BEAUVERIA BASSIANA (BALS.) VUILL. AGAINST RHYZOPERTHA DOMINICA (F.) Ali Arda IŞIKBER1*, Hasan TUNAZ1, Mehmet Kubilay ER1, Özgür SAĞLAM2 1

Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Plant Protection Department, Kahramanmaraş, Turkey 2

Tekirdağ Namık Kemal University, Agriculture Faculty, Plant Protection Department,Tekirdağ, Turkey *

Corresponding author’s e-mail: aaisikber@gmail.com ABSTRACT

In this study, effectiveness of local diatomaceous earth (DE) in combinations with entomopathogenic fungus, a local isolate of Beauveria bassiana (Bals.) Vuill. (EP) against stored grain insect, Rhyzopertha dominica (F.) was determined. Biological tests were carried out to determine insecticidal activity of 250 and 500 ppm concentrations of DE alone, 150 and 300 ppm of EP alone and their binary combinations against R. dominica adults on wheat. The mortality of R. dominica 7 and 14 days after the treatments and their F1 progeny densities 65 days after the complete of each biological test were determined. All treatments of DE alone (250 and 500 ppm DE) resulted in low mortalities of R. dominica adults. After 7 days of the treatments, all combinations of DE and EP resulted in higher mortalities of R. dominica than DE and EP alone. However, treatments did not produce complete mortality of R. dominica adults after 7 days of the treatments. Whereas, all combinations of DE and EP, except treatment at the highest concentration of EP (300 ppm), resulted in significant increase of mortality of R. dominica adults after 14 days of the treatments. The complete mortality of R. dominica adults were obtained at only combination of 300 ppm EP and 500 ppm DE after 14 days of treatment. All combinations of DE and EP significantly reduced F1 progeny production compared with that at control. However, all combinations of DE and EP, except combination of 300 ppm EP and 250 or 500 ppm DE, did not completely prevent progeny production of R. dominica. In conclusion, this study indicated that binary combination of local diatomaceous earth and B. bassiana local isolate would have potential to be used for control of stored grain insects. Keywords: Local diatomaceous earth, entomopathogenic fungus Beauveria bassiana, Rhyzopertha dominica INTRODUCTION The use of diatomaceous earth (DE) is one of the most promising alternatives to insecticides in stored grains. The insecticidal activity of DE currently mined varies depending upon diatom species composition, geological and geographical origin as well as certain physical and chemical characteristics, such as particle size, silicon dioxide content, pH and density (Korunic, 1997). DEs are natural resource-based dry substances that can be used as insecticides (Korunic, 1998). They act on the insect cuticle by absorbing the lipids or cuticular abrasion, resulting in insect death through rapid desiccation. DEs are nontoxic to mammals (acute oral toxicity for the rats; LD50 > 5 g/kg of body weight), leave no toxic residues on the commodity 25

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and according to the US Environmental Protection Agency. Since DEs are classified in the category of GRAS (generally recognized as safe) they are used as food or feed additives (FDA, 1995). Regarding their insecticidal use, there is no specialized equipment required for DE treatment since they can be applied with the same application technology as other grain protectants (Athanassiou et al., 2005). They persist in the treated commodity, providing a longterm protection against insect pests, which is currently a major problem for the use of synthetic pesticides. Eventually, DEs are also completely compatible with organic food production (Subramanyam & Roesli, 2000). Several DE formulations, based on natural deposits, are now commercially available, and have proven to be very effective against stored-grain pests (Subramanyam & Roesli 2000; Athanassiou et al., 2011). However, the investigation to discover new naturally-occurring DEs that are more effective in insect control is still in progress, especially in areas rich in siliceous rocks. Based on the initial evidence and preliminary results, Turkey is considered to have rich natural DE deposits, and there is clear evidence for the existence of large DE deposits in different regions of Turkey (Özbey & Atamer, 1987; Mete, 1988; Sıvacı & Dere, 2006; Çetin & Taş, 2012). Diatomite reserves of Turkey are about 125 Gt. Hırka (Kayseri) in Turkey has the largest diatomite reserve (106 Gt) (Çetin & Taş, 2012). However, there are only a few published reports on the potential use of local DEs against stored-product insect pests. Doğanay (2013) reported that 750 and 1000 ppm of a Turkish DE formulation (Turco 1) applied to wheat and paddy rice resulted in high mortalities of Sitophilus granarius (L., 1758) (Coleoptera: Curculionidae) and Rhyzopertha dominica (F., 1792) (Coleoptera: Bostrichidae) (lesser grain borer) adults and significantly reduced progeny production, while 500 ppm and lower concentrations of Turco 1 had low efficacy against both tested insects and did not prevent reproduction. Akçalı et al. (2018) investigated efficacy of nine local DEs collected from different regions of Turkey against stored-grain insects, Sitophilus oryzae (L., 1763) (Coleoptera: Curculionidae) (rice weevil), Tribolium confusum du Val., 1863 (Coleoptera: Tenebrionidae) (confused flour beetle) and R. dominica and reported that CB2N-1 and BGN-1 local DEs had high efficacy against S. oryzae, T. confusum and R. dominica adults and thus have potential to be successfully used for controlling stored-grain insect pests as a grain protectant. Şen et al. (2019) Turkish DE formulation (ACN-1) provide complete mortality of S. oryzae and T. confusum adults even at its low concentration (500 ppm). However, it did not provide a satisfactory level of efficacy against R. dominica adults even at high concentration (1000 ppm). Sitophilus oryzae was the most DE susceptible, followed by T. confusum and R. dominica. It seems that R. dominica was the most tolerant species to the local DE. Rhyzopertha dominica adults are not very agile (Flinn & Hagstrum, 2011), so the possibility of picking up DE particles is decreased and this might be why this species is among the most DE tolerant species (Korunic, 1998; Fields & Korunic, 2000). Kavallieratos et al. (2005) also reported that 750 ppm DEs (SilicoSec and Insecto) were needed to obtain high mortality of R. dominica adults in wheat and maize. On the other hand, Er et al. (2018) reported that local isolates of B. bassiana resulted low mortalities of S. oryzae and T. confusum adults even at its high concentration (1000 ppm). However, it provided higher mortality levels of R. dominica adults. The key question is to answer if there is an increased efficacy of binary combination of Turkish DE formulation and B. bassiana local isolate against R. dominica adults. Therefore, objective of our study was to test the efficiency of binary combinations of Turkish DE formulation (AGN1) and entomopathogenic fungus (EP), B. bassiana local isolate (isolate code of 139756) against, R. dominica adults under laboratory conditions.

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MATERIALS AND METHOD Test insect Rhyzopertha dominica adults used in the bioassays were taken from a culture that was kept in the laboratory on whole wheat at 30 ± 1ºC, 65 ± 5 % RH. Seven to 10 d-old adults of R. dominica were exposed to DE treatments in bioassays. Wheat cultivar Untreated, clean, low admixture (0.8%) and infestation-free soft wheat (Triticum aestivum L., cv. Elbistan Yazlığı) was used in bioassays. One g of wheat corresponded to 21.3 individual grains. The moisture content of wheat used in bioassays, as determined by a DickeyJohn moisture meter (DickeyJohn Multigrain CACII, DICKEY-John Co., Lawrence, KS), ranged between 11.0 and 11.4%. Local diatomaceous earth formulation A local DE formulation (AGN-1) was used in bioassays. The DE was collected from diatomite reserves in Ankara Province located in Central Anatolia, Turkey. With the DE samples were crashed coarsely with a laboratory type knife hammer mill (LB 160, Mertest, Eskişehir, Turkey), they were oven dried oven (UF260 Memmert, Germany) at 100ºC for 48 h to give 3-4% moisture content. These samples were ground to powder using the same laboratory mill. With the DE powder was sieved by using metal sieve with 140 µm diameter (Retsch, Germany), natural grade local DE was obtained and used in bioassays. Some physical and chemical properties of local DE formulation (AGN-1) used in bioassays are given in Table 1. Table 1. Some physical properties of local diatomaceous earth formulation (AGN-1) DE formulation

SiO2 rate (%)

Mean Particle Diameter (µm)

Adherence of DE on wheat kernels (%)

Colour

AGN-1

81.7

16.2

75.2

Yellowishwhite

Entomopathogenic fungus, Beauveria bassiana local isolate A local isolate of entomopathogenic fungus, B. bassiana (EP) with code of 139756, which was isolated from dead insects found in wheat samples collected from Kahramanmaraş province and proven to be biologically effective against of R. dominica and O. surinamensis adults in preliminary tests carried out in the laboratory was used in the biological tests. Bioassays In bioassays, following treatments were performed with five replicates at 26±1ºC, 65±5% RH.;  250 and 500 ppm concentrations of DE alone (mg DE/ kg wheat)  150 and 300 ppm of EP alone (mg EP conidia spore/ kg wheat)  Binary combinations of:  150 ppm EP+250 ppm DE and 150 ppm EP + 500 ppm DE  300 ppm EP + 250 ppm DE and 300 ppm EP +500 ppm DE For each experiment (DE and EP alone and their combinations), five samples of 50 g wheat were taken. Each grain sample was placed in a small cylindrical self-standing centrifuge tube that was closed, apart from a hole 1.5 cm in diameter (at the top of the tube), and that was covered with muslin cloth to allow sufficient ventilation. The grain samples were treated 27

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individually with the respective DE, EP concentration and their binary combinations and then shaken manually for 5 min to achieve equal distribution of DE and EP dust in the entire grain quantity. Fivee additional tubes containing untreated wheat served as control in each case. Subsequently, 30 1-wk-old adults of R. dominica were introduced into each tube. All tubes containing DE- and EP-treated wheat were placed in the lockable 80 l (26 × 36.5 × 15 cm) plastic container. The plastic containers were then placed in the incubator (IPP55 Plus, Memmert, Germany) set at the desired temperature. Five replicates were used for each trial (DE and EP alone and their combinations). Dead adults of three species were counted 7 and 14 d after DE and EP alone and their binary combination exposure. Temperature and relative humidity during bioassays were monitored by using HOBO digital recorders (HOBO H8, Onset Computers, Bourne, MA, USA). After the last count for mortality (14 d for R. dominica), all adults (dead and alive) were removed from the DE- and EP-treated vials, and the vials were left in the incubators for an additional period of 45 d. Afterwards, the newly emerged R. dominica individuals were counted and recorded as F1 progeny production. Data analysis Generally, for both species, the control mortality was very low, but where it was considered necessary the mortality counts were corrected by using the formula of Abbot (1925). All mortality data for each treatment were normalized using arcsine transformation and then subjected to one-way ANOVA by using the GLM Procedure of SAS/STAT® 12.1 (SAS Institute, 2012). For the progeny production counts, the same procedure was followed. Mean mortality percentages and number of adult progeny for each treatment were separated by using the Tukey multiple range test at 5% significance level. RESULTS AND DISCUSSION Two hunred fifty ppm DE and 150 ppm DE alone treatments resulted in low mortalities of R. dominica. Binary combination of 150 ppm EP and 250 ppm DE resulted in higher mortalities of R. dominica than 250 ppm DE and 150 ppm EP (Figure 1). Athough binary combination of 150 ppm EP and 250 ppm DE resulted in almost 90% mortality of R. dominica after 14 days of treatment, none of the treatments did not achieved complete mortalities of R. dominica (Figure 1).

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Rhyzopertha dominica 100,00 90,00

250 ppm DE 150 ppm EP

Mortality rate (%)

80,00

150 ppm EP + 250 ppm DE

70,00

60,00

B 50,00 40,00

30,00 20,00

B B

10,00 0,00 7. day

14.day

Exposure time (day)

Figure 1. Mortality rate of Rhyzopertha dominica adults exposed to 250 ppm diatomaceous earth (DE) alone and 150 ppm entomopathogenic fungus, Beauveria bassiana (EP) alone and binary combination of 250 ppm diatomaceous earth (DE) and 150 ppm entomopathogenic fungus Beauveria bassiana (EP) Five hundred ppm DE and 150 ppm EP alone treatments resulted in low mortalities of R. dominica. Binary combination of 150 ppm EP and 500 ppm DE resulted in higher mortalities of R. dominica than 500 ppm DE and 150 ppm EP alone treatments (Figure 2). Athough binary combination of 150 ppm EP and 500 ppm DE resulted in over 90% mortality of R. dominica after 14 days of treatment, none of the treatments did not achieved complete mortalities of R. dominica (Figure 2).

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019

Rhyzopertha dominica 100,00 90,00

Mortality rate (%)

80,00

500 ppm DE 150 ppm EP 150 ppm EP + 500 ppm DE

A 70,00

60,00

B 50,00

40,00 30,00 20,00

B B

10,00 0,00 7. day

14.day

Exposure time (day)

Figure 2. Mortality rate of Rhyzopertha dominica adults exposed to 500 ppm diatomaceous earth (DE) alone and 150 ppm entomopathogenic fungus, Beauveria bassiana (EP) alone and binary combination of 500 ppm diatomaceous earth (DE) and 150 ppm entomopathogenic fungus Beauveria bassiana (EP) Two hunred fifty ppm DE alone treatments resulted in low mortalities of R. dominica while 300 ppm EP alone produced the higher mortalities. Binary combination of 300 ppm EP and 250 ppm DE resulted in higher mortalities of R. dominica than 250 ppm DE (Figure 3). However, and 300 ppm EP alone treatment had similiar mortality to combination of 300 ppm EP and 250 ppm DE. Athough 300 ppm EP alone and combination of 150 ppm EP and 500 ppm DE resulted in over 80% mortality of R. dominica after 14 days of treatment, none of the teratments did not achieved complete mortalities of R. dominica. Five hundred ppm DE alone treatments resulted in low mortalities of R. dominica while 300 ppm EP alone produced the higher mortalities. Binary combination of 300 ppm EP and 500 ppm DE resulted in higher mortalities of R. dominica than 250 ppm DE alone (Figure 4). However, and 300 ppm EP alone treatment had similiar mortality to combination of 300 ppm EP and 500 ppm DE. However, only combination of 300 ppm EP and 500 ppm DE resulted in complete mortality of R. dominica after 14 days of treatment (Figure 4).

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Rhyzopertha dominica 100,00

80,00

250 ppm DE 300 ppm EP 300 ppm EP + 250 ppm DE

70,00

Mortality rate (%)

90,00

60,00

50,00 40,00

30,00 20,00

10,00 0,00 7. day

14.day

Exposure time (day)

Figure 3. Mortality rate of Rhyzopertha dominica adults exposed to 250 ppm diatomaceous earth (DE) alone and 300 ppm entomopathogenic fungus, Beauveria bassiana (EP) alone and binary combination of 250 ppm diatomaceous earth (DE) and 300 ppm entomopathogenic fungus Beauveria bassiana (EP)

Rhyzopertha dominica 100,00

90,00

Mortality rate (%)

80,00

500 ppm DE 300 ppm EP 300 ppm EP + 500 ppm DE

A A

70,00 60,00

50,00

40,00 30,00 20,00

C 10,00

0,00 7. day

14.day

Exposure time (day)

Figure 4. Mortality rate of Rhyzopertha dominica adults exposed to 500 ppm diatomaceous earth (DE) alone and 300 ppm entomopathogenic fungus, Beauveria bassiana (EP) alone and binary combination of 500 ppm diatomaceous earth (DE) and 300 ppm entomopathogenic fungus Beauveria bassiana (EP) All binary combinations of DE and EP significantly reduced F1 progeny production compared with that at both DE and EP alone treatments and control treatment (Figure 5). However, all binary combinations of DE and EP did not completely prevent F1 progeny 31

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production of R. dominica. The lowest F1 progeny production (only 3 adults) was obtained in the combination of 500 DE and 300 EP treatment (Figure 5). Rhyzopertha dominica 80

Number of adult proney

B 40

E 10

F G

0 Control

250 DE

500 DE

150 EP

300 EP

250DE+150EP 250DE+300EP 500DE+150EP 500DE+300EP

Treatments

Figure 5. Number of adult progeny in the treatments of diatomaceous earth (DE) alone and entomopathogenic fungus, Beauveria bassiana (EP) alone and their all binary combinations. CONCLUSIONS The mortalities of R. dominica adults at all binary combinations of DE and EP after 7 day of the treatments, were higher than sum of the mortalities of EP and DE alone treatments. After 14 day of the treatments all binary combinations of DE and EP except treatments at the highest concentration of EP (300 ppm) also resulted in significant increase of the mortality of R. dominica adults. The complete mortality of R. dominica adults was obtained at only binary combination of 300 ppm EP and 500 ppm DE after 14 days of treatment. Although all binary combinations of DE and EP significantly reduced F1 progeny production of R. dominica, they did not completely prevent F1 progeny production of R. dominica. This study recommends that the binary combination of 500 ppm Turkish diatomaceous earth and 300 ppm B. bassiana local isolate would be the best option to be used for successful control of R. dominica. Acknowledgments: This study was a part of a project granted by Scientific Research Foundation of Kahramanmaraş Sütçü Imam University) with project number 2015/1-14 YLS. REFERENCES Akçalı S., A. A. Işıkber, Ö. Sağlam, H. Tunaz, M. K. Er, 2018. “Laboratory evaluation of Turkish diatomaceous earths as potential stored grain protectants, 739-743” Proceedings of 12th International Working Conference on Stored Product Protection (IWCSPP), (711 October 2018, Berlin, Germany) 1130 pp. Athanassiou, C. G., B. J. Vayias, C. B. Dimizas, N. G. Kavallieratos, A. S. Papagregoriou, C. Th. Buchelos, 2005. Insecticidal efficacy of diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Tribolium confusum du Val (Coleoptera: 32

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Tenebrionidae) on stored wheat: influence of dose rate, temperature and exposure interval. Journal of Stored Products Research, 41: 47-55. Athanassiou, C. G., N. G. Kavallieratos, B. J. Vayias, Z. Tomanović, A. Petrović, V. Rozman, C. Adler, Z. Korunic, D. Milovanović, 2011. Laboratory evaluation of diatomaceous earth deposits mined from several locations in central and southeastern Europe as potential protectants against coleopteran grain pests. Crop Protection, 30: 329-339. Çetin, M., B. Taş, 2012. A natural mineral with biological origin: Diatomite. Turkish ScienceResearch Foundation (Türk Bilim Araştırma Vakfı (TÜBAV) Science Journal, 5 (2): 2846. (In Turkish, Only abstract in English). Doğanay, Ş. İ., 2013. Determination of Efficiency of Some Diatomaceous Earths against to Stored-Grain Insects, Sitophilus granarius (L.) and Rhyzopertha dominica (F.). Master Thesis. Kahramanmaraş Sütçü İmam University, Institute of Natural and Applied Science, Plan Protection Department, 55p. (In Turkish, Only abstract in English) Er, M.K., A.A. Işikber, F. Aydın, C. Ücük, H. Tunaz, 2018. Rhyzopertha dominica adult mortalities after exposure to indigenous Beauveria bassiana isolates from stored-grain pests: Effects of certain factors in sampling process. Journal of Stored Products Rsearch, 77: 205-210. FDA (Food and Drug Administration, USA), 1995. Specifications for diatomaceous earths as a maximum 2 % animal feed additive. 21 CFR Section 573.340. Fields, P., Z. Korunic, 2000. The effect of grain moisture content and temperature on the efficacy of diatomaceous earths from different geographical locations against storedproduct beetles. Journal of Stored Products Research, 36: 1-13. Flinn, P. W., D. W. Hagstrum, 2011. Movement of Rhyzopertha dominica in response to temperature gradients in stored wheat. Journal of Stored Products Research, 47: 407-410. Kavallieratos, N.G., C. G. Athanassiou, F. G. Pashalidou, N. S. Andris, Z. Tomanovic, 2005. Influence of grain type on the insecticidal efficacy of two diatomaceous earth formulations against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). Pest Management Science, 61: 660-666. Korunic, Z., 1997. Rapid assessment of the insecticidal value of diatomaceous earths without conducting bioassays. Journal of Stored Products Research, 33: 219-229. Korunic, Z., 1998. Diatomaceous earths, a group of natural insecticides. Journal of Stored Products Research, 34: 87-97. Mete, Z., 1988. Enrichment of diatomite reserve in Kutahya-Alayunt region. Journal of The Mediterranean University Isparta Engineering Faculty, 1: 184-201. (In Turkish, Only abstract in English) Özbey, G., N. Atamer, 1987. “Some knowledge on Kizelgur (Diatomite), 493-502”. 10th Turkish Scientific and SAS Institute Inc., 2012. SAS / STATR User’s Guide, Version 12.1, 2nd Ed. SAS Institute Inc., Cary, NC. 21 pp. Sıvacı, R., Ş. Dere, 2006. Seasonal change of Diatomic flora of Melendiz Stream. Ç.U Science and Art Faculty Journal of Science, 27 (1): 1-12. Subramanyam, Bh., R. Roesli, 2000. “Inert Dust, 321-379”. In: Alternatives to Pesticides in Stored-Product IPM (Eds. Bh. Subramanyam & D. W. Hagstrum). Kluwer Academic Publishers, Springer, Boston, USA, 437 pp. Şen, R., A.A. Işıkber, H. Bozkurt, Ö. Sağlam, 2019. Effect of temperature on insecticidal efficiency of local diatomaceous earth against stored grain insects. Turkish Journal of Entomology, 43(4): 441-450.

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MICROWAVE RADIATION TREATMENT FOR CONTROLLING COWPEA WEEVIL (CALLOSOBRUCHUS MACULATUS (FABRICIUS)) ON STORED CHICKPEA Ayşegül TAVUKÇU1, Ali A. IŞIKBER1*, M. Kubilay ER1, Özgür SAĞLAM2, Hasan TUNAZ1 1

Faculty of Agriculture, Department of Plant Protection, Kahramanmaraş Sütçü İmam University, 46060 Kahramanmaraş, Turkey

Faculty of Agriculture, Department of Plant Protection, Namık Kemal University, Tekirdağ, Turkey *Corresponding author’s e-mail: isikber@ksu.edu.tr

ABSTRACT In this study, the experiments were conducted to determine efficiency of microwave radiation treatments in a kitchen-type and industrial conveyor belt type microwave oven against all biological stages of cowpea weevil (Callosobruchus maculatus (Fabricius)) on stored chickpea and their effects on chickpea quality. In biological tests conducted in kitchen type microwave oven, 100 % or close to 100 % mortality of all biological stages of C. maculatus on chickpea was achieved by 720 W 40 s and 900 W 40 s microwave treatments while surface temperatures of chickpea for these treatments were determined to be 64.8 and 69.9 ºC respectively. In biological tests conducted in industrial conveyor belt type microwave oven, all microwave exposure periods (62 s, 72 s, 90 s, 100 s) at 800 W power produced the mortalities ranging from 98.7 % to 100 % of its biological stages with 72.7, 81.5, 90.6 and 97.4 ºC of surface temperature on chickpea respectively, while all microwave exposure periods at 600 W power produced the mortalities ranging from 92.5 % to 100 % of biological stages with 59.8, 72.7, 78.9 and 84.9 ºC of surface temperature respectively. Generally, in both kitchen-type and industrial conveyor belt type microwave oven germination rate of chickpea significantly decreased with increasing both microwave power and exposure time. However, total protein content of chickpea was not affected by microwave treatments. In conclusion, this study indicated that microwave disinfestation of C. maculatus on chickpea could have a great potential to be an alternative for chemical disinfestation methods. Keywords: Microwave radiation, Callosobruchus maculatus, Chickpea, Stored product insects INTRODUCTION Turkey is the largest producer of grain legumes in the world and dry bean continues to be Turkey’s largest crop in terms of both area seeded and production (Anonymous, 2014a). According to the data of 2012, dry beans with 28,780,377 hectares of cultivation area and 23,140,276 tons of production, rank first in the world production of edible legumes (Anonymous, 2014b). A major problem in the marketing of dry beans and their products are infestation by insect pests. The bean weevil (Acanthoscelides obtectus Say.) (Col: Bruchidae) and Cowpea weevil (Callosobruchus maculatus (F.)) (Col: Chrysomelidae) which are quite common in Turkey, are the most important insect pests of beans (Anonymous, 2012). The overall damage caused by these insect pests in grain legumes, worldwide is estimated to be 1034

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40% annually (Upadhyay and Ahmad, 2011). Since losses of grain legumes due to insect infestation are high, disinfestation of grain legumes is very important for the safe storage of grain legumes. Chemical fumigants are used to disinfest grain legumes, but regulatory issues, insect resistance, environmental concerns and the expansion of the organic market have caused the industry to explore non-chemical alternatives (Vadivambal et al., 2010; Abo-El-Saad et al., 2011). Microwave disinfestation is a physical method to control insects in stored grain (Vadivambal, 2009). Microwaves are electromagnetic waves with frequencies ranging from about 0.3GHz to 3GHz and corresponding wavelength from 1 to 0.001m. For common household applications and industrial purposes, only 2.45GHz is permitted (Guo and Zhu, 2009). Microwave radiation, with good penetrability, can kill pests existing in grain kernels. Hazardous impacts could be due to a high oscillation frequency of water molecules in the body of insects. One possible alternation is the use of microwave energy in food processing applications mainly due to its ability to cause fast internal heating products to insecticidal levels, no chemical residues left in the food and hence there are no adverse effects on human beings, on the environment and the insects are unlikely to develop resistance to this treatment. Effects of the thermal process depend on the working parameters of the applied devices and qualitative traits of the processed materials. It is necessary to study the effect of microwave energy on attributes of chickpea quality to establish hypothetical quality changes in order to use microwave energy as an alternative method of pest control. The objectives of this study were to determine efficiency of microwave radiation against all biological stages of cowpea weevil (Callosobruchus maculatus (F.)) on stored chickpea and their effects on chickpea quality in industrial conveyor belt type microwave oven.

MATERIALS AND METHOD Test insect All biological stages (eggs, larvae, pupae and adults) of cowpea weevil (Callosobruchus maculatus (F.)) were used in the biological tests conducted on microwave application (Figure 1). The culture of the bean weevil was made by placing 65 gr chickpea in 1 L glass jars in a dark environment at 65 ± 5% R.H. and at temperature of 26 ± 1 ºC. Industrial conveyor belt type microwave oven The experimental setup used to determine the effectiveness of microwave energy against stored product insects is an industrial conveyor belt type microwave oven microwave dryer with an active band length of 5 m and an active band width of 0.15 m. There are 10 microwave generators located equally spaced in the section of the oven corresponding to the active belt length. The maximum input power of each generator is 800 W. The control of the microwave generators and the belt speed on the belt type microwave oven is provided by the main control panel (Figure 2). With 10 step changers on the control panel, the input power of each microwave unit on the dryer can be adjusted from 50 W to Biological test procedures Fresh harvested chickpeas with 10±1% m.c were used in biological tests carried out in the semi-industrial microwave oven. The chickpeas were previously infested with bean weevils and 40 gr of grain chickpea, each containing three-week mature larvae, 1-2 day old pupae, one week old adult and 1-2 day old eggs, were placed in petri dishes with a diameter of 15 cm and 35

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a height of 1 cm in a single layer. 40 gr of chickpeas infested with each biological stage of cowpea weevil were subjected to 4 different microwave application times (40, 45, 50 and 60 s) at 2 different power levels (800 and 600 W) separately. Microwave application time is regulated by adjusting the band speed. As a result of the preliminary tests, it was determined that the microwave oven band speeds of 55, 50, 45 and 40 mm/s corresponded to 40, 45, 50 and 60 s microwave application periods, respectively. Each microwave treatment was replicated 5 times. After each microwave treatment, chickpea surface temperatures were measured on 5 different points in the petri dish, center, right and left edge, front and rear edge. After each treatment, microwaves energy applied beans in petri dishes, infested with the larvae, pupae and eggs of cowpea weevil, were taken separately into 250 ml glass vials and kept in a dark climate of 65 ± 5% RH and 26 ± 1 ºC until they became adult. Quality analysis Various quality analyses like chickpea protein content and germination rate were applied for the microwave-treated chickpea samples. Data processing and analysis Mortality rates (%) were subjected to Arc-sin transformation and then subjected to twoway variance analysis. The differences between the means were determined according to LSD test at 5% significant level (SAS Institute, 1985). After calculating the quality parameter (germination rate) and product surface temperatures for each microwave treatment, one-way analysis of variance was applied to the data and the differences between the means were determined according to LSD test at 5% significant level (SAS Institute, 1985). RESULTS AND DISCUSSION In biological tests conducted in kitchen type microwave oven, 100 % or close to 100 % mortality of all biological stages of C. maculatus on chickpea was achieved by 720 W 40 s and 900 W 40 s microwave treatments (Table 1) while surface temperatures of chickpea for these treatments were determined to be 64.8 and 69.9 ºC respectively (Figure 1). In biological tests conducted in industrial conveyor belt type microwave oven, all microwave exposure periods (62 s, 72 s, 90 s, 100 s) at 800 W power produced the mortalities ranging from 98.7 % to 100 % of its biological stages with 72.7, 81.5, 90.6 and 97.4 ºC of surface temperature on chickpea respectively, while all microwave exposure periods at 600 W power produced the mortalities ranging from 92.5 % to 100 % of biological stages with 59.8, 72.7, 78.9 and 84.9 ºC of surface temperature respectively (Table 1 and Figure 1).

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100

Temperature (O C)

90 85

C DC

70 65

55 50 600 W 100 s

600 W 90 s

600 W 72 s

600 W 62 s

800 W 100 s

800 W 90 s

800 W 72 s

800 W 62 s

Microwave treatments

Figure 1. Surface temperatures (ยบC) of chickpeas exposed to different microwave treatments in biological tests conducted in semi-commercial trials. The different capital letters on the graph are statistically different according to the LSD test at 5% level.

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Table 1. Mortality rates (%) of all biological stages of Callosobruchus maculatus on chickpeas exposed to different microwave treatments in biological tests conducted in semicommercial trials. Percentage mortality (%)±S.E

Microwave treatments 600 W 60 s 600 W 50 s 600 W 45 s 600 W 40 s

800 W 60 s 800 W 50 s 800 W 45 s 800 W 40 s F and value

Egg

Larva

Pupa

Adult

99.44 ± 0.4

100 ± 0

99.6 ± 0.4

99.7 ± 0.2

100 ± 0

98.9 ± 0.9

98.08± 1.1

99.48 ± 0.3

100 ± 0

99.62± 2.35

92.78 ± 3.1

94.88 ± 1.93

100 ± 0

99.86 ± 0.1

100 ± 0

99.74 ± 0.2

100 ± 0

98.73 ± 0.7

100 ± 0

99.48 ± 0.5

100 ± 0

F7,32 =1.69

F7,32 = 5.96

F7,32 = 8.52

P=0.1463

P=0.0002

P <0.001

Generally, in both kitchen-type and industrial conveyor belt type microwave oven germination rate of chickpea significantly decreased with increasing both microwave power and exposure time (Figure 3). However, total protein content of chickpea was not affected by microwave treatments (Figure 2).

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24 22

20.62

20.95

20.83

20.72

20.59

20.41

21.06

20.83

20.62

Kontrol

600 W 100 s

800 W 100 s

600 W 90 s

800 W 90 s

600 W 72 s

800 W 72 s

600 W 62 s

800 W 62 s

Protein oranı (%)

18 16 14 12 10 8 6 4 2 0

Mikrodalga uygulaması

Figure 2. Crude protein ratio (%) of chickpea exposed to different microwave treatments in biological tests conducted in semi-commercial trials.

100

Çimlenme yüzdesi (%)

60 50 D

40 30 E

20 10 0 Kontrol 600 W 100 s

800 W 100 s

600 W 90 s

800 W 90 s

600 W 72 s

800 W 72 s

600 W 62 s

800 W 62 s

Mikrodalga uygulaması

Figure 3. Germination rates (%) of chickpea exposed to different microwave treatments in biological tests conducted in semi-commercial trials. The different capital letters on the graph are statistically different according to the LSD test at the 5% level. CONCLUSION In conclusion, this study indicated that microwave disinfestation of Callosobruchus maculatus on chickpea could have a great potential to be an alternative for chemical disinfestation methods. Acknowledgments: This study was supported by Kahramanmaraş Sütçü Imam University Scientific Research Foundation (Project Numbers: 2011/4-28 D).

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REFERENCES Anonymous, 2012. Baklagil tohum böcekleri, Bruchus spp. (Col:, Bruchidae) Zirai Mücadele teknikTalimatı.(http://www.gkgm.gov.tr/birim/bitki_karantina/faaliyet/teknik_talimat/y emeklik_baklagiller/b aklagil_tohum_bocekleri.pdf), (August, 2012). Anonymous, 2014a. Food and agriculture organization statistical database, (http://faostat.fao.org/site/567/DesktopDefault. aspx? PageID=567#ancor), (Ağustos, 2014). Anonymous, 2014b. Republic of Turkey Prime Ministry-Turkish Statistical Institute. Crop Production Statistics. (http://www.tuik.gov.tr/itkiselapp/bitkisel.zul), (September, 2014) Abo-El-Saad M.M., Elshafie H. A., Al Ajlan A.M., Bou-Khowh I.A. 2011. Non-chemical alternatives to methyl bromide against Ephestia cautella (Lepidoptera: Pyralidae): microwave and ozone. Agric. Biol. J. North Am. 2: 1222–1231. Guo, W., Zhu, X. 2009. Foreign dielectric property measurement techniques and their applications in agricultural products and food materials. Transactions of the Chinese Society of Agricultural Engineering, 25(2): 308-312. SAS Institute. 1985. User’s Guide: Statistics. SAS Institute, Cary, N.C. Vadivambal, R. 2009. Disinfestation of Stored Grain Insects Using Microwave Energy. Ph.D.Thesis. Faculty of Graduate Studies, University of Manitoba, Canada.197s. Vadivambal R., Deji O.F., Jayas D.S., White N.D.G., 2010. Disinfestation of stored corn using microwave energy. Agr Biol J N Am., 1:18–26. Upadhyay R.K., Ahmad S. 2011. Management strategies for Control of Stored Grain Insect Pests in Farmer Stores and Public Ware Houses. World Journal of Agricultural Sciences; 7:527-549.

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MORTALITY OF SPODOPTERA LITTORALIS LARVAE CAUSED BY EICOSANOID BIOSYNTHESIS INHIBITORS AND TWO NATIVE ISOLATES OF METARHIZIUM ANISOPLIAE Hasan TUNAZ*1 M. Kubilay ER1, Ali Arda IŞIKBER1 1

Kahramanmaraş Sütçü İmam University, Agriculture Faculty, Plant Protection Department, Avşar Campus,46100 Kahramanmaraş, Turkey * Corresponding author’s e-mail: htunaz@ksu.edu.tr ABSTRACT

Mortality responses were changed with two different Metarhizium anisopliae isolates. Although the effect of M. anisopliae (2735) isolate on mortality was lower, the effect of M. anisopliae (3293) isolate was expressed in a time-dependent manner on the mortality of larvae. When M. anisopliae (3293) isolates and eicosanoid biosynthesis inhibitors (phenidone dexamethasone, naproxen, indomethacin, esületin, ibuprofen) were injected to the larvae, increased and faster larval mortality of the larvae was seen. Similarly, increasing dosages of phenidone (eicosanoid biosynthesis inhibitor) were associated with increased mortality activity of the larval insects co-injected with the M. anisopliae (3293) isolate. Nodulation is the predominant cellular reaction to the injection of bacteria and fungi in insects. Treating larvae of S. littoralis with M. anisopliae (3293) isolates induced nodulation reactions and injecting larvae of S. littoralis with eicosanoid biosynthesis inhibitors, immediately before intrahemocoelic injections of M. anisopliae (3293) isolate sharply reduced the nodulation response to fungal challenges. These findings support that the virulent effects of entomopathogen, M. anisopliae (3293) can be enhanced when the S. littoralis immune system is suppressed. Keywords: Eicosanoid, Metarhizium anisopliae, Spodoptera littoralis

INTRODUCTION Two categories of immunity to bacterial and fungal infections, humoral and hemocytic were expressed by insects (Stanley, 2000). Hemocytic immune reactions involve direct interactions between circulating hemocytes and infecting microbes (Stanley and Miller, 2006). These immune functions are well known and the contemporary research frontiers reveal information on the signal mechanisms responsible for mediating and coordinating insect immunity. Tunaz (2006) tested role of eicosanoid biosynthesis inhibitors (EBIs) when co-applied with the pathogenic fungal conidia for insect pest control. They reported increased and faster mortality of Pieris brassicae larvae when B. bassiana (ARSEF-1151) was co-injected with the eicosanoid biosynthesis inhibitors. To continue developing the idea that EBIs can influence mortality in infected insects, the objectives of this study were to determine the influence two native isolates of M. anisopliae on mortality of S. littoralis larvae and to determine whether injecting S. littoralis larvae with EBIs plus M. anisopliae would influence larval mortality.

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MATERIAL AND METHODS Organisms S. littoralis were reared on a culture (38 g agar, 2600 ml distil water, 300 g corn flour, 120 g wheat embryo, 100 g yeast, 20 g casein, 14 g wesson salt, 8 g sorbic acid, 4 g nipagin, 600 mg streptomisin, 18 g ascorbic asid and 80 mg vitamin complex) and maintained in the laboratory at 25±2 °C and 65±5% relatıve humidity (RH). The larvae (5. instars) were tested for each bioassays at 25  2 oC and 65 5 % RH. The entomopathogenic fungus, M. anisopliae (2775, 3293) isolates were used in this study. The isolates were grown at 25 oC on potatoe dextrose agar (PDA) for 30 days. Conidia were harvested from these plates in sterile distilled water containing 0.1 % Tween 80 and vortexed vigorously. Reagents The phospholipase A2 (PLA2) inhibitor dexamethasone {(11, 16)-9- fluoro-11,17,21trihydroxy-16-methylpregna-1,4-dione}, the cyclooxygenase inhibitors naproxen, indomethacin and ibuprofen {O-2-(6-methoxy-naphthyl) propionic acid}, the dual cyclooxygenase and lipoxygenase inhibitor phenidone {1-pheny-3-pryazolidinone}, and the 5and 12-lipoxygenase inhibitor, esculetin {6,7-dihydroxycoumarin} were all purchased from Sigma Chemical Co. (St. Louis, MO). Influence of two different isolates of M. anisopliae on mortality of S. littoralis larvae Larvae were applied with individual isolates(1x107 conidiaspor/ml for each isolates). Control insects were applied with 0.021 % Tween 80 solution. After application, the larvae were kept on room temperature. Mortality was assessed at selected times after injections. Effects of eicosanoid biosynthesis inhibitors on mortality of S. littoralis larvae when coinjected with M anisoplia isolate (3293) S. littoralis larvae were divided into groups and individuals in each group were injected with either the phospholipase A2 (PLA2) inhibitor dexamethasone, three of the cyclooxygenase inhibitor, naproxen, ibuprofen and indomethacin, the dual cyclooxygenase and lipoxygenase inhibitor phenidone, or the lipoxygenase inhibitor esculetin, all in standard dosages of 104 µg in 4 µl EtOH. Control insects were injected with 4 µl EtOH. Following injections, the larvae were injected with 1x107 conidiaspor/ml in 5 µl 0.021 % Tween 80 solution. After injection the larvae were kept on room temperature as described. Mortality was assessed at selected times after injections as described above. Influence of phenidone (eicosanoid biosynthesis inhibitor) dosages on mortality of S. littoralis larvae when co-injected with M anisoplia isolate (3293) Individuals in five groups of larvae were injected with 4 µl of ethanol, or 52, 104, 156, 208 µg of phenidone in 4 µl ethanol, then infected with a standard concentration of M anisoplia isolate (3293). At 24 hpi, mortality was assessed. RESULTS Effects of different isolates of M. anisopliae on mortality of S. littoralis larvae Table 1 indicates that compared to control, two isolates of M. anisopliae caused higher mortality of S. littoralis larvae at each time. At day 9, control caused no mortality of larvae of S. littoralis whereas the isolate (3293) of M. anisopliae caused approximately 63 % mortality of the larvae. 42

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Table 1. The influence of two different isolates of M. anisopliae on mortality of S. littoralis larvae

M.anisopliae Isolates 2735 3293 Control

3.33±3.33 23.33±3,33 0.00±0.00

16.67±3.33 50.00±0.00 0.00±0,00

Time (days) 7 20.00±5.77 63,.3±3.33 0.00±0.00

9 20.00±5.77 63.33±3.33 0.00±0,00

Effects of co-injected M anisoplia isolate (3293) and EBIs on mortality of S. littoralis larvae EBIs strongly enhanced absolute mortality and the speed of kill due to M anisoplia isolate (3293) challenge (fig. 1). Higher mortality obtained in larvae treated with the all inhibitors plus the concentration (1x107 spor/ml ) of M anisoplia isolate (3293) by 48 h PI. Lower mortality was recorded in controls at 48 h PI.

120

100 EtOH+Metarhizium 3293 Dex+Metarhizium 3293 Ind+Metarhizium 3293 Phe+Mertarhizium 3293 Ibu+Metarhizium 3293 Nab+Metarhizium 3293 Esc+Metarhizium 3293

Mortality %

0 0

Time (days)

Figure 1. Effect of eicosanoid biosynthesis inhibitors on mortality S. littoralis larvae infected with of M anisoplia isolate (3293) Influence of phenidone (eicosanoid biosynthesis inhibitor) dosages on mortality of S. littoralis larvae when co-injected with M anisoplia isolate (3293):

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Figure 2 indicates that the mortality due to phenidone was express in a dose-dependent manner. Increasing phenidone (eicosanoid biosynthesis inhibitor) dosages were associated with increasing mortality activity of the larvae co-injected with M. anisopliae (3293) isolate.

100

Mortality %

104

156

208

Phenidone Doses (ď g/4ď l etoh)+107 Metarhizium 3293 consantrations

Figure 2. Influence of phenidone dosages on mortality of S. littoralis larvae when co-injected with M anisoplia isolate (3293) CONCLUSIONS This study has shown that inhibition of the recently associated eicosanoids with insect pathology and thus microbial control leads to an accelerated biological activity and increased mortality, particularly as a result of entomopathogenic fungi applications. Eicosanoid inhibitors accelerated insect mortality and increased mortality when entomopathogenic fungus isolate (Metarhizium 3293) was applied together to Cotton leafworm larvae.

REFERENCES Stanley, D.W. 2000. Eicosanoids in Invertebrate Signal Transduction Systems. Princeton University Press, Princeton, NJ. Tunaz, H. 2006. Eicosanoid Biosynthesis Inhibitors Influence Mortality of Pieris brassicae Larvae Co-Injected With Fungal Conidia. Archives of Insect Biochemistry and Physiology. 63:93-100.

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EFFECT OF FEEDING BEHAVIOR OF RHYZOPERTHA DOMINICA ON ITS SENSITIVITY TO BEAUVERIA BASSIANA INFECTIONS Mehmet Kubilay ER1*, Cebrail BARIŞ1, Hasan TUNAZ1, Ali Arda IŞIKBER1 1

Department of Plant Protection, Faculty of Agriculture, University of Kahramanmaraş Sütçü İmam, Kahramanmaraş, Turkey *Corresponding author’s e-mail: mker@ksu.edu.tr ABSTRACT

Entomopathogenic fungi has been tested against stored-product pests in search for alternative control agents. In many previous studies Beauveria bassiana was found more virulent to Rhyzopertha dominica when it is applied to cereals. The sensitivity of this species was mostly attributed to its morphological and immunity related differences from more resistant species. As R. dominica feeds within grains, this behavior was tested for its contribution to the sensitivity of R. dominica. Twenty adults for each replication were released into either intact or broken wheat and maize kernels (50 g) mixed with B. bassiana conidia (300 ppm in weight) and mortalities were recorded after 7 and 14 days. The experiment was conducted at 25±2oC and 65±5 % relative humidity in darkness with five replications. There was not a statistically important difference between the mortalities when adults were in intact wheat and intact maize kernels. However, mortalities significantly decreased when adults were in broken kernels compared to those in intact kernels. As insects can not completely penetrate inside broken kernels insects were infected less than those in intact kernels. This results illustrate that the sensitivity of R. dominica to B. bassiana treatments is at least partially due to insect’s feeding behavior. This has a significant contribution to sensitivity of R. dominica to entomopathogenic fungi. Keywords: Biological control, Microbial control, Stored-product pest, Cereals

INTRODUCTION As cereals are stored for various times before use, they are under attack by several insect pests during these storage periods. Their control by means of chemicals have disadvantages in terms of human and environmental health issues as well as pest resistance to these chemicals. In search for alternative control strategies, amongst others, entomopathogenic fungi has been tested against stored-product pests and results have been evaluated as promising (Wakil and Ghazanfar, 2010; Shams et al., 2011; Barra et al., 2013; Khashaveh and Chelav, 2013; Sewify et al., 2014). Success of these biological agents varied depending on fungi and targeted pest species. In previous studies Beauveria bassiana was found more virulent to Rhyzopertha dominica comparing to other coleopteran stored-grain pests (Moino et al., 1998; Sewify et al., 2014; Korkmaz, 2017; Öz, 2019). The resistance of other species was mostly attributed to their morphological and immunity related features (Wakefield, 2006; Pedrini et al, 2015). Insect behavior is also an important factor in the process of infections by pathogens (Baverstock et al., 2010; de Roode and Lefevre, 2012). As R. dominica adults enters completely into kernels and feeds within grains, this feeding behavior may contribute to its sensitivity to fungal infections. The purpose of this study was to test whether being completely within kernels contribution to the sensitivity of R. dominica adults to B. bassiana infections.

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MATERIALS AND METHOD Rhyzopertha dominica culture Rhyzopertha dominica culture has been maintained in our laboratory. The culture had been started with insects obtained from surrounding storage facilities. The culture has been maintained on Durum wheat with 12% moisture content. Glass jars of 1 Lt capacity were used with 250 gr of wheat in each jar. Adults of mixed sex were placed into these jars and kept for 3 days for egg laying. After removing the adults, the cultures were left for the emergence of new generation adults. For the experiments, one week old adults were used. R. dominica cultures were at 26±2 ºC and 65±5% relative humidity in darkness. Beauveria bassiana culture The B. bassiana culture used was a single-spore culture (5-4) of a wild isolate (151138) from an infected R. dominica adult (Er et al., 2016). The fungus was initially grown on potato dextrose agar, and spore production was according to production procedure described by Barış (2016). 100 g of rice was soaked overnight with tap water and the excess water was drained. The rice supplemented with 1.5 gr of CaSO4 and CaCO3 was sterilized in a polyethylene bag (25 cm x 38 cm). After cooling, it was inoculated with 10 ml of spore suspension (2x107 spores/ml) and sealed. Following fungal growth at 25±2°C, 12/12 photoperiod for 14 days the culture was dried at 25±2°C. Spores were separated from substrate by using a 500 µm sieve. Before the experiment, the germination success of the fungal spores was determined. A dilute spore suspension was spread on potato dextrose agar and incubated at 25±2°C for 24 hours. The spores were examined under a light microscope and spores with germination tubes at least as long as the diameter of spores were considered germinated. The spores used in the experiment had a germination rate of over 98%. Pathogenicity test Wheat and maize were used as whole or broken kernels. 50 g wheat or maize was mixed with 300 ppm (w/w) B. bassiana conidia and shaken for 5 mins. for homogenous mixture. Twenty R. dominica adults for each replication were released into either intact or broken wheat and maize kernels. The experiment was conducted at 25±2oC and 65±5 % relative humidity in darkness with five replications. The humidity was ensured by placing them in humidity chambers with saturated NaNO2 solution. Insect mortalities were recorded after seven and fourteen days. Mortalities were corrected according to Abbott’s formula (Abbott, 1925). The corrected mortalities were subjected to ANOVA and Duncan multiple comparison tests after arcsine transformation. RESULTS AND DISCUSSION Corrected R. dominica adult mortalities for 7 and 14 days are given in Figures 1 and 2, respectively. There were significant differences amongst the mortalities in both cases (for 7 days F3,16=6.607, P=0.004; for 14 days F3,16=5.692, P=0.008). According to Duncan multiple comparison test (P<0.05) mortality differences in wheat and maize were statistically similar either they are intact or broken. However, mortalities in intact and broken kernels were statistically different for both wheat and maize. Mortalities significantly decreased when R. dominica adults were in broken kernels compared to those in intact kernels regardless of the commodity. As insects can not completely penetrate inside broken kernels, insects were infected less than those in intact kernels. This results illustrate that the sensitivity of R. dominica to B. bassiana treatments is at least partially due to insect’s feeding behavior. This has a significant contribution to sensitivity of R. dominica to entomopathogenic fungi. 46

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100

Corrected mortality (%)

80 70 60

50 40 30 20 10 0

Broken maize

Maize

Wheat

Broken wheat

Substrates

Figure 1. Effect of different substrates on Rhyzopertha dominica adults mortalities in 7 days due to Beauveria bassiana infections (Columns with different letters are different according to Duncan multiple comparison test (P<0.05); bars represent standard errors; n=5) A

100

Corrected mortality (%)

80 70 60

50 40 30 20 10 0 Broken maize

Maize

Wheat

Broken wheat

Substrates

Figure 2. Effect of different substrates on Rhyzopertha dominica adults mortalities in 14 days due to Beauveria bassiana infections (Columns with different letters are different according to Duncan multiple comparison test (P<0.05); bars represent standard errors; n=5) REFERENCES Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18: 265-267. Barış, C., 2016. The use of some cereals on the production of Beauveria bassiana by solid state fermentation technique. MSc Thesis, Graduate School of Natural and Applied Sciences, University of Kahramanmaraş Sütçü İmam, Turkey. Barra, P., L. Rosso, A. Nesci & M. Etcheverry, 2013. Isolation and identification of entomopathogenic fungi and their evaluation against Tribolium confusum, 47

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Sitophilus zeamais, and Rhyzopertha dominica in stored maize. Journal of Pest Science, 86: 217-226. Baverstock, J., Roy, H.E., Pell, J.K., 2010. Entomopathogenic fungi and insect behaviour: from unsuspecting hosts to targeted vectors. BioControl, 55: 89-102. de Roode, J.C. and Lefevre, T., 2012. Behavioral Immunity in Insects. Insects, 3: 789-820. Er, M.K., H. Tunaz, A.A. Işıkber, 2016. Improving the virulence of a native Beauveria bassiana isolate against Rhyzopertha dominica adults. 7th International Scientific Agriculture Symposium (Agrosym 2016). 6-9 October 2016, Jahorina, Bosnia and Herzegovina. pp: 1464-1469 DOI:10.7251/AGRENG1607221. Khashaveh, A. & H. S. Chelav, 2013. Laboratory bioassay of Iranian isolates of entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Ascomycota: Hypocreales) against two species of storage pest. Agriculturae Conspectus Scientificus, 78: 35-40. Korkmaz, H.Y., 2017. Determination of Native Fungal Agents on Stored-Grain Insects and Their Potential Use Against Certain Coleopteran Pests. PhD Thesis, Graduate School of Natural and Applied Scienses, University of Kahramanmaraş Sütçü İmam, Turkey. Moino, Jr A., S. B. Alves & R. M. Pereira, 1998. Efficacy of Beauveria bassiana (Balsamo) Vuillemin isolates for control of stored-grain pests. Journal of Applied Entomology, 122: 301-305. Öz, A., 2019. Efficacy of Entomopathogenic Fungi Obtained From Grain Storages in Kahramanmaraş Against Three Coleopteran Stored-Grain Pest Species. MSc Thesis, Graduate School of Natural and Applied Scienses, University of Kahramanmaraş Sütçü İmam, Turkey. Pedrini, N., A. Ortiz-Urquiza, C. Huarte-Bonnet, Y. Fan, M. P. Juarez, N. O. Keyhani, 2015. Tenebrionid secretions and a fungal benzoquione form competing components of an arms race between a host and pathogen. Proceedings of the National Academy of Sciences, 112: E3651-E3660. Sewify, G. H., H. A. El Shabrawy, M. E. Eweis & M. H. Naroz, 2014. Efficacy of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae for controlling certain stored product insects. Egyptian Journal of Biological Pest Control, 24: 191-196. Shams, G., M. H. Safaralizadeh, S. Imani, M. Shojai & S. Aramideh, 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). African Journal of Microbiology Research, 5: 1192-1196. Wakefield, M. E., 2006. Factors affecting storage insect susceptibility to the entomopathogenic fungus Beauveria bassiana. Proceedings of the 9th International Working Conference on Stored Product Protection, 15-18 October, 2006, Brazil. pp: 855862. Wakil, W. & M. U. Ghazanfar, 2010. Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. Archives of Phytopathology and Plant Protection, 43: 1236-1242.

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EFFECT OF HOST POPULATION ON THE EFFICACY OF BEAUVERIA BASSIANA AGAINST SITOPHILUS ORYZAE Mehmet Kubilay ER1*, Cebrail BARIŞ1, Ali Arda IŞIKBER1, Hasan TUNAZ1 1

Department of Plant Protection, Faculty of Agriculture, University of Kahramanmaraş Sütçü İmam, Kahramanmaraş, Turkey *Corresponding author’s e-mail: mker@ksu.edu.tr

ABSTRACT In management of stored-product pests, alternatives to chemical insecticides have been searched and microbial control by entomopathogenic fungi has been considered as a promising alternative. Previously it was shown that the virulence of Beauveria bassiana to Sitophilus oryzae varies depending on the fungal isolate used. However, fungal infections and further the resulting outcome is determined by the interaction between the pathogen and the host. Therefore, virulence could also be affected by the host, S. oryzae in this study. To test this, two different S. oryzae populations were tested by exposing to two B. bassiana isolates. Twenty adults for each replication were released into wheat (50 g) mixed with B. bassiana conidia at two concentrations, 300 ppm and 500 ppm (w/w). Insect mortalities were recorded after 7 and 14 days. The experiment was conducted at 25±2oC and 65±5 % relative humidity in darkness with five replications. S. oryzae adults from Konya population were significantly more susceptible to both isolates of B. bassiana compared to adults from Kahramanmaraş population. This shows that success of the fungus application as biocontrol agent may vary depending on host populations. Therefore, different host populations should be tested before further development of a fungal agent to realize actual potential of the fungus. Keywords: Biological control, Microbial control, Stored-product pest, Cereals

INTRODUCTION In management of stored-product pests, due to hazardous side effects of chemical insecticide applications, alternative control means have been investigated. Amongst these alternatives, microbial control by using entomopathogenic fungi has been considered as a promising approach (Wakil and Ghazanfar, 2010; Shams et al., 2011; Barra et al., 2013; Khashaveh and Chelav, 2013; Sewify et al., 2014). Although the pathogenicity of Beauveria bassiana to Sitophilus oryzae varied in previous studies, it was usually rather low comparing to other coleopteran stored-product pests (Moino et al., 1998; Sewify et al., 2014; Korkmaz, 2017; Öz, 2019). Variation in virulence is generally attributed to the variation in fungal isolate used in different studies. Although it is well known that virulence of an entomopathogenic fungus species changes depending on isolates, this may not be the only reason for different results. Fungal infections and further the resulting outcomes are determined by the interaction between the pathogen and the host. Therefore, virulence could also be affected by the variation in the susceptibility of host population used in experiments. Research results supporting this has been published for other host and/or fungus species (Keller et al., 1999; Perinotto et al., 2012). The purpose of this study is to test two B. bassiana isolates on the adults from two of S. oryzae populations to detect variation between host populations in terms of fungal virulence.

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MATERIALS AND METHOD Sitophilus oryzae cultures Two Sitophilus oryzae cultures were used in the experiments. These cultures were started with insects collected from two populations from different cities, Konya and Kahramanmaraş. The cultures have been maintained on Durum wheat with 12% moisture content during the study. Glass jars of 1 Lt capacity were used with 250 gr of wheat in each jar. Adults of mixed sex were placed into these jars and kept for 3 days for egg laying. After removing the adults, the cultures were left for the emergence of new generation adults. For the experiments, one week old adults were used. S. oryzae cultures were at 26±2 ºC and 65±5% relative humidity in darkness. Beauveria bassiana cultures Two B. bassiana isolates were used in the experiments. One was a single-spore culture (5-4) of a wild isolate (151138) from an infected R. dominica adult (Er et al., 2016). The other isolate (B11) was obtained from wheat kernels sampled from a storage facility in Hatay. The fungi were initially grown on potato dextrose agar, and spore production was according to production procedure described by Barış (2016). 100 g of rice was soaked overnight with tap water and the excess water was drained. The rice supplemented with 1.5 gr of CaSO4 and CaCO3 was sterilized in a polyethylene bag (25 cm x 38 cm). After cooling, it was inoculated with 10 ml of spore suspension (2x107 spores/ml) and sealed. Following fungal growth at 25±2°C, 12/12 photoperiod for 14 days the culture was dried at 25±2°C. Spores were separated from substrate by using a 500 µm sieve. Before the experiment, the germination success of the fungal spores was determined. A dilute spore suspension was spread on potato dextrose agar and incubated at 25±2°C for 24 hours. The spores were examined under a light microscope and spores with germination tubes at least as long as the diameter of spores were considered germinated. The spores used in the experiment had a germination rate of over 98%. Pathogenicity test Fifty grams of wheat was mixed with 300 ppm and 500 ppm (w/w) B. bassiana conidia and shaken for 5 mins. for homogenous mixture. Twenty S. oryzae adults for each replication were released into the wheat. The experiment was conducted at 25±2oC and 65±5 % relative humidity in darkness with five replications. The humidity was ensured by placing them in humidity chambers with saturated NaNO2 solution. Insect mortalities were recorded after seven and fourteen days. Mortalities were corrected according to Abbott’s formula (Abbott, 1925). A series of t tests were conducted on the corrected mortalities after arcsine transformation. RESULTS AND DISCUSSION S. oryzae adult mortalities caused by B. bassiana isolate 5-4 are given in Figure 1. The isolate killed the adults at significantly higher levels when they are from Konya population regardless of the spore concentration and duration of the experiment (t and P values for 7 days at 300 ppm: 3.60, 0.009; for 7 days at 500 ppm: 5.43, 0.003; for 14 days at 300 ppm: 7.93, 0.001; for 14 days at 500 ppm:4.92, 0.002, respectively). S. oryzae adult mortalities caused by B. bassiana isolate B11 are provided in Figure 2. Similar results were found for B. bassiana isolate B11 (t and P values for 7 days at 300 ppm: 4.58, 0.003; for 7 days at 500 ppm: 4.79, 0.003; for 14 days at 300 ppm: 4.49, 0.003; for 14 days at 500 ppm: 7.77, <0.001, respectively). According to the results, Konya population was significantly more susceptible to both isolates of B. bassiana compared to the Kahramanmaraş population. Different populations of S. oryzae can vary in 50

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their susceptibly to B. bassiana infections. This shows that success of the fungus application as biocontrol agent may vary depending on the susceptibility of host populations. Therefore, different host populations should be tested before further development of a fungal agent to realize actual potential of the fungus. 100

100 Mortality in 7 days at 300 ppm

Mortality in 7 days at 500 ppm

Corrected mortality (%)

90 70 60 50 40 30 20

80 70 60 50 40 30 20

0 Konya

Kahramanmaraş

Konya

Populations

100

100 Mortality in 14 days at 300 ppm

Mortality in 14 days at 500 ppm

Corrected mortality (%)

Kahramanmaraş

70 60 50 40 30 20 10

Konya

Kahramanmaraş

Konya

Populations

Kahramanmaraş Populations

Figure 1. The mortalities of Sitophilus oryzae adults from two populations caused by Beauveria bassiana isolate 5-4 at two spore concentrations (bars represent standard errors, n=5)

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100 90

Mortality in 7 days at 500 ppm

Corrected mortality (%)

100

Mortality in 7 days at 300 ppm

70 60 50 40 30 20 10

80 70 60 50 40 30 20 10

0 Konya

Kahramanmaraş

Konya

Populations

100

100 Mortality in 14 days at 500 ppm

Corrected mortality (%)

Populations

Mortality in 14 days at 300 ppm

Kahramanmaraş

70 60 50 40 30 20 10

80 70 60 50 40 30 20 10

0 Konya

Kahramanmaraş

Konya

Populations

Kahramanmaraş Populations

Figure 2. The mortalities of Sitophilus oryzae adults from two populations caused by Beauveria bassiana isolate B11 at two spore concentrations (bars represent standard errors, n=5) REFERENCES Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. Journal of Economic Entomology, 18: 265-267 Barış, C., 2016. The use of some cereals on the production of Beauveria bassiana by solid state fermentation technique. MSc Thesis, Graduate School of Natural and Applied Sciences, University of Kahramanmaraş Sütçü İmam, Turkey. Barra, P., L. Rosso, A. Nesci & M. Etcheverry, 2013. Isolation and identification of entomopathogenic fungi and their evaluation against Tribolium confusum, Sitophilus zeamais, and Rhyzopertha dominica in stored maize. Journal of Pest Science, 86: 217-226.

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Er, M.K., H. Tunaz, A.A. Işıkber, 2016. Improving the virulence of a native Beauveria bassiana isolate against Rhyzopertha dominica adults. 7th International Scientific Agriculture Symposium (Agrosym 2016). 6-9 October 2016, Jahorina, Bosnia and Herzegovina. pp: 1464-1469 DOI:10.7251/AGRENG1607221. Keller, S., Schweizer, C., and Shah, P. (1999), ‘Differential Susceptibility of Two Melolontha Populations to Infections by the Fungus Beauveria brongniartii’, Biocontrol Science and Technology, 9, 441_446. Khashaveh, A. & H. S. Chelav, 2013. Laboratory bioassay of Iranian isolates of entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Ascomycota: Hypocreales) against two species of storage pest. Agriculturae Conspectus Scientificus, 78: 35-40. Korkmaz, H.Y., 2017. Determination of Native Fungal Agents on Stored-Grain Insects and Their Potential Use Against Certain Coleopteran Pests. PhD Thesis, Graduate School of Natural and Applied Scienses, University of Kahramanmaraş Sütçü İmam, Turkey. Moino, Jr A., S. B. Alves & R. M. Pereira, 1998. Efficacy of Beauveria bassiana (Balsamo) Vuillemin isolates for control of stored-grain pests. Journal of Applied Entomology, 122: 301-305. Öz, A., 2019. Efficacy of Entomopathogenic Fungi Obtained From Grain Storages in Kahramanmaraş Against Three Coleopteran Stored-Grain Pest Species. MSc Thesis, Graduate School of Natural and Applied Scienses, University of Kahramanmaraş Sütçü İmam, Turkey. Perinotto, W.M.S., Angelo, I.C., Golo, P.S., Quinelato, S., Camargo, M.G., Sa, F.A., Bittencourt, V.R.E.P., 2012. Susceptibility of different populations of ticks to entomopathogenic fungi. Experimental Parasitology, 130: 257-260. Sewify, G. H., H. A. El Shabrawy, M. E. Eweis & M. H. Naroz, 2014. Efficacy of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae for controlling certain stored product insects. Egyptian Journal of Biological Pest Control, 24: 191-196. Shams, G., M. H. Safaralizadeh, S. Imani, M. Shojai & S. Aramideh, 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). African Journal of Microbiology Research, 5: 1192-1196. Wakil, W. & M. U. Ghazanfar, 2010. Entomopathogenic fungus as a biological control agent against Rhyzopertha dominica F. (Coleoptera: Bostrychidae) on stored wheat. Archives of Phytopathology and Plant Protection, 43: 1236-1242.

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VARIATION IN THE EFFECT OF BEAUVERIA BASSIANA ISOLATES AGAINST THREE COLEOPTERAN STORED-PRODUCT PESTS: CONCENTRATIONMORTALITY RELATION Mehmet Kubilay ER1*, Ali Arda IŞIKBER1, Hasan TUNAZ1 1

Department of Plant Protection, Faculty of Agriculture, University of Kahramanmaraş Sütçü İmam, Kahramanmaraş, Turkey *Corresponding author’s e-mail: mker@ksu.edu.tr

ABSTRACT Entomopathogenic fungi have been investigated as microbial control agents against storedproduct pests to develop alternative control measures to chemical insecticides. Amongst over a hundred entomopathogenic fungi nine Beauveria bassiana isolates were worth investigating further and thus tested against three stored-product pests, Sitophilus oryzae, Rhyzopertha dominica and Oryzaephilus surinamensis, using five concentrations of the conidia (50, 100, 500, 1000, 5000 ppm in weight). Twenty adults for each replication were released into wheat (50 g) mixed with B. bassiana conidia. The experiment was conducted at 25±2oC and 65±5 % relative humidity in darkness with four replications. Data was evaluated using probit analysis. The results showed that insect mortalities vary depending on host species. Lowest LC50 and LC90 values were calculated for R. dominica and the highest ones for S. oryzae adults, indicating general susceptibilities of pest species to B. bassiana infections. Concentrations needed to kill 90% of the populations of the pests varied enormously amongst B. bassiana isolates and three of them were selected as promising for further investigations. Keywords: Biological control, Microbial control, Stored-product pest, Cereals

INTRODUCTION Cereals are important both for human and livestock in the world. Because they are usually stored for a while before consumption, they are subjected to attacks by insect and mite pests. These pests can cause severe reduction in the weight and value as well as germination of seeds (Moino et al., 1998; Padin et al., 2002; Haq et al., 2005; Stejskal et al., 2015). To control the pests, chemical insecticides have been used (Athanassiou & Palyvos, 2006). These chemicals can cause pest resistance (Arthur, 1996), residue accumulation (Ferizli et al., 2005), and negative side effects on humans and the environment (Michalaki et al., 2007). One alternative to the chemicals that has been considered is the use of entomopathogenic fungi (Moino et al., 1998; Michalaki et al., 2007; Sewify et al., 2014; Wakil & Schmitt, 2014). The potential of entomopathogenic fungi as biological control agents against insect pests of stored products has been demonstrated by several researchers (Cherry et al., 2005; Wakil & Ghazanfar, 2010; Shams et al., 2011; Barra et al., 2013; Khashaveh & Chelav, 2013; Sewify et al., 2014). Entomopathogenic fungi also have potential to be used in combination with diatomaceous earth (Athanassiou & Steenberg, 2007; Athanassiou et al., 2008; Wakil et al., 2011; Riasat et al., 2011, 2013; Shafighi et al., 2014). In a search for potential control agent, over a hundred entomopathogenic fungi have been tested against three stored-product pests, Sitophilus oryzae, Rhyzopertha dominica and Oryzaephilus surinamensis (data not provided). Amongst them nine Beauveria bassiana isolates were considered worth investigating further. In this study, the 54

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concentration-mortality relation for these nine isolates against the three pests was presented, showing variation in the virulence of the fungi.

MATERIALS AND METHOD Insect cultures Rhyzopertha dominica and Sitophius oryzae cultures have been maintained in our laboratory. Starting insects had been originally obtained from surrounding storage facilities. Durum wheat with 12% moisture content was used for the cultures. Glass jars of 1 Lt capacity with 250 gr of wheat were used. Adults of mixed sex were placed into the jars and kept for three days for oviposition. After removing the adults, the cultures were incubated for the emergence of new generation adults. One week old adults were used for the bioassyas. All the cultures were maintained at 26±2 ºC and 65±5% relative humidity in darkness. Oryzaephilus surinamensis, however, was reared on a mixture of broken wheat, oat meal and dry yeast in the ratio of 5:5:1, respectively, in glass jars of 1 Lt capacity. Beauveria bassiana cultures All the B. bassiana isolates were obtained from infected stored-product pests. The fungi were cultured on potato dextrose agar medium at 26±2oC and under 16/8 light/dark conditions. Conidia for the experiments were gathered from these cultures once they complete their sporulation. Before gathering the spores, the Petri dishes left open for one night following Athanassiou and Steenberg’s (2007) procedure to dry. Conidia were gathered by vacuuming and kept on silica gel at +4 oC until using (for 1-2 days). Before the experiment, the germination success of the fungal spores was determined. A dilute spore suspension was spread on potato dextrose agar and incubated at 25±2°C for 24 hours. The spores were examined under a light microscope and spores with germination tubes at least twice as long as the diameter of spores were considered germinated. The spores used in the experiment had a germination rate of over 98%. Concentration-mortality test Centrifuge tubes of 50 ml capacity each with 50 gr of wheat were used for the tests. Wheat in each tube was mixed with a required amount of spores producing a final concentration of 50, 100, 500, 1000, 5000 ppm (w/w) by shaking for 5 minutes. Twenty adults were released in each tube and kept at 25±2oC, 65±5% relative humidity in darkness. Wheat kernels without spores were used as control. This experiment with four replications was conducted on three storedproduct pests, Sitophilus oryzae, Rhyzopertha dominica and Oryzaephilus surinamensis. Data was evaluated using probit analysis by using Polo-PC program. RESULTS AND DISCUSSION The results of the probit analysis for O. surinamensis, S. oryzae and R. dominica are provided in Table1, 2 and 3, respectively. Except for S. oryzae mortality due to isolate 145598, LC values for all tests were calculated. Other few test results were adequate for calculation of confidence intervals. The results showed that insect mortalities vary depending on host species. Lowest LC50 and LC90 values were calculated for R. dominica and the highest ones for S. oryzae adults, indicating general susceptibility of R. dominica adults to B. bassiana infections. Concentrations needed to kill 90% of the populations of the pests varied enormously amongst B. bassiana isolates. In the examination of the statistical parameters, those isolates with data that fit the 55

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probit model, with lower LC values with higher slopes were considered worth investigating further. As all the insect species are stored-product pests, isolates for the purpose of storedproduct pest suppression, B. bassiana isolates 120602, 151138 and 158749 were chosen as promising biological control agents. Table 1.The results of probit analysis for Oryzaephilus surinamensis adults Isolate

intercept

slope (±s.e.) x2 (d.f.:18)

LC50 (%95 CI) (ppm)

LC 90 (%95 CI) (ppm)

120602

-2.89

1.34 ± 0.15

16.78

143.26 (96.86 - 197.38)

1287.42 (874.1 - 2190.13)

137691

-0.35

0.28 ± 0.09

28.52

17.37

661350

138485

-0.62

0.36 ± 0.09

30.09

52.39

196360

139350

-1.79

0.63 ± 0.11

58.44

703.79

76151

139756

-1.24

0.64 ± 0.11

37.18

85.23 (12.39 - 231.19)

8455.3 (2378.8 - 245870)

145598

-2.37

0.88 ± 0.11

22.03

480.23 (272.17 - 809.95)

13592 (5832.3 - 57655)

146432

-1.86

0.76 ± 0.11

19.11

275.48 (143.28 - 470.73)

13284 (5374.3 - 64474)

151138

-1.74

0.89 ± 0.11

10.06

91.37 (48.93 - 143.49)

2525.34 (1427.34 - 6053.81)

158749

-2.14

0.8 ± 0.11

33.26

455.29 (200.92 - 942.25)

17888 (5787.2 - 182660)

Table 2. The results of probit analysis for Sitophilus oryzae adults Isolate

intercept

slope (±s.e.) x2 (d.f.:18)

LC50 (%95 CI) (ppm)

LC 90 (%95 CI) (ppm)

120602

-4.02

1.15 ± 0.23

24.25

3196.2 (1788.6 - 7348)

41912 (14156 - 781820)

137691

-3.51

0.84 ± 0.21

24.69

15026 (5323.2 - 453520)

502160 (56745 - 4425100000)

138485

-4.05

1.23 ± 0.17

28.82

1944.97 (1187.82 - 3510.1)

21320 (9257.3 - 108680)

139350

-1.49

0.37 ± 0.1

53.99

10472

29498000

139756

-2.29

0.56 ± 0.14

24.06

11431 (3221.1 - 396470)

2135570 (118949 - )

145598

-1.23

0.15 ± 0.12

24.32

146432

-8.17

2.52 ± 0.33

23.97

* 1741.21 (1270.16 2344.11)

151138

-8.78

2.65 ± 0.54

29.89

2043.31 (994.11 - 2948.08)

6212 (4156 - 17112)

158749

-2.47

0.82 ± 0.11

24.33

1062.01 (599.85 - 2084.69)

39210 (12850 - 317890)

5613.1 (3887.1 - 10282)

Table 3. The results of probit analysis for Rhyzopertha dominica adults Isolate

intercept

slope (±s.e.)

x2 (d.f.:18)

LC50 (%95 CI) (ppm)

LC 90 (%95 CI) (ppm)

120602

-2.43

1.19 ± 0.13

35.65

122.41 (63.61 - 197.9)

1463.42 (814.19 - 3828.06)

137691

-3.49

1.44 ± 0.17

53.86

267.26 (114.02 - 471.35)

2071.1 (1087.8 - 7084.6)

138485

-4.36

1.83 ± 0.21

27.25

239.73 (148.82 - 344.08)

1198.56 (808.27 - 2137.53)

139350

-2.18

0.96 ± 0.11

51.77

184.74 (71.21 - 357.77)

3965.7 (1589 - 28522)

139756

-3.12

1.42 ± 0.16

42.85

156.38 (75.62 - 261.29)

1247.99 (696.61 - 3381.21)

145598

-1.56

0.89 ± 0.12

26.52

56.57 (20.93 - 103.55)

1539.89 (805.58 - 4664.25)

146432

-2.72

1.44 ± 0.16

34.17

78.58 (42.57 - 120.65)

612.17 (378.1 - 1325.66)

151138

-4.12

1.89 ± 0.19

17.25

150.56 (111.08 - 195.09)

717.29 (531.83 - 1064.04)

158749

-3.85

1.84 ± 0.19

7.12

124.29 (92.37 - 160.39)

618.41 (457.74 - 920.29)

Acknowledgements: This work was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) [Project No: 112O304] 56

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REFERENCES Arthur, F. H., 1996. Grain protectants: current status and prospects for the future. Journal of Stored Products Research 32, 293-302. Athanassiou, C. G. and Palyvos, N. E., 2006. Laboratory evaluation of two diatomaceous earth formulations against Blattisocius keegani Fox (Mesostigmata, Ascidae) and Cheyletus malaccensis Oudemans (Prostigmata: Cheyletidae). Biological Control 38, 350-355. Athanassiou, C. G. and Steenberg, T., 2007. Insecticidal effect of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) in combination with three diatomaceous earth formulations against Sitophilus granarius (L.) (Coleoptera: Curculionidae). Biological Control 40, 411-416. Athanassiou, C. G., Kavallieratos, N. G., Vayias, B. J., Tsakiri, J. B., Mikeli, N. H., Meletsis, C. M., Tomanovic, Z., 2008. Persistence and efficacy of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) and diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Bostrchidae) on wheat and maize. Crop Protection 27, 1303-1311. Barra, P., Rosso, L., Nesci, A., Etcheverry, M., 2013. Isolation and identification of entomopathogenic fungi and their evaluation against Tribolium confusum, Sitophilus zeamais, and Rhyzopertha dominica in stored maize. Journal of Pest Science 86, 217-226. Cherry, A. J., Abalo, P., Hell, K., 2005. A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea. Journal of Stored Products Research 41, 295-309. Ferizli, A. G., Beris, G., Baspinar, E., 2005. Mortality and F1 production of Rhyzopertha dominica (F.) on wheat treated with diatomaceous earth; impact of biological and environmental parameters on efficacy. Journal of Pest Science 78, 231-238. Haq, T., Usmani, N. F., Abbas, T., 2005. Screeening of plant leaves as grain protectants against Tribolium castaneum during storage. Journal of Botany 37, 149-153. Khashaveh, A. and H. Chelav, S., 2013. Laboratory bioassay of Iranian isolates of entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin (Ascomycota: Hypocreales) against two species of storage pest. Agriculturae Conspectus Scientificus 78, 35-40. Michalaki, M. P., Athanassiou, C. G., Teenberg, T., Buchelos, C.Th., 2007. Effect of Paecilomyces fumosoroseus (Wise) Brown and Smith (Ascomycota: Hypocreales) alone or in combination with diatomaceous earth against Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Biological Control 40, 280-286. Moino, Jr A., Alves, S.B., Pereira, R.M., 1998. Efficacy of Beauveria bassiana (Balsamo) Vuillemin isolates for control of stored-grain pests. Journal of Applied Entomology 122, 301-305. Padin, S., Bello, G. D., Fabrizio, M., 2002. Grain loss caused by Tribolium castaneum, Sitophilus oryzae and Acanthoscelides obtectus in stored durum wheat and beans treated with Beauveria bassiana. Journal of Stored Products Research 38, 69-74. Riasat, T., Wakil, W., Ashfaq, M., Sahi, S. T., 2011. Effect of Beauveria bassiana mixed with diatomaceous earth on mortality, mycosis and sporulation of Rhyzopertha dominica on stored wheat. Phytoparasitica 39, 325-331. 57

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Riasat, T., Wakil, W., Yasin, M., Kwon, Y. J., 2013. Mixing of Isaria fumosorosea with enhanced diatomaceous earth and bitterbarkomycin for control of Rhyzopertha dominica. Entomological Research 43, 215-223. Sewify, G.H., El Shabrawy, H.A., Eweis, M.E., Naroz, M.H., 2014. Efficacy of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae for controlling certain stored product insects. Egyptian Journal of Biological Pest Control 24, 191-196. Shafighi, Y., Ziaee, M., Ghosta, Y., 2014. Diatomaceous earth used against insect pests, applied alone or in combination with Metarhizium anisopliae and Beauveria bassiana. Journal of Plant Protection Research 54, 62-66. Shams, G., Safaralizadeh, M. H., Imani, S., Shojai, M., Aramideh, S., 2011. A laboratory assessment of the potential of the entomopathogenic fungi Beauveria bassiana (Beauvarin) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) and Sitophilus granarius (L.) (Coleoptera: Curculionidae). African Journal of Microbiology Research 5, 1192-1196. Stejskal, V., Hubert, J., Aulicky, R., Kucerova, Z., 2015. Overview of present and past and pestassociated risks in stored food and feed products: European perspective. Journal of Stored Products Research 64, 122-132. Wakil, W. and Schmitt, T., 2014. Field trials on the efficacy of Beauveria bassiana, diatomaceous earth and Imidacloprid for the protection of wheat grains from four major stored grain insect pests. Journal of Stored Products Research 64, 160-167. Wakil, W., M. U. Ghazanfar, M. Yasin, 2014. Naturally occurring entomopathogenic fungi infecting stored grain insect species in Punjab, Pakistan. Journal of Insect Science,14: 182. DOI: 10.1093/jisesa/ieu044. Wakil, W., Riasat, T., Ghazanfar, M. U., Kwon, Y. J., Shaheen, F. A., 2011. Aptness of Beauveria bassiana and enhanced diatomaceous earth (DEBBM) for control of Rhyzopertha dominica F. Entomological Research 41, 233-241.

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ENTOMOLOGICAL RISKS AND MANAGEMENT STRATEGIES IN APRICOT ORCHARDS (DROSOPHILA SUZUKII, CERATITIS CAPITATA, LYRISTES PLEBEJUS) Bennur AĞBABA 1*, Hasan TUNAZ2 1 2

Apricot Research Institute, Malatya, Turkey

Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey

*Corresponding author’s e-mail: bennur.agbaba@tarimorman.gov.tr

ABSTRACT Turkey is located among the world's major fruit-producing countries in terms of quantity and the number of species and varieties of fruit production. One of the important agricultural export products grown in Turkey is apricot. Quality and yield losses in apricot production occur due to pests and diseases in apricot production which is extremely important for our country and Malatya economies. Apricot diseases and pest control is one of great importance to obtain more and quality products. It is very important to diagnose the pest correctly and to be able to intervene in time. With this study, current information about the status of Drosophila suzukii, Ceratitis capitata and Lyristes plebejus in apricot will be given and information will be presented about the serious threats that may occur in the future. According to this information, it is aimed to give information about this kind of management opportunities by taking the information in the literature into consideration. Keywords: Apricot, Ceratitis capitata, Drosophila suzukii, Lyristes plebejus.

INTRODUCTION Apricots have always been considered a significant functional food source for human health because of their high content of active compounds such as vitamin A, sugar, and minerals, and because of their phenolic content and dietary fiber. Apricots cover wide areas, Iran, Turkistan, Afghanistan, Middle Asia, and Western China. Turkey is important for apricot producers in the central Eastern Anatolia Region. Malatya is the most important apricot producing center in Turkey with approximately 8 million trees, and fresh fruit production 401,363 t (TUİK, 2018). Apricot is an important source of economic income for Malatya and its districts. In addition to many harmful species that threaten this source of income, species such as Ceratitis capitata, Drosophila suzukii, and Lyristes plebejus have recently been introduced. Drosophila suzukii Matsumura (Diptera: Drosophilidae) is originally an Asian vinegar fly, invasive and serious economic pest in fruit and berry. D. suzukii invaded Southern Europe and the Eastern USA in 2008 (Becher,2017), and it reported the first time in Turkey in 2014 (Tozlu,2014). It damages ripe, undamaged fruit with a female serrated ovipositor. D. suzukii has a wide range hosts (strawberry, blueberry, peach, grape, apricot, etc.), also alternative host plants such as elderberry, honeysuckle, hackberry, and dogwood. Ceratitis capitata Wied. (Diptera: Tephritidae) is a polyphagous fruit fly with more than 200 hosts. This pest is originated east of Africa and tropical regions, its host plant is mainly citrus also include persimmon, peach, nectarine, pomegranate, avocado, fig, and apricot. 59

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Females lay their eggs in ripe fruit. A female can lay about 300 eggs during a lifetime. The larvae are fed fruit and after 3 periods they become pupae. Lyristes plebejus (common cicada) found in allopatry but calling songs easily distinguished by a few acoustic parameters (Sim˜oes and Quartau, 2013). Common cicada found in most of Europe and extends its distribution to the northern and eastern Mediterranean areas up to Turkey. L. plebejus nymphs were fed by absorbing the capillary roots of the plant, especially during the dry summer season so that dryness was observed especially in old and neglected gardens. L. plebejus adult females prefer annual plants that do not have hard tissues for laying eggs, and that females put their eggs by piercing with the ovipositor of the stem of plants. This article was carried out to promising pest management strategies that have been investigated to help understanding the control of three important species for apricot (Drosophila suzukii, Ceratitis capitata, Lyristes plebejus). Therefore, some information was given management program shortly about pests. Management strategies of Ceratitis capitata (Wiedemann) Ceratitis capitata is an important pest for apricot orchards in some religion in Turkey. The control of C. capitata has been relied on chemical, biotechnical or biological control. The chemical control program is based on organophosphate insecticides, especially malathion (Bachrouch, 2003). However, the use of Malathion has been banned in European Union (EU) (MAPA, 2009), because it has resulted in negative effects; in the development of resistant populations, human health concerns and the harmful effects on beneficial insects such as pollinators, natural parasitoids, untargeted insects (Hafsi et al., 2015). Generally, cover spray or bait spray use protection from C. capitata. Studies showed that using protein bait sprays mixed with organophosphates or low toxicity insecticides are successfully integrated pest management programs (Demirel 2019; Vargas et al., 2001; Urbaneja et al., 2009). Biotechnics control generally relies on mass trapping system. They are using Ceratrap®, Tripack® or Moskisan® (Hafsi et al., 2015; Chaaban 2018). If the population is high, studies show that mass trapping is an important control strategy. Another biotechnical control is that using different attractants. Various attractants provide detection, monitoring and controlling of the C. capitata on different host plants. Demirel et al. (2019) used ammonium acetate (AA), ammonium carbonate (AC), trimethylamine (TMA), ammonium bicarbonate (AB), diammonium phosphate (DAP), 1,4-diaminobutane (putrescine) (P) and their different combination. Results showed that the highest catches at traps changed according to years, that reason can be due to ripening and harvesting time. Management strategies of Lyristes plebejus (Scopoli, 1763) Lyristes plebejus was observed in the apricot orchards in the Turkey region where grown apricot economically (Elazığ, Malatya) in 2001-2002 (Öztürk et al., 2004). There is no chemical control against cicadas. Therefore, priority should be given to cultural measures which are alternative methods of control. Identifying the main and alternative host plants, determining the plants in which they lay eggs and removing them from the cultural areas are important measures in the fight against pests. Kaplan and Tezcan (2017) showed that L. plebejus put their eggs woody plants cherry, peach, and almond dry and wet branches; plum, apple, pear, quince, hazelnut, poplar and olive trees dry branches. Also, this study showed that L. plebejus prefers peeled dry poplar branches to lay eggs, therefore during laying eggs of the 60

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pest, it is important to insert the peeled poplar branches into the soil and remove them from the garden before opening the eggs. During the egg-laying period of cicadas (June-July), weeds should not be mowed or left in strips. After the completion of egg-laying, the mowing of the egg-laid herbs or mixing with the soil may be important in terms of decreasing the density of the following years. Management strategies of Drosophila suzukii (Matsumura, 1931) Drosophila suzukii is a quarantine pest and eradication is necessary for Turkey. Culturel control is that harvest should not be delayed and as soon as possible should be harvested. Also, to prevent the spreading of pests, the gardens should be checked and the spilled fruit should be collected and destroyed. Since the pest can fly over long distances, controls must be carried out by all producers in the contaminated area. The control needs to be made before the larva enters the fruit. However, as the time of insecticides application corresponds to the ripening period of the fruits, it is necessary to be very careful about the last application and the harvest interval. Chemical control of this pest is generally using broad-spectrum insecticides with organophosphorus and pyrethroid groups. Classical biological control relies on using pathogenic fungus, viruses, endoparasitoids, and parasitoids. That plays an important role in effective and ecologically friendly control. Also, using mass trapping control is another control method. Using different baits, pheromones, and physical trap design help to monitoring reduces infections and spreading. CONCLUSIONS As it is known, Drosophila suzukii and Ceratitis capitata is a fruit pest and causes serious economic losses all over the world. Recently, studies on this pests have been increased because it needs to more study about control (integrated, chemical and biological). Lyristes plebejus accepted as an important pest, especially by producers. Studies have to focus on this pest because it needs new strategies to control it. This article can be helpful in future studies to understand management strategies about these pests. REFERENCES Abir, Hafsi, H. Ahlem, R. Ridha, B. Chermiti. (2015). Evaluation of the Efficiency of Mass Trapping of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in Tunisian Citrus Orchards Using Two Types of Traps: Ceratrap速 and Tripack速. Acta horticulturae. Acta Hort. (ISHS). 1049-1056. 10.17660/ActaHortic.2015.1065.131. Chaaban, S. (2018). Evaluation of integrated control based on the Cera Trap速 and Moskisan速 mass trapping system against Ceratitis capitata (Diptera: Tephritidae) on citrus fruits in Tunisian oases. J. of New Science. Vol.57 pp.3707-3715. Demirel, N. (2019). Trapping Genders of Ceratitis Capitata (Diptera: Tephritidae) And Other Dipteran With Various Attractants On Pomegranate Fruits In Turkey. Fresenius Environmental Bulletin. 28. 2937-2941. Demirel, N., Y. Ahmet, K. Gamze (2018). Effectiveness of Various Attractants For Mediterranean Fruit Fly, Ceratitis Capitata (Wiedemann) (Diptera: Tephritidae) on Pomegranate Fruits In Turkey. Fresenius Environmental Bulletin. 27.

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Kaplan, C., S. Tezcan (2017). Egg laying plants of Lyristes plebejus (Scopoli, 1763) (Hemiptera: Cicadidae) determined in the province of Izmir, Turkey. Proceedings of the First International Demirci Symposium, 285-291. Öztürk, N., M.R. Ulusoy, L. Erkiliç, S. Bayhan (2004) Pests and predatory species determined in apricot orchards in Malatya province of Turkey. Bitki Koruma Bülteni, 44, 1– 13. Simões, P.C., J.A. Quartau (2013). Distribution of cicadas of the genus Lyristes (Hemiptera: Cicadidae) in the eastern Mediterranean area. Biologia, 68, 961-965. Urbaneja, A., P. Chueca, H. Monton, S. Pascual- Ruiz, O. Dembilio, P. Vanaclocha, Abad- R. Moyano, T. Pina, P. Castanera, (2009) Chemical alternatives to malathion for controlling Ceratitis capitata (Diptera: Tephri-tidae), and their side effects on natural enemies in Spanish citrus orchards. J. Econ. Entomol. 102, 144-151. Vargas, R.I., S.L. Peck, G.T. McQuate, C.G. Jack-son, J.D. Stark, J.W. Armstrong (2001). Potential for areawide integrated mana-gement of Mediterranean fruit fly (Diptera: Tephritidae) with a braconid parasitoid and a novel bait spray. J. Econ. Entomol. 94, 817- 825.

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HOW SEED BUGS SPREAD (HETEROPTERA: LYGAEIDAE) FROM CEREAL CROPS TO FRUITS Bennur AĞBABA *1, Talip YİĞİT1, Rukiye YAMAN1, Hasan TUNAZ2 1 2

Apricot Research Institute, Malatya, Turkey,

Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey,

*Corresponding author’s e-mail: bennur.agbaba@tarimorman.gov.tr

ABSTRACT The family Lygaeidae is represented by 500 genera and 1000 species in the world. The family includes insects commonly known as seed bugs which are elongate, small to medium-sized bugs. The family is sometimes called the chinch bug family because of feeds on the sap of plants. Lygaeid bugs are common and widespread in different habitats. These are also known as ground bugs because most of them are found under leaf litter on the ground. Few also feed on the plants. Some important members of the family include cotton stainer (Oxycarenus hyalinipennis) and the Australian Rutherglen bug (Nysius vinitor), both of which are destructive to fruit trees. Their infestation during grain set and grain fill will reduce yield, oil content and oil quality. In seed crops, they will reduce the germination of seed. Besides field crops, N. vinitor is also a significant pest of a wide range of horticultural crops. Therefore, this study will examine information about the effect and spreading of seed bugs in horticultural crops in the literature into consideration. Keywords: Lygaeidae, Fruits, Seed bugs, Grain

INTRODUCTION Lygaeidae (Heteroptera) is a large family consisting of small and medium-sized, long oval bodies, dark and bright colored species. Today, around 3000 species are known in the world (Lodos, 1982). Most species feed on plants, especially plant seeds. They are usually found on the soil surface, under stones and debris, and on low plants (Lodos and Önder 1986; Zobar and Kıvan 2005). They have known grain seed bugs or ground bugs, but some species are recorded important fruit species. Also, lygaeids are highly sporadic insect pests all over the world. They feed on plants by using their piercing-sucking mouthparts so that damaging plant tissue. Therefore, they caused such as cell split by stylets activity and cell degradation by salivary enzymes, etc. (Hori 2000; Scaccini and Furlan 2019). Seasonal temperature and rainfall are important factors for the pest population spreading. Also, environmental conditions and climate can affect sudden changes in their population. However, the Hemipteran Lygaeidae has a wide host range so that they can adapt easily environment condition (Moradi-Vajargah, 2017). Also, when the main crop is not available, they can pass the non-crop species (Parry et al., 2019). For example, extreme temperatures above 40 ° C and comes from with drought in which weed food sources are limited, can cause the mass distribution of N. vinitor to other avaılable crops especially high water containing. 63

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Family Lygaeidae is one of the important pests of crops, but it is not studying deeply. This article examined the combined effect of different factors on the behavior of different species of Lygaeidae. Genus Nysius (Dallas) The Hemipteran Lygaeidae genus Nysius (Dallas) has economic important pest species because of its extremely wide host range. Nysius is a polyphagous and common genus, as it is found in almost continents. They are known false chinch bugs. Nysius includes more than 100 species (Scaccini and Furlan 2019). Nysius vinitor Bergroth (Hemiptera: Orsillidae) is the common name Rutherglen Bug, which is changed from Lygaeidae to Orsillidae family by Sweet (2000). It has been considered a pest of grain crops such as canola, cotton, linseed, sorghum, safflower, and sunflower; also, it is a significant pest of a many horticultural crops such as potato, tomato, apricot and peach (Merrill and Sweet 2000; Cassis and Gross 2002; Parry et al., 2019; Kehat and Wyndham 1972; Attia 1982; McDonald and Farrow 1988; Merril and Sweet 2000; Murray et al. 2013; MoradiVajargah, 2017). In Australia, sorghum and canola crops economic losses are estimated at 11.4 million AUD (Murray et al., 2013; Parry et al., 2019). Chemical control N. vinitor is limited and generally, rely on broad-spectrum insecticides such as pyrethroids and dimethoate which is effective only infected plants (Moradi-Vajargah and Parry 2017; Parry et al., 2019). Nysius cymoides (Spinola) (Hemiptera: Lygaeidae) is an important pest for various crop species including cotton, alfalfa, clover, canola, almond and apple (Koohpayma et al., 2018). It damages desiccation, wilting and yellowing tissues, necrosis of leaves and then affects crop yield (Scaccini and Furlan 2019). N. cymoides were detected in a different region in Italy and they thought that warm weather and drought can be effective to spread second crops. Oxycarenus hyalinipennis (Costa) It belongs to family Oxycarenidae (Henry 1997), and the common name is cottonseed bug. It causes serious damage to the cotton crop. But they have a wide range of host plants including apple, avocado, corn, dates, figs, grapes, peach, okra, pineapple, and pomegranate, as well as hibiscus (USDA, 2009; Smith and Brambila 2008). It affects the quality of the seed by sucking oil from mature seeds. The pest may damage fruit trees by sucking and excreting toxic saliva and mostly deforming fruits with its feces. Besides, the bugs spread house, and walls of buildings so that they disturb people with an unpleasant odor and an urban nuisance. CONCLUSIONS Finally, the family of Lygaeidae has a wide host range and sporadic insect. These family members of insects are primarily grain crops pest, but they can move fruit crops drought weather or main host not available. Chemical, biological and cultural control are available for this pest. This article hopefully will give observations and bring to the attention of ecologists and hemipterous at their studies. REFERENCES Attia, F.I. (1982). Comparative studies on the biology of Nysius vinitor Bergroth and N. clevelandensis Evans (Hemiptera: Lygaeidae). General and Applied Entomology 14, 15â&#x20AC;&#x201C;21. Cassis, G., Gross, G.F. (2002). Zoological Catalogue of Australia. Volume 27.3B, Hemiptera: Heteroptera (Pentatomomorpha), CSIRO Publishing, Collingwood, Australia. 64

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Henry, T.J. (1997). Phylogenetic analysis of family groups within the infraorder Pentatomorpha (Hemiptera: Heteroptera), with emphasis on Lygaeoidea. Ann. Entomol. Soc. Am. 90: 275-301. Hori K. 2000. Possible causes of disease symptoms resulting from the feeding of phytophagous Heteroptera. in: Schaefer CW, Panizzi AR, editors. Heteroptera of economic importance. Boca Raton (FL): CRC Press; p. 11–35. Kehat, K., Wyndham, M. (1972). The effect of food andwater on development, longevity, and fecundity in the Rutherglen bug, Nysius vinitor (Hemiptera: Lygaeidae). Australian Journal of Zoology 21, 427–434. Koohpayma, F., Bagheri, A., Fallahzadeh, M., Dousti, A. Fazel, & Askari-Seyahooei , M. (2018). Nysius cymoides (Hemiptera: Lygaeidae), a New Economically Important Pest on Acacia tortilis and Its Intracellular Bacterial Endosymbionts. Entomological news, 128, 11-23. doi: 10.3157/021.128.0103. Lodos N. (1982). Türkiye Entomolojisi II (Genel, Uygulamalı ve Faunistik). 591 S. Ege Üniversitesi Ziraat Fakültesi Yay. No. 429, Bornova, İzmir. Lodos N., Önder F. (1986). Heteroptera Türkiye ve Palearktik Bölge Familyaları hakkında genel bilgi. 111 S. Ege Üniversitesi Ziraat Fakültesi Yay. No. 359, Bornova, İzmir. McDonald, G., Farrow, R..A. (1988). Migration and dispersal of the Rutherglen bug, Nysius vinitor Bergroth (Hemiptera: Lygaeidae), in eastern Australia. Bulletin of Entomological Research 78, 493–509. Merril, H., Sweet I. 2000. Seed and chinch bugs (Lygaeoidea). In: Heteroptera of Economic Importance (eds CWSchaefer & AR Panizzi), pp. 143–264. CRC Press, Boca Raton, Florida. Merrill, H., Sweet II. (2000). Chapter 6 Seed and Chinch Bugs (Lygaeoidea). CRC Press, Boca Raton, FL. Moradi-Vajargah, M., Parry, H.R. (2017). Environmental and biological drivers of flight initiation in a sporadic pest, Rutherglen bug, Nysius vinitor Bergroth (Hemiptera: Orsillidae). Austral Entomology 56 (2), 225–234. Murray, D.A., Clarke, M.B. Ronning D.A. (2013). Estimating invertebrate pest losses in six major Australian grain crops. Australian Journal of Entomology 52, 227–241. Parry, Hazel R., M. Anna, M. Sarina, H. Jamie, H. Andrew, N. Mick, B. Felix, J.J.A. Franzmann, B. A. Lloyd, R. J. Miles, M. Zalucki, Myron P. Schellhorn, N. A. (2019). A native with a taste for the exotic: weeds and pasture provide year-round habitat for Nysius vinitor (Hemiptera: Orsillidae) across Australia, with implications for area-wide management. In: Austral Entomology.Vol. 58, No. 2. pp. 237-247. Scaccini, D., Furlan, L. (2019). Nysius cymoides (Hemiptera: Lygaeidae), a potential emerging pest: overview of the information available to implement integrated pest management, International Journal of Pest Management. https://doi.org/10.1080/09670874.2019.1666174. Smith, T.R., Brambila, J.A. (2008). A Major Pest of Cotton, Oxycarenus hyalinipennis (Heteroptera: Oxycarenidae) in the Bahamas. USDA. (2009). Significant Pest Bulletin Cottonseed Bug. United States Department of Agriculture, Animal and Plant Health Inspection Service. 1 p. Zobar H., Kıvan M. (2005). Lygaeus equestris (L.) (Heteroptera: Lygaeıdae)’in Bazı Biyolojik Özellikleri. Trakya Univ J Sci, 6(1): 59-62.

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INVESTIGATION OF SOME AGRICULTURAL TRAITS AND TOLERANCE TO VERTICILLIUM WILT ON COTTON Fatih Mehmet TOK1*, Yaşar AKIŞCAN2 1

Hatay Mustafa Kemal University, Dept. of Plant Protection, Antakya, Hatay, Turkey 2

Hatay Mustafa Kemal University, Dept. of Field Crops, Antakya, Hatay, Turkey *

Corresponding author’s email: ftok@mku.edu.tr

ABSTRACT This study was carried out with 3 cotton varieties (BA-525, Çukurova 1518 and Edessa) under the Amik plain conditions in 2017 growing season in order to investigate of some agricultural traits and tolerance to Verticillium wilt on these cotton varieties. The experiment was established according to randomized complete block design with 4 replications. Seeds were sown in plots each of which has 10m long 4 lines. Verticillium dahliae spore suspension was injected to the first node of plants which located on the outer 2 lines of plots by stem injection method. Just before the harvest, disease symptoms were measured by 0-4 scale according to both leaf and vascular tissue symptoms. Data obtained from experiment were analyzed by SAS statistical analysis software. As a result of the study, variance analysis showed that difference among varieties, were statistically significant in terms of plant height, number of node and boll per plant, seedcotton weight per boll, 100 seed weight, disease severity index according to leaf symptoms, seedcotton weight and ginning ratio traits (P < 0.01), and in terms of disease severity index according to stem tissue symptoms (P < 0.05). According to the results of our study, plant height ranged from 81.78 cm (BA-525) to 58.88 cm (Edessa), number of node per plant 17.15 (Çukurova 1518) to 14.15 (Edessa), number of boll per plant 10.93 (Edessa) to 8.30 (Çukurova 1518), seedcotton weight per boll 5.85 g (Çukurova 1518) to 5.18 g (Edessa), 100 seed weight 12.03 g (Çukurova 1518) to 10.15 g (Edessa), disease severity index according to leaf symptoms 2.33 (Çukurova 1518) to 1.85 (Edessa), disease severity index according to stem tissue symptoms 2.48 (Çukurova 1518) to 2.08 (Edessa), seedcotton yield 440.38 kg/da (Edessa) to 310.50 kg/da (Çukurova 1518) and ginning ratio ranged from 42.35 % (Edessa) to 38.18 % (Çukurova 1518). In the present study, Edessa was more tolerant to Verticillium wilt, and it gave more seedcotton yield and ginning ratio compared to the other varieties used. Keywords: Cotton, Verticillium wilt, Tolerance, Yield components INTRODUCTION Cotton is the most important cash crop in the world as it has unique natural fibers. These fibers harvested from cotton plants are the main raw material of textile sector which provides approximately 20 % of export proceeds in Turkey. During 2015-2016 cotton growing season, 738 thousand tones cotton fiber was produced in 434 thousand hectare areas in Turkey. However, consumption was 1.656 million tones (Özüdoğru, 2017). These situation shows us fibers, which approximately 55 % of consumed in Turkey, are provided by importation. Thus, the studies on increasing the yield and production are important for Turkish economy. The yield is significantly affected by biotic and abiotic stress factors. One of the most important biotic factor which causes severe yield losses in cotton production is Verticillium wilt caused by a soil-borne fungal plant pathogen, Verticillium dahliae Kleb. Fungus can survive up 66

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to 15 years in the soil (Agrios, 2005) as a microscloratia. The fungus, which infects cotton plant, reproduces very fast in the vascular tissues and blocks the xylem transportation, eventually (Gubler, 1996). Cotton harvested from diseased plants has less yield and quality (Korolev et al., 2001; P´erez-Art´es et al., 2000). There is no effective chemical control method for Verticillium dahliae. Thus, growing resistant cotton cultivars to Verticillium wilt is advised to the growers in contaminated areas (Wilhelm et al., 1974; Schnathorst and Cooper, 1975, Akışcan, 2011). The aim of this study was to investigate of some agricultural traits and tolerance to Verticillium wilt on same cotton varieties. MATERIAL AND METHODS The varieties, BA-525, Çukurova-1518 and Edessa were used in this study as plant material. And Verticillium dahliae isolate coded as CotVd17 which determined as highly virulent was used as fungus isolate (Derviş at al., 2008; Akışcan and Tok, 2019) This study carried out during 2017 cotton growing season in experimental fields of Progen Seed Company which is located in the Hatay province of Turkey. Experimental area has typical Mediterranean climate with warm, dry summers and wet, mild winters (Table 1). Table 1. Detailed long term (1940-2017) climate information of experimental area. Temperature ◦C Months Long Term (1940-2017) a Max. Min. Mean Max. June 29.2 20.8 24.8 36.5 July 31.1 23.8 27.1 48.0 August 31.9 24.5 27.8 46.4 September 31.0 21.1 25.6 39.6 October 27.3 15.1 20.6 33.3 a

2017 b Min. 16.2 21.9 20.7 14.1 8.9

Mean 25.6 29.1 28.6 27.0 20.5

2017 precipitation 2017 Relative (mm) b humidity (%)b Total Mean 0.0 77.6 0.2 74.6 0.0 79.4 2.8 73.3 109.8 64.4

Anonim, 2018, bAnonim, 2017.

The experiment was conducted in randomized complete block design with three replications. Plants were sown with 70 cm intra-row and 20 cm inter-row spacing on 01.06.2017. Experimental parcels were 4 rows in 10m length. Drip irrigation system was used in the field and totally 650 mm water was used. First and last 1 m of the lines and outer lines were not used in measurement. Cotton plants were harvested on 13.10.2017 from middle two lines of parcels. Plant height, number of nodes and number of bolls was measured on 10 randomly selected plants and mean numbers were calculated from every parcel. Plant height was measured between cotyledon node and top of the plants. Number of nodes was counted from main stem. Number of bolls was counted from these plants. Mean of twenty randomly selected bolls from each parcel were used to obtain seedcotton weight per plant. Four sets of 100 seeds from cotton plants were scaled to calculate means of 100 seeds weight. Seedcotton yield was measured as kg/da every parcels. After harvest, seedcotton samples was separated into fiber and seed by using saw-gin and ginning turnout was calculated by the equation below. Ginning turnout (%) =

Fiber (g) x 100 Fiber (g) + Seed (g)

In the pathogenicity studies, Verticillium dahliae isolate was transferred to petri dishes amended with antibiotics. Seven days after incubation, spores of V. dahliae were harvested with 67

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sterilized distilled water by using a spatula. Suspension was filtered with 4 layers of cheese cloth and mycelial remains were discarded. Spore concentration was set at 107 with Thoma cell counting chamber. Spore suspension was inoculated to cotton plants from just upper of the first node by stem injecting method. Before injecting, inoculation points on the stem of plants were stabbed with a sharpened nail mounted in a wood. Then spore suspension injected into the stem of cotton plants with a syringe. Disease severity index was calculated with the data obtained from foliar and vascular symptoms. A 0-4 visual scale was used to measure foliar disease severity on the plants; 0= no symptoms, 1= lightly yellowing or wilting, 2-3= severe yellowing, wilting, necrosis, leaf drop and dieback, 4= dead plants. Symptoms on the vascular tissues were measured with a 0-4 disease scale; 0= no discoloration in the vascular tissues, 1= 1-25 % discoloration, 2= 26-50 % discoloration, 3=51-75% discoloration, 4=76-100% discoloration (Bhat and Subbarao, 1999; İrget, 2018; Akışcan and Tok, 2019). Data was analyzed by SAS statistical software according to randomized complete block design and significantly different means were grouped with Duncan’s multiple range test at 5% significance level (SAS Institute Inc., 1998). RESULTS AND DISCUSSION As a result of the study, it was determined that varieties caused statistically difference at P<0.01 level in terms of plant height, node number per plant, 100 seed weight, boll number per plant, seedcotton weight per boll, seedcotton yield, ginning turnout and disease severity index obtained from leaves, also it caused significant difference in disease severity index obtained from vascular tissue at P<0.05 level. Table 2. Plant height, node number per plant and 100 seeds weight of different varieties. Varieties BA-525 Çukurova-1518 Edessa C.V. (%) Probability

Plant height (cm) 81.78 a# 74.15 a 58.88 b 6.72 **

Node number per plant 16.35 b 17.15 a 14.15 c 2.12 **

100 seed weight (g) 10.83 b 12.03 a 10.18 b 3.87 **

** Statistically significant at P<0.01 level. # Means with the same letter are not significantly different.

Table 3. Boll number per plant, seedcotton weight per boll, seedcotton yield, ginning turnout of different varieties. Varieties BA-525 Çukurova-1518 Edessa C.V. (%) Probability

Boll number per plant 9.93 b# 8.30 c 10.93 a 4.50 **

Seedcotton weight Seedcotton yield Ginning turnout per boll (g) (kg da-1) (%) 5.68 b 394.75 b 39.18 b 5.85 a 310.50 c 38.18 b 5.18 c 440.38 a 42.35 a 1.56 5.42 1.76 ** ** **

** Statistically significant at P<0.01 level. # Means with the same letter are not significantly different.

Plant height varied from 58.88 cm to 74.15 cm in the experiment. Varieties were grouped into two different groups. BA-525 (81.78) and Çukurova-1518 (74.15) was in group 68

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“a” which is taller. According to node number per plant three different groups formed. Çukurova-1518 (17.15) was in group “a” having highest node number value, and Edessa was in group “c” having lowest value. As to 100 seeds weight, it varied from 10.18 g to 12.03 g in this study. Çukurova-1518 produced highest seed weight with 12.03 g in group “a” and other group produced lowest 10.18 g (Edessa) and 10.83 g (BA-525) (Table 2). Mean number of bolls per plant varied from 8.30 to 10.93. Varieties were separated into three different groups. Edessa produced the most bolls with 10.93 followed by BA-525 with 9.93 and Çukurova-1518 with 8.30 bolls per plant. Seedcotton weight per boll was between 5.18 and 5.85. Çukurova-1518 was found producing the most seedcotton weight per boll with 5.85 followed by BA-525 with 5.68 and Edessa with 5.18. Varieties were separated into three different groups. Edessa produced highest seedcotton yield with 440.38 kg/da followed by BA525 with 394.75 kg/da, and Çukurova-1518 produced the least seedcotton yield with 310.50kg/da. Ginning turnout was separated into two different groups and Edessa was in first group with highest value (42.35 %), and BA-525 and Çukurova-1518 were in the second group with % 39.18 and 38.18, respectively (Table 3). Table 4. Disease severity index obtained from both vascular and leaves symptoms. Varieties BA-525 Çukurova-1518 Edessa C.V. (%) Probability

Disease severity index obtained from vascular tissue 2.13 b# 2.48 a 2.08 b 7.64 *

Disease severity index obtained from leaves 2.08 b 2.33 a 1.85 b 6.65 **

*, ** Statistically significant at P<0.05 and P<0.01 level respectively. # Means with the same letter are not significantly different.

Disease severity index obtained from vascular tissues varied from 2.08 to 2.48 and varieties were separated into two different groups. Edessa (2.08) and BA-525 (2.13) were in the most tolerant group according to vascular tissue symptoms, and Çukurova-1518 variety was susceptible. Disease severity index obtained from leaves varied from 1.85 to 2.33. Disease severity index obtained from leaves was similar to vascular tissue results. Edessa and BA-525 were in tolerant group, and Çukurova-1518 was susceptible. CONCLUSION As a result of the study, Edessa was more tolerant to Verticillium wilt, and it gave highest seedcotton yield and ginning turnout compared to the other varieties used. Thus Edessa cotton variety can be advised to growers in the areas contaminated with V. dahlia. Acknowledgements: The authors would like to gratefully acknowledge the kind support of Progen Seed Company. REFERENCES Agrios GN 2005. Plant Pathology (5th edition). Department of Plant Pathology, University of Florida, Elsevier Academic Pres. USA, page: 527.

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Akışcan Y 2011. Pamukta (Gossypium hirsutum L.) Verticillium Solgunluğu (Verticillium dahliae Kleb.) Hastalığına Dayanıklılık, Erkencilik, Verim ve Kalite Özelliklerinin Kalıtımı. Çukurova Üniversitesi, Fen Bilimleri Enstitüsü, Tarla Bitkileri ABD, Doktora Tezi. Akışcan, Y. and Tok, F. 2019. Farklı Kökenli Bazı Pamuk Genotiplerinde Verticillium Solgunluğuna Dayanıklılığın Araştırılması. KSÜ Tarım ve Doğa Derg 22(Ek Sayı 2): 354-360. Anonim, 2017. Progen Tohum A.Ş Meteoroloji İstasyonu Verileri, Antakya, Hatay Anonim, 2018. T.C. Tarım ve Orman Bakanlığı, Meteoroloji Genel Müdürlüğü. https//www.mgm.gov.tr Bhat RG, Subbarao KV 1999. “Host range specificity in Verticillium dahliae”, Phytopathology, 89:1218-1225. Derviş S, Kurt Ş, Soylu S, Erten L, Soylu EM, Yıldız M, Tok FM 2008. “Vegetative compatibility groups of Verticillium dahliae from cotton in the southeastern anatolia region of Turkey. Phytoparasitica, 36(1):74-83. Gubler WD 1996. Verticillium wilt. in: Zitter TA, Hopkins LD, Thomas CE [Eds.] Compendium of Cucurbit Diseases. APS Press, St. Paul, MN, USA. İrget, M. 2018. Farklı Orijinli Pamuk Genotiplerinin Tarımsal, Teknolojik ve Verticillium Solgunluğu Hastalığına Dayanıklılık Özelliklerinin Belirlenmesi. Hatay Mustafa Kemal Üniversitesi, Fen Bilimleri Enstitüsü, Tarla Bitkileri ABD, Yüksek Lisans Tezi. Karcılıoğlu A, Onan E, Sezgin E 1992. Bazı pamuk çeşitlerinin Verticillium dahliae Kleb. Fungusunun neden olduğu solgunluk hastalığına karşı duyarlılıklarının saptanması üzerinde araştırmalar. İzmir. Zirai Mücadele Araştırma Yıllığı No: 22-23, s. 138. Koike ST, Subbarao KV, Davis RM, Gordon TR, Hubbard JC 1994. Verticillium wilt of cauliflower in California. Plant Dis., 78: 1116- 1121. Korolev N, Pérez-Artés E, Bejarano-Alcázar J, Rodr´ıguez-Jurado D, Katan J, Katan T, JimenezDiaz RM 2001. Comparative study of genetic diversity and pathogenicity among populations of Verticillium dahliae cotton in Spain and Israel. Eur. J. Plant Pathol., 107:443-456. Krikun J, Bernier CC 1987. Infection of several crop species by two isolates of Verticillium dahliae. Can. J. Plant Pathol., 9: 241-245 Pérez-Artés E, Garc´ıa-Pedrajas MD, BejaranoAlcázar J, Jiménez-D´ıaz RM 2000. Differentiation of cotton-defoliating and nondefoliating pathotypes of Verticillium dahliae by RAPD and specific PCR analyses. Eur. J. Plant Pathol., 106:507-517 Schnathorst WC, Cooper JR 1975. Anomalies in Field and Greenhouse Reaction of Certain Cotton Cultivars in Fected with Verticillium dahliae. In Proc. Beltwide Cotton Prod. Conf., 6-8 January, New Orleans, National Cotton Council, Memphis, p. 148-149. Subbarao KV, Hubbard JC, Greathead AS, Spencer GA 1997. Verticillium wilt. In: Compendium of Lettuce Diseases (Eds.: R.M. Davis, K.V. Subbarao, R..N. Raid and E.A. Kurtz). The American Phytopathological Society, St. Paul, MN, USA, pp. 26-27. SAS Institute Inc. 1998. SAS/STAT User's Guide, Version 6. SAS Institute Inc., Cary, NC, USA Wilhelm S, Sagen JE, Tietz H 1974. “Resistance to Verticillium wilt in cotton: sources, techniques of identification, inheritance trends, and the resistance potential of multiline cultivars”, Phytopathology, 64: 924-931.

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INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) IN POULTRY Kemal KARABAĞ*1, Berkant İsmail YILDIZ2, Mustafa ERSAL2 1

Department of Agricultural Biotechnology, Faculty of Agriculture, Akdeniz University, 07058, Antalya, Turkey

Akdeniz University, Graduate School of Natural and Applied Sciences, 07058, Antalya, Turkey *Corresponding author’s e-mail: karabag@akdeniz.edu.tr

ABSTRACT Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone that structurally related to insulin, which, has multifunctional metabolic activities. IGF-1 contains 70 amino acids with approximately 7600 kDa weights. In birds, IGF-1 which has high molecular similarity to insulin hormone shows significant effects on growth and development. IGF-1 gene plays a key role in the growth of multiple tissues including muscle cells. Many variations in this gene affect gene expression at the transcriptional and translational levels. and these variations can affect the growth and developmental characteristics of animals. The development of molecular biology, especially DNA based markers, during the past three decades has created new approaches for several economically important traits in chickens. Selection according to genotype has the power to increase the productivity of farm animals, as well as to enhance environmental adaptation and maintain genetic diversity. Although recent advances in molecular genetic tools have facilitated the understanding of the functional mechanism of the IGF-1 gene, more information isn’t known about its effects in the regulation of this gene and the nucleotide variation in the gene region promoter. Recently, many studies have been conducted to identify mutations in the IGF-1 gene, especially in poultry, and to understand its relationships with growth and reproduction characteristics. Conclusion, the IGF-1 gene is a candidate gene that can be used in breeding programs. Keywords: Growth, IGF-1, Poultry, Reproduction traits INTRODUCTION Poultry meat is the most consumed meat in the world, especially in OECD countries (OECD, 2014). The global population is expected to reach 9 billion by 2050, as well as an increase in income among poor populations will lead to an unprecedented increase in animal protein demand (King et al., 2017). In this context, the poultry industry has an important role to play in ensuring sustainable food supply, and should be given consideration yield improvement studies in this sector. How growth, development and production regulate in livestock, the relationships between genetic variations and yield traits at various loci can be revealed more clearly through molecular biology techniques and new advances in peptide biochemistry (Kaiya et al. 2007). Candidate gene approach is a prediction used in mapping and identification of genes that affect quantitative properties. A candidate gene can be defined as a gene that is closely linked to a functional gene and has a biological effect on the functional physiology of the particular feature. 71

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On this basis, a number of potential candidate genes have been identified in recent years in relation to the yields of livestock. As candidate genes show physiological or biochemical differences due to changes in their regulator or structural regions, they may lead to differentiation in the quality and quantity of the product. One of these candidate genes is the Insulin-like growth factor-1 (IGF-1) gene with effective physiological responsibilities. In this review, the effects of IGF-1 gene on yield parameters in poultry will be reported. IGF-1 in Poultry IGF-1(Figure 1) is a hormone whose molecular structure resembles insulin, plays important role in the development of growth and reproductive properties. IGF-1 gene is located on chromosome 1 in poultry and consists of 4 exons and 2 introns (Khadem et al., 2010; Tang et al., 2010). IGF-1 is synthesized in the liver under the control of growth hormone (GH) and is involved in various physiological or metabolic processes such as glucose absorption, myogenesis, apoptosis, lipid synthesis, progesterone production in granular cells. All these functions are initiated by signal transduction of IGF-1 to IGF1R, a member of the tyrosine kinase receptor family.

Figure 1. a) Structure of chicken IGF1 protein, b) Location of the mature protein within preproIGF1 and the disulfide bonds (yellow) (Ahiagbe, 2018). IGF-1 has been shown to be a candidate gene for growth in chickens, body composition and metabolism, skeletal properties, and adipose tissue growth (Zhou et al., 2005). Several studies have shown that circulating IGF-1 affects growth rate in poultry (Goddard et al., 1988; Scanes et al., 1989; Ballard et al., 1990). It was observed that in different chickens selected according to their growth rates, IGF expression was higher in chickens with high growth than chickens with low growth (Beccavin et al., 2001). Detection of IGF-1 in chick embryo blood on the sixth day (DePablo et al., 1990) suggests that IGF-1 is directly involved in the early embryonic structure. And many studies have shown that circulating IGF-1 increases with advancing age (Figure 2).

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Figure 2. Profiles of plasma IGF-1 during growth of broiler ( laying ( ) strains of chickens (McMurtry et al. 1997).

), and egg-

Due to its broad biological effects and therapeutic potential, IGF-1 has become the focus of research by many researchers. Tomas et al. (1998) showed that the infusion of recombinant human IGF-1 in chickens increases growth and reduces carcass fat content. Kocamis et al. (1998) reported that in-ovo administration of recombinant IGF-1 affects postnatal growth and development in broiler chickens. Gouda and Essawy (2010) stated that the effects of IGF-1 gene polymorphism on growth characteristics were significant in Egypt chicken breeds. Tang et al. (2010) reported that an Single Nucleotide Polymorphisms (SNP) on IGF-1 gene in chickens was significantly related to body weight, shank length and diameter. Ou et al. (2009) reported that SNP on the IGF-1 gene affect chicken growth and reproductive characteristics in chickens, and that SNPs are effective in achieving early growth and sexual maturity in chickens and can be used for MAS (Marker Assisted Selection). Lei et al. (2005) detected 5 SNPs in selected chickens and reported that the resulting SNP haplotypes were associated with growth and carcass characteristics. Lei et al. (2007) showed that an SNP detected on IGF-1 gene is effective on leg muscle fiber in chickens. Sato et al. (2012) reported that an SNP detected in the promoter region of IGF-1 gene was significantly associated with chest muscle contraction and that the identified polymorphisms could be used in MAS. Kim et al. (2004) reported that IGF1 genotypes can be used as markers for egg production based on egg yield. Li et al. (2009), investigated the relationship between Growth Factor Receptor (GHR), Insulin-like Growth Factor-1 (IGF-1) and Neuropeptide Y (NPY) genes and egg yield, double yolk egg and laying time (day) in chickens, and they found the relationship between IGF-1 polymorphism and egg production important. RESULTS In developing and developed countries, the part of animal protein in people diet living in cities is large. The need for animal protein will increase with increasing population growth. This increase will increase the need for poultry meat because it is cheap and easily accessible. Poultry meat and eggs are high quality sources of protein that are needed by millions of people. In addition to high protein content, it also contains many vitamins and minerals. 73

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Long-term selection programs to improve economic characteristics in poultry include meat and egg yields in general. Meat and egg yields are quantitative characteristics and are under the control of many genes. They are also significantly affected by environmental factors. Therefore, the phenotypic value for quantitative characters often does not represent the genotypic value well. Nowadays, the information obtained from candidate genes can be used as genetic markers and these markers can be transferred to breeding programs in various ways. The use of genetic markers in breeding programs is called marker-assisted selection (MAS). MAS improves the success of breeding programs by helping to select superior genotypes welldirectly and in shorter time. All results show that IGF-1 gene is a potential gene that can be used in breeding programs. The use of IGF-1 in marker-assisted selection appears to be efficient and will lead to improved production performance in animals. REFERENCES Ahiagbe, K. M. J. (2018). Single Nucleotide Polymorphisms in Insulin-Like Growth Factor (IGF) Genes and their Associations with Growth in Local Guinea FOWLS (NUMIDA MELEAGRIS) of Ghana (Doctoral dissertation, University Of Ghana). Ballard, F. J., R. J. Johnson, P. C. Owens, G. L. Francis, F. M. Upton, J. P. McMurtry, J. C. Wallace (1990). Chicken insulin-like growth factor-I: Amino acid sequence, radioimmunoassay, and plasma levels between strains and during growth. Gen. Comp. Endocrinol., 79:459–468. Beccavin, C., B. Chevalier, L. A. Cogburn, J. Simon, M. J. Duclos (2001). Insulin-like growth factors and body growth in chickens divergently selected for high or low growth rate. J. Endocrinol., 168:297–306. DePablo, F., L. A. Scott, J. Roth (1990). Insulin and insulin-like growth factor I in early development: peptides, receptors and biological events. Endocrinonogy Reviews, 11:558–578. Goddard, C., R. S. Wilkie, and I. C. Dunn (1988). The relationship between insulin-like growth factor-1, growth hormone, thyroid hormones and insulin in chickens selected for growth. Domest. Anim. Endocrinol., 5:165–176. Gouda, E. M., G. S. Essawy (2010). Polymorphism of insulin-like growth factor I gene among chicken breeds in Egypt. Zeitschrift für Naturforschung C., 65(3-4), 284-288. Kaıya H., V. M. Darras, K. Kangawa (2007). Ghrelin in Birds: Its Structure, Distribution and Function. The Journal of Poult. Sci., 44, 1-18. Khadem, A., H. Hafezian, G. Rahimi-Mianji (2010). Association of single nucleotide polymorphisms in IGF-I, IGF-II and IGFBP-II with production traits in breeder hens of Mazandaran native fowls breeding station. African journal of Biotechnology, 9(6), 805-810. Kim M. H., Seo D. S., Ko Y (2004). Relationshipi between egg productivity and insulin- like factor- I genotypes in Korean native ogol chickens. Poult. Sci. 83: 1203-1208. King, T., M. J. Osmond-McLeod, L. L. Duffy (2017). Nanotechnology in the food sector and potential applications for the poultry industry. Trends in Food Science & Technology, 72, 62-73. Kocamis, H., D. C. Kirkpatrick-keller, H. Klandrof, J. Killefer (1998). In-ovo Administration of Recombinant Human Insulin-Like Growth Factor-I alters Postnatal Growth and Development of the Broiler Chicken. Poult. Sci., 77: 1913–1919. Lei, M. M., Q. H. Nie, X. Peng, D. X. Zhang, X. Q. Zhang (2005). Single nucleotide polymorphisms of the chicken insulin-like factor binding protein 2 gene associated with chicken growth and carcass traits. Poult. Sci., 84(8), 1191-1198. 74

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Lei, M., C. Luo, X. Peng, M. Fang, Q. Nie, D. Zhang, ... X. Zhang (2007). Polymorphism of growth-correlated genes associated with fatness and muscle fiber traits in chickens. Poult. Sci., 86(5), 835-842. Li, H. F., W. Zhu, K. Chen, X. Wu, Q. Tang, Y. Gao, ... H. Xu (2009). Polymorphism in NPY and IGF-I genes associate with reproductive traits in Wenchang chicken. African journal of Biotechnology, 8(19). McMurtry, J. P., G. L. Francis, Z. Upton (1997). Insulin-like growth factors in poultry. Domestic animal endocrinology, 14(4), 199-229. OECD, (2014). Meat consumption. Retrieved from http://www.oecd-ilibrary.org/content/ indicator/fa290fd0-en. Ou J. T., S. Q. Tang, Y. Zhang (2009). Polymorphisms of Three Neuroendocrine-Correlated Genes Associated with Growth and Reproductive Traits in the Chicken. Poult. Sci., 88, 722-7. Sato, S., T. Ohtake, Y. Uemoto, Y. Okumura, E. Kobayashi (2012). Polymorphism of insulin‐ like growth factor 1 gene is associated with breast muscle yields in chickens. Animal science journal, 83(1), 1-6. Scanes, C. G., E. A. Dunnington, F. C. Buonomo, D. J. Donoghue, P. B. Siegel (1989). Plasma concentrations of insulin like growth factors (IGF)-I and IGF-II in dwarf and normal chickens of high and low weight selected lines. Growth Dev. Aging ,53:151–157. Tang, S., D. Sun, J. Ou, Y. Zhang, G. Xu, Y. Zhang (2010). Evaluation of the IGFs (IGF1 and IGF2) genes as candidates for growth, body measurement, carcass, and reproduction traits in Beijing You and Silkie chickens. Animal biotechnology, 21(2), 104-113. Tomas, F. M., R. A. Pym, J. P. McMurtry, G. L. Francis (1998). Insulin-like growth factor (IGF)-I but not IGF-II promotes lean growth and feed efficiency in broiler chickens. Gen. Comp. Endocrinol, 110:262–275. Zhou, H., A. D. Mitchell, J. P. McMurtry, C. M. Ashwell, S. J. Lamont (2005). Insulin-like growth factor-I gene polymorphism associations with growth, body composition, skeleton integrity, and metabolic traits in chickens. Poult. Sci., 84(2), 212-219.

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DEFOLIANT APPLICATIONS AND EFFECTS FOR COTTON HARVESTER Abdullah SESSİZ*1, Reşat ESGİCİ2, F. Göksel PEKİTKAN1, A. Konuralp ELİÇİN1 1

Dicle University, Faculty of Agriculture, Department of Agricultural Machinery and Technologies Engineering, Diyarbakır, Turkey 2

Dicle University, Bismil Vocational High School, Diyarbakır, Turkey *Corresponding author’s e-mail: asessiz@dicle.edu.tr

ABSTRACT More than half of the cotton (Gossypium hirsutum L.) produced in Turkey is provided by the Southeastern Anatolia Region. Cotton production area and the production amount in the region are constitutes of 58% of Turkey's total cotton production areas at current situation. Harvest period is the most important stage that affects the cotton fiber quality, yield and the gain in cotton production. The product must be collected as soon as possible and with minimal losses for profitability and quality. Hence, to reduce production costs and to improve the fiber quality, completeness of the application must be executed correctly in machine harvesting of cotton. One of the processes affecting cotton harvest is defoliant applications. Farmers who used cotton harvest machine and machine owners in Turkey are making the defoliant application as required and mandatory application. Therefore, the use of chemicals such as deciders, boll openers and leaf desiccants are requires for harvesting of cotton by machine. Defoliant and boll opener application time for machine harvest, application rate and method is very important. So, the defoliant application time and execution of application should be considered for a successful mechanical harvest. With the help of chemicals, leaves are fall earlier and bolls are opened more quickly. The defoliant application time and the defoliant amount are the main factors for machine harvest and to increase the efficiency of the machines used in the cotton harvest depending on the current weather conditions. With these applications, it helps to achieve higher yield and fiber quality by enabling the harvest to be done earlier. However, the deficiencies caused by defoliant application increase the losses of harvest and decrease the fiber quality. The success of the machine harvest depends on defoliant application time, application norm and chemical dose. Therefore, it is essential for a successful machine harvest that the defoliant is made in a timely and appropriate application norm. Keywords: Cotton, Harvest, Defoliant, Quality, Losses, Cotton harvester INTRODUCTION The fact that cotton plant is a fundamental raw material in different sectors such as cotton gin, fiber, textile, oil, paper along with the employment opportunities it provides coupled with the added value opportunities it generates makes the increase of its production and yield value important for Turkey. In addition to being the seventh in the world in terms of cotton production after China, USA, India, Pakistan, Uzbekistan and Brazil, Turkey is also one of the most important textile producers of Europe and is also the leader of the textile sector. More than half of the cotton produced in Turkey comes from the Southeastern Anatolia region. The cotton production areas and cotton production amount in the region makes up 58 % of the cotton production areas and cotton production amount in Turkey. According to 2017 TÜİK data, 76

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cotton agriculture activities have been carried out on a total of 501.000 hectares of land (TUÄ°K, 2018). Even though the current situation is as stated above, this production amount fails to meet the raw material demand of the industrial sectors based on cotton. Therefore, majority of the textile businesses in the region operate at 50 % capacities. Cotton piking or harvesting in the region is mostly based on manual labor (Figure 1). Harvest periods are very long since manual harvesting is difficult and tiring, there are problems related with finding sufficient workers, climate conditions are not suitable and the harvest periods are generally quite rainy which leads to increases in production cost and product losses in addition to reduced cotton fiber quality. Therefore, mechanical harvesting has become a necessity (Figure 1).

Figure 1. Manual and mechanical cotton harvesting Cotton production areas in the region have increased with the gradual increase in the irrigated areas in the GAP region during the past 20-25 years. The increase in production areas has led to an increase in mechanical harvesting as a result of which many different cotton harvesting machines of different brands have started to be used. The primary condition for a successful mechanical harvest is that the bolls should have been opened at the same time if possible and that the leaves have defoliated. Auxiliary chemicals such as defoliants, desiccants and boll openers are used for this purpose. However, product losses increase and fiber qualities are low due to untimely and erroneous (early or late) applications by producers, the use of improper spraying machines, the use of improper adjustments in the spraying machines as well as the excessive or insufficient use of chemicals. These negativities related with the defoliant application process required for a successful harvest decreases the competition strength of the producers in the market. When it is considered that Turkey is ranked number 7 in the world with regard to cotton production, it is necessary that cotton should be produced economically and harvested in a timely manner without decreasing yield or quality in order to ensure that it can continue to complete with cottons from other countries and that the cotton production is continued. Moreover, cotton is a very important plant for ensuring the continuity of the provision of raw materials to the textile industry in Turkey as well as for contributing to the development of the Southeastern Anatolia Region which has the lowest per capita income in Turkey while also enabling the effective use of human resources and providing employment for the rural region. Hence, cotton should be collected in a timely manner in the shortest amount of time possible with minimum losses while preserving quality (Ă&#x2013;z and Evcim., 2002; Sessiz et al., 2016). However, mechanical harvesting cannot be carried out at the desired times due to reasons such as the failure of all cotton bolls to open at the same time or incomplete defoliation. Defoliant applications carried out for mechanical harvesting in the GAP region where cotton production is carried out extensively in Turkey, the problems faced, application issues and the steps that should be taken have been explained in detail in the present study. Are defoliant applications necessary for cotton? Cotton harvesting is one of the most critical processes that has an impact on quality in production and the earnings of the producers. Harvest costs make up about 25 % of the total production costs. Losses increase and quality decreases as a result of late harvesting. Thus, 77

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timely mechanical harvesting has become a necessity. Harvest time, maturation period, harvest date and the settings of the harvester are factors which have direct impacts on cotton fiber yield and quality (Supak, 1996; Larson et al., 2002; Edmisten, 2006; Collins et al., 2006). Therefore, chemicals such as defoliants, boll openers and desiccants have to be used for successful mechanical harvesting (Ball and Grover, 1999). Defoliants, boll openers and desiccants are chemicals used in cotton agriculture for mechanical harvesting. For this purpose, these chemicals known as defoliants are used prior to cotton harvesting to speed up the boll opening process, to improve the harvesting efficiency of the machine and to reduce cotton moisture ratio. Leaves are defoliated earlier and bolls are opened faster with the use of these chemicals (Figure 2). These applications enable early harvesting thereby increasing yield and fiber quality (Larson et al., 2002). Machinery owners and those who apply mechanical harvesting for cotton are forced to implement defoliant applications.

Figure 2. A cotton plant with sufficient boll opening and maturation before and after the defoliant application Defoliant application time and its effects Defoliant effectiveness depends on the application time and amount, plant uniformity, the harvesting method applied, maturation, environmental conditions, defoliant absorption, the amount of the active substance used, the sprayer used, and the operating speed of the machine in the field as well as the mixture uniformity in the sprayer tank (Christenbury, 1989; Burmester et al., 2009; Kerby et al., 1992; Ball and Grover, 1999). Hence, defoliant application should be carried out in a timely and proper manner for successful harvest. Even though defoliant application under normal conditions varies subject to the type of the chemical used, defoliants and boll opening chemicals should be mixed with required dozes generally 15-20 days before harvesting when at least 60-70% of the bolls have opened (Figure 3).

Figure 3. Hydraulic field sprayer that can spray the plants from the top and the bottom for increasing defoliant effectiveness The most important advantage of defoliant applications is to reduce the greenery of the plant as well as its moisture, preserve the whiteness of the fiber and to improve the effectiveness of cotton picker. However, excessive or insufficient defoliant applications in cases when majority of the bolls have not opened lead to late harvest and the overlapping of harvests with rainfall. This results in boll diseases and drying of immature bolls thereby leading to a decrease in cotton yield and fiber quality (Larson et al., 2002; Bednarz et al., 2002; Karademir et al., 2003; Siebert

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and Stewart, 2006). In addition, excessive moisture and reduced chemical effectiveness decreases fiber yield and quality resulting in economic losses. Thus, it is important for the success of mechanical harvesting that the harvest data and its impact on yield are determined subject to the status of the unopened bolls under field conditions. Success of mechanical harvest is subject to defoliant application time, application norm and chemical dose. The state of opened bolls in the field, current and expected climate conditions as well as the harvest program should be taken into consideration when it is decided to carry out defoliant application. It has been observed that application and harvesting carried out before maturity and under excessively moist conditions have adverse impacts on storage conditions and quality while also reducing yield. Moreover, it was determined that rain before mechanical harvest, excessive use of spindle cleaner solution, excessive moisture, green leaf on the plant due to bad defoliant, partial or bad boll opening, immature bolls (Figure 4) have adverse impacts on both machine effectiveness and fiber quality. High volumes of defoliants and boll opener chemicals result in an herbicide effect on the plants thereby leading to incomplete defoliation and dried leaves on the plant yielding decreased product quality. In addition, defoliants are not as sufficient when applied before boll maturation or rainfall and issues such as yield loss and low fiber quality may occur since the growth of the bolls stops.

Figure 4. An example for application before sufficient bolls have opened Harvesting overlaps with autumn rainfalls if cotton in full harvest maturity is not harvested in a very short amount of time and both manual and mechanical harvesting becomes more difficult. Moreover, the fibers that have not yet reached full maturation droop down and fall to the ground. In this case, cotton fiber quality also decreases in addition to yield (Ă&#x2013;z and Evcim, 2002; Siebert and Stewart., 2006). Therefore, cotton producers should determine the harvest schedule by taking into consideration the maturation during the harvest period, the physical conditions of the product and the current weather conditions at the time (Faircloth et al., 2004a; Faircloth et al., 2004b; ). Bolls may go bad, fiber quality may decrease and losses may increase in case the defoliant is applied very late (Figure 5).

Figure 5. Cotton that has not been picked up on time Machines used Different spraying machines are used for defoliant application. In addition to the renting of large capacity self-propelled sprayers, farmers also use their own field sprayers. However, producers may make some changes on their own sprayers in accordance with the structure of 79

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their plant (height, leaf area index etc.) such as raising of the roof, applying thin rubber in order to ensure an effective application (Figure 6). Thin and large tires with a special structure are also used in order to prevent the harming of the plants (Figure 6). However, failure to reach the desired distribution uniformity and coverage ratio on the plants due to spraying from the top is among the significant issues faced. Another issue observed was that sufficient calibration has not been made on the current machines.

Figure 6. Tractors used for spraying and the thin tires used. Environmental impacts of defoliants Excessive or untimely defoliant applications cause chemical residues and drift problems. These erroneous applications have adverse impacts on the environment. It has been observed that wild animals have died to the excessive use of defoliants in the region and significant health problems have been observed in humans and pets. These adversities have also been expressed by animal breeders and cotton producers. It was determined that cattle and ovine have miscarriages due the adverse impacts of the defoliant residues on the plants after being grazed in cotton fields after the harvest period. The most important inadequacy of the producers in the region is that they do not have sufficient knowledge on the proper application time and doses of defoliants. Applications are not made in accordance with the opened boll state and time. For example, it was observed in Bismil and Ă&#x2021;Äąnar districts of DiyarbakÄąr where excessive cotton production is made that chemicals such as defoliants, boll openers are applied based only on their catalogue values rather than factors for mechanical harvest such as product maturity, weather conditions and harvest schedule. These applications have adverse impacts on both cotton yield and fiber quality while also increasing production costs and chemical costs involved. Another problem is the desire of owners of large capacity machinery for spraying and defoliant applications who rent out their equipment to cotton farmers to finish the application as soon as possible without taking into consideration factors such as harvest time, maturity periods, cotton varieties and the boll opening states and by calibrating the machine according to the opened boll ratio. Therefore, more than sufficient chemicals are applied most of the time thereby resulting in residues in the plant, on the soil and the fiber. Errors in application and their impacts Defoliant applications are made before cotton bolls fully mature especially in late harvests. Hence, excessive doses of chemicals applied to speed up the harvest procedure leads to over drying of the leaves and bolls (Figure 7), while insufficient doses lead to failed defoliation.

Figure 7. Dried bolls after defoliant applications before maturation 80

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The application varies subject to the chemical to be used but it is generally applied together with the defoliant and boll opener about 15-20 days before the harvest. Many different brands of defoliants and boll opener chemicals are used in the market. Each company makes suggestions with regard to dosage and application time. This leads to confusions among the producers resulting in improper and untimely applications. Late plantation may generally be problematic for defoliant applications. The weather cools, rain season starts leading to insufficient defoliant effectiveness. Moreover, there are also fundamental issues such as the planting of early varieties not suited for mechanical harvest subject to the plantation applications repeated due to soil crusting, rotting of the seed or insufficient shoot. CONCLUSION AND SUGGESTIONS Significant losses in yield and fiber quality as well as environmental pollution are among issues resulting due to erroneous and insufficient defoliant applications with increasing use of harvesters. Each producer should make defoliant applications by taking into consideration their own field conditions in order to be successful in mechanical harvest. Especially defoliant application time and dose should be determined subject to the state of opened bolls. Harvest procedure should be carried out by taking into account the cotton variety and maturation periods. Application should be based on weather condition and climate date and spraying should be postponed in rainy weather or when there is a possibility of rain. If the bolls are opened using boll openers before sufficient maturation, the fiber and strength are affected adversely. Opened boll ratio and application norm should be taken into consideration in defoliant applications. Defoliant applications can be made starting from when 40 % of the bolls have opened until 100 % (Balkcom et al., 2010). However, early applications lead to decrease in boll weights, fiber thickness and strength, while weak applications lead to decrease in quality. However, majority of the producers apply defoliants as a rule when 60 % of the bolls have opened. It is best for Turkey to apply the defoliant when 60-70 % of the bolls have opened. Spraying nozzles, pump, hoses, manometer and filters should be controlled prior to starting the spraying procedure in order to ensure that the defoliant procedure produces the desired impact. If the nozzles are blocked or if spraying cannot be carried out properly, the required maintenance procedures should be completed, parts should be replaced if necessary and any chemical leaks in the pump or hoses should be repaired immediately. The accuracy of the manometer used for adjusting the operating pressure to the desired value should be controlled and maintenance procedure should be carried out for the filters required for the proper operation of the machinery. According to the observations made in the region, defoliants and boll opener chemicals are applied from the top using a proper field sprayer. Majority of the leaves on the middle and lower sides of plants do not dry due to insufficient spraying and the bolls cannot be opened in a timely manner. It was observed that many of the equipment and machinery used are not maintained well, that there are leakages and drips in various places and that the filters are missing. Therefore, it was observed that a uniform and homogeneous spraying cannot be attained and that the manometers cannot make accurate measurements due to blocked nozzles and non-horizontal. Meteorological conditions at the time of spraying should be taken into consideration in order to eliminate such undesired instances and the machine should be calibrated prior to each application. A more effective mechanical harvest procedure can be carried out with a proper defoliant application. Also, before the cotton harvest begins, pickers should be checked for damaged or worn spindles, moistener pads, doffers, bearings, and bushings. If they are problems such as damaged

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or worn excessively, they should be replaced by operator carefully making repairs and adjustments. Acknowledgement: The author’s express special gratitude to Dicle University Scientific Research Projects Coordinator (DUBAP) for all support received for accomplishment of this research (Bismil-MYO.16.001) REFERENCES Balkcom, K.S., J.S. Bergtold, C.D. Monks, A.J. Price, D.P. Delaney (2010). Planting and defoliation timing impacts on cotton yield and quality. Beltwide Cotton Conferences, New Orleans, Louisiana, January 4-7. Ball, S.T., C. Glover (1999). Defoliants, desiccants, and growth regulators used on New Mexico cotton. New Mexico State University is an equal opportunity/affirmative action employer and educator. NMSU and the U.S. Department of Agriculture cooperating. Bednarz, C.W., W.D. Shurley, W.S. Anthony (2002). Losses in yield, quality, and profitability of cotton from improper harvest timing. American Society of Agronomy. Published in Agron. J.94:1004–1011. doi:10.2134/agronj2002.1004 Burmester, C., C.D. Monk, M.G. Patterson (2009). Cotton defoliation. Alabama Cooperative Extension System, (Alabama A&M University and Auburn University) Web Only, Revised Sept 2009, ANR-0715 Christenbury, G. (1989). Cotton picker management and harvesting effıciency. EC / Clemson University Cooperative Extension Service. Collins, G.D., K.L. Edmisten, D.L. Jordan, R. Wells, J.E. Lanier, G.S. Hamm (2006). Defining optimal defoliation timing and harvest timing for compact, normal, and extended fruiting patterns of cotton (Gossypium Hirsutum L.) achieved by cultivar maturity groups. World Cotton Research Conference-4, Lubbock, Texas, USA, 10-14 September 2007. Edmisten, K.L. (2006). Cotton defoliation. P. 159-178. In K.L. Edmisten (Ed.). North Carolina Cotton Information. Publ. Ag-417. North Carolina Cooperative Ext. Serv., Raleigh, Nc. Faircloth, J.C., K.L. Edmisten, R. Wells, A.M. Stewart (2004a). The influence of defoliation timing on yields and quality of two cotton cultivars. Crop Sci. 44:165-172. Faircloth, J.C., K.L. Edmisten, R. Wells, A.M. Stewart (2004b). Timing defoliation applications for maximum yields and optimum quality in cotton containing a fruiting. Gap. Crop Sci. 44:158-164. Karademir, E., C. Karademir, S. Basbag (2003). Determination the effect of defoliation timing on cotton yield and quality. Journal of Central European Agriculture Vol 8 (3): 357-362. Kerby, T.A., J. Supak, J.C. Banks, C. Snipes (1992). Timing defoliation using nodes above cracked boll. pp:155-156. In Proc. Beltwide Cotton Conf., Nashville, Tn. 6 – 10 Jan. 1992. Natl. Cotton Counc. Am., Memphis, TN. Larson, J.A., C.O. Gwathmey, R.M. Hayes (2002). Cotton defoliation and harvest timing effects on yields, quality and net revenues. The Journal of Cotton Science. 6:13-27. Öz, E, H.Ü. Evcim (2002). Makinalı hasadın pamuk lif teknolojik özellikleri üzerindeki etkilerinin belirlenmesi. Ege Üniv. Ziraat Fak. Derg., 2002, 39(2):119-126, ISSN 10188851 Sessiz, A., R. Esgici., F.G. Pekitkan (2016). The Relationship Between Mechanization and Cotton Ginning Industry. International Scientific Journal. Mechanization in Agriculture. ISSN 0861-9638, Issue 1/2016, Sofia, Bulgaria. Siebert, J.D, A.M. Stewart (2006). Correlation of defoliation timing methods to optimize cotton yield, quality, and revenue. The Journal of Cotton Science 10:146–154

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Supak, J.R. (1996). Overview of U.S. regional cotton defoliation practices – southwest. p. 8891. In Proc. Beltwide Cotton Conf., Nashville, TN 9-12 Jan. 1996. Natl. Cotton Counc. Am., Memphis, TN. TUİK (2018). Turkish Statistical Institute (www.tuik.gov.tr)

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THE EFFECT OF TOP CUTTING HEIGHT OF COTTON STALK AND CHEMICAL APPLICATION ON COTTON YIELD Reşat ESGİCİ*1, F. Göksel PEKİTKAN2, A. Konuralp ELİÇİN2, Abdullah SESSİZ2 1 2

Dicle University, Bismil Vocational High School, Diyarbakır, Turkey

Dicle University, Faculty of Agriculture, Department of Agricultural Machinery and Technologies Engineering, Diyarbakır, Turkey *Corresponding author’s email: resgici@dicle.edu.tr

ABSTRACT The objective of this study was to determine the effect of top cutting height of cotton (Gossypium hirsutum L.) stalk and chemical application on cotton yield. In order to increase the yield and prevent of increase of plant height, the cotton producers in Southeastern part of Turkey are cut the top of cotton plant before bolls open by workers. This process is difficult and tiring. Therefore, it requires mechanical cutting. In accordance with this purpose, BA-440 cotton variety was planted as experimental material on 21 April 2018 by a pneumatic planter at a commercial farm in 2018 at the Diyarbakır province, where cotton production is intensively done. The field experiment was designed according to randomized complete block design with three replications. Experimental field consisted of 18 plots with each measuring 15 m x 6 m with an inter row spacing of 0.7 m distance. Topping cut of cotton stalk is done by worker during the vegetation period. Pix application was done by field sprayer. According to experiment results the cotton yield were not affected by treatments. There were not found significant (p>0.05) differences between the application treatments. However, the highest yield were observed at pix treatment methods as 4.363 kg ha-1, the lowest value were found at 20 cm top cutting height of cotton stalk as 3.821 kg ha-1. Keywords: Cotton, Stalk topping, Yield, Pix application, Turkey INTRODUCTION Cotton (Gossypium hirsutum L.) is known as white gold and it is one of the most important commercial crops in Turkey. Turkey is Europe's largest textile manufacturer and it is the seventh largest producer of cotton in the world. Turkey is divided seven physical region and cotton is cultivated primarily in the Southeast Anatolia Region. Southeast Anatolia Region is known as GAP. With irrigation project of GAP (Southeastern Anatolian Project) in Turkey, the irrigated farmland and cotton production in Southeast Anatolia region has developed rapidly last three decades. Nowadays, more than half of the Turkey’s total cotton production is produces from Southeastern Anatolia region (TUİK, 2018). The increase in cotton production has increased provided the development of the cotton industry (Sessiz et al. 2009; Sessiz and Esgici, 2015). Therefore, cotton has a strategic importance for the region and total production of Turkey. Diyarbakır, Şanlıurfa, Mardin and Batman as agricultural areas with more as well as the producers of the region opportunities for irrigation on the GAP in the provinces of through boreholes have opened their own facilities, irrigated farmland has increased significantly. With water, a significant increase in the area of cotton production has occurred. GAP region produces 84

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61.5 % of Turkey’s total cotton production area in the 2017 (TUIK, 2018). This has led to the development of industries based on cotton in the region. This production ratio in region is important for region’s development, human resources development and rural development. Therefore, increasing cotton production and yield, reducing of cotton losses and protection of fiber quality are very important for sustainability of the production in Diyarbakır province. Labour cost in Turkey is rapidly increasing and therefore mechanization in cotton cultivation will play a key role in keeping the cost under control. Additionally, there will be productivity increase driven by high density planting. However, the farmers still use human labour for many of the operations like top cutting height and picking for these operations. Almost top height operation of cotton plant in region is cutted by worker with use of straight shear. In addition to conventional operations, cotton topping is another cultural practice that should be done during the vegetation period (Aydin and Arslan, 2018). Therefore, the production cost is very high depending on cultural application during the production season. So, to reduce cotton production cost, it is require to use of cutting equipment for top height of cotton plant. It has been reported by cotton producers that the top cutting of cotton the stalks operation is directly related to increase the yield, quality of cotton lint and reduces bollworm infestations without negatively affecting cotton yields. The similar results were reported by Obasi and Msaakpa (2005), Yang et al., (2008), Renou et al., (2011) and Aydin and Arslan (2018), Pekitkan et al. (2018). According to these researchers, the cutting of top section of cotton plants increases the yield and quality of cotton and improves the earliness, limiting the vegetative growth of the plant and improving the development of generative organs of the cotton plants. Cotton farmers in Turkey are interested to mechanize cotton shoots for topping but have limited information and knowledge about cutter process. It is reported that in the literature and by cotton farmers in Southeast part of Turkey the cut of the top part of the cotton plant provides significant advantages to improve cotton yield. The objective of this study was to determine the effect of top cutting height of cotton stalk and chemical (pix) application on fiber cotton yield. MATERIALS AND METHODS The field experiment was conducted at a commercial farm in 2018 during the growing season at the Bismil district, Diyarbakır province (latitude 37°53´N and longitude 40°16´E, 680 m altitude), Southeast part of Turkey. The BA-440 cotton cultivar was planted on 12 April in 2017 by four-row a pneumatic planter. Some mean properties of BA-440 cotton variety are given in Table 1. Table 1. Some properties of cotton plant during the experiment. Properties

Mean values

Plant height, cm

94.00

Boll number on the plant, number

16.00

Number of branch on one the plant

13.00

Average yield (kg ha-1)

4.460

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Experimental area divided 18 plots with each measuring 12 m x 6 m with an inter row spacing of 0.7 m distance. The top cutting operations and pix application were done during the vegetation period before opening the cotton bolls. The top cutting operations were done from top to bottom of plans 5 cm interval at 4 different heights. Top cutting operations were made in twelve plots, pix chemical application was made in three plots and no any application was made in three plots for control. Cotton fiber yield randomly were measured separately at 5, 10, 15 and 20 cm cutting heights from the top of the cotton plant in each experimental plot. In addition, yield has been measured in the control plots and Pix chemical applications plots. The measurements of yields were made in two middle rows and 5 m in-row spacing in each plot (7 m2). The cotton fiber was harvested by hand. Then, it is weighed by an electronic balance and converted to kg ha-1. The obtained yield values were compared each other statistically. Data were analyzed by ANOVA (MSTATC software). One factor randomized complete block design with 5 replications was performed to detect significant differences in the observations. Means were compared at a 5 % level of significance using Duncan's multiple range tests to identify the specific differences among treatments means. RESULTS AND DISCUSSION According to top cutting heights and other applications, the mean cotton yield values are given in Table 2 and Figure 1. As seen in Table 2 and Figure 1, there were not found significant difference between cutting heights, pix chemical application and control plot according to fiber yield. However, the highest yield were obtained at pix treatment methods as 4.363 kg ha-1, the lowest value were found at 20 cm top cutting height of cotton stalk as 3.821 kg ha-1. Similar results were reported by Pekitkan et al. (2018) for the same cotton variety. According to these results, the yield values slightly decreased with the top cutting height increased (Table 1) and when we consider the best result is obtained in the pix method, we can say that there is no need for top cutting of cotton plant. If it is taken into account that the use of chemicals is detrimental, it is more beneficial to top cut height of the plant at 5 cm by mechanical application. Even when we take into consideration the control values, there is no need to do anything process. Because there is no statistical difference between the highest value and the control value according to yield. The relationships between cutting properties and cotton yield depend on top cutting height is very important and valuable a result for decide a new design a top cutting equipment of cotton stalk (Pekitkan et al. 2018). Table 2. The relationship between top cutting heights and cotton yield. Top cutting height of cotton stalk (cm) and other applications

Cotton fiber yield (kg/ha)

4.381 a*

4.360 a

4.307 a

3.821 b

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Top cutting height of cotton stalk (cm) and other applications

Cotton fiber yield (kg/ha)

Pix chemical application

4.363 a

Control (no treatment)

4.253 a

* the same letter in each column are not significantly different by Post Hoc multiple range test at the 5 % level.

Figure 1. The change of cotton fiber yield depend on treatments. CONCLUSION The results showed that the fiber yields of cotton were not affected by the treatments. There were not found significant difference between cutting heights, pix chemical application and control plot according to fiber yield. However, numerically different, the highest yield were observed at pix treatment methods as 4.463 kg ha-1, the lowest value were found at 20 cm top cutting height of cotton stalk as 3.821 kg ha-1. Acknowledgement: The author’s express special gratitude to Dicle University Scientific Research Projects Coordinator (DUBAP) for all support received for accomplishment of this research (Bismil-MYO.16.001) REFERENCES Aydın, İ., S. Arslan (2018). Mechanical properties of cotton shoots for topping. Industrial Crops & Products 112 (2018) 396–401 Dowgiallo, A. (2005). Cutting force of fibrous materials. Journal of Food Engineering 66(1):5761. Obasi, M.O., T.S. Msaakpa (2005). Influence of topping, side branch pruning and hill spacing on growth and development of cotton (Gossypium barbadense L.) in the Southern Guinea Savanna Location of Nigeria. J. Agric. Rural Dev. Trop. Subtrop. 106(2), 155–165.

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Pekitkan, F.G, R. Esgici, A. Sessiz (2018). Effect of top cutting height of cotton stalk on cotton yield. XIX. World Congress of CIGR (Commission Internationale du Génie Rural) “Sustainable Life for Children”22 - 25 April 2018.Antalya, Turkey. Renou, A., I. Téréta, M. Togola (2011). Manual topping decreases bollworm infestations in cotton cultivation in Mali. Crop Prot. 30 (10), 1370–1375. Sessiz, A., R. Esgici (2015). Effects of cotton picker ages on cotton losses and quality. Scientific Papers. Series A. Agronomy, Vol. LVIII, S:417-420. TUİK (2018). Turkish Statistical Institute (www.tuik.gov.tr)

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THE EFFECT OF DIETARY SUPPLEMENTATION OF DIFFERENT MULTIENZYMES ON PRODUCTION PERFORMANCE AND EGG QUALITY CHARACTERISTICS IN LAYING HENS Özgür POLAT1, Muzaffer DENLI1 1

Department of Animal Science, Faculty of Agriculture, Dicle University, 21280, Diyarbakır, Turkey *Corresponding author’s e-mail: muzaffer.denli@gmail.com ABSTRACT

The purpose of this study was to determine the effects of dietary addition of multi-enzymes in different source of grains based diets on performance and egg quality characteristics of laying hens. A total of 225, 24 weeks of old laying hens (Atak-S) were divided into 3 treatments with 5 replicates and 15 hens per replicate for 10 weeks. The control group was fed basal diet (without any supplementation) and treatment groups were fed basal diet supplemented 0.1% Enzyme-A (xylanase, ß-glucanase, cellulase, α-amilase and protease) and 0.05 % Enzyme-B (xylanase, ß-glucanase). Performance and egg quality parameters were checked weekly throughout the experiment. Dietary multi-enzymes supplementation significantly changed the shape index, yolk index and yolk color (L and a) at different weeks of trial (P<0.05). However, daily feed intake, egg production, average egg weight and feed conversion rate were not significantly affected by addition of both multi-enzymes during the experimental period (P>0.05). In addition, dietary addition two types of multi-enzymes did not effect the egg specific gravity, eggshell thickness, eggshell rate, albumen index, Haugh units (P>0.05). As a result, it was determined that the addition of multi-enzyme additions to the laying hens of the hens between 24-33 weeks of age did not affect the performance parameters, but caused limited changes on some egg quality characteristics. Keywords: Multi-enzymes, Laying hens, Performance, Egg quality

INTRODUCTION

Animal products play an important role in providing the adequate and balanced nutrition that people need and maintaining the healthy lives of individuals. Therefore, one of the most consumed animal products is egg. Eggs are the only food source that contains all the nutrients that human needs after breast milk. Especially the rich content of essential amino acids makes the egg an accepted source of quality animal protein (Açıkgöz and Özkan, 1996). As a matter of fact, the biological availability of egg protein is 100%; while this value is 85% in milk, 76% in fish and 74% in beef. With increasing interest in healthy and balanced nutrition in recent years, daily egg consumption per person has also increased. Therefore, with the increase in egg consumption and consumer awareness, the concept of egg quality has started to gain importance. In determining the quality characteristics of the egg, many criteria related to external and internal quality are taken into consideration. Egg quality which is extremely important for the consumer as well as the producer is determined by; egg weight, shell thickness, egg shape, shell pore and shell color (Durmuş, 2014). In addition to this, the shape index, specific gravity, fracture strength, surface area and fracture-crack egg ratio also play a decisive role in external 89

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quality. Although egg weight and size vary depending on genotype, it is also influenced by feeding and environmental factors (Ayyıldız, 2012). Poor egg shell quality is an important problem that negatively affects egg production, reduces hatchability and increases embryo mortality. On the other hand, egg quality with low shell strength and high risk of infiltration of pathogenic bacteria is not preferred by consumers. Therefore, egg production with high shell defects breaks consumer perception of trust and leads to economic losses. In the studies, it has been reported that the use of feed supplementation added to the mixed feed is beneficial in improving egg quality and gives positive results (Świątkiewicz et al. 2015). After commonly used enzyme studies, such as xylanase, amylase, cellulase and phytase, which affect a single substrate in poultry nutrition, it has been reported that the use of multienzyme acting on more than one substrate may further improve feed conversion rate and performance (Kutlu and Özen, 2009; Kutlu and Şahin, 2017). Jia et al. (2008) reported that the addition of multienzyme has positive effects on the rate of feed conversion in laying hens, ether extract digestibility of nonstarch polysaccharides (NSP), eggshell quality and n-3 fatty acid accumulation in the egg. Lima et al. (2012) reported that the addition of enzyme complex to mixed feed improves intestinal health and performance in laying hens and improves internal and external egg quality. The aim of this study is to determine the effects of adding different multienzyme to laying hens mixed feed on performance (feed intake, feed conversion rate, egg production, average egg weight), egg external quality (shape index, shell rate, shell thickness, specific gravity) and egg internal quality (yolk index, albumen index, Haugh unit and yolk color and blood and flesh stain) characteristics. MATERIAL AND METHOD Animal, Diets and Experimental Design In this study 225, 24-week-old Atak-S laying genotypes were used as animal material. The hens were divided into 3 groups according to the randomized plot design, each group consisted of 5 replicates and 15 hens in each replicate. The treatment groups formed in the experiment; control group containing no feed additives, 0.1% enzyme A addition group and 0.05% enzyme B addition group. Each kg of enzyme A used in the assay contains 250,000 U + 790,000 EPU of xylanase, 1,000,000 U + 8,700 EPU of Beta-Glucanase, 350,000 U + 18,000 EPU of Cellulase, 350,000 U + 21,000 EPU of Alpha-Amylase and 7,500,000 U + 8,000 EPU of Protease. Each kilogram of enzyme B contains 200 U of xylanase and 138 U of Beta-Glucanase. The feed raw materials to be used in the study were obtained from a commercial feed factory and the mixed feeds of the experiment were prepared at the feed production facility of the Department of Animal Science, Agricultural Faculty of Dicle University. Enzyme A and B feed additives were added to the main mixed feed after pre-mixing. The nutrient contents of the compound feeds to be used in the experiments were prepared in accordance with the nutrient requirements of laying hens reported in NRC (1994). Laying hens were fed with mixed feed containing 17% CP, 3.8% Ca and 2744 Kcal ME/kg during the 2433 week experiment. The composition (%) and nutrient contents of the mixed feeds used in the study are shown in Table 1. The research was carried out in the Enriched Cage System in the laying hens experimental unit in the Poultry Research and Application Facility, Department of Animal Science, Agricultural Faculty of Dicle University. The enriched cage system has 3 floors and 5 cage sections on each floor. Each cage section is 240 cm in length, 120 cm in width, 77 cm in height and each section has rasp, perch and nest. The experimental room where the enriched cage system is installed has a floor area of 120 m2 and a ventilation height of 4.5 m and a fully automated cooling system. The animals in each cage compartment were group fed, and the feed and water were presented as ad libitum. Illumination of the experimental room was provided by fluorescence and 8 hours of dark and 16 hours of light program was applied daily. 90

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Table 1. Ingredients and chemical composition of experimental diets (as-fed basis) Ingredients (%) Corn 37.00 Soybean Meal (44% CP) 9.500 Full Fat Soybean 17.00 Sunflower Meal (32% CP) 13.00 Wheat 17.00 Dicalcium Phosphate (DCP)a 1.850 Calcium Carbonate 8.600 NaCI 0.300 Vitamin+ Mineral Premixb 0.250 Chemical Analysis (%) Dry Matter 90.70 Crude Ash 10.46 Crude Protein 17.00 Ether Extract 4.10 Calculated values ME (kcal/kg) 2744 Calcium(%) 3.810 Available Phospour (%) 0.400 Na (%) 0.170 L-lysine (%) 0.780 Methionine+cystine(%) 0.590 Treonin(%) 0.610 Tryptophane(%) 0.210 Linoleik asit(%) 2.900 a Premix supplied per 1 kg; Calcium 24,5%, Fosfor; 18%. b Premix supplied per 1 kg: vitamin A; 12.000.000 IU; vitamin D3; 2.500.000, vitamin E; 30.000 mg, vitamin K3;4.000 mg; Vitamin B1; 3.000 mg, Vitamin B2; 7.000 mg, Vitamin B12; 5.000 mg, Vitamin B6; 5.000 mg, Vitamin C; 50.000 mg, Niacin; 30.000 mg, Cal-D-Pantotenat; 10.000 mg, Biotin; 45 mg, Folic acid; 1.000 mg, Choline Chloride; 200.000 mg, xanthate;1.500 mg, Mangan; 80.000 mg, Iron; 60.000 mg, Zinc; 60.000 mg, Co; 5.000 mg, Iodine; 1.000 mg, Cobalt; 200 mg, Selenium;150 mg. Feed Analysis Determination of nutrient contents of feeds used in the experiment (except crude cellulose) was performed according to Weende analysis method (Naumannn and Bassler, 1993) and determination of crude cellulose according to Lepper method (Bulgurlu and ErgĂźl, 1978). In the calculation of metabolic energy content of feeds, regression equation no. 9610 proposed by TSE was used (TSE, 1994). Production Performance Measurement At the beginning of the experiment, all hens were weighed and were left in the experimental group cages as their live weight and egg yield levels same. During the experiment, the egg production, feed intake and egg weight of the animals were measured on a weekly basis and feed conversion rate was calculated using the data obtained. Feed Conversion Rate = Feed Consumption (g)/Total Egg Weight (g) 91

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Determination of Egg Quality Characteristics Internal and external quality analyzes were performed on 15 eggs collected weekly from each group on the same day. Egg weight was determined by weighing with precision balance (0.01g) every other day. Egg Shape Index (ESI): The width and length of the egg were measured by digital caliper and calculated using the formula ESI = (Width of egg / Length of egg) x 100. Egg Specific Gravity was measured with a density analyzer consisting of precision balance, beaker and apparatus. For this purpose, the weight of the eggs which were kept at room temperature for 24 hours was first weighed in the air and then the weight in the water having an average temperature of 20-22 C was calculated to determine the specific gravity of the egg. Shell Thickness: The shells taken from the middle parts of the broken eggshell under laboratory conditions were measured by digital micrometer after drying and separating the membranes. The shell rate was determined by the ratio of the value of the egg shells obtained with the precision balance after the membrane was removed and dried to the egg weight. Egg yolk color was determined by digital colorimeter (Minolta CR-300) in L, a and b. Albumen Index (AI): For this purpose, the eggs were broken on a clean glass so that they could not spread and then the albumen breadth and albumen lengths were measured by means of digital caliper. The height of white was measured with digital foot micrometer and calculated with the formula AI = [albumen height (mm) / ((albumen length (mm) + albumen width (mm)) / 2)] x100. Yolk Index (YI): The diameter of the egg yolk was measured by digital caliper and the height was measured by digital foot micrometer and it was determined by the formula; YI = [(Yolk height / Yolk diameter) x 100]. Haugh Unit: Haugh Unit was calculated by using the egg weight and albumen height and by using formula; Haugh Unit = 100 Log (H + 7.57-1.7G 0.37). H: Albumen height (mm), G: Egg weight (g). Statistical Analysis Statistical analysis of the data obtained at the end of the experiment was performed using SPSS 18.0 package program. The analysis of variance of the averages was performed with General Linear Model (GLM) ANOVA. Tukey's multiple comparison test was used to compare differences between means. RESULTS AND DISCUSSION Performance Parameters In table 2. no statistically important difference was found between the groups in terms of production performance parameters (average feed intake, feed conversion ratio (FCR), egg production and egg weight) in 24-33 week periods (p> 0.05). The findings obtained from the study were found to be consistent with some of the previous literature and it was observed that the addition of a single enzyme or multi enzyme did not change feed intake in laying hens (Polat, 1995; Lee et al., 2014). However, Baghban-kanani et al. (2018) reported that the addition of multi enzyme, Orhan (2016) reported that the addition of phytase increased feed intake. Torki et al. (2016) reported that the addition of Î˛-glucanase and xylanase or Î˛-mannose-containing enzymes reduced feed intake. Joshua (2016) reported that the addition of protease to protein restricted diets did not differ between the control and treatment groups. Since the enzymes do not have an aromatic taste and are not used for appetizing purposes, it can be said that their addition to diets is not expected to have any effect on feed intake. However, it is thought that the discrepancy between the literature is affected by factors that change the efficiency of the enzyme such as laying hens, dietary raw material types, environmental and climatic conditions used in the studies, and it changes the feed intake.

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Table 2. Effects of multi- enzyme supplementation in laying hen diet on performance parameters (24-33. weeks) Groups Parameters

SEM

P value

117.5

0.182

0.310

2.4

0.020

0.779

86.4

87.1

0.771

0.211

55.4

55.8

55.2

0.174

0.335

Control

Enzyme A

Enzyme B

Feed consumption, g/day

117.7

117.1

Feed conservation ratio

2.4

Egg production, % Egg weight, g

The differences between means in the same row with different letters are significant (P < 0.05). SE: Standard Error Mean Attia et al. (2012) and Torki et al. (2016), reported that the addition of multi enzyme improved the feed conversion rate in laying hens; Yalcin et al. (2002) and Scheideler et al. (2005) reported that it did not change. On the other hand, Vieira et al. (2016) found that adding different sources of protease to the mixed feed did not make a difference in feed conversion. As it is known, 'FCR' is the ratio of feed intake to egg weight in laying hens. Therefore, the fact that the addition of multi enzyme in the experiment is not have a significant effect on the 'FCR' may be attributed to the fact that the difference between the groups in terms of feed intake and egg weight was not significant. It is seen that the literature on the effect of enzyme addition to laying hens diets on egg production is incompatible. In related studies; Scheideler et al. (2005), Lee et al. (2014), Vieira et al. (2016), Baghban-Kanani et al. (2018) reported that the enzyme addition did not change egg production, Khan et al. (2011) reported that it increased production. It can be said that the effect of multi enzyme addition on egg production varies depending on the type of enzyme and substrate (dietary composition). When the average egg weight of the treatment groups was considered, no statistically difference was found between the groups (p> 0.05). The findings obtained from the study in terms of egg weight were consistent with the studies of Benabdeljalil et al. (1994), Polat et al. (1995), Lee et al. (2014), Baghban-Kanani et al. (2018). Egg Quality Characteristics When the treatment groups were compared in terms of egg shape index, no statistically difference was found in other weeks except for weeks 27 and 30. (p> 0.05). In the 27th and 30th weeks, the highest shape index was observed in the control group (77.4 and 77.0, respectively), and the difference between the control and enzyme groups was statistically important (p <0.05). In addition, when the overall averages of shape index were examined, the highest control group (77.2) followed by Enzyme A (76.5) and Enzyme B (75.9) groups, respectively, and the results were found to be statistically important (p<0.05). The shape index is expressed by the ratio of egg width to length. Khan et al. (2011), Her (2013), Torki et al. (2016), Baghban-Kanani et al. (2018) reported that adding enzyme to mixed feed does not change the shape index. Accordingly, the findings obtained from the study are consistent with previous literature. In table 3 no statistically important difference was found between the groups in terms of the average shell rate and specific gravities of 24-33 weeks periods (P> 0.05). YÄąldÄąz 93

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et al. (2018) reported that adding enzyme to mixed feed containing different amounts of dried grain solubility (DDGS) had no effect on the shell rate. Khan et al. (2011) reported that egg specific gravity is actually egg thickness and pore index. However, they stated that the egg specific gravity did not change in hens fed with enzyme supplementation in mixed feed.

Table 3. Effects of multi- enzyme supplementation in laying hen diet on external and internal egg quality (24-33. weeks) Groups Control

Enzyme A

Enzyme B

SEM

P value

Shell rate, %

12.0

11.8

12.1

0.090

0.299

Shell thickness, mm

0.37

0.002

0.923

Spesific gravity, g/cm3

1.08

1.07

1.08

0.003

0.085

Shape index

77.2

76.5

75.9

0.170

0.017

Yolk index

48.1

46.9

46.7

0.250

0.050

Albumen index

3.9

3.8

3.7

0.050

0.333

Haugh unit

74.6

73.4

0.520

0.569

L* value

52.4

51.0

49.6

1.068

0.177

a* value

19.4

17.3

17.1

0.643

0.275

b* value

31.2

30.1

29.4

0.504

0.342

The differences between means in the same row with different letters are significant (P < 0.05). SE: Standard Error Mean Scheideler et al. (2005) reported that the specific gravity was significantly affected by the laying hen breed, but that the addition of enzyme to the mixed feed had no effect on the specific gravity regardless of breed. Accordingly, the findings obtained from the study were found to be consistent with previous literature. When the treatment groups were compared in terms of shell thickness, no statistically important difference was found in other weeks except week 25. (P> 0.05). At the 25th week, the highest shell thickness was observed in the control group (0.39) and the shell thickness was more important than the enzyme groups (0.34 and 0.35) (P <0.05). Yaghobfar (2007) reported that the addition of Ă&#x;-glucanase and xylanase to barley-weighted diets reduced the shell thickness. Lee et al. (2014) reported that the addition of the multi enzyme, Orhan (2016) reported that phytase addition and Joshua (2016) reported that protease addition to protein restricted feeds increased the shell thickness. Torki et al. (2016) and Baghban-Kanuni et al. (2018) reported that the addition of multi enzyme to the mixed feed did not change the shell thickness. No statistically important difference was found between the treatment groups in terms of yolk index in the other weeks except 27 and 30 weeks (p> 0.05). The highest yolk index was observed in the control group with the values of 49.2 and 48.4 at the 27th and 30th weeks, respectively, in which statistical difference was observed (p<0.05). In addition, when the 94

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general average of the yolk index is examined, the highest value is in the control group (48.1) followed by Enzyme A (46.9) and Enzyme B (46.7) groups, respectively (P <0.05). Joshua (2016), Souza Resende et al. (2016) found that the addition of enzyme to the laying hens mixed feed increases the yolk index; Him (2013) and Torki et al. (2016) reported that this does not change the yolk index. Abd El-Hack et al. (2019) reported that the addition of enzyme in DDGS supplemented feeds reduces the yolk index. No statistically important difference was found between the treatment groups in terms of the average albumen index and Haugh unit values over 24-33 weeks (Table 3.3). Yörük et al. (2006), reported that the addition of multi-enzyme to corn-soy based diets increases the albumen index. Him (2013) and Souza et al. (2016) reported that the addition of enzyme to mixed feed was not effective on the albumen index. The findings of the Haugh unit obtained from the study are consistent with a significant portion of the previous literature (Benabdeljalil et al., 1994; Yaghobfar et al., 2007; Onu, 2013; Joshua et al., 2016; Lee et al., 2014; BaghbanKanani et al., 2018). In contrast, Khan et al. (2011) reported that the addition of enzyme to mixed feed increases the Haugh unit in laying hens. It was thought that the addition of enzyme in the experiment did not change the Haugh unit due to the fact that there was no difference in egg weight between the groups. When the average L, a and b values of the experimental groups for 24-33 weeks were compared, differences were observed between the weeks (Table 3.4.). No statistically important difference was found between the treatment groups except the 25th, 29th and 32nd weeks when compared in terms of ‘L’ and the other weeks except the 24th, 25th and 32th weeks when compared in terms of ‘a’ (p> 0.05). No statistically important difference was found between the treatment groups in terms of the ratio b between 24-33 weeks (p> 0.05). Scheideler et al. (2005) reported that the addition of enzyme to mixed feed has no effect on brightness (L) and redness (a), but decreased yellowness (b). Accordingly, it can be said that the addition of enzyme to the mixed feed in laying hens has an effect on the color value. There was no difference between the groups in terms of mean blood and meat stains between 24-33 weeks (p> 0.05). When the mean blood and flesh stains of the experimental groups for 24-33 weeks were compared, no difference was observed between the groups (p> 0.05). CONCLUSION As a result, it was observed that the addition of different multi enzyme sources to mixed feeds did not affect the productiın performance of hens aged between 24-33 weeks, decreased the average shape index and yolk index, and changed some egg yolk color values and did not affect egg quality criteria such as albumen index and Haugh unit, specific gravity and shell thickness. According to research results, further studies on the use of multi enzymes in appropriate doses and combinations in different periods are recommended in order to improve performance and improve egg quality. It is predicted that the addition of multi enzyme to cereal-weighted diets may significantly increase the digestibility of non-starch polysaccharides (NSP) in laying hens and improve feed conversion rate by reducing feed intake. In this way, lower cost poultry can be made. Acknowledgements: This research was supported by the Scientific Research Project Fund of Dicle University (DÜBAP) under the project number Ziraat.19.001) REFERENCES Abd El-Hack, M.E., Mahrose, K.M., Attia, F.A.M., Swelum, A.A., Taha, A.E., Shewita, R.S., Hussein, S.O.S., Alowaimer A.N. 2019. Laying performance, physical, and ınternal 95

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egg quality criteria of hens fed distillers dried grains with solubles and exogenous enzyme mixture. Animals, 9: 150. Açıkgöz, Z., Özkan, K. 1996. Yumurta tüketiminin beslenme ve sağlık üzerine etkisi. Hayvancılık’ 96 Ulusal Kongresi.18-20 Eylül, Bornova-İzmir. S, 305-312. Attia, Y.A., El-Tahawy, W.S., Abd El-Hamid, A.E.A., Nizza, A., Al-Harthi, M.A., El-Kelway, M.I., Bovera, F. 2014. Effect of feed form, pellet diameter and enzymes supplementation on carcass characteristics, meat quality, blood plasma constituents and stress indicators of broilers. Archiv Tierzucht, 57 (30): 1-14. Ayyıldız, A. 2012. Yumurta: yumurta kalitesi, besin değeri, saklanması, yumurtanın kısımları. Erişim: [https://www.tavsiyeediyorum.com/makaleler/1645-yumurta.pdf] (03.06.2019). Banghban-Kanani, P., Hosseintabar-Ghasemabad, B., Azimi-Youvalari, S., Seidavi, A., Ayaşan, T., Laudadio, V., Tufarelli, V. 2018. Effect of different levels of sunflower meal and multi-enzyme complex on performance, biochemical parameters and antioxidant status of laying hens. South African Journal of Animal Science, 48 (2): 390-399. Benabdejelil K., Arbaoui M.I. 1994. Effect of enzyme supplementation of barley-based diets on hen performance and egg quality. Animal Feed Science and Technology, 48 : 325- 334. Bulgurlu, Ş., Ergül, M., 1978. Physical, chemical and biological analyses methods of feeds. Izmir, Ege Univ. Agric. Fac. Publish No: 127 (in Turkish). Durmuş, İ. 2014. Effect of egg quality traits on hatching results, Academiz J of Agriculture, 3 (2) :95-99. Joshua, A.O. 2016. Effect of protease supplementation on the performance of laying chickens fed marginally deficient protein diets. Journal for Studies in Management and Planning, 2 (11): 20-29. Jia, W., Slominski, B.A., Guenter, W., Humphreys, A., Jones O. 2008. The effect of enzyme supplementation on egg production parameters and omega-3 fatty acid deposition in laying hens fed flaxseed and canola seed. Poultry Science, 87: 2005-2014. Khan S.H., Atif M., Mukhtar N., Rehman A., Fareed G. 2011. Effects of supplementation of multi-enzyme and multispecies probiotic on production performance, egg quality, cholesterol level and immune system in laying hens. J. Appl. Anim. Res., 39 : 386398. Kutlu, H.R., Özen, N. 2009. Recent advances in animal nutrition, VI. National Animal Science Conference, 24-27 June 2009, Atatürk University., Erzurum, 72p. Kutlu, H.R., Şahin, A. 2017. Recent studies and recommendations for future in poultry nutrition, J of Animal Production, 58(2): 66-79. Lee, K.W., Choi, Y.I., Moon, E.J., Oh, S.T., Lee, H.H., Kang, C.W., An, B.K. 2014. Evaluation of dietary multiple enzyme preparation (natuzyme) in laying hens. Asian Australas. J. Anim. Sci., 7 (12): 1749-1754. Lima, M.R., Costa F.G.P., Goulart, C.C., Pinheiro, S.G., Souza, R.B., Normando Morais S.A. 2012. Nutritional reduction of protein and usage of enzyme in the diet of light layers. R. Bras. Zootec., 41 (9): 2055-2063. Naumann, C., Bassler, R., 1993. Methodenbuch, Band III, Die chemische Untersuchung von Futtermitteln. VDLUFA-Verlag, Frankfurt, Germany NRC, 1984. Nutrient Requirements of Poultry. 8th Rev. Edn., National Academy Press, Washington, DC. Onu, P.N. 2013. Supplementation of exogenous enzyme to laying hens diets containing heat treated sheep manure: effects on performance and egg quality. International Journal of Agriculture Innovations and Research, 1 (6): 188-194. 96

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Orhan, Z.Ç., 2016. The Effects of Phytase Supplementatıon on The Performance and Egg Qualıty Of Free-Range Layers, MSc. Thesis, Namık Kemal University, Tekirdag. Polat, C., Akyürek, H., Konyalı, A., Şenköylü, N. 1995. WPSA Proceedings. 10th European Symposium on Poultry Nutrition, 15-19 October, Antalya, Türkiye, S, 360-362. Scheideler, S.E., Beck, M.M., Abudabos, A., Wyatt, C.L. 2005. Multiple-enzyme (avizyme) supplementation of corn-soy-based layer diets. J. Appl. Poult. Res., 14: 77–86. Souza Resende, V.C., Almeida Brainer, M.M., Modesto, K.P., Sá da Costa Leite, R., Freitas, P.V.D.X. 2016. Effects of enzyme supplementation on diets of medium-heavy laying hens at 28 to 40 weeks. Revista Ciência Agronômica, 48 (4): 683-689. Świątkiewicz, S., Arczewska-Włosek, A., Krawczyk, J., Puchała, M., Józefıak D. 2015. Dietary factors improving eggshell quality: an updated review with special emphasis on microelements and feed additives. World's Poultry Science Journal, 71: 83-94. Torki, M., Mirzaee, M., Habibian, M. 2016. Effects of barley cultivar and dietary supplemental enzyme on performance, egg quality traits, and selected blood parameters of laying hens. Poultry Science Journal, 4 (1): 1-12. TSE, 1994. TS 9610 Animal Feeds- Determintion of Metabolizable Energy (Chemical Method). Vieira, B.S., Pedrozo, S.A., Barbosa, V., Tavares, J.M.N., Beloli, I.G.C., Melo Silva, G.M., Neto, H.R.L., Júnior, J.G.C., Corrêa G.S.S. 2016. Phytase and protease supplementation for laying hens in peak egg production. Ciências Agrárias, Londrina, 37 (6): 4285-4294. Yalçın, S., Kocaoğlu Güçlü, B., Karakaş Oğuz, F., Yalçın, S. 2002. The usage of enzymc, probiotic and antihiotic in laying hen rations, Ankara Üniv Vet Fak Drg., 49: 135141. Yaghobfar, A., Boldaji, F., Shrifi, S.D. 2007. Effects of enzyme supplement on nutrient digestibility, metabolizable energy, egg production, egg quality and intestinal morphology of the broiler chicks and layer hens fed hull-less barley based diets. Pakistan Journal of Biological Sciences 10 (14): 2257-2266 Yıldız, T., Ceylan, N., Atik, Z., Karademir, E., Ertekin, B. 2018. Effect of corn distillers dried grains with soluble with or without xylanase supplementation in laying hen diets on performance, egg quality and ıntestinal viscosity. Kafkas Univ.Vet.Fak.Derg., 24 (2): 273-280. Yörük, M.A., Gül, M., Hayirli A., Karaoglu M. 2006. Multi-enzyme supplementation to peak producing hens fed corn-soybean meal based diets. International Journal of Poultry Science, 5 (4): 374-38.

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IMPROVED NUTRITIONAL QUALITIES OF SUNFLOWER MEAL BY FERMENTATION Ramazan TOSUN1 Sulhattin YAŞAR1, Zeynep SÖNMEZ1* 1

Iğdır University, Faculty of Agrıculture, 7600 Iğdır, Turkey

*Corresponding author’s e-mail: zzzeynep.sonmez@gmail.com

ABSTRACT The study aimed to improve the nutritional quality of sunflower meal (SFM) by Aspergillus ficuum fermentation. Three fermentation experiments were conducted using with or without Aspergillus ficuum. SFM was incubated for 72 hours at fixed conditions of pH, temperature, moisture, aeroation and stirring rates. Samples taken at the hours of 0, 24, 48 and 72 were chemically analysed. The results indicated that fermentation of SFM with Aspergillus ficuum significanly (P<0.05) increased crude ash content by 15% and crude protein content by 11%. The fermentation with Aspergillus ficuum significantly (P<0.05) decreased crude lipit by 32%, the crude fibre SFM by 29%, ADF by 21% and NDF by 13%. There was a remarkable (P<0.05) increase in total reducing sugar by about 341% by A. ficuum fermentation. The fermentation of fungal microorganisms significantly (P<0.05) reduced the acetic acid and butyric acid content of SFM by 78% and 100% respectively. On the other hand the fungal fermentations significantly (P<0.05) increased lactic acids from 0.95% at 0 hours to 16.05% at 24 hours. Fermenting SFM with fungal inoculants significantly (P<0.05) decreased tannin and phytic acid contents. As a result, the fermentation of SFM by fungal microorganism yielded an enrichment of nutritional qualities and added biologically funtional compounds, mainly lactlc acids. The fermented SFM with improved nutritional qualities is holding a great potential in farm animal nutrition. Keywords: Aspergillus ficuum, Functional feed, Solid state fermentation, Sunflower meal

INTRODUCTION Sunflower meal (SFM), the fourth largest oil seed meal produced in the world, also serves as a protein source, mostly in ruminant diets, and has been used in animal nutrition as a second plant protein source after soybean meal, but its use is limited due to its high fibre content and some antinutrional factors (ANFs), especially for feeding young poultry (Sangsoponjit et al., 2017; Yasar et al., 2018). In order to reduce the effect of these limiting factors on animals, poultry diets need to be supplemented with various enzymes, such as cellulase in diets but the addition of enzymes to the diet increases feed costs. Instead, the feed quality of SFM can be improved, and the inherent antınutritional factors can be eliminated or minimized (Haq and Mukhtar, 2004; Karakoç, 2006) by solid state fermentation (SSF). SSF can be defined as the activity of microorganisms in a similar attitude as in their natural environment in the presence of insoluble solid material in which there is little or no free water (Afşin, 2010). There are many studies showing that improvement of the feed quality, organic acid content, enzyme activity, antioxidant capacity of feeds are increased and feed additive or functional feed is obtained by converting into a new form via SSF (Yasar and Tosun, 2019a; Zhong-Tao et al., 2009; Karakurt et al., 2019). This feed additive or functional feed obtained as a result of SSF has been reported to have significant improvements in feed efficiency and 98

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fattening performance of animals using animal feed (Yasar and Yegen, 2017; Yasar and Tosun, 2018). Several microorganisms were successfully used in various conditions of SSF using SFM for he enrichment of nutrients, for reduce ANFs, for improvements of organic acids and antioxidant enzyme activities (Karakurt et al., 2019; Yasar and Tosun, 2019b; Yasar et al., 2018; Hassan et al., 2018; Soltan et al., 2015; Irfan et al., 2014; Azza et al., 2013; Lomascolo et al., 2012; Vendruscolo et al., 2008). In this study, the optimum conditions of pH, fermentation periods, stirring rate and moisture content of substrate selected from the above studies were fixed, optimised and controlled by a modern bioreactor and used to ferment sunflower meal by Aspergillus ficuum (Reichert) Hennings, anamorph (ATCC® 66876™). The objective was to determine the effects of optimised fermentation conditions selected microorganisms on the nutrient fortification of sunflower meal. MATERIAL AND METHODS Sunflower meal (SFM) purchased from a local provider dried and ground to pass a sieve with 3 mm and was further autoclaved at 120 ºC for 15 min. Fungal microorganisms, Aspergillus ficuum (Reichert) Hennings, anamorph (ATCC® 66876™) (A. ficuum) obtained from DSMZ were cultivated according to the supplier instruction to collect inoculating spores. Optimum fermentation conditions fixed in the study (Table 1) were selected from the literature and optimized by using a laboratory bioreactor of 2.5-3 L working capacity, LabforEtOlH 5 (Infors Ltd., Switzerland). A bio-gas detector (SA2Q from Beijing Shi’An Technology Instrument C0., Ltd) was attached to air outlet to monitor the amount of released CO2 with a range of 0 to 100% volume, CH4 with a range of 0 to 100 %volume and H2S with a range of 1 to 1000 ppm. A control fermentation experiment was conducted with no fungal inoculation. The fixed pH values were well optimized by peristaltic pumps using buffer solutions of 0.1 M sodium acetate (pH=1.5) and 0.1 M sodium bicarbonate (pH=9.75). Table 1. Experimental design with optimised fermentation parameters fixed throughout the fermentation period Exp.

Inoculant (spore g-1)

2.4x106

Moisture (%)

Temperature (oC)

Aeration (L dk-1)

Stirring rate (rpm)

Sampling period (h)

5.0

1.5

100

0, 24, 48 and 72

*Control fermentation, a non-pH optimised experiment (initial pH of 5.0 did not change throughout the fermentation period) under sterile fermentation conditions, the same as in experiments II. Exp.: experiment

At each sampling time, 3 sterile independent samples were taken from each of fermentation experiments (Table 1) were analysed in triplicates for the determinations of nutrients, organic acids and antinutritional factors. The data was analysed according to a General Linear Model (GLM) of variance analysis, where the differences between the treatments were separated at 0.05 significance level using a SPSS software. Microbial growth was determined by the method of TSE (2008) and nutrient contents by the methods specifically expressed in AOAC (2005). The content of organic acids were determined by the methods reported by Karabulut and Canbolat (2005). Lignin and metabolic energy (Janssen, 1989) values were calculated. Phytate

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(Raheja et al., 1973) and tannin (Chemesova and Chizhikov, 2004) were spectrophotometrically analysed. All chemical analysis results were expressed as percentage of dry matter. RESULTS SSF process was well optimized by the bioreactor used in this study, particularly for the parameters of pH and temperature throughout the fermentation periods. A constant temperature of 28 °C recorded remained unchanged up to 72 h throughout the fermentation processes, with minor fluctuations. The results of table 2 showed that the growth rate of A. ficuum in experiment II significantly (P<0.05) increased by 3.5 log at the end of 72 hours of fermentation. In parallel to the microbial growth the pH value continously increased during fermantation period. Throughout the experiment II periods, there were statistically significant (P<0.05) changes in pH values of SFM ranging from 5.0 to 6.60. In fermentation with A. ficuum, CO2 output reached 5% at 24 h, and this value decreased by 1% at subsequent fermentation hours. In all fermentation studies, any other gases were not produced, except CO2. In the fermentation studies conducted without the use of microorganisms, no microorganism growth and no statistical change in the pH and CO2 during the fermentation (P>0.05) (Table 2). Table 2. Microbial growth rate, CO2 output and pH fermentation Time, h pH Experiment I 0 5.00±0.11 24 5.05±0.10 48 5.03±0.07 72 5.01±0.05 Experiment II 0 5.00±0.10d 24 5.32±0.05c 48 5.50±0.06b 72 6.60±0.05a

Fungal growth, cfu g-1

CO2, %

0.00±0.05 0.00±0.07 0.00±0.10 0.00±0.14

0.00±0.15 0.00±0.10 0.00±0.08 0.00±0.11

2.4x106±0.05c 1.4x109±0.13b 1.4x109±0.29b 7.5x109±0.15a

0.00±0.10c 5.00±0.15a 5.00±0.12a 1.00±0.09b

Table 3 contained all analytical data of the SFM fermented for 0, 24, 48 and 72 hours. No significant changes (P>0.05) were seen in nutritional composition of SFM fermented with no fungal (Experiment I) inoculations (Table 3). Fermenting SFM with A. ficuum (Experiment II) inoculants significantly (P<0.05) influenced its nutritional composition. The contents of crude ash (CA) significantly (P<0.05) increased by the fungal fermentation. The highest increases in CA content were 15% at the 24 hours of experiment II. There was a 11% increase in the crude protein (CP) content at the 24 hours of experiment II (P<0.05). The crude lipit (CL) contents of SFM were significantly (P<0.05) decreased by about 32% in experiments II. The fermentation of fungal microorganisms significantly (P<0.05) reduced the crude fibre (CF), ADF and NDF content of SFM by 29%, 21% and 13% respectively. In contrary, there was a increase in the lignin content of SFM with the fungal fermantation (from 8.79% at 0 hours to 13% at 72 hours). A remarkable increase in the content of total reducing sugar (TRS) by about 341% of SFM by A. ficuum fermentation at the 24 hours (P<0.05) was seen. There was a 65% increase in the metabolic energy (ME) content at the 24 hours of experiment II (P<0.05).

100

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Table 3. Nutritional composition of SFM affected by experiments I and II fermentation Experiment

Hours

Experiments 0 I 24 (Control) 48 72 Experiments 0 II 24 (A. ficuum) 48 72

CA, %

CP, %

CL, %

CF, %

ADF, %

NDF, %

6.55±0.08 6.38±0.41 6.31±0.25 6.34±0.14 6.55±0.08b 7.55±0.16a 5.95±0.45b 7.16±0.27a

35.25±0.14 35.20 ±0.14 35.15 ±0.11 35.18±0.10 35.25±0.14c 39.10±0.33a 37.52±0.68b 37.84±0.11b

0.28±0.01 0.25 ±0.03 0.25 ±0.05 0.27 ±0.01 0.28±0.01a 0.25±0.04a 0.19±0.01b 0.27±0.01a

25.26±1.80 26.11±0.45 25.24±0.53 25.21±0.52 25.26±1.80a 17.90±0.20c 18.20±0.62bc 19.31±0.07b

34.05±0.97 34.55±0.34 35.01±0.54 34.10±0.65 34.05±0.97a 27.49±0.13c 28.91±0.30b 26.90±0.37c

52.12±1.03 52.04±0.45 52.20±0.68 51.58±0.75 52.12±1.03a 45.40±0.34c 48.69±0.65b 47.77±0.26b

101

Lignin, % 8.79 8.65 8.95 8.68 8.79 10 10 13

TRS, % 13.75±1.81 14.87±0.25 13.63±1.12 14.17±0.85 13.75±1.81c 60.65±0.76a 34.5±3.10b 38.09±2.76b

ME, kcal kg-1 DM 1757 1785 1754 1765 1757 2900 2483 2607

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Yasar et al. (2018) showed that fermentation of the Bacillus subtilis with SFM increased CA and CP. The CA and CP results obtained from this study were in a good agreement with the results in the literature. According to the results obtained in the previous studies, it was found that as a result of fermentation of SFM with bacteria, yeast and fungi, the CL content increased (Soltan et al., 2015) or decreased (Karakurt et al., 2019; Yasar and Tosun, 2019b). The CL content of SFM was decreased by 32% at the end of fermentation in our study. There are many studies reported decreased CF and its fractions by the solid state fermentation (Soltan et al., 2015; Karakurt et al., 2019; Yasar and Tosun, 2019b; Azza et al., 2013; Yasar et al., 2018). In our study, A. ficuum fermentation significantly decreased CF, ADF and NDF which was in good agreement with the previous results. However the lignin content of SFM was increased of fermentation in our study. Canella et al. (1985) reported that TRS content decreased as a result of lactic acid fermentation of SFM. In contrast to these studies, the TRS content of SFM was increased by 341% at the end of fermentation in our study. Vendruscolo et al. (2008) reported that there is a slight decrease in energy values as a result of fermentation of industrial by-products. However, we found the energy content increased as a result of fermentation. These differences between the studies were manly due to the use of different strains of microorganisms and various conditions of fermentations. There was no significant (P>0.05) effect on the contents of organic acids (acetic, butyric and lactic acid) of SFM of the fermention carried out without fungal microorganisms in this study (Table 4). However, fermenting SFM with A. ficuum significantly (P<0.05) influenced these compounds. The fermentation of fungal microorganisms significantly (P<0.05) reduced the acedic acid (AA) and butyric acid (BA) content of SFM by 78% and 100% respectively. Table 4 clearly showed that the fungal fermentations significantly (P<0.05) increased lactic acids (LA) from 0.95% at 0 hours to 16.05% at 24 hours. As a result of fermentation with A. ficuum, the total organic acid (TOA) has increased significantly (P<0.05), the majority of which is due to the increase in LA content. Table 4. Influence of fungal fermentation on the contents of organic acids of SFM Experiment Experiments I (Control)

Experiments II (A. ficuum)

Hours 0 24 48 72 0 24

AA, % 2.10±0.59 2.00±0.15 2.10±0.15 2.05±0.20 2.10±0.59a

BA, % 1.17±0.66 1.00±0.52 1.11±0.32 1.18±0.16 1.17±0.66a

LA, % 0.95±1.30 1.00±0.11 1.01±0.28 0.97±0.10 0.95±1.30d 16.05±0.36

TOA, % 4.23±1.4 4.00±0.25 4.22±0.20 4.20±0.30 4.23±1.40b

a 0.47±0.12c 0.0±0.00b 16.52±0.33a 48 1.43±0.12a 0.0±0.00b 2.98±0.05b 4.51±0.02b b b 72 0.95±0.12 0.0±0.00 2.44±0.09c 4.43±0.13b There are many studies in the literature indicating that organic acid content (especially lactic acid content) increased significantly as a result of fermentation of feed and agricultural by-products with several microorganisms (Karakurt et al., 2019; Yasar and Tosun, 2019b; Yasar et al., 2018; Vendruscolo et al., 2008). In this study, similar results were obtained. It was found that the amount of organic acid, especially the amount of LA, increased significantly. No significant changes (P>0.05) were seen in antinutritional factors (ANFs) of SFM fermented with no fungal inoculations (Table 5). Fermenting SFM with fungal inoculants significantly (P<0.05) influenced ANFs contents. There was an average of 67% decrease in the tannin content

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at the 72 hours of the fermentations carried out experiments II (P<0.05). A remarkable decrease in the content of phytic acid, by about 84%, of SFM by A. ficuum fermentation (P<0.05) was seen. Table 5. Influence of fermentation on the contents of ANFs Experiments ANFs Experiments I Tannin, % (Control) Phytic acid, µg/g Experiments II Tannin, % (A. ficuum) Phytic acid, µg/g

0. hour 3.18±0.52 0.76±0.03 3.18±0.52a 0.76±0.03a

24. hour 3.20±0.10 0.75±0.05 1.27±0.003b 0.36±0.02b

48. hour 3.11±0.21 0.75±0.03 1.71±0.01c 0.23±0.03c

72. hour 3.15±0.12 0.76±0.02 1.04±0.03d 0.12±0.01d

Degradation of tannin and phytic acid in SFM by fermentation with microorganisms was also reported earlier (Yasar et al., 2018; Karakurt et al., 2019; Lomascolo et al., 2012). In our study the tannin and phytic acid content of SFM decreased significantly parallel to the results reported in the literature. CONCLUSIONS As a result, the fermentation of SFM by fungal microorganism yielded an enrichment of nutritional qualities and added some biologically funtional compounds, mainly lactlc acids. The fermented SFM with improved nutritional qualities is holding a great potential in farm animal nutrition. Acknowledgement: We gratefullly acknowledged the TUBITAK, Turkey for their financial support of the study (VHAG: 214O629-1001 project). REFERENCES Afşin M 2010. Production of protease from Bacillus licheniformis ATCC 14580 with the method of solid state fermentation. Dicle Üniversitesi Fen Bilimleri Enstitüsü, Biyoloji Anabilim Dalı, Yüksek Lisans Tezi.Bligh, E. G., W.Y. Dyer (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37(8), 911-917. AOAC 2005. Official Methods of Analysis.18th ed., https://www.aoac.org/AOAC_Prod_Imis/AOAC_Member/Default.aspx?WebsiteKey=2e2 5ab5a-1f6d-4d78-a498-19b9763d11b4&hkey=8fc2171a-6051-4e64-a928-5c47dfa25797. Azza AM, El-Safy SF, Eman HFA 2013. Improvement of nutritional quality and antioxidant activities of fermented wastes by Saccharomyces cervisiae, Bacillus subtilis and Pleurotus salmoneo-stramineus. 3rd International Conference on Biotechnology and Its Applications in Botany and Microbiology, 17-18 April 2018, Cairo, Egypt. Canella M, Rome DM, Capena AB, Monterotondo GS, Grottaferrata GS 1985. Fermented sunflower meal and the method for ıts preparatıon. United States Patent. 4: 551. Chemesova II, Chizhikov DV 2004. Determination of tannin content in rhizomes of Comarum palustre L. and its tincture by spectrophotometric method. Rastitel'nye Resursy, 40: 122-130. Haq IU, Mukhtar H 2004. Biosynthesis of proteases by Rhizopus oligosporus IHS13 in low-cost medium by solid-state fermentation. Journal of Basic Microbiology, 44(4): 280–287. Hassaan MS, Soltan MA, Mohammady EY, Elashry MA, El- Haroun ER, Davies SJ 2018. Growth and physiological responses of Nile tilapia. Oreochromis niloticus fed dietary fermented 103

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sunflower meal inoculated with Saccharomyces cerevisiae and Bacillus subtilis. Aquaculture, 495 (1): 592–601. Irfan. M, Nadeem M, Syed Q 2014. One-factor-at-a-time(OFAT) optimization of xylanase production from Trichoderma viride-IR05 in solid-state fermentation. Journal of Radiation Research and Applied Sciences, 7(3): 317-326. Janssen WMMA 1989. European Table of Energy Values for Poultry Feedstuffs. Spelder holt Institute for Poultry Research and Information Services. ISBN 9071463273. 9789071463273. S: 104. Karabulut A, Canbolat Ö 2005. Yem Değerlendirme ve Analiz Yöntemleri. Uludağ Üniversitesi Yayınları, Bursa, 520 p. ISBN 975-6149-07-8 Karakoç DS 2006. Küflerden kati faz fermantasyon yöntemi ile lipaz üretimi. İstanbul Teknik Üniversitesi Fen Bilimleri Enstitüsü, Gıda Mühendisliği Anabilim Dalı, Yüksek Lisans Tezi. Karakurt Y, Güvercin D, Onder S, Celik C, Tosun R, Baran B, Yasar S 2019. Chemical. enzymatic. and antioxidant enrichments of full-fat soybean and sunflower meal by Bacillus subtilis (ATCC® 6633™) fermentation using a solid-state bioreactor. Turk. J Vet Anim Sci, 43: 8293. Lomascolo A, Uzan-Boukhris E, Sigoillot J, Fine F 2012. Rapeseed and sunflower meal: a review on biotechnology status and challenges. Applied Microbiology and Biotechnology, 95(5): 1105–1114. Raheja RK, Kaur C, Singh A, Batia IS 1973. New colorimetric method for the quantitative of phospholipids without aid digestion. Journal of Lipid Research, 14: 695. Sangsoponjit S, Suphalucksana W, Srikijkasemwat K 2017. Effect of feeding sunflower meal on the performance and carcass characteristics of broiler chickens. Chemical Engineering Transactions, 58: 841-846. Soltan MA, Hassaan MS, Abdella MS, El-Syaad GA, El-Ashry MA 2015. Yeast fermented sunflower meal as a replacer for fish meal in the diet of Nile tilapia, Oreochromis niloticus. Egypt J Aquat Biol Fish, 19(2): 65-72. TSE 2008. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and moulds — Part 2: Colony count technique in products with water activity less than or equal to 0.95; TS ISO 21527-2: 2008. https://www.iso.org/standard/38276.html (Date Accested: 12.07.2016) Vendruscolo F, Albuquerque PM, Streit F 2008. Apple Pomace: A Versatile Substratefor Biotechnological Applications. Critical Reviews in Biotechnology, 28(1): 1–12. Wang XP, Zhang WY 1999. Study on the determination and utilization of the pectin in haw. Quarterly of Forest By-Product and Speciality in China, 9: 13-14. Yasar S, Tosun R 2019a. İki farklı maya karışımı kullanılarak yürütülen katı faz fermantasyon sonucu elma ve domates posasının besin madde içeriğinin artırılması. Umteb 6. Uluslararası Mesleki ve Teknik Bilimler Kongresi 11-12 Nisan 2019, Iğdır. Yasar S, Tosun R 2019b. Trichoderma reesei Simmons. Anamorph (ATCC® 26921) ile Fermente Edilmiş Ayçiçeği Küspesinin Fonsiyonel Yem Olarak Değerlendirilmesi. Mas 5. Uluslar Arası Matematik-Mühendislik-Fen ve Sağlık Bilimleri Kongresi 2-5 Mayıs 2019, Erzurum. Yasar S, Yegen MK 2017. Yeast fermented additive enhances broiler growth. R Bras Zootec 46(10): 814-820. Yasar S, Tosun R (2018) Predicting chemical. enzymatic and nutritional properties of fermented barley (Hordeum vulgare L.) by second derivate spectra analysis from attenuated total

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reflectance-Fourier transform infrared data and its nutritional value in Japanese quails. Archives of Animal Nutrition, 72(5): 407-423. YaÅ&#x;ar S, Uysal C, Tosun R 2018. Nutritional Fortification of Sunflower Meal by Bacillus Subtilis ATCC PTA-6737 Fermentation. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Animal Science and Biotechnologies, Cilt 75(2): 99-104. Zhong-Tao S, Lin-Mao T, Cheng L, Jin-Hua D 2009. Bioconversion of apple pomace into a multi enzyme bio-feed by two mixed strains of Aspergillus niger in solid state fermentation. Electron J Biotechnol, 12(1): 1-9.

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SYNTHESIS OF CARVACROL DERIVATIVES AS ANTIOXIDANT AGENTS Fatih SÖNMEZ*1 1

Sakarya University of Applied Sciences, Pamukova Vocational High School, 54900, Sakarya, Turkey *Corresponding author’s e-mail: fsonmez@subu.edu.tr ABSTRACT

Free radicals in the human body play a pathogenic role in the formation of many chronic degenerative diseases such as cancer, autoimmune, inflammatory and cardiovascular neurodegenerative diseases. In this paper, a series of carvacrol substituted carbamate derivatives was synthesized and their DPPH activities were evaluated as antioxidant property. Among them, 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate exhibited the strongest antioxidant activity with an IC50 value of 18.73 µM, which was 7.7-fold more than that of carvacrol (IC50 = 144.84 µM). Keywords: Carbamate, Carvacrol, DPPH activity INTRODUCTION Many plants and their components have acted an important role in conventional implementation of medicine (Balunas, 2005; Jukic, 2007). Besides the Salvia species are known as a remedy for cognitive disorders, research on Thymus vulgaris essential oil also indicates their protective effects. Carvacrol (5-iso-propyl-2-methyl-phenol) is the main bioactive monoterpen isolated from many medicinal herbs such as Thymus vulgaris and Origanum vulgare (Poulose, 1978; Liolios, 2009). It has been found to possess various pharmalogical and biological properties, such as antioxidant, antiviral, antifungal, antitumor, anti-inflammatory and antibacterial activities (Adam, 1998; Kordoli, 2008; Kumar, 2013; Bhondani, 2014). Reactive oxygen species (ROS) play an important role in the formation of various serious diseases; such as cancer, heart diseases, diabetes, arteriosclerosis and cataracts (Yehye, 2015). The harmful effects of free radicals that cause potential biological damage are called oxidative stress. Free radicals in the human body play a pathogenic role in the formation of many chronic degenerative diseases such as cancer, autoimmune, inflammatory and cardiovascular neurodegenerative diseases (Dai, 2017). In the present study, a series of carvacrol derivatives was synthesized and their DPPH activities were evaluated as antioxidant properties. MATERIAL AND METHODS Synthesis of carvacrol derivatives Carvacrol (1 mmol) was dissolved in CH2Cl2, then Et3N (1.25 mmol) and isocyanate derivatives (1.25 mmol) were added to the solution, respectively. The mixture was refluxed for overnight, cooled and washed with water. The organic layer was dried over Na2SO4, and concentrated in vacuo. The products were purified by coloumn cromotography with Hexane:Ethyl acetate (5:1). 106

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DPPH activity Free radical scavenging activities are determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (Kedare, 2011). 1000 μM stock solutions of the materials were prepared. Of these stock solutions, 2, 5, 10 and 20 μL was taken and completed to 40 μL with ethanol, and then 160 μL of 0.1 mM DPPH solution was added. The absorbance values of the prepared solutions were measured at 517 nm after 30 min of incubation in the dark at room temperature. Inhibition values (%) of the synthesized carvacrol derivatives were calculated from the obtained absorbance values. The absorbance values of the samples were evaluated against the control. RESULTS The synthetic procedures to obtain the carvacrol-carbamate derivatives are depicted in Scheme 1. OH

H N

O R N C O

CH2Cl2, Et3N ref lux, 18h Carvacrol

2a-l

Scheme 1. Synthesis of novel carvacrol derivatives The yields and melting points of the synthesized carvacrol derivatives are given in Table 1. Table 1. The yields and melting points of the synthesized carvacrol derivatives Compound 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l

R 3-OCH3 4-OCH3 4-CH3 3-F 4-F 3,4-di-Cl 4-Cl 4-Br 4-I 3-NO2 4-NO2 4-CF3

107

Yield (%) 58 74 42 70 82 79 53 69 72 79 70 71

Mp. (oC) 71 131 100 111 122 125 117 135 154 123 133 124

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The structures of the synthesized compounds were corrected 1H NMR, 13C NMR and MS. 1H NMR, 13C NMR and MS spectra of the 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate, which has the strongest DPPH activity in this study, are shown in Figure 1-3.

Figure 1. 1H NMR spectra of the 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate

Figure 2. 13C NMR spectra of the 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate

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Figure 3. MS spectra of the 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate The DDPH activity results of the synthesized compounds are given in Table 2. Table 2. IC50 values (μM) of synthesized compounds for DPPH activity Compound

DPPH (IC50,μM)a

2a 3-methoxyphenyl 253.88 ± 2.23 2b 4-methoxyphenyl 187.12 ± 1.41 2c p-tolyl 341.56 ± 2.57 2d 3-fluorophenyl 122.02 ± 0.88 2e 4-fluorophenyl 104.11 ± 0.84 2f 3,4-dichlorophenyl 135.15 ± 1.05 2g 4-chlorophenyl 147.62 ± 1.23 2h 4-bromophenyl 152.08 ± 1.45 2i 4-iodophenyl 117.50 ± 1.21 2j 3-nitrophenyl 66.98 ± 0.52 2k 4-nitrophenyl 18.73 ± 0.12 2l 4-trifluoromethyl phenyl 125.16 ± 1.14 Carvacrol (1) 144.84 ± 1.35 Quercetin 8.69 ± 0.24 a IC50 values represent the mean ± SEM of three parallel measurements (p < 0.05) 109

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DISCUSSION DPPH method is commonly used to measure the ability of antioxidants to sweep free radicals. In this spectrophotometric method, DPPH (2,2-diphenyl-1-picrylhydrazyl), a stable free radical, is used (Kedare, 2011). Antioxidants are based on the ability to degrade the DPPH radical, and when it interacts with radical hydrogen donors, hydrazine is reduced. According to this method, compounds must have strong hydrogen donor groups to exhibit good antioxidant properties. The IC50 values of the synthesized compounds were between 18.73 μM and 341.56 μM for DPPH activity. Among them, only 2k (IC50 = 18.73 μM) showed closed antioxidant activity to quercetin (IC50 = 8.69 μM) and had 7.7-fold more than that of carvacrol (IC50 = 144.84 µM). Other compounds showed lower DPPH activity than quercetin (Table 2). On the other hand, 2d, 2e, 2f, 2i, 2j, 2k and 2l exhibited stronger DPPH activity than carvacrol (IC50 = 144.84 μM). The exchanging methoxy group at para-position of phenyl ring with methyl group decreased the DPPH activity (comparing 2b, R = 4-OCH3, IC50 = 187.12 μM with 2c, R = 4-CH3, IC50 = 341.56 μM). Moving the methoxy and nitro groups and F atom on the phenyl ring from metaposition to para-positions led to an increase in DPPH activities (comparing 2a, R = 3-OCH3, IC50 = 253.88 μM with 2b, R = 4-OCH3, IC50 = 187.12 μM; comparing 2d, R = 3-F, IC50 = 122.02 μM with 2e, R = 4-F, IC50 = 104.11 μM; comparing 2j, R = 3-NO2, IC50 = 66.98 μM with 2k, R = 4NO2, IC50 = 18.73 μM). On the other hand, regarding to halogen series at para-position of phenyl ring, it can be said that DPPH activity depends on the electronegativity, size and polarizability of the halogen substituent (2e, R = 4-F, IC50 = 104.11 μM; 2g, R = 4-Cl, IC50 = 147.62 μM; 2h, R = 4-Br, IC50 = 52.08 μM; 2i, R = 4-I, IC50 = 117.50 μM). Conclusions A series of carvacrol derivatives was synthesized and their DPPH activities were evaluated as antioxidant properties. The structures of the synthesized compounds were corrected 1H NMR, 13C NMR and MS. 5-isopropyl-2-methylphenyl(4-nitrophenyl)carbamate exhibited the strongest antioxidant activity with an IC50 value of 18.73 µM, which was 7.7-fold more than that of carvacrol (IC50 = 144.84 µM). Moreover, seven synthesized compounds (2d, 2e, 2f, 2i, 2j, 2k and 2l) showed better DPPH activity than carvacrol (IC50 = 144.84 μM). According to structure-activity relationships, we can say that the DPPH activity depends on the electronegativity and size of the substituent and the resonance stability of the formed radicals. Acknowledgements: The authors would like to gratefully acknowledge the kind support of Dr. Belma Zengin Kurt and Dr. Zuhal Gunesli.

REFERENCES Adam, K., A. Sivropoulou, S. Kokkini, T. Lanaras, M. Arsenakis (1998). Antifungal activities of origanum vulgare subsp. hirtum, mentha spicata, lavandula angustifolia, and salvia fruticosa essential oils against human pathogenic fungi. J. Agric. Food Chem., 46, 1739–1745. Balunas, M.J., A.D. Kinghorn (2005). Drug discovery from medicinal plants. Life Sci., 78, 431– 441. 110

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Bhandari, S.S., M.P. Kabra (2014). To evaluate anti-anxiety activity of thymol, J. Acute Dis., 3, 136-140. Dai, Y., C. Shao, Y. Piao, H. Hu, K. Lu, T. Zhang, X. Zhang, S. Jia, M. Wang, S. Man (2017). The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical. Carbohyd. Poly., 178, 34-40. Jukic, M., O. Politeo, M. Maksimovic, M. Milos (2007). In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone, Phytother. Res., 21, 259–261. Kedare S.B., R.P. Singh (2011). Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol., 48(4), 412–422. Kordali, S., A. Cakir, H. Ozer, R. Cakmakci, M. Kesdek, E. Mete (2008). Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, carvacrol, thymol and p-cymene. Bioresource Technol., 99, 8788–8795. Kumar, D., D.S. Rawat (2013). Synthesis and antioxidant activity of thymol and carvacrol based Schiff bases. Bioorg. Med. Chem. Lett., 23, 641–645. Liolios, C.C., O. Gortzi, S. Lalas, J. Tsaknis, I. Chinou (2009). Liposomal incorporation of carvacrol and thymol isolated from the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity. Food Chem., 112, 77-83. Poulose, A.J., R. Croteau (1978). Biosynthesis of aromatic monoterpenes: Conversion of γterpinene to p-cymene and thymol in Thymus vulgaris. Arch. Biochem. Biophys., 187, 307-314. Yehye, W.A., N.A. Rahman, A. Ariffin, S.B.A. Hamid, A.A. Alhadi, F.A. Kadir, M. Yaeghoobi (2015). Understanding the chemistry behind the antioxidant activities of butylated hydroxytoluene (BHT): A review. Eur. J. Med. Chem., 101, 295-312.

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PLANT TASTE AND THERAPY IN HAUSA TRADITIONAL MEDICINE Balarabe, S1., Aliyu, B. S*2., Hayatu, M2., Nafisa, M. A3, Haruna, S4. 1

Department of Integrated Science, Federal College of Education Kano 2

3 4

Department of Plant Biology, Bayero University, Kano

Department of Biology, Sa’adatu Rimi College of Education, Kumbotso, Kano

Department of Forestry Technology, Audu Bako College of Agriculture, Dambatta, Kano *Corresponding author’s e-mail: balasidi@gmail.com ABSTRACT

A study was conducted in Kano metropolitan area of Kano State, Nigeria. It covers about 499km² and comprises of eight local government areas with a population of 2,828,861. Tastes of medicinal plants have been used as a basis for plant selection to treat different diseases or disease categories in various traditional medicine practices around the world. In this study some plants locally used as medicine to treat some of the most commonly cited diseases were selected to represent different taste categories from an ethno-botanical survey conducted. The survey revealed that Hausa traditional medicine classifies taste of medicinal preparations into nine different categories as; bitter, astringent, contraption, sweet, pleasant, pungent, salty, sour and Insipid/bland. 199 plants were documented out of which 166 were identified as used in treating 124 different ailment categories. A total number of 24 informants participated in the tasting of plants preparations, most of the participants agreed on tastes of the medicinal plants, but could not actually identify all the plants preparations based on their taste alone. Extraction and phytochemical screening of the selected plants was done following standard procedures described by Vishnoi, 1979; Sofowora, 1993; Yadav and Munin, 2011; Trease and Evans, 2002 (for carbohydrates, monosaccharides, reducing sugars, tannins, free anthraquinones, glycosides, terpenoids, saponins, flavonoids, and alkaloids). The phytochemical screening of the various extracts revealed that the plants contain potent phyto-compounds, that justify the local use of the plants as medicine. Thin Layer Chromatography (TLC), Gas Column-Mass Spectrometry (GCMS) as well as Fourier Transformed InfraRed Spectroscopy (FTIR) analysis of the selected plant extracts indicated the presence of numerous chemical components in the selected plants having various chemical structures. However, the study could not establish a clear scientific relationship between the taste of the plants and their therapeutic activity because there is a great number of phytochemicals in these plants each having a different mode of action on the human body. It was thus concluded that tastes of medicinal plants alone is not enough a criterion for selecting plants to treat diseases, as is done in Hausa traditional medicine. Keywords: Taste, Phytochemicals, Medicinal plants, Ethnomedicine

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INTRODUCTION Plants have been used as food and medicine apart from other uses everywhere in this world. Every ethnic group in Nigeria has its own way of practicing the use of traditional medicine or rather use of plants as medicine. According to Ayitey-Smith (1989), traditional medicine evolved from environmental resources which the people of a community adapted in desperation for survival from disease. Traditional medicine was the sole medical system for health care before the advent of orthodox or modern medicine. Even in this present technological era, traditional medicine is still the predominant means in the third world for the preservation of health of the rural majority who constitute over 70% of the total population (Okoli et al., 2007). According to the World Health Organization, most populations particularly those living in the villages depend mainly on herbal remedies, for their psychological and physical health requirements (Rabe and van Staden, 2000) since they cannot afford the products of modern medicine (Salie et al., 1996). Medicinal plants include various types of plants used in herbalism and some of these plants have some medicinal activities. These medicinal plants are considered as rich resources of ingredients which can be used in drug development and synthesis, besides that these plants play important role in the development of human cultures around the whole world (Rasool, 2012). Every traditional medicine system uses senses in diagnosis and administration of herbal medicine where taste is one of the most important means of providing insight into the properties or activities of medicinal plants. Plant taste lead to better understanding of the biochemistry of a plant and how we can use it to bring balance which is the main target of traditional herbal treatment (Todd, 2013). According to Ayurveda, there are six medicinal tastes in nature which are sweet, sour, salty, pungent, bitter and astringent (Kimmana, 2012). In traditional Chinese medicine there are five tastes; sour, salty, pungent, bitter and sweet while the western science recognizes four tastes namely, sweet, sour, salty and bitter (de la Foret, 2013). Also, Choedon and Kumar (2012) classified tastes into six categories; sweet, bitter, astringent, sour, hot and salty where all these tastes have differing influences on the body. Some researchers believe that taste combined with cognitive and cultural mechanisms allow human to take sufficient amount of phytochemicals to fight parasitic (John, 1990) and other diseases. MATERIALS AND METHOD Ethno-botanical survey was carried out to collect information from knowledgeable people and traditional practitioners regarding some common plants use, taste and relationship between medicinal plants taste and their curative effect. Some plants were selected among those most frequently cited based on different tastes to represent a particular taste group. The selected plants were then extracted using distilled water and methanol and phytochemical screening was carried out. Study Design/Data collection A field research (survey) work was carried out on Hausa traditional herbalist in Kano to get information about common illnesses and the plants used to treat them, the plants taste and relationship between taste and medicinal properties of the plants. Simple questionnaire, participatory observations and semi-structured interviews (Maneenoon et al., 2015) were used for 113

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the data collection as this provided valuable information on real facts, opinions and the attributes of the traditional herbal healers towards the use of plants and relating that to plants taste. Study Area The study was conducted in Kano metropolitan area which covers about 499kmÂ˛ and comprises of eight local government areas namely: Kano municipal, Dala, Tarauni, Fagge, Nasarawa, Gwale, Ungogo and Kumbotso local government areas of the city with a population of 2,828,861 according to the 2006 Nigerian census. Plant Selection based on Taste Test Free lists of some medicinal plants was drawn out (Borgatti,1996) and one (1) plant each or a combination of plants was selected from the list based on different taste (bitter, salty, pungent, sour, astringent and insipid/bland) and illness categories among those that were most frequently cited in the free list as representatives (Casagrande,2000). Plants Preparation and Evaluation of Tastes The selected plants materials were collected and remedies prepared the way that they are commonly prepared by the traditional healers. Volunteers were asked to taste the preparations one at a time (Pushpan and Nishtewsar 2014), then provide a name for the taste and name the source plant (Casagrande, 2000). Volunteers were given water, crackers and sufficient time for tastes to subside between each tasting to avoid one taste masking the next. The Volunteers were also asked to taste other preparations made from bitter leaves (Vernonia amygdalina), lime fruit (Citrus aurantifolia), Guiera senegalensis leaves, chilli (Capsicum cepa) extracts, slight salt solution and water as a control (with no taste). Extraction and phytochemical screening of the selected plants was done following standard procedures as described below. Extraction and Preparation of Plant Materials The selected plant parts were air-dried at room temperature and crushed with mortar and pestle. 100g of each powdered plant material was weighed and extracted exhaustively with 1000ml methanol and 1000ml distilled water for two (2) weeks using maceration technique. The extracts obtained were transferred into clean sterile airtight containers, weighed and kept in desiccators until required for use (Vishnoi, 1979; Sofowora, 1993; Trease and Evans, 2002). Percentage yield of each extract was calculated. Phytochemical Tests Molisch's test for Carbohydrates A few drops of Molisch's reagent was added to each of the extracts dissolved in distilled water, followed by addition of 1 ml of conc. H2SO4 by the side of the test tube. The mixture was allowed to stand for two minutes and then diluted with 5 ml of distilled water. Formation of a red or dull violet colour at the interphase of the two layers shows a positive test (Sofowora, 1993). Barfoed's test for monosaccharides About 0.5 g of each extract was dissolved in distilled water and filtered. One (1) ml of the filtrate was mixed with 1ml of Barfoed's reagent in a test tube and then heated on a water bath for a period of 2 minutes. Reddish precipitate of cuprous oxide shows a positive test (Sofowora, 1993). 114

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Fehling's test for free reducing sugar About 0.5 g of each extracts was dissolved in distilled water and filtered. The filtrate was heated with 5 ml of equal volumes of Fehling's solution A and B. Formation of a red precipitate of cuprous oxide indicates the presence of reducing sugars (Sofowora, 1993). Fehling's test for Combined Reducing Sugars About 0.5 g of each extracts was hydrolysed by boiling with 5 ml of dilute hydrochloric acid and the resulting solution was neutralized with sodium hydroxide solution. Few drops of Fehling's solution was added and then heated on a water bath for 2 minutes. Appearance of a reddish-brown precipitate of cuprous oxide indicates the presence of combined reducing sugars (Sofowora, 1993). Test for Tannins About 0.5 g of each extracts was stirred with about 10 ml of distilled water and then filtered. A few drops of 1% ferric chloride solution were added to 2 ml of the filtrate. Occurrence of a blueblack, green or blue-green precipitate indicates the presence of tannins (Trease and Evans, 2002). Borntrager's Test About 0.2 g of each extracts was shaken with 10 ml of benzene and then filtered. Five ml of 10% ammonia solution was added to the filtrate and shaken. Appearance of a pink, red or violet colour in the ammoniacal (lower) phase indicates the presence of free anthraquinones (Sofowora, 1993). Salkowskiâ&#x20AC;&#x2122;s test Each crude extract was mixed with 2ml of chloroform. Then carefully 2ml of concentrated H2SO4 was added and shaken gently. A reddish brown colour indicates the presence of steroidal ring, i.e., glycone portion of the glycoside (Yadav and Munin, 2011). Keller-kilani test Each crude extract was mixed with 2ml of glacial acetic acid containing 1-2 drops of 2% solution of FeCl3. Then each mixture was poured into another test tube containing 2ml of concentrated H2SO4. A brown ring at the interphase indicates the presence of cardiac glycosides (Yadav and Munin, 2011). Test for terpenoids A little of each extract was dissolved in ethanol. One (1) ml of acetic anhydride was added to each followed by the addition of conc. H2SO4. A change in colour from pink to violet shows the presence of terpenoids (Sofowora, 1993). Test for Saponins One (1) g of each extracts was mixed with 5 ml of distilled water and filtered. To each filtrate, about 3 ml of distilled water was added and shaken vigorously for about 5 minutes. Frothing which persist on warming is an evidence for the presence of saponins (Sofowora, 1993).

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Shinoda's test for flavonoids About 0.5g of each extracts was dissolved in ethanol, warmed and then filtered. Three pieces of magnesium chips were added to each filtrate followed by few drops of conc. HCl. A pink, orange, or red to purple colouration indicates the presence of flavonoids (Trease and Evans, 2002). Ferric chloride test for flavonoids About 0.5ml of each extracts was dissolved in distilled water and then filtered. A few drops of 10% ferric chloride solution were added to 2 ml of each filtrate. A green-blue or violet colouration indicates the presence of a phenolic hydroxyl group (Trease and Evans, 2002). Lead ethanoate test for flavonoids A few quantity of each extracts was dissolved in water and filtered. To 5 ml of each filtrate, 3 ml of lead ethanoate solution was added. Appearance of a buff-coloured precipitate indicates the presence of flavonoids (Trease and Evans, 2002). Sodium hydroxide test for flavonoids A few quantity of each extracts was dissolved in water and filtered. Two (2) ml of 10% aqueous sodium hydroxide was added later to each to produce a yellow colouration. A change in colour from yellow to colourless on addition of dilute hydrochloric acid indicates the presence of flavonoids (Trease and Evans, 2002). Test for alkaloids A few quantities of each extracts were stirred with 5 ml of 1% aqueous HCl on water bath and then filtered. One (1) ml of each filtrate was poured individually into 2 test tubes. To the first portion, few drops of Dragendorff's reagent were added. Occurrence of orange-red precipitate shows positive result. To the second portion 1 ml Mayer's reagent was added and appearance of buff-coloured precipitate indicates the presence of alkaloids (Sofowora, 1993). RESULTS Documented Medicinal Plants from Survey of Eight Local Governments in Kano Metropolitan Area From the 800 questionnaires distributed (100 per Local Government Area) in Kano metropolitan area, 788 were returned. 199 plants were documented out of which 166 were identified as those used in treating 124 different ailment categories. The botanical names of the plants, family name, local name, part used, illness treated and the habit of the plants were all documented. The most commonly cited diseases include; malaria fever, typhoid fever, pile, reproductive ailments, diarrhea, venereal diseases, acute bronchitis, spiritual problems, yellow fever, mouth thrush, etc. These common ailments are treated using different plants preparations as informed by the traditional healers (Table 1). Pile is the most common of all the diseases and mouth thrush the least amongst the common diseases cited.

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Table 1: Common Ailments Frequently Occurring, Most Common Cited Plants Used As Cure, Taste and Number of Citations From Each LGA for Part used

Taste

NSR

DAL

GWL KMC UGG KBT TRN FGG Total citation

Senna occidentalis

Root

Bitter

Diarrhea

Boswellia dalzielii

Stem bark

Astringent

Venereal diseases

Zingiber officinale &

Rhizome

Pungent

Moringa oleifera

Root

Mouth thrush

Nelsonia canescens

Whole plant

Sour

Acute bronchitis

Securidaca longipedunculata

Root bark Salty

Yellow fever

Cochlospermum religiosum & Erythrina senegalensis

Root

Insipid/

Root

bland

Ailment treated

Plants used treatment

Typhoid fever

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A total number of 24 informants participated in the tasting of plants preparations where we have 3 informants from each Local Government Area. Parts of the test plant species Senna occidentalis (root), Boswellia dalzielii (stem bark), Securidaca longipedunculata (root bark), Nelsonia canescens (whole plant), a combination of Zingiber officinale (rhizome) & Moringa oleifera (root) and a combination of Cochlospermum religiosum (root) & Erythrina senegalensis (root) presented different tastes as identified by the taste participants (Table 2). For Senna occidentalis root preparation, 20 participants cited its taste as bitter, 3 as astringent and 1 as contraption. Boswellia dalzielii stem bark extract preparation was identified by 22 participants to have astringent taste and contraption taste by 2 participants. The Securidaca longipedunculata root bark extract preparation was identified to be salty by all the participants. Similarly, Nelsonia canescens whole plant preparation tasted sour to 21 participants, contraption to 2 and astringent to 1 participant. For the preparation of a combination of Zingiber officinale rhizome and Moringa oleifera root 21 participants cited the taste as pungent, 1 as pleasant and 2 as a slightly bitter taste. Lastly, Cochlospermum religiosum root and Erythrina senegalensis root combined preparation was also revealed to have an insipid (bland) taste by 21 participants, a pleasant taste by 1 participant and a contraption taste by 2 participants. For the plants used as control, Vernonia amygdalina leaves preparation was identified to have a bitter taste by all 24 participants, Capsicum cepa fruits preparation was also cited by all participants as pungent. Similarly, Citrus aurantifolia fruits extract preparation was cited as having sour taste by all 24 participants and for Guiera senegalensis leaves preparation 20 participants identified the taste as astringent and 4 as contraption. All 24 participants cited water to have no taste which is insipid or bland and salt solution as salty in taste. There are different types of taste in Hausa traditional medicine, but those considered when describing the quality of a plant or its part are nine (9) types as follows; 1. Bitter known as daci in hausa

6. Pungent known as yaji

2. Astringent known as bauri

7. Salty known as zartsi

3. Contraption known as barci

8. Sour known as tsami

4. Sweet known as zaki

9. Insipid/bland known as salam or lami

5. Pleasant known as gardi These different tastes can be experienced and understood only on the tongue which also has peculiar quality that determines their curative character in organisms. The result of the phytochemical screening of the methanol and aqueous extracts of the selected plants shows that both extracts have a number of phytochemicals as shown in Table 3.

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Table 2: Selected Plant species and control plants used in taste tests and number of informants who cited each taste quality in the Eight Local Government Areas of Kano State Plant species

Plant part

Hausa name

Taste in Hausa

English gloss No. of participants citing taste

Senna occidentalis

Root

Raidore

Daci

Bitter

Bauri

Astringent

Barci

Contraption

Bauri

Astringent

Barci

Contraption

Boswellia dalzielii

Stem bark Ararrabi

Securidaca longipedunculata

Root bark

Sanya

Zartsi

Salty

Nelsonia canescens

Whole plant

Tsamiyar kasa

Tsami

Sour

Barci

Contraption

Bauri

Astringent

Pungent

Zingiber officinale & Moringa oleifera

Cochlospermum religiosum Erythrina senegalensis

Rhizome

Citta

Yaji

Root

Zogale

Galmi/gardi Pleasant

Daci-daci

Slightly bitter 2 Insipid/bland

Root

Rawaya

Salam

Root

Minjirya

Galmi/gardi Pleasant

barci

Contraption

Vernonia amygdalina Leaves

Shuwaka

Daci

Bitter

Citrus aurantifolia

Lemun tsami

Tsami

Sour

Ruwa

Salam

No taste

Sabara

Bauri

Astringent

Barci

Contraption

Gishiri

Zartsi

Salty

Barkono

yaji

Pungent

Fruits

Water Guiera senegalensis

Leaves

Salt solution Capsicum cepa

Fruits

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Table 3: Phytochemical Constituents of the Selected Plant Extracts. Phytochemicals

Carbohydrates

B.dalzielli extract Met Aqu + +

S.longipedunculata extract Met Aqu + +

S.occidentalis extract Met Aqu + +

N.canescence extract Met Aqu + +

M.oleifera/Z.officinale extract Met Aqu + +

Monosaccharide

Reducing sugars

Combined reducing Sugars

Tannins

Free anthraquinones

Cardiac glycosides

Glycosides

Terpenoids

Saponins

Flavonoids

Alkaloids

Phenols

KEY: + = present

- = absent

Met = methanol

Aqu = Aqueous 120

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DISCUSSION Among the most commonly cited diseases are; malaria, typhoid, pile, reproductive ailments, diarrhea, venereal diseases, acute bronchitis (cough), spiritual problems, yellow fever, mouth thrush, etc. Idu et al. (2006) also found out in a study of ethnomedicinal uses of trees among Bachama tribe of Adamawa state, Nigeria a variety of disease conditions such as malaria, typhoid, jaundice, dysentery, gonorrhea, cough, hypertension and fibroid as common diseases treated by the traditional healthcare administrators amongst others. Hausa traditional medicine classified taste of medicinal preparations into nine different categories as; bitter, astringent, Contraction, Sweet, Pleasant, Pungent, Salty, sour and Insipid/bland. This is more than the findings of Jayasundar and Ghatak (2016), Kimmana, (2012) were the taste of medicinal plants in Ayurveda is grouped into six namely, sweet, sour, salty, hot/pungent, bitter and astringent. While, traditional Chinese medicine recognizes only five tastes; sour, salty, pungent, bitter and sweet. Western science recognizes four tastes namely, sweet, sour, salty and bitter (de la Foret, 2013). Choedon and Kumar (2012) in addition classified tastes into six categories; sweet, bitter, astringent, sour, hot and salty. Taste is regarded as a strong quality of a medicinal plant that serves as a guide in its selection to treat diseases or a particular group of disease. Heinrich (2003) also found out the importance of taste in medicinal plants selection among the Mexican Indians. This is also similar to the finding of Kimmana, (2012); Gilca and Barbulescu (2015) about the use of taste in predicting the activity of a medicinal plant in Ayurvedic medicine. In addition, the Amchiâ&#x20AC;&#x2122;s of Dolpo also use taste and potency of plants in selecting medicinal plants (Lama et al., 2001). Taste test for the selected plants The taste test of the selected plants preparations by the participants shows a high agreement about the taste of the different remedies since out of a total of 24 participants from all the Local Government Areas, 20 cited the bitter taste correctly, 22 each for astringent and insipid/bland, 21 as sour and pungent each and lastly 24 as salty. This is in line with Casagrande (2000) who also found out a high agreement about how prepared remedies tasted in a taste test in his research on human taste and cognition in Tzeltal Maya medicinal plants use. For number of correct identification of plant names based on taste alone there were different levels of correct identifications. This indicates that the participants have some knowledge about the taste of medicinal preparations but could not actually identify all the plants preparations based on their taste alone. The findings of this research is in line with that of Casagrande (2000) who also found that taste participants have knowledge about the medicinal plants tasted and the plants name. The finding of this research for S. occidentalis agrees with Egharevba et al. (2010) and Manikandaselvi et al. (2016) who also observed that the aerial part of Senna occidentalis is bitter in taste. The roots are also bitter as reported by Kaur et al. (2014); Rekhah et al. (2016). Fresh leaves of N. canescens have a sour lemon-like taste (powo.science.kew.org, 2019) which agrees with that described by the informants. Acharya, et al. (2012) cited the taste of root of N. canescens as sweet. The taste of the other plants parts could not be found from literature which could be attributed to the fact that most of these parts possess a combination of tastes that can likely not be distinguished. Phytochemicals in selected plants Phytochemical screening of both methanol and aqueous extracts of B. dalzielii stem bark indicates the presence of carbohydrates, monosaccharides, reducing sugars, combined reducing sugars, tannins, free anthraquinones, cardiac glycosides, glycosides, terpenoids, saponins, flavonoids, alkaloids and phenols. This is in agreement with the screening conducted by Hassan 121

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et al. (2009) who also found carbohydrates, tannins, saponins, flavonoids and cardiac glycosides in the methanol extract of B. dalzielii. So also flavonoids, sterols/triterpenes, Saponins and tannins were reported in the stem bark of B. dalzielii (NabĂ¨rĂ¨ et al., 2013). Similarly, Mamza et al. (2018) found reducing sugars and combined reducing sugars in addition to other chemical compounds in the crude methanol stem bark extract of B. dalzielii. The result of this study shows that methanol extract of S. longipedunculata root bark have carbohydrates, monosaccharides, reducing sugars, tannins, cardiac glycosides, glycosides, terpenoids, saponins, flavonoids, and phenols, but combined reducing sugars, free anthraquinones and alkaloids were absent. This is in agreement with Junaid et al. 2008 who also found that the aqueous extract of root of S. longipedunculata have tannins, saponins, cardiac glycosides, steroids, alkaloids and flavonoids and the methanol extract have tannins, cardiac glycosides and steroids. Similar study carried out by Okoli et al. (2005) shows that methanol extract of the root bark of S. longipedunculata tested positive for carbohydrates, reducing sugars, glycosides, flavonoids, terpenoids, sterols and saponins. In another study, its phytochemical screening revealed the presence of cardiac glycosides, flavonoids, tannins, saponins and alkaloids (Auwal et al., 2012; Musa et al., 2013). The result of phytochemical screening carried out by Amina et al. (2017) also revealed the presence of bioactive compounds like alkaloids, glycosides, tannin, saponin, flavonoids, phenols and anthraquinone. The methanol extract of S. occidentalis root shows the presence of carbohydrates, monosaccharides, reducing sugars, combined reducing sugars, tannins, free anthraquinones, cardiac glycosides, glycosides, terpenoids, saponins, flavonoids and phenols but alkaloids were absent while the aqueous extract reveal the presence of carbohydrates, reducing sugars, combined reducing sugars, free anthraquinones, cardiac glycosides, saponins and flavonoids, but monosaccharides, tannins, glycosides, terpenoids, alkaloids and phenols were absent. This is similar to the research conducted by Chinnala et al. (2010) who also reveal that the preliminary phytochemical studies of methanol extract of Senna occidentalis roots show the presence of tannins, saponins, flavonoids, triterpenoids, sterols, cardiac glycosides and carbohydrate but alkaloids are absent. Phytochemical analysis showed that the different parts of S. occidentalis contained different chemical groups which includes alkaloids, steroids, tannins, flavonoids, anthraquinone, saponins, terpenes, carbohydrates, sugars and cardiac glycosides (Ajagbonna et al., 2000; Saganuwan and Gulumbe, 2006; Daniyan et al., 2011; Veerachari and Bopaiah, 2012). In another study by Garba et al. (2015), phytochemical screening of methanol root extract of C. occidentalis revealed the presence of high amount of sterols, flavonoids, tannins, phenols, terpenes and anthraquinones with moderate amount of cardiac glycoside and saponin while the test for alkaloid showed negative result. Similarly, carbohydrates, saponins, terpenes, sterols, flavonoids, alkaloids, cardiac glycosides and anthraquinones were found in the root (Sadiq, et al., 2012). Usha et al. (2007) also reported the presence of alkaloids in the aqueous root extract of the same plant. In N. canescence whole plant both methanol and aqueous extracts show the presence of carbohydrates, monosaccharides, reducing sugars, combined reducing sugars, tannins, cardiac glycosides, glycosides, terpenoids, saponins, alkaloids, phenols and flavonoids while free anthraquinones were absent. David et al. (2016) also found that the aqueous extract of N. canescens is rich in flavonoids, tannins, tripertenes and other secondary metabolites. The results of the preliminary phytochemical analysis of N. canescens also showed the presence of alkaloids, tannins, flavonoids, glycosides and phenols in another study conducted by Bedabati et al. (2011; 2012)

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CONCLUSION In view of the findings of this research, it can be concluded that traditional medicine practitioners in Kano have knowledge of taste of herbal preparations and their uses in disease treatment, however, the herbalists still use plants with a particular taste to treat different disease conditions, indicating that the curative properties of the plants is not restricted to the taste of the plant part used. The phytochemical screening of the various extracts revealed that the plants contain the different phytochemicals tested, this justifies the significant curative properties of the selected plants as described in literatures. It can therefore be concluded that it is difficult to establish a clear relationship between the taste and disease activity of medicinal plants due to the occurrence of a great number of phytochemicals in these plants which have different tastes and modes of action in the human body. Thus the taste of medicinal plants alone is scientifically not enough to select plants for disease treatment as believed in Hausa traditional medicine practice. REFERENCES Acharya, R. N., Padiya, R. H., Patel, E. D., Harish, C. R., Shukla, V. J. and Chauhan, M. G. (2012). Pharmacognostic Evaluation of Nelsonia canescens. (Lam) Spreng. (Acanthaceae) Root. Pharmacognosy Journal. Vol 4:28. Ajagbonna, O.P., Onyeyili, P.A., Osunkwo, V.A, and Muhammed, B,Y. (2000). Acute Toxicity and Phytochemical Studies of Cassia occidentalis Linn Extract in Rats. Sokoto Journal of Veterinary Sciences,2(2), 32-35 Amina, B. A., Yushau, M. and Bukar, A. (2017). Screening Of Securidaca longipedunculata root Extracts for Activity Against Newcastle Disease Virus And Bacterial Isolates From Upper Respiratory Tract Of Human. DUJOPAS Vol. 3 No. 2 Auwal, S.M., Atiku, M.K., Wudil, A.M. and Sule, M.S. (2012). Phytochemical Composition and Acute Toxicity Evaluation of Aqueous Root Bark Extract of Securidaca longipedunculata (Linn) Bajopas Vol. 5(2): 67 â&#x20AC;&#x201C; 72. Ayitey-Smith, E., 1989. Prospects and Scope of Plant Medicine in Health Care. Ghana Universities Press, Accra, pp: 1-2. Bedabati, D., Jogen, C., K., Jibon, K. and Arudyuti C. (2011).Hepatoprotective Activity Of The Methanolic Extract Of Nelsonia Canescens (Lam.) Spreng on Carbon Tetrachloride Induced Hepatic Damage In Albino Rats. Int Journ Of Pharm Sci Rev And Res. Vol 8(2); pp36-37. Bedabati, D., Jogen C. K., Arudyuti, C. and Jibon, K. (2012). Hepatoprotective activity of Nelsonia canescens(Lam.) Spreng on acute hepatotoxicity induced by paracetamol. International journal of Pharmacy and Pharmaceutical Sciences. 4(1): pp107-112 Borgatti, S. P. (1996). ANTHROPAC 4.0 Methods Guide. Naticks, MA: Analytical Technologies. Casagrande, D. G. (2000). Human Taste and Cognition in Tzeltal Maya Medicinal Plant use. Journal of Ecological Anthropology. Vol 4. Chinnala, K. M., Debtha, J., Jenugu, V. S. and Srinivas, A. (2010). Evaluation of antibacterial and antifungal activities of Cassia occidentalis Linn root extracts. Annals of Biol Res, 1(3):81-84 Choedon, T. and Kumar, V. (2012). Medicinal Plants used in the Practice of Tibetan Medicine. RPMP 34 Daniyan, S.Y., Abalaka, M.E., Aransiola, S.A. and Elemba, O.M. (2011). Phytochemical Screening, Proximate Analysis and Mineral Composition of Cassia occidentalis Seed Extract. Asian Journal of Pharmaceutical and health Sciences, 1(3), 145-147. 123

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David, F., Jacqueline, N. K., Anchang, K. Y. and Bum, E. N. (2016). Effects of Nelsonia Canescens (Acanthaceae) on the Stress Induced Behavioral and Reproductive Changes in Female Albino Rats. World Journal Of Pharmacy And Pharmaceutical Sciences. Vol 5(12) 31-49. de la Foret, R., (2013). The taste of Herbs. (The Secret of Herbalism Revealed Through Your Senses of Taste). Pg 1-24. Egharevba, H. O., Odigwe, A. C., Abdullahi, M. S., Okwute, S. K., Okogun, J. I. (2010). Phytochemical Analysis and Broad Spectrum Antimicrobial Activity of Cassia Occidentalis L. (whole plant) New York Science Journal. 3(10) Pp 74-81 Garba, R., Saidu, A.N., Adeyemi, H.R.Y. and Muhammad. H. L. (2015). Effect of Methanolic Extract of Cassia occidentalis L. Root Bark on Body Weight and Selected Biochemical Parameters in Alloxan Induced Diabetic Rats. British Journal of Pharmacology and Toxicology 6(2): 39-49. Gilca, M. and Barbulescu, A. (2015). Taste of medicinal plants: A potential Tool in Predicting Ethnopharmacological Activities? Journal of Ethnopharmacology. Vol 174, Pp 464-473 Hassan, H. S., Musa, A. M., Usman, M. A. and Abdulaziz, M. (2009). Preliminary Phytochemical and Antipasmodic studies of th Stem Bark of B. dalzielii. Nig Journ of Pharm Sci. Vol. 8:1. Heinrich, M. (2003). Ethnobotany and Natural Products: The Search for New Molecules, New Treatments of Old Diseases or a Better Understanding of Indigenous Cultures? Current Topics in Medicinal Chemistry. 3, 29-42 http//powo.science.kew.org/taxon/urn:Isid:ipni.org:names:52558-1, February, 2019. Idu, M., Gill, L. S., Omonhinmin, C. A. and Ejale, A. (2006): Ethnomedicinal uses of trees among Bachama tribe of Adamawa state, Nigeria. Indian J.Trad. Knowledge. 2006; 5: 273-278. Jayasundar, R. and Ghatak, S. (2016). Spectroscopic and E-tongue evaluation of medicinal plants: A taste of how rasa can be studied. Journal of Ayurveda and Integrative Medicine 7 (2016) 191-197. John, T. (1990). With Bitter Herbs They Shall Eat it: Chemical Ecology and the Origins of Human Diet and Medicine. Tucson: University of Arizona Press. Junaid, S. A., Abubakar, A. Ofodile, A. C., Olabode, A. O., Echeonwu, G. O. N., Okwori, A. E. J and Adetunji, J. A. (2008). Evaluationof Securidaca longipedunculata leaf and root extracts for antimicrobial activities.Afri Journ Microbial Res.Vol. 2(12) pp322-325 Karra, N. (2014). Assessment of Rasa (taste) of Non-classical drugs- A Pharmacodynamic Principle. Annals Ayurvedic Med. 3 (1-2) 29 - 35 Kaur, I., Ahmad, S. and Harikumar. S. L. (2014). Pharmacognosy, Phytochemistry and Pharmacology of Cassia occidentalis Linn. Int. Journ of Pharm and Phyto Res. 6(2); 151155. Kimmana, N., (2012). The Six Medicinal Taste of Ayurveda. True Holistic Healing. Retrieved 22/11/2015. http://www.kimmana.com/the-six-medicinal-tastes-of-ayurveda. Lama, Y. C., Ghimire, S. K. and Aumeeruddy-Thomas, Y., (2001). Medicinal Plants of Dolpo (People and Plants). Initiative Amchisâ&#x20AC;&#x2122; Knowledge and Conservation. WWF Nepal Program, Kathmandu. Mamza, U. T., Sodipo, A. O., Abdulrahman, F. I. and Khan, I. Z. (2018). Phytochemical Analysis and In vitro Antimicrobial Assay of the Methanol Stem Bark Extract of Boswellia dalzielii Hutch. (Burseraceae). Chemistry Research Journal. 3(4):161-168. Maneenoon, K., Chunchom K., Yaowalak T., Nisachon S., Supatra P., Nitiphol R., Watid K., Phongsura P. and Winyu W. (2015). Ethnomedicinal Plants used by Traditional Healers in Phatthalung Province, Peninsular Thailand. Journ of Ethnobiol and Ethnomed; 11:43

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Manikandaselvi, S., Vadivel, V.and Brindha, P. (2016). Studies on physicochemical and nutritional properties of aerial parts of Cassia occidentalis L. journal of food and drug analysis. 5 0 8 -5 1 5 Musa, A.A., Oyewale, A.O., Ndukwe, G. I., Yakubu, S. E. and Abdullahi, M. S. (2013). Phytochemical screening and antimicrobial activity of solvent fractions of Securidaca longepedunculata (Fresen) root bark methanol extract. J. Chem Pharm Res 5(10):28-33. Nabèrè, Q., Adama, H., Samson, G., Kiessoum, K., Patrice, Z., Roland, M. N., Moussa, C., Martin, K., Jeanne, M. F. and Germaine, O. N. (2013). Antibacterial and Phytochemical studies of three Acanthaceae species used in Burkina Faso traditional medicine. Journal of Applied Pharmaceutical Science .Vol. 3 (05), pp. 049-055. Okoli, C. O., Akah, P. A. and Ezugworie, U. (2005). Anti-Inflammatory Activity Of Extracts Of Root Bark Of Securidaca Longipedunculata Fres (Polygalaceae). Afr. J. Trad. Cam (2005) 2 (3): 54 - 63 Okoli, R.I., Aigbe, O., Ohaju-Obodo, J. O. and Mensah, J.K. (2007). Medicinal Herbs Used for Managing Some Common Ailments among Esan People of Edo State, Nigeria. Pakistan Journal of Nutrition 6 (5): 490-496. Pushpan, R. and Nishtewsar, K. (2014). Rasa Nirdhārana (assessment of taste) of Leonotis nepetifolia (L.) R. Br.: A preliminary study in healthy volunteers. Ancient Science of Life. Vol 33:3. Rabe, T. and van Staden, J. (2000). Isolation of an Antibacterial Sesquiterpenoid from Warburgia salutaris. J Ethnopharmacol. 73 (1-2):171-4. Rasool, H. B. A. (2012). Medicinal Plants. (Importance & Uses). Pharmaceut Anal Acta, 3;10. Rekhah, U., Thoma,s J., Thomas, V., Tiju, J. M., Prakash, P. and Latha, M. S. (2016). Therapeutic Potencial of the Phytochemicals in Cassia occidentalis. A Review. European Journal of Pharmaceutical and Medical Research. 3(9), 180-188 Sadiq, I. S., Shuaibu, M., Bello, A. B., Tureta, S. G., Isah, A., Izuagie, T., Nasiru, S. and Kamaru, M. B. (2012): Phytochemistry and Antimicrobial Activities of Cassia Occidentalis Used for Herbal Remedies. Journal of Chemical Engineering Vol. 1:1 Saganuwan, A. and Gulumbe, M.. L. (2006). Evaluation of in-vitro antimicrobial activities and phytochemical constituents of Cassia occidentalis. Animal Research International, 3(3), 2006, 566-569. Salie, F., Eagles, P. F. K. and Leng, H. M. J. (1996). Preliminary Antimicrobial Screening of Four South African Asteraceae species. Journal of Ethnopharmacol.52(1):27-33 Sofowora, A. (1993). Medicinal Plants and Traditional Medicine in Africa. 2nd Ed. Sunshine House, Ibadan, Nigeria: Spectrum Books Ltd; Screening Plants for Bioactive Agents; pp. 134–156. Todd, C. (2013). Herbalist in de la Foret, R. (2013). The taste of Herbs. (The Secret of Herbalism Revealed Through Your Senses of Taste). Pg 3. Trease, G.E. and Evans, W.C. (2002). Pharmacognosy. 15th Ed. London: Saunders Publishers; pp. 42–44. 221–229, 246–249, 304–306, 331–332, 391–393. Usha, K., Kasturi, G.M. and Hemalata, P. (2007). Hepatoprotective effect of Hygrophila spinosa and Cassia occidentalis on carbon tetrachloride induced liver damage in experimental rats. Indian J. Clin. Biochem., 22(2): 132-135. Veerachari, U. and Bopaiah, A. K. (2012). Phytochemical investigation of the ethanol, methanol and ethyl acetate leaf ectracts of six Cassia species. International Journal of Pharma and Bio Sciences, 2(2), 260-270. Vishnoi, N.R. (1979). Advanced Practical Chemistry. Ghaziabad-India: Yikas Publication House, PVT Ltd.; 1979. pp. 447–449. Yadav, R.N.S. and Munin, A. (2011). Phytochemical analysis of some medicinal plants. Journal of Phytology, 3(12): 10-14 ISSN: 2075-6240. 125

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EFFECT OF IMAZAMOX USAGE ON PHYTOHORMONE LEVELS IN SUNFLOWER (HELIANTHUS ANNUUS L.) CULTIVATION Hayati ARDA*1, Gülçin ALYÜRÜK1 1

Department of Biology, Faculty of Sciences, Trakya University, 22100, Edirne, Turkey *Corresponding author’s e-mail: hayatiarda@hotmail.com ABSTRACT

In this study, imazamox, an imidazolinone (IMI) herbicide commonly used in sunflower agriculture, and sunflower cultivars thought to be resistant to this herbicide group were used. The effects of different concentrations of herbicide applications on phytohormone levels of sunflower cultivars were investigated. In the study, four different types of sunflower were used as normal group (unresisting to IMI) SN:8 and resistant groups to IMI SN:9, SN:10 and SN:14. Three different concentrations (3.125 ml, 6.25 ml and 9.375 ml) of herbicides were applied to sunflower seedlings which reached to 4-6 leaf phase. Leaf samples were taken 7 days after the herbicide application and hormone analyzes were performed. When the results were evaluated, the highest IAA (23.62 ppb) was observed in 6.25 ml herbicide application in SN: 8 cultivar and the lowest IAA (3.49 ppb) was observed in 6.25 ml herbicide application in SN: 14 cultivar. The highest value for GA3 (19.62 ppb) SN: 8 cultivar 3.125 ml herbicide application, the lowest value (4.75 ppb) SN: 14 was obtained in the control group. The highest value in ABA (42.43 ppb) SN: 8 cultivars of 6.25 ml herbicide application, the lowest value (12.96 ppb) was found in the control group of SN: 10. The highest JA value (174.02 ppb) was obtained in the control group of SN: 9 and the lowest value (15.53 ppb) was obtained in 3.125 ml herbicide application of SN: 8. In terms of SA amount, the highest value (176.13 ppb) was observed in 9.375 ml herbicide application of SN: 9 cultivar and the lowest value (47.59 ppb) was observed in 3.125 ml herbicide application of SN: 9 cultivar. Significant differences were obtained in phytohormone levels of sunflower cultivars treated with different concentrations of herbicides. Keywords: Helianthus annuus L., Imazamox, Phytohormone, Stress

INTRODUCTION Sunflower (Helianthus annuus L.) is an important cultivation plant that is cultivated in many countries of the world and contains high quality oil in its seeds. (Kaya, 2004). One of the most important production problems seen in agricultural fields is weeds. (Cosge and Ulukan, 2005). Weeds competing with sunflower in terms of nutrients, water and light, prevent sunflower growth and lead to loss of yield and quality (Pfenning et al., 2008; Balabanova, 2016). This problem was genetically solved by using Imidazolinone (IMI) herbicide resistant sunflower varieties and the introduction of IMI herbicides. (Kaya, 2015). Imidazolinones are an effective group of herbicides acting on the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS), involved in the biosynthesis of branched chain amino acids (valine, leucine and isoleucine). It inhibits AHAS enzyme in weeds, stops growth and causes the death of the plant, keeping the weeds under control (Duggleby, 2000; Tan et al., 2005; Brecciaa et al., 2013). It is advantageous because of its widespread use in sunflower agriculture, its economy, easy application and quick results. However, depending on the frequency of use of herbicides, it can cause various 126

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environmental and human health problems such as increased environmental pollution, negative effects on non-target organisms, residual problems on soil, water and foodstuff (Galon et al., 2014; Ă&#x2021;ebi et al., 2019). Overdose and misapplication may reveal some undesirable anatomical and physiological damages in sunflower plants and this leads to a decrease of performance in production. In this study, the effects of resistant and non-resistant sunflower cultivars on phytohormone levels were investigated by applying different concentrations of imazamox on the imidazolinone group herbicides. MATERIAL AND METHOD Supplying the Materials The sunflower (Helianthus annuus L.) seeds used in the study were obtained from Trakya Agricultural Research Institute. Four different types of sunflower were used. IMI group not resistant to herbicides SN: 8. The cultivars considered to be IMI resistant to the herbicide are SN: 9, SN: 10 and SN: 14. Formation of Experimental Groups Experimental groups were formed in which sunflower cultivars would be applied herbicides as control, 1 dose (3.125 ml / l), 2 doses (6.25 ml / l), 3 doses (9.375 ml / l). In this application, it was adapted to the study based on the dose used in agriculture (Agricultural use: 125 ml/da). The seeds were planted in the botanical garden and let out to grow. Seedlings coming to 4-6 leaf stage were applied herbicides. Hormone analysis was performed by using LC MS / MS device from the leaf samples taken 7 days after herbicide application. Hormone Analysis with LC MS / MS 1 gram of leaf sample was taken from each experimental group for hormone analysis. The leaf samples were placed in 50 ml falcon tubes and homogenized with 10 ml 50% methanol and 50% isopropyl alcohol in the cold environment with the help of homogenizer. Homogenized leaf samples were taken from falcon tubes to eppendorf tubes and centrifuged at 14000 rpm at 4oC for 10 minutes. The centrifuged samples were stored at -20 Â° C. Then, the supernatants were passed through PTFE syringe filters having a pore diameter of 0.22 UPm, and diluted in phase A with UPW 2 millimolar ammonium formate and 2 millimolar formic acid. After dilution process, samples were placed on LC MS / MS instrument and analyzed. Statistical Analysis SPSS 17 statistical program was used to determine the relationships between the data obtained as a result of hormone analysis. RESULTS Significant changes in phytohormone levels were observed as a result of herbicide applications at different concentrations of sunflower varieties (Table 1). When the average of indole-3-acetic acid (IAA) of the control and experimental groups belonging to SN: 8, SN: 9, SN: 10 and SN: 14 varieties was examined, the highest value observed in the SN: 8 experimental group was 23.62 ppb in which 2 doses of herbicides was applied and the lowest value observed was 3.49 ppb in the experimental group SN: 14 in which 2 doses of herbicide was applied. When the mean of IAA in control and different herbicide applications of SN: 8 cultivars were examined, an increase was observed in all groups compared to the control group. The highest amount of IAA (23.62 ppb) was observed in 2 doses of herbicide application. In SN: 9, an increase was observed in 1 and 2 doses of herbicide 127

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application and a decrease was observed in 3 doses when compared to control. In SN: 10 cultivars, IAA amount decreased in 1 and 3 dose herbicide application and increased in 2 doses. In different herbicide applications within SN: 14 cultivars, the highest amount of IAA (9.28 ppb) was observed in the control group. IAA levels decreased in 1, 2 and 3 dose herbicide applications compared to control. When gibberellic acid (GA) amounts of sunflower cultivars were compared, the highest value (19.62 ppb) was observed in SN: 8 cultivars in 1 dose herbicide application and the lowest value (4.75 ppb) was observed in the control group of SN: 14 cultivars. An increase was determined in GA amounts of SN: 8 compared to control. The highest value (19.62 ppb) was measured in 1 dose of herbicide application. When compared to control group, a decrease was observed in SN: 9 cultivars in 1 dose herbicide application and an increase was observed in GA levels in 2 and 3 dose herbicide applications. In SN: 10, the highest amount of GA (15.00 ppb) was in 2 dose herbicide application. In plants treated with 1 dose of herbicide, a decrease was observed in the amount of GA compared to control, whereas in plants treated with 3 doses of herbicide, some increase was observed. GA of SN: 14 showed a steady increase in all herbicide treated groups compared to the control group. When the amount of absicic acid (ABA) was examined, the highest value (42.43 ppb) was observed in the experimental group of SN: 8 where 2 doses of herbicides were applied and the lowest value (12,96 ppb) was observed in the control group of SN: 10. In SN: 8 cultivars, the highest ABA amount (42,43 ppb) was detected in 2 doses of herbicide application and the lowest amount (14,92 ppb) was detected in 1 dose of herbicide application. In SN: 9, a decrease was observed in ABA amounts of all herbicide treated experimental groups compared to the control group. The highest amount of ABA (35.40 ppb) in SN: 10 cultivars was in 2 dose herbicide application whereas the lowest amount of ABA (12.96 ppb) was in the control group. The ABA amounts of SN: 14 decreased in all herbicide applications compared to the control. When the amount of jasmonic acid (JA) was examined, the highest value (174.02 ppb) was observed in the control group of SN: 9 and the lowest value (15.53 ppb) was observed in the experimental group where 1 dose of SN: 8 was applied. In SN: 8 cultivars, the highest JA amount (59.74 ppb) was in the control group and the lowest JA (15.53 ppb) was in 1 dose of herbicide application. A decrease was detected in JA amounts of SN: 9 cultivar compared to control group. SN: 10 cultivar showed an increase in 1 dose herbicide application and a decrease in 2 and 3 dose herbicide applications compared to control. Among the control and different herbicide applications of SN: 14, the highest JA (107,69 ppb) was detected in the control group. In the groups treated with herbicides, the amount of JA decreased compared to the control group. When the amount of salicylic acid (SA) was examined in sunflower cultivars, the highest value (176.13 ppb) was observed in the experimental group of SN: 9 where 3 doses of herbicides were applied; the lowest value (47,59 ppb) was observed in the experimental group of SN: 9 where 1 dose of herbicides was applied. In the SN: 8 cultivars, the amount of SA decreased in all herbicide treated groups compared to the control. In SN: 9, the highest SA (176,13 ppb) was in 3 doses of herbicide application and the lowest SA (47,59 ppb) was in 1 dose of herbicide application. In SN: 10 cultivar, a decrease was observed in all experimental groups in which herbicide was applied, when compared to the control group. In SN: 14, the highest SA amount (99,52 ppb) was in the experimental group where 1 dose of herbicide was applied and the lowest SA (59,18 ppb) was detected in 2 doses of herbicide application.

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Table 1. Variance table (ppb) Sunflower Cultivars

Control (0)

Imazamox Applications 3.125 ml 6.25 ml

SN8 SN9 SN10 SN14

5.83a 14.40a 8.99a 9.28a

17.16b 15.28a 6.86b 4.01b

SN8 SN9 SN10 SN14

7.06a 8.37a 6.94a 4.75a

19.62b 5.84a 5.32a 10.02a

SN8 SN9 SN10 SN14

31.84a 41.78a 12.96a 25.57a

14.92b 24.78b 13.77a 18.31a

SN8 SN9 SN10 SN14

59.74 174.02a 33.69a 107.69a

15.53 80.21b 45.16c 68.45b

SN8 SN9 SN10 SN14

9.375 ml

IAA 23.62b 18.01a 11.76c 3.49b

17.18b 8.14a 6.38b 3.52b

14.60b 9.51a 15.00b 11.11a

16.41b 11.00a 8.38a 13.82a

42.43c 28.38bc 35.40b 15.89a

32.73a 20.58bd 31.07b 17.12a

57.81a 92.77c 29.61ab 42.91c

16.37b 49.53d 30.79a 21.09d

101.56c 77.67a 140.34a 59.18c

154.99a 176.13c 108.85d 60.05c

ABA

SA 165.15 91.05a 143.49a 79.26a

88.36 47.59b 93.08c 99.52b

Different letters on the same line indicate that the difference between the applied herbicide doses is statistically significant. (p < 0.05).

Corelations Pearson Correlation Analysis was used to determine whether there was statistically significant relationship between the data obtained from the results of the research (p <0.05 or p <0.01). The determined statistical relations and correlation coefficients are given in Table 2. Table 2. Relationships and correlation coefficients between hormones in different dose herbicide applications n =16 IAA GA ABA JA SA

IAA 1 0.384 0.547* 0.177 -0.025

ABA

1 0.102 -0.512* 0.129

1 0.429 0.196

1 -0.271

: correlation is significant at 0.05 (p <0.05); n: number of samples

According to the results of Pearson Correlation Analysis applied by taking into account the data (n = 16); there were significant correlations as of 0.05 between the levels of stress hormones in plants, which are IAA and ABA as positive, and between GA and JA as negative (p <0.05). 129

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Factor Analysis By using correlated data, Factor Analysis was used to determine the factors affecting hormonal differences which were detected in our study. Data that are not correlated or have a low correlation coefficient were not evaluated in order to increase the reliability of Factor Analysis. A total of four hormonal variable data were used to determine the effective factors. The obtained KMO (Kaiser-Meyer-Olkin) Sample Adequacy Test results show that the sample size is adequate and appropriate (0.479). Eigenvalues greater than one were chosen as the criterion for the evaluation of the main components as sources of variance to be explained from the data used and they were described in Scree Plot Figure 1. where the eigenvalues of the principal components are expressed.

Figure 1. Scree Plot Percentage variance values, cumulative percentage variance values and component loads (before and after rotation) are given in Table 3. According to the percentage variances after rotation, two factors account for 83% of the total variance. Table 3. Total variances explained Total Variance Explained

Initial Eigenvalues Component 1 2 3 4

Extraction Sums of Squared Loadings

Rotation Sums of Squared Loadings

% of Cumulative % of Cumulative % of Cumulative Total Variance % Total Variance % Total Variance % 1.791 44.785 44.785 1.791 44.785 44.785 1.781 44.520 44.520 1.563 39.077 83.862 1.563 39.077 83.862 1.574 39.342 83.862 0.390 9.738 93.600 0.256 6.400 100.000

Parameter loads greater than 0.5 (component matrix) determined for the three factors before and after rotation are given in Table 4. The two-dimensional component diagram represents the associated variables of the two factors and is given in Figure 2.

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Tablo 4. Component Matrix

ABA IAA GA JA

Component Matrixa Component 1 2 0.888 0.808 0.575

IAA ABA 0.932 GA -0.738 JA

Rotated Component Matrixa Component 1 2 0.873 0.864 0.881 -0.844

The first factor explains 44.5% of the total variance and this factor is composed of IAA ABA hormones. Both parameters are strongly positively charged for this factor (> 0.75). The second factor explains 39.3% of the total variance and this factor is composed of GA and JA hormones. The GA parameter is strongly positively charged (> 0.75) for this factor and the JA parameter is strongly negatively charged (> 0.75) for this factor.

Figure 2. Component diagram DISCUSSION Phytohormones are organic molecules which are responsible for regulating the growth and development of plants (Peleg and Blumwald, 2011). It is known that phytohormone levels change rapidly in plants that are exposed to biotic and abiotic stress factors. (YĂźrekli et al., 2004; Wang et al., 2005; Ashraf et al., 2008; Iqbalet et al., 2014). Herbicides are an important source of abiotic stress for plants (KosovĂĄ et al., 2011). Studies have reported changes in endogenous IAA, ABA, SA, JA and GA levels in plants under herbicide stress. (Doganlar, 2012; Kaya and Doganlar, 2016). In this study, the effects of imazamox herbicide on phytohormone levels for both resistant and non-resistant sunflower cultivars were investigated. Our results showed that endogenous phytohormone levels increased and decreased in imazamox treated groups compared to control groups. IAA is a phytohormone responsible for the growth and development of plants under both normal and stress conditions (Kazan, 2013). It plays a fundamental role in adaptation of plants to stress conditions. Increase in IAA levels is observed under stress conditions. (Fahad et al., 2015). As a result of the study, an increase was observed in the amount of IAA in SN: 8 131

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cultivars which were applied 1 and 2 doses of the herbicide when compared to the control group, whereas a decrease was seen in 3-dose herbicide application. In SN: 9 cultivar, an increase was observed with 1 and 2 doses of herbicide application and a decrease in 3 doses. In SN: 10 cultivar, a decrease with 1 and 3 doses of herbicide application was observed while an increase was seen with 2 doses. We have the opinion that, in SN: 14, the decrease in the amount of hormones depending on the increase in the dose of the drug may be caused by the stress as a result of the high drug dose that may reduce the physiological activities of the plant and the plant may be switching to dormancy. It can be understood from 1 and 2 doses of herbicide applications of SN: 8 and SN: 9 cultivars, the plants are not adversely affected and their growth and development continue. In the SN: 10 cultivar, the change in the amount of IAA in herbicide doses is inconsistent. Because, while 1 and 3 doses of herbicide application shows a decrease in the development of plants, there is a hormonal change in the direction of increase in plant growth with 2 doses of herbicide application. The highest GA level in SN: 8 cultivar was observed in 1 dose herbicide application and less in 2 and 3 dose herbicide application. In this case, it can be thought that plant growth is not adversely affected in 1 dose herbicide application and it may be negatively affected in 2 and 3 doses. We believe that development may be slow in the control group. In SN: 9, the lowest GA level was observed in 1 dose herbicide application. An increase in 2 and 3 doses was observed. In SN: 10 cultivar, the lowest GA level was observed in 1 dose herbicide application. The highest GA level was observed in 2 doses, with a slight increase in 3 doses. The results that were obtained were not consistent with other groups. A steady increase in GA levels was observed with dose increase in SN: 14 cultivar. This increase is an indication that this kind of plant is not affected by high drug dose applications. ABA is another hormone involved in regulating plant growth and development. ABA is the most studied hormone in biotic and abiotic stress studies because it is called as stress hormone. It regulates stomatal activity, dormancy and other plant activities under different stress conditions (Kirecci and Yurekli, 2018). ABA increase is observed as a common feature in almost all stress conditions. Many researchers have reported that ABA accumulation is observed in plants growing under different stress conditions and that there may be some changes in plant physiological events and behavior (Li et al., 2010; Wani et al., 2016). When ABA values were examined in different types and different dose applications, a decrease in SN: 8 cultivar with 1 dose herbicide application and an increase with 2 and 3 dose herbicide applications were observed. SN: 9 cultivar demonstrated a steady decrease in ABA amount. SN: 10 showed a steady increase in the amount of ABA. SN: 14 cultivar was observed to decrease regularly. When the ABA levels were evaluated, it was thought that SN: 9 and SN: 14 were more resistant to herbicides. It was concluded that SN: 10 was stressed due to high herbicidal applications due to ABA content. JA is a signaling molecule that plays a role in response to biotic and abiotic stresses (Wasternack, 2013). It is known that the amount of endogenous JA increases in plants that were exposed to various stress conditions. (Shan and Liang, 2010; Bankaji et al., 2014). In our study, when the amount of JA in different varieties and different herbicide applications were evaluated, a decrease in JA levels was detected in all groups. SA is a hormone that plays an important role in plant defense responses to local pathogen attack and systemic acquired resistance (Alverez, 2000). It is a plant signaling molecule that plays an important role in the formation of defense responses against abiotic and biotic stress conditions (Cameron, 2000). When the amount of SA was examined at the end of the study, SN: 8 cultivar decreased in all herbicide treated groups compared to the control. In SN: 9 cultivar, SA amount decreased in 1 dose herbicide application and increased in 2 and 3 132

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dose herbicide applications. In SN:10, no regular increase or decrease was detected due to dose increase. In the SN:14 cultivar, the amount of SA showed a significant decrease with increased herbicide applications. It was concluded that this plant cultivar was not adversely affected by the increase in herbicide dose. There were statistically significant differences and results in the change of stress hormones between the experimental groups depending on the herbicide dose changes and the plant varieties used. When the hormonal change values due to dose increase were examined statistically, similarities were observed in the increases and decreases of the IAA and ABA hormones, and the opposite changes were observed between GA and JA. CONCLUSIONS According to the data obtained in the study, it was determined that imazamox applications at different concentrations affect phytohormone levels in sunflower. It was observed that the changes in phytohormones with different doses of imazamox between the experimental groups would not be sufficient as a parameter which can be used alone to determine the resistance of the investigated cultivars to the herbicide. It is thought to be useful if used in conjunction with anatomical, physiological and genetic investigations. Acknowledgements: This study was supported by Trakya University Scientific Research Projects Coordination Unit. Project Number: 2014/13. REFERENCES Alverez, A. L. (2000). Salicylic acid in machinery of hypersensitive cell death and disease resistance. Plant Molecular Biology, 44, 429-442. Ashraf, M., Athar, H. R., Harris, P. J., & Kwon, T. R. (2008). Some prospective strategies for improving crop salt tolerance. Advances in Agronomy, 97, 45-110. Balabanova, D. A. (2016). Photosynthetic performance of the imidazolinone resistant sunflower exposed to single and combined treatment by the herbicide imazamox and an amino acid extract. Frontiers in Plant Science, 7, 1559. Bankaji, I., Sleimi , N., Lo´pez-Climent, M. F., Perez-Clemente, R. M., & Gomez-Cadenas, V. (2014). Effects of combined abiotic stresses on growth, trace element accumulation, and phytohormone regulation in two halophytic species. Journal of Plant Growth Regulation, 33,632-643. Brecciaa, G., Vega , T., Felitti, S. A., Picardi, L., & Nestares, G. (2013). Differential expression of acetohydroxyacid synthase genes in sunflower plantlets and its response to imazapyr herbicide. Plant Science, 208, 28–33. Cameron, R. K. (2000). Salicylic acid and its role in plant defense responses: what do we really know? Physiological and Molecular Plant Pathology, 56, 91– 93. Cosge, B., & Ulukan, H. (2005). Cultivar and sowing date in our sunflower (Helianthus annuus L.) cultivation. Süleyman Demirel University Journal of Natural and Applied Sciences, 9-3, 43-48. Cebi, U., Ozcan, C., Gurbuz, M. A., & Ozer, S. (2019). Residual levels of imazamox herbicide an derivatives in different parts of sunflower plant and honey samples produced under the effect of sunflower flora. Derim, 36(1), 99-107. Doganlar, Z. B. (2012). Physiological and genetic responses to pesticide mixture treatment of Veronica beccabunga. Water, Air Soil Pollution, 223, 6201-6212. Duggleby , R. P. (2000). Acetohydroxyacid synthase. Journal of Biochemistry and Molecular Biology, 33, 1–36. Fahad, S., Hussain, s., Bano, A., Saud, S., Hassan, S., Shan, D., Khan, F. A., Khan, F., Chen, Y., Wu, C., Tabassum, M. A., Chun, M. X., Afzal, M., Jan, A., Jan, M. T., Huang, 133

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J. (2015). Potential role of phytohormones and plant growth-promoting rhizobacteria in abiotic stresses: consequences for changing environment. Environmental Science and Pollution Research, 22, 4907-4921. Galon, L., Lıma, A. M., Guımarães, S., Belarmıno, J. G., Burg, G. M., Concenço, G., Bastıanı, M. O., Beutler, A. N., Zandona, R. R., and Radünz, A. L. (2014). Potential of plant species for bioremediation of soils applied with imidazolinone herbicides. Planta Daninha, 32, 719. Iqbal, N., Umar, S., Khanb, N. A., Iqbal, M., & Khanb, R. (2014). A new perspective of phytohormones in salinity tolerance: regulation of proline metabolism. Environmental and Experimental Botany, 100, 34–42. Kaya, A., & Doganlar, Z. B. (2016). Exogenous jasmonic acid induces stress tolerance in tobacco (Nicotiana tabacum) exposed to imazapic. Ecotoxicology and Environmental Safety, 24, 470–479. Kaya, Y. (2004). The last progresses in sunflower biotechnology and the possible usages in sunflower breeding . Trakya University Journal of Natural Sciences, 5(2), 141-147. Kaya, Y. (2015). Herbıcıde resıstance breedıng ın sunflower, current sıtuatıon and future dırectıons. Journal of ASM. Life Sciences, 2(326). Kazan, K. (2013). Auxin and the integration of environmental signals into plant root development. Annals of Botany, 112, 1655–1665. Kirecci, O. A., & Yurekli, F. (2018). The effect of salts stress, some plant sormones and SNP spplications on signal molecules in Helianthus annuus L. leaves. KSU Journal of Agriculture and Nature, 21(5), 665-671. Kosová, K., Vítámvás, P., Prášil, I. T., & Renaut, J. (2011). Plant proteome changes under abiotic stress contribution of proteomics studies to understanding plant stress response. Journal of Proteomics, 74, 1301–1322. Li, X. J., Yang, M. F., Chen, H., Qu, L. Q., Chen , F., & Shen , S. H. (2010). Abscisic acid pretreatment enhances salt tolerance of rice seedlings: proteomic evidence. Biochimica et Biophysica Acta - Proteins and Proteomics, 1804, 929–940. Peleg, Z., & Blumwald, E. (2011). Hormone balance and abiotic stress tolerance in crop plants. Current Opinion in Plant Biology, 14, 290–295 . Pfenning, M., Palfay, G., & Guillet , T. (2008). The CLEARFIELD® technology – A new broadspectrum post-emergence weed control system for European sunflower growers. Journal of Plant Diseases and Protection, Special Issue. Shan, C., & Liang, Z. (2010). Jasmonic acid regulates ascorbate and glutathione metabolism in Agropyron cristatum leaves under water stres. Plant Science, 178, 130–139. Tan, S., Evans, R. R., Dahmer, M. L., & Singh, B. (2005). Imidazolinone-tolerant crops: history, current status and future. Pest Management Science, 61, 246–257 . Wang, X., Li, X., Meisenhelder, J., Hunter, T., Yoshida, S., Asami, T., & Chory, J. (2005). Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Developmental Cell, 8, 855–865. Wani, S. H., Kumar, V., Shriram, V., & Sah, S. K. (2016). Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants. The Crop Journal, 4, 162176. Wasternack, W. (2013). Action of jasmonates in plant stress responses and development applied aspects. Biotechnology Advances, 32 (1), 31–39. Yurekli, F., Porgalı, Z. B., & Turkan, I. (2004). Variations in absisic acid, indol-3-asetic acid, giberellic acid and zeatin concentrations in two ban species subjected to salt stress. Acta Biologica Cracoviensia Series Botanica, 46, 201–212.

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OBSERVATIONS FOR THE DETERMINATION OF GERMINATION RATE OF JUNIPERUS EXCELSA SUBSP. POLYCARPOS (TURKESTAN JUNIPER) SEEDS Elshad GURBANOV 1, Afag RZAEVA*2 1 2

Department of Biology, Baku State University, Azerbaijan

Department of Chemistry- Biology, Baku Engineering University, Azerbaijan *Corresponding author’s e-mail: afrzayeva@beu.edu.az ABSTRACT

Although juniper has been grown up mainly by shootings, the question of its seed propagation was always in the spotlight. The article is devoted to the improvement of seed propagation methods of juniper plant. Currently, juniper seeding is not considered effective for very low percentage of germination and this fact is explained by the thick and solid seed bark of this plant. In presented article we have proposed a model of complex stratification and scarification for increasing germination rate of juniper seed. Compared to other propagation methods, this method is not very effective but is very important for providing genetic richness. Keywords: Seed propagation, Germination rate, Juniperus, Stratification, Scarification

INTRODUCTION For ensuring the growth of juniper seeds, it is essential to study the quality of seeds first of all. For this purpose, seeds are stored in distilled water using the method proposed by Shelag and Mc Cartan [3]. In the research carried out by us, 100 seeds collected from the trees in the Dendrology Institute in Absheron peninsula and 100 seeds collected from the area of Turianchai State Reserve. All seeds were placed in distilled water and the results were compared. (Table 1.) As can be seen from the table, 19% of the seeds collected from the Dendrology Institute, 25% of the seeds collected from the Turianchay territory have normal endosperm. Seeds without endosperm have always been among the juniper trees. The reason is explained with distortion of pollination (Shelag A. McCartan and Peter G. Gosling 2013). At the table above, we reaffirm this idea once again. Thus, there are more favorable conditions for the pollination of the wind-pollinated juniper in the Turianchay area, and therefore, most of the seeds gathered near the Turianchay are vitabil. MATERIALS AND METHODS We have developed stratification methods proposed by Mulualem Tigabu (Mulualem Tigabu, Joakim Fjellström, Per Christer Odén, Demel Teketay, 2007) and the germination dynamics of juniper seeds were observed using 3D stereomicroscopes. In the course of monitoring of juniper seedlings, the usual agar was used as culture media, and the research, including the application of the stratification and scarification models was performed at the Biological Laboratory of Baku Engineering University. Seeds of Juniperus polycarpos K.Koch were collected from the territory of the Institute of Dendrology of Azerbaijan National Academy of Sciences for studying seed germination 135

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percentage. Seed collection and scarification-stratification measures were carried out in October-March 2016-2017. RESULTS AND DISCUSSION Since juniper seeds are very thick, naturally it is difficult to germinate them. In the nature, juniper seeds are subjected to natural scarring while crossing the digestive tract of animals fed with these pots. We used a graft paper with 0.3 to 0.5 mm teeth to cover the shell of the seeds. After the peeling of the seeds the second stage - stratification begins. Stratification occurs naturally when the juniper seeds pass on the winter season in the mountainous valleys. We kept the seeds in plastic bags in sand and damp soil for a period of 90 days at low temperatures (2-3 ° C). The soil we used was clay podzol type and its moisture was kept under control. The seeds were placed in small-sized Petri dishes (53 mm circle) in agar nutrient to germinate after scarification. Stimulants or mixtures have not been added to medium (Figure2). Petri safes were kept at room temperature at the Biological Laboratory of Baku Engineering University. The first signs of germination were recorded 14 days after seeds were placed in petri dishes. On the 28th day, some of the seedlings have already begun to observe leaves. CONCLUSION As can be seen from the table, germination was observed only in Juniperus polycarpos K.Koch seeds, which were exposed to scarification and stratification processes. Of the 100 seeds exposed to 90-day scarification and stratification 8 (8%) have been germinated, of the 100 seeds, that exposed to stratification only 2 have sprouted (2%). No germination has been recorded in the seeds without any scarification and stratification. After slicing the upper shell, the seeds that are exposed to low temperature stratification also have a higher rate of germination. This can be easily observed from the table. In addition to the stratification and scarification measures, the development dynamics of seeds that are only exposed to low temperature stratification have been presented as diagrammatically REFERENCES Gurbanov E. M., Mamedova Z. C and Rzaeva A. A. Juniperus polycarpos K. Koch (Turkestan Juniper) Species in Turianchai Preserve (Azerbaijan Republic)// Journal of Food Science and Engineering 7 (2017) 458-460 Mulualem Tigabu, Joakim Fjellström, Per Christer Odén, Demel Teketay. Germination of Juniperus procera seeds in response to stratification and smoke treatments, anddetection of insect-damaged seeds with VIS + NIR spectroscopy// New forests, March 2007, Volume 33, Issue 2, pp 155–169 Shelagh A. , McCartan and Peter G. Gosling. //Guidelines for Seed Collection and Stratification of Common Juniper (Juniperus communis L. L.) // Tree Planters’ Notes, Volume 56, No. 1 (2013), page 24-29. Thomas, P. A., El Barghathi, M., and Palwart, A. 2007. Biological Flora of the British Isles: Juniperus communis L. L// J. Ecol. 95: 1404-40

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A RESEARCH ON ELECTRONIC PROPERTIES OF TEMPERATURE SENSORS USED IN ENGINEERING Aydın GÜLLÜ*1, M. Ozan AKI1, Hilmi KUŞÇU,2 1

Trakya University Ipsala Vocational School Electronics and Automations Department 22400 Ipsala, Edirne, Turkey 2

Trakya University Engineering Faculty Mechanical Engineering Department Edirne, Turkey *Corresponding author’s e-mail: aydingullu@trakya.edu.tr

ABSTRACT Temperature is one of the most followed parameters in engineering. It needs to be monitored in many control processes. Some areas where temperature information is used; Food processing, heating and cooling systems, healthcare and production processes. For this purpose, many different methods have been developed to measure the temperature. In this study, the sensors used for temperature measurement will be examined. Parameters such as measuring the working conditions of these sensors, sensitivity of response times will be discussed. Some of the classical and commonly used thermistors (NTC, PTC), RTDs (PT100), thermocouples and semiconductor LM35, DS18B20, and other electronic chips produced in recent years will be discussed and their different aspects will be emphasized. In this way, information about the choice of temperature perceptions that can be used in academic or commercial applications will be presented. Keywords: Temperature Sensors, PTC, NTC, IC Sensor, Thermocouple

INTRODUCTION Temperature is a measure of the temperature or coldness of an object. It is a measure of a molecular kinetic energy. The change of this kinetic energy causes a temperature change in the system. It is necessary to know the temperature value for many scientific events(Childs vd., 2000). It is necessary to measure the temperature directly or indirectly in many fields such as health, chemistry, food, agriculture, engineering. In some processes, the system should be controlled and monitored according to the critical temperature value. Many sensors have been developed to measure temperature information, which is widely used. There are temperature sensors developed for different purposes and temperature range. The temperature is often measured indirectly. The change in the value of the material under the influence of temperature indicates the temperature value. Liquid glass thermometers that we use in our daily life are examples of this. The temperature change creates an expansion of the liquid, which changes the height of the liquid in the graduated vessel and reads the temperature on the preset temperature scale. Bimetal thermometers work similarly. Elongation or shortening of metals with different strain coefficients at temperature are different. The temperature information is read by extending towards each other. However, it is not possible to measure the temperature accurately and to use the measured temperature information in the controller.

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Sensor and controller are required for temperature control. the information obtained by the sensor must be understood by the controller. this is mostly done with electronic circuits. the temperature is detected by the controller and the outputs are controlled. In this study, temperature sensors that can be used for temperature control will be given. Measurement principles, accuracy, operating temperatures and cost information of these sensors will be presented. first, thermistors will be mentioned. then resistance temperature detectors and thermocouples will be described. Next, electronic temperature sensors will be mentioned. The temperature sensor DS1820, STH11 and DHT11 will be examined, followed by a temperature sensor with analog output such as LM35. Finally, examples from other and various sensors will be given. In the conclusion and discussion, the comparison of these sensors to each other will be discussed. Temperature Sensors Thermistors Thermistors are commonly used temperature measurement sensors. The resistance value of the thermistors changes with temperature change. The temperature value is detected. There are two types as positive temperature coefficient (PTC) and negative temperature coefficient (NTC). shows a non-linear resistance value versus a linear temperature change.(Steinhart ve Hart, 1968) The thermistor curve is shown in figure 1. Resistance temperature tables are published by the manufacturers. Users find the temperature value corresponding to the resistance they read by looking at these values. NTC is a thermistor whose resistance decreases with increasing temperature. In PTC, the resistance value increases as the temperature increases. The changes in both thermistors are non-linear. NTC is more preferred in industrial applications.(Feteira, 2009) Cheaper than other temperature sensors. An uncoated NTC is approximately $ 0.08.(Direnc, 2019).

Figure 1. Resistance temperature curve for NTC and PTC They can measure from -55℃ to 150℃. Accuracy value for Standard Sensor: ± 0.2 ℃ (0℃ to 70 ℃). Thermistor values are classified and sold as resistors. 10kΩ NTC means 25kΩ 138

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resistance value 10k. Some manufacturers produce more sensitive thermistors. Figure 2 shows NTC and PTC. Where the sensitivity is ± 0.1 ℃ (0℃ to 70 ℃). It can be produced with protective sheath and ready cable according to the usage areas. It is not possible to connect the thermistors directly to the controller. Additional electronic circuit should be made according to the measurement temperature and sensitivity. Temperature measurement can be made in the desired range with the resistance measurement circuit.(Becker vd., 1946)

Figure 2. Thermistors (NTC and PTC) Resistance Temperature Detectors (RTD) Another sensor such as thermistors is RTD whose resistance varies with temperature change. In addition to the non-linear variation of thermistors, RTD shows a more linear variation. High reproducibility and precision. Temperature can be calculated with a formula from the measured resistance value. RTDs are mostly made of platinum, nickel and copper. Operating temperatures are between -200℃ and 500℃ on average. they are produced from different materials for different temperature values. Temperature resistance tables are published by the manufacturers. The most widely used type is platinum PT100 (J. Liu vd., 2010). The PT100 resistance temperature curve is shown in figure 3. Others RTDs are produced from different metals such as PT1000, Ni100 and different values. Production is made in special cases according to the place of use. It is a widely used sensor due to its high accuracy, low slip value and wide temperature range. The price is about $ 1.15. Price may vary depending on cable length and sheath type. It needs an additional electronic circuit to be used in the controller as in thermistors.

Figure 3. PT100 resistance temperature curve A formula for resistance temperature variation at 0℃-100℃ was determined. in this formula, the intermediate values can be calculated with the coefficient to be obtained. A formula 139

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(1) for resistance temperature variation at 0â&#x201E;&#x192;-100â&#x201E;&#x192; degrees was determined. In this formula, the intermediate values can be calculated with the coefficient to be obtained. The alpha of the standard platinum pt100 is 0.00385. đ?&#x2018;&#x2026; â&#x2C6;&#x2019;đ?&#x2018;&#x2026; đ?&#x203A;ź = 100 0 (1) 100 Â°đ??ś đ?&#x2018;&#x2026;0 đ?&#x2018;&#x2026;0 = Resistance at 0â&#x201E;&#x192; đ?&#x2018;&#x2026;0 = Resistance at 100â&#x201E;&#x192; PT100 shows 100 â&#x201E;Ś resistance at 0â&#x201E;&#x192;. Increases in proportion to alpha for each degree.(Honggang ve Applications, 2011; Lang vd., 2009; Quilty vd., 2007) Thermocouple Thermocouple is a transducer that generate electricity by temperature change. It does not need an external energy source. The structure is produced by combining two different metals. It causes electrical interaction between metals with temperature change. There is a potential difference between the electron flow and the metals. A different voltage value occurs at the cable terminals at different temperatures. There is a relationship between voltage change and temperature change. Temperature measurement is made using this. It is used in many places because of its simple structure. They can measure up to the melting temperature of metals at the measuring point. This allows measurement of very high temperatures. Type T can measure between -200â&#x201E;&#x192; and 350â&#x201E;&#x192;, while type B can measure between +200â&#x201E;&#x192; and 1700â&#x201E;&#x192;. They can generally measure from -200â&#x201E;&#x192; to + 2350â&#x201E;&#x192;.(Elimko, 2019)

Figure 4. Voltage Temperature curve of the Thermocouples The voltage generated in the thermocouples during the temperature change is in millivolts. The voltage temperature curve of the thermocouples is shown in the figure 4. This 140

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value must be increased to be processed by the controller. An additional electronic circuit is required for other sensors. It has two ends and the polarity must be observed in the connection. Cable colors from world standards are coded according to thermocouple type. The price of a type K thermocouple that can measure up to 400℃ is about $ 2.63.(Direnc, 2019) The integrated sensors have been manufactured for easy communication of these commonly used sensors with the controller. The max 6675℃ integrated is a digital SPI communication integrated for the K-type sensor and transmits information of 12-bit resolution. High temperatures can be easily measured if such and such devices are used.(Farrow vd., 1982; Stephenson, 1993) LM 35 The LM35 is the simplest of the integrated temperature measuring sensors. It has a 3-pin connection. The 2 pin is the energy pin. It has a wide supply between 4-20V. It can measure from -55 ℃ to 150 ℃. The output signal is analog. gives a linear output according to the temperature change. 10mV / C output for each degree centigrade. as such, it can be connected directly to a microcontroller. However, an additional electronic circuit is required for a precise output. The accuracy is ±0.2℃ at 25 ℃ s and ±0.3℃ at -10 ℃. Widely used for temperature measurement on the electronic circuit.(Instrument, 2019; Instruments, 1999; C. Liu vd., 2011; Silva vd., 2014) The LM35 can be used without microcontroller thanks to its analog output. The price is $ 1.9 (Direnc, 2019). The LM35 and pin connections are as shown on figure 5.

Figure 5. The LM35 and pin connections DS18B20 The DS18B20 is a digital integrated temperature sensor. The resolution can be set from 8 bits to 12 bits. It can measure temperatures between -10℃ and 85℃. A microcontroller is required for the measurement presentation. It sends temperature information via single wire serial communication(Maxim, 2019). With one wire communication, multiple sensors can be read at the same time. Multiple DS18B20 connections are shown in the figure6. It requires a power supply between 3v and 5.5v.(CHEN vd., 2008; Degang, 2006; Jun, 2010; Maxim, 2019; ZHAO ve XU, 2008) There are case options according to the place of use. price is around $ 1.02 (Direnc, 2019).

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Figure 6. Multiple DS18B20 Connections

Other Temperature Sensors In recent years, with the advances in electronic technology, different temperature sensors have been produced by different companies. These sensors are available in different configurations to meet specific requirements. Basically, the common feature is that it presents the temperature data digitally. SHT11 is one of them. The fully calibrated sensor is ready for use and measures both temperature and air humidity simultaneously. It can measure at temperatures between -40 ℃ and 120 ℃. sensitivity is ±0.3 ℃. The price is about $ 10. Response time is 5 sec. SHT11 is as shown on figure 7.

Figure 7. SHT11 Another is DHT11. It can also measure temperature and humidity. The price is $ 1.5. Temperature sensitivity is +2. The measuring range is between 0-50 degrees. Response time is 6 sec. SHT11 is as shown on figure 8.

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Figure 8. DHT11

The number of sensors built for various purposes is increasing day by day. The most common and used integrated temperature sensors are described above. RESULTS AND DISCUSSION Temperature is one of the most used properties. It is a necessary parameter to measure the temperature and check the measured value. It is critical in air conditioning, production and health sectors. A variety of sensors have been developed to measure this value. In this study, the oldest and most widely used temperature measurement sensors and digital sensors produced in recent years, are discussed. Information is given about the sensors that can be used in engineering applications. It is more suitable to use conventional thermistors, RTDs and thermocouples in sensitive and industrial applications. They are designed to work in industrial environments. If high temperatures are to be measured, thermal pairs should be preferred. At temperatures above 400 ℃, thermocouples are the most efficient sensors. The thermistors are suitable for use if the measurement is not very precise and the temperature is between 0-100 ℃. It is the most cost effective sensor. If sensitivity is important, RTDs should be preferred. direct measurement of these sensors is unlikely. Integrated temperature measurement sensors can be used if the application is a simple measurement or an academic study. In academic studies, temperature measurement can be done quickly rather than dealing with the circuit in order to obtain experimental data quickly with IC sensors. IC sensors can provide many other advantages. Multi-link serial communication, measurement without calibration are some of them. Digital sensors are also available at different prices depending on the location. REFERENCES Becker, J., Green, C., ve Pearson, G. J. E. E. (1946). Properties and uses of thermistors— Thermally sensitive resistors. 65(11), 711-725. CHEN, M., CHEN, M., ve QIU, C. J. M. E. T. (2008). Design and Realization of Digital Temperature Sensor Based on DS18B20 [J]. 8. Childs, P. R., Greenwood, J., ve Long, C. J. R. o. s. i. (2000). Review of temperature measurement. 71(8), 2959-2978. Degang, M. J. J. o. G. U. (2006). The Application of DS18B20 at the MCU System of Temperature Measure and Control [J]. 1. Direnc. (2019). Electronic Component Sales Web Site. Retrieved from https://www.direnc.net/ 143

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Elimko.

(2019).

Termokupllar

ilgili

genel

bilgiler.

Retrieved

from

http://www.elimko.com.tr/files/TermokupllarGenelBilgiler.pdf

Farrow, R. L., Mattern, P., ve Rahn, L. A. J. A. o. (1982). Comparison between CARS and corrected thermocouple temperature measurements in a diffusion flame. 21(17), 31193125. Feteira, A. J. J. o. t. A. C. S. (2009). Negative temperature coefficient resistance (NTCR) ceramic thermistors: an industrial perspective. 92(5), 967-983. Honggang, S. J. C. D., ve Applications. (2011). Application of PT100 Temperature Sensor to Real-time Temperature Monitoring System [J]. 4. Instrument, T. (2019). LM35 PrecisionCentigradeTemperatureSensors. Retrieved from LM35 PrecisionCentigradeTemperatureSensors Instruments, T. J. L. d., Aug. (1999). LM35 Precision Centigrade Temperature Sensors. Jun, Z. J. I. T. (2010). Smart temperature sensor DS18B20 and its application. 4, 68-70. Lang, Q., Zhang, Y., ve Xiu, W. (2009). Dynamic compensation of Pt100 temperature sensor in petroleum products testing based on a third order model. Paper presented at the 2009 International Workshop on Intelligent Systems and Applications. Liu, C., Ren, W., Zhang, B., ve Lv, C. (2011). The application of soil temperature measurement by LM35 temperature sensors. Paper presented at the Proceedings of 2011 International Conference on Electronic & Mechanical Engineering and Information Technology. Liu, J., Li, Y., ve Zhao, H. (2010). A temperature measurement system based on pt100. Paper presented at the 2010 International Conference on Electrical and Control Engineering. Maxim. (2019). DS18B20 Datasheet. Retrieved from https://datasheets.maximintegrated.com/en/ds/DS18B20.pdf

Quilty, J., Robinson, J., Appleby, G., ve Edgar, A. J. R. o. S. I. (2007). Thermoluminescence apparatus using PT100 resistors as the heating and sensing elements. 78(8), 083905. Silva, J. L. d. S., Melo, M., Camilo, R. d. S., Galindo, A. L., ve Viana, E. C. J. X. E. R. d. C. B., Alagoas e Sergipe . Feira de Santana, BA. (2014). Plataforma Arduino integrado ao PLX-DAQ: Análise e aprimoramento de sensores com ênfase no LM35. Steinhart, J. S., ve Hart, S. R. (1968). Calibration curves for thermistors. Paper presented at the Deep Sea Research and Oceanographic Abstracts. Stephenson, D. (1993). Tool-work thermocouple temperature measurements—theory and implementation issues. ZHAO, Y., ve XU, Y. J. M. E. T. (2008). Temperature Measurement System Based on DS18B20 [J]. 10.

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NANOTECHNOLOGICAL STRATEGIES TO IMPROVE STABILITY AND BIOAVAILABILITY OF ASTAXANTHIN Osman Kadir TOPUZ*1 1

Fisheries Faculty of Akdeniz University, Antalya, Turkey

*Corresponding authorâ&#x20AC;&#x2122;s e-mail: oktopuz@akdeniz.edu.tr

ABSTRACT Astaxanthin is a carotenoid pigment and commonly used as coloring and antioxidative agent in nutraceutical, cosmetics, food and feed industries. Astaxanthin is synthesized by algae and distributed in aquatic animals. Astaxanthin is available as a food supplement, but, like other carotenoids, is a very lipophilic compound and has low bioavailability within the human gastrointestinal tract. Bioavailability of lipophilic compounds has been received the growing interest in food and pharmaceutical areas, during few past decades. Since the solubility of bioactive compounds determines their bioavailability, the slow dissolution or solubilization of lipophilic compounds in the aqueous based systems, causes their low absorption rate and consequently, their low bioavailability. In this case, nanotechnological colloidal systems such as such as nanoemulsions, liposomes, nanogels, nanoparticles, nanoencapsulation etc., may increase bioavailability of astaxanthin. Nanoscale systems have considerable potential within the food industry, but they must be carefully designed to ensure that they are safe, economically viable and effective. This study highlights the challenges associated with nanotechnological incorporating of astaxanthin into nanoscale food systems to improve stability and bioavailability of astaxanthin. Keywords: Food nanotechnology, Astaxanthin, Bioavailability, Bioactive compounds

INTRODUCTION Astaxanthin is a ketocarotenoid with stronger antioxidant activity than vitamin E and coenzyme Q10, and with pharmacological effects against cancer, cardiovascular disease, diabetes, inflammation, and others. Increased astaxanthin supplementation in foods, nutraceuticals, and cosmetic products has resulted in tremendous growth in the demand for natural astaxanthin (Fang et al., 2019). Astaxanthin also has pharmacological effects in many diseases and is safe for humans thus it has received great attention across the world. Astaxanthin is widely used in aquaculture, nutraceutical and cosmetic industries, and food supplements, and has broad market prospects (Wang et al., 2019). Synthetic astaxanthin as well as the astaxanthin from Phaffia yeast and Paracoccus bacteria are predominantly used in aquaculture. The market size for astaxanthin was > USD 550 million in 2017 and is estimated to exhibit a compound annual growth rate (CAGR) of > 4.8% during the forecast period 2018 to 2024 (Fang et al., 2019). In recent years, there has been a growing trend to use astaxanthin in foods, nutraceuticals, and cosmetics which leads to safety concerns from consumers and regulatory issues over the synthetic astaxanthin entering human food chain. Therefore, recent interest in natural 145

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astaxanthin sources has increased substantially, especially in the nutraceutical industry (Shah et al., 2016). The primary source of natural astaxanthin for human applications, such as dietary supplements, cosmetics, foods and beverages, is the alga (Haematococcus pluvialis also referred to as Haematococcus lacustris) and red shrimp processing wastes. Because astaxanthin is easily altered in the presence of light, oxygen or high temperatures, it is important to define adequate procedures or formulations that preserve the quality of astaxanthin enriched extracts against their oxidation. Bioaccessibility and bioavailability of some hydrophobic bioactives (e.g., carotenoids, polyphenols, fat-soluble vitamins, phytosterols and fatty acids) are limited due to their low water solubility, and in some instances low chemical stability. Nanotechnology could be used to improve the solubility of hydrophobic bioactives and thus enhance the bioaccessibility and bioavailability of hydrophobic compounds. Nanometric delivery systems, derived from food grade phospholipids and biopolymers adopt many forms, including liposomes, micelles, nanoemulsions, particles, polyelectrolyte complexes, and hydrogels. The small particle sizes and customized materials used to create delivery systems confer their unique properties such as higher stability and/or resistance to enzymatic activity in the gastrointestinal tract (Jones et al., 2019). Nanotechnological approaches to improve bioavailability Nanotechnology has been utilized to create a variety of delivery systems for the encapsulation, protection and controlled release of bioactives and nutraceuticals such as astaxanthin. Nanoemulsions Nanoemulsions are thermodynamically unstable colloidal dispersions typically formed from emulsifiers, oil, and water. Oil-in-water (O/W) nanoemulsions consist of spherical lipid nanoparticles dispersed within an aqueous medium, with then a noparticles having a core shell structure. The hydrophobic core consists of oil molecules and the non-polar parts of emulsifiers, whereas the polar shell consists of the polar parts of the emulsifiers. Nanoemulsions can be distinguished from emulsions based on their dimensions: r<100 nm for nanoemulsions; r>100 nm for emulsions (McClement and Rao 2011). Two different approaches to fabricate nanoemulsions can be distinguished, i.e. low-energy and high-energy methods. Low-energy methods are based on the spontaneous formation of nanosized oil droplets in surfactant-oilwater mixtures when the composition or temperature of the system are changed. High-energy methods use mechanical devices that generate intense disruptive forces to break up the oil phase into tiny droplets that are then dispersed within the aqueous phase (McClement and Rao 2011). The main advantage of using low energy methods is their relative simplicity and the reduced equipment costs, however, they typically require high levels of synthetic surfactants. The main advantage of high-energy methods is that they require lower surfactant levels and a wider variety of ingredients can be used, but they are more expensive to implement. In general the functional attributes of nanoemulsions can be tailored by control-ling the concentration and size distribution of the lipid nanoparticles, as well as the composition, thickness, and electric characteristics of the interfacial layer (Joye et al., 2014).

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Nanoliposomes Nanoliposomes are spherical liquid structures smaller than 200 nm with an aqueous core surrounded by a single or several lipid bilayers. The primary advantage of liposomes is their ability to deliver both hydrophilic and lipophilic bioactive compounds, even simultaneously; and their similarity to natural cell membranes is an additional advantage (Livney, 2015). Liposomes may consist of single or multiple bilayers mainly composed of phospholipids, and may vary in diameter range from tens of nanometers to tens of micrometers depending on the composition, preparation method, and environmental conditions. Different methods can be employed to form liposomes: solvent evaporation, electro formation, thin film dehydratation/rehydratation, proliposome, membrane extrusion, dialysis, ultrasonication and high-pressure homogenization. Whatever method is used care should be taken to heat the phospholipids above their thermotropic phase transition temperature to homogeneously distribute them in the suspension medium and display sufficient flexibility to align themselves in vesicular structures (McClement and Rao 2011). Usually high-energy methods result in unilamellar liposomes, while low energy methods result in multilamellar or multivesicular liposomes. Liposomes have an aqueous interior that has similar characteristics as the aqueous medium in which they were formed. Hydrophilic compounds can be passively or actively loaded in the aqueous liposome interior (Zucker et al., 2009). Nanomicelles Nanomicelles are thermodynamically stable systems that form spontaneously when surfactants and other components are added to water under appropriate environmental conditions. Micelles (d < 20 nm) are usually composed of only surfactants. They are spherical particles with a hydrophobic core. Lipophilic micronutrients can be encapsulated within the hydrophobic core of micelles, while hydrophilic micronutrients can be encapsulated in the core of reverse micelles (Joye et al., 2014). Solid lipid nanoparticles Solid lipid nanoparticles and nanostructured lipid carriers are O/W emulsions in which the lipid phase has been either fully or partially solidified (McClements et al., 2007). As with conventional emulsions, the size and concentration of the lipid particles can be controlled, as well as the nature of the interfacial layer surrounding the lipid phase. Solid lipid nanoparticles are usually created by preparing an O/W nanoemulsion at a temperature above the melting point of the lipid phase, and then cooling it down to induce lipid crystallization. Solid lipid nanoparticles can be made by different strategies, such as high shear homogenization, high pressure homogenization, ultrasound dispersion, cold homogenization and solvent emulsification/evaporation the stability of the lipid nanoparticles formed can be controlled by selecting the number and type of lipids present, the nature of the emulsifier(s) used to stabilize the initial oil droplets, the initial droplet size and concentration, and the cooling conditions. Solid nanoparticles may be able to protect encapsulated lipophilic components from chemical degradation due to the ability of the solidified lipid phase to inhibit molecular diffusion processes. n this case, the lipophilic component should be trapped within the solid matrix formed after lipid crystallization(and not expelled to the particle surfaces). Typically, less ordered crystalline phases lead to enhanced encapsulation and retention, while highly ordered crystalline phases led to low encapsulation and high expulsion of micronutrients (Guri et al., 2013). 147

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Nanostructured lipid carriers Nanostructured lipid carriers are modified solid lipid nanoparticles in which the lipidic phase contains both solid (fat) and liquid (oil) lipids at room temperature. These are nano vehicle particles with mean size 100nm. The partially solid material creates interesting nanostructures which enhance stability of entrapped bioactives, enables high loading capacities, and offers sustained release profiles (Muller et al., 2002). Nanostructured lipid carriers can be made by various traditional dispersion methods, including solvent diffusion method, homogenization technique, double emulsion, supercritical fluid technology, etc. Large-scale production of nanostructured lipid carriers is easily possible (Selvamuthukumar and Velmurugan, 2012). Hydrogel nanoparticles Hydrogel nanoparticles or polymeric nanogels have gained considerable interest as one of the most potential nanoparticulate drug delivery systems that combines both the properties of a hydrogel system (e.g., hydrophilicity and large affinity for water absorption) and a nanoparticle (Salatin et al., 2016). One of the superior features of nanogels includes high drug/bioactive loading without chemical reactions and release of incorporated agents in a controlled behavior at the target site. CONCLUSIONS This review outlines current developments associated with research on nanotechnological strategies for improving of bioavailability of bioactive compound such as astaxanthin. Nanotechnological methods may aid in the sustained release of orally administered astaxanthin in the gastrointestinal tract, which in turn could improve their stability and enhance bioavailability. Stability and bioavailability of the bioactive compounds including astaxanthin could be minimized or prevented through nanotechnological strategies. Many nanotechnological systems have been developed for their potential to protect micronutrients and increase their bioavailability. Nevertheless, there are fewer studies on determining the capacity of these systems to be incorporated into complex food matrices and to with stand realistic food processing conditions. Future work and reviews should also be directed to studying the effect of incorporation of tailor-made astaxanthin nanoparticles in food systems on human and environmental health. REFERENCES Fang, N., Wang, C., Liu, X., Zhao, X., Liu, Y., Liu, X., ... & Zhang, H. (2019). De novo synthesis of astaxanthin: from organisms to genes. Trends Food Sci Technol., 92, 162-171. Guri, A., GĂźlseren, I. Corredig, M (2013). Utilization of solid lipid nanoparticles for enhanced delivery of curcumin in cocultures of HT29-MTX and Caco-2 cells. Food Funct, 4 (9), 1410-1419. Jones, D., Caballero, S., Davidov-Pardo, G (2019). Bioavailability of nanotechnology-based bioactives and nutraceuticals. Adv Food Nutr Res, 88, 235-273. Joye, I. J., Davidov-Pardo, G., McClements, D. J. (2014). Nanotechnology for increased micronutrient bioavailability. Trends Food Sci Technol, 40(2), 168-182. Livney, Y. D. (2015). Nanostructured delivery systems in food: Latest developments and potential future directions. Curr Opin Food Sci, 3, 125-135. 148

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McClements, D. J. Decker, E. A., Weiss, J (2007). Emulsionâ&#x20AC;?based delivery systems for lipophilic bioactive components. J Food Scie , 72(8), R109-R124. McClements, D. J., Rao, J (2011). Food-grade nanoemulsions: formulation, fabrication, properties, performance, biological fate, and potential toxicity. Crit Rev Food Sci Nutr., 51(4), 285-330. Muller, R. H., Radtke, M., & Wissing, S. A. (2002). Nanostructured lipid matrices for improved microencapsulation of drugs. Int J Pharm., 242, 121â&#x20AC;&#x201C;128. Salatin, S., Barar, J. Barzegar-Jalali, M. Adibkia, K. Milani, M. A. Jelvehgari, M (2016). Hydrogel nanoparticles and nanocomposites for nasal drug/vaccine delivery. Arch Pharm Res, 39, 1181-1192. Selvamuthukumar, S., Velmurugan, R (2012). Nanostructured lipid carriers: A potential drug carrier for cancer chemotherapy. Lipids Health Dis., 11, 159-159. Shah, M. M. R., Liang, Y. Cheng, J. J. Daroch, M (2016). Astaxanthin-producing green microalga Haematococcus pluvialis: From single cell to high value commercial products. Front Plant Sci., 7, 531-531. Zucker, D., Marcus, D. Barenholz, Y. Goldblum, A (2009). Liposome drugs' loading efficiency: a working model based on loading conditions and drug's physicochemical properties. Drug Deliv Transl Res., 139 (1), 73-80. Wang, J., Liu, S., Wang, H, Xiao, S, Li, C, Li, Y., et al. (2019). Xanthophyllomyces dendrorhous-derived astaxanthin regulates lipid metabolism and gut microbiota in obese mice induced by a high-fat diet. Mar Drugs.,17(6), 337.

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FOOD-GRADE EMULSIFIERS: SOURCE, PROPERTIES AND APPLICATIONS Osman Kadir TOPUZ*1 1

Fisheries Faculty of Akdeniz University, Antalya, Turkey

*Corresponding authorâ&#x20AC;&#x2122;s e-mail: oktopuz@akdeniz.edu.tr

ABSTRACT Emulsion-based systems are being increasingly studied for their potential applications within the food and pharmaceuticals industries to encapsulate, protect, and release lipophilic bioactive compounds, such as polyunsaturated fatty acids, flavonoids, carotenoids, vitamins, and drugs. Emulsifiers play a key role in the development of emulsion formulations because they facilitate the formation of small droplets during homogenization by adsorbing to the oil-water interface and reducing the interfacial tension, and also protect the droplets from aggregation after their formation by generating repulsive interactions between the droplets. A large number of emulsifiers are available for the formulation of emulsions and emulsions, including polysaccharides, proteins, phospholipids, natural extracts, and synthetic surfactants. The selection of an emulsifier for a particular application depends on its physicochemical properties, as well as its ease of utilization, legal status, and cost. This study provides a review of recent studies on the identification, characterization, and utilization of food grade emulsifiers used in emulsions. Keywords: Emulsifiers, Food ingredients, Emulsions, Bioactive compounds INTRODUCTION Increasing consumer demand for food and beverage products containing natural ingredients is a major trend in the food industry. For this reason, many food manufacturers are searching for commercially natural ingredients that have similar or better functional attributes than synthetic ones. Emulsions represent a major category of foods and beverages, including products such as milk, creams, sauces, dressings, soups, desserts, beverages, and creamers (Chung et al., 2017). Emulsions are ubiquitous in foods, and decades of research work have led to advanced, although often empirical, control over the formulation and functionality of those systems. However, the conventional strategies to make food emulsions have to be revised, due to the trends in the food sector area that have emerged in recent years. This includes a strong focus on naturalness, health and sustainability, which promotes the use of natural ingredients, ideally obtained from mild processing, and thus, by sense, far from pure and well-characterized (Berton-Carabin & Schroen, 2019). The manufacture of high quality emulsion-based products depends on identifying emulsifiers that possess the appropriate emulsifying and stabilizing properties required for the specific food application. The emulsifiers should: (a) rapidly adsorb to the oil droplet surfaces during homogenization so as to reduce the interfacial tension and facilitate droplet disruption; (b) form a stable protective layer around the oil droplets that prevents them from aggregating during manufacture, transport, and storage; (c) be easy to use, and available in a reliable quantity and quality; and, (d) be economically viable (McClement et al., 2015). In this study we focus on natural food grade emulsifiers that have considerable potential for replacing synthetic emulsifiers in many food products. This study provides a review of recent studies on the identification, characterization, and utilization of food grade natural emulsifiers used in emulsions. 150

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Overview of natural food-grade emulsifiers Emulsifiers vary considerably in their molecular and physicochemical properties depending on their origin. An understanding of how combinations of emulsifiers behave depends on understanding how individual emulsifiers behave, and so brief overview of the most important properties of individual emulsifiers are given below. Synthetic surfactants, proteins, phospholipids, and polysaccharides are common natural emulsifiers used in food industry. Synthetic surfactants Most synthetic surfactants such as Tweens, Spans, Citrem and Datem used in the food industry are small molecule surfactants, but recently some natural versions of small molecule surfactants have been identified from bark of quillaja saponins (McClements and Gums, 2016) and approved for use in commercial products (Dickinson, 2003. Synthetic surfactants with small molecule surfactants typically have a non-polar tail group and polar head group, although this is not always the case. For example some surfactants have ellipsoid structures with a hydrophobic side and hydrophilic side, such as bile salts and saponins (McClements and Jafari et al., 2018). Proteins Many proteins are surface active compounds since they contain a mixture of hydrophilic and hydrophobic amino acids along their polypeptide chains. To be a good emulsifier a protein should have an appropriate balance of polar and non-polar groups on its surface, so as to produce good water-solubility and good surface activity. If the surface of protein is too nonpolar then it will not dissolve in water, but if it is too polar then it will not be surface active (Dickinson, 1994). The incorporation of proteins at the oil-water interface has allowed scientists to utilize them to form emulsions, which may be used in food formulations, drug and nutrient delivery. The systematic study of the proteins at the interface and the factors that affect their stability (i.e., conformation, pH, solvent conditions, and thermal treatment) has allowed for a broader use of these emulsions tailored for various applications (Lam and Nickerson, 2013). Phospholipids Phospholipids are a lipid class molecule, which are a key component of all cell membranes and have amphiphilic characteristic so it can form lipid bilayers. In the structure of phospholipids molecules, consists of two hydrophobic fatty acid “tails” and a hydrophilic phosphate “head”, joined together by an alcohol or glycerol molecule. The 'head' is hydrophilic (attracted to water) that contains the negatively charged phosphate group and glycerol while the hydrophobic 'tails' consists of 2 long fatty acid chains which are repelled by water and are forced to aggregate. When placed in water, phospholipids form a variety of structures depending on the specific properties of the phospholipids. These specific properties allow phospholipids to play an important role in the lipid bilayer (Singh et al., 2017). The nature of functional groups attached to the phosphate group may also vary considerably depending on the origin of the phospholipids. Some of the most common phospholipids with different head groups are phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidic acid. The functional performance of a phospholipid as an emulsifier depends strongly on the nature of the head group and tail groups attached to the glycerol backbone. The phospholipids used in food and beverage applications are usually referred to as lecithin, which consist of a complex mixture of acetone-insoluble phospholipid and non-phospholipid components (McClement and Jafari, 2018). 151

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Polysaccharides Many polysaccharides used as food ingredients are highly hydrophilic molecules with poor surface activity, and are therefore unsuitable as emulsifiers. However, a number of natural or modified polysaccharides have hydrophobic groups attached to a hydrophilic chain, which makes them amphiphilic, e.g., gum Arabic modified starch, modified cellulose, and beet pectin (Dickinson, 2003). Gum Arabic is commonly used as an emulsifier in the production off flavor oil-in-water emulsions for application in the beverage industry. It is a tree gum exudate obtained from the stems and branches of acacia trees which grow in Africa, notably in Sudan, Chad and Nigeria. It is a complex, highly branched polysaccharide consisting of a core of β-1,3 linked galactose residues with branches consisting of galactose, arabinose, rhamnose and glucuronic acid (Littoz and McClements., 2008). There has been considerable interest in the food industry in recent years in the use of pectins for the stabilization of oil-in-water emulsions. Pectin is a complex polysaccharide and consists of linear chains of 1,4 linkedα-D-galacturonic acid residues, interrupted by 1,2 linked Lrhamnose residues. There are branches, consisting of neutral sugars linked to the rhamnose producing ‘hairy regions’ along the otherwise ‘smooth’ galacturonic acid main chain (Endress, 2011). There has been particular interest in recent years in the use of sugar beet pectin as an emulsifier. Sugar beet pectin constitutes ~20% of sugar beet pulp which is obtained as a byproduct during the extraction of sugar and in the EU amounts to ~400 K tons p.a. A distinct feature of sugar beet pectin is that it contains ferulic acid which is not the case for citrus pectins (Evans and Williams, 2013). Solubility and emulsifying properties of emulsifiers The solubility of emulsifiers have a pronounced impact on their functional performance in emulsions. The formation of oil-in-water emulsions usually requires that an emulsifier has good water solubility, and preferentially partitions into the water phase (rather than the oil phase). However, this is not always the case, as it is better to disperse certain types of phospholipids (lecithin) in the oil phase prior to the formation of oil-in-water emulsions (Magnusson et al., 2016). For small molecule surfactants and phospholipids, the hydrophile-lipophile balance (HLB) number is often used to de-scribe their solubility and partitioning characteristic surfactants with low HLB numbers (2-6) partition into the oil phase and stabilize water-in-oil emulsions, whereas surfactants with high HLB numbers(8-18) partition into the water phase and stabilize oil-in-water emulsions. For mixed surfactants, an effective HLB can be calculated from knowledge of the concentrations and HLB numbers of the individual emulsifiers. The HLB number does not always give a good prediction of the performance of individual or mixed surfactants in emulsions, e.g., two systems with the same HLB number may perform quite differently because other factors are also important (such as molecular geometry or specific interactions). For amphiphilic biopolymers, there is no equivalent to the HLB system, but solubility and partitioning characteristic are still important. The vast majority of surface active biopolymers are predominately hydrophilic, and should be dissolved in the aqueous phase prior to forming an oil-in-water emulsion (Dickinson, 2003). Differences in the solubility characteristics of emulsifiers can be utilized when forming emulsions from combinations of emulsifiers. For example, a water-soluble emulsifier can be dissolved in the aqueous phase (such as a biopolymer) and an oil-soluble emulsifier can be dispersed in the oil phase (such as a lecithin) prior to homogenization. Alternatively, two watersoluble emulsifiers could be dissolved in the aqueous phase prior to forming an emulsion, e.g., an ionic and a non-ionic surfactant. The combination of two different emulsifiers may lead to the formation of emulsions with different droplet sizes and stabilities than either of the individual emulsifiers used separately (Liu et al., 2011). 152

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There is often a relatively high interfacial tension at an oil-water interface because of the thermodynamically unfavorable contact between the oil and water molecules associated with the hydrophobic effect. Emulsifiers reduce the interfacial tension by adsorbing onto the interface and screening the unfavorable interactions between the oil and water molecules. Typically, the lower the molecular weight, the greater the entropy of mixing effect opposing adsorption (Fainerman et al., 1998). Emulsion formation factors Some physicochemical factors affect the emulsion formation:  Surface activity: Emulsifiers must be capable of adsorbing to oil-water interfaces, which means that they must have an appropriate ratio of polar and non- polar groups on their surfaces.  Adsorption kinetics: Emulsifiers must rapidly adsorb to droplet surfaces during homogenization so they can quickly reduce the interfacial tension and prevent droplet aggregation.  Interfacial tension reduction: Adsorbed emulsifiers should effectively decrease interfacial tension as this facilities droplet disruption within homogenizers.  Stabilization: Adsorbed emulsifiers should protect droplets from aggregating during droplet–droplet encounters by generating strong repulsive interactions, such as steric or electrostatic repulsion.  Surface coverage: The amount of emulsifier required to stabilize an emulsion depends on the surface load, which is the mass of emulsifier per unit surface area at saturation. The higher the surface load, the more emulsifier required to stabilize a given emulsion. These factors affect the forming efficiency of emulsions. An effective emulsifier must have a number of physicochemical characteristics if it is going to be effective at forming small droplets during homogenization (Öztürk and McClements, 2016). CONCLUSION In the food industry, these emulsifiers are usually selected from either high molecular weight biopolymers (such as proteins or polysaccharides) or low molecular weight surfactants (such as lecithin, saponins, Tweens, or Spans). Each type of emulsifier has its own unique molecular and physicochemical properties that can be used to modulate the interfacial properties of emulsion droplets. The utilization of mixed emulsifiers may be either beneficial or detrimental to emulsion formation and stability, and therefore the type and level of emulsifiers employed must be carefully selected. A better understanding of the molecular basis emulsifier performance will enable the selection of the most appropriate natural emulsifier for a particular application, as well as facilitating the identification of new natural materials that may prove to be highly effective emulsifiers. REFERENCES Berton-Carabin, C., Schroën, K (2019). Towards new food emulsions: designing the interface and beyond. Curr. Opin Food Sci., 27, 74-81. Chung, C., Sher, A., Rousset, P, Decker, E. A, McClements, D. J (2017). Formulation of food emulsions using natural emulsifiers: Utilization of quillaja saponin and soy lecithin to fabricate liquid coffee whiteners. J. Food Eng., 209, 1-11. Dickinson, E (1994). Protein-stabilized emulsions. J. Food Eng., 22, 59-74.

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Dickinson, E (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17(1), 25-39. Endress, H. U. (2011). Pectins: Production, properties and applications. In Renewable resources for functional polymers and biomaterials (pp. 210-248). Royal Society of Chemistry Publishers Cambridge. Evans, M., Ratcliffe, I., & Williams, P. A. (2013). Emulsion stabilisation using polysaccharideprotein complexes. Curr. Opin. Colloid Interface Sci., 18(4), 272-282. Fainerman, V. B., Lucassen-Reynders, E. H, Miller, R (1998). Adsorption of surfactants and proteins at fluid interfaces. Colloids Surf., A, 143 (2-3), 141-165. Lam, R. S., Nickerson, M. T (2013). Food proteins: a review on their emulsifying properties using a structure–function approach. Food Chem, 141(2), 975-984. Littoz, F., McClements, D. J.(2008). Biomimetic approach to improving emulsion stability: Cross-linking adsorbed beet pectin layers using laccase. Food Hydrocolloids, 22(7), 1203-1211. Liu, Y., Wei, F., Wang, Y., & Zhu, G. (2011). Studies on the formation of bifenthrin oil-inwater nano-emulsions prepared with mixed surfactants. Colloids Surf., 389 (1-3), 90-96. Magnusson, E., Nilsson, L., & Bergenståhl, B. (2016). Effect of the dispersed state of phospholipids on emulsification: Part 1. Phosphatidylcholine. Colloids Surf., A., 506, 794-803. McClements, D.J., 2015. Food Emulsions: Principles, Practices, and Techniques. CRC press. McClements, D. J., Gumus, C. E (2016). Natural emulsifiers-biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance. Adv. Colloid Interface Sci., 234, 3-26. McClements, D. J., Jafari, S. M (2018). Improving emulsion formation, stability and performance using mixed emulsifiers: A review. Adv. Colloid Interface Sci., 251, 55-79. Özturk, B., McClements, D. J (2016). Progress in natural emulsifiers for utilization in food emulsions. Curr. Opin. Food Sci, 7, 1-6. Singh, R. P., Gangadharappa, H. V, Mruthunjaya, K (2017). Phospholipids: unique carriers for drug delivery systems. J. Drug Delivery Sci. Technol., 39, 166-179.

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MARINE PHOSPHOLIPIDS: IMPORTANCE, CHARACTERIZATION AND APPLICATIONS IN FOOD INDUSTRY Osman Kadir TOPUZ*1 1

Fisheries Faculty of Akdeniz University, Antalya, Turkey

*Corresponding author’s e-mail: oktopuz@akdeniz.edu.tr

ABSTRACT Marine phospholipids have different properties from other type of phospholipids such as soybean, egg yolk, etc. Phosphatidylcholine is the most abundant phospholipids (PL) found in marine sources, including salmon, tuna, mackerel, bonito, herring, trout etc. The second abundant marine PL is phosphatidylethanolamine and followed by phosphatidylinositol, phosphatidylserine, sphingomyelin, and lysophosphatidylcholine. Phospholipids derived from marine sources have been the focus of much attention, since polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in phospholipid forms has better bioavailability compared to PUFA in triglyceride form. Nevertheless marine PL is sensitive to lipid oxidation since it contains higher content of PUFA. Marine PLs have amphiphilic properties and therefore they are natural surfactants for emulsion and liposome preparation. Due to the numerous advantages of marine PL, there is a growing interest of using marine PL as ingredient for food fortification. In this review study, the attention is focused to give a basic idea about its sources, characterization techniques, importance in food fortification and applications in food systems. Keywords: Emulsion, Marine phospholipid, Polyunsaturated fatty acid, Phosphatidylcholine

INTRODUCTION Phospholipids consist of a glycerol, two fatty acids, a phosphate group, and a polar head group. They are the structural and functional components of all cell membranes. Glycerol contains three carbon atoms and is the main carbon backbone of the phospholipid molecule. The hydroxy groups in glycerol at the sn-1 and sn-2 positions are esterified with fatty acids, whereas phosphoric acid is almost always linked to the sn-3 position of the glycerol. Beneficial effects of dietary phospholipids have been mentioned since the early 1900's in relation to different illnesses and symptoms, e.g. coronary heart disease, inflammation or cancer (Küllenberg et al., 2012). The phospholipid content of plant raw materials is usually in the range of 0.3–2.5% (on dry weight basis) while animal sources have substantially higher levels, ranging from 2% to 14% (Schneider and Virmani, 2001). Egg yolk is one of the most abundant animal sources of phospholipids, accounting for approximately 10% of the wet weight of the egg yolk, corresponding to approximately 22% of the egg yolk total solids and 50% of lipids. The milk fat globule membrane is a byproduct of the cheese industry and has been receiving a great deal of attention recently as it is a rich source of dairy phospholipids. The phosphoric acid residue can be further esterified with polar head groups such as choline, serine, ethanolamine, or inositol to give phosphatidylcholines (PC), phosphatidy-lethanolamines (PE), phospahtidylserines (PS), phosphatidylinositols(PI), or phosphatidic acid (PA) in small proportions (Chojnacka et al., 2012). Unlike the plant source phospholipids, phospholipids derived from aquatic sources have long fatty acid chains with 20 and 22 carbon atoms containing four to six double bonds, as found in fatty acids of DHA and EPA. As far as aquatic 155

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sources are concerned, PC and PE arethe predominant phospholipids, whereas PI, PS, sphingomyelin, and lysophosphatidylcholine (LPC) are usually found in smaller amounts in aquatic sources (Lu et al., 2011). One important source of aquatic phospholipids is fish, in particularly cold water fish such as salmon, herring, mackerel, anchovies, and sardines. A typical characteristic offish phospholipid is their enrichment of n-3 PUFAs, such as DHA and EPA. However, the n-3 PUFA composition in the phospholipids depends on the growing environment and the feeding composition of fish. Moreover, the phospholipid and fatty acid composition of fish is also affected by other factors, such as the species and the body parts offish. In fish species such as big eye, bonito, frigate mackerel, skip jack, and yellow fin, the main phospholipids are PC, PE and LPC, accounting for more than 80% of the total phospholipids. By contrast, LPC makes up only a minor part of the phospholipids of other fishes such as salmon and rainbow trout, even though PC and PE remained the majority (Lu et al., 2011). As far as the fish body parts are concerned, phospholipids are found relatively abundantly in roe, fish heads, and offal such as viscera and other visceral organs. Among these, fish roe is a rich source of n-3 PUFAs in the form of phospholipids with PC being the predominant lipid class (Burri et al., 2012 ). Hence, fish represents a potential source of aquatic phospholipids, but the production of aquatic phospholipids from fish has so far been limited. However, the by-products from fish processing could be a valuable source of aquatic phospholipids (Sun et al., 2018). Sources of of marine phospholipids Fish meat The main dietary source of EPA and DHA is fish, in particular cold water oily fish like salmon, sardine, anchovy, herring, or mackerel. Depending on the species of fish, up to one third of the EPA and DHA content might exist in the form of phospholipids. Studies have shown that in salmon, EPA and DHA are bound to phospholipids in a % 40. Hence, fish represents a potential source of marine phospholipids, but the production of marine phospholipids from fish meat has so far been limited. Moreover, the amount of byproducts from fish is significant and thus further represents a valuable source of marine phospholipids (Burri et al., 2012). Fish roe (egg) One by-product of the fish industry that is particularly interesting in respect to marine phospholipids is fish roe. The word â&#x20AC;&#x2DC;roeâ&#x20AC;&#x2122; stands for the eggs and the ovaries full of eggs of seafood. Fish roe is used for human consumption and is a rich source of n-3 PUFAs in phospholipid form. Fish roe from herring, salmon, pollock, and flying fish contain between 38 %-75% of their lipids in the form of phospholipid with PC being the predominant lipid class (Shirai et al., 2006.). In salmon, that had the highest total lipid content of the four fish examined, 56% of the lipids were in triglycerides form, whereas the other roes had values below 20 % (Burri et al., 2012). Fish heads and offal As far as the fish body parts are concerned, phospholipids are found relatively abundantly in roe, fish heads, and offal such as visceral organs (Sun et al., 2018). Antarctic krill Antarctic krill (Euphausia superb) is also a rich source of aquatic phospholipids containing PUFAs. About 5% of the natural weight of Antarctic krill is extractable lipids, 75% of which are in the form of phospholipids-PC (80.4% of the phospholipids), PE (14.9%), and PI (0.7%) 156

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(Ali Nehari et al., 2012). The EPA formed the most abundant molecular species in PE, but not in PC. Currently, lipid extract rich in phospholipids from Antarctic krill (Euphausia superba) has been authorized by the European Commission as a novel food/food ingredient (Sun et al., 2018). Health effects of marine phospholipids During the latest years, more and more focus has been laid on the beneficial health effects of phospholipids in both animals and humans. Marine phospholipids however, that come in a particular blend of phospholipids molecules and n-3 PUFAs bound to it, have been studied to a lesser extent. Due to the dual hydrophilic and hydrophobic properties of phospholipids, they are mainly known for their role as building blocks for cell membranes in almost all known living beings. In addition to their role in cellular structure and function, they play an important part in the formation of lipoproteins, which transport lipids to tissues via the blood stream. Additionally, certain phospholipid metabolites serve as important molecules with in several signaling systems. The studies with health effect of marine phospholipids are listed in Table 1. Table 1. Overview over clinical studies with marine phospholipids (Burri et al., 2012). Study area

Target

Main findings

References

Cardiovascular

Dyslipidemia

Improved blood lipids

Bunea et al. (2004)

Obesity

Normal to obese

Changed endocannabinoid levels

Banni et al. (2011)

Inflammation

Arthritis

Reduced arthritic symptoms

Deutsch (2007)

Brain

Memory complains

Improved word recall

Sampalis et al. (2003)

Eye

ADHD children

Improved attention

Vaisman et al. (2008)

Liver

Chronic liver disease

Improved lipid parameters

Hayashi et al. (1999).

Bioavailability

Healthy

Increased n-3 FA blood levels

Maki et al. (2009), Schuchardt et al. (2011)

CONCLUSIONS This review outlines current developments associated with research on marine derived phospholipids. In recent decades, phospholipids have been isolated and characterized from various sources, including plant, animal, and aquatic sources. Phospholipids play an important role in food manufacturing and are currently used in a wide range of food products such as baked goods, instant drinks, dairy products, chocolate and margarine. The most widely used application of marine phospholipids in foods is as an emulsifier, where it is utilized in both water-in-oil emulsions, as well as in oil-in-water emulsions. These phospholipids are significantly different in their structure, owing to variations in the properties of the polar and apolar groups, the length of the fatty alkyl chain, and the degree of unsaturation of lipids. Concerning the antioxidant properties of phospholipids, current research has mainly focused on the antioxidant effects of marine phospholipids in the food and bevarege systems. 157

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More data on the effect of phospholipids on their bioavailability are necessary for functional ingredient industry. REFERENCES Banni, S., Carta, G, Murru, E. Cordeddu, L. Giordano, E. Sirigu, A.R. Berge, K. Vik, H. Maki, K.C. Di Marzo, V. et al. (2011). Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects. Nutr. Metab. (Lond), 8, 7. Bunea, R., El Farrah, K. Deutsch, L. (2004). Evaluation of the effects of neptune krill oil on the clinical course of hyperlipidemia. Altern. Med. Rev. 2004, 9, 420-428. Burri, L., Hoem, N, Banni, S, Berge, K (2012). Marine omega-3 phospholipids: metabolism and biological activities. Int. J. Mol. Sci., 13(11), 15401-15419. Chojnacka, A., Gladkowski, W, Kielbowicz, G, Wawrzenczyk, C (2012). Isolation of egg‐yolk phospholipids and enzymatic modification of their acyl chains. Lipid Tech., 24 (2), 3335. Deutsch, L (2007) Evaluation of the effect of neptune krill oil on chronic inflammation and arthritic symptoms. J. Am. Coll. Nutr., 26, 39–48.. Lu, F. H., Nielsen, N. S, Timm-Heinrich, M, Jacobsen, C (2011). Oxidative stability of marine phospholipids in the liposomal form and their applications. Lipids, 46 (1), 3-23. Hayashi, H., Tanaka, Y. Hibino, H. Umeda, Y. Kawamitsu, H. Fujimoto, H. Amakawa, T. (1999) Beneficial effect of salmon roe phosphatidylcholine in chronic liver disease. Curr. Med. Res. Opin., 15, 177-184. Küllenberg, D., Taylor, L. A., Schneider, M., Massing, U (2012). Health effects of dietary phospholipids. Lipids Health Dis., 11(1), 3. Li-Nehari, A., Chun, B. S (2012). Characterization of purified phospholipids from krill (Euphausia superba) residues deoiled by supercritical carbon dioxide. Korean J. Chem. Eng., 29 (7), 918-924. Maki, K.C.; Reeves, M.S. Farmer, M. Griinari, M. Berge, K. Vik, H. Hubacher, R. Rains, T.M. (2009). Krill oil supplementation increases plasma concentrations of eicosapentaenoic and docosahexaenoic acids in overweight and obese men and women. Nutr. Res., 29, 609615. Sampalis, F., Bunea, R. Pelland, M.F. Kowalski, O. Duguet, N. Dupuis, S (2003) Evaluation of the effects of neptune krill oil on the management of premenstrual syndrome and dysmenorrhea. Altern. Med. Rev., 8, 171–179. Schneider, M (2018). Phospholipids. In Lipid Technologies and Applications (pp. 51-78). Routledge. Schuchardt, J.P., Schneider, I. Meyer, H. Neubronner, J. von Schacky, C. Hahn, A (2011) Incorporation of EPA and DHA into plasma phospholipids in response to different omega-3 fatty acid formulations—A comparative bioavailability study of fish oil vs. krill oil. Lipids Health Dis., 10, 145. Shirai, N., Higuchi, T. Suzuki, H (2006). Analysis of lipid classes and the fatty acid composition of the salted fish roe food products, Ikura, Tarako, Tobiko and Kazunoko. Food Chem., 94 (1), 61-67. Sun, N., Chen, J., Wang, D., & Lin, S. (2018). Advance in food-derived phospholipids: Sources, molecular species and structure as well as their biological activities. Trends Food Sci. Technol., 80, 199-211. Vaisman, N., Kaysar, N., Zaruk-Adasha, Y. Pelled, D. Brichon, G. Zwingelstein, G. Bodennec, J, (2008) Correlation between changes in blood fatty acid composition and visual sustained attention performance in children with inattention: Effect of dietary n-3 fatty acids containing phospholipids. Am. J. Clin. Nutr., 87, 1170-1180. 158

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ANTIOXIDANT ACTIVITY OF TOTAL ANTHOCYANINS EXTRACTED FROM SWEET CHERRY CULTIVARS Fatih SÖNMEZ*1 1

Sakarya University of Applied Sciences, Pamukova Vocational High School, 54900, Sakarya, Turkey *Corresponding author’s e-mail: fsonmez@subu.edu.tr

ABSTRACT Fruits and vegetables are important in terms of nutrition and human health due to their low energy content, high content of minerals and vitamins. Sweet cherry (Prunus avium L.) is one of the most popular table fruits and a member of the Rosaceae family. Sweet cherry cultivars contain polyphenols consist of flavonoids, flavan-3-ols, and flavonols and non-flavonoid compounds, such as hydroxycinnamic and hydroxybenzoic acids. In this paper, total anthocyanins were extracted from eleven sweet cherry cultivars grown in Sakarya, Turkey. The DPPH activities of the extracts were investigated as antioxidant properties. The results exhibited that ‘Churchill’ and ‘Merton Premier’ have high antioxidant activity with 74.102% and 73.503% DPPH activity, respectively. Keywords: Anthocyanins, Cherry, DPPH activity

INTRODUCTION Fruits and vegetables are important in terms of nutrition and human health due to their low energy content, high content of minerals and vitamins (Sezgin, 2014). The regular consumption of fruits and vegetables are widely recommended in dietary guidelines worldwide because of their richness in nutrients (Seraglio, 2018). The consumption of sufficient amount of vegetables and fruits decreases the risk of many chronic diseases, such as cancer, cardiovascular diseases, hypertension, and digestive system diseases. It also provides strengthening of the immune system and delays aging. Vegetables and fruits are the best source of vitamin A, vitamin C and beta-carotene, especially for antioxidants. Sweet cherry (Prunus avium L.) is one of the most popular table fruits (Correia, 2017) and a member of the Rosaceae family (Hewitt, 2017). Sweet cherry cultivars contain polyphenols consist of flavonoids, flavan-3-ols, and flavonols and non-flavonoid compounds, such as hydroxycinnamic and hydroxybenzoic acids (Shirasawa, 2017). Additionally, cherries are rich in vitamin C, A, E, and K, carotenoids, especially β-carotene, lutein, and zeaxantine. It has been reported the carotenoids (β-carotene, β-cryptoxanthin, and α-carotene) are found in sweet cherry fruit (Sezgin, 2014). Important quality characteristics of cherry cultivars are weight, color, firmness, sweetness, sourness, flavour, and aroma. Free radicals in the human body play a pathogenic role in the formation of many chronic degenerative diseases such as cancer, autoimmune, inflammatory and cardiovascular neurodegenerative diseases (Dai, 2017). In the present study, total anthocyanins were extracted from eleven sweet cherry cultivars grown in Sakarya, Turkey. The DPPH activities of the extracts were investigated as antioxidant properties.

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MATERIAL AND METHODS Materials The sweet cherry (‘Stella’, ‘0900 Ziraat’, ‘Bing’, ‘Regina’, ‘Noble’, ‘Beyaz Anodolu’, ‘Merton Premier’, ‘Berriyassa’, ‘Churchill’, ‘Lambert’ and ‘Kuş Kirazı’) samples were analyzed in an appropriate stage of maturity. The samples (0.5 kg) were packed in plastic bags, frozen and kept at -18oC before extraction of anthocyanins. Extraction of total anthocyanins Seeds of deep-frozen cherry samples were separated by hand. Cherry samples (about 100 g) were blended twice with 250 mL of methanol acidified with 1.0 N HCl (85:15, v/v) for 2 minutes in a Waring blender. Extract was filtered under vacuum on a Buchner funnel using Whatman No. 1 paper. Filtrate was concentrated to a small volume at 40 ºC using rotary evaporator under vacuum for purification of total anthocyanins. Total anthocyanins were extracted from all sweet cherry cultivars three times and the mean value was calculated. Purification and measurement of total anthocyanins The anthocyanins were purified according to the methodology of Zhang et al. (2004). The filtrate containing sugars, organic acids, polyphenolics and anthocyanins etc. were loaded onto an Amberlite XAD-7 resin column. Firstly, it was washed with distilled water (about 1000 mL) to remove sugars, acids and other water-soluble compounds and then washed with ethyl acetate (about 500 mL) to remove polyphenolics compounds. Finally, total anthocyanins were recovered with methanol containing 0.1% trifluoroacetic acid (v/v). This acidified methanol fraction was combined and evaporated under vacuum to dryness. 20 mg of total anthocyanins was dissolved with 0.1% TFA-MeOH (v/v) and submitted to spectrophotometric analysis (200– 600 nm) using a UV–Vis spectrophotometer. All measurements were taken in duplicate. DPPH activity Free radical scavenging activities are determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical (Kedare, 2011). 1000 μM stock solutions of the materials were prepared. Of these stock solutions, 2, 5, 10 and 20 μL was taken and completed to 40 μL with ethanol, and then 160 μL of 0.1 mM DPPH solution was added. The absorbance values of the prepared solutions were measured at 517 nm after 30 min of incubation in the dark at room temperature. Inhibition values (%) of the extracts derivatives were calculated from the obtained absorbance values. The absorbance values of the samples were evaluated against the control. RESULTS Average amounts of total anthocyanins from sweet cherry cultivars were between 45 and 305 mg/100 g (Table 1). The highest total anthocyanins amount was in the ‘Regina’ cultivar (305 mg/100 g), followed by the ‘Noble’ (219 mg/100 g) and ‘Lambert’ (179 mg/100 g). The lowest total anthocyanins amount was recorded in the ‘Berriyessa’ cultivar (45 mg/100 g). λmax values and DPPH activity of total anthocyanins were presented in Table 1 and Table 2.

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Table 1. Average amounts and maximum absorbances (λmax) of total anthocyanins in sweet cherry cultivars Sample No Sweet Cherry Cultivars

λmax (nm)

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11

283.0, 532.5 281.5, 520.0 284.5, 528.5 280.5, 528.5 285.0, 529.0 286.5, 529.5 288.0, 522.5 281.5, 528.0 282.5, 531.0 283.0, 529.5 282.5, 530,5

Total anthocyanins amounts (mg/100 g, average) Stella 150 0900 ziraat 98 Bing 102 Regina 305 Noble 219 Beyaz Anadolu 141 Merton premier 58 Berriyessa 45 Churchill 70 Lambert 179 Kuş kirazı 168

Table 2. DPPH activity (%) of total anthocyanins in sweet cherry cultivars

Sample No

Sweet Cherry Cultivars

DPPH Activity (%)

N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11

Stella 0900 ziraat Bing Regina Noble Beyaz Anadolu Merton premier Berriyessa Churchill Lambert Kuş kirazı

7.859 50.674 58.982 19.611 28.368 67.365 73.503 19.611 74.102 58.159 35.778

DISCUSSION DPPH method is commonly used to measure the ability of antioxidants to sweep free radicals. In this spectrophotometric method, a stable free radical, DPPH (2,2-diphenyl-1picrylhydrazyl), is used (Kedare, 2011). Antioxidants are based on the ability to degrade the DPPH radical, and when it interacts with radical hydrogen donors, hydrazine is reduced. According to this method, compounds must have strong hydrogen donor groups to exhibit good antioxidant properties. The DPPH activities of the extracts were between 7.859% and 74.102% (Table 2). Among them, ‘Churchill’, ‘Merton Premier’ and ‘Beyaz Anadolu’ exhibited high antioxidant activity with 74.102%, 73.503% and 67.365%, respectively. The UV-Visible absorption spectra of total anthocyanins in sweet cherry cultivars were recorded between 200 and 600 nm. The spectrophotometric analysis of extracts obtained from these cultivars showed maximum absorption bands around 280 nm and 528 nm. Taking into account that anthocyanins display two distinct absorption bands, one in the UV-region (260– 161

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280 nm) and another in the visible region (490–550 nm) (Simoes et al., 2009). These results provided evidences of the anthocyanic nature of the extracts produced in the sweet cherry cultivars grown in Sakarya, Turkey. CONCLUSIONS In conclusion, total anthocyanins were extracted from eleven sweet cherry cultivars (‘Stella’, ‘0900 Ziraat’, ‘Bing’, ‘Regina’, ‘Noble’, ‘ Beyaz Anodolu’, ‘Merton Premier’, ‘Berriyassa’, ‘Churchill’, ‘Lambert’ and ‘ Kuş Kirazı’) grown in Sakarya, Turkey. Their DPPH% activities were examined. ‘Regina’ (305 mg /100 g) has the highest total anthocyanins amounts, whereas ‘Berriyessa’ (45 mg /100 g) was the lowest total anthocyanins amounts among all cultivars. ‘Churchill’ and ‘Merton Premier’ have high antioxidant activity with 74.102% and 73.503% DPPH activity, respectively. These sweet cherry cultivars could be used as natural antioxidant agents. Acknowledgements: The authors would like to gratefully acknowledge the kind support of Dr. Taki Demir and Dr. Zuhal Gunesli.

REFERENCES Correia S., R. Schouten, P.A. Silva, B. Gonçalves (2017). Factors affecting quality and health promoting compounds during growth and postharvest life of sweet cherry (prunus avium l.). Front. Plant Sci., 8(2166), 1-15. Dai, Y., C. Shao, Y. Piao, H. Hu, K. Lu, T. Zhang, X. Zhang, S. Jia, M. Wang, S. Man (2017). The mechanism for cleavage of three typical glucosidic bonds induced by hydroxyl free radical. Carbohyd. Poly., 178, 34-40. Hewitt S., B. Kilian, R. Hari, T. Koepke, R. Sharpe, A. Dhingra (2017). Evaluation of multiple approaches to identify genome-wide polymorphisms in closely related genotypes of sweet cherry (Prunus avium L.). Comput. Struct. Biotechnol. J., 15, 290-298. Kedare S.B., R.P. Singh (2011). Genesis and development of DPPH method of antioxidant assay. J. Food Sci. Technol., 48(4), 412–422. Seraglio T.K.S., M. Schulz, P. Nehring, D.F. Betta, C.A. Valese, H. Daguer, V.L. Gonzaga, R. Fett, O.C.A. Costa (2018). Nutritional and bioactive potential of Myrtaceae fruits during ripening. Food Chem., 239, 649-656. Sezgin C.A., (2014). Fruit, vegetable and our health. J. Tour. Gastr. Stud., 2(2), 46-51. Shirasawa K., K. Isuzugawa, M. Ikenaga, Y. Saito, T. Yamamoto, H. Hirakawa, S. Isobe (2017). The genome sequence of sweet cherry (Prunus avium) for use in genomicsassisted breeding. DNA Research, 24(5), 499–508. Simoes C., C.H.B. Bizarri, L.S. Cordeiro, T.C. De Castro, L.C.M. Coutada, A.J.R. Da Silva, N. Albarello, E. Mansur (2009). Anthocyanin production in callus cultures of Cleome rosea: Modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS. Plant Physiol. Biochem., 47, 895–903. Zhang Z., P. Xuequn, C. Yang, Z. Ji, Y. Jiang (2004). Purification and structural analysis of anthocyanins from litchi pericarp. Food Chem., 84, 601–604.

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COLOR CHANGES IN DIFFERENT OILS USED IN A CANNED EEL L. GÓMEZ-LIMIA1, I. FRANCO1, H. VILLAR1, S. MARTINEZ1* 1

Food Technology, Faculty of Science, University Campus as Lagoas s/n, 32004 Ourense, University of Vigo, Spain CITACA - Clúster de Investigación y Transferencia Agroalimentaria del Campus Auga, Universidad de Vigo, 32004-Ourense, España *Corresponding author’s e-mail: sidonia@uvigo.es

ABSTRACT Canned fishes are products of economic importance in many countries. These products are normally canned in vegetable oils, having a preserving effect and contributing to make the product more palatable. Processing can cause changes in the sensory and physical properties of the oils. Colour is one of the most important parameter to determine changes in quality food. The aim of this work was to study the effect of different treatment and storage on the colour of different oils used as filling medium during European eels (Anguilla anguilla) canning. Sunflower oil and olive oil were selected as filling mediums. In addition, a canned eels was made using olive oil with garlic in the frying, and then pepper and chilli pepper was added to the inside of the canned during the sterilization process. Samples of the different oils were sampled, raw, after the frying of the fish and after the sterilization treatment. Samples were also sampled after two months and one year of storage. The CieLab system was used to determine the colour parameters. The type of oil, the type of treatment and the storage time conditioned the changes experienced in the colour parameters of the oils used during the preparation of an eel can. In canned eels with spicy olive oil, greater changes in colour parameters were observed than when using sunflower or olive oil. Keywords: Olive oil, Sunflower oil, Canned eel, Oxidation, Colour

INTRODUCTION Canning is one of the oldest food processing methods. Canned fishes are products of economic importance in many countries, supporting a significant market demand because of its convenient and affordable use. The quality of canned products varies with different factors like quality of raw fish, processing conditions, packaging material and filling ingredients (Morsy, 2016). The sensory and nutritional characteristics are result of complex interactions between fish and filling medium. Fish is normally canned in vegetable oils, having a preserving effect and contributing to make the product more palatable. Olive and sunflower oils are two of the most common filling media used in canning manufacturing. Filling oil can cause dilutions, partial extraction of some components, and different heat transfer in fish muscle (Medina et al., 1998). Processing can also cause changes in the sensory and physical properties of the oils. Colour is one of the most important parameter to determine changes in quality food, and it has an important impact on the acceptability of canned fish. Eels have a great commercial value in Europe (mainly Spain, Portugal, Italy and Netherlands), and Asian countries (mainly Japan, China, Korea and Taiwan). The eels can be processed in different forms such as freezing, smoking, canning and the preparation of jellied 163

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eels. Although canned eel is a commercially important product in some countries, available information on its quality parameter is scarce. The aim of this work was to study the effect of different treatment and storage on the colour of different oils used as filling medium during European eels (Anguilla anguilla) canning. MATERIAL AND METHODS Samples and fish preparation European eel were caught by professional eel fishermen operating in the River Ulla (Galicia, NW Spain), during the authorized fishing season (winter). The fish were transported to tanks connected to freshwater recirculation modules, where they were held until slaughter by ice water immersion. They were then purchased and transferred to the laboratory. Eels with weights between 200 and 400 g were used for canning. The fish were eviscerated, washed, packed in vacuum bags and stored at -20 °C until use. Before canning, frozen eels were defrosted in 12% brine. The head and skin were removed, and the fish was cut in small pieces. The pieces were fried in sunflower oil, olive oil, and olive oil with garlic. Then, the portions were placed in jars. Three filling medium were tested: olive oil, sunflower oil and olive oil with pepper and chilli pepper. The jars were vacuum-sealed and sterilized at 118ºC for 30 min. Then, the cans were cooled and storage at room temperature. Samples of the different oils were sampled, raw, after the frying of the fish and after the sterilization treatment. Samples were also sampled after two months and one year of room storage. In the case of canned fish, the cans were opened and the liquid part was carefully drained off gravimetrically, filtered by means of a filter paper and collected. Colour determination Colour was analysed by the CIEL*a*b* system and using a portable colorimeter (Chroma Meter CR-400, Konica Minolta Sensing, Inc., Osaka, Japan) to determine the L* (lightness), a* (balance between red and green) and b* (balance between yellow and blue) coordinates. The colorimetric parameters hue angle (Hº) (colour of sample as defined by its location in a 360º axis; 0 or 360º= red, 90º = yellow, 180º = green and 270º = blue), chroma (C*) (colour saturation starting from 0 and increasing), and total changes in colour (ΔE) were calculated as follows: Hº = 180º + arctg (b*/a*), for a*<0 and b*>0 ΔE = √ [(L0-L)2 + (a0-a)2 + (b0-b)2], colorimetric coordinates of fresh kales are designated by “0”. All determinations were made at least in triplicate. Statistical analysis All analyses have been done at least in triplicate. The statistical treatment of the data was carried out by analysis of variance (ANOVA). The least squares test (LSD) was applied for a 95% confidence interval (P ≤ 0.05) for the comparison of the mean values, using the statistical software Statistica version 7.1 of Statsoft © Inc. (Tulsa, OK, USA). RESULTS Color parameter values, L*, a* and b*, are reported in Figures 1, 2 and 3 for sunflower oil, olive oil and spicy olive oil. 164

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100

Raw Oil fried Oil canned 2 months storage 12 months storage

20 a

-20

Figure 1.- Color parameters (L*, a* and b*) of sunflower oil (means ± standard deviation) (a-c values with different letters are significantly different, p < 0.05). 100

a b

Raw Oil fried Oil canned 2 months storage 12 months storage

c a

-20

Figure 2.- Color parameters (L*, a* and b*) of olive oil (means ± standard deviation) (a-c values with different letters are significantly different, p < 0.05).

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100

Raw Oil fried

c 80

Oil canned d

2 months storage 12 months storage

40 a

b c

20 a

-20

Figure 3.- Color parameters (L*, a* and b*) of spicy olive oil (means ± standard deviation) (ad values with different letters are significantly different, p < 0.05). It was observed that L* increased during the frying and sterilization treatment and remained constant during storage for the sunflower oil. On the other hand, the initial luminosity of the olive oil, was higher than in sunflower oil, however, in this oil, L* decreased with frying and the sterilization treatment. Using only olive oil, during storage, there was an increase in luminosity. With regard to spicy olive oil the luminosity increased during the first months of storage, but decreased in the 12-month preserve. In sunflower oil, a* values decreased with frying and sterilization. During storage, it remained stable at the beginning (2 months), but it decreased again after 12 months of storage. The olive oil and spicy olive oil showed higher a* values after frying and sterilization, remained stable at the beginning of storage and decreased after 12 months of storage of the preserves. The parameter b* was lower in sunflower oil than in olive oil. In respect of sunflower oil, it increased during frying, but not during sterilization. It was stable during the first months of storage and increased to 12 months. In olive oil, b* value remained stable during frying and sterilization treatment, decreased at the beginning of storage of the preserved and increased to 12 months of storage. However, the spicy olive oil, the b* value decreased with the two heat treatments under study and at the beginning of storage, but increased after 12 months of storage of the preserves. Hº, C* and ΔE parameters are reported in Figures 4, 5 and 6 for sunflower oil, olive oil and spicy olive oil.

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360

Sunflower oil Olive oil

310

Spice olive oil

260

H°

210

160

a b

c b

110

10 Raw

Oil fried

Oil canned

2 months storage 12 months storage

-40

Figure 4.- Hº parameter of sunflower oil, olive oil and spicy olive oil (means ± standard deviation) (a-d values with different letters are significantly different, p < 0.05). 60 Sunflower oil Olive oil Spice olive oil

e 40

b,d b,d

d f

c a

0 Raw

Oil fried

Oil canned

2 months storage

12 months storage

Figure 5.- C* parameter of sunflower oil, olive oil and spicy olive oil (means ± standard deviation) (a-f values with different letters are significantly different, p < 0.05).

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Sunflower oil 100

Olive oil Spice olive oil

ΔE

d a

a b

a,c

a a,b

0 Raw

Oil fried

Oil canned

2 months storage

12 months storage

Figure 6.- Colour variations (ΔE) of sunflower oil, olive oil and spicy olive oil (means ± standard deviation) (a-d values with different letters are significantly different, P < 0.05). The fresh sunflower oil showed H° values higher than olive oil. The frying treatment decreased this parameter in sunflower oil, but not in olive oil. There were no changes in any of the oils under study after sterilization. After 12 months of storage, it decreased in the three coverage fluids. The parameter C* was lower in sunflower oil than in fresh olive oil. Frying produced an increase in C* in sunflower oil, but did not significantly change the C* values in olive oil and spicy olive oil. During storage, the C* values increased. ΔE were greater in spicy olive oil and during storage. DISCUSSION In the food industry, the common process used to achieve a stable product is canning. In this process, the fish can be adversely affected resulting in undesirable sensorial and nutritional changes and specifically loss of heat-sensitive nutrients. Colour is an important factor influencing consumer acceptance of foodstuffs in general, so there is a commercial interest in studying the modifications of this parameter because of processing. Colour can be used to evaluate composition and chemical changes, being one of the indicators of product quality (Maskan, 2003; García and Yousfi, 2005; Sikorska et al. 2007). In canned fish, filling medium may produce a different dilution, partial extraction of some components and different heat transfer in fish muscle as well, which cause change in colour parameters (Medina et al., 1998). On the other hand, the degradation of these constituents during processing affects the filling medium colour. For canned fish, various filling media are used like brine, oil and natural pack. Some vegetable oils are widely used for canning fish. The colour of canned products are developed by a combination of reactions and compounds absorbed by the filling oil. The main factors affecting the changes in quality parameters during the process are the oil type, storage and thermal changes, interfacial tension between the oil and the product, temperature and length of 168

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processing, moisture content, size and characteristics of fish surface and pre-frying treatments (Fellows, 2006). Vegetable oils also are susceptible to oxidation. Oxidation is an important cause of oil quality deterioration during processing and storage (Morello et al., 2004). Browning reactions, which are caused through oxidation products in the frying oil, can change the colour in the oil (Pokorny, 1981). The colour of oils is chiefly influenced by two groups of constituents, carotenoids and chlorophyll. Sánchez-Gimeno et al. (2008) pointed out that colour changes indicate the degradation of the chlorophyll by loss of the magnesium of the molecule during frying. A significant difference (p < 0.05) was observed in canned eel samples at different filling medium compared to the control sample in most colour parameters. According to the raw eel values, in canned eels with different filling medium, there was a deterioration of the colour of oil. The change were higher in spicy olive oil. These more pronounced changes in spicy olive oil may be due to migrations of some of the components of the pepper and/or the chilli pepper towards the oil. Thermal stability of oils depends on their chemical structure. Reda (2004) reported that saturated oils are more stable than unsaturated ones. Moreover, during storage, the colour of the oil changes considerably with storage time. These may due to a dilution and partial extraction of some components (Medina et al., 1998). CONCLUSIONS The changes in colour of fried and sterilized oils used in canned eels, and the modifications during storage was related to the heating process and the filling medium (sunflower, olive and spicy olive oil). In general, it was found that frying caused more changes in colour than sterilization process. The colour was also changed during storage. The use of spices (pepper and/or the chilli pepper) in the eel canning caused higher changes in the colour of the filling medium. The changes in the colour in the filling medium during the manufacture of canned fish products have an important effect on its quality. Acknowledgements: Thanks to CITACA Strategic Partnership ED431E 2018/07 (Xunta de Galicia, Spain). REFERENCES Fellows, P. J. (2006). Food processing technology. Principles and Practice, Artmed, São Paulo, Brazil. 608. Garcia J. M., K. Yousfi (2005). Non-destructive and objective methods for the evaluation of the maturation level of olive fruit. Eur. Food Res. Technol., 221, 538-541. Maskan, M. (2003). Change in colour and rheological behaviour of sunflower seed oil during frying and after adsorbent treatment of used oil. Eur. Food Res. Technol., 218, 2025. Medina, I., R. Sacchi, L. Biondi, S. P. Aubourg, L. Paolillo (1998). Effect of packing media on the oxidation of canned tuna lipids. Antioxidant effectiveness of extra virgin olive oil. J. Agric. Food Chem., 46, 1150−1157. Morello J.R., M.J. Motilva, M.J. Tovar, M.P. Romero (2004). Changes in commercial virgin olive oil (cv. Arbequina) during storage, with special emphasis on the phenolic fraction. Food Chem., 85, 357-364.

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Morsy, M. K. (2016). Quality enhancement of canned little tunny fish (Euthynnus alletteratus) by whitening solutions, pre-cooking time and filling medium. J. Food Process. Technol., 7, 1-8. Pokorny, J. (1981). Browning from lipid-protein interactions. Prog. Food Nutr. Sci., 5, 421428. Reda, S. Y. (2004). Comparative study of vegetable oils subjected to the thermal stress. Master Dissertation, Universidade Estadual de Ponta Grossa, Brasil. Sánchez-Gimeno, A. C., A.I. Negueruela, M. Benito, A. Vercet, R. Oria (2008). Some physical changes in Bajo Aragón extra virgin olive oil during the frying process. Food Chem., 110, 654–658. Sikorska, E., F. Caponio, M. T. Bilancia, C. Summo, A. Pasqualone, I. V. Khmelinskii, M. Sikorski (2007). Changes in colour of extra-virgin olive oil during storage. Pol. J. Food Nutr. Sci., 57, 495-498.

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THE DETERMINATION OF NUTRITIVE VALUE OF ALFALFA AND CORN SILAGES AT DIFFERENT RATES Mehmet ALAGÖZ*1, Mevlüt TÜRK1, Kadir Emre BUĞDAYCI2 1

Isparta Applied Science University, Agricultural faculty, Department of Field Crops, Isparta, Turkey 2 Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Department of Animal Nutrition and Diseases, Burdur, Turkey *Corresponding Author: mehmetalagoz@isparta.edu.tr ABSTRACT

This research was carried out in Isparta University of Applied Sciences, Faculty of Agricultural, Education, Research and Application Farm and in field crops laboratories in 2018-2019. In this study, it was aimed to determine the silage quality characteristics alfalfa (A), maize (M) and three different alfalfa+maize mixtures (75% A+25% M, 50% A+50% M and 25% A+75% M). In this research, ‘bilensoy’ alfalfa cultivar and ‘DKC721’ hybrid corn cultivar were used as study materials. The alfalfa was harvest at the 50% blooming stage, while the silage maize was harvested at milk-dough stage. The experiment was established in a randomized complete parcels design with three replications. After waiting 60 days jars were opened for dry matter content (DM), crude protein content (CP), acid detergent fiber content (ADF), neutral detergent fiber content (NDF), silage pH, physical characteristics (PC) and flieg score of silages were determined in this study. As a results of this research, as the amount of alfalfa in the silage increases DM content, CP ratios and pH volues increased, acid detergent fiber content and neutral detergent fiber content values also decreased. Alfalfa in terms of nutritional values gave the best values. But the smell, color, structure, total score and Flieg Score as physical evaluation parameters in alfalfa silage were significantly improved with increasing level of corn. Keywords: Alfalfa, Corn, Silage, Crude protein, Dry matter, Fleig score, ADF

INTRODUCTION Ensiling is a common preservation method for moist forage crops. It is based on lactic acid bacteria converting water-soluble carbohydrates into organic acids, mainly lactic acid, under anaerobic conditions. As a result, pH decreases and the forage is preserved from spoilage microorganisms (McDonald et al., 1991, Filya, 2004). Whole crop maize is the major crop ensiled in Turkey. Corn silage is normally a highenergyforage with high dry matter (DM) yield relative to other forage crops (Coors, 1996). However, maize silage is poor in protein content at approximately 7.5% crude protein (CP) on a dry matter basis when maize is cut at the hard dough stage (Topps and Oliver, 1993; Titterton and Maasdorp, 1997). Protein supplementation is required for maintenance and production of low yielding dairy cows on a maize silage ration (Dube, 1995). Mustafa and Seguin (2005) reported that forage-type alfalfa cultivars were well preserved as silages. Alfalfa and their legumes are usually difficult to ensile because of low sugar content and high buffering capacity. No information is available regarding the feeding value of forage alfalfa silage for dairy cows (Vargas-Bello-Perez, 2008).

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Mixtures in different rates among various silage plants not only increase silage quality but also decrease negative properties in silages. Thus, many investigations have been made related to the topic by many researchers in recently (Demirel et al 2008; Zhu et al 2011; Souma et al 2011). The objective of the present study was to evaluate silage quality of corn and alfalfa silages at different rates. MATERIAL AND METHODS This research was carried out in Isparta University of Applied Sciences, Faculty of Agricultural, Education, Research and Application Farm and in field crops laboratories in 20182019. In this research, ‘bilensoy’ alfalfa cultivar and ‘DKC721’ hybrid corn cultivar were used as study materials. The plots were harvested when the corn was at milk-dough stage for silage and plants were chopped and filled into 2 liters of jars without having any air (Altınok et al., 2005). The experiment was established in a randomized complete parcels design with three replications and totally 15 jars silages were made. After waiting 60 days jars were opened. Some physical observations (colour, smell and structure), dry matter (DM) ratio, pH, fleig score, crude protein (CP) ratio, acid detergent fibre (ADF) and neutral detergent fibre (NDF) were determined in silage samples. At the end of the fermentation period, jars were opened and chemical analyses were carried out to determine the feed quality and silage quality of the silage. Some physical observations (colour, smell and structure) were done according to Alçiçek and Özkan (1997). pH values of silage samples were measured according to Polan et al. (1998) using a pH-meter. Dry matter content and crude protein analyses were carried out following the method reported in AOAC (1990). The ANKOM Fibre Analyser was used for NDF and ADF analysis (Anonymous, 2006). In determining the quality of the silage samples, the following Fleig equation was used (Kilic, 1986; Nauman and Bassler, 1993): Fleig score = [220 + (2×silage dry matter content (%) – 15)] - 40 × pH value The data from 20018–19 were analysed together using the SAS program (SAS 1998). Means were separated by LSD at the 5% level of significance. RESULTS AND DISCUSSION Silages rates systems had significant effects on physical observations such as colour, smell and structure. Sole corn and corn-alfalfa mixtures had significantly better colour and structure than sole alfalfa (Table 1). According to physical observations, sole alfalfa silage had medium quality, while 50% Corn+50% Alfalfa and 75% Corn+25% Alfalfa silages had excellent quality. Table 1. The Physical Observations on Corn and Alfalfa Silages. Silages Mixtures Sole Corn 75% Corn+25% Alfalfa 50% Corn+50% Alfalfa 25% Corn+75% Alfalfa Sole Alfalfa LSD (%5)

Colour 2.00 a 2.00 a 1.67 b 1.33 c 1.00 d 0.22

Smell 14.00 a 12.00 b 11.00 b 8.33 c 4.00 d 1.42

Structure 4.00 a 3.50 b 3.50 b 3.00 c 2.00 d 0.43

Means followed by the same letter was not significantly different at 0.05 level using LSD test

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Total 20 17.5 16.2 12.7 7.00

Quality Class Excellent Excellent Excellent Good Medium

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Sole alfalfa silage had the highest DM content and pH values, while had the lowest fleig score (Table 2). Increased pH value may have caused by relatively higher nitrogen content of alfalfa (Lujia et al. 2004; Saricicek and Kılıc, 2011; Saruhan et al. 2011; Carpici et al. 2017). According to fleig scores, sole alfalfa silage had medium quality, the other silages mixtures had excellent quality. The CP content of sole alfalfa (17.1 %) was significantly higher than the other group silages. Sole corn silage had poor CP content (6.27%). The reduction in CP content of silage was attributed to the extensive proteolysis during the ensiling process. Although legume silage usually has a high protein content (Jacobs and McAllan, 1991), legume material on its own is extremely difficult to ensile because of its high buffering capacity and low levels of fermentable carbohydrate (Harrison et al. 1994; De Figueiredo and Marais, 1994). Similar results were reported by Demirel et al. 2003; Aykan and Saruhan, 2018; Gelir, 2018. Table 2. The Dry Matter, pH, CP, ADF, NDF and Fleig Scores on Corn and Alfalfa Silages. Silages Mixtures Sole Corn 50% Corn+50% Alfalfa 75% Corn+25% Alfalfa 25% Corn+75% Alfalfa Sole Alfalfa LSD (%5)

DM (%) 21.98 cd 22.45 c 22.38 c 24.29 b 27.15 a 1.19

pH 3.37 d 3.72 c 3.81 c 4.18 b 5.02 a 0.33

CP (%) 6.27 d 7.54 c 8.13 c 10.33 b 17.1 a 1.27

ADF (%) 27.11 a 25.35 b 24.17 b 21.84 c 17.14 d 1.51

NDF (%) 47.15 a 45.22 b 43.31 b 38.15 c 30.23 d 1.52

Fleig Score 114.2 a 101.1 a 97.4 ab 86.4 b 58.5 c 16.2

Quality Class Excellent Excellent Excellent Excellent Medium

DM: Dry Matter, CP: Crude Protein, ADF: Acid Detergent Fiber, NDF: Neutral Detergent Fiber

Sole corn silage had the highest ADF and NDF contents. Corn silage is a major source of NDF for many dairy cows. Increasing the concentration of NDF of corn silage could mean that lesser amounts of other forages would have to be grown or purchased by the dairy farmer to meet NDF requirements (NRC, 2001). Similar results were reported by Tan et al. (2015), Demirel et al. (2008), Arslan et al. (2016); Çarpıcı et al. (2017). CONCLUSIONS In conclusion, according to averages of this research, the quality of silages which was found the better then other silages mixtures included corn. The silages made by sole alfalfa were found on medium quality. The crude protein rates of silage were increased by increasing of alfalfa rate in the mixtures. REFERENCES Alçiçek, A., K. Özkan (1997). Silo yemlerinde fiziksel ve kimyasal yöntemlerle silaj kalitesinin saptanması. Türkiye I. Silaj Kongresi, s: 241246, 16-19 Eylül, Bursa. Altınok, S., A. Genç, İ. Erdoğdu (2005). Farklı Ekim Şekillerinde Yetiştirilen mısır ve soyadan elde edilen silajlarda kalite özelliklerinin belirlenmesi. Türkiye VI.Tarla Bitkileri Kongresi, Cilt II, s.1011-1016. Anonymous (2006). Acid detergent and neutral detergent fiber using ANKOM’s fiber analyzer F200. Ankom Technology Corporation Fairport, NY. http://www.ankom.com/00_products/product_a200.shtml. Virginia. AOAC (1990). Official Methods of Analysis. 15 th edn. Association of Official Analytical Chemists. Arligton, Coors, J.G., 1996. Findings of the Wisconsin corn silage consortium. In: Proc. of Cornell Nutr. Conf.Feed Manuf., Rochester, NY. Cornell Univ., Ithaca, NY, pp. 20-28. 173

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Arslan, M., C. Erdurmuş, M. Öten, B. Aydınoğlu, S. Çakmakçı (2016). Mısır ile soyanın farklı oranlarda karıştırılmasıyla elde edilen silajlarda besin değerinin belirlenmesi. Anadolu Tarım Bilimleri Dergisi, 31(3), 417-422. Aykan, Y., V. Saruhan (2018). Farklı Oranlarda Silolanan yem bezelyesi (Pisum sativum L.) ve arpa (Hordeum vulgare L.) karışımlarının silaj kalite özelliklerinin belirlenmesi. Dicle Üniversitesi Veteriner Fakültesi Dergisi. 11(2):64-70. Çarpıcı, E. B., N. Tatar, Y. Öztürk, S. Erol, Ö. Arslan (2017). Farklı Oranlarda Mısır ve Şeker Mısırı Atığı ile Karıştırılan Yonca Silajında Kalitenin Belirlenmesi. KSÜ Doğa Bilimleri Dergisi, 20, 65-67. De Figueiredo, J. P. Marais (1994). The effect of bacterial inoculants on kikuyu silage quality. J.Agric. Sci. Cambridge 122, 53-60. Demirel, M., D. Bolat, S. Celik, Y. Bakici, S. Eratak (2008). Determination of fermentation and digestibility characteristics of corn, sunflower and combination of corn and sunflower silages. Journal of Animal and Veterinary Advences 7: 707-711. Demirel, M., F. Cengiz, S. Erdoğan, S. Çelik (2003). Farklı karışım oranlarında yapılan macar fiği ve sudan otu silajlarının kalitatif özellikleri ve rumende parçalanabilirlikleri üzerine bir çalışma. Turk J. Vet. Anim. Sci., 27 :853-859 Dube, D. (1995). The role of high quality dry season forage from mixed crop silages in the small-holder dairy farming sector of Zimbabwe: Dairy Development Programme perspective. Paper presented to a workshop on proposals for a mixed crop silage research project in the Southern African region. University of Zimbabwe, Harare. Filya, I. (2004). Nutritive value and aerobic stability of whole crop maize silage harvested at four stages of maturity. Animal Feed Science and Technology. Gelir, G. (2018). Diyarbakır koşullarında yetiştirilen yem bezelyesi (Pisum sativum subs arvense L.), tritikale ve karışımlarıın silaj kalite özelliklerinin belirlenmesi. Dicle Üniversitesi Fen Bilimleri Enstitüsü. Yüksek Lisans Tezi. Harrison, J.H., R. Blauwiekel, M. R. Stokes (1994). Fermentation and utilisation of grass silage. In:Symposium on Utilisation of Grass Silage. J Dairy Sci 77, 3209-3235. Jacobs, J.L., A. B. McAllan (1991). Enzymes as silage additives silage quality, digestion, digestibility and performance in growing cattle. J Grass and Forage Sci 46,63-73. Kilic, A. (1986). Silo Feed (Instruction, Education and Application Proposals). Bilgehan Pres, p. 327. Lujia, H., L. Xian, H. Shinichiro, N. Kazuhisa (2004). Effect of different additives on the quality of alfalfa silage. J.China Agric.Univ., 9:25-30. McDonald, P., A. R. Henderson, S. J. E. Heron (1991). Microorganisms. In: McDonald, P., Henderson, A.R., Heron,S.J.E. (Eds.), The Biochemistry of Silage, 2nd ed. Chalcombe Publications, Abersytwyth, UK, pp. 81–151 (Chapter 4). Mustafa, A., P. Seguin (2005). Effects of variety on chemical composition and ruminal nutrient degradability of forage soybean. J. Dairy Sci. 88(Suppl. 1):385. (Abstr.) National Research Council. (2001). Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC. Nauman, C., R. Bassler, (1993). Die Chemische Untersunhung von Futtermittein. Methodenbuch, Band III. VDLUFA-Verlag, Darmstadt. Polan, C.E., D. Stieve, J. Garrett (1998). Protein preservation and ruminal degradation of ensiled forage treated with heat, formic acid, ammonia or microbial inoculants. J. Diary Sci., 81: 765-776. Saricicek, Z., U. Kılıc (2011). Effect of Different Additives on the Nutrient Composition, in vitro Gas Production and Silage Quality of Alfalfa Silage. Asian Journal of Animal and Veterinary Advances.

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Saruhan, V., R. Demirel, M. S. Baran, D. Ş. Demirel (2011), Sarı Çiçekli Gazal Boynuzu (Lotus corniculatus) ve Arpanın (Hordeum vulgare) Farklı Düzeylerdeki Karışımlarının Silolanma Özelliklerinin Belirlenmesi, Anadolu Tarım Bilimleri Dergisi, 26(1):4045. Souma, K., P. Wang, F. Kanda, H. Igarashi, T. Masuko (2011). Influences of combination feeding of high moisture low quality grass-dominant mixture silage with corn silage on nutritive value and feed intake in sheep. Grassland Science 57: 18-22. Tan, M., H. Yolcu, Z. D. Gul (2015). Nutritive Value of Sunflower Silages Ensiled with Corn or Alfalfa at Different Rate. Journal of Agricultural Sciences. 21 (2015) 184-191. Titterton, M., B. V. Maasdorp (1997). Nutritional improvement of maize silage for dairying:mixed crop silages from sole and intercropped legumes and a long season variety of maize. 2. Ensilage. Animal Feed Science Technology 69:263-270. Topps, J. H., J. Oliver (1993). Animal Foods of Central Africa. Zimbabwe Agric. J. Tech. Handbook No.2. PublDick Elliott Memorial Trust, Harare, Zimbabwe. p.135. Vargas-Bello-Perez, E., A. F. Mustafa, P. Seguin (2008). Effects of Feeding Forage Soybean Silage on Milk Production, Nutrient Digestion, and Ruminal Fermentation of Lactating Dairy Cows. J. Dairy Sci. 91:229–235. Zhu, Y., C. S. Bai, X. S. Guo, Y. L. Xue, K. Ataku (2011). Nutritive value of corn silage in mixture with vine peas. Animal Production Science 51: 1117-1122.

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OXIDATIVE STRESS RELATED KIDNEY TOXICITY OF A GLYPHOSATEBASED HERBICIDE Elif ORUÇ1*, Derya KOCAMAZ1, Merve INCEMAN2, Gülfiliz GONLUSEN2 1

Cukurova University, Faculty of Arts and Science, Department of Biology, 01330, Adana, Turkey

Cukurova University, Medical Faculty, Department of Pathology, 01330, Adana, Turkey *Corresponding author’s e-mail: eozcan@cu.edu.tr, eliforuc@gmail.com

ABSTRACT Glyphosate is an efficient herbicide widely used worldwide in aquatic weed control in pools, canals and lakes. However, its toxicity to non-target organisms has not been fully elucidated. In this study, the toxicity of glyphosate-based herbicide in Oreochromis niloticus and the pathological alterations were evaluated. Fish were exposed to 4.6 ppm, 9.2 ppm and 18.4 ppm glyphosate concentrations for 96 hours. Biochemical parameters such as superoxide dismutase and catalase activities; glutathione, malondialdehyde and protein contents were measured in kidney tissues. Glutathione content increased at 18.4 ppm concentration while it remained at the control level in fish exposed to 4.6 ppm and 9.2 ppm glyphosate concentrations. Superoxide dismutase and catalase activities increased at 9.2 ppm and 18.4 ppm glyphosate exposed groups. Malondialdehyde increased at all exposure concentrations. Protein content decreased after herbicide exposures in order to compensate for metabolic stress or energy demand except for 4.6 ppm exposure. Remarkable damage, such as vacuolization and infiltration were found. The elevated lipid peroxidation and antioxidant activities in the present study suggest that reactive oxygen species-induced oxidative damage may be one of the main toxic effects of glyphosate in kidney tissue. This study also reveals that malondialdehyde content can be used as a biomarker in assessment pesticide toxicity in aquatic environments due to its early response at even low concentration levels. Keywords: Pesticide, Oxidative Stress, Oxidative Damage, Protein Carbonyl, Fish

INTRODUCTION Glyphosate-based herbicides are the most widely used commercial formulations worldwide in agriculture, particularly genetically modified soy and corn plants, to increase tolerance to pesticides (EPA, 1995). Glyphosate is the active ingredient of more than 750 different broad-spectrum pesticides (Mesnage et al., 2015). The half-life of glyphosate in aquatic systems is 7-14 days (Caux, 1998). Concerns about the safety of the aquatic environment and glyphosate toxicity in aquatic organisms have increased due to the high water solubility of glyphosate and its intensive use in agriculture (Ma et al., 2015). Roundup 96 hours LC50 value was determined as 22.19 ppm in Cyprinus carpio. It has been reported that glyphosate persistence is high in the environment and therefore more data are needed in human and environmental health risk assessments than acute toxicity (Bai and Ogbourne, 2016). 73.6 ± 28.2 g/L (Aris and Leblanc, 2011) in the blood tissue of people who were not directly exposed to pesticide effect, glyphosate poisoning was found in the blood 0.6- 7480 mg/L (Zouaoui et al., 2013) glyphosate. 176

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Glyphosate acts in 5-enol pyruvyl shikimate-3-phosphate synthase (EPSPS) inhibition in the shikimate pathway in amino acid metabolism in plants (Boocock and Coggins, 1983). Inhibition of EPSPS by glyphosate causes protein depletion and death. Since the lack of this biochemical pathway in vertebrates, glyphosate is thought to be safe in human and other mammals and the use of glyphosate-based herbicides has continued to increase. However, there are studies showing that it is present in contaminated soil and water, bioaccumulated in animals, poses risk and intoxication in humans (Ma et al., 2015). Furthermore, the use of glyphosatebased herbicides continues with the production of genetically modified organisms tolerant to these pesticides, and residues are found in non-genetically modified foods and feeds. Genetically modified plants tolerant to glyphosate-based herbicides cannot metabolize glyphosate and accumulate during development (Arregui et al., 2004). Oreochromis niloticus is an economically important nutrient source produced worldwide. It is often preferred as a model organism in ecotoxicology studies. Indiscriminate use, uncontrolled discharge or accidental spillage of herbicides in the aquatic environment causes harmful effects on fish populations and other organisms. Fish that are affected by pesticides are also important risk factors for human health because they are consumed as nutrients (Acquavella et al., 2004). The aim of this study was to investigate the potential neurotoxicity of glyphosate and the mechanism of its toxic effects in Oreochromis niloticus, an important component of the food chain in the aquatic environment. MATERIALS AND METHODS O. niloticus samples, 30-35 g in body weight and 10-12 cm in total length were taken from the local fish farm. Before the experiment fish were acclimated at least two weeks to the laboratory conditions. Water quality parameters were: temperature 20±20 ⸰C, pH 8.01±0.73, dissolved oxygen 7.40±0.22 mg/L, alkalinity 261 mg/L CaCO3 and hardness 285 mg/L CaCO3. Fish were fed twice a day with commercial fish food (2% of body weight, Pinar, Turkey). Sublethal environmental relevant concentrations of commercial formulation of glyfhosate (N(Phosphonomethyl) glycine, Roundup, Star, EC: 441 g/mL) were used in the experiment. Semistatic toxicity test was performed. The experiment was run for 96 hours. The experiments were carried out in three replications with 4 aquariums containing dechlorinated tap water and 18 fishes. At the end of 96 h expesoure period, kidney tissues were dissected out and stored at -80 C until analyses. Tissues were homogenized for 1,5 minutes in 0.1 M sodium-phosphate buffer pH 7.4 containing 1.17% KCl (Ultra-Turrax T 25, Janke & Kunkel). The supernatant obtained after centrifugation of homogenates at 10000 rpm for 30 minutes at 4 oC was used to determine the amount of biochemical parameters. AChE activity was meausured according to the method described by Ellman et al. (1961), SOD activity was meausured according to the method described by Mc Cord and Fridovich (1969), CAT activity was meausured according to the method described by Beutler (1975), GST activity was meausured according to the method described by Habig et al. (1974), the amount of GSH was meausured according to the method described by Beutler (1975), the amount of MDA was meausured according to the method described by Ohkawa et al. (1979), the amount of protein was measured by the method of Lowry et al. (1951). Data analysis were performed using SPSS soft ware for WINDOWS. Analysis of Variance (ANOVA) and SNK tests were used to examined the biochemical parameters in the treatment and control groups. o

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RESULTS The results of the histopathological examination of the fish kidneys after Roundup treatment is shown in Fig. 1. Remarkable damage, such as vacuolization and infiltration were found in the kidney of the glyphosate-exposed fish.

Figure 1. the histopathological examination of kidney tissue of O. niloticus following exposure to glyphosate for 96 hours. While the amount of protein, and SOD and CAT activities in the kidney tissue did not change compared to the control following exposure to 4.6 ppm glyphosate, it was found to be significantly higher in fish exposed to 9.2 ppm and 18.4 ppm concentrations than control group (Table 1).

Table 1. Protein content and, SOD, CAT activities in kidney tissue of O. niloticus following exposure to glyphosate for 96 hours.

Control

Protein mg/mL X±S 0,970.05 x

SOD U/mg protein X±S 19.510.41x

CAT U/mg protein X±S 49.314.01x

4.6 ppm

1.030.04 x

20.160.77x

49.462.67x

9.2 ppm

0.740.01 y

24.650.99y

78.462.69y

18.4 ppm

0.730.02 y

28.140.62z

80.532.14y

X±S: Results are given as meanstandard error. *: Letters x, y, and z show differences between glyphosate concentrations. p < 0:05 Interestingly, the opposite effect was determined on the amount of MDA as an indicator of lipid peroxidation and AChE activity as an indicator of neurotoxicity. It was determined that AChE activity decreased while malondialdehyde amount increased in all applications (Table 2). 178

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Glutathione S-transferase activity and GSH amount increased by about 30% in the kidney tissue of the fish exposed to 18.4 ppm concentration compared to the control (Table 2). Table 2. MDA and GSH contents and, AChE, GST activities in kidney tissue of O. niloticus following exposure to glyphosate for 96 hours. MDA nmol/mg protein

AChE U/mg protein

GSH U/mg protein

GST U/mg protein

X±S

Control

5.670.33x

0.0200.0006x

0.2330.01x

43.792.57x

4.6 ppm

6.750.22y

0.0180.0004y

0.2260.015x

45.260.86x

9.2 ppm

7.260.15y

0.0160.0006z

0.2150.003x

50.742.75x

18.4 ppm

8.450.38z

0.00150.0005z

0.3120.02y

60.732.63y

X±S: Results are given as meanstandard error. *: Letters x, y, and z show differences between glyphosate concentrations. p < 0:05 DISCUSSION Investigation of toxic effects of pesticide pollution is important for environmental risk assessment. Recently, studies that determine glyphosate toxicity are needed because of their indirect or direct health effects. High levels of glyphosate have been identified even in the blood tissue of people directly or indirectly exposed to the pesticide effect. Because fish develop similar biochemical responses to mammals and other vertebrates, they have become important model organisms for the assessment of toxic effects of chemical substances in the aquatic environment. these mechanisms should be elucidated with the study. Herbicides can cause oxidative stress in aquatic organisms by increasing the production of reactive oxygen species (ROS). Oxidative stress is defined as the imbalance between antioxidant defense activity and ROS production (Aruoma, 1998). Glyphosate is thought to induce oxidative stress in specific regions of the brain, such as the cerebral cortex and hippocampus (Cattani et al., 2014). Studies have shown that acute or chronic exposure to glyphosate triggers symptoms similar to Parkinson's disease (Barbosa et al., 2001; Wang et al., 2011). Changes in AChE activity, which is the key enzyme of the nervous system in fish, are widely used as biomarkers in determining the neurotoxicity of herbicides (Rodríquez-Fuentes and Bouchot, 2004). It was determined that AChE activity decreased while malondialdehyde amount increased in all applications. Various biomarkers are used to evaluate the ecotoxicological effects of herbicides on fish. Among these, the most commonly used antioxidant enzymes responsible for the detoxification and biotransformation of herbicides. Superoxide dismutase (SOD), catalase (CAT) and glutayon Stransferase (GST) are the most widely used antioxidant and biotransformation enzymes among the extensible fields required (Di Giulio and Hilton, 2008). Herbicide-induced stress can cause an induction in the activity of antioxidant enzymes and, the biochemical responses generated in the organism may vary depending on the duration and intensity of pollutant exposure and may vary between different species (Bonifacio et al., 2016). SOD and CAT activities in the kidney tissue did not change compared to the control following exposure to 4.6 ppm glyphosate, 179

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it was found to be significantly higher in fish exposed to 9.2 ppm and 18.4 ppm concentrations than control group (Table 1). Glutathione (GSH) is the most important cellular non-enzymatic antioxidant that plays a central role in the detoxification of herbicides. Glutathione S-transferase activity and GSH amount increased in kidney and liver tissues of the fish exposed to 18.4 ppm concentration compared to the control. Herbicides that induce GSH consumption by GSH conjugation or oxidation of GSH to GSSG cause a change in the cellular defense mechanism of the organism against contaminants (Della Morte et al., 1994). Oxidized free radicals can attack polyunsaturated fatty acids and proteins, causing loss of function or damage to cells and tissues (Ayala et al., 2014). Malondialdehyde (MDA) is widely used as a biomarker of lipid peroxidation, which is considered an important molecular mechanism of pesticide toxicity. CONCLUSIONS The use of Roundup, commercial formulations of glyphosate, can induce neurotoxicity, and oxidative stress that causes tissue damage in O. niloticus due to oxidative stress in kidney tissue. This is a serious issue as it can affect the entire food chain and produce many unwanted changes. Assessing the sublethal effects of herbicides will help to advise environmental organizations on the effects of herbicide application on non-target species. Acknowledgements: This study was supported by a grant from Cukurova University (FBA2018-11335). REFERENCES Acquavella, J.F., Alexander, B.H., Mandel, J.S., Gustin, C., Baker, B., Chapman, P., Bleeke, M. (2004). Glyphosate Biomonitoring for Farmers and Their Families: Results from the Farm Family Exposure Study. Env. Health Pers., 112: 3, 321-326. Aris A., Leblanc, S. (2011). Maternal and fetal exposure to pesticides associated to genetically modified foods in Eastern Townships of Quebec, Canada. Reprod Toxicol. 2011 May;31(4):528-33 Arregui, M.C., Lenardón, A., Sanchez, D., Maitre, M.I., Scotta, R., Enriqui, S. (2004). Monitoring Glyphosate residues in transgenic Glyphosate‐resistant soybean. Pest Manag. and Sci., 60:163-166. Aruoma, O.I. (1998). Free Radicals, Oxidative Stress, and Antioxidants in Human Health and Disease. JAOCS, 75:2. Ayala, A., Munoz, M.F., Arguelles, S. (2014). Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydoxy-2-Nonenal. Oxidative Medicine and Cellular Longevity, 1-31. Bai, H.S. and Ogbourne, S.M. (2016). Glyphosate: Environmental Contamination, Toxicity and Potential Risk to Human Health via Food Contamination. Env. Sci. and Pollut. Res., 23, 18988-19001 Barbosa, E.R., Leıros Da Costa, M.D., Bacheschi, L.A., Scaff, M., Leite, C.C. (2001). Parkinsonism after Glycine-Derivate Exposure. Mov. Dis. J., 16: 565-568. Beutler, E. (1975). Red Cell Metabolism. A Manual of Biochemical Methods. Grune and Stration, New York, London, 67-69. Bonifacio, A.F., Cazenave, J., Bacchetta, C., Ballesteros, M.L., Bistoni, M.L.A., Ame, M.V., Bertrand, L., Hued, A.C. (2016). Alterations in the General Condition, Biochemical Parameters and Locomotor Activity in Cnesterodon decemmaculatus Exposed to 180

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Commercial Formulations of Chlorpyrifos, Glyphosate and Their Mixtures. Ecol. Indic., 67: 88-97. Boocock, M.R., Coggins, J.R. (1983). Kinetics of 5-enol pyruvyl shikimate-3-phosphate synthase Inhibition by Glyphosate. FEBS Letters, 154: 127-133. Cattani, D., Cavalli, V.L.O, Rieg, C.E.H., Domingues, J.T., Dal-Cim, T., Tasca, C.I., Silva, F.R.M.B., Zamoner, A. (2014). Mechanisms Underlying the Neurotoxicity Induced by Glyphosate-Based Herbicide in Immature Rat Hippocampus: Involvement of Glutamate Excitoxicity. Toxicol., 34-45. Caux, P.Y., Kent, R., Fan, G., Grande, C. (1998). Canadian Water Quality Guidelines for Linuron. Env. Toxicol. W. Quality, 13:1-41. Della Morte, R., Villani, G.R., Di Martino, E., Squilacioti, E., De Marco, L., Vuotto, P., Belisario, M.A., Staiano, N. (1994). Glutathione Depletion Induced in Rat Liver Fractions by Seven Pesticides. Bolletino della Societa Italiano di Biologica Sperimentale, 70: 185-192. Di Giulio, R.T., Hilton, D.E. (2008). The Toxicology of Fishes. CRC Press, Taylor and Francis, pp. 1096. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity. Biochem. and Pharmacol., 7: 88-95. EPA, 1995, In: EPA (Ed.), Linuron: Reregistration Eligibility Decision (RED). US EPA. Habig, W.H., Pabst, M.J., Jakoby, W.B. (1974). Glutathione S-transferases. The First Enzymatic Step in Mercapturic Acid Formation. J. Biochem., 25(249): 7130-7139. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (1951). Protein Measurement with Folin Phenol Reagent. J. Biol. Chem., 193: 265-275. Ma, J., Bu, Y., Li, X. (2015). Immunological and Histopathological Responses of the Kidney of Common Carp (Cyprinus carpio L.) Sublethally Exposed to Glyphosate. Env. Toxicol. . Pharmacol., 39: 1-8. McCord, J.M., Fridovich, I. (1969). Superoxide Dismutase; an Enzymatic Function for Erythrocuprein (Hemocuprein). J. Biochem., 244: 6049-6053. Mesnage, R., Defarge, N., Spiroux de Vendomois, J., Seralini, G.E. (2015). Potential Toxic Effects of Glyphosate and its Commercial Formulations. Food Chem. Toxicol., 84, 133-135. Ohkawa, H., Ohishi, N., Tagi, K. (1979). Assay for Lipid Peroxides in Animal Tissues by Thiobarbituric Acid Reaction. Anal. Chem., 95: 351-358. Rodriquez-Fuentes, G., Gold-Bouchot, G. (2004). Characterization of Cholinesterase Activity from Different Tissues of Nile Tilapia (Oreochromis niloticus). Mar. Env. Res., 58: 505-509. Wang, G., Fan, X.N., Tan, Y.Y., Cheng, Q., Chen, S.D. (2011). Parkinsonism after Chronic Occupational Exposure to Glyphosate. Parkinsonism Relat. Disord.. 17: 486-487. Zouaoui, K., Dulaurent, S., Gaulier, J.M., Moesch C., Lachatre, G. (2013). Determination of Glyphosate and AMPA in Blood and Urine from Humans: About Cases of Acute Intoxication. Forensic Sci. Int., 226, pp. 20-25.

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THE EFFECTS OF DIFFERENT NITROGEN DOSES ON SOME AGRICULTURAL CHARACTERISTICS OF PHASELIA (PHACELIA TANACETIFOLIA BENTHAM) Mevlüt TÜRK1, Mehmet ALAGÖZ1* 1

Isparta Applied Science University, Agricultural faculty, Department of Field Crops, Isparta, Turkey *Corresponding Author: mehmetalagoz7@gmail.com ABSTRACT

This research was conducted to investigate the effects of five different nitrogen doses (0, 40, 80, 120 and 160 kg ha-1) on some agricultural characteristics of phaselia (Phacelia tanacetifolia Bentham) at the experimental area of Agricultural Faculty of Isparta University of Applied Science under Isparta ecological conditions in 2017. This research was conducted in randomized block design with three replications. Plant height, first flowering time, 50% flowering time, number of racemes per plant, number of flowers per raceme, 1000 seed weight and seed yield were investigated in this research. According to the results of the research, nitrogen doses had a significant effect on all properties. Nitrogen applications increased plant height, beginning of flowering period, 50% flowering, number of racemes per plant, number of flowers per raceme, 1000 seed weight and seed yield. Keywords: Phaselia, Nitrogen doses, Seed yield, 1000 seed weight

INTRODUCTION Phacelia (Phacelia tanacetifolia Benth) is an annual herbaceous plant belonging to the family Hydrophyllaceae. The flowers are violet-blue color. Floral scents can be good attractants that attract bees. The whole plant is covered with dense, short hairs, which is resistant to drought. One plant has more than 5000 flowers. In addition to production in order to obtain seeds is used for silage, green manure (Sağlamtimur ve Baytekin, 1993). Phacelia is an excellent bee pasture. Under bee pasture is understood the whole flora of a place visited by bees. Phacelia is one of the honey plants (Kumova ve Korkmaz, 2002; Gilbert, 2003; Leszczyńska, 2012). Phacelia has a large agro-technical importance. Strong spindly root penetrates over 1 m depth, which improves soil quality, and dying leaves significant amounts of organic matter and nitrogen bound, which makes it an excellent choice for improving poor soils. At the end of flowering, the plant can be plowed making it suitable for green fertilizers. Many studies have have phacelia declared nematocidal plant, because it is very useful for the preservation of soil hygiene. It is suitable for growing in organic production. Seed production phacelia is very profitable and can serve as a generator of economic development. Phacelia has great significance for beekeeping, because it is flowering, when the period without pasture, which lasts until the sunflower. With sowing phacelia beekeepers to have a safe pasture. For successful commercial beekeeping honey plants are very important condition for achieving high yields. The objective of this research was to determine the effects of different rates of nitrogen fertilizers on some agricultural characteristics of phaselia (Phacelia tanacetifolia Bentham).

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MATERIAL AND METHODS The research was performed at the experimental area of Agricultural Faculty of Isparta University of Applied Science under Isparta ecological conditions in 2017. The major soil characteristics, based on the method described by Rowell (1996) were as follows: the soil texture was clay-loam (clay: 31.2%, silt: 45.1%, sand: 23.7%); organic matter was 1.1% by the WalkleyBlack method; total salt was 0.3%; lime was 7%; sulphur was 12 mg kg–1; extractable P by 0.5N NaHCO3 extraction was 3.3 mg kg–1; exchangeable K by 1N NH4OAc was 119 mg kg–1; pH was 7.1 in soil saturation extract. The experiments were evaluated in a randomized complete block design with three replications. Sowing was done by hand on 11 March in 2017. Seeding rates were 15 kg ha-1. Plot sizes were 1.8 x 5 m = 9 m2. Five different nitrogen doses (0, 40, 80, 120 and 160 kg N ha1 ) were applied. Plant height, first flowering time, 50% flowering time, number of raceme per plant, number of flower per raceme, 1000 seed weight and seed yield were investigated in this research. The data was analysed together using the Proc GLM (SAS 1998). Means were separated by LSD at the 5% level of significance. RESULTS AND DISCUSSION Nitrogen applications had significant effects on plant height, first flowering time, 50% flowering time, number of raceme per plant, number of flower per raceme, 1000 seed weight and seed yield (Table 1). Increasing N fertilization rates resulted in an increase in plant height of phacelia. The highest plant heights were obtained by the 120 and 160 kg ha-1 nitrogen doses. Similar results were reported by Dağ (2013), Erturk (2019). First flowering time was delayed as nitrogen doses increased. The first flowering was realized 46.7 days after sowing in control application, but it was 55 days after sowing in 120 kg nitrogen application. Similar results were reported by Dağ (2013). Table 1. Means plant height, first flowering time, 50% flowering time, number of raceme per plant, number of flower per raceme, 1000 seed weight and seed yield. Nitrogen doses (kg ha-1)

Plant height (cm)

First flowering time (day)

0 51.1 d 46.7 c 40 55.3 c 50.0 b 80 63.4 b 51.7 b 120 69.2 a 55.0 a 160 68.4 a 55.0 a Sources of variations Block ns ns Nitrogen ** ** Doses

50% flowering Time (day)

Number Number of 1000 Seed Seed yield of raceme flower per weight (g) (kg ha-1) per plant raceme

55.8 c 60.7 b 61.8 b 65.9 a 66.1 a * **

4.1 c 4.8 b 4.9 b 5.4 a 5.3 a * **

183

16 c 19 b 22 a 21 a 22 a ns **

1.51 d 1.68 c 1.82 b 1.96 a 1.94 a * **

129.3 e 157.2 d 179.2 c 192.4 b 208.3 a * **

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The 50% flowering time was realized 55.8 days after sowing in control application, but it was 65.9 and 66.1 days after sowing in 120 and 160 ha-1 kg nitrogen applications. Similar results were reported by Dağ (2013). Number of raceme per plant increased as nitrogen doses increased. Although average 4.1 raceme per plant were detected in the control application, an average of 5.4 and 5.3 raceme per plant were obtained in 120 and 160 kg ha-1 nitrogen applications. Similar results were reported by Dağ (2013). Number of flower per raceme increased as nitrogen doses increased. Although average 16 flower per raceme were determined in the control application, an average of 22, 21 and 22 flower per raceme were obtained in 80, 120 and 160 kg ha-1 nitrogen applications. Similar results were reported by Dağ (2013). The lowest 1000 seed weight was obtained by the control treatment, while the highest 1000 seed weights were observed in the 120 and 160 kg ha-1 nitrogen applications. Increasing N fertilization rates resulted in an increase in 1000 seed weight of phacelia. The 1000 seed weights we obtained are similar to those obtained by Kızılşimşek and Ateş (2004). The highest seed yield (129.3 kg ha-1) was obtained by the 160 kg ha-1 nitrogen treatment, while the lowest seed yield (208.3 kg ha-1) was observed in the controls treatment. Increasing N fertilization rates resulted in an increase in seed yield of phacelia. Seed yield in our study was found to be lower than other studies (Başbağ et al. 2001; Kızılşimşek and Ateş, 2004; Genç Lermi and Palta, 2016). The most important reason is that we do planting in the spring. CONCLUSION According to the results of the research, nitrogen doses had a significant effect on all properties. Nitrogen applications increased plant height, beginning of flowering period, 50% flowering, number of raceme per plant, number of flower per raceme, 1000 seed weight and seed yield. At the end of the this research conducted in Mediterranean conditions of Turkey, 120 and 160 kg ha–1 nitrogen fertiliser are recommended for high seed yield in phaselia. REFERENCES Başbağ, M., V. Saruhan, İ. Gül (2001). Diyarbakır koşullarında farklı tohumluk miktarlarının arıotu (Phacelia tanacetifolia Benth.)’nda bazı tarımsal özellikler üzerine etkisi, GAP 2. Tarım Kongresi, 24-26 Ekim 2001, Şanlıurfa, s: 985-992. Dağ, V. (2013). Farklı azot dozlarının arıotu (Phacelia tanacetifolia Bentham )’nda verim ve bazı tarımsal karakterlere etkisi. Yüzüncü Yıl Üniversitesi Fen Bilimleri Enstitüsü Tarla Bitkileri Anabilim Dalı, 48s. Ertürk, A. (2019). Kahramanmaraş Koşullarında Farklı Azot ve Fosfor Dozlarının Arı Otu (Phacelia tanacetifolia Bentham) 'nda Verim Ve Bazı Tarımsal Karakterlere Etkisi. Kahramanmaraş Sütçü İmam Üniversitesi Fen Bilimleri Enstitüsü. Yüksek Lisans Tezi. 32s. Genç Lermi A. and Ş. Palta, (2016). Arı Otu Bitkisinin Sonbahar Ekim Periyodunda Farklı Ekim Zamanlarının Tohum Verimi ve Verim Komponentleri Üzerine Etkileri. YYÜ TAR BİL DERG (YYU J AGR SCI) 2016, 26(3): 366-371. Gilbert, L. (2003). Phacelia tanacetifolia: what we know about its suitability as an insectary plant and cover crop in the Mid-Atlantic region. Small Farm Success Project, Sustainable Agricultural Systems Lab, USDA, USA, 1. 184

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Kızılşimşek, M., F. Ateş (2004). Kahramanmaraş şartlarında arı otunun (Phacelia tanacetifolia Bentham) değişik ekim zamanlarındaki çiçeklenme seyri ve arı merası olarak değerlendirilmesi. KSÜ Fen ve Mühendislik Dergisi 7(1):96-103. Kumova, U., and A. Korkmaz (2002). Arıcılık Açısından Arıotu (Phacelia tanacetifolia Bentham) Bitkisinin Önemi ve Bu Konuda Ülkemizde Yapılan Çalışmalar. Uludağ Arıcılık Dergisi. Leszczyńska, D. (2012). Facelia w poplonie. Farmer, 9, 76-78. Sağlamtimur, T. and H. Baytekin (1993). Arıcılık İçin İdeal, Silaj Üretimine Uygun Bir Bitki: Arıotu, Teknik Arıcılık Dergisi, 40: 16-17. SAS institute (1998). iNC SAS/STAT users’ guide release 7.0. Cary, NC, USA.

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TRADITIONAL CHEESE DESSERTS AND KÜNEFE Nuray GÜZELER1, Ahmet Refik ÇAY1, Firuza KOBOYEVA*1 1

Cukurova University Faculty of Agriculture Department of Food Engineering, Adana, Turkey *Corresponding author’s e-mail: firuza.koboyeva@gmail.com

ABSTRACT Cheese which has rich nutrient content is used in making various desserts in Turkish cuisine. Traditional cheese desserts made in our country can be listed as Höşmerim, Kemalpaşa (also known as a Hayrabolu, Curd and Biga dessert), Cheese Halva, Kadayıf, and Künefe dessert. Antakya Künefe which has an important place among these desserts received the geographical indication certificate in 2007. On the other hand, an application was made in 2018 for the geographical indication of Künefe cheese used in the traditional dessert of Antakya. Unsalted fresh cheese is generally used in desserts. For example, Künefe cheese used in making the Künefe dessert is a soft type cheese that contains no additives and which consumed fresh and without ripening. The most important features to be considered in the structure of this cheese are: creep ratio, odor, smooth and bright structure, hardness-softness rates. The national adoption of the Künefe also contributed to an increase in the demand of Künefe cheese. This cheese is mostly produced in the Eastern Mediterranean Region, especially in Antakya. The requirement for this cheese in other regions of Turkey is provided from Hatay. According to the studies, besides the production of fresh Künefe cheese made from raw milk, there are also the production of salted Künefe cheese (chestnut cheese), pipe type Künefe cheese, made with adding melting salt and culture added Künefe cheese made from pasteurized milk, in Hatay. Pipe type Künefe cheese is usually used in the production of frozen Künefe dessert. However, Künefe cheese made from pasteurized milk is not too much preferred in the dessert because of the negative melting feature. Apart from Hatay, there are also some dairy factories produced Künefe cheese in other cities. In these plants, for the production of Künefe cheese pipe type Künefe cheese and culture added Künefe cheese production methods are generally preferred. These cheeses are produced by dry boiling of curd with adding melting salt and it is generally used in making desserts and patties in restaurants. In this research, the production and characteristics of cheese desserts made in Turkey were given. Also, different production methods of Künefe cheese which is used in the making of the most important dessert of Hatay such as Künefe were investigated. Keywords: Turkey, Traditional desserts, Künefe, Künefe cheese INTRODUCTION Cheese is the food with the most variety among milk and dairy products in the world. When considering the definition of cheese in Turkish Food Codex: cheese; is obtained by coagulating milk using an appropriate coagulant and separating whey from the curd, with different hardness and fat contents, unsalted or salted with brine or dry salting, with or without starter culture, with cooked curd or without cooked curd, flavored or without flavor, and it is produced in accordance with the necessary techniques. It is a dairy product that is consumed before or after ripening and has its own characteristics according to its type (TFC, 2015). According to various sources, there are about 4000 varieties of cheese around the world, this number is around 200 186

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in Turkey (Say, 2008). Each cheese type has its own production method. The raw material used in the production of cheese, technological process applied to this raw material and due to factors such as ripening time the general physical properties and quality values of cheese may change (Elmalı and Uylaşer, 2012). Among these 200 cheese, the most consumed cheeses are White, Kashar and Tulum (Say, 2008). However, there are many types of cheese that are traditionally produced and consumed. In Turkey, as is done all over the world, in addition to the simple consumption of cheese, it is used by adding to food and desserts. The cheeses produced by traditional methods are generally produced in small dairy or family factories, are limited to the region where they are produced and are limited in the national and international markets because they are not well-known (Akpınar-Bayizit et al., 2009; Kayış and Yaman, 2018). Therefore, the desired quality and standards in these cheeses cannot be obtained (Tan and Erturk, 2002). The cuisine of a country is one of the most important cultural indicators of that country. It is possible to find different types of food due to regional differences in Turkish cuisine, which has a history of thousands of years, is among the three richest cuisines in the world and holds the distinction of being the world’s most nutritious cuisine. Especially the climate of the region and the agricultural products obtained shape the culinary culture of the region and the richness of the culinary variety. This has led to the development of the concept of local cuisine (Şanlıer et al., 2008; Akpınar-Bayizit et al., 2010; Cömert, 2014; Anon., 2018a). For example, while herbal diet mainly in the Mediterranean region of Turkey is much more widespread, meals made with meat and meat products are consumed more in Eastern and Southeastern Anatolia (Birer, 1991; Baysal, 1993; Sürücüoğlu and Akman, 1998). Although most of the Turkish cuisine consists of soups, salads, appetizers, drinks, and other foods, traditionally made desserts also have a great role in the worldwide recognition of Turkish culture. In Turkey, custard, sütlaç (rice pudding), kazandibi (white pudding with the blackened surface), keşkül (milk and almond pudding), profiteroles and gullac (güllaç, rose pudding) stand out as some important desserts obtained from milk, while some desserts are also made using cheese (Seçim, 2017). Some of the desserts with cheese have come from the Ottoman Empire to the present day, while some of them have been converted to Turkish cuisine from other cultures and are still consumed. Studies have reported that desserts obtained from milk and dairy products are an important factor in reducing the risk of various diseases with their rich nutrients. The most important cheese desserts are Höşmerim, Kemalpaşa (Kemalpasha) dessert known as Hayrabolu dessert, Lor and Biga dessert; Cheese halva; Kadayıf and Künefe desserts. In addition, desserts such as cheese lokma, sweet curd with honey or jam are also made. While these are naturally consumed extensively in the country, some of the desserts are also made and consumed in some parts of the Middle East and Europe (Özer et al., 2012). Unsalted fresh cheese is generally used in dessert production. For example, Künefe cheese used in Künefe dessert is a soft type cheese that does not contain any additives and is consumed without ripening. General information is given below about cheese desserts produced in our country and Künefe cheese used in the production of Künefe dessert, which has the most important place among these desserts. The Cheese Desserts Produced in Turkey Höşmerim It is estimated that the first Höşmerim production was made by Anatolian nomads coming from the migrations from Central Asia (Ünsal, 2003). This sweet dessert, which has also decorated the tables of Ottoman cuisine in history, is one of the leading Turkish desserts. In particular, it is produced in Turkey’s Aegean, Black Sea, and Central Anatolia regions (Cokal et al., 2012; Arı and Güzeler, 2015). Today, it is mostly produced in Balıkesir, Bursa, 187

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Çanakkale, Tekirdağ, Kırklareli and İzmit provinces (Ünsal, 2003). Unsalted fresh cheese, sugar, semolina and egg yolk are used in making, while unsalted fresh cheese, sugar, and flour are used in Tekirdağ region. In the following, the production technique of the Höşmerim is described in detail. There are different types of Höşmerim dessert. For example, the Höşmerim, which is usually light yellow in the vicinity of Balıkesir, is baked before being served in the Çanakkale region and a crispy crust forms on it (Anon., 2018b). While the large hammered hazelnuts or walnuts are sprinkled on unbaked Höşmerim, the oven-baked Höşmerim is served with ice cream. Traditional Höşmerim production; the milk is fermented at 25-30 ℃ (2 tablespoons of rennet is added to 5 kg of milk). After the formation of the curd (1-1.5 hours later), it is broken with a wire whisk and egg is added. The curd is continuously mixed and cooked until it boils and sugar is added in the meantime. When the temperature reaches 85-90 ℃, the semolina is added and the stirring and cooking continue until it thickens. When it stiffens, the cooking process is ended and filled in packages and cooled (Aydın et al., 2009; Seçim, 2017). However, Höşmerim production technique varies according to the regions (Anon., 2017; Anon., 2019a). “Höşmerim” is called “cheese halva” in some regions, and for making this cheese dessert, 1 cup of fat unsalted fresh cheese, 1 teaspoon of baking powder and 1 egg yolk are cooked on low heat until the fat comes out and gets honey color. After consistency, 1 tea glass of semolina, 1 tea glass of flour and 1 glass of sugar are added and mixed until the sugar melts. The dessert is divided into bowls or poured into a heat-resistant oven. Optionally, it can be cooked at 180℃ until it is browned or it can be consumed when the Höşmerim is cooled slightly. In this kind of Höşmerim, fat unsalted Dil cheese is used as fresh cheese. Small amounts of saffron can also be added to make the Höşmerim more yellow (Anon., 2018b). Cheese Halva Cheese halva is a traditional dessert consumed in large amounts in the Aegean, Marmara, Thrace, and Central Anatolia regions, especially in the provinces of Tekirdağ and Çanakkale (Kurultay et al., 2008a; Güven and Demir, 2010; Arı and Güzeler, 2015). It is generally consumed with or without bread at breakfast (Sengul and Ertugay, 2006). It is called cheese halva because it is made from sweet flour and milk cream (Sengul and Ertugay, 2006). The main components of cheese halva are fresh, unsalted and plain cheese as in Höşmerim dessert (Kurultay et al., 2008a). It is reported to have an important place in human nutrition in terms of the serum proteins it contains (Eralp, 1974; Arı and Güzeler, 2015; Seçim, 2017). There are differences in the production techniques of this dessert according to the regions too. In general, cow or sheep milk is pasteurized at 90 – 95 ℃ in cheese halva production. The milk cooled to 35 ℃ is fermented with rennet enzyme in 40 minutes to coagulate. The resulting curd is broken and rested for a while. Then the curd is filtered and cooked for 5 minutes. The cheese, which are left to boil over low heat, begin to stretch and melt. When the cheese starts to boil, 200 grams of flour is added for each 1 kg of cheese. The mixture is stirred continuously during the cooking process. Finally, 300 grams of sugar per kg of cheese is added during cooking. When sugar is added, it is continuously mixed to make the mixture homogeneous. After mixing the mixture for 4-5 minutes, the cheese halva becomes ready (Kurultay et al., 2008b; Aydın et al., 2009; Seçim, 2017). The production method used in Adıyaman differs from the method described above. 1.5 kg of sugar is added to 2 liters of water to prepare the sherbet. Approximately 1 kg of semolina is added to the boiling sherbet. To prevent agglomeration, the sherbet is continuously mixed while adding the semolina. After cooking the semolina, 0.5 - 1 kg of cheese is added to the mixture. The cheese used here is the special Çeçil cheese produced in Adıyaman. After adding this type cheese, the mixture is mixed and cooked over low heat. After the cheese is melted, butter is 188

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added and the cheese halva is cooked on low heat for 5 minutes. Thus, cheese halva becomes ready. Cheese halva is served with ice cream or Pistachio in various restaurants in Adıyaman (Anon., 2018c). In making Thracian cheese halva, first 0.5 kg of unsalted fresh crumbly cheese is put to a cooking pot and then 250 g of butter is added. After a little stirring, 1 egg yolk is added and mixed continuously until dissolved. When the ingredients are thoroughly melted, 400 g of flour, 100 g of semolina and 750 g of sugar are added and stirring must be continued. After boiling, the lid is kept closed for 15 minutes. After 15 minutes the cheese halva is ready to be served. Ice cream can be served on request (Anon., 2016a). It has been reported that the addition of salt in the production of cheese halva facilitates melting and provides a standardized effect (Kurultay, 2005). Sengul and Ertugay (2006) investigated some chemical and microbiological properties of 9 cheese halva samples collected from various villages of Erzurum province. The values found by the researchers are shown in Table 1. As a result of microbiological analysis, 44.4% of cheese halva samples did not contain coliforms and 53.6% of samples were reported to have 5.1×103 cfu/g coliform bacteria. Table 1. Chemical and microbiological properties of Cheese halva (Sengul and Ertugay, 2006). Chemical properties Dry matter Fat Protein Carbohydrate NaCI Titratable acidity (%) Ash (%) pH Microbiological properties Total aerobic mesophilic bacteria (TAMB) Yeast and mould Yeast Lactic acid bacteria (LAB) Proteolytic bacteria Lipolytic bacteria

(kg/100 kg) 84.32 37.44 13.75 38.56 0.58 0.21 1.49 5.30 cfu/g 4.9 × 107 6.4 × 104 3.5 × 103 7.6 × 106 4.6 × 103 8.6 × 103

Kemalpaşa Dessert Kemalpaşa dessert is also a traditional Turkish dessert and it was first produced in the Kemalpaşa district of Bursa province between 1960-1962 (Uzel et al., 2017). This dessert, which is an important food for the local people’s economy, was later described and produced by the surrounding provinces and districts (Uçurum et al., 2016). There are 13 production facilities defined as SME (Small and Medium Size Enterprises) for the production of Mustafakemalpaşa cheese dessert (Uzel et al., 2017). According to the Turkish Patent Institute, the Kemalpaşa dessert is prepared by adding the wheat flour and pastry type, semolina, fresh unsalted cheese, egg, drinking water, baking powder, and any other additive when necessary, shaping and cooking of the dough. This dessert is a semi-product known as sweet not boiled with sugar sherbet (TPI, 2002).

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This dessert can be produced by adding oil to the egg and using fresh yogurt instead of saltfree cheese. Vanilla is also used to enhance the sweet smell when baking soda is added. TS 12102 Kemalpaşa Dessert standard canceled in 2011 and the same year it was replaced by TS 13470 Dough Desserts-Desserts Ready to Add to Sherbet standard. In 2017, amendments were made to this standard (TSI, 2011; TSI, 2017). However, the definition of various desserts is very general in this standard and the raw materials that should be used in the traditional production of the Kemalpaşa dessert are not clearly stated. In other words, it is seen that the chemical properties specified in the standard are not sufficient to determine the quality criteria of the dessert. This situation causes unfair competition for the producers, and the consumers have to consume products that are not of the same quality and do not have their characteristics (Uçurum et al., 2016). The most important feature of Kemalpaşa dessert, which has been finalized and registered as of 13.01.2002 as per Article 12 of the Decree-Law No. 555 on the Protection of Geographical Indications published in the Official Gazette No. 24639, distinguishes it from other cheese desserts; the cheese used in this dessert is unsalted cheese produced by special methods from cow’s milk grown in Mustafakemalpaşa region (TPI, 2002). According to the Turkish Patent Institute, the production method of salt-free cheese used in this dessert is as follows; the milk of cows raised in Mustafakemalpaşa region is heated to 65℃ in stainless steel and double-walled boilers and then cooled to 34 ℃ through closed system coolers circulating cold water in the pipes. The cooled milk is taken to the fermentation table and then it is fermented by adding rennet. After 1 – 1.5 hours, the resulting cheese is filtered and the whey is removed. The remaining portion is brought to the minced cheese section to make additions (TPI, 2002). Kemalpaşa dessert is a semi-processed dessert type as stated in TPI and its commercial importance is increasing in our country. According to TPI, the production technique and components of Kemalpaşa dessert are as follows. The dough is obtained by adding flour, egg, semolina, baking soda, baking powder into the cheese obtained by the method described above. After that the dough is shaped using a cutting machine. According to the geographical registration document (TPI, 2002), the ratio of the diameter of the desserts in the package to the thickness should be between 0.5 and 1.5 cm. The shaped dough is then baked in a oven heated to 300 ℃. The baked desserts are removed from the oven and kept outside for a while and then dried in an oven set to 100 – 150 ℃. Desserts made in this way should be stored in humid conditions and the sunless environment with a temperature of 20-25 ℃. Today, many companies produce fresh dessert (single baked) in the district boundaries, and in other provinces, the dessert is prepared by double baked for long-term durability. While the single baking process is carried out at 280 – 300 ℃ for 15-20 minutes, the double baking process is cooked at 280 - 300 ℃ for 15 – 20 minutes as well as 45 - 60 minutes at 130 - 150 ℃. The shelf life of desserts under double baking is 6 months, while the shelf life of desserts under single baking is 3 days. However, consumers prefer single baked desserts more because of their freshness (Guldas et al., 2010). Recipe of Kemalpaşa dessert made at home among the people: 1 egg, 1 glass of oil is transferred into the mixing bowl. Then 1 cup of yogurt is added and 1 cup of semolina, one pack of vanilla and baking soda is added. Then 1 cup of flour is added and the mixture is mixed. Knead until a non-stick paste is obtained. Walnut-sized balls are taken from the dough and transferred to baking paper spread film tray and cooked at 180 ℃ in oven for 25 minutes until golden brown. After that cooled dessert is boiled in boiling sherbet for 5 minutes or preprepared cold sherbet poured over the hot dessert. The Kemalpaşa dessert is sprinkled with coconut or served with clotted cream in winter and ice cream in summer (Anon., 2014; Anon., 2016b). 190

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Guldas et al. (2010) studied the shelf life of Mustafakemalpaşa cheese desserts (single baking at 280 - 300 ℃ degree) coated with edible films such as k-carrageenan, chitosan, corn zein and whey protein concentrate. As a result of the research, the researchers determined deterioration in the samples coated with chitosan and k-carrageenan during 3 days storage as determined in uncoated samples. The normal shelf life of single baked desserts was extended to 10 days in samples coated with whey protein concentrate or corn zein without significant loss of sensory properties. In another study, some quality characteristics of 60 different Kemalpaşa desserts from 13 different dairy factories in 3 different times in Mustafakemalpaşa district, and 30 Kemalpaşa desserts from 10 different dairy factories operating in different regions were examined. The researchers found that the protein content of Kemalpaşa desserts was in the range of 15.2128.8%, the total fat content was 6.96-24.08%, the total acidity was 0.83-1.21%, the peroxide value was 4.94-6.57 meq/kg, the starch value was 39.75-61.06% and reported that β-sitosterol levels reach up to 15% (Uçurum et al., 2016). Curd Cheese Dessert It is known that curd cheese dessert culture was carried by families who came from the Greek island of Crete in the 1920’s and settled in Ayvalık, Balıkesir. Curd cheese dessert is one of the most famous recipes of Ayvalık and Aegean cuisine with its light consistency and mouthspreading flavor (Anon., 2019b). For the production of curd cheese dessert: 500 grams of unsalted curd cheese, which is passed through a mixer, is whipped with 4 eggs. Then one cup of sugar, one cup of flour, one cup of rice flour, one lemon grated shell, one pack of vanilla and baking soda are added and mixed with kneading. Then, the mixture, which is taken to the hole bag with one end, is squeezed by hand and poured into the tray in certain dimensions and then baked in the oven at 180 - 200 ℃ for 1 hour. It is then boiled in boiling sherbet for 5 minutes, or the pre-prepared cold sherbet is poured on the dessert (Anon., 2016c; Anon., 2019b). Some producers mix curd cheese with eggs and then add two teacups yogurt and one teacup of oil. Hazelnut, peanut, walnut, coconut, clotted cream, ice cream, whipped cream or cream can be used to decorate the dessert when served (Anon., 2018d). Kadayıf Kadayıf anywhere in Turkey is one of the desserts consumed with admiration (Seyyed Cheraghi, 2014; Seyyed Cheraghi et al., 2019). The history of this dessert goes back to the Seljuk period. There are many different desserts under the name Kadayıf. Two main types of these are Tel Kadayıf (wire Kadayıf) and Tray Kadayıf. In addition, there are varieties such as Bread Kadayıf and Yassı/Taş Kadayıf. All four can be prepared with walnuts or Pistachios (Aydın and Çakmakçı, 2014; MEGEP, 2018). In TS 10344 Tel Kadayıf Standard, Tel Kadayıf is defined as follows: It is a semi-product obtained by pouring and baking the dough prepared by adding the drinking water (TS 266) to the sifted wheat flour (TS 4500) and cooking according to the technique and frying when necessary on a hot plate (TSI, 1992). This dessert, purchased in semi-finished form, is subjected to frying or to put sherbet to the fried Kadayıf in the kitchen. During this process, according to the consumer or the taste of the local palate, various raw materials such as walnut, hazelnut, peanut, milk, and grape molasses are put in Kadayıf (Pekak, 2006). Tel Kadayıf is offered in two types as fried and unfried (Çakmakçı and Aydın, 2001). The sweet desserts prepared from Tel Kadayıf are walnut Tel Kadayıf and Kadayıf custard desserts. Aydın and Çakmakçı (2014) investigated the possibilities of using whey in the production of Tel Kadayıf dessert. The researchers investigated the microbiological, technological and 191

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sensory properties of Kadayıf dessert on the 1st, 3rd, 5th and 7th days of storage by adding whey instead of 0%, 25% and 50% water to the Kadayıf dough. According to the results of the analysis, it was reported that nutritional value and sweetness increased and cooking time decreased with the increasing amount of whey used in the production of Tel Kadayıf. In addition, it was concluded that the quality characteristics of Tel Kadayıf desserts would provide the most positive 25% whey addition and vacuum packaging. Tray Kadayıf is a semi-product obtained by baking the dough prepared following the technique by adding drinking water, cooking salt, bread yeast and additives if necessary to the baklava and pastry type of wheat flour. Tray Kadayıf is offered in raw and fired varieties in markets (Seyyedcheraghi et al., 2019) In Başar’s (2017) study, the effects of different oil and sugar sherbet on the sensory and textural properties of Tray Kadayıf were investigated. Three different levels of sunflower oil (0%, 25%, 50%), three different levels of hazelnut oil (0%, 25%, 50%), and sucrose and glucose syrup were used in the production of Tray Kadayıf. The control sample was prepared using 100% butter. As a result of the analysis, the researcher reported that sunflower oil and hazelnut oil variables had a significant effect on the L, +a and +b color values of the Tray Kadayıf samples. In addition, it was reported that sunflower oil increased the hardness values of Kadayıf samples higher than hazelnut oil, and that the highest score was obtained with 75% butter and 25 % oil. However, it was determined that panelists preferred Tray Kadayıf samples containing sucrose syrup. The Bread Kadayıf is available in Creamy Bread Kadayıf type (MEGEP, 2018). In the Hatay region, Yassı Kadayıf made with walnut is defined as a Taş (Tash) Kadayıf dessert in folk language. The production method of this dessert is described below. Making of Taş Kadayıf: Kadayıf is made with milk product/cheese which is obtained with the colostrum or heating of milk with improved acidity and called milk cottage or milk junket (also known as “ekşimik” or “kesik” among the local people). Yassı Kadayıf is placed on the undercooked side, the edge is closed with milk junket or milk cottage and sprinkled with sugar. Then, it is closed by pressing the edges inward to make a semicircle. If desired, after baking for 15 – 20 minutes the prepared cooled sherbet can be spread over. When Tel (wire) Kadayıf is used instead of Yassı Kadayıf, the same method is used for making Künefe dessert, only colostrum or cottage cheese (milk cottage) is used instead of Künefe cheese. Kadayıf is also widely used in the production of Künefe dessert (Seçim and Uçar, 2017). Künefe Künefe, which is the regional flavor of Hatay, is one of the most popular desserts of Turkish cuisine, which is among the richest cuisines in the world (Anon., 2018a). Particularly in Hatay province, the Künefe dessert is also produced in Mersin, Adana, Gaziantep, Kilis, Şanlıurfa, Diyarbakir and Mardin in different ways. Künefe is a kind of Tel Kadayıf and it is one of the most famous desserts in Syria (Satouf and Köten, 2019). Antakya Künefe which has an important place among Turkish desserts received the geographical indication certificate in 2007 (TPI, 2007). Künefe is a dessert that is made from melting, extensible fresh white cheese in fresh Tel Kadayıf (between upper and lower two layers of Tel Kadayıf), browning of Kadayıf in a hot oven (at this stage, the cheese melts into gum consistency) and which is served warmly (Arı and Güzeler, 2015; Anon., 2019c). The milk used for the production of cheese and the type of the milch animal, species of the milch animal, nutritional conditions of the milch animal and fat content of the cheese, butter, clarified butter and similar oils used in the production of the dessert play an important role in the product to have an admirable taste and aroma (Arı and Güzeler, 2015; Anon., 2019c). 192

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Seçim (2017) examined the effect of cheeses obtained from cow, goat and sheep’s milk used in the production of Künefe dessert. The characteristics of Künefe desserts were analyzed on 0, 7 and 14 days of storage. Looking at the properties of all Künefe desserts; dry matter ratios between 69.66 – 73.12%, fat values between 40.00 – 42.00%, ash ratios between 0.98 – 1.14%, acidity values between 0.49 – 0.94%, pH values between 5.07 – 5.34, L* values of 35.93 – 67.63, a* values of 10.65 – 13.12, b* values ranged between 12.81 – 25.11 and texture values ranged from 2 to 56.44. It was reported that there were no significant differences in the dry matter, fat, ash, acidity and pH values of the Künefe samples, and the differences in color determination L*, a*, b* values, texture, and sensory analysis were determined. However, it was found out that the Künefe dessert lost its flavor as a result of waiting for 14 days under appropriate conditions. In addition, the researcher reported that there were no microorganisms sought after the microbiological evaluation of Künefe desserts. Künefe Cheese Künefe cheese is traditionally produced in Turkey’s Hatay province and is a type of soft cheese that is consumed without ripening (Say et al., 2016). This cheese is also the main component of cheese varieties such as Yuvalama, Dil, and Sünme cheeses produced in the same region (Say et al., 2016; Karaca and Ocak, 2016). Künefe cheese should have a consistency close to Dil, but less salty than Dil cheese. It is suitable for diets because of low salt characteristics. A good Künefe cheese; should be full-fat cheese. In general, the most important features to be considered in the structure are as follows: creep rate, odor, smooth and bright structure, hardness-softness ratios. Another feature that should be in this cheese is that it does not burn quickly, does not stretch too much and does not break immediately (Anon, 2018b). The production of Künefe cheese is almost the same as the production of white cheese and goat milk, cow milk or mixtures thereof are used in its production. Nowadays, cow milk is generally preferred by producers. This cheese is fresh, unsalted, white in color, and melts and stretches when heated (Onur and Biber, 2015). Çayır (2019) has investigated that there are four kinds of the production of Künefe cheese in Hatay. The first three types are fresh Künefe cheese, salted Künefe cheese, and pipe (boru) type Künefe cheese. The 4th kind of this cheese is Künefe cheese made from pasteurized milk and with culture addition. As can be understood from its name, this type of Künefe cheese is produced by using the starter culture. In Adana, the dairy factories are able to produce pipe type Künefe cheese and Künefe cheese with culture addition and then sell to various restaurants (Anon., 2016d). Say et al. (2016) reported in their research that there are two types (as winter and summer method) of the production of salted Künefe cheese. The difference in the production methods of this type cheese is due to seasonal changes in milk temperature and milk composition. The chemical properties of fresh and salted Künefe cheeses are given in Table 2. Nowadays, freshly produced Künefe cheeses are frozen in the deep freeze and delivered safely to markets and restaurants. Due to the fact that Künefe cheese is made without salt and used daily, difficulties in transporting to other cities have started to emerge. Therefore, the production method was changed to extend the shelf life of the cheese and to facilitate transportation. The production of salted Künefe cheese is based on dry salting and has been widely used by producers in recent years (Kamber and Terzi, 2008; Say et al., 2016). According to the Turkish Patent and Trademark Authority, Antakya Künefe cheese known as fresh and salty in the market were filled with the C2018/173 file number on 28.08.2018 with the protected geographical indication (TPI, 2018).

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Table 2. The chemical properties of Künefe cheeses Properties pH Titratable acidity (l. a. %) Dry matter (DM) (%) Fat (%) Fat in DM (%) Protein (%) Protein in DM (%) Ash (%) Salt (%) Salt in DM (%) Number of samples

Fresh Künefe Cheese Karaca et al. (2008) 5.36 0.63 46.43 24.12 51.88 19.60 42.29 2.20 0.24 0.53 22

Salted Künefe Cheese Say et al. (2016) 5.86 0.81 60.11 19.64 32.68 19.95 33.19 10.90 9.29 15.46 21

In his study, Aslan Günay (2019) investigated the chemical and microbiological characteristics of a total of 30 Künefe cheeses produced by three different methods (fresh, salted and pipe type Künefe cheeses) and sold in Hatay province. According to the results of the research, when the cheeses were evaluated in terms of composition, it was reported that the differences between the analyzed characteristics of the Künefe cheese groups were found to be statistically significant. In particular, the high salt content of salty Künefe cheese affects many composition ratios, microbiological quality and volatile component profile in the cheese. The chemical properties of Künefe cheese samples obtained from the factories are presented in Table 3 and microbiological properties of the same cheese samples are showed in Table 4. Table 3. Chemical properties of fresh Künefe cheese, pipe type Künefe and salted Künefe cheeses (Aslan Günay, 2019). Properties pH Dry matter (%) Fat (%) Fat in Dry Matter (%) Ash (%) Protein (%) Salt (%) Salt in Dry matter (%) Salt in Moisture (%)

Fresh Künefe Cheese 4.99±0.12 40.59±4.98 20.30±4.35 49.54±5.96 2.22±0.24 16.34±1.31 0.21±0.15 0.50±0.34 0.35±0.31

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Pipe Type Künefe Cheese 4.87±0.06 43.59±2.61 22.60±1.66 51.82±1.38 2.17±0.59 17.02±1.09 0.10±0.05 0.22±0.12 0.17±0.10

Salted Künefe Cheese 5.08±0.29 64.46±3.74 28.20±1.79 43.80±2.47 12.79±1.36 22.38±3.15 9.31±2.52 14.62±4.64 26.08±5.95

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Table 4. Microbiological properties of fresh Künefe cheese, pipe type Künefe and salted Künefe cheeses (log kob/g) (Aslan Günay, 2019). Properties Total aerobic mesophilic bacteria Total coliform Escherichia coli Total yeast-mold

Fresh Künefe Cheese

Pipe Type Künefe Cheese

Salted Künefe Chese

8.67±0.05

8.41±0.10

8.48±0.07

6.43±0.11 6.28±0.07 5.05±0.09

5.63±0.12 4.97±0.11 4.79±0.16

4.78±0.24 4.21±0.36 4.27±0.05

Production of Künefe Cheese Künefe cheese is one of the traditional cheese varieties in Turkey that is produced in Antakya (Hatay), most particularly in the Eastern Mediterranean Region (Kamber, 2015; Karaca and Ocak, 2016). The majority of the demand is also provided for other parts of Turkey from Hatay. Since the composition of the milk, especially the temperature of the milk, varies seasonally, the fermentation temperature and other processes applied to the cheese are different in winter and summer (Say et al., 2016). The summer period varies according to winter. Milk with a high pH or low temperature in winter is delivered to the factory at low pH and high temperature in summer. It is usually fermented directly in the summer months after milking (at 27±1 ℃) without any heat treatment. Compared to winter, milk fermentation temperature and fermentation values are quite low. The temperature of the milk is 33±1 ℃ in winter and decreases to 26 ℃ in summer, and the coagulant rate can drop to 10% cc. The most important disadvantage of this is the increased yield loss due to low coagulant rate and the clot being soft (Çetinkaya, 2005; Karaca et al., 2008; Say et al., 2016). Künefe cheese has four different production methods as follows. 1. The milk used in fresh Künefe cheese is subjected to the necessary controls and then taken to the fermentation tanks and fermented at 34±1 ℃ with rennet from 10-15 liters per 100 liters of milk. After the curd is formed, the curd breaks and it is left in the tanks for a while to separate the whey. After removing the whey, the curd is put under pressure and left for ripening for 1-2 hours. The curd formed after-ripening is cut into 10 cm molds and offered for retail sale and used directly by the producer of desserts in Hatay for making the Künefe dessert. Fresh Künefe cheese, which is formed for the production of salted Künefe and pipe Künefe cheeses, is processed in different processes. 2. The curd for the production of salted Künefe cheese is cut into chestnut size and salted by using 2 kg salt for 14 kg cheese and left for 72 hours fermentation. Dry salting is repeated the next day before the cheese is packed. At the end of 72 hours, the cheeses are filled with 18-21% brine, their lids are closed and left to ripen in cold storage. After the ripening process is completed, cube (chestnut) cheeses which are got out of brine are vacuum packed. In other regions close to Hatay, the confectioners who use Künefe cheese mostly prefer this cheese. In addition, when milk production is high, the dairy factories produce this cheese and make stock. When needed, salt of this cheeses can be taken, and processed into pipe type Künefe cheese. 3. For pipe type Künefe cheese, fresh Künefe cheese is melted by the addition of melting salts. The obtained curd is passed through round pipes and directed to the weighting unit. Produced cheeses are packaged and stored at +4 ℃. Pipe type Künefe cheese, which is produced by adding melting salt, is cut into round slices and mostly used in the production of the frozen Künefe dessert. The production method of this cheese is not traditional and it is not included in the application of Geographical Indication. The production flow chart of fresh, salted and pipe type Künefe cheeses is shown in Figure 1.

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INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 Raw Milk Clarification Rennet Addition and Coagulation (at 34±1 ℃, 10-15 ml/100 kg) Curd Cutting and Whey Removal Pressing and Ripening of Curd (1-2 hours) Fresh Künefe Cheese

Fresh Cheese Cutting and Dry Salting Curd Cutting

Salting in 18-21% Brine (after 72 hours) Cheese Removal from Brine

Malting (by adding malting salt)

Salted Künefe Cheese Packaging and Storage (at +4 ℃)

Pipe Type Künefe Cheese Packaging and Storage (at +4 ℃)

Figure 1. Flow diagram of production process of fresh, salted and pipe type Künefe cheeses

4. The milk that is brought into the factory for the production of the Künefe cheese with culture addition is clarified and pasteurized at 72±1 ℃ for 15 seconds. Pasteurized milk is transferred to fermentation tanks and cooled to 34 ℃ and 1 g of starter culture is added for 100 kg milk. After waiting for 15 - 20 minutes, and when the milk temperature is 34±1 ℃, 10-15 ml of rennet is added to 100 liters of milk and then it is allowed to clot for 1 hour. The clot formed is cut with special clot breakers and steam is fed to the process tank wall to heat the curd. The whey is then removed. After a portion of the whey is removed, the pressure is applied to the filtered curd. The curd which is formed during the 1-2 hour pressing process is ripened until the pH value reaches 5.2-5.3. The ripened curd is then cut and melted at 72 ℃. Then, the obtained cheese is weighed, shaped and kept in a pasteurized cold water bath at 6±2 ℃ for 10 minutes. The cheeses extracted from the water bath are dried in the drying room (1-2 hours) and packaged. The production flow chart of Künefe cheese made by adding the culture is given in Figure 2. Künefe cheese obtained from pasteurized milk is not preferred as much as fresh Künefe cheese by dessert producers in Hatay province due to its negative physical properties in Künefe dessert. However, in order to eliminate microbiologically harmful bacteria and in order to transfer the product to other regions, this type of Künefe cheese is produced in some dairy factories operating in Hatay and Adana provinces as previously described (Karaca et al., 2008; Say et al., 2008; Say et al., 2016).

196

INTERNATIONAL BIOLOGICAL, AGRICULTURAL AND LIFE SCIENCE CONGRESS 2019 Raw Milk Clarification and Standardization Pasteurization (at 72±1 ℃ for 15 sec) Cooling and Culture Addition (1g / 100 kg) Rennet Addition and Coagulation (at 34±1 ℃; 10-15 ml/100 l) Curd Cutting and Whey Removal Pressing and Ripening of Curd (1-2 hours) Melting (10-15 minutes at 72 ℃ degree) Wheighting and Shaping Keeping in Water (at 6±2 ℃ for 10 minutes) Drying Culture Added Künefe Cheese Packaging and Storage (at +4 ℃) Figure 2. Flow diagram of production process of culture added Künefe cheese

CONCLUSION It is possible to find many desserts in Turkish cuisine, which is among the richest cuisines in the world. Also, Turkey has attracted attention with its varieties of cheese in the world. Approximately 200 types of cheese produced in Turkey are known. However, there are many kinds of cheese produced traditionally. As is done all over the world, in addition to the simple consumption of cheese, it is used by adding to food and desserts in country. Traditional cheese desserts made in Turkey are Höşmerim, Kemalpaşa dessert, Cheese Halva, Kadayıf and Künefe desserts. Differences in the production methods of these desserts may also be observed by region. What they have in common is the use of unsalted fresh cheese. One of the traditional cheeses produced in Turkey is the unsalted Künefe cheese, which is used by dessert producers mostly in Künefe dessert and is available for other desserts. Today, several production methods of Künefe cheese have been developed. To transfer to nearby regions, cheeses started to be produced by salting, freezing and pasteurizing milk and adding starter culture. Also recently, Künefe cheese is produced in dairy factories operating in other provinces and sold to restaurants in these provinces. REFERENCES Akpinar-Bayizit, A., Ozcan, T., & Yilmaz-Ersan, L. (2009). Milk-based traditional Turkish desserts. Mljekarstvo, 59(4): 349. Akpinar-Bayizit, A., Ozcan, T., & Yilmaz, L. (2010). A Turkish cheese-based dessert. Bulgarian Journal of Agricultural Science, 16(4): 493-499.

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Anonymous, (2014). Kemalpaşa tatlısı tarifi. https://yemek.com/tarif/kemalpasa-tatlisi/. Accessed on date: 10.10.2019. Anonymous, (2016a). Trakya Peynir Helvası tarifi. https://www.neoldu.com/trakya-peynirhelvasi-tarifi-15541h.htm. Accessed on date: 10.10.2019. Anonymous, (2016b). Mustafakemalpaşa Tatlısı. http://www.mustafakemalpasa.bel.tr/ilcemiz/kemalpasa-tatlisi.html. Accessed on date: 10.10.2019. Anonymous, (2016c). Peynirin En “Tatlı” Halleri - Lor Tatlısı - Balıkesir. https://www.haberler.com/peynirin-en-tatli-halleri-lor-tatlisi-balikesir-8387226-haberi/. Accessed on date: 09.09.2019. Anonymous, (2016d). Çay Çiftlik Ürünleri San. Tic. Paz. Ltd. Şti. http://www.cayciftlik.com.tr/. Accessed on date: 10.10.2019. Anonymous, (2017). Höşmerim (Ankara yöresi). https://www.nefisyemektarifleri.com/hosmerim-ankara-yoresi/. Accessed on date: 10.10.2019. Anonymous, (2018a). Kaya Çiftliği’nden Künefe Peyniri. Gıda 2000. Gıda Teknolojisi ve Tarım Dergisi. http://www.gida2000.com/kaya-ciftliginden-kunefe-peyniri.html. Accessed on date: 06.10.2019. Anonymous, (2018b). Höşmerim nasıl yapılır? Püf noktalarıyla höşmerim tarifi. http://www.hurriyet.com.tr/lezizz/hosmerim-nasil-yapilir-puf-noktalariyla-hosmerimtarifi-41067300. Accessed on date: 16.10.2019. Anonymous, (2018c). Peynir helvası nasıl yapılır - Tatlı tarifleri - Yemek tarifleri. https://www.youtube.com/watch?v=bAWq5JlycTA. Accessed on date: 16.10.2019. Anonymous, (2018d). Hürriyet. Lor tatlısı nasıl yapılır? Lor tatlısı tarifi. http://www.hurriyet.com.tr/kelebek/gurme/lor-tatlisi-nasil-yapilir-lor-tatlisi-tarifi40999385. Accessed on date: 10.10.2019. Anonymous, (2019a). Höşmerim tatlısı (Balıkesir yöresi). https://ye-mek.net/tarif/hosmerimtatlisi. Accessed on date: 10.10.2019. Anonymous, (2019b). Enfes Lor Tatlısı Tarifi. http://www.yasemin.com/yemek/haber/2843818-enfes-lor-tatlisi-tarifi. Accessed on date: 10.10.2019. Anonymous, (2019c). Künefetek. “Tadıyla Tek.” http://www.kunefem.net/siparis/. Accessed on date: 10.10.2019. Arı., E., Güzeler, N. (2015). Cheesy desserts from Turkish cuisine. The 3rd International Symposium on “Traditional Foods from Adriatic to Caucasus”, 01-04 October 2015, Sarajevo, Bosnia and Herzegovina, p. 234. Aydın, A., Aksu, H., Taskanal, N. & Gunsen, U. (2009). Microbiological, physico-chemical and toxicological quality of traditional Turkish cheese desserts. Journal of Food Quality, 32: 590 - 606. Aydın, F., Çakmakçı, S. (2014). Possibilities for the use of whey in tel Kadayıf (a Turkish dessert) production. Turkish Journal of Agriculture and Forestry, 38(2): 250-257. Başar, B. (2017). Effects of different oil and sugar surbines on the functional and documentary characteristics of tray Kadayıf. Master Thesis. Atatürk University, Institute of Social Sciences, Department of Tourism and Hotel Management, p.44. Baysal, A. (1993). Changes in Turkish culture of cuisine, evaluation in sight of health and nutrition. Researches in Turkish Culture of Cuisine. Türk Halk Kültürünü Araştırma ve Tanıtma Vakfı Yayın, (3): 12-241. Birer, S. (1991). The dia chronic change of Turkish cuisine and its current aspect. National Culture (Milli Kültür), 87, 47-50.

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Çakmakçı, S., Aydın, F. (2001). Determinatıon of some quality characteristics of fresh and storaged tel Kadayıf (special Turkish dessert) manufactured with different levels of whey addition. Atatürk University, Journal of Faculty of Agriculture, 32 (2): 181-188. Çayır, M.S. (2019). Interview about Künefe Cheese. Ministry of Agriculture and Forest. Food Engineer at Hatay Provincial Directorate of Agriculture and Forestry. Çetinkaya, A. (2005). Our local cheeses (Yöresel Peynirlerimiz). 1st Edition. Academic Book Production. p. 209. Cokal, Y., Dagdelen, A., Cenet, O. & Gunsen, U. (2012). Presence of L. monocytogenes and some bacterial pathogens in two Turkish traditional foods, Mihalic cheese and Hosmerim dessert. Journal of Food Control. 26: 337 - 340. Cömert, M. (2014). The importance of regional cuisine in tourism marketing with a case study: Hatay Cuisine. Journal of Tourism and Gastronomy Studies, 2(1): 64 - 70. Guldas, M., Bayizit, A. A., Yilsay, T. O., & Yilmaz, L. (2010). Effects of edible film coatings on shelf-life of Mustafakemalpasa sweet, a cheese based dessert. Journal of food science and technology, 47(5): 476-481. Güven, S., Demir, H. (2010). Recipe development for production of a Turkish dessert, cheese halva. International Journal of Dairy Technology, 63 (4): 581 - 586. Elmalı, G., Uylaşer, V. (2012). Production and properties of Cecil cheese as a traditional food. Uludağ University, Journal of Faculty of Agriculture, 26 (1): 83-92. Eralp, M. (1974). Cheese technology. Ankara, Ankara University Press. Kamber, U., Terzi, G. (2008). The traditional cheeses of Turkey: Southeast Anatolia Region. Food Reviews International, 24: 62-73. Kamber, U. (2015). Traditional Turkey cheeses and their classification. Van Veterinary Journal, 26(3): 161-171. Karaca, O.B., Güven, M., Mutluer, U. & Başar Saydam, İ. (2008). Production and properties of Hatay Künefe cheese. 10th Food Congress of Turkey, 21-23 May, Erzurum, pp. 745748. Karaca, O.B., Ocak, S. (2016). Dairy goat products of east Mediterranean region of Turkey: künefe and sünme cheeses. Scientific Papers. Series D. Animal Science. Vol. LIX, 288 – 295. Kayış, V., Yaman, H. (2018). Examining the sector representatives’ opinions regarding on the place of Turkish Traditional Turkish cheese as an alternate international cheese in tourism sector. Journal of Tourism and Gastronomy Studies, 105: 124. Kurultay, S. (2005). Investigation of usage possibilities of melting salt in production of Tekirdağ cheese halva (Höşmerim). Scientific Research Projects of Trakya University (TÜBAP 485), Edirne. Kurultay, Ş., Öksüz, Ö., Taş, M. (2008a). Optimization of fat content and pH level of unsalted fresh cheese used in the manufacturing of cheese halva (Höşmerim). International Journal of Food Science and Technology, 43: 330 - 332. Kurultay, Ş., Öksüz, Ö., Kaptan, B. (2008b). Proving of cheese halva (Hosmerim) manufacturing process. International Journal of Dairy Technology, 62, 63-67. MEGEP, (2018). Varieties of Kadayıf. Ministry of National Education of Turkey, Ankara. Onur, B.B., Biber, N.A. (2015). Peynir aşkına. Turkey Is-bank Cultural Publications, İstanbul, p. 291. Özer, M. S., Kola, O., Altan, A., Duran, H., & Zorlugenç, B. (2012). Acrylamide content of some Turkish traditional desserts. Journal of Food, Agriculture & Environment, 10(1): 74-77. Pekak, R. (2006). A study on the optimization of Kadayıf flour production in a commercial mill. Master thesis. Selcuk University Institute of Natural and Applied Sciences. p. 51.

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Şanlıer, N., Cömert, M. & Durlu-Özkaya, D. (2008). Türk mutfağındaki geleneksel tatlı ve helvaları gençlerin tanıma durumu. Türkiye 10. Gıda Kongresi, 21-23 Mayıs, Erzurum. p. 1123 - 1127. Satouf, M., Köten, M. (2019). The favored Syrian desserts. 3rd International Conference on Agriculture, Food, Veterinary and Pharmacy Sciences. 16-18 Nisan 2019. Trabzon. Say, D. (2008). Effects of salt concentration of scalding solution and storage period on properties of Kaşar cheese. Ph.D. thesis, Cukurova University Institute of Natural and Applied Sciences, Adana, p.150. Say, D., Çayır, M.S., Güzeler, N. (2016). Production method and some properties of salted Künefe cheese. Journal of Agricultural Faculty of Uludag University, 30: 454 - 457. Seçim, Y. (2017). Quality criteria of Hosmerim, Kunafah and Cheese Halva produced from goat, sheep and cow based cheese products. Ph.D. thesis, Selcuk University, Institute of Health Sciences, Department of Food Hygiene and Technology, Konya, p.101. Seçim, Y., Uçar, G. (2017). Evaluation of the desserts; which are Hosmerim, Cheese Halva, Kunafah produced in Turkish cuisine -in aspect of tourism. International Journal of Social Sciences and Education Research, 3(5): 1478-1484. Seyyed Cheraghi, K. (2014). Effect of frying temperature and duration of tray Kadayıf dessert on amount of HMF and acrylamide. Master Thesis. Atatürk University, Institute of Natural and Applied Sciences, Department of Food Engineering, p. 54. Seyyed Cheraghi, K., Kotancilar, H.G. & Karaoglu, M.M. (2019). Determination of acrylamide and hydroxymethylfurfural (HMF) values as affected by frying duration and temperature levels during the preparation of tray Kadayıf dessert. Applied Ecology and Environmental Research, 17(2): 4007-4017. Sengul, M., Ertugay, M. F. (2006). Microbiological and chemical properties of cheese Helva produced in Turkey. International Journal of Food Properties, 9(2): 185-193. Sürücüoğlu, M.S., Akman, M. (1998). The dia chronic change of Turkish cuisine and the current reasons for this change. Standart 37, 439, 42-45. Tan, S., Ertürk, E.Y. (2002). Cheese. Agricultural Economics Research Institute, 1(11). TFC, (2015). Turkish Food Codex Communique on Cheese. Official gazette. Number: 29261. Ministry of Food, Agriculture and Livestock. Communique No: 2015/6. TPI, (2002). Turkish Patent Institute. Geographical indication registration certificate. Kemalpaşa dessert. Date of publishing: 13.01.2002. Registration No: 51. Application No: 2001/010, Bursa TPI, (2007). Turkish Patent Institute. Geographical indication registration certificate. Antakya Künefe. Date of publishing: 27.12.2007. Registration No: 101. Application No: 2006/006, Antakya TPI, (2018). Turkish Patent Institute. Antakya Künefe Cheese. https://www.turkpatent.gov.tr/TURKPATENT/geographicalRegisteredList/. Application No: C2018/173. TSI, (1992). Turkish Standards Institution. TS 10344 Tel Kadayıf Standart. Necatibey street No.112 Bakanlıklar/Ankara. p.9. TSI, (2011). Turkish Standards Institution. TS 12102 Kemalpaşa dessert (Cheese dessert). Necatibey street No.112 Bakanlıklar/Ankara, p.14. TSI, (2017). Turkish Standards Institution. TS 13470 Dough Desserts-Desserts Ready to Add to Sherbet standard. Necatibey street No.112 Bakanlıklar/Ankara, p. 17. Ünsal, A. (2003). When milk sleep (Süt uyuyunca). Turkey’s Cheeses. 1st Edition, Istanbul, Yapı Kredi Publications, p. 221. Uçurum, H. Ö., Kaygısız, M. & Uğur, N. (2016). Determination of quality characteristics of Kemalpaşa dessert (cheese dessert). Food, 41 (4): 221 – 225.

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Uzel, G., Turhan, Ş., Mayiyoh, A., Çil, N. (2017). Socio-economic structure of businesses producing Mustafakemalpaşa cheese dessert in Mustafakemalpaşa district, Bursa province. Journal of Food and Feed Science - Technology, 18: 11-17.

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ACUTE TOXICITY OF LOW DOSE DIAZINON PUPA STAGE OF ROSE TORTRIX ARCHIPS ROSANA (LINNAEUS, 1758) (LEPIDOPTERA: TORTRICIDAE) Utku GÜNER1*, Mitat AYDOĞDU1, Fulya Dilek GÖKALP1 1

Department of Biology, Faculty of Science, Trakya University, Edirne, Turkey *Corresponding author’s e-mail: uguner@trakya.edu.tr

ABSTRACT The European leafroller (ELR) Archips rosana (Linnaeus, 1758) is native to the Palearctic region, but it is distributed all over the world. The rose tortrix is a moth of the family Tortricidae (Lepidoptera). This species plays an important role in plant protection due to a large number of harmful species and frequent occurrence on different cultivations. In modern agriculture, obtaining crops of sufficient quantity and quality without pesticides is an important problem today. Therefore, pesticides in agriculture are increasingly used more and more. Basudin is one of the pesticides used against rose tortrix Archips rosana. Basudin active ingredient diazinon (active substance content: 630 g / l) was used as test substance. Diazinon, prepared in different doses, was sprayed on organophosphorus pesticides cherry leaves. The concentrations were selected according to active ingredient Diazinon recommended dose (r.d) (441 μm) in agricultural use and half of r.d and 10-1, 10-2, 10-3, 10-4 fold diluted concentrations of r.d. were exposed in laboratory conditions on pupae of A. rosana. After single dose application, mortality ratios were obtained after 7, 12 and 15 days. The 7-day LC50 value is 0.137 ppm daily while the 12-day LC50 value decreases by 0.001 ppm. Keywords: Diazinon, Pest-control, LC50, Archips rosana INTRODUCTION The rate of pesticide use in agricultural areas is continuing to rise in many countries. Due to its harmful effect to living organisms, pesticide use should be reduced, or provide biological control to avoid or minimize the exposure of living organisms (Muranlı, Aydoğdu, & Güner, 2015). In order to prevent product losses in agricultural products, It is known that chemical drugs used in some way cause some important problems. The most important of these problems are the harm to human health and the environment (Kapidani & Duraj, 1991). In order to eliminate the negativity of the chemicals, the pest populations, which are defined as integrated struggle in recent years, considering all the population dynamics of the pest species and their relations with the environment, use all appropriate control methods and techniques in a harmonious way(Polat & Tozlu, 2010). Cherry orchards are one of the major crops in northwestern part of Thracian region of Turkey. In the last decade, the application of ecological cherry production methods has been studied in a project in the important cherry production areas of western Turkey (M Aydoğdu, Gökalp, & Güner, 2017; Doganlar, 2007). In order to avoid potential serious side effects of conventional agriculture on human health and environment, organic agriculture practices have been initiated in all the countries of the world (Mitat Aydoğdu, 2014).The importance of parasitic wasps in ecological agricultural systems cannot be underestimated. For all these reasons, this paper has been produced to reveal the beneficial parasitic insect fauna in cherry orchards of Turkey. 202

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Archips species spend the winter, eggs and thick branches on the trunk. The larvae hatching in the spring in the first two periods of the ends of the newly sown leaves; they are fed by eating the inner and outer parts of the eyes from the sides and bottom (H Piekarska-Boniecka, Wilkaniec, & Dolańska-Niedbała, 2008; Polat & Tozlu, 2010). Large amounts of the Diazinon insecticides are used against A. rosana, which is one of the major orchard pests in the Thrace region in the north west of Turkey. Its short life span makes its control rather difficult, if not monitored regularly. Effective doses are important for selection of insecticides, which must be tested at different life-stages of insects. The life-cycle times of pupae are known, so it may be useful to know the effective concentrations at these different stages. Moreover, this knowledge may be useful in decreasing the use of insecticides in agriculture. In the present study, we aimed to investigate both lethal/effective concentrations of Diazinon on pupae stages of A. rosana. MATERIAL AND METHODS We investigated the toxicity of Diazinon against pupae of A. rosana under laboratory conditions (Figure 1). We collected larvae and pupae from almond, apple, plum and cherry trees in the vicinity of Edirne (north west Turkey) in the early mornings, ensuring that we kept hand contact with the larvae and pupae to a minimum (Muranlı et al., 2015). The localities from which we made our collections are chemical free areas, and not used for agricultural purposes, and we also collected from different areas of the Edirne region, which is known for being unexposed to any kind of pesticides (Mitat Aydoğdu, Güner, & Muranlı).

Figure 1. Pupa of Archips rosana (Lepidoptera: Tortricidae) We used commercial formulations of Basudin active ingredient Diazinon as test chemicals. Diazinon (IUPAC name: O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate, INN - Dimpylate), a colorless to dark brown liquid, is a thiophosphoric acid ester developed in 1952 by Ciba-Geigy, a Swiss chemical company (later Novartis and then Syngenta). It is a nonsystemic organophosphate insecticide formerly used to control cockroaches, silverfish, ants, and fleas in residential, non-food buildings. Diazinon was heavily used during the 1970s and early 1980s for general-purpose gardening use and indoor pest control (El-Sherif, Ahmed, El-Danasoury, & El-Nwishy, 2009). The commercial names, CAS numbers and amount of active substance in the commercial forms are given in figure 2.

Figure 2. Chemical structure of diazinon We studied the recommended doses and diluted concentrations of the recommended doses. Exposures of insecticides were expressed as concentrations of the active ingredient All pupa 203

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were collected in selected study areas (pesticides unused land, Edirne-Thrace) and pupae were collected in Petri dishes with artificial diet. We maintained each individual pupae in large Petri plates (10 cm diameter) containing a 50% solution of diluted honey embedded in cotton pieces, which we used as the food source during the experiments. Diazinon, prepared in different doses, was sprayed on organophosphorus pesticides cherry leaves. These leaves made discs with a diameter of 5 cm and dipped into the test solutions for 10 seconds and allowed to dry laboratory conditions at 25 ± 2°C, 16:8 hours Light: Dark cycle and 70% relative humidity. The concentrations were selected according to active ingredient diazinon recommended dose (r.d) (441 μm) in agricultural use and half of r.d and 10-1, 10-2, 10-3, 10-4 fold diluted concentrations of r.d. were exposed in laboratory conditions on pupae of A. rosana(Güner, Aydoğdu, & Gökalp, 2018a). After single dose application, mortality ratios were obtained after 24, 48 and 72 hours. Basudin active ingredient diazinon (active substance content: 630 g / l) was used as test substance. Diazinon, prepared in different doses, was sprayed on organophosphorus pesticides cherry leaves. The concentrations were selected according to active ingredient diazinon recommended dose (r.d) (441 μm) in agricultural use and half of r.d and 10-1, 10-2, 10-3, 10-4 fold diluted concentrations of r.d. were exposed in laboratory conditions on pupae of A. rosana. Mortality ratios were calculated after 24, 48 and 72 hours (see table 1). LC50 values were calculated for diazinon at the end of the experiment. The 7-day LC50 value is 0.137 ppm daily while the 12-day LC50 value decreases by 0.001 ppm. RESULTS Pupae of Rosaceae pest A. rosana reached 100% mortality after r.d. Pupae mortality was observed 100% after and 100 fold (1.10-4 r.d.) diluted concentration application after 72 hours (Table 1). Table 1. Applied concentrations of Diazinon on pupae stage of A. rosana (r.d. recommended dose) Mortality % ± S.E.Times after application Dose / Time - control 10 µM (r.d.)

24 h 0±0.0 100±0.0***

48 h 0.0±0.0 100.0±0.0***

72 h 0.0±0.0 100.0±0.0***

100.0±0.0***

1.10-1 µM

75±11,2***

1.10-2 µM

80±9,4***

90±10***

100.0±0.0***

1.10-3 µM

75±11,2***

90±10***

100.0±0.0***

1.10-4 µM

60±10***

85±10***

95±5***

The results showed that diluted concentrations of Basudin active ingredient Diazinon are still effective on pupae forms of A. rosana. 100 fold diluted concentration (1.10-2 r.d.) application induced 100% mortality at 72 hours. 1000 fold diluted concentration (1.10-3 r.d.) induced 90% mortality at 48 hours and 100% mortality at 48 hours. In this study; even at a much lower concentration than the recommended doses, the insecticide is effective. DISCUSSION In many studies using this method, a high mortality rate was observed even at doses much lower than the recommended doses. In most cases, the recommended dose was affected not only pests 204

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but also beneficial organisms (such as parasitic wasps)(Mitat Aydoğdu & Güner, 2012; Hanna Piekarska-Boniecka, Rzanska-Wieczorek, Siatkowski, & Zyprych-Walczak, 2019). The use of pesticides below the recommended dose leads to insignificant reduction in pest control, while reducing the risk of pesticide residues. It is seen that pesticides below the recommended dose cause effective death even in long periods (15 days). CONCLUSIONS The use of pesticides under the recommended dose (r.d.) may be important for the survival of beneficial organisms (Güner, Aydoğdu, & Gökalp, 2018b; Kapidani & Duraj, 1991). A pesticide applied under the recommended dose (r.d.) is sufficient to kill target organisms. The use of low concentrations of pesticides not only provides economic benefits, but also has a lower impact on non-target organisms. Using a low concentration in reducing the use of pesticides can prevent damage to the pesticide environment. New application methods can help reduce these problems. It is important to study the concentrations that are effective on pests. Therefore, this approach may be helpful for integrated pest management programs for to save environment and may contribute to reducing the use of pesticides. REFERENCES Aydoğdu, M. (2014). Parasitoid abundance of Archips rosana (Linnaeus, 1758)(Lepidoptera: Tortricidae) in organic cherry orchards. North-Western Journal of Zoology, 10(1), 4247. Aydoğdu, M., Gökalp, F., & Güner, U. (2017). Toxic Effects Of Pyrethroids LambdaCyhalothrin And Alpha-Cypermethrin On Pest Archips Rosana (Lepidoptera: Tortricidae) And Its Common Parasitoid. Fresen. Environ. Bull, 26, 2436-2445. Aydoğdu, M., & Güner, U. (2012). Effects of 5 differet insecticides on mortality of the leafroller parazitoid Itoplectis maculator (Fabricius, 1775)(Ichneumonidae, Hymenoptera). Türkiye Entomoloji Bülteni, 2(4), 243-250. Aydoğdu, M., Güner, U., & Muranlı, F. D. G. Effects of Two Different Pyrethroid Group Insecticides on Mortality Itoplectis Maculator (Fabricius, 1775) Obtained from Archips Rosana (Linnaeus, 1758) Pupae in Laboratory Conditions. The Turkish Journal Of Occupational/Environmental Medicine and Safety, 1(2 (2)). Doganlar, O. (2007). Distribution of European leaf roller, Archips rosanus (L.)(Lep.; Tortricidae) egg masses on different apple cultivars. Asian journal of plant sciences, 6(6), 982-987. El-Sherif, M., Ahmed, M., El-Danasoury, M., & El-Nwishy, N. H. (2009). Evaluation of diazinon toxicity on nile tilapia fish (O. niloticus). J Fish Aquat Sci, 4(4), 169-177. Güner, U., Aydoğdu, M., & Gökalp, F. D. (2018a). Method of observing the effect of the recommended doses on family rosaceae pest larvae. MESMAP-4 ABSTRACT BOOK, 120. Güner, U., Aydoğdu, M., & Gökalp, F. D. (2018b). Reduction of recommended dose value of DDVP 550 ec on Archips rosana (linnaueus, 1758)(lepidoptera: Tortricidae). MESMAP-4 ABSTRACT BOOK, 119. Kapidani, A., & Duraj, N. (1991). Investigations on the biology and control of the tortricid Archips rosana L. in Albania. Pflanzenschutzberichte, 52(2), 79-88. Muranlı, F. D. G., Aydoğdu, M., & Güner, U. (2015). Mortality and LD50 Concentrations of Pyrethroid Insecticide Isoldesis on Larvae of Archips Rosana (Linnaeus, 1758). The Turkish Journal Of Occupational/Environmental Medicine and Safety, 1(2 (2)). 205

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Piekarska-Boniecka, H., Rzanska-Wieczorek, M., Siatkowski, I., & Zyprych-Walczak, J. (2019). Controlling the abundance of the rose tortrix moth [Archips rosana (L.)] by parasitoids in apple orchards in Wielkopolska, Poland. Plant Protection Science, 55(4), 266-273. Piekarska-Boniecka, H., Wilkaniec, B., & Dolańska-Niedbała, E. (2008). Parasitoids of Ichneumonidae family (Hymenoptera, Apocrita) limiting abundance of rose tortrix moth Archips rosana (L.) in selected orchards in Wielkopolska. Progress in Plant Protection, 48(4), 1319-1322. Polat, A., & Tozlu, G. (2010). Erzurum'da Archips rosana (Linnaeus, 1758)(Lepidoptera: Tortricidae)'nın kısa biyolojisi, konukçuları ve parazitoitleri üzerinde araştırmalar. Türkiye Entomoloji Dergisi, 34(4), 529-542.

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FACTORS INFLUENCING CONSUMPTION OF MEAT IN TURKEY Güliz HASKARACA1*, Bengisu KANDIL1, Kübra Nazli INÖZÜ1, Nisanur KOÇANLAR1, Sinem SANCAKTAR1 1

Sakarya University, Faculty of Engineering, Department of Food Engineering, Sakarya, Turkey *Corresponding author’s e-mail: gyaldirak@sakarya.edu.tr

ABSTRACT This study was aimed to investigate the meat (red and poultry meat) consumption habits of individuals who lives in Turkey and the factors that affect their meat consumption habits. For that aims, survey study was carried out with the participation of 717 people from Turkey. Consumption habits of people were evaluated with the data obtained from 27 questions by comparing the demographic properties, consumption frequencies, purchasing habits and knowledge levels. It was determined that the most important factor that positively influence the consumers red or poultry meat consumption preference is its taste while high prices of red meat and low quality assurance of poultry meat are the most important factors that reduced the consumers demand. Among the surveyed individuals 66% of them think that there are not enough controls about hygiene and food safety at the slaughterhouse, processing plants and in meat sales points and 62% of them believe that the meat offered for consumption is now unhealthier comparing to previous years. It was observed that individuals did not trust the meat they consumed in terms of safety and animal diseases and had low knowledge about the health effects of meats. To ensure this trust and to raise awareness of consumers, accurate and detailed information should be delivered to the consumers by experts such as food scientists, food engineers and veterinary surgeons. Keywords: Meat consumption habits, Consumer preferences, Red meat, White meat, Healthy diet INTRODUCTION Nutrition is not suppressing the feeling of hunger or feeding the stomach, or eating and drinking the things that we want. Nutrition is an action that needs to be done consciously in order to maintain health and improve the quality of life by taking the nutrients that the body needs in sufficient amounts and at appropriate times (Anonymous, 2015). The daily amount of energy an adult person needs is 12000 kJ, of which 15% must be supplied from proteins. Protein is found in the structure of all living cells, in the structure of enzymes that catalyze reactions in the body, and in some hormones. When sufficient protein is not taken into the body; growth and development slows down, the immune system weakens, diseases last longer, the health of tissues deteriorates and the function of organs weakens. At least half of the protein that the human body needs must be of animal origin. For this reason, foods of animal origin have been the main component of human nutrition with its flavors and contributions to health. Meat and meat products contains all the essential amino acids in required quantity and ratio which are necessary for the growth, survival and physiological functions. Red meat contains plenty of protein, B6, B12 vitamins, iron and zinc. Thus, red meat advised to be included in the weekly diet at least once. Also, poultry meat is very important for physical and mental 207

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development. It contains high level of proteins that the body needs and all the amino acids required for human nutrition. It is rich in vitamins such as B2, B6, and B12 that develop and protect the nervous system. And also contains high unsaturated fatty acids. Thus, these important protein sources must be take their place in meals (Biesalski 2005, Nohr and Biesalski 2007, McAfee, McSorley and Cuskelly, 2010). But on the other hand, high consumption of meat and meat products can cause some health problems such as obesity, cardiovascular disease due to high cholesterol level or cancer such as colorectal cancer (Ferguson 2010, Salter 2018). Therefore, an adequate and balanced consumption of meat and meat products are very important in terms of maintaining healthy life. The aims of the study were; to reveal the meat (red and poultry meat) consumption habits of people live in Turkey, to investigate the knowledge level of people about the effects of meat consumption on health, to find out recent perspectives of consumers' on the sector and on marketing and to understand the consumersâ&#x20AC;&#x2122; meat purchasing habits. MATERIAL AND METHODS The survey was done by using multiple choice method and Likert scaling methods between March and April months of 2019 in Turkey mostly from Marmara Region. The number of participants from Marmara Region is high because the cities in this region have the highest immigration rates compared to other regions due to the high job and education opportunities wide social activity opportunities. Also, this region has the most cosmopolitan structure that can represent the whole of Turkey. The questionnaire was both available online and in paperand-pencil format, and 717 people participated to the survey. In total 27 different questions were asked to participant consumers to determine the consumer profile, meat consumption habits, meat buying habits, and the effects of meat on health. RESULTS AND DISCUSSION Demographic characteristics of the responders were shown in Figure 1. As can be seen from the figures, the majority of the participants were women (62%). Fifty-seven percent of the responders were single and others were married. Most of the responders were between the 18 and 55 years old, the percentages of the responders whose age was between 18 and 24, 25 and 34, 35 and 44, 45 and 54 were 41%, 23%, 19% and 14%, respectively. The majority of the responders had 3, 4 or 5 people in their family.

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Figure 1. Demographic structure of the responders

In figure 2, the consumption rate and the consumption frequency of red and poultry meat was given. Ninety-nine percent of individuals stated that they are consuming red meat and 70% of them stated that they are consuming once or twice a week. These levels were found 96% and 76% for poultry meat consumption, respectively. It seems that, the majority of participant are consuming enough meat.

Figure 2. Consumption rate and consumption frequency of red and poultry meat 209

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Figure 3 shows the reasons for consumption of red and poultry meat. When looking to the reasons for consumption of red meat, it can be seen that the biggest reason is liking its’ taste. It is followed by habit and easy to provide. Besides, it was observed that the most important reasons for consumption of poultry meat are liking its’ taste, easily accessing and being a healthy meal. According to these results, especially for red meat, it is possible to say that people prefer to choose what they eat according to their taste and habits rather than being healthy.

Figure 3. The reasons for consumption of red and poultry meat Those who do not consume red meat stated that the biggest reasons for not consuming red meat is that “meat is expensive and unreliable” (Figure 4). On the other hand, for poultry meat, these reasons are stated as they are not reliable, not healthy or do not have good taste.

Figure 4. The reasons for not consuming red or poultry meat

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Consumers were asked to evaluate red and poultry meat in terms of their health risks. Results, obtained from that question are given in Figure 5. The majority of participants found red meat low-risky while they found poultry meat risky and low-risky. It was observed that poultry meat was found more risky than red meat by the consumers most probably due to the peoplesâ&#x20AC;&#x2122; thoughts have been influenced by the news about antibiotics and hormones given to chickens or food poisoning events, in recent years.

Figure 5. Evaluation of red and poultry meat in terms of health risks

Table 1 and 2 shows the respondents' views on the health effects of red and poultry meat, respectively. It was found that the biggest concern about the health effect of red meat was cholesterol content. As can be seen from the Table 1, lack of bacteriological hygiene or carrying animal diseases are found the other major concerns. On the other hand, serious concerns are observed about the effects of poultry meat on human health such as lacking of hygienic conditions, carrying harmful bacteria and animal disease.

Table 1. Respondents' views on the effects of red meat on human health Neither Strongly Somewhat agree nor agree agree disagree

Strongly No idea disagree

Effect of calorie value

142*

356

152

Effect of oil content

167

373

130

Effect of cholesterol content

221

334

Effect of having of harmful bacteria to health

143

305

197

Effect of deficiency of hygiene conditions

182

314

158

Effect of having animal disease

190

311

148

* Number of responders who choose related parameter

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Table 2. Respondents' views on the effects of poultry meat on human health Neither Strongly Somewhat agree nor agree agree disagree

Strongly disagree

No idea

Effect of calorie value

61*

282

299

Effect of oil content

273

314

Effect of cholesterol content

259

313

Effect of having harmful bacteria to health

311

269

Effect of deficiency of hygiene conditions

325

254

Effect of having animal disease

299

267

101

* Number of responders who choose related parameter

According to the answer of the question â&#x20AC;&#x153;where are you consume your meat mostly?â&#x20AC;? it was determined that people prefer to consume meat mostly at their home. In this question, individuals had the right to mark more than one option. It was observed that the number of individuals who consume meat only at their home, increase with the increasing in age and their marriage while the number of individuals who consuming meat only at fast-food points, were between the ages of 18-24 and were single. Besides, it was determined that the consumers prefer to buy their meat mostly from the butcher and then from the market. The proportion of consumers who buy from farmers and local producers, or who grown their own animal themselves is quite low. The most likely reason for this low rate is thought to be owing to the carrying out of the survey mostly in Marmara region. Because the Marmara Region has less agriculture and animal husbandry due to its large industrial areas. Therefore, consumers are less likely to have their own cultivation or purchase from farmers. In addition, both changes in socio-economic structure and non-compliance with ethical values have caused people to lose confidence in big cities. The percentage of answers to these questions may vary in different regions of Turkey. On the other hand, still, there were a group of people who do not want to buy their meat from market or butcher. The reasons of these people for not buying meat (red or poultry meat) from market and butcher were given in Figure 6 and 7, respectively. The most popular answer of this question was that they did not find the market products reliable. As seen from Figure 6, other reasons for not buying from markets were; not knowing the composition of meat and meat products, and lacking of hygienic conditions. It is a very important and a big problem that the trust to market is so low. Thus, markets should have find solutions to give confidence to the consumer.

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Figure 6. The reasons for not buying meat (red or poultry meat) from market

It was determined that the reasons for not buy meat from the butcher were; high prices, unreliability, inaccessibility and unknown ingredients. Consumers reported that they find that meat prices in butchers are more expensive than markets.

Figure 7. The reasons for not buying meat (red or poultry meat) from butcher In the current survey, the answers of what people mostly pay attention to when buying products from the market was also examined. According to the answer of this question it was determined that the first thing that they pay attention is expiration date, followed by brand and being packaged of the product. In addition, pictures and label information on the package, and price were found as the other most attention-grabbing parameters. In line with these results, it can be said that brand and expiration date affects their purchase habit more than price. At this point, the importance of trust in brands emerges. Also, with the current study, it was wanted to determine how much organic and halal label will affect people's purchasing preferences. It was observed that the presence of organic label affect the purchasing preference of the consumer positively by 79% while this ratio was 74% for halal label (Figure 8). Today, people tend to eat healthy and additive-free foods, and parallel to this, the fact that foods are organic has gained importance among consumers.

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Figure 8. Effects of organic and halal label on purchasing preferences Table 3 shows individuals' perceptions of their confidence in the meat and meat product industry. As seen from table, consumers are thinking that meats are unhealthier now comparing to past years, and they find slaughterhouse conditions more insufficient. In addition, they find meat sale points unsafe, they think that producers make cheatings in production and that the information on the packaging is not reliable. Also, it was observed that consumers feel insecure and worried about the consumption of meat especially regarding hygienic conditions and the use of appropriate production methods. And it was seen that many concerns about meats came to sight due to the inadequate and inaccurate news which are broadcasted in media such as television and internet. Table 3. Meat and meat industry assessments of consumers Agree

Disagree

No idea

Meat is more healty recently

118*

445

The supervisions of slougther house and production factory are enough

142

486

Hygine and food safety preventions are enough

164

473

There are so many deception in food processing

486

164

The informations on the package are true

248

370

* Number of responders who choose related parameter As regards meat safety and health, consumers were asked which occupational group and institutions they trust the most (Table 4). According to the answers, the Ministry of Agriculture and Forestry is located at the top of most reliable group while it was followed by nutrition experts and doctors, and then producers. It is thought that the most probable reason for finding nutritionists and doctors more reliable comparing to researchers and academicians may that, in Turkey, nutrition experts and doctors are showed themselves much more than researchers and academicians, in the media.

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Table 4. Ranking of trust level to occupational group and institutions First

Second

Third

Forth

Fifth

Sixth

Seventh

Ministry of agriculture and forestry

193*

132

Producter factory

151

127

130

104

Product’s label

118

112

149

105

101

Customer groups

117

142

115

105

News

133

131

118

116

Researchers and academicians

133

146

114

100

Dieticians and doctors

175

122

105

116

* Number of responders who choose related parameter CONCLUSIONS As a conclusion, it is apparent that meat and meat products consumed highly in individuals’ daily diet but there are serious concerns about the hygiene conditions and application of good manufacturing processes in production and sale steps. In order to reduce the consumers’ concerns about meat and meat industry: the relevant units of the Ministry of Agriculture and Forestry, the institution consumers trust most, can be informed consumers about production, slaughterhouse and sales conditions. Also, it should be avoided to present the news in the media to people without relying on strong and reliable sources. Information on nutrition, animal health and food consumption published in mass media should be provided by experts in field and awareness of consumers should be increased.

REFERENCES Anonymous (2015) Turkey Specific Food and Nutrition Guide/Türkiye’ye Özgü Besin ve Beslenme Rehberi, Hacettepe University faculty of health sciences Department of Nutrition and Dietetics, Merdiven advertising and promotion. Internet address: http://eyupihl.meb.k12.tr/meb_iys_dosyalar/34/09/160116/dosyalar/2019_02/131 34741_TYRKYYEYE_YZGY_BESLENME_REHBERY.pdf Bee, G., G. Guex, W. Herzog (2004). Free-range rearing of pigs during the winter: adaptations in muscle fiber characteristics and effects on adipose tissue composition and meat quality traits. J. Anim. Sci., 82, 1206–1218. Biesalski, H. K (2005). Meat as a component of a healthy diet are there any risks or benefits if meat is avoided in the diet? Meat Sci., 70(3):509-524. Bligh, E. G., W.Y. Dyer (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37(8), 911-917. Briggs, M. A., K. S. Petersen, P. M. Kriss-Etherton (2017). Saturated fatty acids and cardiovascular disease: replacements for saturated fat to reduce cardiovascular risk. Healthcare, 5(2), 29. 215

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Castellini, C., C. Mugnai, A. Dal Bosco (2002). Effect of organic production system on broiler carcass and meat quality. Meat Sci., 60 (3), 219-225. Ferguson L. R (2010). Meat and Cancer. Meat Sci., 84(2):308-313. McAfee A. J., E. M. McSorley, G. J. Cuskelly et al. (2010). Red meat consumption: an overview of the risks and benefits. Meat Sci., 84(1):1-13. Nohr, D., H. K. Biesalski (2007). ‘Mealthy’ food: meat as a healthy and valuable source of micronutrients. Animal., 1(2):309-316. Salter A. M (2018). The effects of meat consumption on global health. Rev. Sci. Tech. Off. Int. Epiz., 37 (1):47-55.

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AMINO ACID PROFILES OF WHITE WINES FROM THREE AUTOCHTHONOUS GALICIAN VARIETIES José M. MIRÁS-AVALOS1,2, Yolanda BOUZAS-CID1, Emiliano TRIGO-CÓRDOBA1, Ignacio ORRIOLS1,3, Elena FALQUÉ*4,5 1

Estación de Viticultura e Enoloxía de Galicia (EVEGA-AGACAL), Leiro – Ourense, Spain 2 3

Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Murcia, Spain

Dirección Xeral de Innovación e Industrias Agrarias e Forestais. Santiago de Compostela Spain 4

Facultad de Ciencias, Universidade de Vigo, Ourense, Spain

CITACA – Clúster de Investigación y Transferencia Agroalimentaria del Campus Auga, Ourense, Spain *Corresponding author’s e-mail: efalque@uvigo.es

ABSTRACT Amino acids play a relevant role in wine aroma and sensory characteristics and several studies proved that amino acid profiles have been useful for classification of white wines. In the current work, the amino acid profiles of white wines from three grapevine varieties (Albariño, Godello and Treixadura) grown in Galicia (NW Spain) were characterized over three consecutive vintages. The varieties were grown on the same vineyard and were harvested at optimum maturity. Wines were made following the same protocol for the three varieties. The concentrations of primary amino acids were determined by high-performance liquid chromatography with DAD detection. Wine amino acid contents from the three varieties were within the range of values reported for other European wines. Treixadura showed the highest concentration of amino acids, while wines from Albariño variety were characterized by substantially lower amounts of free amino acids. Aspartic acid, glutamic acid, lysine, arginine, asparagine, alanine and histidine were the most abundant amino acids after proline, which appeared in extremely high concentrations due to yeast release. Principal component analysis was able to separate wines by variety according to their amino acid profiles. Therefore, these profiles could be useful for classifying wines according to grape variety by means of statistical methods. Keywords: Nitrogen fraction, Principal component analysis, Vitis vinifera (L.), Wine classification INTRODUCTION Amino acids represent up to 40% of the total nitrogen in wines. Those present in grapes are consumed by yeasts during fermentation and might yield higher alcohols, aldehydes, esters and other volatile compounds (Bell and Henschke, 2005), influencing wine aroma (Robinson et al., 2014). Despite the wide range of factors affecting the nature and concentrations of the amino acids present in wines, some researchers have successfully employed the amino acid composition for differentiation of the product (Soufleros et al., 2003; Garde-Cerdán et al., 2009), classifying either grapes or wines from different varieties, management conditions and growing regions. 217

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In Galicia (NW Spain), white wines are produced with autochthonous varieties from which the most relevant are Albariño, Godello and Treixadura, with different aroma and sensory properties (Versini et al., 1994; Falqué et al., 2002). Recent studies determined the effect of irrigation on the amino acid composition of the musts from these varieties (Bouzas-Cid et al., 2018; Mirás-Avalos et al., 2019); however, the amino acid profile of wines made from these varieties has never been characterized in order to differentiate the product. Since amino acids have a relevant role in the progress of alcoholic fermentations and in the formation of aroma compounds, the current research aimed at: (1) identifying amino acid profiles of wines from the three main grapevine white varieties grown in Galicia (NW Spain), and (2) characterizing the wines from each variety according to their amino acid profiles. MATERIAL AND METHODS Description of the experimental vineyard The experiment was conducted over three years (2012-2014) in a 0.2 ha vineyard within the farm of the Estación de Viticultura e Enoloxía de Galicia in Leiro (42º 21.6’’ N, 8º 7.02’’ W, elevation 115 m), Ourense, Spain, within the Ribeiro Designation of Origin. Climate is warm-temperate, moderately dry and with cold nights. During grape maturation (August and September), the highest temperatures were recorded in 2013. No rainfall events occurred in August 2013, while more than 20 mm rainfall were registered in 2012 and 2014. September was less rainy in 2012, while in 2013 and 2014 more than 100 mm were recorded, mainly after harvest (Table 1). Table 1. Mean air temperature and total rainfall at the studied vineyard during the maturation period (August and September) in 2012, 2013 and 2014. Year Mean temperature (ºC) Total rainfall (mm) August September August September 2012 19.8 19.0 22.8 40.0 2013 21.8 19.6 0.0 108.6 2014 19.9 18.9 29.0 100.2 Soil at this site is sandy textured (64% sand and 19.6% clay), pH is 6.3, and 2.7% organic matter. The available water capacity is 100 mm m-1, approximately. The vineyard was planted with three white grapevine (Vitis vinifera L.) varieties autochthonous from NW Spain: Albariño, Godello and Treixadura. All of them were grafted in 1998 on 196-17C rootstock. Vines were vertically trellised on a single cordon system (10-12 buds per vine). Rows were East-West oriented; spacings were 1.25 and 2.4 m between vines and rows, respectively (3,333 vines ha-1). Sampling and winemaking The different grapevine varieties were manually harvested at their optimal maturity. Winemaking was performed separately on lots of 40 kg, approximately, per replicate (3 per variety and year) as detailed in Mirás-Avalos et al. (2019). Briefly, grapes from each replicate (three lots per variety) were separately destemmed, crushed and pressed. Pectolytic enzymes were added to favour settling (4 g per 100 kg of grapes). Then, SO2 (50 mg L-1) was added to avoid oxidation and for microbiological control. After 24 h, musts were racked and moved to 35-L stainless steel tanks. Commercial yeast (Excellence FW, Lamothe-Abiet, France) was added at 20 g hL-1. Once alcoholic fermentation 218

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was finished, wines were racked and sulphited to 35 mg L-1 free sulphur dioxide, clarified at 4 ºC for one month and, finally, wines were filtered, bottled and stored. Analytical methods Basic parameters of wines (alcohol content, pH, total acidity and tartaric and malic acids) were determined by Fourier transform infrared spectroscopy (FTIR) using a WineScan FT120 analyzer (FOSS Electric, Barcelona, Spain) calibrated according to the official methods (OIV, 2009). Determinations were carried out in triplicate five months after bottling. The determination of amino acids present in the wines from the three grapevine varieties was carried out through high-performance liquid-chromatography (HPLC) following a method based on a derivatization reaction in a basic methanolic medium (Bouzas-Cid et al., 2018). Determinations were carried out in triplicate. Statistical evaluation Two-way analysis of variance (ANOVA) considering grapevine variety, year and their interaction as factors was used for analyzing data. When needed, mean separation was carried out using the Tukey’s Honest Significance Difference test. Principal Component Analysis (PCA) using the amino acid concentrations allowed us to separate the wines according to variety. Statistical procedures were performed using R software v3.4.1 (R Core Team, 2017). RESULTS General parameters of wines Except for alcohol content, the general parameters of wines differed significantly among varieties (Table 2). Wines from Albariño had the highest acidity, whereas those from Treixadura had the lowest, and the contrary occurred for pH. The concentration of malic acid was lower in wines from Godello and that of tartaric acid was higher in Albariño wines. Year exerted a significant effect on pH and malic acid concentration. No significant interactions between year and variety were detected for any wine parameter (Table 2). Table 2. General parameters of the wines from the three white grapevine varieties studied. Data are averages for the three years ± standard errors. The P-values for the factors variety, year and their interaction are shown. Variety

Alcohol (% Vol.) 13.6 ± 2.2 14.1 ± 0.2 13.9 ± 0.3

Total acidity Tartaric acid Malic acid -1 -1 (g L as tartaric acid) (g L ) Albariño 3.03 ± 0.30 a 9.1 ± 1.8 b 4.8 ± 1.9 b 2.7 ± 1.3 b Godello 3.15 ± 0.04 a 7.1 ± 0.2 a 3.0 ± 0.4 a 2.0 ± 0.1 a Treixadura 3.45 ± 0.05 b 6.9 ± 0.2 a 2.5 ± 0.3 a 2.7 ± 0.1 b Factors Variety 0.300 < 0.01 < 0.01 < 0.01 < 0.01 Year 0.105 0.047 0.769 0.088 < 0.01 Variety x Year 0.466 0.773 0.079 0.094 0.356 Different letters in the column indicate significant differences among varieties for a given parameter according to the Tukey’s HSD test.

Amino acids in wines On average for the three studied years, the concentration of free amino acids in the wines from the three varieties studied ranged from 59.5 mg L-1 to 159.8 mg L-1, when proline was not 219

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accounted since yeasts excrete this amino acid. Aspartic acid was the most abundant amino acids in wines from the three varieties, accounting for 11-13% of the total free amino acids (Figure 1). Arginine, asparagine and alanine percentages were similar among varieties. Albariño wines had the lowest and Treixadura the highest concentrations of amino acids. The percentage of GABA and leucine is around 7% in Treixadura and 6% in Albariño and 4.5% and 7%, respectively, in Godello wines (Figure 1). The percentages of glycine, threonine and tryptophan were higher in Albariño, whereas those of valine, ornithine and glutamine were higher in Treixadura wines. Phenylalanine, serine and tyrosine appeared in percentages slightly higher in wines from Godello variety. The percentages of the rest of the amino acids detected in the wines were similar among the three white varieties (Figure 1).

Figure 1. Percentage of each amino acid over the total free amino acids in Albariño, Godello and Treixadura wines. Data are averages for the three studied years. Bars indicate standard errors. Abbreviations: Aspacid (aspartic acid), Gluacid (glutamic acid), Lys (lysine), Arg (arginine), Asp (asparagine), Ala (alanine), His (histidine), GABA (aminobutyric acid), Leu (leucine), Gly (glycine), Val (valine), Phe (phenylalanine), Thr (threonine), Ser (serine), Ile (isoleucine), Tyr (tyrosine), Met (methionine), Try (tryptophan), Orn (ornithine), Glu (glutamine), Cys (cysteine). The PCA considering the percentages of free amino acids except proline revealed a clear separation of the wines from each variety (Figure 2). The first two principal components accounted for 100% of the total variance in the data set: PC1 explained 67.2% and PC2 explained 32.8%. Treixadura wines appeared on the positive side of both PC1 and PC2 due to their high concentrations in glutamine, cysteine, GABA and ornithine. Albariño wines appeared on the negative side of PC1 and on the positive side of PC2 due to their high concentrations in methionine, asparragine, threonine and tryptophan. Finally, Godello wines appeared on the

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positive side of PC1 and on the negative side of PC2 due to their high concentrations in serine, tyrosine and phenylalanine (Figure 2).

Figure 2. Principal component analysis (PCA) of Albariño, Godello and Treixadura wines averaged for the three years studied (2012-2014). Bi-plot for the first two components (PC) for free amino acids except proline. DISCUSSION On average for the three years studied, the concentration of amino acids was 59.5, 89.0 and 159.8 mg L-1, respectively for Albariño, Godello and Treixadura wines. These concentrations are slightly lower than the range reported for white wines from different regions (Soufleros et al., 2003) and much lower than those for red wines (Ali et al., 2010; Martínez-Pinilla et al., 2013). Aspartic and glutamic acids, lysine, arginine, asparragine, alanine and histidine were the most abundant amino acids present in the studied wines, representing approximately 58% of the total primary amino acid content. The effects of grapevine variety, year and region of production (including agricultural and oenological practices) are relevant for the final concentration of amino acids in white wines (Soufleros et al., 2003; Ali et al., 2010). In the current study, we removed the effect of the region, agricultural and oenological practices by employing three white varieties grown on the same vineyard, under the same agricultural practices and using the same winemaking procedure. Therefore, the differences in the concentration of amino acids among wines from the different varieties considered in this study will be exclusively originated by the variety. Wines from Albariño and Godello presented similar concentrations of most amino acids, whereas wines from Treixadura were characterized by higher amino acid concentrations. However, when compared to other white varieties, the amino acid concentrations in wines from Albariño, Godello and Treixadura are much lower. For instance, wines produced with Roditis (a Greek variety) showed 430 mg L-1 of free amino acids (Soufleros et al., 2003). Similarly, Chardonnay wines showed amino acid concentrations up to 600 mg L-1 (Soufleros et al., 2003), significantly higher than those observed in the wines studied here. The 22 amino acids determined appeared in the wines from the three varieties studied, with slight differences in the relative proportion of major amino acids, except for aspartic and glutamic acids and lysine. In contrast, the profiles of minor amino acids differed clearly among varieties, which could lead to a means for wine classification and a tool for authentication of 221

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wine origin, confirming previous studies (Soufleros et al., 2003; Garde-Cerdán et al., 2009; Martínez-Pinilla et al., 2013). CONCLUSIONS This work showed, for the first time, the amino acid profiles of wines made from the Galician autochthonous varieties Albariño, Godello and Treixadura. The amino acid concentrations of white wines from these varieties were lower than the ranges reported for other white wines, confirming the influence of grape variety, geographic location and vintage on the amino acid composition of wine. When principal component analysis was applied, amino acids were able to differentiate wines by variety. Consequently, the amino acid profile could be used as a tool in wine classification. Acknowledgements: This study was ﬁnanced by Instituto Nacional de Tecnología Agraria y Alimentaria (INIA) project RTA2011-00041-C02-01, with 80% FEDER funds. Y. Bouzas-Cid and E. Trigo-Córdoba thank INIA for their PhD. scholarships. J.M. Mirás-Avalos thanks Xunta de Galicia (08.A3.561A.432.0) for his “Isidro Parga Pondal” contract. E. Falqué-López acknowledges CITACA for the financial support to assist at the Congress. REFERENCES Ali, H. S. M., R. Pätzold, H. Brückner. (2010). Gas chromatographic determination of amino acid enantiomers in bottled and aged wines. Amino Acids, 38, 951-958. Bell, S. J., P. A. Henschke. (2005). Implications of nitrogen nutrition for grapes, fermentation and wine. Aust. J. Grape Wine Res., 11, 242-295. Bouzas-Cid, Y., E. Falqué, I. Orriols, J. M. Mirás-Avalos (2018). Effects of irrigation over three years on the amino acid composition of Treixadura (Vitis vinifera L.) musts and wines, and on the aromatic composition and sensory profiles of its wines. Food Chem., 240, 707716. Falqué, E., E. Fernández, D. Dubourdieu (2002). Volatile components of Loureira, Dona Branca, and Treixadura wines. J. Agric. Food Chem., 50, 538-543. Garde-Cerdán, T., C. Lorenzo, J. F. Lara, F. Pardo, C. Ancín-Azpilicueta, M. R. Salinas (2009). Study of the evolution of nitrogen compounds during grape ripening. Application to differentiate grape varieties and cultivated systems. J. Agric. Food Chem., 57, 2410-2419. Martínez-Pinilla, O., Z. Guadalupe, Z. Hernández, B. Ayestarán (2013). Amino acids and biogenic amines in red varietal wines: the role of grape variety, malolactic fermentation and vintage. Eur. Food Res. Technol., 237, 887-895. Mirás-Avalos, J. M., Y. Bouzas-Cid, E. Trigo-Córdoba, I. Orriols, E. Falqué (2019). Effects of two different irrigation systems on the amino acid concentrations, volatile composition and sensory profiles of Godello musts and wines. Foods, 8, 135, doi: 10.3390/foods8040135 OIV (Office International de la Vigne et du Vin) (2009). Recueil des méthodes internationales d’analyse des vins et des moûts. Ed. OIV, Paris, France. R Core Team (2017). R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. https://www.R-project.org Robinson, A. L., P. K. Boss, P. S. Solomon, R. D. Trengove, H. Heymann, S. E. Ebeler (2014). Origins of grape and wine aroma. Part 1. Chemical components and viticultural impacts. Am. J. Enol. Vitic., 65(1), 1-24.

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Soufleros, E. H., E. Bouloumpasi, C. Tsarchopoulos, C. G. Biliaderis (2003). Primary amino acid profiles of Greek white wines and their use in classification according to variety, origin and vintage. Food Chem., 80, 261-273. Versini, G., I. Orriols, A. Dalla Serra (1994). Aroma components of Galician AlbariÃ±o, Loureira and Godello wines. Vitis, 33, 165-170.

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BACTERIOPHAGES AS COMPLEMENTARY AGENTS IN THE MANAGEMENT STRATEGIES OF FIRE BLIGHT DISEASE Ayşegül GEDÜK1, Kubilay Kurtuluş BAŞTAŞ1* 1

Selçuk University Faculty of Agriculture Dept. of Plant Protection, Campus, Konya, Turkey *Corresponding Author e-mail: kbastas@selcuk.edu.tr ABSTRACT

Fire blight disease, is caused by Erwinia amylovora, is a very destructive and economically significant disease of apples, pears and many other Rosaceous plants. Specific bacteriophages for E. amylovora play an important role in epidemiology of fire blight disease and they have a great potential to disease management. Phages of E. amylovora have usually been found on above ground parts and in soil associated with fire blight infected apple and pear trees. The identified bacteriophages have highly specific for the pathogen and also complete genomes of these phages have been determined. Control of the blossom blight stage of fire blight is crucial in the combat program of the disease. The bacterium colonizes on the stigmatic surfaces of blossoms before blossom infection. It was determined that E. amylovora specific phages present on the blossoms, they might be suppressed the growth of the pathogen on the stigmatic surfaces. However, natural phage populations are below desirable levels during the bloom period. Therefore, the blossoms applications of E. amylovora phages would be required to be with similar methods and time, as with bacterial and fungal biological preparations. Obtaining data showed that prior colonization of the host blossoms by an avirulent strain of E. amylovora was important for the establishment and maintenance of phage populations. Some phages reduced the infection of pear and apple blossoms between 50% and 80% ratios, comparing to streptomycin. Without such being a prior colonization on host the population of phage importantly declined, due to UV light, desiccation, temperature and the other climatically effects.It is considered that E. amylovora phages may show the potential to confer effective the disease control as part of an sustainable, organic and integrated management strategy. Keywords; Bacteriophage, Control, Pome fruits, Eco-friendly agriculture

INTRODUCTION Fire blight caused by Erwinia amylovora (Burrill) Winslow et al., is the most destructive bacterial disease of plants within the Rosaceae family (Zwet and Keil, 1979; Zwet and Beer, 1991). E. amylovora which causes significant economic losses in pome fruit, is the first bacterial disease identified in plants in the world. After the disease was first discovered in the United States, spread to about 40 countries, including North America, Europe, the Middle East and New Zealand in the late 1700s (Bonn and Zwet, 2000). The disease can cause severe economic losses by causing a high epidemic in some years (Vanneste, 2000).In the USA, fire blight losses and control costs per year are estimated to be more than $100 million (Norelli et al., 2003). Conventional management of fire blight relies on cultural practices and preventive copper and antibiotic sprays (Norelli et al., 2003).

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Successful fire blight disease management is dependent on controlling the epiphytic pathogen populations. Fire blight management has focused on investigating plant resistance inducers and biological control agents as potential alternatives to antibiotics and copper (Johnson and Stockwell, 1998; Maxson-Stein et al., 2002; Norelli et al., 2003; Sundin et al., 2009; Johnson and Temple, 2013; Acimovic et al., 2015). The use of bacteriophages as new and promising biological agents in controlling of the pathogen is getting common in practical applications. In this study, the bacteriophages which are promising to be used in the control of E. amylovora and their usage possibilities are presented to growers and beneficiaries. Use of Bacteriophages in Control of Erwinia amylovora Bacteriophages used in the control of Erwinia amylovora take over the mechanism of the cell to make phage components. They then destroy, or lyse, the bacteria cell, releasing new phage particles. Many bacteriophage isolates have been obtained for use in the control of E. amylovora. Gill et al. (2003) collected 50 bacteriophage isolates from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Schnabel and Jones (2001), among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages fEa1 and fEa7 and 3 novel phages named fEa100, fEa125,and fEa116C, have identiﬁed based on differences in genome size and restriction fragment pattern. The first and successful studies for phage based control of fire blight have been shown by Erskine who has isolated the phages for E. amylovora and Pantoea agglomerans (Erskine, 1973). In subsequent studies since the 1970s, different phages have been isolated and characterized in detail (Erskine, 1973; Kim et al., 2004). Müller et al. (2011), characterized E. amylovora bacteriophages collected from North America and Germany and they determined that Фea104 and Фea116 phages reduce the fire blight symptoms in blossoms and immature pears compairing to Фea1h and Фea100. Lytic bacteriophages have been used for control many bacterial pathogens causing disease in plants (Buttimer et al., 2017; Doffkay et al., 2015). Park et al. (2018) have characterized lytic bacteriophage phiEaP-8, which is effective on both E. amylovora and E. pyrifoliae, which causes severe diseases in apple and pear. The main strategy of controlling fire blight with biocontrol agents is to prevent the accumulation of E. amylovora populations on the nutrient-rich stigmatic surfaces of the blossom in spring (Thomson, 1986; Johnson et al., 1998). Lehman (2009) researched the effect of the phages he obtained from blossoms of pear and apple trees in Canada. Identified phages decreased the pathogen population on Gala cultivar by 50% ratios (Ritchie et al., 1977; Ritchie et al., 1979). In addition, it was determined that the phages were low effective when applied individually, but they were more successful when they applied the Φea1, Φea7 and Φea116c phages as a mixture. Field based trials have demonstrated that phage-based biopesticides are effective in reducing E. amylovora in blossom populations (Lehman, 2007). Schwarczinger et al. (2011) determined that the effect of phages on E. amylovora populations depends on the pathogen susceptibility of plants. The researchers tested two selected phage on three apple varieties which exhibited different susceptibility to E. amylovora and found that the number of bacteria decreased significantly compared to controls. The best results were obtained from the application of phage H6 on moderately resistant apple cv. Freedom and it was determined that the bacterial population decreased by 90% (Figure 1). Nagy et al. (2015) investigated the uptake and transport of E. amylovora-specific bacteriophages in apple plants. In the study, H5K and ФEa104 phages were used (Figure 2). E. amylovora-specific H5K and ΦEa104 phages were 225

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found to be able to penetrate plants and reduced the degree of symptoms by 45% and 67%, respectively.

Fig. 1 Effect of phages on Erwinia amylovora infection on apple (Malus domestica Freedom) flowers (Schwarczinger et al., 2011).

Fig. 2 Plaques of bacteriophage H5K formed on soft agar top layer medium containing E. amylovora (Nagy et al., 2015)

In Hungary, two species of E. amylovora phage (PhiEaH1 and PhiEaH2) belongs to the Siphoviridae family have been identified and found successful in the struggle (Dömötör et al., 2012; Meczker et al., 2014). These two phages are biological control agents of the biopesticide ERWIPHAGE FORTE, which is registrated in Hungary since 2012 for fire blight control (Anonymous, 2019) (Table 1). Ritchie and Klos (1979) isolated E. amylovora phages from the aerial parts of the apple tree in Michigan, and subsequently described their properties. Schnabel et al. (1999) reported 26–37% control of fire blight on apple blossoms with a mixture of three phage isolates and demonstrated that high populations of phage were dependent on the presence of E. amylovora. Schnabel and Jones (2001) characterized eight E. amylovora phages by plaque morphology, restriction fragment polymorphisms, PCR analysis, pulsed field gel electrophoresis and host range studies. Using these techniques along with transmission electron microscopy, Gill et al. (2003) and Lehman (2007) studied 42 and 56 E. amylovora phage isolates, respectively, and assessed their potential as biological control agents. At the present days, three commercial preparations containing bacteriophages are used successfully in the biological control of Erwinia amylovora in orchard conditions (Table 1). Table 1. Commercial Bacteriophage Preparates of Erwinia amylovora in the World Pathogen / Disease

Commercial name AgriPhage™Fireblight

E. amylovora (Fire Blight)

Erwiphage Forte Erwiphage Plus

Application Type 2 weeks intervals

Recommended Rate 2,4-4,7 L ha-1

3 times during flowering

component "A" 0.1 L ha-1+ component "B" 1.0 L ha-1 (1000 L)

3 times during flowering

component "A" 0.1 L ha-1 + component "B" 1.0 L ha-1 (1000 L).

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RESULTS AND DISCUSSION Since the latest four decades, we have a substantial amount of information on the biology and ecology, including the molecular characterization of bacteriophages that infect plant pathogens. Therefore, the potential for use bacteriophages as biological control agents in agriculture is highly remarkable (Okabe and Goto, 1963; Civerolo, 1972; Ackermann and DuBow, 1987; Calendar, 2004; Gill et al., 2003; Lehman et al., 2009; Jones et al., 2007). Due to their simple structure, specific relationships with host bacteria, easy and cheap production, widespread in nature and ability to renew themselves, they can be used effectively in integrated struggle. To be successful in biological control of E. amylovora, it is necessary to prevent the accumulation of the pathogen populations on the nutrient-rich stigmatic surfaces of the blossom in spring. Therefore, Svircev et al. (2005) and Lehman et al. (2009) used a pear blossom model to determine the control effect of bacteriophages, as well as setting up initial parameters for field experiments and selecting the best phage isolates for orchard trials. Svircev et al. (2005) obtained a similar effect to streptomycin, while Lehman (2009) suppressed the pathogen by 50%. Although the use of bacteriophages against E. amylovora is promising, there are several drawbacks. For example, the use of a single phage to control is extremely risky in terms of genetic diversity, susceptibility to phages, and the ability of bacteria to resist a single phage. Schnabel et al. (1999) applied the mixture of E. amylovora phages and obtained very successful results. Another major challenge to be considered in the struggle is the negative effects of various environmental factors such as exposure to sunlight, radiation, especially UV rays, drought and temperature. Born et al. (2015) considered that bacteriophages used in the control of E. amylovora should be adequately protected against UV rays. For this, natural extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances such as astaxanthin, aromatic amino acids, and Tween 80 were prepared and tested as natural sunscreens for phage. They have found that all these compounds significantly increase the halflife of the UV-exposed phage particles and do not adversely affect the infectivity of the phages. All of these factors should be taken into account in order to maintain the high phage populations in the target region for a longer period of time and phages should be used as part of an integrated fire blight disease management strategy. REFERENCES Ackermann H-W., DuBow M.S., (1987). Viruses of Prokaryotes (vol. I): General Properties of Bacteriophages, CRC Press, Boca Raton, Florida Anonymous, (2019). Erwiphage Forte, Access Date: 03.11.2019, Address: http://www.erwiphage.com/ Basım, H., (1999). Erwinia amylovora İzolatlarının Streptomisin ve Bakır’a Dayanıklılıkları ve Plazmid Profilleri. S. Ü. Ziraat Fakültesi Dergisi 12 (17): 22-30. Baysal Ö., Zeller W. (2001). Studies on control of fire blight with the plant activator BION®, In: De Boer S. H. (Ed.), Plant pathogenic bacteria, Kluwer Academic Publishers, Dordrecht, The Netherlands, 324–328 Bonn W.G., Zwet T. (2000). Distribution and economic importance of fire blight, In: Vanneste J.L. (Ed.), Fire blight: the disease and its causative agent, Erwinia amylovora, CABI Publishing, Wallingford, United Kingdom, 37-53 Born, Y., Bosshard, L., Duffy, B.,L oessner, M.J., Fieseler, L., (2015). Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds, Basıc Research Paper, 1-2 227

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Bubán T., Sallai P. (2002). Obzsut-Truskovszky E., Hertelendy L., Trials with applying chemical agents other than bactericides to control fire blight in pear orchards, Acta Hortic., 590, 263–267 Buttimer, C., McAuliffe, O., Ross, R. P., Hill, C., O’Mahony, J. and Coffey, A. (2017). Bacteriophages and bacterial plant diseases. Front Microbiol. 8:34. Calendar R., (Ed.), (2004). The Bacteriophages, Oxford University Press, New York. Civerolo E.L., (1972). Interaction between bacteria and bacteriophage on plant surfaces and in plant tissues, 25-37, In: Maas Geesteranus H.P. (Ed.), Third Int. Conf. Plant Pathogenic Bacteria, Proc., Centre Agric. Publ. Doc. (PUDOC), Wageningen, Netherlands, [53] Billing E., An association Doffkay, Z., Dömötör, D., Kovács, T. and Rákhely, G. (2015). Bacteriophage therapy against plant, animal and human pathogens. Acta Biol. Szeged. 59:291-302. Erskine J.M. (1973). Characteristics of Erwinia amylovora bacteriophage and its possible role in the epidemiology of fire blight, Can. J. Microbiol., 19, 837-845 Gill, J.,& Abedon, S. T. (2003). Bacteriophage ecology and plants. APSnet Feature. doi:10.1094/APSnetFeature-20031103. Hanke V., Geider K., Richter K. (2003). Transgenic apple plants expressing viral EPSdepolimerase: Evaluation of resistance to the phytopathogenic bacterium Erwinia amylovora, In: Vasil I.K., (Ed.), Plant Biotechnology 2002 and Beyond, Kluwer Academic Publishers, Dordrecht, Netherlands, 153-157 Johnson K.B., Stockwell V.O., (1998). Management of fire blight: a case study in microbial ecology, Annu. Rev. Phytopathol., , 36, 227–248 Jones J.B., Jackson L.E., Balogh B., Obradovich A., Iriarte F.B., Momol M.T. (2007). Bacteriophages for plant disease control, Annu. Rev. Phytopathol., 45, 245-262 Jones J.B., Jackson L.E., Balogh B., Obradovich A., Iriarte F.B., Momol M.T. (2007). Bacteriophages for plant disease control, Annu. Rev. Phytopathol., 45, 245-262 Kim W.S., Salm H., Geider K. (2004). Expression of bacteriophage ФEa1h lysosyme in Escherichia coli and its activity in growth in inhibition of Erwinia amylovora, Microbiology, 150, 2707-2714 Lehman, S.M. 2007. Development of a bacteriophage-based biopesticide for fire blight.Brock University: PhD Thesis. Lehman S .M., Kropinski A.M., Castle A.J., Svircev A.M. (2009). Complete genome of the broad-hostrange Erwinia amylovora phage Ea21-4 and its relationship to Salmonella phage Felix O1, Appl. Environ. Microb., 2009, 75, 2139–2147 Momol M.T., Norelli J.L., Aldwinckle H.S. (1996). Field evaluation of fire blight resistance of an attacin E-transgenic M.7. apple rootstock, Phytopathology, 86, S92 Moller, W. J., Schroth, M. N., and Thomson, S. V., (1981). The Scenario of Fire Blight and Streptomycine Resistance. Plant Disease, Vol.65:563-568. Müller I., Kube M., Reinhardt R., Jelkmann W., Geider K. (2011). Complete genome sequences of three Erwinia amylovora phages isolated in North America and a bacteriophage induced from an Erwinia tasmaniensis strain, J. Bacteriol., 193, 795–796 Okabe N., Goto M., (1963). Bacteriophages of plant pathogens, Annu. Rev. Phytopathol., 1, 397-418 Orlova, E. (2012). Bacteriophages and their structural organisation. In: Bacteriophage, ed. by I. Kurtboke, pp. 3-30. InTech, London, UK. Öktem Y, Benlioğlu K. (1988). Studies on fire blight (Erwinia amylovora (Burr) Winslow et al.) of pome fruits. Journal of Turkish Phytopathol., 5th. Turkish Phytopatholological Congress., Antalya, Turkey, 17: 3.

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Park, J., Lee, G. M., Kim, D., Park, D. H., Oh, C. (2018). Characterization of the Lytic Bacteriophage phiEaP-8 Effective against Both Erwinia amylovora and Erwinia pyrifoliae Causing Severe Diseases in Apple and Pear, Plant Pathol. J. 34(5) : 445-450. Ritchie D.F., Klos E.J., (1977). Isolation of Erwinia amylovora bacteriophage from aerial parts of apple trees, Phytopathology, 67, 101-104 Ritchie D.F., Klos E.J., (1979). Some properties of Erwinia amylovora bacteriophages, Phytopathology, 69, 1078-1083 Schnabel E.L., Fernando W.G.D., Meyer M.P., Jones A.L., Jackson L.E. (1999). Bacteriophage of Erwinia amylovora and their potential for biocontrol, Acta Hortic., 489, 649–654 Schnabel E.L., Jones A.L., (2001). Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora, Appl. Environ. Microb., 67, 59-64 Schwarczinger I., Kiss E., Süle S., Tóth M., Hevesi M., (2011). Control of fire blight by bacteriophages on apple flowers, Acta Hortic., 896, 457-462 Svircev A.M., Lehman S.M., Kim W.S., Barszcz E., Schneider K.E., Castle A.J., Control of the fire blight pathogen with bacteriophages, In: Zeller W., Ullrich C., (Eds.), Proceedings of the 1st International Symposium on Biological Control of Bacterial Plant Diseases, (2326 October 2005, Darmstadt/ Seeheim, Germany), Arno Brynda, Berlin, 2006, 408, 259261 Thomson S.V., The role of stigma in fire blight infections, Phytopathology, 1986, 76, 476–482 Vanneste, J. L. (2000). Fire blight, the disease and its causative agent, Erwinia amylovora. New York: CABI. Vanneste, J.L., 2000. Fire Blight: The Disease and Its Causative Agent, Erwinia amylovora. CABI Publishing, Wallingford, UK., Pp.370. Winslow, C. E., Broadhurst, J., Buchanan, R. E., Krumwiede Jr., C., Rogers, L. A., & Smith, G. H. (1920). The families and genera of the bacteria: final report of the committee of the Society of American Bacteriologists on characterization and classification of bacterial types. Journal of Bacteriology, 5(3), 191. Wright S.A.I., Beer S.V., (1996). The role of antibiotics in biological control of fire blight by Erwinia herbicola strain Eh318, Acta Hortic., 411, 309–311 Zeller W., Status of biocontrol methods against fire blight, Phytopathol. Pol., 2006, 39, 71-78 Zwet, T.,&Beer, S. V. (1991). Fire blight, its nature, prevention and control - a practical guide to integrated disease management. Washington: Agriculture information bulletin. U.S. Department of Agriculture. Zwet, T., Keil, H. L. (1979). Fire blight, a bacterial disease of Rosaceous plants .Agriculture Handbook, Science and Education Administration. Washington: U.S. Department of Agriculture.

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SCREENING OF RESISTANCE GENES AND SOME REACTIVE OXYGEN SPECIFIC ENZYMES AGAINST XANTHOMONAS AXONOPODIS PV. PHASEOLI AND PSEUDOMONAS SAVASTANOI PV. PHASEOLICOLA IN BEAN VARIETIES Tibebu BELETE1&2 Badel UYSAL SAHIN3 1

Kubilay Kurtulus BASTAS1*

Selçuk University Faculty of Agriculture Dept. of Plant Protection, Campus, Konya, Turkey 2

Rwanda Institute for Conservation Agriculture (RICA), Bugesera Campus, Bugesera, Rwanda

Igdir University Faculty of Agriculture Dept. of Plant Protection, Campus, Igdir, Turkey *Corresponding Author’s e-mail: kbastas @selcuk.edu.tr ABSTRACT

Common bacterial blight (CBB) and halo blight (HB) of bean are the most destructive bacterial diseases that cause reduction of yield and quality of bean production and the use of resistant cultivars is the most effective control. Identifying the presence of resistance gene in a given bean variety and determine the activity of enzymes after and before bacterial inoculation is very important to develop a resistant breeding line. Therefore, the objectives of this study was to investigate the presence of resistance genes against Xanthomonas axonopodis pv. phaseoli (Xap) and Pseudomonas savastanoi pv. phaseolicola (Psp) in eight bean varieties (Ozmen, Noyanbey, Akman, Alberto, XAN159, Vax1, Aras 98 and 36K) and to determine the activities of the antioxidant enzymes like Peroxidase (POX) and Catalase (CAT) against to Xap and Psp in two bean varieties (Aras 98 and 36K). The resistant genes (SAP6, BAC6, BC420 and R7313 for Xap and SR13, ST8, SH11 and SB10 for Psp) using SCAR markers, the above eight varieties were screened at green house conditions. A four week-old bean seedlings were inoculated with different Xap and Psp isolates for enzyme analyses. To determine the above enzyme activities, one gram of bean leaves were collected from each pathogen-inoculated and control bean plants at 0, 12th, 24th, 48th, 72nd hours. The lowest disease incidence and severity for CBB and HB diseases were observed under greenhouse conditions from the variety XAN159, Vax1, Ozmen, Noyanbey and 36K (p≤ 0.01). SAP6 and BAC6, SR13 resistance genes were determined in all cultivars except cv. Alberto and cv. Aras 98, respectively and SR13 resistance gene was detected on all cultivars except cv. Noyanbey and cv. Aras 98. POX enzyme activity increased by 198.40% in 36K cultivar at 36th hr. and CAT enzyme activity found as the highest (0.4087 Ugr-1/ FW) in 36K cultivar at 72hr after Psp inoculation (p≤ 0.01). Increase of antioxidant enzymes and resistance genes activities might be an important component in the defense strategy of resistance and susceptible bean genotypes against the bacterial infection. Keywords: Bean, Enzyme, SCAR, Xanthomonas axonopodis pv. phaseoli, Pseudomonas savastanoi pv. phaseolicola

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INTRODUCTION Common bean (Phaseolus vulgaris L.) is an important legume in the world as a source of protein, dietary fibre, and minerals such as potassium, thiamine, vitamin B6 and folic acid in diets (Garden-Robinson and McNeal, 2013; Chekanai et al., 2018). Also the presence of phytochemicals such as polyphenolic compounds in common bean prevents various human diseases and they show high antioxidant activity (Hayat et al., 2014). The production of beans (green and dried) worldwide in 2017 was about 56 million tons, and they were harvested on 38 million hectares (Anonymous, 2019). In Turkey sowing area and production of beans (green and dried) is 1 382 613 decares and 869 347 tons respectively (Anonymous, 2019a). Bacterial diseases that cause yield and quality losses in bean production lead to severe epidemics in warm and humid conditions and yield losses of up to 45% depending on the durability of bean variety and environmental conditions (Singh and Schwartz, 2010; FélixGastélum et al., 2016). The damage by halo blight (HB) caused by Pseudomonas savastonoi pv. phaseolicola (Psp) and common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli (Xap) in the bean plants is of great economic importance worldwide due to the significant losses it generates in the crop (Hall, 1994; Singh et al., 1995).HB and CBB are difficult to control and the effectiveness of existing strategies for the treatment of existing diseases is limited due to the absence of an effective chemical. Genetic resistance and using of disease-free seed provides the most effect viable control of these two disease (Miklas et al., 2014; Ferreira et al., 2003).Plant resistance is highly effective in controlling crop loss from bacterial pathogen infection. Plants have evolved varied defense mechanisms to protect themselves against pathogen attacks (Waller et al., 2005). Antioxidant enzymes such Peroxidase (POX) and Catalase (CAT) play as an important role in the plant response mechanism to pathogen invasion, by the detoxification of superoxide and H2O2 (Bernal‐Vicente et al., 2015). In this study, it was focused on antioxidant activities of POX and CAT enzymes in eight bean varieties and the resistance genes in these bean varieties for Xap and Psp were scanned using SCAR (sequence characterized amplified region) markers. MATERIALS AND METHODS Materials Bean cultivars were procured from Dept. of Field Crop, Selcuk University (Ozmen, Noyanbey, Akman, Alberto) and Ataturk University (36K, resistant) and Eastern Anatolia Agricultural Research Institute in Turkey (Aras98, susceptible,) and International Centre for Tropical Agriculture (CIAT) (XAN159, Vax1). Bacterial strains, Xanthomonas axonopodis pv. phaseoli (Xap (120-x, 145-x)) and Pseudomonas syringae pv. phaseolicola(Psp (510-p,522-p)) culture was provided by Dr. M. F. Donmez, Dept. of Plant Protection, Iğdır University. Methods Enzyme Assays All bean cultivars seed samples used in the experiment were surface sterilized with 3% (v/v) sodium hypochlorite solution for 3 min and washed with distilled water. Plants were grown in 10 cm pots in a soil mix containing sand, perlite, and peat compost under greenhouse conditions. Greenhouse-grown 36K and Aras98 bean seedlings leaves were used to determine POX and CAT enzymes activities.

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Two days old cultures of Xap and Psp were harvested from NA plates, suspended in sterile deionized water, and adjusted to a concentration of 108 CFU mL-1 determined by aspectrophotometer at 660 nm and OD: 0.15. Plants of the 36K bean genotype and Aras98 bean variety were sampled 0, 12th, 24th, 48th, 72nd hours post inoculation (hpi) and without pathogen inoculation. The samples were stored at -80 oC prior to the enzyme assay (Terenashi et al., 1974). Fresh leaf material (1.0 g) from bacterial pathogen treated and control bean plants was homogenized with a mortar and pestle in 6 ml of ice-cold 50mM sodium potassium phosphate pH 7. After the homogenate was centrifuged at 10,000g for 25 min and the supernatant (crude extract) used as the source of POX and CAT enzymes. All the steps were carried out at 0-4 oC (Jebara et al., 2005). Peroxidase (POX, EC: 1.11.1.7)Assay:The activity of peroxidase was determined by adding 100 μlof the crude enzyme preparation to 2mL of a solution containing 10 mmol/L KH2PO4, pH 7, 20 mmol/LGuiacol and 40 mmol/L H2O2. After change of absorbance at 470 nm was record for 1 min with a spectrophotometer (Lin and Kao, 1999). Catalase (CAT; EC: 1.11.1.6) Assay:The activity of catalase was determined by adding 100 μL of the enzyme extract to 900 µL of a solution containing 50 mM potassium phosphate buffer and 30 mM H2O2. After change of absorbance at 240 nm was record for 1 min with a spectrophotometer (Aebi, 1984). Determination of Resistance Genes bySCAR Markers in Bean Varieties Plant material Ozmen, Noyanbey, Akman, Alberto, XAN159, Vax1, Aras 98 and 36K bean seeds were planted into pots (diameter 15 cm)filled with a mixture of sterilized soil,sand and farmyard manureand grown at 25–35˚Cin the greenhouse. DNA isolation Total genomic DNA extractions were prepared from fresh bean leaves. 200-300 mg powdered plant materials for each bean varieties was transferred to a 2 mL eppendorf tubes and 1 mL freshly prepared extraction buffer was added (Doyle and Doyle,1987). PCR amplifications PCR amplification for SCAR marker primers (Table 1) from genomic DNA was performed in a total reaction volume of 20 μL containing 4 μL (5 ng/μL) of template bean DNA, 2 μL 10XTaq polymerase reaction buffer, 7 μL dH2O, 2 μL MgCl2, 0,5 μLof each dNTPs (dATP, dCTP, dGTP, and dTTP), 0.2 mM primers and 0,5 μLof Taq DNA polymerase (Applied Biological Materials (ABM, Canada)).

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Table 1. The SCAR markers used in PCR amplifications SCAR Markers

SR13/Ps p

Size (bp)

1150 bp

ST8/Psp

1350 bp

SH11/Ps p

800 bp

SB10/Ps p

525 bp

SAP6/Xa p

820 bp

BAC6/X ap

1250 bp

BC420/X ap

R7313/X ap

900 bp

700 bp

Amplification Protocol

Primers

95oC 5 min for 1 cycle, 5’GGACGACAAGGAACATA 30 cycles of 60s at 94oC, TTCA 3’ 60s at 60oC, and 60s at 5’GGACGACAAGGCTGCAA 72oC; followed by one GAACCAT 3’ cycle of 7 minutes at 72oC 95oC 5 min for 1 cycle, 5’AACGGCGACATCAGTGT 30 cycles of 10 s at AAAGG 3’ 94oC, 40s at 65oC, and 5’AACGGCGACAACCGACC 120 s at 72oC; followed ATGTTTTAC 3’ by one cycle of 7 minutes at 72oC 95oC 5 min for 1 cycle, 5’CTTCCGCAGTCGAGAGA 30 cycles of 10s at T 3’ 94oC, 40s at 67oC, and 120s at 72oC; followed 5’CTTCCGCAGTAGCAC C 3’ by one cycle of 7 minutes at 72oC 94oC 5 min for 1 cycle, 5’CTGCTGGGACAATCACC 30 cycles of 10s at AAGTC 3’ 94oC, 40s at 65oC, and 120s at 72oC; followed 5’CTGCTGGGACTCTCTTAC by one cycle of 5 3’ minutes at 72oC 34 cycles of 10s at 5’GTCACGTCTCCTTAATAG 94oC, 40s at 55oC, and TA 3’ 120s at 72oC; followed 5’GTCACGTCTCAATAGGC by one cycle of 5 AAA 3’ minutes at 72oC 34 cycles of 10s at 94 5’TAGGCGGCGGCGCACGT o C, 60s at 70 oC, and TTTG 3’ 120s at 72 oC; followed 5’TAGGCGGCGGAAGTGGC by one cycle of 5 GGTG 3’ minutes at 72 oC 5’GCAGGGTTCGAAGACAC 35 cycles of 30s at 94 o ACTGG 3’ C, 30s at 50 oC, and 60s at 72 oC; followed 5’GCAGGGTTCGCCCAATA by one cycle of 5 ACG 3’ minutes at 72 oC 5’ATTGTTATCGTCGACACG 34 cycles of 10s at 94 o 3’ C, 40s at 60 oC, and 120s at 72 oC; followed 5’AATATTTCTGATCACACG by one cycle of 5 AG 3’ minutes at 72 oC

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References

Miklaset al., 2000

Miklaset al., 2009

Fourieet al., 2004

Miklas et al., 2000

Jung et al.,1999

Yu et al., 2000

Beattie et al.,1998

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RESULTS AND DISCUSSION Enzyme Assays P. s. pv. phaseolicola and X. a. pv. phaseoli were reported as a causal agent of halo blight and bacterial blight of beans, respectively. They cause serious losses in different regions of Turkey and the World (Kahveci and Maden, 1994). There is no precisely effective chemical against CBB and HB diseases. Therefore, use of resistant bean varieties is the most convenient management for these diseases. The objective of study was to research the defense-related responses of some bean cultivars to Psp and Xap infections. According to the results of enzyme assays, POX enzyme activity occurred in the both bean (resistant and susceptible) defense system after Xap and Psp pathogen inoculation. POX enzyme activity was higher in resistant cultivar 36Kinoculated with the Xap and Psp pathogen than susceptible cultivar Aras 98. POX enzyme activity increased by 198.40% in 36-K cultivar at 36th hr. But the highest increase for Aras 98 variety occurred at 72 hours (Figure 1). CAT enzyme activity found as the highest (0.4087 Ugr-1/ FW) in 36K cultivar at 72hr after Psp inoculation. The 36K genotype showed a higher CAT activity than the control plants at 24 and 36 hours after Xap bacterial inoculation. CAT activity was generally low compared to the control in Aras 98 variety (Figure 2). Several studies have reported that CAT and/or POX enzymes are associated with plant defense mechanism (Milosevic and Slusarenko, 1996; Chandrashekar and Umesha, 2012; de Freitas and Stadnik, 2012; Keshavarz-Tohid et al., 2016). CAT and POX enzymes is part of a group of antioxidative enzymes that are most important in amelioration the damage caused by oxidative stress (Mhamdi et al., 2010). The catalase (CAT) enzyme is found in peroxisomes of almost all aerobic cells. CAT is a protective antioxidant enzyme that prevents damage to cells. Catalase enzyme hydrogen peroxide decomposes into water and molecular oxygen (Nicholls et al., 2000; Cavalcanti et al., 2006). Peroxidase enzyme is involved in cell wall lignification process using hydrogen peroxide which is formed during oxidative combustion and also oxidizes phenols to quinones, which are toxic to the pathogen (Brisson et al., 1994; Kawano, 2003). Thus, our result agrees with Chandrashekar and Umesha, (2012) who observed an earlier increase of CAT enzyme activity in resistant tomato plants than susceptiple plants after pathogen inoculation. Similarly, Baysal et al., (2003) and Kavitha and Umesha, (2008) were examined POX activity in resistant and susceptible tomato variety after bacterial pathogen inoculation. They found that POX activity increased higher in resistant variety than susceptible after bacterial pathogen inoculation.

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POX Activity(Ugr-1/FW)

1000 900 800 700 600 500 400 300 200 100 0

800 700

600 500

400 300

200 100

0 0

A-98/522

A-98/510

A-98/Kontrol

36-K/522

36-K/510

36-K/Kontrol

800

700

600

500

400

300

200

100

0 0

A-98/120

A-98/145

A-98/Kontrol

36-K/120

36-K/145

36-K/Kontrol

Figure 1. Peroxidase (POX) enzyme activity, A; Aras 98 variety after Psp (522-p and 510-p) inoculation, B; 36K genotype after Psp (522-p and 510p) inoculation, C; Aras 98 variety after Xap (120-x and 145-x) inoculation, D; 36K genotype after Xap (120-x and 145-x) inoculation B

CAT Activity (mmol/g-1FW/da-1)

A 0,45

0,2 0,18 0,16 0,14 0,12 0,1 0,08 0,06 0,04 0,02 0

0,4

0,35 0,3

0,25 0,2

0,15 0,1

0,05 0 0

A-98/522

A-98/510

36-K/522

A-98/Kontrol

36-K/510

36-K/Kontrol

0,4

0,5 0,45 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0

0,35

0,3

0,25

0,2

0,15

0,1

0,05 0 0

A-98/120

A-98/145

A-98/Kontrol

36-K/120

36-K/145

36-K/Kontrol

Figure 2. Catalase (CAT) enzyme activity, A; Aras 98 variety after Psp (522-p and 510-p) inoculation, B; 36K genotype after Psp(522-p and 510p) inoculation, C; Aras 98 variety after Xap(120-x and 145-x) inoculation, D; 36K genotype after Xap (120-x and 145-x) inoculation SCAR Analysis Before investigating of the resistant genes by using SCAR marker, the bean varieties were tested at greenhouse conditions for their susceptibility reaction to the pathogens. According to results, it was obtained highly significant differences (p â&#x2030;¤ 0.01) among common bean cultivars on disease incidence and severity of Xap and Psp agents under greenhouse conditions. The 235

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lowest disease incidence and severity for both pathogens were observed from the variety XAN159, Vax1, Ozmen, Noyanbey and 36K. After testing of the eight bean cultivars as pathological against Xap and Psp in the greenhouse condition, the presence of resistance genes were detected in most of the bean varieties. With the SCAR markers, 820 bp band was obtained for SAP6 on all cultivars except Alberto, 1250 bp band for BAC6 on all cultivars except Aras 98 and 1150 bp band yield for SR13 marker on all cultivars except Noyanbey and Aras 98 cultivars with PCR amplification. We did not obtain any result for resistance genes by BC420, R7313, ST8, SH11 and SB10 markers in examined bean cultivars. Similarly, Poyraz et al., (2017) were screened ten resistance genes against Xap and Psp in 12 local bean cultivars using molecular markers (SCAR). They found that there were a presence/absence of the tested resistance genes in bean varieties against Xap and Psp. Molecular markers are very important for the detection of resistance against plant diseases becausenowadays in plant breeding programs including disease resistance are being used by incorporating molecular markers techniques like SCAR (Meziadi et al., 2016). Our studies indicate that the defense enzymes POX and CAT are actively involved in imparting resistance to halo blight caused by P. s. pv. phaseolicola and common bacterial blight (CBB) caused by X. a. pv. phaseoli in beans. Development of resistant bean cultivars to halo and common blight diseasescaused by bacterial pathogens; it will provide high added value to the economy of the country, reducethe chemical input for the farmer, and will have great benefits on the environment and human health. REFERENCES Aebi, H. (1984). Catalase in vitro. Methods in enzymology. Academic Press, 105, 121-126. Anonymous (2019). FAO- Faostat Statistics Division, http://www.fao.org/faostat, (Access Date: 24.09.2019). Anonymous (2019a). TÜİK, https://biruni.tuik.gov.tr/medas, (Access Date: 24.09.2019). Baysal, Ö., E. M. Soylu, S. Soylu (2003). Induction of defence‐related enzymes and resistance by the plant activator acibenzolar‐S‐methyl in tomato seedlings against bacterial canker caused by Clavibacter michiganensis ssp. michiganensis. Plant pathology, 52(6), 747753. Bernal‐Vicente, A., J. A. Pascual, F. Tittarelli, J. A. Hernández, P. Diaz‐Vivancos (2015). Trichoderma harzianum T‐78 supplementation of compost stimulates the antioxidant defence system in melon plants. Journal of the Science of Food and Agriculture, 95(11), 2208-2214. Beattie, A., T. E. Michaels, K. P. Pauls (1998). An efficient, reliable method to screen for common bacterial blight (CBB) resistance in Phaseolus vulgaris L., Bean. Improvement Cooperative (USA), 41, 53-54. Brisson LF, R. Tenhaken, C. Lamb (1994). Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance. The Plant Cell, 6, 1703-1712. Cavalcanti, F. R., M. L. V. Resende, J. P. M. S. Lima, J. A. G. Silveira, J. T. A. Oliveira (2006). Activities of antioxidant enzymes and photosynthetic responses in tomato pre-treated by plant activators and inoculated by Xanthomonas vesicatoria. Physiological and Molecular Plant Pathology, 68(4-6), 198-208. Chandrashekar, S., S. Umesha (2012). Induction of antioxidant enzymes associated with bacterial spot pathogenesis in tomato. Int. J. Food Agric. Vet. Sci, 2, 22-34. Chekanai, V., R. Chikowo, B. Vanlauwe (2018).Response of common bean (Phaseolus vulgaris L.) to nitrogen, phosphorus and rhizobia inoculation across variable soils in Zimbabwe. Agriculture, ecosystems & environment, 266, 167-173.

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De Freitas, M. B., M. J. Stadnik(2012). Race-specific and ulvan-induced defense responses in bean (Phaseolus vulgaris) against Colletotrichum lindemuthianum. Physiological and molecular plant pathology, 78, 8-13. Doyle, J. J., J. L. Doyle (1990). Isolation of plant DNA from fresh tissue. Focus, 12(13), 39-40. Félix-Gastélum, R., I. E. Maldonado-Mendoza, R. Navarrete-Maya, N. G. Olivas-Peraza, H. Brito-Vega, J. A. Acosta-Gallegos (2016). Identification of Pseudomonas syringae pv. phaseolicola as the causal agent of halo blight in yellow beans in northern Sinaloa, Mexico. Phytoparasitica, 44, 369–378. doi: 10.1007/s12600-016-0530-5. Ferreira, C. F., M. G. Pereira, A. D. S. Dos Santos, R. Rodrigues, R. E. Bressan-Smith, A. PioViana, R. F. Daher (2003). Resistance to common bacterial blight in Phaseolus vulgaris L. recombinant inbred lines under natural infection of Xanthomonas axonopodi spv. phaseoli. Euphytica, 134(1), 43-46. Fourie, D., P. Miklas, H. Ariyaranthe (2004). Genes conditioning halo blight resistance to races 1, 7, and 9 occur in a tight cluster. Annual Report-Bean Improvement Cooperative, 47, 103-104. Garden-Robinson, J., K. McNeal(2013). All about beans: Nutrition, health benefits, preparation and use in menus. NDSU Extension Service, N. Dakota State University. Hayat, I., A. Ahmad, T. Masud, A. Ahmed, S. Bashir (2014). Nutritional and health perspectives of beans (Phaseolus vulgaris L.): an overview. Critical reviews in food science and nutrition, 54(5), 580-592. Hall, R. (1994). Compendium of Bean Diseases (Edt). APS press, The American Phytopathological Society, 73. Jebara, S., M. Jebara, F. Limam, M. E. Aouani (2005). Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress. J. of plant physiology, 162(8), 929-936. Jung, G., P. W. Skroch, J.Nienhuis, D. P. Coyne, E. Arnaud-Santana, H. M. Ariyarathne, J. M. Marita (1999). Confirmation of QTL associated with common bacterial blight resistance in four different genetic backgrounds in common bean. Crop Science, 39(5), 1448-1455. Kahveci, E. S. Maden (1994). Detection of Xanthomonas campestris pv. phaseoli and Pseudomonas syringae pv. phaseolicola by bacteriophages. Journal of Turkish Phytopathology, 23, 79-85. Kawano, T. (2003). Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction, Plant cell reports, 21 (9), 829-837. Kavitha, R. S., Umesha (2008). Regulation of defense-related enzymes associated with bacterial spot resistance in tomato. Phytoparasitica, 36(2), 144. Keshavarz-Tohid, V., P. Taheri, S. M. Taghavi, S. Tarighi (2016). The role of nitric oxide in basal and induced resistance in relation with hydrogen peroxide and antioxidant enzymes. Journal of plant physiology, 199, 29-38. Lin, C. C., C. H. Kao (1999). NaCl induced changes in ionically bound peroxidase activity in roots of rice seedlings. Plant and Soil, 216(1-2), 147. Meziadi. C., M. M. S. Richard, A. Derquennes, A. Thareau, S. Blanchet, A. Gratias, S. Pflieger, V. Geffroy (2016) Development of molecular markers linked to disease resistance genesin common bean based on whole genome sequence. Plant Science, 242: 351-357. Mhamdi, A., G. Queval, S. Chaouch, S. Vanderauwera, F. Van Breusegem, G. Noctor (2010). Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. Journal of experimental botany, 61(15), 4197-4220. Miklas, P. N., R. Delorme, V. Stone, M. J. Daly, J. R. Stavely, J. R. Steadman, J. R. Beaver (2000). Bacterial, fungal, and viral disease resistance loci mapped in a recombinant inbred common bean population (Dorado'/XAN 176). Journal of the American Society for Horticultural Science, 125(4), 476-481. 237

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Miklas, P. N., D. Fourie, J. Wagner, R. C. Larsen, C. Mienie (2009). Tagging and mapping Pse1 gene for resistance to halo blight in common bean differential cultivar UI-3. Crop science, 49(1), 41-48. Miklas, P. N., D. Fourie, J. Trapp, J. Davis, J. R. Myers (2014). New loci including Pse-6 conferring resistance to halo bacterial blight on chromosome Pv04 in common bean. Crop Sci., 54, 2099–2108. Milosevic, N., A. J. Slusarenko (1996). Active oxygen metabolism and lignification in the hypersensitive response in bean. Physiological and Mol. Plant Pathol., 49(3), 143-158. Nicholls, P., I. Fita, P. C. Loewen (2000). Enzymology and structure of catalases. Advances in Inorganic Chemistry, 51, 51-106. Poyraz, I., B. Şahin, E. Atmaca (2017). Detection of Ten Resistance Genes Against P. syringae pv. Phaseolicola and X. axonopodis pv. phaseoliin Twelve Local Bean Varieties Using SCAR Markers SCAR Markörler Kullanarak On İki Yerel Fasulye Çeşidinde P. syringae pv. Phaseolicola ve X. axonopodis pv. phaseoli’ye Karşı On Direnç Geninin Tespit Edilmesi. J. Inst. Sci. & Tech.,7(2): 143-150 Singh, U. S., R.P. Singh, K. Kohmoto (1995). Pathogenesis and host specificity in plant diseases: histopathological, biochemical, genetic and molecular bases. Pergamon Press. Singh, S. P., H. F. Schwartz (2010). Breeding Common Bean for Resistance to Diseases: A Review, Crop Science, 50(6), 2199-2223. Teranishi, Y., A. Tanaka, M. Osumi, S. Fukui (1974). Catalase activities of hydrocarbonutilizing Candida yeasts. Agricultural and Biological Chemistry, 38(6), 1213-1220. Waller, F., B. Achatz, H.Baltruschat, J. Fodor, K. Becker, M. Fischer, P. Franken (2005). The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proceedings of the National Academy of Sciences, 102(38), 13386-13391. Yu, K., S. J. Park, V. Poysa (2000). Marker-assisted selection of common beans for resistance to common bacterial blight: efficacy and economics. Plant breeding, 119(5), 411-415.

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RESOLVING OF CERTAIN CONSERVED MIRNA IN OLEA EUROPAEA Sultan Cansu ILGIN1, Hatice Nur AYDIN2, Nehir Özdemir ÖZGENTÜRK*2 1

Department of Vocational School of Health Services, Programs of Anesthesia, University of Altinbas University, Bakırköy, İstanbul, Turkey 2

Department of Molecular Biology and Genetic, Yildiz Technical University, Esenler, İstanbul, Turkey *Corresponding author’s e-mail: nehirozdemir@yahoo.com

ABSTRACT Micro RNAs were first discovered in 1993. These are 21-23 nucleotides in length and are defined as short RNA sequences. This short interfering RNA sequences has the role of gene expression control. Olive plant is an important food source for countries in terms of economy and health. In this study, conserved micro RNAs were investigated to show their expresion level in olive plant. First of all, RNAs were isolated from young olives leaves (Olea europaea). RT PCR (Reverse Transcriptase Polymerase Chain Reaction) was performed by using primers to transformed into cDNA which is more stable nucleotid than RNA. The real time PCR method was used to detect these miRNAs (miR-159, miR-160, miR-171, miR-396, miR-2919 and miR8123) that are specific to the olive plant. A total of six miRNAs were tested. Real Time PCR results were show those five miRNA expressed well (miR-159, miR-160, miR-396, miR-2919 and miR-8123). micro RNA types associated with growth and development were observed in young olive leaves. Keywords: miRNA, Olea europaea, Olive, RT-PCR, cDNA, Real-Time PCR

INTRODUCTION Olive tree (Olea europaea), olives (Oleaceae) belongs to the family. Olive tree (Olea europaea) is a kind of tree that grows in Mediterranean climate. There are up to 27 genera and 600 species of the family of olives (1). A Gemlik olive is variety from the Gemlik, Zeytinbağı area of northern Turkey. They are small to medium-sized blackolives with a high oil content. Gemlik olives are considered as the best pickled olives in the world due to their taste and structure. Close to 80% of the olive trees produced by government agencies and private organizations in Turkey are Gemlik Olive saplings (2). In the previous study of our group, two cDNA libraries were established by using leaf and fruit samples of Gemlik olive plant. EST collection for olives was made by sequence analysis of 3840 clone samples randomly selected from cDNA libraries (3). To understand the molecular structure of olives, regulation of gene expression should be examined. MicroRNA (miRNA) are short (average 22 nucleotides in length) sequences that regulate gene expression by acting differently on mRNA (messenger RNA) (4). miRNAs are non-coding RNAs. This means that, they are encoded by genes that are transcribed from DNA (RNA production) but not translated (translated into protein). The precursor transcripts, called pri-miRNA, are processed and then converted into short stem-loop structures called premiRNA and then into functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNAs (mRNAs). Its main function is to regulate gene expression (4). 239

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The chromosomal localizations of miRNAs affect their expression and function. Therefore, it is important to know the genomic organization of miRNA (5). About half of the miRNAs show cluster placement. Cluster-shaped miRNAs have an operon-like structure and are transcribed as polysistronic (having multiple genetic codes) (6) (7). An important feature of clustered miRNAs is that they are conserved between species and homologous miRNAs are observed in all species (8). miRNAs are grouped by intergenic / intragenic and intronic / exonic localization (9) (10). These small RNAs, play an important role in the growth and development of plants. Rapidly increasing evidence suggests that plant miRNAs work to suppress the translation of their target genes. The roles of these genes in the developmental process; stem cell identity, hormone signaling, developmental patterns (meristem cell identity, leaf organ morphogenesis and polarity, flower differentiation and development, auxin signaling, border formation / organ separation), miRNA biogenesis (biological formation) and stress response (4) (11) (12). Based on previous studies, miRNAs preserved in plants are selected from experimentally determined miRNAs. These types of miRNA (miR-159, miR-160, miR-171, miR-396, miR2919 and miR-8123) observed in mulberry, poppy were preferred to examine whether the olive plant also maintained the presence of this miRNAs (13) (14). The miR159 family represses conserved GAMYB-like genes encoding the R2R3 MYB region in transcription factors. Said transcription factor acts on the expression of giberallin hormone genes in the plant. This is a plant hormone associated with signaling in the anthers and germination of the seed. The two major miR159 family members for the Arabidopsis plant are miR159a and miR159b. Both show specific functionality for the MYB33 and MYB65 genes, GAMYB-like genes (15). The polymorphisms of miR159 show 187 different sequences (16). miR160 is a predicted and experimentally verified and conserved micro RNA type in plant species such as Arabidopsis thaliana and Oryza sativa (17). miR-160 is estimated to match and bind to untranslated regions of the auxin response factor genes to regulate its expression (18). When one of these targets, the ARA 17 gene, is confronted with a miRNA resistance, a number of developmental disorders can be observed in the plant (19). In particular, A. Thaliana's auxin response factor genes are thought to be regulated by mir-160 as a post-transcriptional gene (19) (20). The polymorphisms of miR160 show 141 different sequences (21). miR171 has a role in suppressing the expression transcription factors in Arabidopsis and suppressing the differentiation of meristems (22). miR171 has been shown to negatively regulate scarecrow-like proteins (SCL6 / 22/27), which are the targets of chlorophyll biosynthesis, by an unknown mechanism (23). miR171 is present in four plants, and there are three miR171 genes in A. thaliana, nine in rice and grapes, and thirteen in poplar (24). The polymorphisms of miR171 have 212 different sequences (25). miR396 is one of the old conserved miRNAs in Arabidopsis leaves that regulates conserved targets belonging to the family of growth regulating factors (GRF), which are known transcription factors to control cell proliferation (26). As arabidopsis, miR396 encodes two different types of miRNA (miR396a and miR396b) and shows that three ceramidase-like genes (ATceramidase-like1, AT ceramidase-like 2, and AT ceramidase-like 3) may be targeted to miR396 (26). The polymorphisms of miR396 have 134 different sequences (27). mir2919 is present Oryza sativa, Prunus persica, opium poppy or some other monocotyledonous species (14) (29) (30). Prunus persica and Solanum tuberosum, has two varieties of miR8123 are called miR8123a and miR8123b (28) (29). In this study, it was thought that preserved miRNAs previously detected in plants could be preserved for olive plant and it was aimed to detect these preserved miRNAs in G20 olive line. Thus, miRNAs that are effective in regulating gene expression can illuminated in olive.

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MATERIAL AND METHODS Plant Material Seedlings of Olea europaea Gemlik cultivars Ministry of Agriculture of the Republic of Turkey Yalova Atatürk Garden Culture were obtained from the Central Research Institute. Preparation of Materials Because of the -OH group of ribose sugar in the structure of RNA molecules, it is broken down by ribonuclease enzymes that are widely available in biological environments. Therefore, DEPC solution is used to remove RNase contamination in the medium prior to RNA isolation. Mortars and pestles to be used before RNA isolation, all plastic and glass materials are kept in 0.1% DEPC water overnight, then autoclaved for 60 minutes at 121 ° C under 1 atm pressure. Likewise, 0.1% DEPC is added to the pure water to be used in RNA studies. It is allowed to stand overnight and autoclaved for 60 minutes at 121 ° C under 1 atm pressure. RNA Isolation miRNA isolation is performed with the Exicon miRCURY ™ RNA Isolation Kit. Nanodrop spectrophotometer is used to detect total RNA isolated. Thus, the amount and quality of RNA is determined. cDNA synthesis miRCURY LNATM cDNA synthesis steps were performed following Universal RT microRNA PCR kit protocol. Each target RNA sample is adjusted to 5 ng / µl construction using nuclease-free water. 5X reaction buffer: 5X reaction buffer is slowly dissolved with nuclease-free water. Total volume was completed to 10 µl by adding 2 µl 5x reaction buffer, 5 µl nuclease-free water, 1 µl enzyme mixture and 2 µl of RNA samples. The samples are incubated at 42 ° C for 60 minutes and then at 95 ° C for 5 minutes. Reverse Transcription (RT PCR) For these conserved miRNA samples, miR-159, miR-160, miR-171, miR-396, miR2919 and miR-8123, previously identified in plants, reverse transcription reactions were performed using six different primer pairs, back and forth (Table 1).

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Table 1: Sequence of the primers Primer Name

Primer Sequence

GSP1 miR-159f

5’-GTGGGTTTGGATTGAAGGG - 3’

GSP1 miR-159r

5’-GTGCAGGGTCCGAGG - 3’

GSP2 miR-160f

5’-TTGTGCCTGGCTCCCTGT - 3’

GSP2 miR-160r

5’-GTGCAGGGTCCGAGGT - 3’

GSP3 miR-171f

5’-GTTTTGATTGAGCCGTGCC - 3’

GSP3 miR-171r

5’-GTGCAGGGTCCGAGGT - 3’

GSP4 miR-396f

5’-GTTGGGGTTCCACAGCTTT - 3’

GSP4 miR-396r

5’-GTGCAGGGTCCGAGGT - 3’

GSP5 miR-2919f

5’-TTTTTTTCCCCCCCCCCC - 3’

GSP5 miR-2919r

5’-GTGCAGGGTCCGAGGT - 3’

GSP6 miR-8123f

5’- GTTTGGGAACACGGTAAC- 3’

GSP6 miR-8123r

5’- GTGCAGGGTCCGAGG- 3’

Real Time PCR (qPCR) Real Time PCR was performed by applying the AriaMX Real-Time PCR System (Agilent Technologies, Inc.) and the appropriate PCR protocol (Table 2). 1 µl master cDNA samples, 12.5 µl master mix, primer and RNase-free water are prepared for qPCR. Standard cycle is: 95˚C-10 min; 40 cycles of 95˚C-10 sec; 58-60˚C-15 sec, 72˚C- 30 and 1cycle 95˚C-30 sec; 65˚C-30 sec, 95˚C- 30 sec. RESULTS Reverse Transcription (RT PCR) RNA isolation from Gemlik G20 young olive leaf samples was performed according to the protocol of Exion miRCURY ™ RNA Isolation Kit . Quality and presence of RNA obtained by isolation were measured by Nanodrop spectrophotometer. The sample with a high amount of RNA was selected as the sample to be used in the conversion to cDNA. After cDNA synthesis protocol, cDNAs were generated by polymerase chain reaction (PCR) with six different primer pairs (miR-159, miR-160, miR-171, miR-396, miR-2919 and miR8123). The resulting PCR products and the negative control (NC), the PCR product without cDNA, were electrophoresed in the presence of dye and ladder. As a result of the irradiation detected under UV light only, the image of Figure 1 was obtained. Polymerase chain reaction (PCR) of cDNAs was generated by six different primer pairs. All of the resulting PCR products gave bands below the 100 bp marker DNA band. Since these cDNAs were amplified from miRNAs, the band was observed in less than 100 bp on the agarose gel.

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Figure 1: cDNA PCR agarose gel walking image Real Time PCR Results As a result of Real Time PCR of cDNA with miR 159 primer, Cq: 19.27 and Tm: 79.50 were measured (Table 4) The amplification graph (Figure 2) shows the sample in purple and the negative control in blue. Melting curve (Figure 3) shows the sample in purple and the negative control in blue. Table 4: miR159 Real Time PCR Cq and Tm values

Figure 2: miR 159 Real Time PCR Reproduction Graph

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Figure 3: miR159 Real Time PCR Melting Curve The negative control (NC) a PCR product without cDNA, showed no bands. This indicates that the bands seen are not the primer-dimers formed by the coupling of the primers to each other. It also indicates that the amplification formed is not non-specific binding. As a result of Real Time PCR of the cDNA with miR159 primer; In the amplification graph (Figure 2), the sample is expressed in purple and the negative control in blue. The purple line shows a smooth exponential graph, while the blue negative control line indicates no growth. This indicates a correct Real Time PCR and that miR159 has been replicated. In the graph of the melting curve (Figure 3), the sample was expressed in purple and the negative control in blue. There is also a single peak here. Tm: 79.50 at the peak of miR159. Amplification with this primer was measured as Cq: 19.27 and Tm: 79.50 (Table 2). The same procedure was repeated for other five primers (miR-160, miR-171, miR-396, miR2919 and miR-8123). Real Time PCR data and graphs were obtained (Figure 4).

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Figure 4: miR 159, miR-160, miR-171, miR-396, miR-2919 and miR-8123 Real Time PCR Reproduction Graph DISCUSSION The threshold value is the value determined according to the graph in order to eliminate the small movements that may occur in the graph and should be ignored in the graph. The threshold value appears as a thick dark blue line on the amplification graph (Figure 2). Looking at the Cq and Tm charts, there was no proliferation in the negative control (Table 4). Based on these data, the replication of miR159 is said to be successful.

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From the plots for the miR160 primer, the reproduction plots; the product gives a proper peak, the negative control, we say no growth. This indicates that the desired product has multiplied. But; Looking at the melting graph; There are two peaks in the product, one large (Tm: 82.00) and one small (Tm: 65.00). The small peak contamination in the product suggests a non-specific binding. However, the same small peak in the negative control shows that a primary dimer is formed. Looking at the Cq and Tm tables, there is no remarkable increase in the negative control. As a result, the desired sample was multiplied in this plate, but a small amount of primary dimer formation was observed (Figure 4). According to the amplification plot for primer miR171; After the 35th cycle, the negative control increased. This indicates contamination of the plate. The melting curve graph also shows two peaks. The first peak (Tm: 81.50) refers to the binding with external DNA as a result of contamination, and the large peak at the end (Tm: 88.00) refers to the desired binding. Here, when the Cq and Tm tables are examined, the negative control has increased. The experiment must therefore be repeated (Figure 4). Looking at the graphs of miR396 and miR 2919, miRNAs proliferate smoothly without proliferation in negative controls (Figure 4). For miR8123, the amplitude of the peak is smooth, but the melting curve is not clear and initially produces a small peak (at Tm: 83). This experiment can be repeated (Figure 4). CONCLUSION As a result, in this study, successful results were obtained for five of the six miRNAs (miR159, miR-160, miR-396, miR-2919 and miR-8123). According to miR171 data, proliferation was observed in the negative control. This indicates contamination. Therefore, the assay has to be repeated for miR171. Acknowledgements: This study was supported by Yıldız Technical University Scientific Research Projects Coordination Project 2015-01-07-YL02. REFERANCE Alonso M. ve diğ., (August 10, 2010). "The miR159 regulated GAMYB-like genes inhibit growth and promote Programmed Cell Death in Arabidopsis" ,Plant Physiology, 110:160630 doi: http://dx.doi.org/10.1104/pp.110.160630. Barakat, A., et.al., (2012). "Genome wide identification of chilling responsive microRNAs in Prunus persica", BMC Genomics, 13: 481. Bartel, D.P, (2004) "MicroRNAs: genomics, biogenesis, mechanism, and function", Cell, 116:281-297. Boke, H., Mine, Turktas, E., Parmaksiz I. , Ozcan S. ve Unver T., (2015). "Regulation of the alkaloid biosynthesis by miRNA in opium poppy", Plant Biotechnology Journal, 13, pp. 409–420. Curaba, J., Talbot, M., Li, Z. and Helliwell, C., "Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley", BMC Plant Biology, 2013:13:6 DOI: 10.1186/1471-2229-13-6. Debernardi, J.M., Rodriguez, R.E., Mecchia, M.A. and Palatnik, J.F., (January 5, 2012). "Functional Specialization of the Plant miR396 Regulatory Network through Distinct MicroRNA–Target Interactions" http://dx.doi.org/10.1371/journal.pgen.1002419. Engels B, Hutvagner G, (2006). Principles and effects of microRNA-mediated posttranscriptional gene regulation. Oncogene 25: 6163–6169 Gemlik Zeytini, http://www.gemlikzeytini.net/, 3 Nisan 2016.

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Jia, L., Zhang, D., Qi, X., Ma, B. and Xiang Z., (August 13, 2014) "Identification of the Conserved and Novel miRNAs in Mulberry by High-Throughput Sequencing" http://dx.doi.org/10.1371/journal.pone.0104409. Jones-Rhoades, M.W., Bartel, D.P., (2004). "Computational identification of plant microRNAs and their targets, including a stress-induced miRNA", Mol. Cell, 14:787–799. Kim, VN, Han J, Siomi MC, (2009). Biogenesis of small RNAs in animals. Nature Rev. Mol. Cell Biol 10:126–139 Kim, YK, Kim VN, (2007). Processing of intronic microRNAs. EMBO J. 26: 775–783. Lee RC, Ambros V, (2001). An Extensive Class of Small RNAs in Caenorhabditis elegans. Science 294: 862-64. Lee Y, Kim M, Han J., Yeom KH., Lee S. And Baek SH. et al, (2004). MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23:4051-60 Liu, X., Huang, J., Wang, Y., Khanna, K., Xie, Z., Owen, H.A., Zhao, D., (May 2010). "The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a, in organogenesis and the mechanism regulating its expression.", The Plant Journal: for cell and molecular biology, 62 (3): 416–28. doi:10.1111/j.1365313X.2010.04164.x. PMID 20136729. Ma, Z. and et. al., (2014 Aug). "Arabidopsis miR171-Targeted Scarecrow-Like Proteins Bind to GT cis-Elements and Mediate Gibberellin-Regulated Chlorophyll Biosynthesis under Light Conditions", PLoS Genet., 10(8): e1004519. Mallory, A.C., Bartel, D.P. and Bartel, B., (May 2005). "MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes.", The Plant cell, 17 (5): 1360–75. doi:10.1105/tpc.105.031716. PMC 1091760. PMID 15829600. Mirbase, http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0026147, 3 Nisan 2016. Mirbase, http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000010, 4 Haziran 2016. Mirbase, http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000032, 4 Haziran 2016. Mirbase, http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000032, 4 Haziran 2016. Mirbase, http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000030, 4 Haziran 2016. Mirbase, http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000047, 4 Haziran 2016. Ozdemir Ozgenturk, N., Oruç, F., Sezerman, U., Kuçukural, A., Vural Korkut, S., Toksoz, F. ve Un C., (2010). "Generation and Analysis of Expressed Sequence Tags from Olea europaea L.", Comp Funct Genomics, 2010:757-512. doi: 10.1155/2010/757512. Researchgate.https://www.researchgate.net/figure/279751435_fig3_Figure-3-Potatosenseantisense-miRNA-gene-The-newly-explored-potato-miRNA-miR8123-was, 3 Nisan 2016. Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B. and Bartel, D.P., (2002). "Prediction of plant microRNA targets", Cell, 110 (4): 513–520. doi:10.1016/S00928674(02)00863-2. PMID 12202040. Ruvkun G, (2001). Molecular biology. Glimpses of a tiny RNA world. Science 294: 797–799. Sunkar, R., Kapoor, A. and Zhu , J.K., (2006). "Posttranscriptionalinduction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance" ,Plant Cell, 18: 2051 – 2065. Zeytin Ağacı ve Zeytin Hakkında Bilgi, http://www.arsivbelge.com/yaz.php?sc=2554, 3 Nisan 2016. 247

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Zhu, X., Leng, X., Sun, X., Mu, Q., Wang, B., Li, X., (2015). "Discovery of Conservation and Diversification of miR171 Genes by Phylogenetic Analysis based on Global Genomes" ,The Plant Genome, 8(2):1. doi:10.3835/plantgenome2014.10.0076

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GENERATION OF RNASEQ DATA FOR CORYLUS EVELLANA L. Nehir Özdemir ÖZGENTURK*1, Zehra Ömeroğlu ULU1, Aysun AKAR2, Salih ULU1 1

Yıldız Teknik University, Faculty of Art and Science, Department of Moleculer Biology and Genetics, Istanbul, Turkey 2

Republic of Turkey Ministry of Agriculture and Forestry Hazelnut Research Institute, Giresun, Turkey *Corresponding author’s e-mail: nehirozdemir@yahoo.com ABSTRACT

Turkish Tombul hazelnut (Corylus avellana L.) is the most widespread hazelnut species in our country and has high impact on industry and global agricultural sector. It has high rich nutrients such as oleic acid, phytosterols and vitamin E. In this study, young leaves, flowers (male and female), bud, husk shoot of Turkish Tombul hazelnut (Corylus avellana L.) obtained the collection of Agriculture and Forestry Hazelnut Research Institute. Total RNA from these material was extracted with RNeasy Plant Mini Kit (Qiagen), separately. After total RNA extraction, RNA pool was composed after cleaning with RNA MinElute Kit (Qiagen). The concentration of RNA was measured with Bio-spec-nano UV-VIS Specthrophometer. Pair-end (2x100 bp) sequencing was performed using an Illumina HiSeqTM 4000 sequencing system (Illumina) at Beijing Genomics Institute (BGI). RNA-Seq resulted in an average of ~42 million clean reads. We used the Trimmomatic software for quality trimming. And then the quality of clean reads was checked using FastQC program. After analysing the de novo assembly from this RNA-seq via Trinity platform, we will get comprehensive information about gene expression profile of Turkish Tombul hazelnut (Corylus avellana L.) species with this study. Keywords: Corylus avellana L., RNASeq

INTRODUCTION Hazelnut has become a valuable nutrient throughout the history. As it is used in cake and chocolate products as well as appetizers, its economical value is high since oil extraction from its plant. More than 25 species have been identified in Coryleae subfamily and Betulaceae family in taxonomical order. Commercially most preferable ones are grown in Anatolia region. It is quite important plant and has high economicall value for Turkey that 70% of world need is supplied from Turkey. Among the most widely grown hazelnut species in our country, the most important species are plump hazelnut (Corylus avellena L.) and Turkish hazelnut (Corylus colurna L.) which are used as rootstocks (1, 2, 3). For the transcript profiling and discoverying new genes RNA sequencing (RNA-seq) is one of the applications of the next generation sequencing (NGS) technologies (4-9). Also, RNAseq has been successfully used for annotation, gene expression levels, single-nucleotide polymorphism (SNP) discovery, and detecting alternatively spliced RNA forms (10-12). In this study, after the RNAseq data is obtained, the cleaning steps are completed. This data can givecomprehensive information about gene expression profile of Turkish Tombul hazelnut (Corylus avellana L.) species. 249

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MATERIAL METHOD RNA Isolation Isolation of total RNA was extracted from Turkish filberts young leaves, flowers (male and female), bud, husk, shoot from the collection of Agriculture and Forestry Hazelnut Research Institute, Giresun. Total RNA from these material was isolated with RNeasy Plant Mini Kit (Qiagen), separately. After total RNA, RNA pool was composed after cleaning with RNA MinElute Kit (Qiagen). RNAseq Pair-end (2x100 bp) sequencing was performed using an Illumina HiSeqTM 4000 sequencing system (Illumina) at Beijing Genomics Institute (BGI). Cleaning the RNAseq Data We used the Trimmomatic software for quality trimming. Trimmomatic is a flexible read trimming tool for Illumina NGS data (13). Trimmomatic performs a variety of useful trimming tasks for illumina paired-end and single ended data. The selection of trimming steps and their associated parameters are supplied on the command line. java -jar trimmomatic-0.35.jar PE -phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 The quality of clean reads was checked using FastQC program (14). FastQC aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing pipelines. RESULTS All RNA isolations were collected in one pool for RNAseq. RNA quality is very important as starting material. The concentration of each sample of RNA was measured with Bio-spec-nano UV-VIS Specthrophometer (Shimadzu, Kyoto, Japan) (Table 1).

Table 1. The Results of Nanodrop

Sample Name

Nucleic Acid Conc (ng/ÎźL)

OD260 /280

OD260 /230

OD 260

OD 280

OD 230

avellana1

43,11

1,76

0,18

1,289

0,824

6,366

avellana2

63,16

1,58

0,27

2,776

2,196

7,103

avellana3

63,34

1,95

0,41

3,109

2,336

5,365

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RNA-Seq resulted in an average of ~42 million clean reads (Table 2). Reads statistic results are shown in Table 2. We have aproximately 41 million clean reads. Table 2. Reads statistic results

C. avellana

Clean Reads

Clean Bases

Read Length (bp)

41,854,426

4,185,442,600

100

Q20(%)

GC(%)

98.13%

47.87%

We used the Trimmomatic(2) software for quality trimming. And then the quality of clean reads was checked using FastQC (3) program (Figure 1). Per Base Sequence Quality This view shows an overview of the range of quality values across all bases at each position in the FastQ file. The central red line is the median value. The yellow box represents the interquartile range (25-75%). The upper and lower whiskers represent the 10% and 90% points. The blue line represents the mean quality.The y-axis on the graph shows the quality scores. The higher the score the better the base call. The background of the graph divides the y axis into very good quality calls (green), calls of reasonable quality (orange), and calls of poor quality (red). The our RNAseq data for hazelnut (Corylus avellana L.) lay into green line, so our data shows very acceptable high quality depens on per base sequence quality (Figure 1)

Figure 1. Per base sequence quality for RNAseq data for hazelnut (Corylus avellana L.)

Per Sequence GC Contentâ&#x20AC;¨ This module measures the GC content across the whole length of each sequence in a file and compares it to a modelled normal distribution of GC content. We would expect to see a roughly 251

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normal distribution of GC content where the central peak corresponds to the overall GC content of the underlying genome. The modal GC content is calculated from the observed data and used to build a reference distribution. An unusually shaped distribution from reference could indicate a contaminated library or some other kinds of biased subset. The our RNAseq data for hazelnut (Corylus avellana L.) has a normal distrubition as we expect to see a roughly normal distribution of GC content (Figure 2).

Figure 2. Per sequence GC content for RNAseq data for hazelnut (Corylus avellana L.) Per Base Sequence Contentâ&#x20AC;¨ Per Base Sequence Content plots out the proportion of each base position in a file for which each of the four normal DNA bases has been called. In a random library you would expect that there would be little to no difference between the different bases of a sequence run, so the lines in this plot should run parallel with each other. The lines for each bases in our RNAseq data run parallel with each other. We can ignore the read start position which is normal read polution (Figure 3).

Figure 3. Per base sequence content for RNAseq data for hazelnut (Corylus avellana L.) 252

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DISCUSSION Trimmomatic is a very common read trimming tool for Illumina paired-end and single ended NGS data. Our RNAseq data was trimmed by selected trimming steps and their associated parameters. FastQC aims to provide a simple way to do some quality control checks on raw sequence data. It provides a modular set of analyses which you can use to give a quick impression of whether your data has any problems of which you should be aware before doing any further analysis. FastQC is provide a quick overview to tell in which areas there may be problems. we were assesed our data by using FastQC summary graphs and tables. The graphs and tables obtained by FastQC analysis showed that our RNAseq data are of good quality. The analysis of de novo assembly from this RNA-seq of hazelnut data will done via Trinity platform (15), so we can get comprehensive information about gene expression profile of Turkish Tombul hazelnut (Corylus avellana L.). Acknowledgements: This study was supported by Yıldız Technical University Scientific Research Projects Coordination Project FBA-2017-3134. REFERENCES Alasalvar C1, Shahidi F, Liyanapathirana CM, Ohshima T. (2003) Turkish Tombul hazelnut (Corylus avellana L.). 1. Compositional characteristics. J Agric Food Chem. 2003 Jun 18;51(13):3790-6. Andrews S., FastQC: 2010 http://www.bioinformatics.babraham.ac.uk/projects/fastqc Benjamini Y., Yekutieli D. The control of the false discovery rate in multiple testing under dependency. The Annals of Statistics. 2011;29:1165–1188. Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170. Costa V., Angelini C., De Feis I., Ciccodicola A. Uncovering the complexity of transcriptomes with RNA-Seq. Journal of Biomedicine and Biotechnology. 2010;2010:19. doi: 10.1155/2010/853916.853916 Fındık Tanıtım Grubu, Fındık ve Sağlık, ttp://www.ftg.org.tr/tr/turk-findigi-findik-vesaglik.html, 25 October 2017. Haas BJ, et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc. 2013 Aug;8(8):1494512. Open Access in PMC Liu G. F., Cheng H. J., You W., Song E. L., Liu X. M., Wan F. C. Transcriptome profiling of muscle by RNA-Seq reveals significant differences in digital gene expression profiling between Angus and Luxi cattle. Animal Production Science. 2015;55(9):1172–1178. doi: 10.1071/an14096. Mortazavi A., Williams B. A., Mccue K., Schaeffer L., Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 2008;5(7):621–628. doi: 10.1038/nmeth.1226. Ozsolak F., Milos P. M. RNA sequencing: advances, challenges and opportunities. Nature Reviews Genetics. 2011;12(2):87–98. doi: 10.1038/nrg2934. Ren S., Peng Z., Mao J. H., et al. RNA-seq analysis of prostate cancer in the Chinese population identifies recurrent gene fusions, cancer-associated long noncoding RNAs and aberrant alternative splicings. Cell Research. 2012;22(5):806–821. doi: 10.1038/cr.2012.30. T.C. Gümrük ve Ticaret Bakanlığı Kooperatifçilik Genel Müdürlüğü. (2017). 2016 Yılı Fındık Raporu, March 2017, Ankara. 253

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Wang Z. Y., Fang B. P., Chen J. Y., et al. De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweet potato (Ipomoea batatas) BMC Genomics. 2011;11:p. 726. doi: 10.1186/14712164-11-726. Wang Z., Gerstein M., Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics. 2009;10(1):57â&#x20AC;&#x201C;63. doi: 10.1038/nrg2484 Yang S. S., Tu Z. J., Cheung F., et al. Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems. BMC Genomics. 2011;12(1):p. 199. doi: 10.1186/1471-2164-12199.

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PRODUCTION AND CHARACTERIZATION OF BIODIESEL FROM SCENEDESMUS QUADRICAUDA (TURPIN) ISOLATED FROM KANYE WATER RESERVOIR IN KANO STATE, NIGERIA *Mustapha, Y., Indabawa, I.I., Jibril, A.I. Department of Plant Biology, Bayero University, Kano, Nigeria *Corresponding author’s e-mail: ymustapha116@gmail.com

ABSTRACT In this study, S. quadricauda was isolated from Kanye water reservoir in Kano state, Nigeria and cultivated in the laboratory for the extraction and characterization of biodiesel. The algal culture was scaled-up in a photo bioreactor designed to provide optimum light intensity and aeration which yielded 1000ml of the algal culture . Algal oil was extracted from the harvested algal biomass and it was subjected to esterification and purification protocols. Characterization of the oil using FT-IR revealed the presence of ester, alkane and aliphatic functional groups. The oil was further subjected to ASTM standard tests which revealed that the oil has essential energy properties whose values fall within set limits as follows: Saponification value (193mgKOH/g); Acid value(0.6mgKOH/g); Free fatty acid(0.3%); Molecular weight(168); Refractive index(1.5%); pH(7.2) and Iodine value(83mgKOH/g). The findings of this investigation indicate that S. quadricauda biomass is a good feedstock for biodiesel production which has the potential to serve as a good alternative to fossil fuels when produced on a large scale. Keywords: Biodiesel, S.quadricauda, Kanye water reservoir

INTRODUCTION Lipid production from microalgae and it’s corresponding biodiesel production have been studied since the late 1970s as an alternative fuel source (Weldy, 2007). Potential benefits of biofuels include carbon neutrality and carbon sequestration, biodegradability and renewability. The overconsumption of fossil fuels in the last few decades may soon lead their exhaustion in the near future and the resultant energy crisis would become one of the greatest challenge of the 21st century (Vasudevan and Briggs, 2008). One promising source of biomass for alternative fuel production is microalgae that have the ability to grow rapidly and synthesize and accumulate large amounts (20%−50% of dry mass) of neutral lipid (mainly in the form of triacylglycerol, TAG) stored in cytosolic lipid bodies (Duffy ,2009; Hu et al., 2008 and Day, 1999). One algal species that shows promise as a potential biodiesel feedstock due to its high oil content and adaptability is Scenedesmus quadricauda. S. quadricauda is one of the most common green algae found in the shallow/freshwater lakes in the tropical countries including Nigeria. The current study attempts to examine the capacity of S. quadricauda biomass to serve as a good feedstock for biodiesel production. The study also aimed at evaluating the quality of the biodiesel obtained with a view to establishing it’s suitability to serve as an alternative source of fuel. 255

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MATERIALS AND METHODS Isolation and cultivation of microalga (Scenedesmus quadricauda) Scenedesmus quadricauda was isolated from the water samples collected from Kanye water reservoir and cultured in BG-11 medium using 1000ml flask and scaled up in a photobioreactor as described by Rippka et al. (1979). Measurement of algal growth The growth pattern of S. quadricauda was measured during the cultivation period using UVspectrophotometer as described by Sanchez (2003).The culture was allowed to grow for 25 days and the optical density (OD) was taken from the 5th day after inoculation at 540nm according to the method described by Ilavarasi (2012), up to the 25th day and a growth curve was generated accordingly. Harvesting of algal biomass S. quadricauda biomass was harvested by flocculation method. The pH of the culture medium was adjusted from a range of 7.3-7.8 to 9.8-10.4 by the addition of 1M KOH/NaOH. The entire algal biomass settled slowly and was filtered using micro filter. Extraction of algal oil The dried algal biomass was used for oil extraction and it was performed in a soxhlet apparatus using hexane as solvent. The oil extracted was purified using distillation method in which the hexane in the oil was removed as vapor using rotary evaporator. Transesterification procedure (Production of fatty acid methyl ester-FAME) The FAME preparation was performed using the oil extracted from the algal biomass using methanol, while KOH was used as catalysts in accordance with standard protocol (Meher et al., 2006). Physicochemical characterization of algal biodiesel The physicochemical properties of S. quadricauda biodiesel (Saponification value, acid value, free fatty acid, molecular weight, refractive index, pH and iodine value) were determined according ASTM D6751standard procedures. Characterization of algal biodiesel using FT-IR The structural property of the biodiesel was characterized using Fourier Transform Infrared Spectroscopy (FTIR) by means of Agilent Technologies Cary 630 FTIR spectrophotometer in the wave number range 1000-5000cm-1.

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RESULTS Measurement of growth of S. quadricauda Growth of the microalga was determined by plotting the optical densities against number of days as shown in Figure 1. It could be seen that the optical density increased with increase in number of days from the 5th day after inoculation (log phase) to the 23rd day (lag phase). A slight decrease (1.80) was however observed on the 25th day. 1,85

Optical Density

1,8 1,75 1,7 1,65 1,6 1,55 1,5 5

Days

Figure 1: Growth curve of S. quadricauda Harvesting algal biomass Scenedesmus quadricauda shows a promising growth rate in both the flask and the designed photobioreactor and a total of 5g of algal biomass was obtained, which is quite high. Physicochemical characterization of algal biodiesel Table1 shows the values obtained for the physicochemical characterization of the biodiesel obtained from S.quadricauda. The results indicated that saponification value obtained was 193mgKOH/g, acid value was obtained at 0.6mgKOH/g, free fatty acid at 0.3%, molecular weight at 168, refractive index at 1.5Â°C, pH at 7.2, and Iodine value at 83mgKOH/g. The values were found to fall within the ASTM standard limits.

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Table 1: Values obtained for the physicochemical characterization of S. quadricauda biodiesel Properties of biodiesel

Values for algal biodiesel

ASTM standards Remarks

Saponification value

193mgKOH/g

191-202 (mgKOH/g)

Within acceptable limit

Acid value

0.6mgKOHg-1

0.80mgKOHg-1 (max)

Within limit

Free fatty acid

0.3%

0.45

Below limit

Molecular weight

168

Refractive index

1.5%

1.479 (max)

Within limit

7.20

7-9

Within limit

Iodine value

83mgKOHg-1

82-98mgKOHg-1

Within limit

ASTM: American Society for Testing and Materials. NA = Not available FTIR Characterization of algal biodiesel From the FTIR spectrum, peaks at 2924 and 2855cm-1 are typical C-H bond stretching vibration. This was further supported by peaks at 1460 and 1380cm-1 as C-H bond bending vibration. A peak at 1745cm-1 can be assigned to C=O bond stretching vibration typical for an ester. The ester linkage was confirmed by the presence of a peak at 1182cm-1. From the available peaks observed, the molecule is suspected to be a saturated fatty acid. The result of the FT-IR spectra revealed that the fatty acids of S.quadricauda biodiesel when KOH was used as catalyst was converted to fatty acid methyl ester at 1745 C=O, while standard value of ester bond formation is obtained at 1744.

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Figure 2. FTIR Spectrum of S.quadricauda biodiesel DISCUSSION The growth curve of the optical densities against number of days showed that Scenedesmus quadricauda grows rapidly at the beginning of cultivation followed by a stationary phase. After the maximum cultivation time of 25 days, the growth curve indicated that the maximum cell density could be obtained in this period. The main reason for the decrease of cell growth rate at the end of cultivation was due, in part, to the poor light penetration, availability of nutrients, stability of pH and temperature (Wang et al., 2010). The microalga color became darker with increasing cultivation time, which could prevent light from penetrating through the container thereby inhibiting cell growth. Aeration is also an important factor in the growth of algae. Kaewpintong (2004) reported a better growth for an airlift photobioreactor than for an unaerated column. This is because aeration leads to a better mixing of the algal cultures, which prevents sedimentation, maintains homogenous conditions and help for a better contact between cells and nutrients. The amount of biomass harvested (5g) is higher than that of other microalgae reported by previous workers. Griffths and Harrison (2009) recorded a biomass value of 1.65g for Dunaliela salina. Chlamydomonas reinhardtii and S. quadricauda were cultivated in municipal wastewater by Kong et al. (2010) and a maximal biomass productivity of 2.0g and 2.4g were obtained. The total lipid content and net biomass productivity in microalgae vary greatly from one species to another. The amount of lipid obtained in this study (9ml from 5g of biomass) suggests that S.quadricauda has strong potential to be used as an economically valuable source for biodiesel 259

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production. Yeesang and Cheirslip (2011) found relatively high level of lipid content when the culture medium was exposed to high light intensity. Unsaturated fatty acid shows high yield at low temperature and saturated fatty acid at high temperature (Hu et al., 2008). Light intensity also influences the composition of lipids. Jiang et al. (2011) found a biomass concentration of 2.23g/L and lipid content of 59.9% under high light and nutrients deprieved condition. From the findings of Liu et al. (2011), S.quadricauda produced 15.61ml of oil which was higher than the result obtained in this study. Chlorella, Scenedesmus, Neochloris and Nanochloropsis strains are repeatedly reported to give an average lipid content of 40-60% in dry cell mass in laboratory (Hu et al., 2008). In a previous study by Mata et al. (2012), S.quadricauda was evaluated for lipid accumulation, biomass yield, total photosynthetic pigments and fatty acid profiling under nitrogen limited condition. The results revealed the increase in lipid accumulation under nitrogen starved conditions. One of the most important decisions in obtaining oil from microalgae is the choice of species. From the work of Rodolfi et al. (2009) and Reda et al. (2011), fifteen purified strains of microalgae were screened for their lipid content and mass productivity. Among all the isolates C. vulgaris, S. quadricauda and T. oblonga showed the most potential. The dry weight of the three isolates were recorded as 1.23, 1.09 and 0.9g/l while the lipid contents were 37%, 34% and 29% respectively,which qualifies them to be considered as very promising in terms of biomass production and lipid content. From FTIR spectrum, peaks at 2924 and 2855 cm-1 are indicative of a typical stretching vibrations of C-H alkane group. They could be methyl (CH3) or methylene groups in the ester chains of the biodiesel and they require high energy to cause stretching vibrations within their bond when compared to the ordinary C-H bending vibration of alkene groups detected at low energy and frequency region (Shiu et al.,2010) . This was further supported by peaks at 1460 and 1380cm-1 as C-H bond bending vibration. The peak at 1745cm-1 can be attributed to C=O indicative of the presnce of a carbonyl functional group in the biodiesel.This bond stretching vibration is typical of an ester.The ester linkage was confirmed by the presence of a peak at 1182cm-1 indicative of stretching vibrations of C-O and C-O-C. They can also indicate the bending vibration of O-CH3 in the spectrum (John,2000). From the available peaks observed, the molecule is suspected to be a saturated fatty acid. The physico-chemical characterization of the fuel was compared with the ASTM standards. The result revealed that the saponification value, acid value, free fatty acid, refractive index, pH, iodine value and molecular weight were found to be within the ASTM standard range. The saponification value(SV) is expressed by mgKOH required to neutralize one(1) gram of fat depending on the kind of fatty acid contained in the fat. It allows the comparison of the average fatty acid chain length. The saponification value obtained in this study was 193mg/KOH/gm which was compared with the ASTM standard and found to be within the limits. Onyeisue( 2011) carried out a research on the physicochemical characteristics of the oil of Chlorella specie and the SV was obtained at 167.83mg/KOH/g. Indabawa et al. (2014) carried out a similar research on the oil of D. salina and found the S.V of the algal oil to be 192.57mg/KOH/g. Algal oil with SV of 200 and above have been reported to possess low molecular weight (Abaye et

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al., 1998). The molecular weight of S. quadricauda oil in this research was obtained at 168. The lower the molecular weight the higher the saponification value and vice versa. The acid value(AV) obtained was 0.6 which is lower than the standard(0.8). If acid value is higher than 0.8 it can cause corrosion to the engine. The acid value represent free fatty acid content due to enzymatic activity and is usually an indication of spoilage. Acid value can be used to check the level of oxidative deterioration of the oil by enzymatic or chemical oxidation. The free fatty acid (FFA) of S.quadricauda biodiesel was found to be 0.3%as against the standard value of 0.45%. The higher the FFA the lower the quality of diesel fuel as it can cause deposition in conbustion systems which would lead to a reduction o the life span of fuel pumps and filters (Kwangdinata et al.,2014). Ananadhi and shaleesha (2012) carried out a research on the FFA of chlorella vulgaris and found the FFA of the oil to be 0.71%. The refractive index obtained for S.quadricauda is 1.5, this value agrees with ASTM value of 1.479 maximum limit. The refractive index which is the ratio of the velocity of light in vacuum to the velocity of light in a medium is an indication of the level of saturation of the oil (Oderinde et al., 2009). The iodine value(IV) is a measure of the degree of unsaturation of oil and this value could be used to quantify the amount of double bonds present in the oil, which reflects the susceptibility of oil to oxidation. The iodine value for the algal oil in this study is 83mgKOHg-1 was found to be within the acceptable limit when compared with the ASTM standards. S.quadricauda usually has a good adaptation to pH change. They grow well in pH range from 6.5 to 8.5 with the best growth rate obtained at initial values of 7.5 to 8.0. In this study, the pH for the microalga was obtained at 7.2, this result is similar to the study of Kong et al., (2010). The alga S.quadricauda can change the pH value of the culture medium to suit their growth by means of their own physiological metabolism. This is the reason why S. quadricauda can grow well at different pH conditions. CONCLUSIONS The microalga S.quadricauda was successfully isolated from from Kanye water reservoir which confirms it to be a native strain of the study area. The algal biomass and lipid yield were promising thus making it a good feed stock for biodiesel prduction.The quality of the biodisel extracted was good as it met most of the standard values which makes it a good candidate to serve as an alternative to fossil fuel when produced on a large scale. REFERENCES Abaye, O. J. Alna, E. A. and Okuonghea, C. O. (1998). Oil content and quality characteristics of some Nigerian oil seeds. Journal of Pure and Applied Science, 1(1). 17-23. Ananadhi, P.M.R. and Shaleesha, A.S. (2012). Microalgae as an oil producer for biodiesel applications. Journal of Recent Sciences. 1(3).57-62.

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Day, J.G. Benson, E.E. and Fleck, R.A. (1999). In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research. Vitro Cellular & Developmental Biology of Plant. 35(2): 127-136. Duffy, J.E. Canuel, E.A. and Adey, W. Swaddie, J. (2009). Biodiesels: Algae Journal of Science, 326(5958): 1345-1345. Griffiths, M. and Harrison, S. T. L. (2009). Lipid productivity as a key characteristic for choosing algal species for biodiesel production. Journal of Applied Phycology, 21(5):493-507. Hu, Q. Sommerfeld, M. Jarvis, E. Ghiradhi, M. Posewitz, M. and Seibert, M. (2008). Microalgal triacylglycerols as feedstocks for biodiesel production: perspectives and advances. Plant Journal, 54:621-39. Ilavarasi, A. MubarakAli, D. Parveez, A. Hameed, A. and Hajuddeen, N.T. (2012). Production of FAME from freshwater microalgae and profiling of fatty acids for biodiesel feedstocks, Department of microbiology, School of life sciences, Barathidasan University, 42(1): 10-14(2012). Indabawa, I.I. (2014). Bioinstrumentation lecture note for postgraduate students, Bayero University, Kano. (Unpublished). John, C.(2000). Interpretation of infrared spectrum, a practical approach. Encyclopedia of analytical chemistry. R.A .Meyers (Ed).John Wiley and Sons Ltd. Chichester. 1081510837. Kaewpintong,K.(2004). Cultivation of Haematococcus pluvialis in Airlift bioreactor. M.Sc. Thesis in Chemical Engineering. Dept. of Chemical Engineering Chulalongkorn University,Thailand. Kong, Q.X. Li, L. and Ruan, R. (2010). Culture of microalgae Chlamydomonas reinhardtii in wastewater for biomass feedstock production. Journal of Applied Biochemistry and Biotechnology, 160(6):9-18. Kwangdinata, R., Inda R. and Muhammad, Z. (2014). Production of Biodiesel from lipid of phytoplankton Chaetocerus calcitrans through ultrasonic method. Scientific World Journal. Vol 2004.Article ID 23136, 5 pages. www.dx.org/10.1155/2014/231361. Li, W. Guo, Y. and Fu, K. (2011). Enclosure experiment for influence on algae growth by shading light. Proceedings of Environmental Science (Part B), 10:1823â&#x20AC;&#x201C;8. Mata, T.M., Junaina Santo, Adelio M. Mendes and Nidia S. Caetano (2012). Sustainability evaluation of biodiesel produced from microalga Chlamydomonas sp grown in brewery wastewater. Chemical Engineering Transactions.37:823-28. Meher, L.C. Vidya, S. and Naik, S.N. (2006). Technical Aspects of Biodiesel Production by Transesterification A review. Journal of Renewable and Sustainable Energy Review, 10: 248-268. Oderinde, R. A. Ajayi, I. A. and Adewale, A. (2009). Characterisation of seed and seed oil of Huracrepitans and the kinematics of degradation of the oil during heating. Electronic Journal of Environmental, Agricultural and Food Chemistry, 8(3):201-208. Reda, A. I. Abou- shanab, C. Yunchul, M. Booki, J. and Byong, H. (2011). Applied Energy. 88(2011): 3300-3306.

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Rippka, R. Deruelles, J. Waterbury, J.B. Herdman, M. and Stainer, R.Y. (1979). Generic assignment, strain histories and properties of puren cultures of cyanobacteria. Journal of Microbiology. Vol 175(1): pp 469-476. Rodolfi, L. Zitelli, G.C. and Bassi, N. (2009). Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Journal of Biotechnology and Bioengineering, 102(1): 100-112 (16). Sanchez, M. (2003).Bio-fuels: Alternative feed stocks and conversion process. Science and Technology Part A. 25(1-2): 31-43. Vasudevan, P.T. and Briggs, M. (2008). Biodiesel production-current state of the art and challenges. Journal of Industrial Microbiology and Biotechnology. 35(5):451-30. Wang, X., Feng, Y., Liu. J., Lee, H., Li, C. and Li, N. (2010). Sequestration of CO2 discharged from anode by algal cathode in microbial carbon capture cells (MCCs). Biosens Bioelectron. 25 ;273-94. Weldy, C.S., (2007). Lipid production by Dunaliella salina in batch culture; Effects of nitrogen limitation and light intensity .US Department of Energy Journal of Undergraduate Research.www.researchgate.net ID 236354765. Yeesang, C.and Cheirslip, B. (2011). Effect of light supply and carbon source in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresource Technology. 102:3034-40.

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INVESTIGATION OF THE POTENTIAL PROTECTIVE EFFECT AGAINST DNA DAMAGE OF SOUR CHERRY PEELS WASTES FROM FRUIT JUICE PRODUCTION Uğur VURAL*1, İbrahim Halil KILIÇ2, İslam ŞAHBUDAK2, Işık Didem KARAGÖZ2 1 2

Aksaray University, Aksaray, Turkey

Gaziantep University, Department of Biology, Gaziantep, Turkey *Corresponding author’s e-mail: Vuralugur332@gmail.com

ABSTRACT From the birth of humanity, plants have been used for nutritional purposes and have also been the first source of treatment ever. Raw materials of most treatment drugs are plant-based. In addition to medicinal purposes, plants are among the main substances that are frequently used in the cosmetics and dyestuff industry. Every part of the plant (fruit, root, leaf, etc.) is considered an important source for industry. It is also known to be used as a source of aroma in recent years. For example, lemon, mint, lavender, blackberries, strawberries are used extensively in cosmetic field as the main aroma sources. The sunscreens used for protection against harmful rays of the sun contain oxybenzone and retinol palmitate. Studies have shown that these two compounds have a lot of side effects. Therefore, the discovery of new components with the least side effects that can protect our skin from harmful rays of the sun has gained importance. For this purpose, in this study, we investigated whether the sour cherry fruit peels discarded after fruit juice production have a protective effect against UV radaition. The sour cherry juice purchased from the market was squeezed and the fruit peels were collected. Fruit peels were then lyophilized, and water and methanol extracts were obtained. The potential protective effect against UV radiation of these extracts was examined using plasmids pBR322, H2O2, and UV-B. According to the results of this study, fruit peels of sour cherry which are considered as waste from fruit juice production, have a potential protective effect against UV radiation. Keywords: Sour cherry, UV, Protective effect, pBR322, Biological activity

INTRODUCTION Large amounts of organic wastes are produced during food production and processing. During fruit juice production, a significant amount of pulp consisting of fruit peels and seeds are discarded as waste. The recycling of these wastes is very important to reduce both environmental pollution and economic costs. The identification of the chemical contents of organic wastes is valuable to investigate their potential bioactivity and to provide the industry sector with raw materials. Turkey is in the 6th rank of the world fruit production with 16.3 million tonnes, which meets approximately 3% of the world's needs. Turkey is at the top rank of sour cherry juice production. Sour cherry differs from sweet cherry by its sour taste and health benefits.It is a great source of vitamins and minerals. It contains high amounts of strong antioxidants such as beta carotene and vitamin E (Karataş et al.,2016) . Sour cherry is also rich in vitamin A. There are 58 calories and 14.3 g carbohydrates in 100 g cherry (Tanver et al.,2015). Several studies have reported the antioxidant, antiallergic and antimicrobial effects of sour cherry due to the presence of anthocyanins (Kaftanoğlu et al.,2013). Other groups such 264

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as hydroxycinnamates, flavonoids, flavan-3-ols, and procyanidins also found in sour cherry (Bonerz et al., 2007; Çapanoğlu et al., 2011). The contents of methanol and water extracts of sour cherry peel were analyzed and it was shown that they contain anthocyanin, punicalagin, gallic acid, ellagic acid, and other polyphenols. Studies also reported their antimicrobial activity and therapeutic effects against many diseases (Wojdyło et al.,2014). Although many studies have been conducted to investigate the antioxidant, antimicrobial effects, and to analyze the chemical content of sour cherry peel, to our knowledge, no study has been found in the literature that investigated the DNA protective properties. Therefore, the potential protective effect of the sour cherry peels against DNA damage caused by UV radiation and oxidative stress was investigated in this study.

MATERIAŁ AND METHODS Preparation of fruit shell and leaf extracts Sour cherry fruits were purchased from a local market in Gaziantep. Sour cherry fruits were squeezed to obtain the juice and the pulp. Fruit peels obtained from the pulp were placed in a 50 ml Falcon tube, frozen at -80oC and lyophilized for 48 hours. The fruit peels were lyophilized, then methanol and water extracts were obtained using Gerhardt's SOXTHERM® rapid extraction system. The leaves were dried in the shade, then milled to obtain methanol and water extracts using the Gerhardt's SOXTHERM® rapid extraction system. Analysis of DNA Protective Activity The pBR322 Plasmid DNA (vivantis) was used to study the potential activity of the bark, leaf and fruit peel extracts of sour cherry to protect DNA from UV and oxidative stress damage. Plasmid DNA was damaged by H2O2 and UV-C treatment in the presence of the extracts. Then, using the method established by Russo et al. (2000), 50 mg of the dry extracts were weighed and dissolved in 1000μl of distilled water. The control pBR322 plasmid DNA was diluted 1: 3 with distilled water. 1.5% agarose gel was prepared. 5μl of the prepared extract was added to 5μl of orange G loading dye and 10μl was loaded to each well. Then, gel electrophoresis was performed at 100 volts for 60 min..The control and experimental groups were as follows: K1: Control1: Plazmid DNA (3μl) + dH 2 O (6μl), K2: Control2: Plazmid DNA (3μl) + dH 2 O (6μl) + UV (5min) + H 2 O 2 (1μl), KS: Plazmid DNA (3μl) + Water extract of fruit peels 5μl + UV (5min ) + H 2 O 2 (1μl), SS: Plazmid DNA (3μl) + Water extract of fruit stalks 5μl + UV (5min) + H 2 O 2 (1μl) YS: Plazmid DNA (3 μl) + Water extract of leaves 5μl + UV (5min) + H 2 O 2 (1μl), KM: Plazmid DNA (3μl) + Methanol extract of fruit peels 5μl + UV (5min) + H 2 O 2 (1μl), SM: Plazmid DNA (3μl) + Methanol extract of fruit stalk 5μl + UV (5min) + H 2 O 2 (1μl), YM: Plazmid DNA (3 μl) + Methanol extract of leaves 5μl + UV (5min) + H 2 O 2 (1μl) RESULTS The potential protective activity of sour cherry extracts against DNA damage was evaluated by exposing of plasmid DNA to UV radiation (5 min.) and H2O2 (1 μl). The gel electrophoresis results are shown in Figure 1.

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K1: Control1: Plazmid DNA (3μl) + dH 2 O (6μl), K2: Control2: Plazmid DNA (3μl) + dH 2 O (6μl) + UV (5min) + H 2 O 2 (1μl), KS: Plazmid DNA (3μl) + Water extract of the fruit peel 5μl + UV (5min ) + H 2 O 2 (1μl), SS: Plazmid DNA (3μl) + Water extract of the fruit stalk 5μl + UV (5min) + H 2 O 2 (1μl) YS: Plazmid DNA (3 μl) + Water extract of the leaf 5μl + UV (5min) + H 2 O 2 (1μl), KM: Plazmid DNA (3μl) + Methanol extract of the fruit peel 5μl + UV (5min) + H 2 O 2 (1μl), SM: Plazmid DNA (3μl) + Methanol extract of the fruit stalk 5μl + UV (5min) + H 2 O 2 (1μl), YM: Plazmid DNA (3 μl) + Methanol extract of the leaf 5μl + UV (5min) + H 2 O 2 (1μl) Figure 1. Electrophoretic pattern of pBR322 plasmid DNA after treatment with UV and H2O2 in the presence or absence of sour cherry extracts

The samples prepared by adding the water and methanol extracts of sour cherry were compared to control samples. As shown in the gel electrophoresis image, water extract of leaves, methanol extract of a fruit peel , methanol extract of fruit stalk and methanol extract of leaves showed DNA protective activity, whereas water extract of fruit peel and water extract of fruit stalk did not show a protective effect. The methanol extracts of the fruit peel, fruit stalk, and leaves of sour cherry showed the highest DNA protective effect. As shown in the figure, the control 2 DNA sample was damged by oxidative stress and UV radiation and no band was observed. DISCUSSION Several studies have investigated the potential antioxidant and antimicrobial effects of sour cherry. However, as far as we know, no studies on the potential protective activity against DNA damage of sour cherry have been found in the literature. In this study, we have investigated the potential protective effect of the bark, fruit peel, fruit stalk and leaf extracts obtained from sour cherry against DNA damage caused by toxic and mutagenic activities of UV and H2O2. It is known that the UV radiation reaching the earth as a result of ozone depletion, has negative effects on living creatures. Antioxidants protect against the harmful effects of UV radiation. Exposure to UV radiation cause serious diseases such as skin cancer and skin aging. The topical application of enzymatic and non-enzymatic antioxidants is an effective approach to protect the skin from the harmful effects of UV radiation (Gutteridge et al., 1984). Human skin has a number of mechanisms to reduce the harmful effects of visible light and UV radiation. However, high exposure to UV radiation can lead to a reduction in the number of cellular antioxidants and ultimately lead to UV-induced oxidative DNA damage caused by reactive oxygen species (Bülbül, 2014). In addition to UV radiation, free radicals can also cause DNA damage. The results of the DNA protective activity demonstrated that there were no linear DNA bands in the water extracts of the bark, fruit peel, fruit stalk, and leaves as well as the 266

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methanol extracts of the bark, fruit peel, fruit stalk, and leaf samples. In other words, both water and methanol extracts were found to be protective against DNA damge caused by UV and H2O2. Oxybenzone and retinyl palmitate sunscreens, which are assumed to be protective against UV radiation, are widely used as effective components in the personal care products, hand, and body creams (Benevenuto et al.,2015; Downs et al., 2015). However, these components which are indispensable for cosmetic products have been reported to have side effects. In addition, Oxybenzone has been reported to have carcinogenic effects on the endocrine system (Hopkins et al., 2017). Recently, extensive studies have been carried out on extracts from medicinal and aromatic plants to obtain new compounds that control cancer-causing oxidative DNA damage (Bayil Oguzkan et al., 2016).

CONCLUSION The high DNA protective activity of bark, fruit stalk, fruit peel and leaf parts of sour cherry as shown by the results of this study, make its use more convenient as an alternative product to the products currently available in the market. These results provide a basis for further investigation of the protective effect of sour cherry against UV radiation. REFERENCES Bayil Oguzkan, S., Uğraş, S., Aksoy, E. S., Ülger, S., Üzmez, Ş., Karagül, B., & Uğraş, H. İ. (2016). Biological activity analysis of hazelnut nutshell extracts. International Journal of Chemical and Natural Science, 4(5), 481-485. Benevenuto, C. G., Guerra, L. O., & Gaspar, L. R. (2015). Combination of retinyl palmitate and UV-filters: phototoxic risk assessment based on photostability and in vitro and in vivo phototoxicity assays. European Journal of Pharmaceutical Sciences, 68, 127-136. Bonerz, D., Würth, K., Dietrich, H., & Will, F. (2007). Analytical characterization and the impact of ageing on anthocyanin composition and degradation in juices from five sour cherry cultivars. European Food Research and Technology, 224(3), 355-364. Bülbül, B. (2014). Bioactivity studies on some natural mycorrhizal fungi in Gaziantep, M.Sc., Gaziantep University, Gaziantep. Downs, C. A., Kramarsky-Winter, E., Segal, R., Fauth, J., Knutson, S., Bronstein, O., ... & Pennington, P. (2016). Toxicopathological effects of the sunscreen UV filter, oxybenzone (benzophenone-3), on coral planulae and cultured primary cells and its environmental contamination in Hawaii and the US Virgin Islands. Archives of environmental contamination and toxicology, 70(2), 265-288. Gutteridge, J. M. (1984). Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide. FEBS letters, 172(2), 245-249. Hopkins, Z. R., Snowberger, S., & Blaney, L. (2017). Ozonation of the oxybenzone, octinoxate, and octocrylene UV-filters: Reaction kinetics, absorbance characteristics, and transformation products. Journal of hazardous materials, 338, 23-32. Kaftanoğlu E. (2013). Taze ve ticari vişne sularının (Prunus Cerasus) toplam antosiyanin, toplam fenolik ve şeker miktarlarının tayini ve karşılaştırılması / Determination and comparison of the amount of total anthocyanin, total phenolics and sugar content of fresh and commercı̇ al cherry (Prunus Cerasus) juices, İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, Kimya Anabilim Dalı. 267

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Karataş F., Çöteli E. (2016). Kırmızı ve Koyu Kırmızı Bazı Meyvelerdeki A, E Vitamini, Beta Karoten ve Likopen Miktarlarının Araştırılması, Fırat Üniversitesi, Fen Fakültesi, Kimya Bölümü, 23119, Elazığ, Türkiye. Russo, A., Acquaviva, R., Campisi, A., Sorrenti, V., Di Giacomo, C., Virgata, G., ... & Vanella, A. (2000). Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors. Cell biology and toxicology, 16(2), 91. Tanver, A., Huang, M. H., Luo, Y., Khalid, S., & Hussain, T. (2015). Energetic interpenetrating polymer network based on orthogonal azido–alkyne click and polyurethane for potential solid propellant. RSC Advances, 5(79), 64478-64485. Wojdyło, A., Nowicka, P., Laskowski, P., & Oszmiański, J. (2014). Evaluation of sour cherry (Prunus cerasus L.) fruits for their polyphenol content, antioxidant properties, and nutritional components. Journal of agricultural and food chemistry, 62(51), 1233212345.

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INDUCTION MUTATION IN BREAD WHEAT (TRITICUM AESTIVUM L.) GENOTYPES FOR IMPROVE YIELD COMPONENT AND QUALITY PARAMETERS İrfan ÖZTÜRK*1, Ayşe ŞEN2 1 2

Trakya Agricultural Research Institute, Edirne, Turkey

Istanbul University, Faculty of Science, Department of Biology, Istanbul, Turkey *Corresponding author’s e-mail: ozturkirfan62@yahoo.com

ABSTRACT In the study, conventional gamma ray mutagenesis was used to develop mutant lines, which have such characters moderate or high yielding, high quality, semi dwarf and early maturing. In the study cultivar Saban and seven advanced genotypes were irradiated with 170, 200, and 250 Gy gamma ray. In M1 population, observation were recorded for days of heading, plant height, peduncle length, spike length, number of spikelet per head, number of kernel per spike, 1000-kernel weight, test weight, and protein ratio. To compare non-treatment (control) and mutant lines, the highest plant height, peduncle length and spike length was determined in nontreatment. The increase in the dose of gamma rays, reduced the plant height, peduncle length and spike length. The spike number and kernel number in spike in M1 populations was less than over the control (non-treatment) population (non-treatment). The similar results were also obtained in 1000-kernel weight and test weight in genotypes. The data showed that in comparison with the control population protein ratio in the mutagen treated population was higher than non-treatment. The phenotypic and agronomic correlations revealed that days of heading were negatively associated with plant height (r=-0.984*), and 1000-kernel weight (r=0.975*). Various positive correlation was determined between plant height with peduncle length (r=0.968*), flag leaf area (r=0.984*), spikelet number per spike (r=0.962*), and 1000kernel weight (r=0.983*). In the present study, a strong positive correlation was determined between flag leaf area with spikelet number (r=0.989*) and kernel number (r=0.956*) per spike and TKW (r=0.989*). The results of correlation showed that protein ratio was negative association with plant height, peduncle length, flag leaf area, spike length, spikelet number per spike, kernel number per spike, and 1000-kernel weight. Results of the study showed that protein ratio was higher in the mutagen treated population. Keywords: Bread wheat, Gamma ray mutagenesis, Yields component, Quality characters

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INTRODUCTION Turkey is one of the centers of origin of wheat and wheat has been grown on about 8.5 million ha with production of around 20 million metric tons annually (Anonymous, 2015). Bread wheat (Triticum aestivum L.) is one of the most important cereal crops in the world due to adapting to diverse environments from -35ºC in the vegetative stage to 40ºC during grain filling period (Shewry, 2009). Mutation breeding is one of the breeding tools based on artificially inducing hereditary changes in plants using either physical or chemical mutagen, which has been successfully used to develop diverse and valuable materials in several crops with agronomical important traits (Thapa, 2004; Borzouei et al., 2010; Shah et al., 2012). Mutation breeding is a significant breeding tool which has been used successfully in several crops for breeding agronomically important traits (Maluszynski et al., 1995). Therefore, mutagenesis is applied to amend few blemishes in a cultivar that has several agronomic traits preferable by farmer. In wheat breeding, Sakin et al., (2004, and 2005) obtained superior mutant types having better agronomic values in term of yield and yield components. Mutation breeding is relatively a quicker method for improvement of crops (Ilirjana et al., 2007). Hundreds of useful mutants have been induced for various plant characters in variety of crops including wheat through treatment with physical and chemical mutagens (Ram Din et al., 2003; Morten et al., 2006). Induced mutation help to develop of many agronomical important traits such as shorter growing period, suitable for rotation, increased tolerance or resistance to abiotic and biotic stresses use in major crops such as wheat, rice, barley, cotton, peanuts and beans (Ahloowalia et al., 2001; Maluszynski et al., 2002). Mutation is a heritable change in genes and chromosomes, and occurs at random, either spontaneous or induced with mutagenic agents (Şen et al., 2017). Induced mutations are necessary to enhance rate of genetic variability since spontaneous mutation rate is very low and that prevents breeders to exploit them in plant breeding programmes. Selecting new cultivar(s) against tolerance to drought stress is generally evaluated using phenotypic observations. Thus, effective stress related biomarkers are needed to identify and implement in breeding programmes for screening of drought tolerant genotypes (Şen and Öztürk, 2018). Gamma radiation can be useful for changing physiological characteristics (Kiong et al., 2008). Induced mutagenesis and its breeding strategies are potential tools for improving both quantitative and qualitative traits in crops within a much shorter period of time than conventional breeding. Because of its relative simplicity and low cost, mutagenic treatment of seeds and other parts of the plant remains a useful tool for isolating the desired variants and developing resistance to biotic and abiotic stresses in various crops (Oladosu et al., 2014). Gamma rays in particular, is an important physical mutagen which is well known with their effects on the plant growth and development by inducing morphological, cytological and physiological changes in cells and tissues (Thapa, 2004; Borzouei et al., 2010; Shah et al., 2012). Gamma rays in particular, is well known physical mutagen and often used in mutation breeding program to induce desirable mutants with different genotypes (Konzak, 1987; Knott, 1991). Irradiation of seeds may cause genetic, variability that enables plant breeders to select new genotypes with improved characteristics such as salinity tolerance, grain yield and quality (Ashraf et al., 2003). Due to limited genetic variability among the existing plant genotypes opened a good era for crop improvement and now mutation induction has become an established tool in plant breeding that can improve cultivars in certain specific traits as well (Oladosu et al., 2016). The objectives of this work were to apply gamma ray irradiation to obtain mutant bread wheat lines among the 270

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advanced genotypes and cultivar, which have superior characters, such as moderate or high yielding, good quality, short plant height, and early maturing. MATERIALS AND METHODS Experimental Design Eight winter bread wheat (Triticum aestivum L.) genotypes were subjected to 170, 200, and 250 Gy gamma irradiation in 2017. Treated genotypes cross and pedigree number was given in Table 1. Table 1. Cross and pedigree number of the parent genotypes used in this study No

Genotypes

Parents Genotypes Cross

Pedigree

Saban

Trakia/3/MvC41090/GkKalaka//MvC410-90/Ftm-II

TE6060-1T-1T-1T-0T

BBVD72014

Admis//Milan/Ducula

CMSW01WM00331S030YE-30E-1E-0E-1E-0E

BBVD252016

Rumba//Chirya.3/GK Othalon

TE6627-0T-0T-20T-2T1T-0T

ÖVD1-142016

Tekirdağ//GK Kalasz/Beaubourg

TE6748-0T-0T-9T-1T-0T

BBVD222016

Rumba//Chirya.3/GK Othalon

TE6627-0T-0T-4T-1T-3T0T

BBVD122016

15.99/3/Pehl//Rpb868/Chrc/4/Chatelet

TE6411-2T-0T-9T-5T-3T0T

BBVD172016

Flm85/7/Kç66/Bez//Sup/3/Wrm/2*Hu a/4/

TE 6295-2T-0T-13T-1T6T-0T

Dons/5/Kate/MvM/6/Presl 8

BBVD32015

90Zhong150//Mex65/Momt/3/Trakia

TE6217-3T-1T-1T-3T-0T

Treated seeds were grown in row spacing of 30 cm to produce the M1 population. The untreated seeds of mother varieties (parental line/variety) were also planted to comparison with the M1 population. The planting was implemented in 2017-2018 growing season. The experiment was conducted with 8 genotypes in randomized completely blocks design (RCBD) with 3 replications. Each genotype was sown with three rows 5m long and 30 cm apart, being plot size of 4.5m2, and sowing density was 500 seed per square meter. Sowings of the experiment were performed by hand.

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Table 2. Climate condition in 2016-2017 cropping years in Edirne/Institute Temperature (oC)

Precipitation (mm)

Moisture (%)

Min.

Max.

Mean

September 2016

9.2

57.5

5.0

33.8

20.8

October 2016

44.4

69.5

1.3

28.8

14.3

November 2016

3.2

72.9

-9.9

15.4

0.7

December 2016

3.2

72.9

-9.9

15.4

0.7

January 2017

67.8

83.7

-17.0

8.4

-1.9

February 2017

43.4

80.0

-8.4

20.6

5.3

March 2017

51.0

73.0

-1.9

25.5

10.2

April 2017

65.6

63.1

-1.6

28.6

12.5

May 2017

85.0

65.4

4.4

30.0

17.9

June 2017

44.4

74.4

12.9

40.0

21.2

Total/Mean

417.2

71.2

-17.0

40.0

10.2

Months

During 2016-2017 growing season, the mean annual rainfall was 417.2 mm. It was lower than long year rainfall (589.1 mm) (Öztürk and Korkut, 2017). The mean values of the relative humidity and mean, maximum and lower temperature was recorded in growing years and given in Table 2. Measurement of Agronomic Parameters Data on seed germination and surviving plants were recorded considering whole plots of M1 population. Data on yield components; peduncle length (cm), spike length (cm), spikelet number per spike, and spike number per spike were taken from 10 randomly selected plants of each treatment. Also, days of heading, plant height (cm), thousand kernel weight (g), test weight (kg), protein ratio (%), and hardness were investigated. Protein ratio, 1000-kernel weight, test weight and hardness were determined according to Anonymous, (1980); Blakeney et al., (2009); Perten, (1990), Pena, (2008), and Köksel et al., (2000). Statistical Analysis The data for the four replications were combined and statistically analyzed using analysis of variance (Gomez and Gomez, 1984). The significance of differences among means was compared by using Least Significant Difference test (L.S.D. at P<0.05) test (Kalaycı, 2005). Coefficients of the regression equations (R2) were calculated according to (Finlay and Wilkinson, 1963; Eberhart and Russell, 1969). Regression graphs were drawn to predict adaptability of genotypes.

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RESULTS AND DISCUSSION In M1 population, observations were recorded for days of heading, plant height, peduncle length, spike length, number of spikelet per head, kernel number per spike, 1000-kernel weight, test weight, and protein ratio (Table 1). The ANOVA results for physiological characteristics of wheat genotypes were given in Table 3. Analysis of variance for 2016-2017 seasons indicated statistically significant differences (P<0.01 and P<0.05) for all traits measured in presented study (Table 3). To compare non-treatment (control) and mutant lines, the highest plant height, peduncle length and spike length was determined from the non-treatment and the increase in the dose of gamma rays, reduced the plant height, peduncle length and spike length. The spike number and kernel number in spike in M1 populations was less than over the control (non-treatment) population (Non-treatment). The similar results were also obtained in 1000kernel weight and test weight in wheat genotypes. The data showed that in comparison with the control population protein ratio in the mutagen treated population was higher. Table 3. The mean value of treatment in terms of agronomic and quality characters Treatment DH

SPL

SNS

KNS

TKW

PRT

Control

145.1c

75.9a

23.0a

8.6a

19.0a

40.3a

34.5a

73.0a

14.8c

Mut170

150.0b

66.7b

20.1b

8.5a

17.8b

38.4ab

34.1a

71.5b

15.6b

Mut200

151.1b

64.6b

19.6b

7.9b

16.6c

35.1ab

33.8a

71.8ab

15.7b

Mut250

154.7a

61.3c

19.6b

7.9b

16.4c

34.3b

33.7a

71.2b

16.7a

Mean

150.3

67.1

20.6

8.2

17.4

37.0

34.0

71.9

15.7

LSD (0.05)

1.57

2.64**

1.7

0.43

0.78

5.7

2.15

1.24

0.31

54.94**

47.58** 7.86** 5.96** 20.3**

2.02ns

0.22ns

3.56*

48.56**

Note: Significance at *: P<0.05; **: P<0.01; DH: days of heading, PH: Plant height (cm), PL: Peduncle length (cm), SPL: Spike length (cm), SNS: Spikelet number per spike, SNS: Spike number per spike, TKW: Thousand kernel weight (g), TW: Test weight (kg), PRT: Protein ratio (%) The aim of wheat breeding programs is to improve the genotypes adaptation to target environment. In most case, grain yield and its components are the most important economic traits in wheat improvement. However, yield is a polygenic trait with low inheritance. Induced genetic variability with mutation technique brings about heritable changes in plants and offer new genetic varieties to plant breeders. Kenzhebayeva et al., (2017) used gamma radiation to generate genetic variation in wheat and tested the linkage between various important grain parameters in fifth generation mutants. In this study, we have created the mutant wheat populations using 170 Gy, 200 Gy and 250 Gy gamma irradiation and data and parameters scored from M1 generation mutant populations. The highest TKW (equivalent to 34.5 g) was obtained under non-treatment conditions (control), while the lowest TKW (equivalent to 33.7 g) were obtained under Mutant 250 that was applied 250 grey gamma dosses. The mutant treatments were given the highest value based on protein content (16.7%) in mutant 250 Gy gamma irradiation, and followed by 16.7% in mutant 200, and 15.6% in mutant 170. Mean 273

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protein ratio in non-treatment (control) was 14.8%. Kenzhebayeva et al. (2017) reported that gamma ray mutagenesis increased the protein content in some advanced mutant lines than parent line. Length of the spike of main tiller was measured in centimetres from the base of spike to the upper most spikelet excluding awns. The highest plant height (75.9 cm), peduncle length 23.0 cm), and spike length (8.6 cm) was measured in non-treatment condition. At the time of maturity, the height of each selected plant was measured in centimeters from the surface of soil to the tip of ear-head excluding awns. Plant height is a major agronomic parameter in bread wheat because of its association with lodging. Plant height was 66.7 cm in mutant 170, 64.6 cm in mutant 200, and 61.3 cm in mutant 250 applications. Peduncle length based on mutant application varied 19.6 cm in mutant 200 Gy gamma irradiation and mutant 250, 20.1 cm in mutant 170. The numbers of spikelets in the primary tiller of each selected plant were counted based on mutant treatment. Spikelet number per spike was 17.8, 16.6, and 16.4 application of mutant 170, mutant 200 and mutant 250, respectively (Table 3). The main spike of primary tiller of each selected plant was threshed separately, numbers of grains were counted. In presented work, there was no promising result based on kernel number per spike. The kernel number per spike was 40.3 in non-treatment, 38.4 in mutant 170, 35.1 in mutant 200 and 34.3 in mutant 250 Gy gamma irradiation. Table 4. The mean value of the genotypes in terms of agronomic, physiological and quality characters Entry

Genotypes

SPL

SNS

Saban

151.0c

61.8e

18.77cd

8.41b

18.08b

BBVD7-2014

143.5f

75.5a

27.82a

8.51b

15.80c

BBVD25-2016

146.0e

74.0a

20.24bc

7.31c

16.55c

Ă&#x2013;VD1-14-2016

156.8a

66.8cd

18.20cd

8.32b

17.95b

BBVD22-2016

145.3ef

54.5f

18.73cd

9.42a

16.70c

BBVD12-2016

156.5a

71.8ab

21.77b

8.47b

20.43a

BBVD17-2016

154.3b

68.5bc

17.15d

8.47b

18.13b

BBVD3-2015

148.8d

64.3de

21.73b

6.89c

15.85c

Mean

150.3

67.1

20.55

8.22

17.43

LSD (0.05)

2.21**

3.74**

2.40**

0.62**

1.11**

CV (%)

1.0

3.8

7.9

5.1

4.3

Note: * and ** represented significance at: P<0.05 and: P<0.01, respectively. DH: days of heading, PH: Plant height (cm), PL: Peduncle length (cm), SPL: Spike length (cm), SNS: Spikelet number per spike

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The ANOVA results for agronomic and quality characteristics of wheat genotypes were given in Table 4 and 5. Analysis of variance for 2016-2017 seasons indicated statistically significant differences (P<0.01) for all traits except kernel number in spike measured in presented study (Table 4 and 5). Based on mean performance, genotype BBVD7-2014 was very early, and had highest plant height (75.5 cm) and peduncle length (27.82 cm). Line BBVD222016 had the longest spike (9.42 cm). Genotype BBVD12-2016 produced maximum spikelet number per spike (20.43), gave more kernel number per spike (38.95) after BBVD25-2016 (39.20). BBVD25-2016 and BBVD7-2014 gave highest TKW with 38.15 g and 37.93 g, respectively. The highest test weight (78.25 kg) was measured in BBVD7-2014. The data regarding mean performance of seven parental lines and one cultivar evaluated for quality parameters in bread wheat are summarized in Table 5. Most of the genotypes gave higher mean values for protein ratio. Cultivar Saban (16.45%) and BBVD17-2016 (16.33%) had the highest protein ratio. Table 5. The mean value of the genotypes in terms of agronomic and quality characters Entry

Genotypes

KNS

TKW

PRT

HARD

Saban

38.08a

34.80b

72.00de

16.45a

50.00bc

BBVD7-2014

38.18a

37.93a

78.25a

15.58cd

51.75ab

BBVD25-2016

39.20a

38.15a

74.20bc

15.50cd

51.75ab

Ă&#x2013;VD1-14-2016

37.35ab

31.23c

66.75f

15.90bc

37.75e

BBVD22-2016

38.33a

38.05a

70.38e

14.73e

46.00d

BBVD12-2016

38.95a

28.13d

64.85g

15.73cd

48.50c

BBVD17-2016

36.83ab

32.90bc

75.45b

16.33ab

51.25ab

BBVD3-2015

29.30b

31.03cd

73.13cd

15.40d

52.50a

Mean

37.03

34.02

71.87

15.7

48.68

LSD (0.05)

8.20ns

3.04**

1.75**

0.44**

2.46**

CV (%)

15.1

6.1

1.7

1.9

3.4

Note: Significance at *: P<0.05; **: P<0.01; KNS: Kernel number per spike, TKW: Thousand kernel weight (kg), TW: Test weight (g), PRT: Protein ratio (%), HARD: Hardness

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Table 6. The correlation coefficients among agro-physiological parameters Traits

FLA

SNS

KNS

TKW

-0.984*

-0.907

0.968*

FLA

-0.950

0.984* 0.974*

SPL

-0.802

0.829

0.805

0.903

SNS

-0.942

0.962*

0.929

0.989* 0.950*

KNS

-0.927

0.927

0.866

0.956* 0.965*

0.989*

TKW

-0.975* 0.983*

0.935

0.989*

0.910

0.993**

0.980*

-0.928

0.948

0.931

0.889

0.610

0.826

0.760

0.874

PRT

0.984*

-0.937 -0.820 -0.881 -0.732

-0.884

-0.886

-0.930 -0.891

Note: * and ** represented significance at: P<0.05 and: P<0.01, respectively. DH: days of heading, PH: Plant height (cm), PL: Peduncle length (cm), FLA: Flag leaf area (cm2), SL: Spike length (cm), SNS: Spikelet number per spike, KNS: Kernel number per spike, TKW: 1000kernel weight (g), TW: Test weight (kg), PRT: Protein ratio (%). Correlation coefficients were done between parameters combination. The phenotypic and agronomic correlations revealed that days of heading were negatively associated with plant height (r=-0.984*), and 1000-kernel weight (r=-0.975*). Various positive correlation was determined between plant height and peduncle length (r=0.968*), flag leaf area (r=0.984*), spikelet number per spike (r=0.962*), and 1000-kernel weight (r=0.983*). In the present study, a strong positive correlation was determined between flag leaf area with spikelet number (r=0.989*) and kernel number (r=0.956*) per spike and TKW (r=0.989*). The results of correlation showed that protein ratio indicated negative association with plant height, peduncle length, flag leaf area, spike length, spikelet number per spike, kernel number per spike, and 1000-kernel weight. Thus estimation of correlation and regression analysis among yield and yield components may provide effective selection criteria to improve wheat grain yield. Selection for grain yield can only be effective if desired genetic variability is present in the genetic stock. Genotypic and phenotypic correlations are important in determining the degree to which various yield contributing characters are associated. Several researchers have reported their findings regarding the correlation studies. Sandhu and Mangat (1985) and Gupta et al., (1999) reported positive correlation of grain yield with a number of grains per spike, plant height and 1000 grain weight. However, Gupta et al., (1999), and Chowdhry et al., (2000) conducted such studies and concluded that yield components like tillers per plant, grains per spike and 1000 grain weight are main contributors to grain yield in wheat. Keeping in view the present situation, this research is designed to meet the objectives to estimate the correlation between yield components and seed yield in wheat and to work out regression of grain yield on its components.

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Protein ratio and TKW

Protein ratio and days of heading

Flag leaf area and TKW

Flag leaf area and KNS

Flag leaf area and SNS

Test weight and plant height

Figure 1. Regression analysis among investigated parameters The results of regression equation (R2) were given in Figure 1. In presented study, the highest significant negative association was found between protein ratio and TKW (R2=0.864). On the other hand, regression analysis indicated that protein ratio was positively associated with days of heading (R2=0.968). This result showed that late genotypes had higher protein ratio. Flag leaf area of the genotypes positively affected yield component and it was found positive relation between flag leaf area and TKW (R2=0.977), kernel number per spike (R2=0.914), and spikelet number per spike (R2=0.977). In this study it was found positively relationship between plant height and TW (R2=0.898). These results suggested that flag leaf was the critical factor to increase yield component of the wheat genotypes.

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CONCLUSIONS To compare non-treatment (control) and mutant lines, the highest plant height, peduncle length and spike length was determined in non-treatment. The increase in the dose of gamma rays reduced the plant height, peduncle length and spike length. The spike number and kernel number in spike in M1 populations was less than over the control (non-treatment) population. The similar results were also obtained in 1000-kernel weight and test weight in genotypes. The data showed that in comparison with the control population protein ratio in the mutagen treated population was higher than non-treatment. The phenotypic and agronomic correlations revealed that days of heading were negatively associated with plant height and 1000-kernel weight. Various positive correlations were determined between plant height with peduncle length, flag leaf area, spikelet number per spike, and 1000-kernel weight. In the present study, a strong positive correlation was determined between flag leaf area with spikelet number and kernel number per spike and TKW. The results of correlation showed that protein ratio was negative association with plant height, peduncle length, flag leaf area, spike length, spikelet number per spike, kernel number per spike, and 1000-kernel weight. Results of the study showed that protein ratio was higher in the mutagen treated population.

REFERENCES Ahloowalia, B.S., and M. Maluszynski (2001). Induced mutations - A new paradigm in plant. Euphytica, 118 (2): 167-173. Anonymous, (2015). FAO, 2015. Wheat Landraces in Farmers’ Fields in Turkey: National Survey, Collection, and Conservation, 2009-2014, by Mustafa Kan, Murat Küçükçongar, Mesut Keser, Alexey Morgounov, Hafiz Muminjanov, Fatih Özdemir, Calvin Qualset. Anonymous. (1980). ICC-Standart No: 105. Method for the determination of crude protein in cereals and cereal products for food and for feed. Ashraf, M., A.A. Cheema, M. Rashid, and Z. Qamar (2003). Effect of gamma rays on M1 generation in Basmati rice. Pak. J. Bot., 35 (5): 791-795. Blakeney, A.B., R.L. Cracknell, G.B. Crosbie, S.P. Jefferies, D.M. Miskelly, L. O'Brien, J.F. Panozzo, D.A.I. Suter, S. Solah, T. Watts, T. Westcott, and R.M. Williams (2009). Grains Research and Development Corporation, Understanding Australian Wheat Quality. Borzouei, A., M. Kafi, H. Khazaei, B. Naseriyan, and A. Majdabadi (2010). Effects of gamma radiation on germination and physiological aspects of wheat (Triticum aestivum L.) seedlings. Pak. J. Bot., 42(4): 2281-2290. Chowdhry, M.A., M. Ali, G.M. Subhani, and I. Khaliq (2000) Path Coefficient Analysis for Water Use Efficiency, Evapo-Transpiration Efficiency, Transpiration Efficiency and Some Yield Related Traits in Wheat. Pakistan Journal of Biological Sciences, 3, 313-317. http://dx.doi.org/10.3923/pjbs.2000.313.317 Eberhart,, S.A., and W.A. Russell (1969). Yield stability for a 10-line diallel of single-cross and double-cross maize hybrids. Crop Sci. 9, 357-361.

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Finlay, K.W., and G.N. Wilkinson (1963). The Analysis of Adaptation in a Plant Breeding Programme. Aust. J. Agric. Res., 14: 742-754. Gupta A. K., R.K. Mittal, and A. Ziauddin (1999) Association and Factor Analysis in Spring Wheat. Annals of Agricultural Research, 20, 481-485. Gomez, K.A., and A. A. Gomez (1984). Statistical Procedures for Agricultural Research. 2nd Ed. John Willey and Sons, Inc. New York. 641. Kalaycı, M. (2005). Örneklerle Jump Kullanımı ve Tarımsal Araştırma için Varyans Analiz Modelleri. Anadolu Tarımsal Araştırma Enst. Müd. Yayınları, Yayın No: 21, Eskişehir. (Using Jump with Examples and Variance Analysis Models for Agricultural Research. Anadolu ARI Pub. No: 21, Eskişehir Turkey) Kenzhebayeva, S.S., G. Doktyrbay, N.M. Capstaff, F. Sarsu, N.Z. Omirbekova, T. Eilam, D.K. Tashenev, and A. J. Miller (2017). Searching a spring wheat mutation resource for correlations between yields, grain size and quality parameters. J Crop Improvement. 31: 209-228. Knott, D. R. (1991). What determines the success of mutation breeding? IAEA, V.1. p. 111118. Konzak, C. F. (1987). Mutations and Mutation Breeding. In: E.G. Heyne (ed.), Wheat and Wheat Improvement, Second Edition, 428-443, ASA. CSSA. SSSA. Inc. Press, Madison, WI, USA. Köksel, H., D. Sivri, O. Özboy, A. Başman, H. D Karacan (2000). Hububat Laboratuarı El Kitabı. Hacettepe Üni. Müh. Fak. Yay. No: 47, Ankara. (Handbook of the Cereal Laboratory. Hacettepe Uni. Fac. of Eng. No: 47, Ankara, Turkey). Kiong, A., A. L. Pick, S. H. G. Lai, and A. R. Harun (2008). Physiological responses of Orthosiphon stamineus plantlets to gamma irradiation. Am-Eurasian j. Sustain. Agric., 2 (2): 135-149. Ilirjana, S., Y. Ariana, and D. Andon (2007). Induced Mutations for Improving Production on Bread and Durum Wheat. Sixth International Conference of the Balkan Physical Union. AIP. Smithsonian/NASA ADS Physics Abstract Service. Conference Proceedings, 899: 747-747. Maluszynski, M., and K. J. Kasha (Eds.), (2002). Mutations, In vitro and Molecular Techniques for Environmentally Sustainable Crop Improvement. Kluwer Academic Publishers, Dordrecht/Boston/London. ISBN 1-4020-0602-0. Maluszynski, M., B.S. Ahloowalia and B. Sigurbjornsson (1995). Application of In vivo and In vitro mutation techniques for crop improvement. Euphytica, 85: 303-315. Morten, L., C. Feng, X. Xianchun, W. Manilal, J. P. Roberto, R. Trethowan and Z. He (2006). Puroindoline grain hardness alleles in CIMMYT bread wheat germplams. J. Cereal Sci., 44(1): 86-92. Oladosu, Y., M. Y. Rafii, N. Abdullah, G. Hussin, A. Ramli, H. A. Rahim, G. Miah and M. Usman (2016). Principle and application of plant mutagenesis in crop improvement: A review, Biotechnology and Biotechnological Equipment, 30 (1): 1-16. Oladosu, Y., M. Y. Rafii, N. Abdullah, M. Abdul Malek, H. A. Rahim, G. Hussin, M . Abdul Latif, and I. Kareem (2014). Genetic variability and selection criteria in rice mutant lines as revealed by quantitative traits (Internet). The Scientific World J.:190531. http://dx.doi.org/ 10.1155/2014/190531 279

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Öztürk, İ., Z. K. Korkut (2017). Response of Bread Wheat Genotypes to Various Drought Stress Conditions. 2nd International Balkan Agriculture Congress, 16-18 MAY 2017. Electronic Book. p: 199-208. Tekirdağ, Turkey. ISBN : 978-605-4265-49-7. http://agribalkan2017.nku.edu.tr/ and http://ziraat-en.nku.edu.tr/ Singh, B.D. (1993). Plant breeding Principles and Methods. Kalyani Publishers, Ludhiana, pp: 227-257. Şen, A., İ. Öztürk, O. Yaycili, S. Alikamanoğlu (2017). Drought Tolerance in Irradiated Wheat Mutants Studied by Genetic and Biochemical Markers, Journal of Plant Growth Regulation, DOI: 10.1007/s00344-017-9668-8. Şen, A., and İ. Öztürk (2018). Drought tolerance level in BC2F2 wheat individuals using stress related biomarkers. Proceeding Book. International Congress on Agricultural and Animal Science. P: 642-645. 7-9 Nov 2018. Alanya Turkey. Wiersinski, N. (1984). Mutation research in triticale. Comparative studies on the susceptibility of wheat, rye and triticale to mutagens. Archiv, Zuchtunge, 14: 181-192. Shewry, P. R. (2009). Wheat. J. Exp. Bot. 60:1537-1553. Sandhu, B. S., and N. S. Mangat (1985) Interrelationships in Some Quantitative Traits in Wheat. Indian Journal of Agricultural Research, 19, 98-102. Perten, H. (1990). Rapid Measurement of Wheat Gluten Quality by the Gluten Index. Cereal Foods World, 35: 401-402. Pena, R. J. (2008). Improving or preserving bread making quality while enhancing grain yield in wheat. International Symposium on Wheat Yield Potential: Challenges to International Wheat Breeding. Mexico, D.F.: CIMMYT. p: 171-174. Sakin, M. A., A. Yildirim and S. Gokmen (2004). The evaluation of agronomic traits of durum wheat (Triticum durum Desf.) mutants. Pak. J. Biol. Sci., 7(4): 571-576. Sakin, M. A., A. Yildirim and S. Gokmen. (2005). Determining some yield and quality characteristics of mutants induced from a durum wheat (Triticum durum Desf.) cultivar. Turk. J. Agric. For., 29: 61-67. Shah, T.M., B. M. Atta, J. I. Mirza, and M. A. Haq (2012). Radio-Sensitivity of various chickpea genotypes in M1 generation II-field studies. Pak. J. Bot., 44(2): 631-634. Thapa, C.B. (2004). Effect of acute exposure of gamma rays on seed germination and seedling growth of Pinus kesiya Gord and P. wallichiana A.B. Jacks. Our Nature, 2: 13-17.

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IRRIGATION SCHEDULING OF COOL AND WARM SEASON TURFGRASSES IRRIGATED WITH SPRINKLER METHOD* A. Halim ORTA, Büşra TÜRK Tekirdağ Namık Kemal University, Agricultural Faculty, Biosystem Engineering Department, Land and Water Source Division, Tekirdağ, Turkey Corresponding author’s e-mail: horta@nku.edu.tr ABSTRACT The aim of this study is to determine the irrigation scheduling of cool-season (CS) and warmseason (WS) turfgrass species under sprinkler irrigation method. Field experiments were conducted in the experimental fields of Silivri municipality in Gümüsyaka village located between boundaries of Tekirdag and İstanbul city-TURKEY (41°03ʹN; 28°00ʹE; 46 m a.s.l.), during the summer of the 2018. In the study, three different irrigation strategies were applied on cool season and warm season turfgrass species. Experimental design was split-plots in randomized blocks design with three replications. In the experimental area, Bermudagrass (Cynodon dactylon, cv tifway) was used as a warm season turf type and a four-part mixture (30% Lolium perenne, 25% Festuca rubra rubra, 35% Festuca arundinacea and 10% Poa pratensis) was used as cool season turf type. Irrigation scheduling was planned according to changes of available soil moisture level in root zone. Soil moisture was monitored via PR2 Probe and HH2 Soil Moisture Meter, which are Time Domain Reflactometry (TDR) based. Soil moisture values of 0-30 cm soil depth were used to determine the amount of irrigation water, and that of 0-60 cm soil depth were used for measuring of actual evapotranspiration. Irrigation water was applied when approximately (I30)30%, (I50)50%, and (I70)70% of readily available water at effective root zone of 30 cm until the soil moisture content reached field capacity. At the end of the study, for cool season turfgrass types; the total amount of irrigation water applied in different irrigation strategies varied between 523.5-275.3 mm, seasonal evapotranspiration values varied between 754.8-521.9 mm, and daily evapotranspiration values varied between 5.2-3.7 mm/day. As for warm season turfgrass types; same values varied between 423.8-186.2 mm; 590.1-521.5 mm; and 4.1-3.6 mm/day, respectively. The results indicated that colour, quality, fresh yield, dry matter yield, irrigation water use efficiency, water use efficiency, vegetation height and mowing were significantly different in terms of irrigation strategies for both species. When factors such as amount of irrigation water applied, cutting frequency and quality under the region's conditions are evaluated together; it is suggested to start irrigation when (CS I50) 50% of the available water in effective root zone is consumed in cool season turfgrass and when (WS I70) 70% is consumed in warm season turfgrass. At this irrigation timing, crop water stress index values before irrigation were determined as 0,52 and 0,65 for CS I50 and WS I70, respectively. When suggested irrigation strategies are compared, it can be said that warm season turfgrass demands 63% less irrigation water and consumes 28% less water than cool season turfgrass. Besides, the most suitable reference evapotranspiration estimation method under the experimental condition is Penman-FAO modification (P-FAO) and Penman-Monteith (P-M) method for cool season and warm-season turfgrass species, respectively, and crop coefficient (kc) curves are prepared for both turfgrass species. Keywords: Landspace irrigation, turfgrass species, CWSI, irrigation method, irrigation timing *The study is summarized from MSc thesis

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INTRODUCTION Irrigation scheduling methods are generally based on measurement of soil water content or meteorological parameters for modelling or computing evapotranspiration. In addition, methods based on crop monitoring have become widely used, including porometers, pressure chambers, etc. In general, these approaches rely on direct contact with the crop; however, methods based on remote sensing, such as infrared thermometers (IRTs), have become the preferred methods for detecting crop water stress. This remote sensing through measurement of a crop's surface temperature can be accomplished with no damage. The correlation between surface temperature and water stress is based on the assumption that, as the crop goes under water stress, the stomata close, transpiration decreases and leaf temperature increases. In practice, leaf/canopy temperature obtained by hand-held IRTs could be used in the Crop Water Stress Index (CWSI) method, defined by Idso et al., 1981. The idea of CWSI is first to determine minimum water stress (lower baseline) under well irrigated conditions, maximum water stress (upper baseline) under rain-fed conditions and then periodically monitor leaf/canopy temperature between the minimum and maximum stress conditions with respect to water deficit. Technically, lower baseline has been based on empirical linear relationships between canopyair temperature difference (Tc-Ta) and vapour pressure deficit (VPD); however, upper baseline depends on the (Tc-Ta). Ta and VPD have been obtained in several ways, including use of a psychrometer to get dry and wet bulb temperatures, or use of other temperature and humidity measuring devices and accompanying software built into an infrared thermometer and data logging system. Many studies have reported on determination of CWSI for different turfgrasses. For example, (Jalali-Farahani, Slack, Kopec, Matthias, & Brown, 1994) reported that crop water stress index (CWSI) values of Bermuda grass should be 0.16 to start irrigation to maintain quality in Tucson, AZ city (USA). Similar CWSI value of Bermuda grass, 0.15, was found by Bijanzadeh et al. 2013 for maintaining quality of turfgrass in Shiraz city. Also, (Emekli et al., 2007) suggested that, for good quality, the CWSI value should be 0.10 to start irrigation for Bermuda grass in Antalya city (TURKEY). The main purpose of this study was to determine irrigation scheduling of warm-season and cool-season turfgrass under sprinkler irrigation method in the western part of Turkey. Specific objectives are to measure actual ETc (crop Evapotranspiration) values for both warm and cool season turfgrass species, to compare cool and warm season turfgrass species in the concept of actual ETc and their response to different irrigation threshold, to determine CWSI values for both turfgrass species under different irrigation scheduling and opportunity of using CWSI in irrigation timing, to focus on crop growing and irrigation management parameters under sprinkler irrigation method. MATERIALS AND METHODS The field experiment was conducted in the Agricultural Production and Research Centre (TURAM) of Silivri municipality, Istanbul,-Turkey (41°03ʹN; 28°00ʹE; 46 m a.s.l.) during the summer period of 2018 (Figure 1). Climate in this region is classified as semi-arid and the averages of annual temperature, relative humidity, wind speed, sunshine duration per day and total annual precipitation are 14.8 °C, 74.0 %, 2.4 m s , 6.5 h and 643.8 mm, respectively. In the experimental area slope was between 2%-7% from east to west and soil type was clay loam, deep profile, no shallow underground water table and the available water holding capacity within 0.60 m of the soil profile is about 80 mm. The electrical conductivity (EC) of irrigation water was 0.56 dS m'1 and the sodium absorption rate was 1.7, which was classified as C2S1 according to U.S Salinity Lab. (US Salinity Lab. Staff, 1954).

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Figure 1. View of the study area The experiment was designed in Split-Plots (SP) in a Randomized Complete Block Design (RCBD) with three replications. Main treatments were two different turfgrass type: CoolSeason (CS) turfgrass (10% Poa pratensis, 25% Festuca rubra var. rubra, 30% Lolium perenne, 35% Festuca arundinacea) and Warm-Season (WS) (Cynodon dactylon L. Pers. Cv tifway) turfgrass. Sub-treatments were three different irrigation strategies as a threshold (I30, I50, and I70). These thresholds corresponds 30%, 50% and 70% of total available soil moisture depletion at the effective root zone of 30 cm and irrigation until the soil moisture content reached field capacity. Each plot was 2.5 m * 2.5 m, totally 6.25 m2 and there was a gap of 2 m within blocks and plots in order to avoid the side effects of seepage. Sprinkler popup heads were located at the corners of each plot. Discharge of each head is 87.25 L.h-1 and irrigation radius is 2.5 m at the operating pressure of 210 kPa (Figure 2). The soil water level was monitored daily via soil moisture profile probe (PR2/6, Delta company, UK) and a soil moisture meter (HH2, Delta company, UK) for 0.60 m depth during the whole growing season (May-August). The amount of soil water in the effective root zone of 0.30 m was used to initiate irrigation. Evapotranspiration for a 10-day period was calculated via TDR data by using water balance method for 0.60 m soil depth (Heerman, 1985). By this this way, possible deep percolation could be monitored at 0.30-0.60 m soil depth for trustable calculation of actual crop evapotranspiration.

Figure 2. Field layout of experimental plots 283

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CWSI was calculated according to (Idso et al. 1981) using canopy temperature (Tc). Canopy temperature measurements were carried out via a hand-held IRT (Model 574 precision, Fluke Corporation, Washington, USA) with a 3° field view and equipped with an 8–14 µm spectral band-pass filter. Air temperature (Ta) and vapour pressure deficit (VPD) were taken daily from an automatic meteorological station. The IRT was operated with emissivity adjustment set at 0.98. The IRT data was collected between the dates of 1st July (DOY 182) and 15th August (DOY 227), four times a day during the solar noon time (11:00 a.m. to 02:00 p.m.) at four directions (North, East, South, and West) under clear sky conditions (Figure 3 & Figure 4). The lower baseline (Non-stress) was determined via Tc data gained from well-irrigated plots (I30) for both species. It is impossible to grow turfgrass under rain-fed condition in our region. however, two small plots (0.50 m * 0.50 m) were established separately on 30th June and never irrigated for both types to determine the upper baseline (fully stressed).

Figure 3. IRTs measurement under a clear sky in a plot of cool-season turfgrass

Figure 4. IRTs measurement under a clear sky in a plot of warm-season turfgrass

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Figure 5. Small plots for determining of non-transpiring baseline The warm-season turfgrass and cool-season turfgrass were mowed leaving nearly 5-6 cm vegetation height on the ground after mowing (Kopec & Umeda, 2015) when they reached approximately 9-11 cm and 14-16 cm height, respectively, (Figure 6). After mowing, fresh yield and dry matter were determined in g m-2 (Brede & Duich, 1984); (Avcıoğlu 1997). Besides some quality parameters such as; vegetation height, quality, covering, colour were observed for each plot and evaluated periodically with a scale of 1 to 9 (1: poor, 6: acceptable, 9: excellent) (Brede & Duich, 1984);(Avcıoğlu, 1997), and water use efficiency (WUE) and irrigation water use efficiency values for each treatment (IWUE) were calculated (Zhang & Owesis, 1999).

Figure 6. Mowing process of plots Daily and seasonally actual evapotranspiration (ETc) were calculated by applying the water balance method and reference evapotranspiration values (ETo) were estimated by 285

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different methods such as; Jensen-Haise (JH), Penman FAO modification (P-FAO), PenmanMonteith FAO modification (PM-FAO), Class A Pan by FAO (A-FAO), and Blaney-Criddle (BC) (Jensen 1973, Doorenbos and Pruitt 1977), during the study for cool-season turfgrass (CS) and warm-season turfgrass (WS). Three assessments were referred to determine the most appropriate ETo method for the region. The first assessment was the sum of squares of the differences (SS) between the measured actual evapotranspiration (ETc) and the reference evapotranspiration (ETo) estimated by different equations. The second evaluation was Seasonal ETc Coverage Rate % (CR ETc ), and third one was correlation values (r) between measured evapotranspiration and calculated reference evapotranspiration (Orta, 1994). Crop Coefficients (kc) were calculated as the ratio of ETo to ETc for the growing period. Data were analysed with software package “Jump 5.0.1”. Data gained from field such as; Vegetation Height, Colour (Cr), Quality (Qy), Coating (Co), Fresh Yield (FY) and Dry Matter Yield (DMY), Irrigation Water Use Efficiency (IWUE) and Water Use Efficiency (WUE) were compared by using “LSD test” (Fisher, 1935). RESULTS AND DISCUSSIONS The total number of irrigations, total amount of irrigation water, rainfall during whole growing periods and seasonal evapotranspiration for each treatment are given in Table 1. The seasonal evapotranspiration in treatment I30 for both turfgrass type were the highest; suggesting that the irrigation water applied was adequate to meet the full crop water requirements. This treatment was used, therefore, to determine the non - stressed CWSI baseline. Other treatments underwent water deficits and gave lower seasonal ET. The lowest ET occurred in treatment I70 because of maximum water deficit in the root zone. In all species, shorter irrigation interval increased evapotranspiration (ET) and fresh yield (Y). Table 1. Total number of irrigations, total amount of irrigation water, rainfall, seasonal evapotranspiration for treatments

Treatment

SubTreatments

Number of irrigation

Irrigation water applied (mm)

I30 I50 I70 I30 I50 I70

36 23 11 31 20 8

523,5 506,0 275,3 423,8 368,2 186,2

Rainfall (mm)

Seasonal evapotranspiration (mm)

290,0

754,8 636,0 521,9 590,1 560,7 521,5

Colour (Cr) and Quality (Qy), Vegetation Height and Mowing (VHM), Fresh Yield (FY) and Dry Matter Yield (DMY), Irrigation Water Use Efficiency (IWUE) and Water Use Efficiency (WUE), and Crop Water Stress Index (CWSI) are given in Table 2. These values given in Table 2 could be used to decide the proper irrigation scheduling for turfgrass. Concerning CS turfgrass, the highest water consumption and the most frequent mowing were obtained by CSI30 treatment. In addition, CSI30 showed the darkest green colour, the densest 286

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texture, higher resistance to broad-leaf weeds, and the highest FY and DMY. However, the IWUE and the WUE were inefficient. Contrarily, the CSI50 treatment needed less irrigation water and consumed less water. So, in landscape areas, although CSI30 treatment provides a good visual quality, it will create high cost in terms of fuel and labour. Whereas CSI50 treatment that have acceptable quality with less cost. Concerning the WS turfgrass, the different irrigation threshold did not affect significantly the quality parameters as in the case of CS turfgrass. The WSI70 treatment which had less number and quantity of irrigation provided good visual appearance, with higher WUE and IWUE. In the case of cool-season turfgrass, the CSI50 treatment was irrigated 23 times, applied 506,0 mm irrigation water, and consumed 636,0 mm (ETc) water during the growing period. However, in the case of warm-season turfgrass, the WSI70 treatment was irrigated 8 times, applied 186,2 mm irrigation water, and consumed 521,5 mm (ETc) water (Table 1).

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Table 2. Some growth and quality Characteristics of plots Morphological and Agronomical Characteristics Colour (Cr)

Sub-Treatments CS

Quality (Qy)

Vegetation (VHM)

height

Fresh Yield (FY)

Dry Matter (DMY)

Yield

Irrigation Water Use Efficiency (IWUE)

Water Use Efficiency (WUE)

LSD,**: P<0.05

P<0.01,

Main Treatments WS Mean

I30 8,033a 5,600c 6,817a** I50 7,633b 5,600c 6,617a I70 5,700c 5,767c 5,733b Mean 7,122a** 5,656b I30 8,567a 8,567a 8,567a** I50 5,567b 8,767a 7,167b I70 5,233c 8,533a 6,883c Mean 6,456b** 8,622a I30 13,60 11,40 12,50a* I50 12,63 10,50 11,67b I70 12,63 10,37 11,50b Mean 12,96a* 10,82b I30 160,133a 157,333a 158,733a** I50 79,767b 150,567a 115,167b I70 64,567b 154,367a 109,467c Mean 101,489b* 154,089a I30 40,500cd 55,700ab 48,100a* I50 29,733de 47,400bc 38,567b I70 24,700e 61,833a 43,267ab Mean 31,644b* 54,978a I30 0,307bc 0,373b 0,340b** I50 0,160d 0,407b 0,283b I70 0,240cd 0,830a 0,535a Mean 0,236b** 0,537a I30 0,210b 0,267a 0,238a* I50 0,123c 0,267a 0,195b I70 0,123c 0,297a 0,210b Mean 0,152b** 0,277a Levels not connected by the same letter are significantly different.

Non-Water-Stress baselines with their equations based on treatments CSI30 and WSI30 and Maximum stress baselines of Cool-season and Warm-season turfgrass based on little plots are given in Figure 7 and Figure 8, respectively. The CWSI values were calculated for the period of 01.07.2018 (DOY: 182)-15.08.2018 (DOY: 227) (during 45 days) for all sub-treatments of both turfgrass type. CWSI values for Cool-Season (CS) turfgrass species ranged from 0.21 to 0.75, and corresponding values for Warm season turfgrass (WS) species were 0.17 and 0.45. During the measurement period, CWSI values showed a variation within 0,17 and 0,75. In other words, WSI30 treatment realized the smallest CWSI value because of no water stress; in spite of that, CSI70 treatment gave the greatest CWSI value because of water deficit throughout the period. The means of CWSI values before irrigation are given in Figure 9. In all treatments, 288

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CWSI values increased with soil moisture deficit and reached peak values just before irrigation. The mean CWSI before irrigation, by other words irrigation threshold was 0.52 for CSI50 and 0.65 for WSI70. 8

Full stressed

Tc-Ta ( C)

Tc-Ta = -2,037VPD + 7,529 R² = 0,73** Syx=0,17

Well watered

0 0,00

1,00

2,00

3,00

VDP (kPa)

Figure 7. The upper non-transpiring baseline and the lower non-water-stressed baseline for CS 9,0

Full stressed

8,0 7,0

Tc-Ta C

6,0 5,0 4,0 3,0

Well watered Tc-Ta = -1,8785VPD + 7,3583 R² = 0,78** Syx=0,21

2,0 1,0 0,0 0,00

1,00

2,00

3,00

VDP kPa

Figure 8. The upper non-transpiring baseline and the lower non-water-stressed baseline for WS

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0,70

0,65

0,60

0,65

0,53

0,52

CWSI

0,50 0,40 CS

0,30

0,26

C2 WS

0,20 0,10 0,00 S0.30 S30

S0.50 S 50

S0.70 S 70

Treatments

Figure 9. Average CWSI values just before irrigation for all treatments Daily and seasonally actual evapotranspiration (ETc) calculated by applying the water balance method and reference evapotranspiration values (ETo) estimated by different methods such as; Jensen-Haise (JH), Penman FAO modification (P-FAO), Penman-Monteith FAO modification (PM-FAO), Class A Pan by FAO (A-FAO), and Blaney-Criddle (BC) (Jensen 1973, Doorenbos and Pruitt 1977), during the study for Cool-season turfgrass (CS) and warmseason turfgrass (WS) are given in Table 3.

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Table 3. Actual evapotranspiration (ETc) and reference evapotranspiration (ETo) values estimated by different methods

Main treatments

Period

Actual evapotranspiration (Etc) (mm.d-1)

Reference evapotranspiration (ETo) estimated by different methods (mm.d-1)

2.5-10.5

4,50

B-C 2,90

11.5-20.5

4,47

4,15

3,89

4,21

2,84

3,00

21.5-31.5

4,30

3,40

4,19

5,12

2,98

3,08

1.5-10.6

5,60

3,20

6,02

5,46

3,09

4,48

11.6-20.6

5,67

5,05

5,58

5,57

3,19

4,25

21.6-30.6

4,93

3,80

3,61

6,79

3,31

2,90

1.7-10.7

3,90

5,10

6,82

5,49

3,44

4,18

11.7-20.7

3,57

5,00

5,93

5,74

3,56

4,00

21.7-31.7

3,53

5,30

3,79

5,67

3,76

4,06

1.8-10.8

5,37

7,10

6,44

6,09

4,07

4,74

11.8-20.7

6,30

6,50

6,80

6,03

4,32

4,62

21.8-31.8

6,13

5,50

6,63

5,67

3,89

4,08

1.9-13.9

3,63

4,35

5,29

4,15

3,41

2,82

14.9-24.9

0,13

4,80

0,00

4,02

2,80

2,90

2.5-10.5

4,53

2,90

0,34

4,02

2,55

1,65

11.5-20.5

4,47

4,15

3,89

4,21

2,84

3,00

21.5-31.5

4,70

3,40

4,19

5,12

2,98

3,08

1.5-10.6

3,93

3,20

6,02

5,46

3,09

4,48

11.6-20.6

4,90

5,05

5,58

5,57

3,19

4,25

21.6-30.6

3,80

3,61

6,79

3,31

2,90

1.7-10.7

2,73

5,10

6,82

5,49

3,44

4,18

11.7-20.7

3,83

5,00

5,93

5,74

3,56

4,00

21.7-31.7

3,07

5,30

3,79

5,67

3,76

4,06

1.8-10.8

3,70

7,10

6,44

6,09

4,07

4,74

11.8-20.7

4,60

6,50

6,80

6,03

4,32

4,62

21.8-31.8

4,43

5,50

6,63

5,67

3,89

4,08

1.9-13.9

4,83

4,35

5,29

4,15

3,41

2,82

14.9-24.9

0,40

4,80

0,00

4,02

2,80

2,90

291

A-FAO 0,34

P-FAO 4,02

P-M 2,55

J-H 1,65

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Table 4. Some statistical parameters to determine suitable estimation method (ETo) for the region. Sum of Squares (SS)

Correlation coefficent, (r)

Seasonal ETc Coverage Rate % (đ??śđ?&#x2018;&#x2026;đ??¸đ?&#x2018;&#x2021;đ?&#x2018;? )

B-C A-FAO P-FAO P-M J-H

3,00 2,73 2,24 2,90 2,48

0,70 0,08 0,88 0,88 0,58

106 105 119 75 81

B-C A-FAO P-FAO P-M J-H

3,78 4,44 3,79 1,63 2,09

0,77 0,23 0,88 0,89 0,68

122 122 137 87 94

MainTreatment

Methods

P-FAO and P-M methods gave the lowest value of Sum of Squares difference (SS) with high correlation coefficient for CS and WS, respectively.(Table 4). So, these estimation methods of reference evapotranspiration can be suggested for turfgrass ETo in experimental region. In addition, kc equations and graphs have been prepared for suggested methods (P-FAO and P-M) and tested statistically by t-test for reliability (Table 5, Figure 10 & Figure 11).

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Table 5. kc crop coefficients and crop coefficient equations for estimated reference crop evapotranspiration Main treatments

Number of days from beginning, T 9 19 30 40 50 60 70 80 91 101 111 122 135 146 Seasonal Mean Equation

Correlation, r 9 19 30 40 50 60 70 80 91 101 111 122 135 146 Seasonal Mean

kc B-C 1,55 1,08 1,26 1,75 1,12 1,30 0,76 0,71 0,67 0,76 0,97 1,12 0,84 0,06

A-FAO 13,39 1,15 1,03 0,93 1,01 1,37 0,57 0,60 0,93 0,83 0,93 0,93 0,69 0,00

P-FAO 1,12 1,06 0,84 1,03 1,02 0,73 0,71 0,62 0,62 0,88 1,04 1,08 0,87 0,07

P-M 1,76 1,57 1,44 1,81 1,78 1,49 1,13 1,00 0,94 1,32 1,46 1,58 1,07 0,10

J-H 2,72 1,49 1,39 1,25 1,33 1,70 0,93 0,89 0,87 1,13 1,36 1,50 1,29 0,09

1,00

1,74

0,84

1,32

1,28

kc = -5E05T2 + 0,0067T + 0,6756 0,41 1,13 1,06 0,92 0,72 0,88 0,56 0,50 0,67 0,54 0,61 0,76 0,78 1,16 0,10

kc = 7E05T2 0,0119T + 1,7407 0,33 1,78 1,57 1,58 1,27 1,54 1,15 0,79 1,08 0,82 0,91 1,06 1,14 1,42 0,14

kc = 0,0002T2 0,0254T + 2,065 0,71 2,74 1,49 1,52 0,88 1,15 1,31 0,65 0,96 0,76 0,78 1,00 1,09 1,71 1,14

kc = 7Ekc = -3E-05T2 05T2 +0,0017T + 0,0126T + 1,0562 1,3493 0,59 0,52 1,56 13,49 1,08 1,15 1,38 1,12 1,23 0,65 0,97 0,88 1,00 1,05 0,54 0,40 0,77 0,65 0,58 0,81 0,52 0,57 0,71 0,68 0,81 0,67 1,11 0,91 0,08 0,00 0,88

1,65

0,74

1,16

Equation

kc = 7E05T2 0,0133T + 1,4511

kc = 5E-05T2 - 0,0099T + 1,2636

kc= 0,0001T2 0,017T + 1,3131

kc = 0,0002T2 0,0366T + 0,5188

kc = 0,0002T2 0,0368T + 2,2722

Correlation coeefficient (r)

0,68

0,52

0,85

0,75

0,72

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1,2

Crop coefficient, kc

1 0,8 0,6 0,4

kc = -5E-05T2 + 0,0067T + 0,6756 RÂ˛ = 0,1696**

0,2 0 0

100

120

140

160

Number of days from the begining, T

Crop coefficient, kc

Figure 10. Crop coefficient curve (kc) of the Penman FAO Modification (P-FAO) Method for Cool-Season turfgrass (CS)

Number of days from the begining, T

Figure 11. Crop coefficient curve (kc) of the Penman - Monteith (P-M) Method for WarmSeason turfgrass (WS) CONCLUSIONS The results suggest that cool season turfgrass needs irrigation nearly every five days when approximately 50% of the total available soil moisture at 30 cm depth is consumed (CSI50) and if monitoring of soil moisture level is not possible. 60% of evaporation from Class A pan can be applied as irrigation water. Warm-season turfgrass needs irrigation nearly every 10 days when approximately 70% of total available soil moisture at 30 cm depth is consumed (WSI70) and if monitoring of soil moisture level is not possible, 50% of evaporation from Class A pan 294

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can be applied as irrigation water. When the suggested scheduling is compared, it can be seen that the seasonal amount of irrigation water applied for warm-season turfgrasses is 63% less than that of cool-season turfgrasses, and similarly 28% less in terms of ETc. At this scheduling, CWSI values of 0,52 and 0,65, could be used as a threshold to start irrigation for CS and WS, respectively. The most suitable reference evapotranspiration (ETo) estimation methods are Penman-FAO modification (P-FAO) and Penman-Monteith (P-M) method for CS and WS species, respectively. In addition, crop coefficient (kc) curve has been prepared for those methodologies. REFERENCES Avcıoğlu, R. (1997). Çim Tekniği - Yeşil Alanların Ekimi Dikimi ve Bakımı. Ege Üniversitesi, Ziraat Fakültesi Tarla Bitkileri Bölümü. Ege Üniversitesi Matbaası, Bornova-İzmir. Brede, A.D., J.M. Duich (1984). Establishment Characteristics of Kentucky BluegrassPerennial Ryegrass Turf Mixtures Affected by Seeding Rate and Ratio. Argonomy Journal, 76: 875-879. Doorenbos, J., W. Pruitt (1977). Guidelines for Predicting Crop Water Requirements. Irrigation and Dranaige, No:24, Food and Agriculture organization of the United Nations, 144pp, Rome. Emekli, Y., R. Bastug, D. Buyuktas, N.Y. Emekli (2007). Evaluation of a crop water stress index for irrigation scheduling of bermudagrass. Agricultural Water Management 90: 205- 212. Fisher, R.A. (1935). The Design of Experiments. Oliver & Boyd, Edinburgh. Heerman, D. F. (1985). ET in irrigation management. In Proceedings of the National Conference on Advances in Evapotranspiration (pp. 323–334). ASAE Publication., Idso, S.B., R.D. Jackson, P.J. Pinter, J.L. Hatfield (1981). Normalizing the stress-g-degreeday parameter for environmental variability. Agric. Meteorol. 24: 45-55. Jalali-Farahani, H. R., D. C. Slack, D. M. Kopec, A. D. Matthias, P. W. Brown (1994). Evaluation of Resistances for Bermudagrass Turf Crop Water Stress Index Models. Agronomy Journal, 86(3), 574. Jensen, M. (1973). Consumptive Use of Water and Irrigation Water Requirements. ASCE, Irrig. Drain. Div. 215pp, New York. Kopec, D. M., K. Umeda (2015). Mowing Turfgrasses in the Desert (No. az1681-2015). Arizona. Retrieved from https://extension.arizona.edu/pubs/mowing-turfgrasses-desert Orta, A.H. (1994). Farklı Sulama Yöntemlerinin Biber (Capsicum annuum L.) Verimine Etkisi. Doktora Tezi, Ankara Üniversitesi Tarımsal Yapılar ve Sulama Anabilim Dalı, Ankara. Zhang, H., T. Owesis (1999). Water - yield relations and optimal irrigation scheduling o wheat in the Mediterranean region. Agric. Water Manag., 38(395–311). U.S. Salinity Laboratory Staff, (1954). Diagnosis and improvement of saline and alkali soils. U.S. dept. Agric., Handbook 60.

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EFFECT OF ALOE VERA AND MAP TREATMENTS ON SENSORY EVALUATION OF JUJUBE FRUITS DURING COLD STORAGE AND SHELF LIFE Ali İSLAM1*, Burhan ÖZTÜRK1 1

Ordu University, Department of Horticulture, Ordu, Turkey *Corresponding author: islamali@hotmail.com

ABSTRACT This study was carried out in Ordu University using jujube (Zizyphus jujube Mill.) fruits which harvested from Jujube (Zizyphus jujube Mill.) trees planted in a producer orchard in Amasya. Effect of Aloe vera and MAP (Modified Atmosphere Packaging) treatments on sensory evaluation of jujube fruits (Ziziphus jujuba M.) during cold storage and shelf life were investigated. Fruits were divided into 4 different groups, while the first group was not treated (control group), the second group was modified atmosphere package (MAP), the third group was Aloe vera gel (30% concentration), and finally, Aloe vera + MAP applications. They were stored at 0 ° C and 90% + 5% humidity for 35 days and measurements were taken per week. Changes on sensory evaluation was evaluated during cold storage and shelf life. Keywords: Sensory evaluation, Modified Atmosphere Packaging, Ziziphus jujube INTRODUCTION The jujube (Ziziphus jujube Mill.) fruit belonging to the Rhamnaceous family grows mostly in Europe, southern and eastern Asia, and Australia, especially the inland region of northern China. Jujube consists of more than 170 species and distributes from the temperate to tropical regions. It is becoming increasingly popular because of its outstanding advantages including early bearing, rich in nutrition, multi-use, long flowering season, as well as high tolerance to drought and barren soils (Liu and Zao, 2009). The most production areas are India and China, Shandong region. The physico-chemical properties and antioxidant capacity of the five main jujube varieties (Junzao, Lingbaozao, Jinzao, Zanhuangzao and Lizao) in China were determined (Goa et al, 2011). It was investigated the effects of preharvest application of putrescine, gibberellic acid, salicylic acid, naphthalene acetic acid, cytofex and calcium chloride on fruit yield, ripening date and quality at harvest and after cold storage at 5°C and 85 to 90% relative humidity. The results was showed the latest harvest was obtained with NAA followed by GA3 and CPPU treatments. The CaCl2 and NAA gave the shortest harvest period. Vitamin C, total soluble solids, acidity, firmness and quality grade increased significantly by all treatments (Al-Obeed, 2012). Although the tree adapts to many climates, it is need hot summers to produce good fruit. It is locally grown in Marmara, Western and Southern Anatolia in Turkey. In addition, different species are observed in the Black Sea region and especially in the Çoruh Valley Basin. Fruits resemble spindle, sweet and juicy, egg-shaped, first olive green, then dark red-black color and single seed. The outer surface is thin and thin, the soft part of the fruit is yellow and sweet taste (Yaltirik, 1997; Young, 2005; Yucel, 2005). 296

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Jujube fruit, which has high nutritional content, is used as dietary product and dried product as well as table consumption. The fresh consumption period is short. therefore, longer storage is required for table consumption. The number of detailed studies on the quality changes of jujube fruit after harvest and during shelf life is very low. The effects of Aloe Vera Gel and MAP (Modified Atmosphere Packaging) applications on post-harvest storage and shelf life were investigated in order to determine sensory characteristics and to preserve the fruit quality after harvesting in jujube. Thus, it is aimed to present products to the table of the consumer for a long time. MATERIAL AND METHOD Material Fruits was collected by hand from Jujube (Zizyphus jujube Mill.) Trees from Amasya province. Pruning and other cultural processes (spraying, irrigation, fertilizing, etc.) were carried out regularly. Method The fruit collected by chance from the jujube fruit trees were harvested and brought to the Pomology Laboratory of the Department of Horticulture, Faculty of Agriculture, Ordu University. Considering the losses that may occur during storage, approximately 20% more fruit was harvested. Fruits were divided into 4 different groups. The first group was the control group, the second group was modified atmosphere package (MAP), the third group was Aloe vera gel (30% concentration), and the fourth group was Aloe vera + MAP. The fruits of the applications (control, MAP, Aloe vera and Aloe vera + MAP) were stored for 5 weeks at 0 ± 0.5 ° C and 90 ± 5% relative humidity. Sensory characteristics were evaluated after 4 days in storage period 0, 7, 14, 21, 28 and 35 days. Fruits taken from the cold storage were kept on the shelf for 4 days which is considered as marketable period. Appearance, sweetness, taste, crunchiness, aromatic and hardness were characterized as sensory characteristics. In the examination of sensory properties, 1-10 point was applied by experts who know jujube fruit (Very poor 1-2 points, poor 3-4, moderate 5-6, good 7-8, very good 9-10). Statistical analysis: The experiment was established in randomized plot design with 3 replications. The data were analyzed with JMP 10.0 statistical package program. Mean values were compared with Tukey at 5% significance level. RESULTS Data on sensory properties of jujube fruits during cold storage are presented in Table 1. On the 35th day of cold storage, MAP applications were optimal in all aspects. Table 1 shows that sensory properties decrease over time for all properties. The highest decrease was in control and A. vera application in terms of vision. Appearance begins to change color after 14 days. Taste and aroma in terms of 28 days is appropriate. Hardness was more acceptable in all applications than control. 7th day, crunchiness and aroma gave the best value in all applications. Sweetness, taste and aroma properties were found acceptable in Aloe vera, Aloe vera + MAP and MAP treated fruits compared to control. After 0, 7, 14, 21, 28, 35 days from the cold storage, fruits were kept on shelf and sensory properties were examined. Jujube fruits were kept on the shelf for 4 days. Sensory properties related to shelf life are presented in Table 2. Considering all the features, all MAP applications worked best on day 35 of the cold storage. 14 4 days appearance Aloe vera gave the best value in MAP and MAP applications. 297

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Table 1. Effect of Aloe vera and MAP treatments on sensory evaluation of jujube fruits (Ziziphus jujuba M.) during cold storage Sensory

Treatments

evaluation

Cold Storage Harvest

7 day

14 day

21 day

28 day

35 day

Control

8.22

8.33 a

7.33 b

5.00 c

4.00

4.00 c

A.vera

8.22

6.67 c

7.33 b

6.00 b

4.00

4.00 c

A.vera+MAP

8.22

7.34 b

6.67 c

7.00 a

4.00

6.00 b

MAP

8.22

7.44 b

8.67 a

6.00 b

4.00

7.00 a

Control

8.56

8.11 a

6.67 b

7.00 b

6.00 c

4.00 b

A.vera

8.56

7.56 b

6.67 b

8.00 a

7.00 b

6.00 a

A.vera+MAP

8.56

7.68 b

6.40 b

8.00 a

6.00 a

MAP

8.56

8.33 a

8.00 a

7.00 b

8.00 a

6.00 a

Control

8.33

8.33 a

7.00 b

6.00 c

4.00 b

A.vera

8.33

7.22 b

7.00 b

8.00 a

7.00 b

6.00 a

A.vera+MAP

8.33

7.36 b

6.33 c

8.00 a

6.00 a

MAP

8.33

8.33 a

7.00 b

8.00 a

6.00 a

Control

8.11

7.36

6.13 b

5.00 c

4.00 b

5.00 c

A.vera

8.11

7.11

6.00 b

7.00 b

4.00 b

6.00 b

A.vera+MAP

8.11

7.33

6.00 b

8.00 a

6.00 a

7.00 a

MAP

8.11

7.33

7.33 a

5.00 c

6.00 a

7.00 a

Control

7.22

7.33 b

7.00 b

4.00 b

A.vera

7.22

7.19

6.30 c

7.00 b

7.00 a

6.00 a

A.vera+MAP

7.22

7.16

6.33 c

8.00 a

7.00 a

6.00 a

MAP

7.22

8.00 a

7.00 a

6.00 a

Control

7.44

6.56 b

7.00 b

5.00 c

4.00 b

5.00 b

A.vera

7.44

7.30 a

6.67 c

7.00 b

4.00 b

7.00 a

A.vera+MAP

7.44

7.56 a

7.00 b

8.00 a

7.00 a

MAP

7.44

6.78 b

7.67 a

5.00 c

7.00 a

Appearance

Sweetness

Taste

Crunchiness

Aromatic

Hardness

n= 9 for the sensory evaluation. Means in columns with the same letter do not differ according to Tukey's test at P<0.05. 298

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Table 2. Effect of Aloe vera and MAP treatments on sensory evaluation of jujube fruits (Ziziphus jujuba M.) during shelf life Sensory

Treatments

evaluation

Shelf life Harvest+4 day

7+4 day

14+4 day

21+4 day

28+4 day

35+4 day

Control

6.93

8.40 a

8.00

6.00 b

4.00 d

2.00 b

A.vera

6.93

8.60 a

8.00

6.00 b

5.00 c

2.00 b

A.vera+MAP

6.93

8.53 a

8.00

8.00 a

7.67 a

5.00 a

MAP

6.93

7.53 b

8.00

8.00 a

6.67 b

6.00 a

Control

7.53

8.27 a

7.00 b

6.17 b

3.00 c

A.vera

7.53

7.20 b

8.00 a

7.00 b

6.00 b

5.00 b

A.vera+MAP

7.53

7.40 b

7.33 b

8.00 a

7.67 a

5.00 b

MAP

7.53

7.93 a

6.67 c

8.00 a

7.67 a

6.00 a

Control

7.60

8.20 a

5.00 c

6.00 b

6.21 b

2.00 c

A.vera

7.60

7.07 c

5.33 c

6.00 b

5.00 b

A.vera+MAP

7.60

7.40 b

7.33 a

8.00 a

7.67 a

5.00 b

MAP

7.60

7.53 b

6.00 b

8.00 a

7.67 a

6.00 a

Control

7.93

8.13 a

6.00 c

5.00 b

6.67 b

3.00 c

A.vera

7.93

7.93 a

8.00 a

5.00 b

6.33 c

5.00 b

A.vera+MAP

7.93

8.07 a

8.00 a

6.00 a

7.00 a

5.00 b

MAP

7.93

7.67 b

7.00 b

6.00 a

6.67 b

6.00 a

Control

6.93

7.60 a

5.00 b

6.00

6.07 b

3.00 b

A.vera

6.93

6.87 b

5.33 b

6.00

6.00 b

3.00 b

A.vera+MAP

6.93

7.13 b

7.00 a

6.00

7.67 a

5.00 a

MAP

6.93

7.73 a

5.33 b

6.00

7.60 a

5.00 a

Control

7.27

7.80 a

6.00 c

6.00 b

6.67 b

3.00 b

A.vera

7.27

7.33 b

8.00 a

6.00 b

6.67 b

3.00 b

A.vera+MAP

7.27

7.80 a

8.00 a

7.00 a

8.00 a

5.00 a

MAP

7.27

7.73 a

7.00 b

7.00 a

7.33 b

6.00 a

Appearance

Sweetness

Taste

Crunchiness

Aromatic

Hardness

n= 9 for the sensory evaluation. Means in columns with the same letter do not differ according to Tukey's test at P<0.05.

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CONCLUSION According to the findings, it can be stated that the jujube fruit can be kept in the cold for 35 days. It was observed that all applications except control had positive effects on sensory characteristics. Jujube fruits can be stored in MAP for 35 days in terms of sensory properties. In addition, MAP and Aloa vera + MAP were generally better than other application for shelf life. Jujube fruits had a short harvest period. The storage and marketing period should be extended. Thus, manufacturers and consumers will have longer period for table jujube fruit. Acknowledgements: The authors would like to gratefully acknowledge the kind support of ODU BAP TF-1530.

REFERENCES Liu M.J., Zhao Z.H. 2009. Germplasm Resources and Production of Jujube in China. 1st Int’l Jujube Symp. Acta Hort. 840, 23-29 Al-Obeed, R. S. 2012. Jujube post-harvest fruit quality and storagability in response to agrochemicals preharvest application African Journal of Agricultural Research7(36), 50995107 DOI: 10.5897/AJAR12.151 Gao, QH., Wu, PT., Liu, JR., Wu, CS., Parry, JW., Wang, M. 2011. Physico-chemical properties and antioxidant capacity of different jujube (Ziziphus jujuba Mill.) cultivars grown in loess plateau of China. Scientia Horticulturae 130(1): 67-72 Yaltırık, F., Efe, A., & Uzun, A. (1997). Tarih Boyunca İstanbul’un Park Bahçe ve Koruları Egzotik Ağaç ve Çalıları. İsfalt Yayını, 4

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IN-SILICO SHOTGUN METAGENOMICS DATA COMPARISON EXPANDS THE NUMBER OF POSSIBLE CANDIDATES TO BE USED FOR BIOCONTROL OF FIRE BLIGHT DISEASE Ömür BAYSAL*1, Kubilay Kurtuluş BAŞTAŞ*2 1

Muğla Sıtkı Koçman University, Faculty of Science Dept. of Molecular Biology and Genetics, Muğla, Turkey 2

Selcuk University Faculty of Agriculture Dept. of Plant Protection, Konya, Turkey

*Corresponding Authors e-mails: omurbaysal@mu.edu.tr; kbastas@selcuk.edu.tr

ABSTRACT Shotgun metagenomics is an advanced tool that makes it possible to study onunculturable microorganisms of which analyses are difficult or impossible. Unlike capillary sequencing or PCR-based approaches, next-generation sequencing (NGS) allows us to sequence hundreds of microorganisms in parallel. In view of comparing bio-control properties of different microorganism having capacity, metagenomics data that suggest a guide role for very quickly choosing of strains belongs to same species to control of some major disease in fields. In-silico analysis on different strains of same genus bacteria, which have antagonistic effect on Erwiniaamylovora, we have provided great easiness on scanning of possible candidates to thepathogen causing fire blight disease. In this concept, a designed genome viewer program using artificial intelligence approach has been designed considering sequence-based differences considering genes playing role in bio-control. Major gene sequences of Pseudomonas fluorescens and Pantoeaagglomerans strains were compared with each other and similar gene patterns were also found in different bacterial strains some of which are unculturable genus strains. These findings indicate sequence data of hundreds microorganisms showing parallel property on specific genes –same mutational ratio, sequence encoding proteins with high similarity -can be used effectively to find new bio-control agents and these gene sequences can be converted to cassette form by cloning technology to increase performance of present biocontrol candidates. Keywords: Shot gun sequencing, Metagenomics, in-silico analysis, Erwiniaamylovora, Protein database, Bioinformatics, Biocontrol

INTRODUCTION Metagenomic sequence can be taxonomically classified to identify the microbes, or functionally classified to identify the functional potential of the community. There exist two general approaches for characterizing the taxonomic content of environmental samples.Firstly sequencing of PCR amplicons corresponding to phylogenetic marker genes and second shotgun sequencing whereby all genomic DNA in the community is sequenced. Furthermore, a notable subset of taxa cannot be captured by traditional 16S sequencing owing to divergent 16S rRNA gene sequences (Brown et al., 2015). Fire blight caused by Erwiniaamylovora, is the most serious bacterial disease of apple, pear, hawthorn, cotoneaster and other plant species in the family Rosaceae (Vanneste, 2000). The pathogen is included among quarantine organisms in many countries around the world and very strict quarantine measures are enforced (Smith et al., 1997). The development of E. 301

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amylovoraresistance to the antibiotic streptomycin, the greater desire of the society on safe production in agriculture and the prohibition of antibiotics for use in fruit growing in most countries of the European community and other states. Thus, several of these aspects have promoted the need for alternative control strategies (Zeller and Laux 2001, 2002). In the following, the main activities within biocontrol of fire blight on the basis of microbial antagonists, natural compounds and resistance elicitors will be presented. Microbial diversity is a key factor in avoiding pathogen outbreaks (van Elsas et al., 2012). Therefore, biocontrol of plant pathogens is a promising solution to control plant pathogens (Berg et al., 2013) because it was also shown that it enhances general microbial diversity (Erlacher et al., 2015). However, the ecology of naturally occurring antagonistic microorganisms is only partly understood and not yet exploited. Comparison of antagonistic microorganisms in view of efficiency should be detected and determined and the best ones promising as biological agents can be considered for further experiments to be carried out in in vitro and vivo conditions. Efficient property of any microorganism can be followed depending on their lytic enzymes able to degrade of cell wall. In this study, we have designed a new strategy using bioinformatics tools to select effective antagonistic microorganism considering their genetic property and genes encoding cell wall degrading enzymes. By this way, in silico analysiswe can predict their capacity with high precisely ratio of possible new candidates to control of fire blight disease in practice. MATERIAL AND METHODS We constructed our dataset to contain taxa with a variety of levels of divergence from one another, including closely related species. The latter helps evaluate the ability of methods to handle taxa prediction when closely related taxa are present. Because there is such a large difference in microbial communities in terms of number of organisms, which organisms are present, their taxonomic novelty, and diversity in terms of abundance distribution, it is not possible to simulate communities that will be appropriate for all environmental communities. Additionally, all metagenome-based analyzes were carried out on the assembled dataset using NCBI databases. The effector proteins of each antagonistic bacteria and genes encoding enzymes able to degrade of cell wall enzymes has been tagged using UniProt and classified then separated according to functions and domains. Tagged proteins in UniProt were aligned using Clustal Omega, T-COFFEE and MEGA 7. Then phylogenetic trees were constructed by obtained results. In these classifications, DNA binding domains, membrane proteins, chaperons and enzymatic pathways of effector proteins were aligned and clustered to compare their similarities according aminoacid sequences. We predicted functions using SWISS-MODEL of effector proteins and assessed depending on coverage index and correlated with protein network through STRING. RESULTS AND DISCUSSION Results showed similarity within amino acid sequence of effector proteins and genes encoding enzymes of bacteria. These similarities are given in Figure 1 and 2, and Table 1. Even we have no detailed information on functional properties of proteins; we were able to do prediction on their role depending on their amino acid sequence similarity using in-silico. Correlated proteins were by 9% DNA binding domain, 5% enzymatic pathways, and 3% membrane proteins. 7% of proteins were related to leucine rich repetitive proteins playing role in nuclear localization it could not be related to any functional proteins. Further studies will be carried out to clarify these remained questions on mutated bacterial cells using gene cloning.

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Expression level of common genes in antagonistic bacteria DNA binding proteins Uncharacterized proteins CTP sythesis Chemotaxis

Figure 1. Expression level of common genes in antagonistic bacteria

Figure 2. Alignment of genesencodinglyticenzymes Table 1. Genetic similarity of sequences encoding genes conferring enzymatic property of antagonistic microorganisms Pseudomonas flouresans

0,02 80 0,03 Pseudomonas spp. 42 Bacillus subtilis var subtilis 0,07 10 Bacillus subtilis var. 0,11 72 amyqualifaciensis 0,11 Streptomyces spp. 64 Pseudomonas aerufaciensis 0,11 88 0,12 Streptomyces griseoviridis 03 0,12 Pantoneaagglomerans 89

0,02 44 0,06 86 0,11 89 0,11 68 0,11 93 0,11 70 0,13 34

0,03 55 0,09 62 0,09 57 0,09 72 0,09 53 0,10 52

0,08 63 0,08 70 0,08 59 0,08 70 0,09 45

0,00 41 0,00 45 0,00 70 0,04 80

0,00 22 0,00 50 0,05 01

0,00 67 0,05 47

0,04 92

All of the methods analyzed performed very well in terms of sensitivity and precision when the query sequences were in the reference databases. Microbial communities in certain environments are very complex andthese environments, which are very diverse and contain a large number of organisms, would have a large proportion of the microbes found at less than 1 303

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% of the total abundance of the community, and thus a 1% filtering threshold would filter out many of the microbes actually in the metagenome. There has been a real need for a comprehensive evaluation of metagenomics classification methods, due to the notable number of new methods being released. As new methods are developed, further evaluations will need to be performed, including with a reference dataset like MetaSimHC, and/or the CAMI approach. Robust metagenomic data analysis is absolutely critical at this stage of the development of microbiome research as a key research area. CONCLUSION With the fast improvement of NGS sequencing, the gigantic measures of bacterial DNA information may be useful for selection of exact antagonistic bacteria of bioinformatic database. Specifically, the exponential development pace of natural information has presented an issue of capacity and the executives for compelling examination and information sharing on biocontrol. Our data showed that in silico analysis will provide big opportunity to predict and select best antagonistic candidate to control of bacterial disease caused by E. amylovora. REFERENCES Berg G., Zachow C., Müller H., Phillips J., Tilcher R. (2013). Next-generation bio-products sowing the seeds of success for sustainable agriculture. Agronomy 3 648–656. Brown CT, Hug LA, Thomas BC, Sharon I, Castelle CJ, Singh A, et al. (2015). Unusual biology across a group comprising more than 15 % of domain Bacteria.Nature.523(7559):208– 11. advance online publication. Erlacher A., Cardinale M., Grube M., Berg G. (2015). Biotic stress shifted structure and abundance of enterobacteriaceae in the lettuce microbiome. PLoS ONE 10:e0118068 Smith J.M.,McNamara D.G., Scott P.R., Holderness M., 1997. QuarantinePestsfor Europe. 2nd Ed. EPPO/CABI, Wallingford, UK. Van Elsas J. D., Chiurazzi M., Mallon C. A., Elhottova D., Kristufek V., Salles J. F. (2012). Microbial diversity determines the invasion of soil by a bacterial pathogen. Proc. Natl. Acad. Sci. U.S.A. 109 1159–1164. Vanneste J.L.,McLaren G.F., Yu J., Cornish D.A., Boyd R.(2005). Copperandstreptomycinresistance in bacterialstrainsisolatedfromstonefruitorchards in New Zealand. New ZealandPlantProtection58: 101-105. Zeller, W.,Laux, P.(2001).Newestresults on thebiocontrol of fire blight in Germany. Actahorticulturae 590(590):243-246. Zeller, W.,Laux, P.(2002).PerspektivenzurBekämpfungdesFeuerbrandes mit biologischenundanderenalternativenPräparaten. GesundePflanz. 54: 86–91.

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ORGANIC DAIRY FARMING AND ANIMAL HOUSINGS Sedat KARAMAN*1, Serkan YAZAREL1 1

Gaziosmanpasa University, Department of Biosystem Engineering, 60250, Tokat, Turkey *Corresponding author’s e-mail: sedat.karaman@gop.edu.tr

ABSTRACT Parallel to rapidly growing population worldwide, demand for plant and animal products has also increased. As a result of these pressures, conventional agricultural techniques using various synthetic and chemical materials were used to obtain more products from the unit area and animal. The negative effects of conventional agriculture on human and animal health are emerging day by day. In intensive livestock operations, animals kept in a closed confinement cannot continuously exhibit their natural behaviors enough and animal diseases such as mastitis, lameness, acidosis and ketosis occur in animals. In intensive farming, animals are constantly encouraged to eat more for more products. In order to meet this need of agricultural enterprise, inputs such as fertilizer and chemicals are used intensively in conventional plant production. These inputs cause soil deterioration, leach into groundwater through irrigation and pollute water resources and generate serious damages on environment and human health. In intensive production systems, antibiotics and hormones are also used in order to accelerate the growth and get more efficiency. As a result of consuming these products, various diseases such as immune system diseases, asthma, allergy and anemia occur in humans. Today, due to these problems, the importance of organic livestock operations is increasing day by day. Therefore in this study, conditions of organic dairy farming and design principles of animal housings were explained. Keywords: Organic Farming, Animal barn

INTRODUCTION Organic farming: It is a transparent process with its own rules, whereby the use of chemicals such as pharmaceuticals, fertilizers, growth regulators is limited as much as possible, consumer health is prioritized, soil fertility is regulated by the food chain between soil-plant-animal (Demiryürek, 2011). In recent years, the search for healthy products by the conscious consumers has accelerated, and the tendency towards the healthier organic agricultural products has increased and this increase has led to the development of the organic product market at a faster rate than the other agricultural sectors. The amount of land used for organic farming in Turkey is increasing every year and some non-governmental organizations are established to support organic farming (Yüceboy, B., 2018). Organic livestock operation: According to the regulation published in the Official Gazette of 18 August 2010; Production of animals using breeding animals or semen, production of animal products, animal food and animal nutrition products from animal products, supplying organic raw materials to the industries and scientific studies receiving the raw material from agriculture, each stage includes production activity which is controlled and certified by the authorized organization according to this Regulation. 305

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According to this regulation, production rules in organic livestock (Anonymous, 2010) In breeding or production of organic livestock, select breeds with high adaptability to environmental conditions and resistant to diseases, giving priority to native breeds and hybrids adapted to the region to be cultivated, - Use of animals that are brought from organic enterprises and fed completely with organic feeds, whose genetic structure is not changed, resistant to environment, climate conditions and diseases, as breeds, - Natural insemination is essential in organic animal breeding. Artificial insemination can be done with semen obtained from the breeding animals by using natural methods, stored and used. No embryo transfer, - Animals should be able to access pastures or open-air navigation areas or open spaces. The number of animals per unit area in pastures and open areas is not limited enough to provide sufficient animal manure for plant production in the production unit, - Breeding all animals in the same production unit according to the provisions of this regulation, However, conventionally reared animals, shelters and lands where they are reared can be in the same facility provided that they are clearly separate from organically reared units and that there are separate species, - The lands used as pastures must have completed the transition process, must be approved by a control and certification body or control body, and the animals must have undergone extensive production, - If the conditions are treated in accordance with the regulations for three years, the animals are not in the pasture with the same breed animals at the same time as the conventional breeding animals, organic breeding animals will be grazed in the common areas with conventional breeding animals in case the conditions of organic animal products are distinguished from conventional products, - Animals without health problems are selected as breeds and in the case of milk production, transition to organic agriculture may be provided after the 6th month, - Regular records of animals' entry-exit and treatment practices should be kept. Barns in Organic Dairy Operations Animal barns generally aim to ensure that the shelter is located within the comfort zone, which is the most comfortable environment for animals, aiming to improve the welfare and health of animals by protecting them from the adverse weather conditions. Dairy barns are generally planned in three different ways as tie-stall, free-stall and free systems. Tie-stalls are undesirable because the animals are tied to restrict the natural behavior of the animals. Therefore, barns of organic dairy operations should be planned as either free-stall or free open systems. Organic dairy barns should be planned in such a way as to provide sufficient natural light and fresh air needed by the animals, while also protecting them from the adverse outdoor conditions. As in all shelters, climate and topography of the region, stalls, stall dimensions, bedding, animal compartments and areas where animals can easily walk and perform their natural behavior should be taken into consideration. The condition of feeders and waterers, quantity

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and quality of ventilation and suitability of shelter floor are important. Each of these conditions affects performance in organic dairy operations (Curtis, 1987). Shelter Areas; The floors are of great importance in organic dairy cattle breeding. Stress conditions cause hormonal changes in animals, directly affect the immune system of the animals and will cause the animals to become more easily ill. Therefore, animals need adequate shelter and floor spaces. In dairy barns, the net area to be allocated to each cattle should be 6 m 2 and the free circulation area outside the pasture should be 4.5 m2. Bedding; As in all shelter systems, the barn floor is of great importance in organic dairy cattle shelters. In a study conducted by Han et al. (2017), animal foot diseases and nail deformation rates were determined according to the type of shelter floor. It was indicated that floors played a role in nail and foot diseases in animals due to lack of hygiene in the ground conditions, inadequate urinary canals and bedding material. Stalls; GĂśncĂź et al. (2016) reported that there were significant yield losses due to an increase in foot and breast diseases in standing dairy cattle instead of lying in stalls due to adverse stall conditions. They stated that ideal herd management could not be ensured due to the stall sizes that were not suitable for the breed and the untrained employees since the animals could not exhibit their natural behaviors. Munksgaard and Lovendahl (1993) reported that the amount of growth hormone in their blood was lower in cows that were prevented from reaching out at certain times of the day compared to unrestricted cows. It is also important to note that more blood passes through the udders and tends to ruminate in the stretching cows. Feeders and Waterers; Different reactions and health problems can be seen in feed consumption behaviors. First of all, enough feeder space must be set for each animal in a shelter. Rather than the amount of space left here, it is also important to make available space calculations. Designing feeders so that the cattle can take the feed as in the grassland is very important in feed consumption. The materials used in shelters such as feeders and waterers should be selected according to the animal race, size and age, and should be placed in accordance with the nature of the animal. The size and number of these materials should be adjusted according to the herd size, and the animals should be placed in such a way that they can easily reach all the food and water what they need. Milking Parlors; One of the biggest problems in organic dairy farming is mastitis disease and it is one of the biggest problems because of the limited use of chemicals such as antibiotics in these farming type. Therefore, hygiene is one of the most important issues in milking units. Disease spread should be prevented by identifying sick animals early, by alternative treatment methods or by separating them from the herd. The equipment in milking units should not be cleaned with chemicals and contamination of milk should be prevented (Bal, 2011). Stress of animals in the milking unit can lead to hormonal changes, which may adversely affect the quality and yield of milk. Therefore, a milking unit should be planned in such a way that animals can behave in a comfortable manner and milking parlor workers should be competent in animal behavior.

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CONCLUSION It was concluded that organic dairy barns and shelter should be so planned to provide suitable conditions for these operations; animal welfare conditions are of great importance. The conditions of the shelter and the tools, equipment and elements to be used should be planned in such a way that they allow the natural behavior of the animals and prevent the animals from getting stressed. Hormonal activity of animals is negatively affected by the stress in low welfare environment and this will lead to decrease in organic milk yield and quality where quality is of great importance. Due to the limited use of chemicals in organic dairy farming, it is necessary to carry out disease follow-up procedures frequently and timely, to take preventive measures and to select the barns and shelters in accordance with these measures.

REFERENCES Anonymous (2010). Regulation On The Principles And Applicatıon of Organic Agriculture, Resmî Gazete, Number : 27676, 18 Augst 2010. Bal, Y (2011). The Approaches to The Mastitis Problem in Organic Dairy Farm, Journal of the Turkish Veterinary Medical Society, Number: 82(2): 7-14. Curtis, S.E (1987). Animal well-being and Animal care. The veterinary clinics of north America, Food Animal Practice, July, 1987, Volume 3 Number 2. Demiryürek, K (2011). The Concept of Organic Agriculture and Current Status of in the World and Turkey, Journal of Agricultural Faculty of Gaziosmanpasa University (JAFAG), 2011, 28(1), 27-36. Göncü, S., N. Koluman, U. Serbester, M. Görgülü (2016). Animal Welfare Issues and Critical Control Points in Dairy Cattle Farming, Çukurova Journal of Agriculture and Food Sciences, Number 31: 09-20. Han M .C., M. Sağlıyan, E. Polat (2017). Investigation of Cattle in the Diseases of Stable Ground Foot Type and Nail on the Effects of Deformation, Harran University Journal of the Faculty of Veterinary Medicine, Number: 6 (1,) 19-24. Munksgaard L., P. Lovendahl (1993). Effects of social and physical stressors on growth hormone levels in dairy cows. Can J Anim Sci. 1993;73: 847-853. Yüceboy, B (2018). The Position of Turkish Organic Products in The World Market (Postgraduate Thesis), İstanbul Ticaret Üniversitesi, Dış Ticaret Enstitüsü Uluslararası Ticaret Ana Bilim Dalı, İstanbul.

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EFFECTS OF DIFFERENT APPLICATIONS AND TEMPERATURE LEVELS TO CUTTINGS ROOTING IN KIWIFRUIT (ACTINIDIA DELICIOSA A. CHEV.) Ali İSLAM*1, Cemal KARABULUT1, Selim KARAGÖL1 1

Department of Horticulture Faculty of Agriculture Ordu University 52100 Ordu, Turkey *Corresponding author: islamali@hotmail.com

ABSTRACT This study was conducted in 2016. Hardwood cuttings of Hayward varietes were used. The cuttings taken from the plants was belong to 16 years-old Hayward kiwifruit plant in Altınordu province of Ordu. They were taken from 28 February to 1 March 2016. 0, 2000, 4000, 6000 ppm IBA, 50, 100, 200 ppm SA and 4000+50 ppm (IBA+SA), 4000+100 ppm (IBA+SA), 4000+200 ppm (IBA+SA) doses were applied the cuttings before planting. That cuttings were planted in the rooting media at 21 ºC and 26 ºC degrees at March 15. IBA+ SA and SA applied cuttings were planted at March 16 after waiting 24 hours in SA doses. Rooting cuttings were removed from rooting media after 90 days. Live cutting ratio, callus ratio, number of roots, root length and root quality were investigated. As a result, rooting ratio, number of roots and root quality were found better in 2000 ppm IBA doses. SA and combination applications were not good enough like IBA applications. Keywords: Kiwifruit, Hardwood cutting, Rooting, IBA, Salicylic Acid

INTRODUCTION Commercial cultivation of kiwifruit and scientific studies were insufficient until the early 1900s. However, paleobiology studies have shown that kiwi is at least 20 million years old (Lee, 1990). Kiwi cultivars grown in commercial gardens outside of China come from two females and one male plants produced from seeds taken to New Zealand in 1904 and the origin of these seeds is unknown (Ferguson and Bollard, 1990). The first cultivation known in kiwifruit began with selection in 1910. Today, the most cultivated Hayward cultivar in the world among the Actinidia deliciosa species was obtained by selection. According to the data of 2017, the world kiwi production is 4038 thousand tons and the highest production is made in China with 2024 thousand tons. Italy, New Zealand and Chile are the leading countries in kiwi production. Turkey wants to enter the major producing countries with between 56 364 tonnes in 2744 hectares in last 30 years (FAO, 2017). In kiwi production, the certified plants must be obtained and presented to the producer. Kiwi can be grown with generative and vegetative methods. However, vegetative (tissue culture, grafting and cuttings) methods are preferred for propagation. İn Kiwi wood cutting method gives easier and more successful results than other propagation methods (Lawes and Sim, 1980). One of the factors influencing the rooting of cuttings is the time to take the cuttings. Wood cuttings are selected from well-wooded, moderately strong, disease-free year-old branches starting from leaf casting until spring (January-March). Various applications are made to increase the success rate in cutting reproduction. Growth regulators are the most important of these practices. The most widely used growth regulating agent in rooting is IBA from auxin group. The aim here is to accelerate the root formation of cuttings and to increase the number and quality of roots per plant (Gerçekcioğlu,

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2009). In order to obtain a successful rooting, growth regulators, temperature of the rooting medium, light conditions and water relations are also effective (Yılmaz, 1992). Salicylic acid activates root and shoot growth in plants. Light, sweating, ion uptake and transfer, the level of chlorophyll and carotenoid pigments and the increase in the activities of some important enzymes, causes significant changes in leaf anatomy and chloroplast structure (Gallal, 2005). Many studies have been conducted with IBA and similar auxin group hormones on rooting of kiwi cuttings (Connor, 1982; Sivritepe and Eriş, 2000; Ercişli et al., 2002; Zenginbal et al. 2006; Zenginbal and Özcan, 2014) but no studies on salicylic acid have been found. They have been conducted many studies with IBA hormones on rooting of kiwi cuttings. The aim of this study was to investigate the effect of IBA and Salicilic acid applications on the rooting of kiwi wood cuttings at different doses and at different temperatures. MATERIAL AND METHODS The study was carried out in 2016, in a high plastic tunnel with under misted and thermostatcontrolled pans in perlite media. Wood cuttings of Hayward kiwi cultivar were used in the study. And Cuttings were taken from 15 years old orchards. Cuttings were wrapped in wet cloths and kept in the cooling room up to March 15. the cuttings were disinfected with fungicides and treated with different IBA and SA treatments. Treatments were 0, 2000, 4000, 6000 ppm of IBA; 0, 50, 100, 200 ppm of SA; 4000 + 50, 4000 + 100 ppm and 4000 + 200 ppm of IBA + SA. Two different temperatures, 21 ℃ ± 0.5 ℃ and 26 ℃ ± 0.5, were used termostad-controlled heating. Perlite (Morini and Isoleri, 1986) was used as the rooting medium. The rooting was done in high plastic tunnel, under heated and temperature-controlled rooting pans. In the greenhouse, the relative humidity was 70-90%. Rooted cuttings were removed from the rooting media on June 16 Observations and measurements; live cutting ratio, callus ratio, rooting rate, number of roots, root length, root quality, shoot length, number of leaves and dry root weight. The experiment was established in a randomized plot design with 3 replications and 30 cutting. JUMP 10.0 software was used for statistical analysis. RESULTS AND DISCUSSION Live Cutting Ratio The average rooting rate for all applications was 99%. 4000 + 100 ppm (IBA + SA) and 200 ppm SA remained 100% alive at both temperatures. It was 98.33% live cutting obtained in the control. The lowest live cutting ratio was found to be 96.67% at 4000 + 50 ppm (IBA + SA) and 50 ppm SA at 26 ℃ rooting medium. Zenginbal, (2002) obtained the highest live cutting ratio from Hayward cuttings (97%) obtained 4000 ppm BA on September 1. Zenginbal and Ozcan, (2002); obtained the highest viability rate from Hayward wood cuttings on February 1, 4000 ppm IBA application (79.8%) and they said for better viability and rooting rates most appropriate time to take the cuttings from plant is from January 1 to February 1. Zenginbal et al., (2005); Özcan, (1993) and Ercişli et al. (2002) stated that IBA applications increased live cutting rates in wood cuttings.

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Table 1. Live Cutting Ratio Temperature Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C

26 0C

Average ( % )

98.33 98.33 100.00 100.00 98.33 100.00 98.33 100.00 100.00 100.00 99.33

98.33 100.00 98.33 98.33 96.67 100.00 100.00 96.67 98.33 100.00 98.67

Mean 98.33 99.17 99.17 99.17 97.50 100.00 99.17 98.33 99.17 100.00 99.00

Callus Rate There was no statistical difference in callus rate in kiwi cuttings for all applications. The average callus rate was 98.75%. Callus formation is not mandatory for rooting but can be encouraging. It also creates scar tissue and then tissue differentiation. Table 2. Callus Rate Temperature 21 0C Applications Average ( % ) Control 96.67 IBA 2000 ppm 98.33 IBA 4000 ppm 98.33 IBA 6000 ppm 100.00 4000+50 ppm (IBA+SA) 98.33 4000+100 ppm (IBA+SA) 100.00 4000+200 ppm (IBA+SA) 98.33 50 ppm SA 100.00 100 ppm SA 100.00 200 ppm SA 100.00 99.00 Average

26 0C Average ( % ) 98.33 98.33 98.33 98.33 96.67 100.00 100.00 96.67 98.33 100.00 98.50

Mean 97.50 98.33 98.33 99.17 97.50 100.00 99.17 98.33 99.17 100.00 98.75

Rooting Rate A significant difference has emerged between the applications in kiwi cuttings. The highest rooting rate was 95.0% at IBA 2000 ppm. On the other hand, 89.67% rooting was obtained at 210C. In the studies (Lawes and Sim, 1980; Costa and Baraldi, 1984; Morini and İsoleri, 1986; Covatta and Borscak, 1991; Özcan, 1993; Ercişli et al., 2002; Üçler et al., 2003; Zenginbal et al., 2005) indicate that the best results in rooting kiwi cuttings are obtained from 4000 and 6000 ppm IBA applications. In this study, the results are in parallel with similar studies. 311

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Table 3. Rooting Rate Temperature Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average ( % ) 90.00 95.00 93.33 95.00 86.67 90.00 86.67 88.33 88.33 83.33 89.67

26 0C Average ( % ) 78.33 95.00 93.33 91.67 90.00 88.33 93.33 83.33 71.67 83.33 86.83

Mean 84.16 bc 95.00 a 93.33 a 93.33 a 88.33 ab 89.16 ab 90.00 ab 85.83 bc 80.00 c 83.33 bc 88.25

Number of roots There are statistically significant differences between different temperatures and applications in term of number of roots. the number of roots was 4.98 in 21℃, whereas this data was 5.29, in 26 ℃. These values were found to be highest with 7.17 root counts at 4000 ppm IBA dose and the lowest root count was 3.72 with 100 ppm SA dose at 21 ℃ rooting medium. Zenginbal et al., (2002-2003); found 4000 (5.26) and 6000 (4.22) ppm IBA application in Hayward and Matua varieties. Various researchers (Anvari et al., 1991; Özcan, 1993; Mattiuz and Fachinello, 1996; Sivritepe and Eris, (2000) state that the most suitable cutting is picked up January 1. Rathore, 1984; Özcan, 1993 reported that 4000-6000 ppm IBA increases the number of roots. The results are in parallel with the literature studies. Table 4. Number of Roots Temperature Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm(IBA+SA) 4000+200 ppm(IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average (quantity)

26 0C Average (quantity)

6.01 a-d 6.09 abc 5.72 bcd 5.12 c-g 5.83 bcd 4.81 d-h 4.90 c-h 3.81 h 3.72 h 3.83 h 4.98

4.03 fgh 6.48 ab 7.17 a 5.22 c-f 5.39 bcd 5.95 a-d 5.12 c-g 5.15 c-g 3.97 gh 4.40 e-h 5.29

312

Mean 5.02 bc 6.28 a 6.45 a 5.17 bc 5.61 ab 5.38 b 5.01 bc 4.48 cd 3.85 d 4.11 d 5.14

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Root Lengths The average root length was 61.31 mm. Root lengths were showed differentation between applications. The highest root length was obtained with 96.39 mm at 4000 ppm IBA in 26 ℃ rooting media. The lowest root lengths were obtained from SA applications. Zenginbal et al., (2002 - 2003); Sivritepe and Eris, (2000), Ercişli et al., (2002) reported similarity with the findings. Table 5. Root lengths Temperature

21 0C

26 0C

Applications

Average ( mm )

50.73 70.06 77.86 70.96 62.21 61.01 43.56 34.93 36.24 38.61 54.62 b

59.42 82.78 96.39 72.50 86.95 79.30 65.49 50.93 41.05 42.81 67.76 a

Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

Mean 55.08 c 76.42 b 87.13 a 71.74 b 74.58 b 70.15 b 54.53 c 42.93 d 38.64 d 40.72 d 61.31

Shoot Lengths It was obtained Significant differences between all application and temperature in shoot lengths. The average length of shoots was 52.79 mm in different applications in 21 ℃ rooting media and 52.43 mm in 26 ℃ rooting media. Overall average shoot length was determined as 52.61 mm. Table 6. Shoot lengths Temperature Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average (mm) 44.45 58.31 67.87 64.62 46.48 44.25 45.16 61.81 50.03 44.92 52.79

gh b-e a ab gh gh gh abc e-h gh

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26 0C Average (mm) 45.59 60.12 55.79 60.81 59.75 51.99 53.22 46.71 42.51 47.80 52.43

Mean gh a-d b-f a-d a-d d-g c-g fgh h fgh

45.02 59.22 61.83 62.71 53.12 48.12 49.19 54.26 46.27 46.36 52.61

d ab a a bc cd cd bc d d

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Number of Leaves The highest leaf number was obtained with 8.53 at 2000 ppm IBA. The lowest leaf number with 6.02 at 50 ppm SA in the 26 ℃ rooting media. In 21 ℃ rooting medium, 8.17 leaves were counted at 4000 ppm IBA dose and the lowest number of leaves was obtained at 6.29 with 100 ppm SA. The average number of leaves is 7.22 for all applications. Table 7. Number of Leaves Temperature Application Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average (adet) 7.47 7.83 8.17 7.95 7.10 6.95 7.08 7.23 6.29 7.72 7.38

b-e a-d ab abc c-h d-j c-ı b-h hj a-e a

26 0C Average (adet) 6.12 8.53 7.92 7.32 7.72 7.38 6.38 6.02 6.43 6.80 7.06

Mean j a a-d b-g a-e b-f g-j j f-j e-j b

6.79 8.18 8.04 7.63 7.41 7.17 6.73 6.62 6.36 7.26 7.22

def a ab abc bcd cde def ef f cde

Dry Root Weight Root weight in dried samples, the highest root weight was obtained with 5.04 g at 4000 ppm IBA and the lowest dry root weight was obtained with 0.34 g at 100 ppm SA. The dry matter accumulation was 2.22 g in the samples for all applications Table 8. Dry Root Weight Temperature Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average (g) 0.80 2.62 4.11 3.92 1.68 2.01 1.07 0.53 0.27 0.37 1.74

f-ı cd b b ef de e-ı hı ı ı b

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26 0C Average (g) 1.37 6.04 5.97 3.18 3.99 3.32 1.52 0.64 0.42 0.44 2.69

Mean e-h a a bc b bc efg ghı ı hı a

1.08 4.33 5.04 3.55 2.83 2.66 1.29 0.58 0.34 0.40 2.22

ef b a c d d e fg g g

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Root Quality The IBA 4000 ppm application gave the highest value with 3.63. It can be said that IBA applications increase the root quality and IBA 4000 and 2000 ppm doses give very good results in both temperature applications. The root quality averages obtained in 50, 100, 200 ppm of SA applications were lower than the control group averages. Zucherelli and Zucherelli (1985), Caldwel et al., (1988), Rana, (1991), Zenginbal et al., (2005) reported similar results as root quality. Table 9. Root Quality Temperatures Applications Control IBA 2000 ppm IBA 4000 ppm IBA 6000 ppm 4000+50 ppm (IBA+SA) 4000+100 ppm (IBA+SA) 4000+200 ppm (IBA+SA) 50 ppm SA 100 ppm SA 200 ppm SA Average

21 0C Average 2.96 3.80 3.62 3.18 2.92 2.63 2.49 1.98 1.90 1.83 2.73

26 0C Average 1.87 3.30 3.64 2.76 2.76 2.92 2.68 2.45 1.61 2.07 2.61

Mean 2.41 3.55 3.63 2.97 2.84 2.77 2.59 2.22 1.76 1.95 2.67

cde a a b bc bc bcd def f ef

CONCLUSIONS AND RECOMMENDATIONS In conclusion, IBA 2000 ppm is recommended for 95% rooting rate and 6.28 root number in the both temperatures. The highest average values were obtained IBA 4000 ppm in 26 ℃ rooting media in terms of root number (7.17), root length (96.39 mm) and root weight (47.87 g). In 50, 100 and 200 ppm of Salisilic acid applications, it was obtained 88.33% rooting rate, in all applications of SA in 21 ℃. As a result of the study, the highest values were obtained from IBA applications. According to these results, it was sait that SA did not have an expected effect on rooting. But it should be conducted some research on Salisilic acid with other doses and combinations. In addition, it is recommended to continue working in different media and wood or semi-wood cuttings with different SA concentrations, in order to understanding the effectiveness of SA. Acknowledgements: The authors would like to gratefully acknowledge to the kind support of ODU BAP AP-1732. REFERENCES Connor, D.M., 1982. Cutting propagation of Actinidia chinensis (kiwifruit). Combined Proceedings of the International Plant Propagators Society, 32: 329333. Costa, G., Baraldi, R. 1984. Studies on the propagation of Actinidia chinensis from wood cuttings. Universita Di Bologna, Italy. Horticultural Abstract. 67(2):123128. Ercişli, S. Anapalı, Ö., Esikten, A., Şahin, Ü. 2002. The effect of IBA, rooting media and cutting collection time on rooting of kiwifruit. Gartenbauwissenschaft, 67 (1):34-38. FAO, 2014. Statistical databese. http://fao.org (Erişim tarihi: 27/04/2017) 315

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Ferguson, A.R., Bollard, E.G. 1990. Domestication of the kiwifruit. p. 165–246 + 3 plates. In: I.J. Warrington and G.C. Weston (eds.), Kiwifruit: Science and management. Ray Richards, Publisher in association with the New Zealand Soc. Hort. Sci., Auckland. Gerçekcioğlu, R. 2009. Çeliklerde kök oluşumu: Genel Meyvecilik, Editörler: Gerçekcioğlu, R., Bilginer, Ş., Soylu, A., Nobel yayınları, 247-250. Lawes, G.S., Sim, B.L. 1980. An analysis of factors affecting the propagation of kiwifruit. The Orchordist of New Zealand. Massey University. 53(3) Palmerston North. New Zealand. Lee, S.H. 1990. Kiwifruit researches and productions in China. Acta Horticulture 282:57-63 Sivritepe, N., Eriş, A. 2000. Farklı çelik alma zamanları ve büyümeyi düzenleyici madde uygulamalarının kivi çeliklerinin köklenmesi üzerine etkileri. Bahçe 29: 27-38 Üçler, A. Ö., Parlak, S., Yücesan, Z. 2004. Kivi Actinidia deliciosa, A. Chev.)’ de yarı odunsu çeliklerin köklenmesi üzerine IBA ve çelik alım zamanının etkisi. Turk J Agric For 28:195-201 Yılmaz, M. 1992. Bahçe Bitkileri Yetiştirme Tekniği. Çukurova Üniversitesi Basımevi, Adana. Zenginbal, H., Özcan, M. 2013. Hayward ve Matua kivi çeşitlerinin odun çelikleri ile çoğaltılmasında farklı uygulamaların etkileri. Anadolu Tarım Bilim Dergisi, 28(3):115-125 Zenginbal, H., Özcan, M. 2014. Kivide çelik alma zamanı, çelikteki göz sayısı ve IBA uygulamalarının çeliklerin köklenmesi üzerine etkileri. Anadolu Tarım Bilim Dergisi, 29(1):1-11 Zenginbal, H., Özcan, M., Haznedar, A. 2006. Hayward kivi çeşidinde farklı koşullarda muhafaza edilen odun çeliklerinin köklenmesi üzerine IBA’ nın etkisinin belirlenmesi. OMÜ Ziraat Fakültesi dergisi, 21(1):20-26 Zenginbal, H., Özcan, M., Haznedar, A. 2006. Kivi (Actinidia deliciosa, A. Chev.) odun çeliklerinin köklenmesi üzerine IBA uygulamalarının etkisi. OMÜ Ziraat Fakültesi dergisi, 21(1):40-43

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ADAPTATION OF THE SELECTED CHERRY LAUREL GENOTYPES IN ORDU (TURKEY) Ali İSLAM*1, Selim KARAGÖL1, Hale ORTA SEÇMEN1 1

Ordu University, Agricultural Engineering, Department of Horticulture, Ordu, Turkey *Corresponding author: islamali@hotmail.com

ABSTRACT In this study; It is aimed to determine the adaptation of 23 different genotypes of the naturally grown cherry laurel (Prunus laurocerasus) in the Ordu, Black Sea region. 23 genotypes selected by selection from Samsun, Ordu, Giresun, Trabzon, Rize and Artvin were planted in the orchard in 2012 and their development was followed. Cluster length, cluster weight, fruit dimensional properties, fruit weight, stone dimensional properties, stone weight, petiole length and thickness, leaf width and length, TSS, pH, acidity, astringency, taste, a sample, fruit flesh hardness, fruit shape, cluster fullness measurements were carried out in Ordu University Horticulture pomology laboratories and orchard. As a result; T-303 has the heaviest fruits with 5.98 g. The R-27 genotype had the lowest fruit weight average with 1.83 g. The R-27 genotype also had the lowest value with a cluster weight which average is 22.38 g. The highest cluster weight average was measured in T-303 genotype with 125.70 g. In the study, the highest TSS rate was found %15.6 with T-303 genotype and the lowest TSS rate was found to be %6.8 with Keller genotype. Keywords: Cherry laurel, Pomology, Adaptation, TSS, Laurocerasus officinalis

INTRODUCTION Turkey is one of the world's most important gene centers because of geographic structure and different ecological conditions. Due to the suitability of ecological conditions, the Black Sea Region is one of the important places with rich natural resources and diversity. There are various fruits and nuts in the region. One of the most important of these is cherry laurel, the species that grows naturally in the region and is used as a fruit and ornamental plant. Cherry Laurel (Laurocerasus officinalis Reomer) belongs to Rosaceae family (Özbek, 1978). The other common name of this species is ‘taflan’ in Turkey (Islam, 2005). Cherry laurel forms are found in different parts of the world, especially in the southeast of Europe, the Balkans and Northern Iran. The natural spread of the plant in the world is the eastern regions of the Black Sea, the Caucasus, the Toros Mountains, the North and the Eastern Marmara (Zeybek 1960; Anşin and Özkan 1993). In Turkey, this species is used both fruit and ornamental plants. Studies on the value of cherry laurel as fruit are limited (İslam and Bostan (1996); İslam (2002); Macit 2008). On the other hand, it has different uses in pharmacy (Güven & Geçgil, 1961; Baytop, 1999; Koç, 2003). In our country, this species is used both as fruit species and ornamental plants. Studies on the value of cherry laurel as fruit are limited (İslam and Bostan (1996); İslam (2002); Macit 2008). Cherry Laurel trees can be 5-20 m long. These types differ from each other in terms of leaf size and shape, flower boards, fruit color and size. The leaves of the cultivated types are larger and different in terms of fruit color, taste and size (İslam and Bostan, 1996; Turna and Güney, 2006; İslam, 2002). Islam et al. (2010) were found leaf length 13.8 cm, width 5.5 cm, fruit width between 9.72-20.52 mm, fruit length between 10.55-24.41 mm, fruit weight between 0.69 and 317

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7.82 g and average fruit weight 4.85 g in a study between 2007-2009 with the support of TUBITAK 'Selection of natural cherry laurels in the Black sea region'. In the same study, cluster weights were determined between 4.74-126.54 g and the number of fruits in cluster was between 2.0-30.6. A study conducted to determine the pomological characteristics of 17 cherry laurel types grown in the central district of Trabzon, cluster weight 19.79-103.28 g, average fruit number in cluster 7.80-22.85, fruit weight 2.06-6.79 g, fruit weight 0.27-0.52 g, stone weight 0.27-0.52 g soluble solids 13.50-26.67% and total acidity in terms of citric acid was found to vary between 0.1270.291% (Bostan and Islam, 2003). It was also stated that the identified types were evaluated as table, jam, pickle and dried in the region. Akbulut et al. (2007), In their study of 28 cherry laurels in Samsun, cluster weight, the number of fruits in cluster and fruit weights found respectively between 5.84-57.82 g, 3.6-18.3, 1.40-5.39g. The fruit meat / core ratio was found 1.08-1.43, TSS was 8.6-21.3% and acidity was found between 0.36-1.21%; 14 fruits shape was seen round type, 8 types of conical, 3 types of flat, 3 types of oval; 15 types of fruits color were dark red, 9 types were black and 4 types were red. Ä°slam and Vardal (2009) stated that the fruit weights of local black types in Pazar were 2.635.56 g; The TSS was 15.7-23.1%; the number of fruits in cluster ranged from 7 to 16 and that the fruits were consumed fresh, dried or pickled. Today, it is seen that many countries attach more importance to the research of new fruit species; determination of the characteristics of the species found in their flora, production techniques, researching areas of use, cultivation studies and spreading is becoming increasingly important. Mankind, by seeking diversity in taste, examines the medical values of nutrients to try to eat more conscious (Ayaz et al., 1997a; 1997b; Kadioglu and Yavru, 1998). It is aimed to determine the adaptation of 23 different genotypes which are natural plants of Black Sea Region in Ordu province. In addition, the determination of pomological properties of cherry laurel genotypes and detection of superior genotypes were also aimed. MATERIALS AND METHODS Material Genotypes which are naturally grown in the Black Sea region and selected from Samsun, Ordu, Giresun, Trabzon, Rize and Artvin by selection were planted in Ordu University Faculty of Agriculture Research and Application Orchard in 2012 and necessary cultural practices were applied until the fruit started to yield. The 23 genotypes that started to yield after their development were harvested between July and August. METHODS 10 cluster samples were taken from different parts of the tree to represent the tree.fruit, seed and clusters weighed with scales sensitive to 0.01 g, fruit and seed sizes, leaf width and length, fruit stem length and thickness and cluster length (width, length) with caliper sensitive to 0.01 mm, Number of fruit in cluster was determined by counting. In addition, fruit color with Minolta CR400 chromameter, acidity by titration juice pH and soluable solids were measured. Fruit hardness, cluster fullness, astringency, and taste were determined by 3 people who were familiar with the fruit and received taste training in sensory random fruits. At the end of the measurements, the meaning of the data was made by using Minitab 17 with Tukey 5 at 5% significance level.

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RESULTS Table 1. Determined genotypes fruit weight, fruit width and length, stone weight and stone width and length. TYPE A19 KELLER O26 O29 O44 R135 R24 R25 R27 R5 S16 S21 S22 S23 S37 S39 SARI 3 T193 T203 T214 T217 T219 T303

FW (g) 3.53 c-f 4.71 a-d 3.4 def 5.26 abc 4.74 a-d 4.48 a-d 4.48 a-d 4.17 bcd 1.83 f 3.89 b-e 2.14 ef 4.24 a-d 4.92 a-d 4.38 a-d 5.14 a-d 4.71 a-d 4.78 a-d 5.24 abc 5.12 a-d 4.85 a-d 5.49 ab 5.48 ab 5.98 a

FW (mm) 16.06 b-f 17.17 a-f 17.30 a-f 21.53 a 16.44 a-f 18.38 a-e 18.61 a-d 17.80 a-f 12.98 ef 15.30 c-f 12.90 f 18.23 a-f 19.55 abc 19.00 a-d 20.89 ab 19.29 abc 13.67 def 19.17 abc 17.98 a-f 19.134 abc 19.97 abc 20.93 ab 19.60 abc

FL (mm) 18.32 a-d 20.77 ab 15.98 bcd 22.24 a 19.70 abc 18.29 a-d 20.34 ab 19.29 abc 13.60 d 15.97 bcd 14.17 cd 19.56 abc 19.50 abc 19.05 a-d 20.72 ab 19.85 ab 14.17 cd 20.41 ab 19.53 abc 18.562 a-d 20.90 ab 20.59 ab 23.10 a

SW (g) 0.365 b-f 0.425 a-e 0.310 ef 0.466 a-d 0.487 a 0.432 a-d 0.355 def 0.395 a-e 0.249 f 0.417 a-e 0.387 a-e 0.449 a-d 0.460 a-d 0.397 a-e 0.470 a-d 0.361 c-f 0.431 a-d 0.484 ab 0.415 a-e 0.359 c-f 0.435 a-d 0.447 a-d 0.478 abc

SW (mm) 7.73 abc 7.17 abc 7.50 abc 8.49 ab 7.64 abc 9.34 ab 8.41 abc 7.17 abc 6.98 abc 5.53 bc 7.57 abc 8.04 abc 9.31 ab 8.85 ab 9.34 ab 9.53 a 4.60 c 8.66 ab 7.07 ab 8.64 ab 6.30 abc 9.59 a 9.90 a

SL (mm) 11.30 ab 10.77 bc 11.37 ab 11.53 ab 14.00 a 11.96 ab 11.17 ab 11.89 ab 10.13 bc 10.40 bc 10.50 bc 12.97 ab 12.30 ab 11.92 ab 12.74 ab 12.96 ab 8.07 c 12.85 ab 12.29 ab 11.93 ab 10.40 bc 12.32 ab 12.93 ab

Fruits weights are important in Cherry laurel, and between 1.83 g (R-27) and 5.98 g (T-303) (average: 4.46 g) were determined. Genotypes T-303, T-217 and T-219 were 5.98 g, 5.49 g and 5.48 g respectively. The R-27 genotype was the lowest weight genotype with 1.83 g. In the measurements, the heaviest stone was detected in O-44 genotype with 0.487 g and the least weight stone was determined in R-27 genotype with 0.25.

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Table 2. Cluster weight and length, Leaf width and length, Fruit stalk length and thickness. TÄ°P NO A19 KELLER O26 O29 O44 R135 R24 R25 R27 R5 S16 S21 S22 S23 S37 S39 SARI 3 T193 T203 T214 T217 T219 T303

CW (g) 62.37 b-e 89.71 ab 31.74 de 79.76 a-d 74.40 bcd 65.08 b-e 67.7 b-e 65 b-e 22.38 e 94.18 ab 38.90 cde 95.77 ab 69.01 b-e 85.12 abc 78.93 a-d 55.00 b-e 93.40 ab 69.51 b-e 86.76 abc 88.56 abc 80.45 a-d 62.23 b-e 125.70 a

CL (mm) 146.82 ab 154.82 ab 172.63 a 116.02 ab 178.20 a 12.80 ab 121.90 ab 127.90 ab 91.23 b 113.61 ab 138.61 ab 89.17 b 101.67 b 127.37 ab 118.08 ab 118.62 ab 127.50 ab 122.97 ab 125.04 ab 112.00 ab 124.44 ab 127.20 ab 129.33 ab

LW (mm) 26.25 b 43.87 ab 47.57 a 55.00 a 52.67 a 49.75 a 40.50 ab 44.80 ab 35.32 ab 41.73 ab 39.08 ab 44.60 ab 44.17 ab 55.20 a 43.04 ab 44.10 ab 40.42 ab 53.60 a 42.33 ab 35.21 ab 55.40 a 42.92 ab 44.67 ab

LL (mm) 103.00 def 95.8 ef 129.90 b-f 142.25 a-f 129.3 b-f 132.92 b-f 93.29 f 133.20 b-f 120.55 c-f 123.30 b-f 125.00 b-f 151.57 a-d 100.83 def 176.10 ab 149.00 a-e 157.77 abc 140.40 a-f 163.54 abc 175.47 ab 146.71 a-f 194.00 a 150.90 a-d 166.20 abc

FSL 6.88 ab 6.80 ab 4.59 ab 5.26 ab 7.70 a 6.38 ab 4.81 ab 5.30 ab 3.47 b 7.78 a 5.39 ab 3.39 b 5.70 ab 5.58 ab 6.25 ab 6.03 ab 5.00 ab 5.38 ab 4.87 ab 5.63 ab 5.67 ab 5.90 ab 6.47 ab

FST 1.37 cde 1.20 de 2.29 a-e 2.53 abc 1.48 cde 1.73 b-e 1.98 a-e 1.82 a-e 1.58 b-e 1.06 e 2.39 a-d 1.81 a-e 1.75 b-e 1.83 a-e 1.89 a-e 2.34 a-d 3.00 a 2.46 abc 1.81 a-e 1.62 b-e 1.78 a-e 2.13 a-e 2.73 ab

The average weight of clusters of genotypes was measured as 73.1 g. According to the measurements; the highest cluster weight was 125.70 g (T-303) and the lowest 22.38 g (R-27). The cluster lengths of the genotypes were examined between 178.20 mm (O-44) and 89.17 mm (R-27), with an average of 126.68 mm. The leaf measurements, the longest leaf was determined as T-217 genotype with 194.00 mm and the widest leaf was 55.40 mm wit T-217 again. The longest stalk was found on genotype R-5 with 7.78 mm and the thickest stalk was found to be Yellow-3 genotype with 3.00 mm.

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Table 3. TSS, pH, and Acidity TYPE A19 T193 R135 KELLER T214 T203 T217 O44 S21 R24 T219 S39 S37 S16 R27 O26 SARI 3 S22 S23 R25 R5 O29 T303

TSS 8.8 8.4 7.6 6.8 8.8 9.6 7.6 8.8 7.6 11.6 8.4 10.4 7.2 8.0 12.4 11.2 10.0 8.4 8.0 10.4 9.2 8.8 15.6

pH 4.65 5.00 5.18 5.02 4.99 5.07 5.06 4.73 4.87 5.36 4.87 4.98 5.07 4.51 4.94 5.09 4.91 5.17 5.00 4.89 4.77 5.03 4.54

Acidity 0.608 0.296 0.258 0.246 0.498 0.406 0.344 0.494 0.424 0.530 0.412 0.356 0.300 0.588 0.742 0.344 0.524 0.332 0.232 0.410 0.516 0.408 0.936

The performance of selected genotypes under Ordu conditions was determined. Total solible solid amounts of genotypes ranged between 15.6 (T-303) and 6.8 (Keller) and the average was 9.2. The highest pH value was 5.36 with R-24 type and the lowest pH was 4.51 with S-16 type. Acidity in the genotypes were ranged 0.936 (T-303) to 0.232 (T-203). DISCUSSION The fruit weights determined in the study was ranged 1.83 g (R-27) to 5.98 g (T-303) (average: 4.46 g). The previous studies the measurements were; Islam and Deligöz (2012) 1.47 - 6.24 g, Akbulut et al. (2007) 1.40 - 5.39 g, Macit (2008) 3.48 - 4.81 g. Our stats were similar with other literatures. The highest cluster weight was between 125.70 g (T-303) and lowest was 22.38 g (R-27) (average 73.1). The cluster lengths of the genotypes were between 172.63 mm and 89.17 mm (average: 126.68). Macit (2008), were determined cluster weights were between 13.31- 40.20 g and cluster lenghts were 7.67 - 12.50 cm in Samsun region. The weight and length data obtained in our study are higher compared to Macit (2008). Karadeniz and Kalkışım (1996) studied in cherry laurel types in Akçaabat and the cluster weight varied from 62.7 to 123.9. İslam and Deligöz (2012) were found that the cluster weight were found 17.28 - 70.69 g; cluster lengths were determined as 99.10 cm and Bostan (2001) determined 46.75 g in ‘Su’ cherry 321

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laurel types grown in Trabzon; Islam (2002) reported that the cluster weight of cherry laurel was 67.9 g in ‘Kiraz type’. In our study, the cluster weights was obtained similar to those of Karadeniz and Kalkışım (1996) and the others were higher. Ecological and genetical differences is an important for it. The total soluble solid content of the genotypes was found between 6.8 and 15.6%. Akbulut et al. (2007) found TSS contents 8.6 - 21.3%. Some studies carried out in Trabzon, the TSS content of cherry laurel was found 13.50 - 26.67% (Bostan, 2001; Bostan and Islam, 2003). İslam and Deligöz (2008) found the TSS rates 13.00 - 29.00%. The obtained results were similar to the literature. In the study, fruit width ranged between 21.53 and 12.90 mm and fruit length ranged between 23.10 mm and 13.60 mm. İslam and Deligöz (2008) found fruit width 17.52-20.26 mm and fruit length 18.49 - 21.25 mm, Islam (2002) was found fruit width and length of 20-21 mm respectively, Islam and Odabaş (1996) in their study carried out in the Vakfıkebir determined 8-16 mm and 11 - 20 mm, Bostan and Islam (2003) in their study in Trabzon, 1.48-235 cm and 1.44-2.26 cm have determined. The results obtained are similar to the literature. CONCLUSIONS AND RECOMMENDATIONS As a results of the obtained data from 23 genotypes of cherry laurel, it can be recommented as fruit weight: T-303, T-219 and T-217 Genotypes, as TSS: T-303, R-27, R-24 and O-26. Genotypes, as fruit taste: S-22, T-214, T-203, R-24, T-303 and O-29, as cluster properties: T303, Yellow-3, Keller and R-5 Genotypes. Cherry laurel is an important in Turkey as different and alternative taste. It was consumed as fresh and processed product, especially in the Black Sea Region, it was a good popular fruit and processed as jam, marmalade, pickles for different purposes. We don’t have cherry laurel orchards, the plants grow naturally at the edges of the hazelnut orchards or single plant and was benefited from the fruits of this. It is important to increase the yield and quality of the cherry laurel whose economic potential is not known clearly. It is recommended that this study will be continued in more detail and with different aspects. And also the taste for humans’ changes over time. It is important for future generation because of different taste. REFERENCES Akbulut, M., Macit, İ., Ercilsi, S., Koç, A., 2007. Evaluation of 28 cherry laurel (Laurocerasus officinalis) genotypes in the Black Sea region, Turkey. New Zealand Journal of Crop and Horticultural Science, 35: 463–465. Anşin, R. ve Özkan, Z.C., 1993. Tohumlu Bitkiler (Spermatophyta). Odunsu Taksonlar. K.T.Ü. Orman Fak. Genel Yayın No: 167, Fakülte Yayın No: 19, Trabzon, 512 s. Ayaz, F.A., Kadıoğlu, A., Reunanen, M., Var, M., 1997a. Sugar composition in fruits of the Laurocerasus officinalis Roem. and its tree cultivars. Journal of Food Composition and Analysis. 10:82-86 Ayaz, F.A., Kadıoğlu, A., Reunanen, M., Var, M., 1997b. Phenolic acid and fatty acid composition in the fruit of the Laurocerasus officinalis Roem. and its cultivars. Journal of Food Composition and Analysis. 10:350-357 Baytop, T., 1999. Türkiye’de bitkiler ile tedavi. Nobel Tıp Kitabevi. ISBN: 975-420-021-1 480s Bostan, S. Z., 2001. Pomological traits of “Su” cherry laurel. Journal American Pomological Society, 55(4): 215-217

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Bostan, S. Z., İslam, A., 2003. Trabzon'da yetiştirilen mahalli karayemiş (Prunus laurocerasus L.) tiplerinin pomolojik ve fenolojik özellikleri. Ondokuzmayıs Üniversitesi Ziraat Fakültesi Dergisi, 18(1): 27-31. Güven K.L, Geçgil, T.H., 1961. Taflan suyu hazırlanması hakkında. Eczacılık Bülteni No:3, s:117 İslam, A., 2002. 'Kiraz' cherry laurel (Prunus laurocerasus). New Zealand Journal of Crop and Horticultural Science, 30: 301-302. İslam, A., 2005. Karayemiş yetiştiriciliği ve önemi. Ege Karadeniz Dergisi, 28(4): 25-32. İslam, A., Bostan, S. Z., 1996. Ümitvar bir meyve: Karayemiş. Ziraat Mühendisliği Dergisi, 291:21. İslam, A., Vardal, E., 2009. Pomologıcal characteristics of cherry laurel (Prunus laurocerasus L.) grown in Rize. Acta Horticulturae, 818:133-136. Kadıoglu, A., Yavru, I., 1998. Changes in the chemical content and polyphenol oxidase activity during development and ripening of cherry laurel fruits. Phyton 37(2):241-251 Koç, H., 2003, Lokman Hekimden Günümüze Bitkilerle Sağlıklı Yaşama. Kültür Bakanlığı Yayınları 2883, Kültür Eserleri Dizisi 373, Ankara. Macit, İ., 2008. Karadeniz Bölgesi Karayemiş (Prunus laurocerasus L.) Seleksiyonu II. Aşama. Ondokuzmayıs Üniversitesi Fen Bilimleri Enstitüsü, Yüksek Lisans Tezi (Basılmamış), Samsun, 47 s. Özbek, S., 1978. Genel Meyvecilik. Çukurova Üniversitesi Ziraat Fakültesi Ders Kitabı No:31, Adana, 386 s. Turna, İ., Güney, D., 2006. Karayemiş (Laurocerasus oficinalis Roemer)'in genel özellikleri ve odun dışı orman ürünü olarak değerlendirilmesi. 1st International Non-Wood Forest Products Symposium (01-05 Kasım 2006, Trabzon), Bildiriler Kitabı, 56-62. Tosun,F., Sağsöz, S., 1998. Bitki Islahı. Atatürk Üniversitesi Ziraat Fakültesi Yayınları No:172, Erzurum, 225 s. Zeybek, N., 1960. Türkiye’nin Tıbbi Bitkileri. Kuzeydoğu Anadolu Bölgesi. Ege Üniversitesi Tıp Fakültesi Yayınları, İzmir.

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EVALUATION OF ERWINIA AMYLOVORA STRAINS BY CRISPR TECHNOLOGY Mustafa Alparslan UMARUSMAN1, Kubilay Kurtuluş BAŞTAŞ2* 1

Konya Food and Agriculture University Faculty of Agriculture and Natural Sciences Dept. of Plant Production and Technology, Konya, Turkey

Selcuk University Faculty of Agriculture Dept. of Plant Protection, Campus, Konya, Turkey *Corresponding Author e-mail: kbastas@selcuk.edu.tr

ABSTRACT Erwinia amylovora is the causal agent of fire blight, a devastating disease of apple, pear, quince and other plants in the Rosaceae family. The strains of E. amylovora exhibits very low levels of genetic diversity. The diversity of E. amylovora habitats could potentially increases the ecological context of CRISPR spacer evolution with exposure to distinct microbiomes. Analysis of CRISPR spacer sequences and patterns revealed considerably more genetic diversity in the pathogen than had been known previously. The pathogenicity effector (DspE) of E. amylovora interacts with apple DIPM protein genes. CRISPR/Cas9 system is utilized to target DIPM genes in apple protoplast to develop resistance against fire blight disease. The experiments are showed successful direct delivery of CRISPR/Cas9 ribonucleoproteins into plant protoplasts which has several benefits like rapid targeting efficiency, improved on target and reduced off-target activity. Genome editing by CRISPR/Cas9 may yield planned disease resistant traits within a very short period which cannot be achievable by traditional breeding methods. CRISPR technology will help new insights into fire blight spread, strain genetic diversity, phage resistance, plasmid content/ evolution, host pathogen interactions and control strategies. Keywords: Fire blight, CRISPR, Genome, Strain identification, Resistance, Apple

INTRODUCTION Fire blight caused by Erwinia amylovora is a destructive disease in a wide variety of host species in the Rosaceae family, especially apples, pears and quinces (Bonn et. al. 2000; Van der Zwet et al. 2012). Under suitable climatical conditions, it causes different necrotic symptoms and sometimes the host's death (Dellagi et. al. 1998). Due to the systemic movement of the pathogen throughout the plants' vascular system and the ability of the pathogen to cause infections in flowers and actively growing shoots, and the sensitivity of rootstocks, it is difficult to control the pathogen (Norelli et. al. 2003; Koczan et. al. 2011). There are many virulance factors that E. amylovora has causing diseaeses mechanism in host plant. These factors are; exopolysaccharide (EPS) has been described including amylovoran, Type III Secretion System (TTSS), biofilm and lipopolysaccharide (LPS) (Malnoy et. al. 2012; Zeng and Sundin, 2014). The molecular size of amylovoran is influenced by various environmental conditions and factors related to cell metabolism (Vrancken et al. 2013). In addition, E. amylovora strains which are not capable of producing amylovoran are not pathogenic and do not spread in the plant vascular system (Bellemann and Geider, 1992; Vrancken et al. 2013). Levan is another EPS produced by E. amylovora, which is admitted a virulence factor (Koczan et al. 2011). Deficiency of levan synthesis has been reported to cause 324

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slow development of symptoms in the host plant (Geier and Geider, 1993; Vrancken et. al. 2013). The TTSS is an important virulence factor used by E. amylovora to infection the host plant (Vrancken et. al. 2013; Khokhani et. al. 2013). E. amylovora uses this secretory system to deliver effector proteins to the cell fluid of the host plant via a pilus-like structure (Vrancken et al. 2013). The TTSS in phytopathogenic bacteria consists essentially of Hrc proteins encoded by pathogenicity (hrp) genes. The hrc and hrp genes are composed of four regions in E. amylovora (Oh and Beer, 2005; Vrancken et. al., 2013). The bacterium has the DspA/E effector protein involved in the host-pathogen interaction. These effector protein interacts with DIPM proteins found in host plants and play a role in the infection of the pathogen (Meng et al. 2006). Inactivation of these genes found in apples and other host species is also important in the management of the pathogen. Borejsza-Wysocka et al. (2006) silenced genes that encoding DIPM proteins in apple and they have achieved to prevent the development of disease by preventing the interaction of E. amylovora with DspA/E proteins. CRISPRs are a bacterial defense system that provides the bacterial host to gain resistance or immunity to bacteriophages and plasmids (Yagubi et. al. 2013). Bacterial CRISPRs are sites containing conserved short direct repeats separated by sequences called ''spacers''. These short repeat regions are used by the CRISPR system to recognize or silence bacteriophages and other exogenetic structures (McGhee and Sundin, 2012). In this work, we focused on characterization of E. amylovora strains and using as a new tool in management of the pathogen by the CRISPR method. Principals of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR system consists of a genetic locus which spaced sequences and adjacent genes encoding CRISPR-associated (Cas) proteins (Shabbir et. al. 2016a; Shabbir et. al. 2016b). Some researchers have begun to use the CRISPR-Cas system for biotechnological applications and for the production of phage-resistant dairy bacterial cultures (Hsu et al. 2014). The CRISPRCas system blocks exogenic genetic material in three steps (Figure 1). Adaptation or spacer acquisition (Garneau et al. 2014) is the first stage in which the spacer sequence is integrated into the CRISPR sequence after recognition of the exogenic genetic structure. At this stage, Cas proteins, which change according to CRISPR types, cut the target gene region from the exogenic structure and transfer it to the short repetitive gene region located in the CRISPR region. The second step is the biosynthesis step in which CRISPR RNA (crRNA, pre-CRISPR RNA) is replicated by the RNA polymerase. This pre-crRNA is then cleaved into small crRNA by specific endoribonucleases. CrRNA is referred to as guide RNA (Brouns et. Al. 2008; Carte et. Al. 2008). The third stage of interference (Deveau et. Al. 2010) is the stage in which crRNAs recognize the base pair specific to exogenic RNA or DNA. This structure serves as the endonuclease that cleaves the target site in a second attack by the same phage. The CRISPR-Cas system is classified as types I, II and III. This classification is based on the specific genes available in each type. Type I has Cas3, type II has Cas9 and type III has the Cas10 gene. All types and subtypes of the CRISPR system have Cas1 and Cas2 proteins. The CRISPR-Cas type I system is found in most bacteria and archaea (Makarova et. al. 2011). This system is further divided into six subtypes (A - F) encoding the Cas3 gene. Cas3 is a multidomain protein which has helicase and nuclease activity (Sinkunas et. al. 2011). The Cas3 protein contains two effect domains. These are an N-terminal HD phosphohydrolase for cleavage of DNA and a C-terminal DExH helicase site to denaturation double-stranded DNA. E. amylovora is also a bacterium with the CRISPR-Cas Type I system (Shabbir et. al. 2016a; Shabbir et. al. 2016b) (Figure 2).

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Figure 1. Three stages of the mechanism of CRISPR-Cas system (Pak, 2014). (Stage 1: Adaptation, stage 2: Production of CRISPR RNA, stage 3: Targeting)

Figure 2. Genetic map of the CRISPR locus of E. amylovora ATCC 49946. The Cas and cse (CRISPR Cascade complex) genes and the CR1, CR2 and CR3 regions indicate the positions, while the A-B regions indicate the non-CRISPR-related gene regions (McGhee and Sundin, 2012). CRSPR-Cas systems are used in areas such as understanding the genetic diversity of bacteria, population structure, distribution and evolution, definition of antibiotic resistance mechanisms and prevention of these resistance mechanisms. In Identification and Control of Erwinia amylovora using CRISPR Technology Understanding the diversity of E. amylovora population and the transition to different geographical areas is important for future control of the disease. According to a study of extended comparative genomic analysis of the Spiraeoideae E. amylovora population in North America and Europe, the genetic diversity of the 30 E. amylovora genomes examined was 30 times higher than the genetic diversity of the strains previously identified (Zeng et al. 2018). The genome of E. amylovora consists of a circular chromosome of 3.805.874 bp and two plasmids. These plasmids; AMYP1 (28,243 bp) reported as PEA29 and the larger plasmid AMYP2 (71,487 bp) called pEA72 (Sinkunas et. al. 2011). The environmental conditions, the resistance mechanisms of the host plant, the antibiotic or other chemicals used, may result in variation in the genetic structure of the pathogen during the adaptation of the agent to adverse conditions. This diversity can be identified by using molecular approaches such as a variable number of tandem repeats (VNTR) or CRISPR (Rezzonico et. al. 2011 and 2012). The number of CRISPR sequences in a bacterial chromosome is variable, and the content of each sequence may vary both in the number of repeats and in the presence or absence 326

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of certain ranges. A total of 85 E. amylovora isolates from different geographies were studied for CRISPR sequence variability. Of these isolates, 588 unique ranges were identified in three CRISPR sequences (CRISPR 1-3) found in E. amylovora. Spacer content analyzes separated most of the apple and pear isolates isolated from the eastern USA from western isolates. It was stated that E. amylovora isolates from Rubus and Indian hawthorn contain more unique ranges compared to apple and pear isolates and 79 % of the isolates coming from the loquat are the same as apple and pear isolates (McGhee and Sundin, 2012). To determine the genetic diversity of the pathogen, isolates collected from orchards located in different regions of Kyrgyzstan were identified by 16S rRNA analysis and pathogen collection was made. Genetic diversity among the isolates was characterized by single sequence repeat (SSR) marker system. The CRISPR analysis included in the study indicated that the Kyrgyz E. amylovora isolates had a close similarity with the species present in Europe and the Middle East (Doolotkeldieva et. al. 2019). In Tunisia, to determine the variability in the genetic constructs of 54 isolates, including the reference isolate CFBP 1430, which was isolated between 2012 and 2014, PCR analysis were made that using primers specific for CRISPR and variable number of tandem repeats (VNTR). In the CRISPR analysis performed with all primers used, 49 isolates had similar genetic structure as CFBP 1430 reference race, whereas four isolates showed genetic structure different from reference strains. All VNTR sequences used had a similar result to CRISPR analysis. 49 isolates showed the same genetic structure as the reference isolate CFBP 1430, while four isolates were found to have significantly different genetic structure. Till now, several methods such as RFLP and AFLP used in epidemiological studies of E. amylovora have shown a low genetic variability within this species. This suggests that the analysis of CRISPR regions showing pathogen-specific structures will yield more specific results in determining the genetic structure of pathogens (Dardouri et al. 2017). E. amylovora isolates from different host species, 18 different CRISPR structures were identified in 37 isolates. Isolates from Spiraeoideae hosts were clustered in three major CRISPR groups. Both group II and group III were obtained from bacteria from the USA only, while the isolates in group I were generally originating from Europe, New Zealand and the Middle East. The researchers concluded that the fire blight isolates at Pomaceae were affected by the wider genetic population than those found in host plants in North America (Rezzonico et. al. 2011). Streptomycin is the most effective and widely used chemical in the USA against fire blight diseases caused by E. amylovora. However, since 2002, the pathogen's resistance to antibiotics has become a problem in New York. With this aim, genetic analysis was conducted to understand the origins and diversity of pathogen that are streptomycin-resistant (SmR) E. amylovora isolates from various apple orchards from 2011 to 2014 by using CRISPR sequences. Twenty-seven streptomycin resistant SmR E. amylovora isolates and 76 streptomycin susceptible SmS E. amylovora isolates were examined by comparing CR1, CR2 and CR3 regions. According to CRISPR sequence analysis, New York SmR and SmS E. amylovora isolates were grouped into 19 separate CRISPR profiles divided into three groups. As a result of these analyzes, it is stated that there is 68% variation among genomes. The majority of SmR E. amylovora isolates were reported to have the same CRISPR profile as the SmR E. amylovora isolates discovered in 2002. Furthermore, various CRISPR profiles for SmR E. amylovora isolates showed similar structure with SmS E. amylovora isolates collected from the same orchards in New York (Tancos & Cox, 2016). In California pear orchards, it was reported that strA/B streptomycin resistance genes were found in E. amylovora MR-L isolates which is moderately resistant to streptomycin. However, IS1133, which enables activation of these strA/B genes, is not unable to resist streptomycin due to lack of promoter region. The gene region in which StrA/B was found was determined for 26 isolates using tnpR-F and strA59out primers by PCR analysis. In addition, 327

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clustered regularly CRISPR sequence analysis was performed to determine the sensitivity of the isolates collected from the region. These ranges consist of plasmid and other DNA sequences and are typically 32 bp in E. amylovora (Sorek et al. 2013). In this study, two MRL strains of 96 isolates showed the same CRISPR structure as a streptomycin-susceptible strain (Förster et al. 2015). Fire blight resistance genes DIPM-1, DIPM-2 and DIPM-4 to Golden apple cultivar were tranfered by using purified CRISPR/Cas9 ribonucleoproteins. Recombinant Cas9 protein (160 kDa) and sgRNAs were commercially available from ToolGen, Inc., and were transferred to protoplasts under in vitro conditions. After transfer, total genomic DNA was izolated from the transferred protoplast, and its accuracy was determined by amplifying target regions in CRISPR/Cas9, MLO-7, DIPM-1, 2 and 4 with region-specific primers. In this study, the direct transmission of related genes to apple protoplasts was successfully tranfered using CRISPR/Cas9 system (Malnoy et. al. 2016). RESULT AND DISCUSSION Erwinia amylovora is a bacterial pathogen that causes serious problem and continues to be a major economic problem in the world. Different methods should be developed in the struggle of this factor which is a considerable problem in many pome fruits, ornamental plants and other plant species in the Rosaceae family. For this reason, understanding the genetic structure of the pathogen, geographical distribution and antibiotic resistance mechanisms are of great importance in the emergence of new methods of struggle. In this context, there are some researchs about understanding the genetic diversity of E. amylovora using CRISPR-Cas systems (Dardouri et. al. 2017, Doolotkeldieva et. al. 2019), determination of population structure and distribution (Rezzonico et. al. 2011, McGhee and Sundin 2012), identification of antibiotic resistance mechanisms (Förster et. al. 2015, Tankos et. al. 2016) and studies on control of these resistance mechanisms. Genetic analysis by CRISPR-Cas technology is of great importance in developing alternative control strategies for the control of this pathogen. Furthermore, it is possible to carry out studies on pathogen-resistant cultivars using CRISPR technology at host-pathogen interaction point. For this purpose, some phenotypic character genes and resistance genes against E. amylovora were transferred to the pome fruit groups by using CRISPR technology and successful results were obtained (Malnoy et. al. 2016, Nishitani et. al. 2016, Charrier et. al. 2019). In our country and in the world, the number of pear varieties with high resistance to this pathogen and at the same time superior to fruit quality parameters is insufficient (Mertoğlu, 2016). In addition to classical breeding methods, it is possible to develop superior varieties with CRISPR technology in order to obtain new varieties that are superior in terms of fruit quality and resistant to E. amylovora.

REFERENCES Bellemann, P., Geider, K., (1992). Localization of transposon insertions in pathogenicity mutants of E. amylovora and their biochemical characterization. J. Gen. Microbiol. 138, 931–940. Bonn, W.G., van der Zwet, T., (2000). Distribution and economic importance of fire blight. Vanneste, J.L., Ed.; CAB International: Wallingford, CT, USA, pp. 37–53. Borejsza-Wysocka EE, Malnoy M, Aldwinckle HS, Meng X, Bonasera JM, Nissinen RM, Kim JF, Beer SV., (2006). Fire blight resistance of apple clones in which DspEinteracting proteins are sliend. Acta Hort., 704: 509-513. Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, et al., (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science (80−). 321:960–4. 328

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Carte J, Wang R, Li H, Terns RM, Terns MP., (2008). Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22: 3489–96. Charrier, A., Vergne, E., Dousset, N. J. P., Richer, A., Petiteau, A., Chevreau, E., (2019). Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPRCas9 system. Frontiers in plant science, 10, 40. Dardouri, S., Chehimi, S., Murillo Martínez, J., Hajlaoui, M. R., (2017). Molecular characterization of Tunisian strains of Erwinia amylovora. J. of Plant Pathology, vol. 99, n. 2 (2017), p. 331-337. Dellagi, A.; Brisset, M.N.; Paulin, J.P.; Expert, D., (1998). Dual role of desferrioxamine in E. amylovora pathogenicity. Mol. Plant Microbe Interact. 11, 734–742. Deveau H, Garneau JE, Moineau S., (2010). CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol. 64: 475–93. Doolotkeldieva, T., Bobushova, S., Schuster, C., Konurbaeva, M., Leclerque, A. (2019). Isolation and genetic characterization of E. amylovora bacteria from Kyrgyzstan. Eu. J of Plant Pathol., 1-10. Förster, H., McGhee, G. C., Sundin, G. W., Adaskaveg, J. E., (2015). Characterization of streptomycin resistance in isolates of E. amylovora in California. Phytopathology, 105(10), 1302-1310. Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R, Boyaval P, et al. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 468: 67–71. Geier, G.; Geider, K., (1993). Characterization and influence on virulence of the levansucrase gene from the fireblight pathogen E. amylovora. Phys. Mol. Plant Pathol. 42, 387–404. Hsu PD, Lander ES, Zhang F., (2014). Development and applications of CRISPR–Cas9 for genome engineering. Cell. 157:1262–78. Khokhani, D.; Zhang, C.; Li, Y.; Wang, Q.; Zeng, Q.; Yamazaki, A.; Hutchins, W.; Zhou, S.S.; Chen, X.; Yang, C.H., (2013). Discovery of plant phenolic compounds that act as type III secretion system inhibitors orinducers of the fire blight pathogen, E. amylovora. Appl. Envi. Microbiol. 79, 5424-36. Koczan, J.M.; Lenneman, B.R.; McGrath, M.J.; Sundin, G.W., (2011). Cell surface attachment structures contribute to biofilm formation and xylem colonization by E. amylovora. Appl. Environ. Microbiol. 77, 7031–7039. Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, et al., (2011). Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol. 9: 467–77. Malnoy, M., Viola, R., Jung, M. H., Koo, O. J., Kim, S., Kim, J. S., Nagamangala Kanchiswamy, C., (2016). DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Frontiers in plant science, 7, 1904. Malnoy, M.; Martens, S.; Norelli, J.L.; Barny, M.A.; Sundin, G.W.; Smits, T.H.; Duffy, B., (2012). Fire blight: Applied genomic insights of the pathogen and host. Annu. Rev. Phytopathol. 50, 475–494. McGhee, G. C., & Sundin, G. W., (2012). Erwinia amylovora CRISPR elements provide new tools for evaluating strain diversity and for microbial source tracking. PLoS One, 7(7), e41706. Meng XD, Bonasera JM, Kim J, Nissinen RM, Beer SV., (2006). Apple proteins that interact with DspA/E, a pathogenicity effector of E. amylovora, the fire blight. Mol. Plant Mic. Interact., 19: 53-61. Mertoğlu, K., (2016). Armutta melezleme yoluyla ateş yanıklığına (Erwinia amylovora) dayanıklılık konusunda elde edilen F₁ melez populasyonunun fenolojik ve meyve özelliklerinin belirlenmesi (Master's thesis, ESOGÜ, Fen Bilimleri Enstitüsü).

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Nishitani, C., Hirai, N., Komori, S., Wada, M., Okada, K., Osakabe, K., Osakabe, Y., (2016). Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific reports, 6, 31481. Norelli, J.L.; Jones, A.L.; Aldwinckle, H.S., (2003). Fire blight management in the twenty-first century: Using new technologies that enhance host resistance in apple. Plant Dis., 87, 756–765. Oh, C.-S.; Beer, S.V., (2005). Molecular genetics of E. amylovora involved in the development of fire blight. FEMS Microbiol. Lett. 253, 185–192. Pak, E., (2014). CRISPR: a game-changing genetic engineering technique. Science in the News. Rezzonico, F.; Braun-Kiewnick, A.; Mann, R.A.; Goesmann, A.; Rodoni, B.; Duffy, B.; Smits, T.H.M., (2012). Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of E. amylovora. Mol. Plant Pathol. 13, 975–984. Rezzonico, F., Smits, T.H., Duffy, B., (2011). Diversity, evolution, and functionality of clustered regularly interspaced short palindromic repeat (CRISPR) regions in the fire blight pathogen E. amylovora. Appl. Environ. Microbiol. 77, 3819–3829. Shabbir MAB, Hao H, Shabbir MZ, Hussain HI, Iqbal Z, Ahmed S, et al., (2016). Survival and evolution of CRiSPR–Cas system in prokaryotes and its applications. Front Immunol. 7: 375 (a) Shabbir MAB, Hao H, Shabbir MZ, Wu Q, Sattar A, Yuan Z., (2016). Bacteria vs. bacteriophages: parallel evolution of immune arsenals. Front Microbiol. 7: 1–8. (b) Sinkunas T, Gasiunas G, Fremaux C, et al., (2011). Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30: 1335–42. Sorek, R., Lawrence, C. M., and Wiedenheft, B., (2013). CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 82: 237-266. Tancos, K. A., Cox, K. D. (2016). Exploring diversity and origins of streptomycin-resistant E. amylovora isolates in New York through CRISPR spacer arrays. Plant Dis., 100(7), 13071313. Van der Zwet, T.; Orolaza-Halbrendt, N.; Zeller, W., (2012). Fire Blight: History, Biology, and Management; APS Press: St. Paul, MN, USA. Vrancken, K.; Holtappels, M.; Schoofs, H.; et al., (2013). Pathogenicity and infection strategies of the fire blight pathogen E. amylovora in Rosaceae: State of the art. Microb., 159, 823– 832. Yagubi, A. I., Castle, A. J., Svircev, A. M., (2013). Sequence analysis of CRISPR arrays of E. amylovora isolates from Canada. XIII Int. Workshop on Fire Blight 1056 (pp. 149-153). Zeng, Q., Cui, Z., Wang, J., Childs, K. L., Sundin, G. W., Cooley, D. R., Yuan, X., (2018). Comparative genomics of Spiraeoideae‐infecting E. amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low‐virulence strain. Mol. plant pathology, 19(7), 1652-1666. Zeng, Q., Sundin, G.W., (2014). Genome-wide identification of Hfq-regulated small RNAs in the fire blight pathogen E. amylovora discovered small RNAs with virulence regulatory function. BMC Genomics,15, doi:10.1186/1471-2164-15-414.

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CYTOTOXICITY OF NATURAL AND SYTNHETIC COLOURANTS USED IN TRADITIONAL MARDIN BLUE ALMOND TOFFEE Nurcan ARSLAN1, Mehmet OZASLAN1, Isik Didem KARAGOZ*1, Ibrahim Halil KILIC1 1

Department of Biology, University of Gaziantep, 27310, Gaziantep, Turkey *Corresponding author’s e-mail: karagoz@gantep.edu.tr ABSTRACT

Colourant (Indigo blue) used in blue almond toffee is obtained from hydrolysis and oxidation of Indicane B from Isatis tinctoria folia. All ground surface tissues produce indicane B when plant grows under sun. The cost of natural indigo is too expensive, hard and taking long time so researchers investigated the molecular structure and produced as synthetically. Since then natural indigo blue gives its place to the synthetic. Indigo blue is used as blue colourant in textile and medical sectors together with its benefits. Approximately 17.000 tons synthetic colourant have produced every year in world. Besides its usage in textile and medical industry, it has not used in food industry. However, famous traditional blue almond toffee has been stained with it and it has not known potential risk for human health. In this perspective, we aimed to investigate the cytotoxic potential of natural and synthetic indigo blue colourants by MTT. As a result, natural indigo blue is safer than synthetic for food industry. Keywords: Isatis tinctoria, Indigo blue, Cytotoxicity.

INTRODUCTION The flora of Turkey includes a large number of plants with natural dyes. Various parts of these plants such as flowers, leaves, barks, and roots contain dyes that can be used in dyeing. Natural dyes derived from these plants are used in foods, textiles, cosmetics, and pharmaceuticals (Piccaglia and Venturi 1998). Blue is one of the rare natural dye colours. There are two main natural sources of blue color: Isatis tinctoria (Dyer’s woad) ve Indigofera tinctoria (True Indigo). There are 25 species of the genus Isatis in Anatolia (Baytop, 1984). I. tinctoria L. belongs to the Brassicaceae family. It is biennial or perennial plant. The stem is green or purple. The leaves are hairy or hairless. The flowers are clustered and hairless most of the time (Tezcan ve Suyunu, 1991). I. tinctoria L. is grown naturally in the roadsides of Turkey (Davis, 1965). Indigo, a blue-colored dye is formed as a result of the oxidation of the hydrolysis product of an indoxylβ-d-glucoside called indican found in the leaves of I. tinctoria L. (Baytop, 1984; Enez, 1987). The history of the use of natural dyeing in the textile industry in India and Mesopotamia dates back to 4000 BC. (Karadağ, 2007). I. tinctoria L. is one of the first plants that are widely cultivated in Europe in the middle ages for dyeing purposes. In 1905, Adolf von Bayear was awarded the Nobel Prize for discovering the molecular structure of Indigo and its synthetic production. However, since that date, natural dyes have been replaced by synthetic dyes in a rapid pace (Kaufman and Rúveda, 2005). Although natural dyeing is a costly and timeconsuming process, its products are much more valuable and long-lasting compared to synthetic dyeing. Moreover, synthetic dyes are mostly toxic, carcinogenic and its production-wastes 331

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could be damaging to the environment, therefore, the use of natural dyeing has been reconsidered (Nagar et al., 2005; Ali et al., 2007; Karadağ, 2007). Besides its use as a natural blue dye for dyeing purposes, I. tinctoria L. was used as a medicinal plant with healing effects for wound healing, constipation, and liver diseases till the 13th century in Europe (Baytop, 1984; Farah, 1987; Jing-xian, 1999). I. tinctoria L. is widely used in traditional Chinese medicine nowadays (Condurso et al., 2006). In recent years, a considerable amount of in vivo and in vitro studies has been carried out to investigate its phytochemical and biological activities. Several compounds such as alkaloids, glucosinolates, flavonoids, indigo precursors, carotenoids, fatty acids, porphyrins, lignans, cyclohexanone, and phenylpropanoids have been identified as in this species (Hamburger, 2002; Mohn et al., 2009). Several studies have focused on the anti-inflammatory and anticancer properties of lipophilic extracts and isolated compounds from I. tinctoria L.; in particular, some alkaloids including tryptanthrin, indirubin, and indolinone have been widely studied for their beneficial health effects (Ronald Steriti, 2002; Eisenbrand et al., 2004; Recio et al., 2006a, 2006b). Nevertheless, very few studies have been carried out addressing the biological activities of the crude extract and the synthetic dye form derived from this species. Furthermore, Indigo dye is used as a food colouring in the production of Blue Almond, a widely produced candy in the Mardin region that draws the attention of local and foreign tourists. Therefore, this study aimed to investigate the cytotoxic effects of Indigo dye in both its natural and synthetic forms on two different cell lines such as H1299 lung adenocarcinoma and HUVEC human umblicial vein cells. MATERIALS AND METHODS Two dyes were used in this study; one is the leaf extract of I. tinctoria L. (Dyer's Woad) and the second one is the extract of synthetic indigo dye (Lahor). 1. Collection of the plant: I. tinctoria L. plant was collected from the region of Huzurlu plateau, Islahiye, Gaziantep. All the fresh parts of the plant (root, stem, leaves and flowers) were dried in the shade according to herbarium guidelines. Green leaves of the dried plant were used to prepare the extract. 2. Preparation of leaf extract: The green leaves part of the dried I. tinctoria L. plant was homogenized to a fine powder. 2/3 of a beaker was filled with distilled water and allowed to boil on the Bunsen burner, then the dried leaves of I. tinctoria L. were added to the boiled water and left to boil for one minute. After cooling, Isatan b containing solution was obtained. Isatan B is an unstable compound; therefore, ammonia was added at 1% of the extract volume. After the addition of ammonia, the color of the solution changed from brown to green and an Indoxyl-containing solution was obtained (Farusi, 2007). 3. Preparation of Indigo dye solution: 100 mg/ml stock solution was prepared from synthetic Indigo dye powder (Lahor). It was dissolved in dimethyl sulfoxide (DMSO) to obtain the extract and then, stored at 4 °C. The concentrations to be added to the cells were 6.25,12.5,25,50, and 100 µg/ml. 1: 1 dilution with the medium was prepared. 4. Cell culture: H1299 and HUVEC cells were cultured in DMEM: Ham’s F12 (1:1) medium supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. The cells were allowed to grow in a humidified incubator at 37°C and 5% CO2. Cells were maintained in 25 cm2 tissue culture flasks and passaged by trypsinization with 0.25% trypsin-EDTA when they reached 80% confluence. 5. Cytotoxicity assay (MTT): HUVEC and H1299 cells were counted and 5×103 cells in 100μl of medium were seeded in each well. Cells were incubated for 24 hours at 37oC 332

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and %5 CO2. Five different concentrations (6-100 µg/ml) from the leaf extract of I. tinctoria L. and synthetic Indigo dye (6-100 µM) (Lahor) were added to the cells in a 100 μl volume to each well of 96-well plates, and incubated for 48 hours. Cells cultured in medium without any addition of extracts were used as a negative control for the measurement of maximum viability (MO). 100μl of the medium was added to the blank control wells. The cells were then incubated for 48 hours at 37oC and %5 CO2. 5mg/ml in PBS buffer, pH = 7.2 of MTT stock solution, was prepared. Then, the prepared MTT stock solution was filtered and sterilized. At the end of the 48 hours incubation period, 40μl of MTT solution was added to each well. Then, cells were incubated at 37oC for 4 hours (Watjen and Beyersmann, 2004). 80μl of 10% SDS solution was added to each well to dissolve the formed formazan crystals and incubated for 18 hours at 37 oC in a 5% CO2 incubator. At the end of the incubation period, the color intensity of the wells was measured using a spectrophotometer (Thermo Scientific) at 570nm wavelength and the viability of the cells was calculated using the measured absorbance values. 6. Calculation of viability: The viability of the extract-treated cells was calculated using the following formula, assuming that the viability of the non-treated control cell (MO) is 100%. Each concentration was repeated three times independently for each experiment. Viability (%) = [(Extract-treated cell absorbance mean-blank absorbance mean)/ (Control cells absorbance mean-blank absorbance mean) ×100]. RESULTS The MTT method is a colorimetric assay that measures the activity of mitochondrial dehydrogenase enzyme in viable cells (Mossman et al., 1983). MTT (tetrazolium (3- (4,5dimethylthiazole-2) -2,5-diphenyl tetrazolium bromide) is a yellow water-soluble tetrazolium salt that is taken up by viable cells and converted by mitochondrial succinate dehydrogenase enzymes into water-insoluble dark blue-violet formazan crystals. These formazan crystals are then solubilized in water using 10% SDS in 37% HCl and their colour intensity is measured by spectrophotometer at 570nm wavelength. The colour intensity in the untreated control cells (MO) and in the drug-treated cells are calculated to measure the viability (percentage) of the drug-treated cells. The reduction of MTT and formation of formazan crystals are considered a sign of cell viability as indicated by mitochondrial respiration. Formation of formazan crystals after treatment with MTT is observed only in viable cells with intact mitochondrial function. Accordingly, viable cells with intact mitochondrial function are stained with blue-violet color, while dead cells with dysfunctional mitochondria are not stained. Cell death by cytotoxic compounds decreases the reduction of MTT tetrazolium to formazan. The measured absorbance values correlate with the number of viable cells. The high number of formazan crystals reflects the high number of viable cells present in the sample (Denizot and Lang, 1986; McGahon et al., 1995; Horakova et al., 2001). Synthetic Indigo dye (Lahor) and the leaves extract of I. tinctoria L. (Dyer's woad) were prepared for cell culture studies. The cytotoxic effects of these dyes on H1299 and HUVEC cells were assayed by MTT. Positive (max. viability) and negative (min. viability) control cells were used to evaluate the changes in cell viability after the treatments and to minimize the error rates. The microscopic images of H1299 and HUVEC cells are shown in Figure 1. The formazan crystals formed after treatment with MTT are shown in Figure 2.

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(A)

(B) Figure 1: (A), HUVEC; (B), H1299 cells.

(A)

(B)

Figure 2: Formazan crystals formed in (A), HUVEC; (B), H1299 cells. The cell viability of the cell lines after the treatment with synthetic Indigo dye (Lahor) and leaf extract of Dyer's Woad for 48 hours is shown in the graphs given in Figure 3.

(A)

(B)

Figure 3: (A), The cytotoxicity of Synthetic Indigo dye (Lahor); (B), of natural Indigo dye on H1299 and HUVEC cells. Each data point represents the average of 3 independent wells. The leaf extract of I. tinctoria L. (natural) did not show any cytotoxic effect at all the concentrations (6.25-100 µg/ml) on HUVEC cells; show slight cytotoxicity at high dose (100 µg/ml) on H1299. However, synthetic Indigo dye (Lahor) did not effect H1299 cell viability, whereas a significant cytotoxic effect was observed in HUVEC cells at higher doses 50 µg/ml. 334

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DISCUSSION AND CONCLUSION Nowadays, plant-derived products are more preferred in synthetic, cosmetic, soap and textile sectors than synthetic products. I. tinctoria L. leaves are used as traditional medicine for the treatment of respiratory tract infections and gastroenteritis. Extracts obtained from the roots have been reported to have antiviral, antimicrobial and antiparasitic effects (Herbal, 1993). In another study, anticancer and anti-inflammatory effects, as well as side effects such as runny nose, diarrhea, and thrombocytopenia were observed (Mingzhu and Bangyuan, 1983). Recently, the antioxidant, antiproliferation and antifungal effects, as well as anthelmintic activities of plant-derived Indigo dye have been investigated (Anusuya and Manian, 2013; Kameswaran and Ramanibai 2008; Tapsoba and Deschamps, 2006). In this study, the cytotoxic effects of the natural Indigo dye obtained from leaf extract of I. tinctoria L. and synthetic Indigo dye were investigated on H1299 and HUVEC cell lines. According to our results, natural Indigo dye had no significant cytotoxic effect on cells. However, the synthetic Indigo dye had cytotoxic effect on HUVEC cells especially. In conclusion, we suggested that natural dye should be preferred in food and medicine industries instead of synthetic one. Synthetic dye is more suitable for textile industry because of its low cost and stability, but it is harmful for healthy cells. Further studies should be conducted to investigate the health hazards of Indigo dye in detail.

REFERENCES Ali, S., Nisar, N., & Hussain, T. (2007). Dyeing properties of natural dyes extracted from eucalyptus. Journal of the Textile Institute, 98(6), 559-562. Anusuya, N., & Manian, S. (2013). Antioxidant and free radical scavenging potential of different solvent extracts of Indigofera tinctoria L. leaves. Int J Pharm Pharm Sci, 5(1), 142-147. Baytop, T. (1984). Treatment with plants in Turkey. Istanbul Univ. Publ, 3255. Condurso, C., Verzera, A., Romeo, V., Ziino, M., Trozzi, A., & Ragusa, S. (2006). The leaf volatile constituents of Isatis tinctoria by solid-phase microextraction and gas chromatography/mass spectrometry. Planta medica, 72(10), 924-928. Davis, P. H. (1965). 1988. Flora of Turkey and the East Aegean Islands. Vols. 1-10. Edinburgh: Edinburgh UniversityPress. Denizot, F., & Lang, R. (1986). Rapid colorimetric assay for cell growth and survival: modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of immunological methods, 89(2), 271-277. Eisenbrand, G., Hippe, F., Jakobs, S., & Muehlbeyer, S. (2004). Molecular mechanisms of indirubin and its derivatives: novel anticancer molecules with their origin in traditional Chinese phytomedicine. Journal of cancer research and clinical oncology, 130(11), 627635. Enez, N. (1987). Doğal boyamacılık: Anadolu'da yün boyamacılığında kullanılmış olan bitkiler ve doğal boyalarla yün boyamacılığı. Marmara Üniversitesi. Farusi, G. (2007). Monastic ink: linking chemistry and history. Science in School, 6, 36-40. Hamburger, M. (2002). Isatis tinctoria–from the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical. Phytochemistry Reviews, 1(3), 333. Herbal, B. D. G. A. C. (1993). Medicine: Materia Medica. Revised edition. Seattle Eastland Pr. 335

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Horáková, K., Šovčı́ková, A., Seemannová, Z., Syrová, D., Bušányová, K., Drobná, Z., & Ferenčıḱ , M. (2001). Detection of drug-induced, superoxide-mediated cell damage and its prevention by antioxidants. Free Radical Biology and Medicine, 30(6), 650-664. Kameswaran, T. R., & Ramanibai, R. (2008). The antiproliferative activity of flavanoidal fraction of Indigofera tinctoria is through cell cycle arrest and apoptotic pathway in A549 cells. Journal of Biological Sciences, 8(3), 584-590. Karadağ, R. (2007). Doğal boyamacılık. Dösim. Kaufman, T. S., & Rúveda, E. A. (2005). The quest for quinine: those who won the battles and those who won the war. Angewandte Chemie International Edition, 44(6), 854-885. McGahon, A. J., Nishioka, W. K., Martin, S. J., Mahboubi, A., Cotter, T. G., & Green, D. R. (1995). Regulation of the Fas apoptotic cell death pathway by Abl. Journal of Biological Chemistry, 270(38), 22625-22631. Mohn, T., Plitzko, I., & Hamburger, M. (2009). A comprehensive metabolite profiling of Isatis tinctoria leaf extracts. Phytochemistry, 70(7), 924-934. More, G., Tshikalange, T. E., Lall, N., Botha, F., & Meyer, J. J. M. (2008). Antimicrobial activity of medicinal plants against oral microorganisms. Journal of Ethnopharmacology, 119(3), 473-477. Nagar, M., Ogunyomade, A., O’brart, D. P. S., Howes, F., & Marshall, J. (2005). A randomised, prospective study comparing selective laser trabeculoplasty with latanoprost for the control of intraocular pressure in ocular hypertension and open angle glaucoma. British Journal of Ophthalmology, 89(11), 1413-1417. Piccaglia, R., & Venturi, G. (1998). Dye plants: a renewable source of natural colours. Agro Food Industry Hi-Tech, 9(4), 27-30. Recio, M. C., Cerdá-Nicolás, M., Hamburger, M., & Rios, J. L. (2006b). Anti-arthritic activity of a lipophilic woad (Isatis tinctoria) extract. Planta medica, 72(08), 715-720. Recio, M. C., Cerdá-Nicolás, M., Potterat, O., Hamburger, M., & Ríos, J. L. (2006a). Antiinflammatory and antiallergic activity in vivo of lipophilic Isatis tinctoria extracts and tryptanthrin. Planta medica, 72(06), 539-546. Ronald Steriti, N. D. (2002). Nutritional support for chronic myelogenous and other leukemias: a review of the scientific literature. Alternative medicine review, 7(5), 404-409. c, R. L., Miller, K. D., & Jemal, A. (2016). Cancer statistics, 2016. CA: a cancer journal for clinicians, 66(1), 7-30. Tapsoba, H., & Deschamps, J. P. (2006). Use of medicinal plants for the treatment of oral diseases in Burkina Faso. Journal of Ethnopharmacology, 104(1-2), 68-78. TEZCAN, İ., & SUYUNU, A. (1991). İpek Halılarda Doğal Boyarmaddeler. Tekstil ve Mühendis, 5(26). Wätjen, W., & Beyersmann, D. (2004). Cadmium-induced apoptosis in C6 glioma cells: influence of oxidative stress. Biometals, 17(1), 65-78.

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YIELD AND YIELD COMPONENTS OF DIFFERENT SWEET POTATO (IPOMOEA BATATAS) GENOTYPES Yasin Bedrettin KARAN*1, Özlem Gültekin ŞANLI1, Şevket ÖZDEMİR1 1

Tokat Gaziosmanpasa University, Faculty of Agriculture, Department of Field Crop, Tokat, Turkey *Corresponding author’s e-mail: yasinb.karan@gop.edu.tr

ABSTRACT The experiment was conducted in 2017 under net greenhouse conditions in the Turkey-TokatKazova. Altitude of Tokat-Kazova was 630 meters, soil type is loamy, lower salt content, and slighty alkaline. Variabilities in total yield, marketable yield and components of yield (tuber numbers and mean tuber weights) were studied in four sweet potato cultivars. Yield variability was high, particularly in marketable tubers, and was related to either or both components of yield. The contribution of yield components to variability in total yield was evaluated and sources of yield variation were attributed to planting material, tuber development and season. The highest total tuber yield from Havuç genotype (7604.93 kg/da). In this study, the total tuber yield of the genotypes ranged from 1283.95 – 7604.93 kg/da. Keywords: Sweet potato, Yield, Local genotype INTRODUCTION Sweet potato (Ipomoea batatas (L.) Lam.) is a dicotyledonous plant belonging to the family Convolvulaceae. This family includes about 55 genus and more than 1000 species. However, only Ipomoea batatas is of economic importance as a source of food (Purseglove, 1968; Woolfe,1992). Sweet potato (Ipomoea batatas (L.) Lam.), known as a potato, is well known long-term species in a warm and hot climate zone and an annual plant (spring) in temperate zone. This species has moist and delicate tubers with a sweetish taste, pleasant and aromatic smell. It also has a high nutritional value – about 50% higher than the potato. Therefore, it plays an important role in the diet of the world’s population (Ofori et al., 2005). The storage roots are rich sources of carbohydrates, vitamins and bioavailable β-carotene. Sweetpotato is used for food, industrial purposes and feed. The improvement of this crop is very important in achieving increased production and productivity in developing countries (Laurie et al., 2015). The aim of this study was to determine yield potential of some sweetpotato local genotypes in Tokat Kazova condition.

MATERIAL AND METHODS The experiment was conducted in 2017 in Turkey-Tokat-Kazova. 36.360 East longitude, 40.330 North latitudes 570 meters’ altitude. The experiments was laid out in Randomized Complete Block Design, with three replications. The planting process was carried out in early May in Kazova. 337

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Sweetpotato seedlings were planted as 90 cm row spacing and 45 cm plant spacing (Yurtsever, 1984). In this study, four different local genotype were used. Table 1. Plant material used in the experiment Hatay Kırmızısı

Kalem

Havuç

Hatay Yerlisi

The trial was fertilized 12 kg/da 15:15:15 (N:P:K) at the time of planting. 8 kg/da N was applied 45 days after planting. The sweet potato seedlings were irrigated as required to maintain adequate moisture levels by drip irrigation. When cultural and chemical methods were needed to combat diseases and pests, necessary applications were made. RESULTS AND DISCUSSIONS Storage Root Weight/Plant (g) In this study, the storage root/plant of the sweet potato genotypes ranged from 520 to 3080 g. The highest storage root weight/plant was obtained from Havuc genotype.

Storage Root Weight/Plant (g) 3500 3000 2500 2000 1500 1000 500 0 Havuç Patates

Hatay Kırmızısı

Kalem

Hatay Yerlisi

Storage Root Number/Plant Figure shows the storage root number/plant. The highest storage root number/plant produced was from Hatay Kırmızısı (8.9). In this study, the storage root number of the genotypes ranged from 5.04 to 8.87.

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Storage Root Number/Plant 9 8 7 6 5 4 3 2 1 0 Havuç Patates

Hatay Kırmızısı

Kalem

Hatay Yerlisi

Average Storage Root Weight (g) According to figure mean comparison among storage root weight indicated that Havuc had higher average storage root (445.7 g) than other genotypes.

Average Storage Root Weight (g) 450 400 350 300 250 200 150 100 50 0 HATAY YERLİSİ

KALEM

HAVUÇ

HATAY KIRMIZISI

The lowest average storage root weight was obtained from Kalem genotype as 111.2 g. The average storage root weight of the genotypes ranged from 111.2 to 445.7 g.

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Total Storage Root Yield (kg/da) The highest total tuber storage root yield was produced by Havuç (7604.9 kg/da). The results showed that the lowest total tuber storage root yield belonged to Kalem genotype (1284 kg/da). The highest total storage root yields were produced by Havuç, followed by Hatay Yerlisi, Hatay Kırmızı and Kalem genotypes.

Total Storage Root Yield (kg/da) 8000 7000 6000

5000 4000 3000 2000 1000 0 HATAY YERLİSİ

KALEM

HAVUÇ

HATAY KIRMIZISI

REFERENCES Laurie SM, Van Heerden SM., 2015. Consumer acceptability of four products made from betacarotene-rich sweet potato. Afr J Food Sci. 2012;6:96–103. http:// dx.doi.org/10.5897/AJFS12.014 Ofori G, Oduro I, Elis, HK Dapaah. 2005. Assessment of vitamin A content and sensory attributes of new sweet potato (Ipomoea batatas) genotypes in Ghana. AfricanJournal of Food Science 3, 184−192. Purseglove, J.W., 1968. Tropical Crops: Dicotyledons, Longman Scientific and Technical. John Wiley and Sons Inc., New York. Woolfe, J.A., 1992. Sweetpotato: An untapped food resource. Cambridge, UK: Cambridge University, Press and the International Potato Center (CIP). ISBN 9780521402958. Yurtsever, N., 1984. Deneysel İstatistik Metotları. Toprak ve Gübre Araştırma Enstitüsü Yayınları, Genel Yayın No: 121, Teknik Yayın No:56, Ankara.

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ROLE OF PROCESSING ON FLAVOR DEVELOPMENT IN MEAT Semanur YILDIZ1, Güliz HASKARACA1* 1

Sakarya University, Faculty of Engineering, Department of Food Engineering, Sakarya, Turkey *Corresponding author’s e-mail: gyaldirak@sakarya.edu.tr

ABSTRACT Flavor is one of the leading sensorial impressions of meat quality that affects the consumer purchase decision towards meat products. Fat and low molecular weight water soluble compounds play a major role in the formation of meat flavor. Some processes that applied to meat after slaughtering can generate some changes in these precursors and lead to the further development of meat flavor. Since consumers demand safe meat products with high quality, extended shelf life as well as maintained natural flavor and taste, nonthermal technologies or alternative mild processing methods have been implemented for preservation and decontamination of meat products to satisfy the consumers’ requests. High pressure processing (HPP), irradiation, bio-preservation and use of natural antimicrobials are some of the alternative technologies that promise natural attributes and flavor in addition to safety. Thus, this study was focused on the recent advances on development of meat flavor after processing by thermal and nonthermal technologies. Keywords: Nonthermal technologies, Mild processing, Meat flavor, Flavor formation, High pressure processing, Irradiation, Cold storage, Biopreservation

INTRODUCTION Meat Flavor Flavor is one of the most important indicators of food quality that highly affect the consumer acceptance and preferences (Khan et al., 2015) since consumers have a demand on consumption of food with desired flavor. Flavor is generally defined with the combined effect of smell and taste. Although the taste and smell are separate senses and have their own receptor organs, they are intertwined senses in flavor formation. During eating, chemical components that formed taste are detected by taste buds, which consist of special sensory cells. After the stimulation, these cells send signals to specific areas of the brain and make us aware of taste perception. Besides, for perception of smell, specialized cells pick up odor molecules in the air. Odorants stimulate receptor proteins found on hair like cilia at the tips of the sensory cells, and trigger off a neural response. Finally, the combined responses of taste and smell are perceived as aroma (Ba et al. 2012; Anonymous, 2012). In fact, flavor is not only combined perception of taste and smell but also it as a complex sensation that involves odor, taste, texture, temperature and pH (Pegg and Shaidi, 2014). In a broad sense, raw meat is consisting of 75% water, 18.5% protein, 3% fat, 1.5% soluble, non-protein substances, 1% carbohydrates and 1% minerals. Although the nutritional composition has been well defined for meat, the nature formation and development of flavor in meat are more complex. While the raw meat can be characterized with a metallic, bloody, serum-like sweet and slightly salty flavor, generation of volatile compounds during cooking contributes to the meat flavor as a result of the complex interaction of precursors derived from 341

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both the lean and fat compositions of meat (Oztan, 2008; Pegg and Shahidi 2014). Since further preservation technologies could have a potential influence on formation of flavor precursors, it is of great interest to evaluate their effect on the characteristics of meat flavor. Meat Flavor Precursors and Flavor Formation In the earlier studies on meat flavor, researchers recognized that the low molecular weight, water soluble compounds such as free sugars, free amino acids, peptides, vitamin, sugar phosphate, nucleotide-bound sugars and nucleotides, and fats in meat constituents are the most important precursor of aroma and flavor characteristics of cooked meat. Some processes that applied to meat after slaughtering can generate some changes in these precursors and lead to the development of meat flavor. As a result of these treatments, chemical reactions such as glycolysis, proteolysis, lipolysis, oxidation, degradation and pyrolysis occur individually or simultaneously, new components form and create the final aroma flavor characteristics of cooked meat (Khan, 2015). Since the precursors participate to the flavor formation reactions, the factors that affects the type and quality of these precursors play an important role on the flavor formation. These factors can be pre- or post-slaughtering factors such as variety, slaughtering type, gender, cooking and ageing type, age, cooking time-temperature combination, diet of the animal, storage type, fatness, storage parameters, the composition of fat and stress (Lawrie and Ledward, 2006; Khan et al., 2015). Although meat is highly desirable and nutritious, it is also highly perishable because of its nature. The physical, chemical, microbial or sensorial quality attributes reduce easily during storage/agining until cooking/processing due to the effects of O2, water, light, temperature, enzymes and pathogens. Since consumers demand safe meat products with high quality, extended shelf life, natural flavor and taste; nonthermal technologies or alternative mild processing methods have been implemented for preservation and decontamination of meat products in order to satisfy the consumers’ requests. In order to prevent the aforementioned quality changes, some thermal and non-thermal technologies have been used such as cold storage, heating (pasteurization or sterilization), high pressure processing (HPP), irradiation, bio-preservation, use of natural antimicrobials, active packaging, lyophilization or drying. All these technologies have significant effects on quality characteristics of meat as well as the meat flavor. The effects of cold storage, bio-preservation and use of natural antimicrobials, irradiation and HPP on the formation of meat flavor were discussed in the following sections. Effect of Preservation Technologies on Meat Flavor Cold Storage Cold storage have been one of the preservation methods that is used for protecting of meats from microbial spoilage for many years. Cold storage can be applied as either chill or frozen according to the purpose (chilling for slow down or frozen for stop development of microbial growth) (Aidani et al., 2014). The chill storage of meat generally conducted at the temperatures between (+4°C) - (-1°C) while temperatures below -10 °C are used for the frozen storage. However, these temperatures are recommended as +4°C for chill storage and below -18 °C for frozen storage (CRS, 2016). After slaughtering, meat flavor is enhanced by the effects of endogen enzymes throughout the agining process that occurs during the cold storage. For instance, meat flavor precursors such as free sugars (e.g. ribose), and free amino acids (e.g. phenylalanine, methionine, lysine, leucine and isoleucine) can alter during storage by the effect of oxygen and enzymes, leading to the final meat flavor (Imafidon and Spanier, 1994). The better taste was observed in beef samples of M. longissimus lumborum stored at 0–2 °C for 10 342

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to 14 days than those conditioned for 3 to 7 days which proved the positive effect of aging on the organoleptic properties such as flavor, tenderness, and juiciness. (Daszkiewicz, Wadja, and Matusevicius, 2003). But, unfortunately, lipid oxidation is the major cause of chemical deterioration in meat which limited the shelf life of meat during storage. Oxidation is an autocatalytic reaction conducted between oxygen and fat (Amaral et al., 2018). Once the reaction starts, it continue faster and form primary and secondary oxidation products such as malonedialdehyde, hexanal, etc. These products results in unacceptable “off” or “rancid” flavors and affect the meat flavor adversely (Wójciak & Dolatowski, 2012; Aidani et al., 2014). But, this oxidation process can be significantly decelerate in frozen meat if oxygen is completely eliminated and the storage temperature is excessively reduced (i.e., under -60 °C) (Pérez Chabela and Mateo - Oyague, 2006). Bio-preservation In the food industry, bio-preservation have been extensively used in order to maintain the microbial and sensorial quality of the product for many years. Meat biopreservation stands for the enhanced food safety and extended shelf life by utilizing natural or controlled microflora or their antimicrobial products. It refers to the control of pathogenic and spoilage microorganisms by a competitive microflora of desired indigenous microorganisms or starter and protective cultures. Protective cultures show their protective effects by two fundamental ways: (i) hindering the growth activity of microorganisms, especially by affecting the product pH and (ii) inactivating the other microorganisms by their metabolites such as organic acids, molecular compounds or bacteriocins (Hugas, 1998; Serdaroğlu and Sapanci Özsümer, 2000; Gálvez et al., 2014). Lactic acid bacteria (LAB) are the main players among these protective or starter cultures. However, special attention should be paid to guide the fermentation into the desired direction, to promote the development of the desired microorganisms and to suppress the development of undesired microorganisms. Improper use of LAB can result in undesired or low quality outcomes because of the different metabolites of homofermentative and heterofermentative species. While homofermantative species produce almost exclusively lactic acid from fermentable carbohydrates present in meats, which is relatively mild and palatable, heterofermantative species produce significant amounts of less desirable fermentation end products such as CO2, ethanol, acetic acid, butanoic acid and acetoin. For instance, Lactobacillus spp., especially heterofermentative species, may produce significant amounts of acetic acid, cause ropy slime and discoloration (greening), and deteriorate the meat flavor under certain conditions. Natural Antimicrobials Addition of antimicrobial additives have been one of the commonly using methods in order to ensure the microbial safety and increase shelf life of meats, for long years. Some synthetic and natural additives have been using for that aims. However, the claims that many of the additives may have negative effects on consumer health have led consumers to gain a negative view, especially about synthetic additives. For that reason, in recent years, consumers have begun to prefer foods which are produced with additives obtained from natural sources. This tendency is resulted to direct the manufacturers to produce products with natural ingredients. In order to meet this demand, natural chemical preservatives such as sodium or potassium salts of lactic acid or acetic acid have been using in food preservation for years (Bingol and Bostan, 2012; Akarca, Gök and Tomar, 2014; Paulsen and Smulders, 2003). These lactates and acetates have been categorized among the GRAS (generally recognized as safe) additives which can be directly use in foods (FDA, 2019). Although the antimicrobial and shelf life enhancing effects of lactates and acetates have been proven by several researchers, it has also been reported that they can affect or change sensorial quality parameters such as color, odor/flavor (leaving a sour 343

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mouthfeel) and drip loss (Bell et al., 1986; Greer and Dilts, 1992; Smulders, 1995; Shelef et al., 1997; Maca et al., 1999; Vasavada et al., 2003). For instance, as a result of increased concentration of K-lactate (in combination of 50% molar substitution of KCl (0–50%) and potassium lactate (0–50%)), the acid taste and saltiness were reduced while the sweetness and K-lactate flavor were increased in small calibre fermented sausages. This finding has been attributed to the higher pH and the reduction in growth of lactic acid bacteria (Guàrdia et al., 2008). On the other hand, Williams and Phillips (1998) reported that the antimicrobial effect of sodium lactates increases with the increasing in concentration, however, off-flavor development problems occur with increasing concentrations of sodium lactate above 2.0%. Irradiation Food irradiation refers to a physical process where food is subjected to ionizing energy through Gamma radiation from Cobalt (60Co) or Cesium (137Cs) radioisotopes, accelerated electrons, or machine-generated X-rays (Farkas, 2004). As shown in Figure 1, the wavelength and energy of rays has reciprocal proportion. While wavelength decreases from the radio rays to gamma rays, the corresponding energy increases. Thus, the X- and gamma-rays are very short wavelength radiations that have very high energy levels.

Figure 1. Electromagnetic spectrum (Anonymous, 2019a)

The irradiation facilitates the reduction of pathogens, inactivation of spoilage microorganisms, extension of product shelf life, disinfestation of insects and delaying of sprouting and ripening. Irradiation possess a several advantages for foods. Several practical advantages of irradiation reported by Robert et al. (2014) includes (i) minimal influence on nutritional components since it is a cold process, (ii) high efficiency against non-sporing bacteria, insects, and many other pests, (iii) avoiding chemical residues or use of chemicals, (iv) immediate distribution of foods after processing. FDA has approved irradiation of several foods such as beef and pork, crustaceans, fresh fruits and vegetables, poultry, seeds for sprouting, shell eggs, shellfish – molluscan, spices and seasonings (FDA, 2016). Irradiation dose is represented by Gray (Gy) which is equal to 100 rads (rad refers to the radiation absorbed dose). One gray (Gy) corresponds to the absorption of one joule of radiation energy by one kilogram of matter (1 Gy = 1 J/kg). According to the Joint Expert Committee on Food Irradiation, an overall average dose of 10 kGy exhibits no toxicological hazard and introduces no nutritional or microbiological problems (JECFI, 1981). In the literature, there are several studies about the irradiation of meat and meat products. Despite the several advantages 344

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of irradiation, there might be some drawbacks of this technology in terms of oxidation and volatile profile of meat and meat products. The characteristic odor of irradiated meat products have been reported as hot fat, burned oil, burned feathers, bloody and sweet, barbecued cornlike odor (Hashim et al.,1995; Heath et al.,1990; O’Bryanetal., 2008). Table 1 gives some examples of earlier studies related to the changes in lipid oxidation and volatile compounds after irradiation. Feng et al. (2017) reported that the lipid oxidation was accelerated by the irradiation process at 4.5 kGy in raw turkey breast meat comparing to control samples. Also dimethyldisulfide and hexanal were found responsible for the irradiation off-odor in irradiated raw turkey breast meat. Li et al. (2017) reported that application of irradiation higher than 7 kGy could make the fresh pork samples produce obvious irradiation off-odor and added that lipid oxidation was also promoted by irradiation. In another study about cured turkey meat products, it was found that protein oxidation increased at 4.5 kGy and the amount of sulfur compounds increased linearly as the doses increased (Feng and Ahn, 2016). In the same study, the authors stated limited effects of irradiation on volatile profile of turkey sausage up to 1.5 kGy. Therefore, it is relevant to suggest that irradiation doses should be optimized in order to avoid any possible off-flavor formation after irradiation. Table 1. Effect of irradiation on volatile profile of meat Treatment conditions raw turkey 1.5 - 4.5 kGy breast meat Product

Main findings

Reference Feng et al. (2017)

fresh pork

3 - 7 kGy

• Lipid oxidation was accelerated from 0.026 to 0.055 mg malonaldehyde per kilogram meat by irradiation at 4.5 kGy. • Dimethyldisulfide and hexanal are responsible for the irradiation off-odor. • Irradiation ≥7 kGy could make the samples produce obvious irradiation off-odor. • After 7 days storage, irradiation off-odor was reduced. • Lipid oxidation was also promoted by irradiation. • Benzyl methyl sulfide was produced newly and significantly increased (p< 0.05) by irradiation.

cured turkey meat products

1.5 – 4.5 kGy

• Up to 1.5 kGy irradiation had limited effects on the volatile profile of turkey sausage. • Protein oxidation increased at 4.5 kGy. • The amount of sulfur compounds increased linearly as doses increased in RTE turkey meat products.

Feng ve Ahn, (2016)

Li et al. (2017)

High Pressure Processing High pressure processing (HPP) is a commercially applicable technology that employs pressures in the range of 100-800 MPa with or without addition of heat (Balasubramaniam, Martínez-Monteagudo & Gupta, 2015). The fundamentals of HPP is based on the two principles i.e. Le Chatelier’s principle and isostatic principle. The Le Chatelier’s principle states that “the pressure shifts the system equilibrium toward the state that occupies the smallest volume” while isostatic principles refers to the uniform distribution of the pressure throughout the entire sample Balasubramaniam et al., (2008). Thus, the processing time is independent of sample size and shape, assuming uniform distribution within the sample. Figure 2 shows a schematic representation of a HPP treatment system. Briefly, the system is composed of pressure vessel, closure(s) for sealing the vessel, high-pressure intensifier pump(s), control system monitoring 345

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the pressure and (optionally) temperature, product-handling system for transferring product to and from the pressure vessel (Balasubramaniam et al., 2008). In this kind of application, packed foods are inserted to the pressure vessel and pressure is applied to the product in liquid body.

Figure 2. Schematic representation of HPP treatment system (Anonymous, 2019b) Figure 3 shows the processing line of HPP. In this system, packed product enter to the treatment chamber through the conveyor band, the treatment chamber fills with pressure transmitting water, and then pressurization is applied at the pressure and time combinations. HPP can be employed for different types of foods including fresh cut fruits, vegetables, and juices, dressings, soups, seafood and shellfish, dips, spreads, salsa, and meat-based products. There are some advantages of HPP to foods such as elimination of pathogens, extension of shelf-life, producing a clean label and an innovative fresh products (Hogan et al., 2005).

Figure 3. The continuous application system of HPP (revised from Anonymous 2019c)

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HPP could provide the opportunity to preserve the characteristic meat flavor. Effect of HPP on meat flavor has been investigated in terms of TBARS, hexanal, and many other volatile compounds as represented in Table 2. For instance, Beltran et al. (2003) reported that HPP resulted in less oxidation compounds compared to the thermally treated minced chicken breast. The authors also found out lower hexanal content at pressures less than 500 MPa. It should be taken into consideration that HPP could also increase the levels of methanethiol and sulfur dioxide as reported in the study of Martinez-Onandi et al. (2016) where HPP was applied to Serrano dry-cured ham at 600 MPa for 6 min at 21 Â°C. On the other hand, higher levels of ethyl acetate, ethyl butanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, dimethyl disulfide and dimethyl trisulfide were reported for untreated control samples. Regarding the refrigerated storage of HPP treated and untreated meat products, Rivas-CaĂąedo et al. (2011) achieved fewer changes in pressurized ground beef samples compared to the untreated ones indicating a volatile profile closer to that of control ground beef samples. In addition to volatile compounds, Lou et al. (2018) observed that HPP at 150 MPa increased the taste intensity of vinasse-cured ducks compared to the controls while no further improvement was obtained at 300 MPa. Thus, the pressure levels should be optimized for different types of food matrices in order to avoid undesired oxidation compounds and volatiles as well as to preserve the natural meat flavor compared to untreated meat products.

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Table 2. Effect of high pressure processing on flavor of different types of meats Product

Treatment conditions

Analysed property

Chicken HPP: 300 and 500 TBARS breast MPa for 30 min at 20 hexanal (minced) °C.

Main findings

Reference

HPP at 300 MPa resulted in lower hexanal compared to HPP Beltran et al. (2003) at 500 MPa.

Pressurized samples presented less oxidation compounds than cooked samples.

Thermal: 90 °C for 15 min. Storage: 9 days at 4 °C Serrano drycured ham

HPP: 600 MPa for 6 volatile min at 21 °C compounds

· higher levels of methanethiol and sulfur dioxide in HPP- MartínezOnandi et treated samples al. (2016) · Untreated control samples contained higher levels of ethyl acetate, ethyl butanoate, ethyl 2methylbutanoate, ethyl 3methylbutanoate, dimethyl disulfide and dimethyl trisulfide. · NaCl concentration significantly influenced the volatile profile. ·

vinassecured ducks

HPP: 0.1, 150 and taste 300 MPa, 15 min.

ground beef

HPP: 400 MPa, 10 volatile min at 12 C. compounds

HPP at 150 MPa increased the taste intensity of products Lou et al. (2018) compared to the controls.

· HPP at 300 MPa did not further improve the taste.

Refrigerated storage: 3 days

· Pressurized samples underwent fewer changes than non- Rivaspressurized ones during Cañedo et refrigerated storage, leading to a al. (2011) volatile profile closer to that of control beef

CONCLUSION This review has summarized the formation and development of meat flavor by focusing on the applications of physical preservation methods (i.e. cold storage, biopreservation, natural antimicrobials, irradiation, and HPP). It is remarkable to indicate that these technologies 348

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provide opportunities to maintain the characteristic meat flavor; however, their excessively intense applications may further result in undesired off-flavor formation. Optimized conditions should be considered in order to benefit the preservation effect of such technologies on the fresh-like attributes of meat compared to the untreated products.

REFERENCES Aidani E., B. Aghamohammadi, M. Akbarian, A. Morshedi, M. Hadidi, N. Ghasemkhani, A. Akbarian (2014). Effect of chilling, freezing and thawing on meat quality: a review. Int. J. Biosci., 5(4):159-169. Akarca G., V. Gök, O. Tomar (2014). Gıda Muhafazasında Kullanılan Bazı Doğal Antimikrobiyaller. Kocatepe Vet. J., 7(1): 59-68. Amaral A. B., M. V. Silva, S. C. S. Lannes (2018). Lipid oxidation in meat: mechanisms and protective factors – a review. Food Sci. Technol, Campinas, 38(Suppl. 1): 1-15. Anonymous (2012). BrainFacts/SfN. Taste and Smell. Web site: https://www.brainfacts.org/thinking-sensing-and-behaving/taste/2012/taste-and-smell Anonymous (2019a). Web site: https://sites.google.com/site/chempendix/em-spectrum. Accessed date: 01 Nov 2019. Anonymous. (2019b). Web site: http://en.gazkhodro.ir/food-high-pressure-processing-hpp/ Accesssed date: 21 Nov 2019. Anonymous. (2019c). Web site: https://www.hiperbaric.com/en/high-pressure. Accessed date: 01 Nov 2019. Ba V. H., I. Howang, D. Jeong, A. Touseef (2012). Principle of Meat Aroma Flavors and Future Prospect (Chapter 7), in book: Latest Research into Quality Control, Isin Akyar (ed). Intech Open. DOI: 10.5772/45955, ISBN: 978-953-51-0868-9. Balasubramaniam V. M., D. Farkas, E. J. Turek (2008). Preserving foods through high-pressure processing. Food technology. Balasubramaniam, V. M., S. I. Martínez-Monteagudo, R. Gupta (2015). Principles and application of high pressure–based technologies in the food industry. Annu Rev Food Sci. T., 6:435-462. Bell M. F., R. T. Marshall, M. E. Anderson (1986). Microbiological and Sensory Tests of Beef Treated with Acetic and Formic Acids. J. Food Prot., 49(3): 207-210. Beltran E., R. Pla, J. Yuste, M. Mor-Mur (2003). Lipid oxidation of pressurized and cooked chicken: role of sodium chloride and mechanical processing on TBARS and hexanal values. Meat Sci., 64(1):19-25. Bingöl E. B., K. Bostan (2012). Usage of lactates in meat and meat products as a food additive. J. Fac. Vet. Med. İstanbul Üniv., 38 (1), 79-88 (article in Turkish with an English abstract). CRS (2016). Guide to meat storage and handling. https://www.crscoldstorage.co.uk/knowledge/meat-cold-storage.html#freezing Accessed date: 02 Nov 2019. Farkas J. (2004). Food irradiation. In A. Mozumder, & Y. Hatano (Eds.), Charged particle and photon interactions with matter (pp. 785 - 812). New York, Basel: Marcel Dekker, Inc. FDA (2016). Food and Drug Administration, Food Facts, Food Irradiation: What You Need To Know. https://www.fda.gov/media/81259/download FDA (2019). Food and Drug Administration, CFR - Code of Federal Regulations. Title 21, Volume 3, Part 184 - Direct Food Substances Affirmed as Generally Recognized as Safe. Revised as of April 1, 2019.

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Feng X., S. H. Moon, H.Y. Lee, D. U. Ahn (2017). Effect of irradiation on the parameters that influence quality characteristics of raw turkey breast meat. Radiat Phys Chem., 130: 4046. Feng X., D. U. Ahn (2016). Volatile profile, lipid oxidation and protein oxidation of irradiated ready-to-eat cured turkey meat products. Radiat Phys Chem., 127:27-33. Gálvez A., R. L. López, R. P. Pulido, M. J. G. Burgos (2014). Biopreservation of Meats and Meat Products. In: Food Biopreservation. Springer Briefs in Food, Health, and Nutrition. Springer, New York, NY. Online ISBN 978-1-4939-2029-7. Guàrdia M.D., L. Guerrero, J. Gelabert, P. Gou, J. Arnau (2008).Sensory characterisation and consumer acceptability of small calibre fermented sausages with 50% substitution of NaCl by mixtures of KCl and potassium lactate. Meat Sci., 80(4):1225-1230. Greer G. G., B. D. Dilts (1992). Factors affecting the susceptibility of meatborne pathogens and spoilage bacteria to organic acids. Food Res. Int., 25(5): 355-364. Hashim I. B., A. V. A. Resurreccion, K. H. McWalters (1995). Descriptive sensory analysis of irradiated frozen or refrigerated chicken. J Food Sci., 60(4):664-666. Heath J. L., S. L. Owens, S. Tesch, K. W. Hannah (1990). Effect of high-energy electron irradiation of chicken meat on thiobarbituric acid values, shear values, odor, and cooked yield. Poultry Sci., 69(2):313-319. Hogan E., A. L. Kelly,D. W. Sun (2005). High pressure processing of foods: an overview. In Emerging technologies for food processing (pp. 3-32). Academic Press. Hugas M. (1998). Bacteriocinogenic lactic acid bacteria for the biopreservation of meat and meat products. Meat Sci., 41(1):139-150. Imafidon, G.I., A.M. Spanier (1994). Unraveling the secret of meat flavor. Trends Food Sci. Tech., 5: 315-321. JECFI, Joint FAO/IAEA/WHO Expert Committee on Food Irradiation,1981. Wholesomeness of Irradiated Food. Technical Report Series no. 659. World Health Organization, Geneva,Switzerland. Khan M. I., C. Jo, M. R. Tariq (2015). Meat flavor precursors and factors influencing flavor precursors-A systematic review. Meat Sci., 110:278–284. Lawrie R.A., D. A. Ledward (2006). Lawrie’s Meat Science (7th edition). Woodhead Publishing Limited and CRC Press LLC. P 461. Li C., L. He, G. Jin, S. Ma, W. Wu, L. Gai (2017). Effect of different irradiation dose treatment on the lipid oxidation, instrumental color and volatiles of fresh pork and their changes during storage. Meat Sci., 128:68-76. Lou X., Y. Ye, Y. Wang, Y. Sun, D. Pan, J. Cao (2018). Effect of high-pressure treatment on taste and metabolite profiles of ducks with two different vinasse-curing processes. Food Res. Int., 105:703-712. Maca J.V., R.K. Miller, M.E. Bigner, L.M. Lucia, G.R. Acuff (1999). Sodium lactate and storage temperature effects on shelf life of vacuum packaged beef top rounds. Meat Sci., 53(1): 23-29. Martínez-Onandi N., A. Rivas-Cañedo, M. Nuñez, A. Picon (2016). Effect of chemical composition and high pressure processing on the volatile fraction of Serrano dry-cured ham. Meat Sci., 111:130-138. O'bryan C. A., P. G. Crandall, S. C. Ricke, D. G. Olson (2008). Impact of irradiation on the safety and quality of poultry and meat products: a review. Crit Rev Food Sci., 48(5): 442457. Oztan A. (2008). Meat Science and Technology/ Et Bilimi ve Teknolojisi (in Turkish). TMMOB Gıda Mühendisleri Odası Yayınları, publication no:1, Ankara, Turkey. p 256. ISBN: 975- 395- 632-0

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Paulsen P., F. J. M. Smulders (2003). Combining Natural Antimicrobial Systems With Other Preservation Techniques: The Case of Meat. In eds: Zeuthen and Bøgh-Sørensen. Food Preservation Techniques, Woodhead Publishing Limited and CRC Press, p 581. Pegg R. B., F. Shahidi (2014). Cooking of meat, in book: flavor development. M. Dikeman, C. Devine (Eds.), Encyclopedia of meat sciences (Second edition), Academic Press, Oxford, p. 377-384. Pérez Chabela M. L., J. Mateo - Oyague (2006). Frozen meat: Quality and shelf life . In eds Y. H. Hui. Handbook of Food Science, Technology, and Engineering, CRC Press, Taylor and Francis Group. Rivas-Cañedo, A., C. Juez-Ojeda, M. Nuñez, E. Fernández-García (2011). Volatile compounds in ground beef subjected to high pressure processing: A comparison of dynamic headspace and solid-phase microextraction. Food Chem., 124(3):1201-1207. Roberts, P. B. (2014). Food irradiation is safe: Half a century of studies. Radiat Phys Chem., 105:78-82. Serdaroğlu M, M. Sapanci Özsümer (2000). The Importance of Bacteriocins in Meat and Meat Products. Pamukkale Univ Muh Bilim Derg., 6(3): 211-217. Shelef L. A., S. Mohammed, T. Wei, M. L. Webber (1997) Rapid optical measurements of microbial contamination in raw ground beef and effects of citrate and lactate. J. Food Prot., 60:673-676. Smulders F. J. M. (1995). Preservation by microbial decontamination; the surface treatment of meats by organic acids. In eds: Gould G. W. New Methods of Preservation, London, Blackie Academic & Professional, 253-282. Vasavada M., C.E. Carpenter, D.P. Cornforth, V. Ghorpade (2003). Sodium levulinate and sodium lactate effects on microbial growth and stability of fresh pork and turkey sausages. J. Muscle Foods, 14:119-129. Williams S. K., K. Phillip (1998) Sodium Lactate Affects Sensory and Objective Characteristics of Tray-Packed Broiler Chicken Breast Meat. Poultry Sci., 77:765-769. Wójciak, K. M., Z. J. Dolatowski (2012). Oxidative stability of fermented meat products. Acta Scientiarum Polonorum. Technologia Alimentaria, 11(2): 99-109.

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REMOVAL OF METHYLENE BLUE DYE BY ADSORPTION ON AN ALGERIAN MONTMORILLONITE CLAY Belal SALIHA*1, Mokhtari FATIHA1, Benaissa KHEIRA2, Lambert STEPHANIE3 1

Laboratoire des Eco-Matériaux et Nanostructurés, Faculté de Chimie, Département de Génie des Matériaux, Université des Sciences et de la Technologie d’Oran Mohamed Boudiaf, ElM’naouar, Oran, Algeria 2 3

University Centre of Tamanghasset, BP 10034 Serssouf, 11000 Tamanrasset, Algeria

Laboratory of Chemical Engineering, Faculty of Applied Sciences, Applied Chemistry Department, University of Liège, Belgium *Corresponding author: saliha.belal@univ-usto.dz

ABSTRACT The capacity of an Algerian montmorillonite clay to remove methylene blue was investigated. This research paper is articulated on the physicochemical characterization of natural clay sampled from the region of Maghnia city (Western of Algeria). Several techniques were used such as X-ray diffraction, infrared spectroscopy, and electronic scan microscopical board, in addition to the determination of some physicochemical properties as CEC and PZC. Kinetic experiments were carried out to study the effect of contact time, stirring speed initial dye concentration and adsorbent mass on the adsorption processes. This enabled us to establish a series of data with these different properties of our materials: AMN, AMN Intercaled, AMNCa2+ saturated and Intercaled. The obtained adsorption isotherm indicates high affinity between the adsorbent and the adsorbate. The use of the studied Algerian montmorillonite clay may have great potential for the removal of methylene blue dye. Keywords: Algerian montmorillonite, AMN, Methylene blue, Cationic dye, Ca2+, Adsorption, X-ray diffraction, FTIR, MEB, CEC, PZC

INTRODUCTION Nowadays, the potable water resources are strongly reduced following the demographic increase accompanied by a strong industrialization and the intensive development of agriculture. Rejections of micro-pollutants (pesticides, dyes, phenols, heavy metals…etc.) in the environment is increasingly important. These non-degradable and toxic micro-pollutants are in general the origin of many diseases. They also directly affect the ecosystems balance following the deterioration of the quality of the various mediums of the environment (ground, water, air). Therefore, the concern of developing processes which work with the safeguarding of the water resources against pollution by the treatment the polluted water source. The application of the natural and abundant adsorbents as the clay in water treatment is a legitimate way to preserve the hydrous resources. To that end, the present research paper was articulated around the chemical and the physicochemical characterization of a natural Montmorillonite clay (AMN) sampled from the region of Maghnia (Western Algeria). Several techniques were employed; X-ray diffraction, infrared spectroscopy, and electronic scan microscopical board in addition to the determination of some physicochemical properties as the 352

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CEC and the PZC. This enabled us to establish a series of data with different properties of these materials: AMN, AMN Intercaled, AMNCa2+ saturated and Intercaled. The main aim of this study is to investigate the capacity of this Algerian montmorillonite to remove a typical cationic dye, methylene blue, from aqueous solutions by changing initial mass, contact time, stirring rate, temperature, and dye concentration. MATERIALS AND METHODS Materials The raw montmorillonite used in this study is from Maghnia city (Hammam Boughrara, Western Algeria). The material was ground because its grain size was not adequate for test purposes (i.e. ≤ 10 µm); the cation exchange capacity (CEC) is 86.61meq/100g of clay. The montmorillonite is natural and all reagents are analytical grade and used without further purification. The clay was air dried, first at ambient temperature (25°C) then at 100°C in an oven during 24 hours, according to (Mouni et al., 2018) with slight modifications. Chemical properties of the raw AMN sample were measured using Philips (Magi XPRO) spectrophotometer and are shown in Table 1. As it is mentioned, SiO2 and Al2O3 are the main components of the clay (60.06% and 15.9%, respectively), with other oxides presents in much smaller amount. This raw clay sample will be denoted AMN–Argile de Maghnia Naturelle in the following discussion. Table 1. Composition of the raw AMN sample Constituent SiO2 Al2O3 Fe2O3 Cao MgO SO3 K 2O Cl Na2O LSF Silica ratio Aluminium ratio

Content 60.06% 15.9% 4.48% 0.62% 5.83% 0.09% 0.37% 0.197% 2.16% 0.33 2.95 3.55

+ CH3 N

CH3 +

H3C

N CH3

Figure 1. Chemical structure of MB

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Methods Characterization Clay Electronic Scan Microscopical Board (MEB) The texture of the AMN sample was studied using a Philips ESEM XL 30 board scanning electron microscope equipped with an X-ray energetically dispersion spectrophotometer (EDX), used in the morphological observation of clays and in the qualitative determination in chemical elements composition. As it is shown in Figure 2, the board of electronic scan microscopy (MEB) of AMN presents the organization of the clay particles in the form of an assembly of aggregates, which is due to the presence of impurities.

Figure 2. Electronic scan microscopical board (MEB) photo of AMN X-ray diffraction (XRD) Powder X-ray diffraction (XRD) analyses were made in order to characterize the interlayer distance of the clay particles. Minerals in the montmorillonite were identified using an X-ray diffractometer (PANalytical X'PERT PRO, ®Poland). PANalytical X'PERT PRO has an automatic system of charger and uses the detector X' CELERATOR (standard linear, detects the X-ray along a line) which allows a fast and precise study. The incident beam was Cu-Kα radiation (λ=1.5406 Å) from a rotating X-ray anode at 40 mA and 45 kV. The instrument was equipped with a diffracted beam monochromator. The scan range was 4-70º2Ɵ. Fourier Transform Infrared (FTIR) Fourier transform infrared (FTIR) spectrum of the samples were recorded using FTIR-8400 Infrared Spectrophotometer of SHIMADZU. Samples were placed in a sample holder as KBr pellets on a Bruker (Model Alpha) spectrometer at room temperature scanning over a 400-4000 cm-1 range with 2cm-1 resolution.

RESULTS AND DISCUSSION Characterization of The Raw AMN Clay Sample Chemical Composition In the literature, authors are presenting the report (SiO2/AlO3) as being a qualitative index (Mansri, 2011). In the Table 1, it has shown that our Algerian montmorillonite clay is associated 354

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with impurities and it is noted that the presence of iron (Fe3+) and magnesium (Mg2+) content is significant. The quantity of sodium is more important than that of calcium within our clay. The substituent report silica / alumina is about 3.77 (maximum substitution of Si4+ by Al3+) and 2.73 (maximum substitution of the ion Al3+ by Mg2+). The molar report silica/alumina is estimated at 6.46, this value indicates that our clay is not a pure montmorillonite, which is not in the previously mentioned interval0 [2-5.5].

According to the results of the X-rays diffraction, the major crystalline phases contained in the raw AMN are the following minerals: Montmorillonite, Quartz and the Illite. The crystalline impurities (non argillaceous minerals) are composed primarily of Quartz, calcite and dolomite. The DRX of AMN shows that it consists of a mixture inter laminated of Montmorillonite–Illite. It also contains many impurities such as Dolomite, Calcite, Cristobalite, and Quartz, whose results are summarized on the Table. 4. The examination of the DRX of a raw AMN sample, shows the presence of peak relating to Montmorillonite, in particular with (d = 4.47 Å, 2Ɵ = 19,84°) is indexed as being the line (020) characteristic of Montmorillonite. On the other hand the peaks with (d= 2.58 Å, d= 1.69 Å) for the plan HKL (003) are allotted to the presence of the illite. Certain peaks were identified as impurities. The presence of Quartz with (d= 3.35 Å, d = 4.23 Å) is selected to the plans HKL (101) and (111) respectively containing impurities 355

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such as Quartz and Cristobalite, respectively. It is noted also the presence of Calcite with (d = 2.99 Å, d = 2.27 Å) which exists in very small quantity in the plan HKL (011). The reflection with (d = 2.89 Å)) on the spectrum prove the presence of Dolomite in the plans HKL (011). The DRX of modified clay AMNCa2+ make it possible to follow the evolution of the interfoliaceous distance. This distance is due to the presence of the cations Ca2+. This phenomenon is due to the delamination of the argillaceous layers and by comparing the evolution of this distance to different intercaled materials. Table 1. Inter-articular distance of AMN Minerals

d(Å)

Plan HKL

Impacts

001 020 004 200 060

Quartz

Montmorillonite

15.62 4.45 3.77 2.57 1.49 9.42

003

Illite

d(Å) 3.34 1.54

Plan HKL 101 211

Cristobalite

3.25

111

Dolomite

2.23

113

Calcite

3.039

011

CONCLUSION SEM scanning (chemical composition) microscopy presents an organization of the clay particles in the form of an aggregate assembly (presence of impurities). The mineralogical study of the chemical analysis of our sample affected by X-ray fluorescence according to some authors the ratio (SiO2 / AlO3) varies between 2 and 5.5, indicating that our material is actually a montmorillonite clay associated with impurities. The characterization results show that the saturated and intercalated materials display an increase in interlayer distance. The behavior study of natural, saturated and intercalated materials in the adsorption of methylene blue revealed remarkable structural properties. The kinetic study showed that the process of adsorption of the MB on montmorillonite takes place in a rapid step.

REFERENCES Almeida, C. a. P., Debacher, N.A., Downs, A.J., Cottet, L., Mello, C. a. D., 2009. Removal of methylene blue from colored effluents by adsorption on montmorillonite clay. J. Colloid Interface Sci. 332, 46–53. G. Mc Kay, 1996. Use of adsorbents for the removal of pollutants form. wastewaters. J Gregg, K.S.W. Sing, 1982. Adsorption, surface area and porosity, 2nd edition. ed. Academic Press, New York - USA. Mansri, A., 2011. VANADIUM SUPPORTE SUR MONTMORILLONITE INTER CALEE AU TITANE V/TI -PILC: PREPARATION, CARACTERISATION ET REACTIVITE EN EPOXYDATION DU CYCLOHEXENE (Working Paper). Mouni, L., Belkhiri, L., Bollinger, J.-C., Bouzaza, A., Assadi, A., Tirri, A., Dahmoune, F., Madani, K., Remini, H., 2018. Removal of Methylene Blue from aqueous solutions by adsorption on Kaolin: Kinetic and equilibrium studies. Appl. Clay Sci. 153, 38–45. https://doi.org/10.1016/j.clay.2017.11.034 Tong, D.S., Wu, C.W., Adebajo, M.O., Jin, G.C., Yu, W.H., Ji, S.F., Zhou, C.H., 2018. Adsorption of methylene blue from aqueous solution onto porous cellulose-derived

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carbon/montmorillonite nanocomposites. Appl. Clay Sci. 161, 256–264. https://doi.org/10.1016/j.clay.2018.02.017 Wang, G., Wang, S., Sun, Z., Zheng, S., Xi, Y., 2017. Structures of nonionic surfactant modified montmorillonites and their enhanced adsorption capacities towards a cationic organic dye. Appl. Clay Sci. 148, 1–10. https://doi.org/10.1016/j.clay.2017.08.001

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THE EFFECT OF USING SPICES ON PHYSICAL, SENSORIAL PROPERTIES AND VITAMIN C CONTENT OF RED BEET CHIPS Sezin ERDOĞAN1, Pelin Günç ERGÖNÜL1* 1

Manisa Celal Bayar University, Faculty of Engineering, Food Engineering Department, Sehit Prof Dr Ilhan Varank Campus, Manisa, Turkey *Corresponding author’s e-mail: pelin.ergonul@cbu.edu.tr

ABSTRACT Beetroot which widely consumed in our country, is root vegetable rich in bioactive components because of its high antioxidant content. Red beet that contains iron has regulatory effect on anemia and on blood pressure. It also has betaxanthin and betacyanin pigments and it is rich in phenolic substances. Because of these properties, it was aimed to produce beetroot as chips. At this point of view, this new healthy snack product has been developed without any pretreatment and any chemical additive. In this study, the effects of spices added to beetroot chips on vitamin C, color values, sensory and textural properties of beet chips were investigated. For this purpose, fresh beets were supplied from a local producer in Manisa during its own season. Red beetroots were sliced into 5 mm thickness and flavored with 3% olive oil, 0.6% salt and 0.2% in different spices (thyme, coriander, garlic powder, red pepper powder). Then they were dried at a rotary tray dryer at 60oC/390 minutes. While the highest L values were found in beet chips aromatised with garlic and highest a and b values were found in beet chips with red pepper. Also, it was found statistically significant differences between a values of spicy chips (p<0.05). According to the results of vitamin C, the significant difference was determined between the control group and beet chips with garlic (p<0.05). Similarly, the effect of using spices was found statistically significant on textural properties (p<0.05). According to sensory analysis results the beetroot chips containing garlic powder received the highest score in terms of overall acceptability. Keywords: Antioxidant, Beet, Chips, Drying, Spices INTRODUCTION Beta vulgaris (red beet), which is belonging to the Chenopodiaceae family, grown in north and south of Europe, and on the coastline of our country. The regions that have highest production and consumption are the Aegean and Marmara (Er, 2011). Red beet contains 12-20% dry matter, 4-12% sugar, 1.5% protein, 0.1% fat, 0.8% fiber and sodium, potassium, phosphorus, calcium and iron as minerals. It also contains a small amount of vitamins (Janiszewska, 2014). Red beet is the most important source of betalain color pigment divided into two groups as betacyanine (red-purple) and betaxanthin (yellow) (Nemzer et al., 2011). Betalain pigment is used as a natural additive in cosmetic, pharmaceutical and food industries. For this purpose, beet can be dried or evaluated in concentrated form. Betalaines have been found to be more effective antioxidants than other antioxidant compounds such as ascorbic acid, routine and catechin (Gengatharan, Dykes and Choo, 2015). In addition, these color pigments have the ability to inhibit cervical ovarian and bladder cancer cells in vitro (Ravichandran et al., 2013). It prevents hypertension, helps to preserve heart health and ensures the healthy functioning of the kidneys (Er, 2011). 358

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In this study, it was aimed to produce different forms of red beet that is frequently produced and consumed in our country. In this respect, red beet slices flavored with various spices were produced as beet chips. Thus, an alternative healthy product was developed in terms of different taste and ease of consumption without any pretreatment MATERIAL AND METHODS Material Red beets, olive oil, wheat starch, lemon and salt used in this study was purchased from the local market and spices (thyme, garlic powder, coriander and sweet red pepper) purchased from the herbalist in Manisa. Productions were carried out with a total 3 replicates as one control sample and 4 aromatised samples. The samples were coded as follows: K: Control (without spices), KB: Blended product with red chili powder, KIS: Blended product with coriander, KEK: Blended product with thyme and S: Blended product with garlic powder Production Steps of Red Beet Chips Beets are cleaned, peeled and sliced at 5 mm thickness. Then they soaked in 2% citric acid solution for 5 minutes after this slices dipped in a 0.5% starch solution, blended with olive oil and salt. Finally, slices were blended with spices separately and dried at 60oC/390 min rotary tray dryer. Physical Analysis of Beet Chips The color of chips was measured with Bench-top Colorimeter (CR-5) as a L (black/white), a (red/green), b (blue/yellow) values (Bhaduri, S., 2013). The hardness of chips was measured with TA-XT-2 Texture Analyser (Texture Technologies Corp., Scarsdale, NY) according to the Salvador et al. (2019). Speeds modified before, during and after test as 10 mm/s, 1 mm/s and 10 mm/s, respectively. Sensorial Analysis of Beet Chips Scoring test was conducted with 7 trained panelists. They evaluated 4 different parameters as color, crispness, aroma and general acceptability by giving a score between 1-5 for each parameter and sum of these scores for each sample was determined as a total score value (Onoğur and Elmacı, 2011). Vitamin C Analysis on Beet Chips In this analysis, the method of Cemeroğlu, (2013) was used. In the preparation stage, 5 g of the solid sample was weighed and homogenized with the same amount of 6% oxalic acid. 3 g of the sample taken from the mixture was transferred to a 100 ml flask and filled to the volume line with 3% oxalic acid and filtered. 20 ml of the sample was transferred to the flask. After dropping 2-3 drops of 1% starch solution as an indicator, titration was made with 0.001 N iodine solution in the burette. Ascorbic acid concentration in the sample was calculated by the following formula: Ascorbic Acid (mg / 100g) = (V / M) * 8.806 (3.4) V: Net amount of 0.001 N iodine solution spent in titration, ml M: Sample amount, g 359

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Statistically Analysis All values given are the average of three replicates. Data were subjected to the variance analysis using by Statistically Analyses Programme (SAS, 2001). Statistically significant differences were determined (p<0.05) and Duncan test was used for different samples. RESULTS Color analysis results As seen as in the Table 1, L values of the samples ranged between 20.35 and 20.95 and b values ranged between 1.42 and 2.01. It was concluded that the effect of spice addition was not statistically significant on the L and b values of chips (p>0.05). The highest a value was measured in the control sample as 12.35, the lowest a value was measured in beet chips containing thyme as 8.45. Adding thyme, garlic powder and coriander to the control samples was significantly affected a values (p<0.05). Table 1. Color values of beet chips Samples L a b a** a* a** K 1.97±0.25 20.55±1.02 12.35±1.95 a ba a KB 2.01±0.17 20.58±0.33 10.27±1.43 a b a KEK 1.42±0.52 20.35±1.42 8.45±1.01 a b a KIS 1.63±0.21 20.51±1.39 8.59±1.02 a

1.50±0.25 20.95±0.61 9.05±1.00 Values are mean ± standard error of the means of triplicate determinations. *Different letters indicate significance at p<0.05. **Same letters indicate not significance at p>0.05. Textural analysis results When the hardness values of chips were examined, the highest result was obtained with garlic flavored beet chips as 17.76 N (Table 2). The lowest value was found in the beet chips flavored with red pepper as 11.25 N. It was observed that adding spices had a significant effect on the hardness values of the samples (p<0.05). Table 2. Hardness values of beet chips (N) Samples Hardness cb* K 12.54 c KB 11.25 KEK

17.61

KIS

14.72

b a

17.76 Values are mean ± standard error of the means of triplicate determinations. *Different letters indicate significance at p<0.05. 360

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Sensorial analysis results As seen as from the Table 3, the highest score was obtained from beet chips containing garlic powder (4.71). There was no statistically significant difference between the control group and thyme beet chips (p>0.05). As regards to the score of color and crispness, the control sample has a highest score as 4.67 in both criteria. No statistically significant difference was observed between color and sharpness values (p>0.05). General acceptability scores ranged between 4.19 - 4.67. The highest score was obtained from beet chips containing garlic powder and the lowest score was obtained from beet chips red pepper and coriander as 4.19. Table 3. Sensorial values of beet chips Samples Color Crispness Aroma General Acceptability a** a** a* a* K 4.67±0.30 4.67±0.10 4,43±0,14 4,62±0,16 a a b b KB 4.43±0.43 4.43±0,08 4,10±0,22 4,19±0,22 a a a ba KEK 4.52±0.22 4,52±0,41 4,43±0,25 4,48±0,51 a a b b KIS 4.33±0.22 4,33±0,14 4,05±0,08 4,19±0,08 a a b a S 4.62±0.22 4,62±0,16 4,71±0,25 4,67±0,08 Values are mean ± standard error of the means of triplicate determinations. *Different letters indicate significance at p<0,05. **Same letters indicate not significance at p>0,05. Vitamin C analysis results According to the results, the highest vitamin C content was determined in the control sample as 13.19 mg AAE/100 g of dry matter (Table 4). Fresh red beets should contain approximately 3.60 mg/100 g of vitamin C. As known, drying process caused a decrease of vitamin C amounts in fruits and vegetables. A significant difference was found between the control sample and garlic flavored beet chips (p<0.05). Table 4. Vitamin C amounts of beet chips (mg AAE/100 g dry matter) Samples K

Amount

13.19±0.95 * ba

11.75±0.71

KEK

12.07±0.48

KIS

11.73±1.00

ba b

11.64±0.60 Values are mean ± standard error of the means of triplicate determinations. **Same letters indicate not significance at p>0.05.

DISCUSSION Chandran et al. (2014) examined the L, a, b values of the beet purees dried at different times (20, 40, 60 min.) and at a constant temperature (60oC). They found that L, a, b values ranged 361

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between 6.02 and 6.66, 8.76 and 11.57, 1.33 and 1.45. In another study, Hamouia (2018) found that L, a, b values ranged between 22.98 and 24.61, 15.35 and 17.63, 4.18 and 5.22 in fresh red beet pickles containing different amounts of salt (2.5%, 5.0%, 7.5%). When our results were compared with these results, it has been seen that a and b values decreased due to drying process. Oyedeji et al. (2017) examined the effect of frying on the color values of yellow fleshed cassava root slices (40mm Ă&#x2014; 25mm Ă&#x2014; 3 mm). 3 different frying methods were applied to the samples at 60â&#x2C6;&#x2DC;C, 70â&#x2C6;&#x2DC;C and 80â&#x2C6;&#x2DC;C temperatures (vacuum frying, atmospheric frying and equivalent thermal driving forces). They determined that at all the frying treatments, đ??żâ&#x2C6;&#x2014; values decreased, redness increased and yellowness value protected with increase of temperature. Peng et al. (2018) have studied instant controlled pressure drop (DIC). The effects of freezing pretreatments with different freezing time on the texture of DIC dried carrots in cubic form (10 mm Ă&#x2014; 10 mm Ă&#x2014; 40 mm) were investigated. They placed the carrots at 4 Â°C for 12 h as control sample and they freezed other samples at â&#x2C6;&#x2019;18, â&#x2C6;&#x2019;40, â&#x2C6;&#x2019;80 and â&#x2C6;&#x2019;196 Â°C for 12 h, respectively. They demonstrated a significant decrease in hardness (34.75â&#x20AC;&#x201C;39.69) in frozen samples at â&#x2C6;&#x2019;18, â&#x2C6;&#x2019;40 and â&#x2C6;&#x2019;80 Â° C. They found maximum hardness value (69.45 N) in the samples frozen by Liq.N2. Tuta and PalazoÄ&#x;lu (2017), fried and baked 1.4 mm thick potato chips at 170, 180, 190 oC and examined their texture and color values. They found the highest hardness value in the chips produced with frying at 180oC (2.85 N / mm) and in the chips produced with baking at 170oC (3.90 N/mm). When they examined the color values, they defined oven-baked chips as orangeyellow and fried samples as bright yellow. Also they found that color parameters of cooked and fried potato chips were significantly different at all temperatures (P<0.05). Nemzer et al. (2011) applied spray drying, air-drying and freeze-drying methods to the fresh red beet. They found the highest amount of Vitamin C as 557 mg AAE / 100 g in the sample obtained by spray drying method and the lowest amount was found to be <0.1 mg AAE / 100 g in the sample dried by air. Olumese ve Oboh (2016) were determined the amount of vitamin C in fresh red beet juice, red beet boiled in water and in fresh red beets dried in the oven and they found them as follows; 44.34 mg AAE / g, 30.18 mg AAE / g, and 31.14 mg AAE / g. Kaya et al. (2010), have dried kiwi fruit at different temperatures (35, 45, 55 and 65 â&#x2014;ŚC), velocities (0.3, 0.6 and 0.9 m sâ&#x2C6;&#x2019;1) and relative humidities (40%, 55%, 70% and 85%). According to the results the maximum loss in vitamin C content occurred at the lowest value of the relative humidity (Ď&#x2022; = 40%) and the highest value of the drying temperature (T = 65 â&#x2014;ŚC). Conclusions Fresh red beet is a food with high nutritional value, which is quite common consumption in various forms. In this study, fresh red beet flavored with spices and the effect of these spices on the physical, chemical, sensory and textural qualities of fresh red beet were investigated. According to the sensory analysis results, the consumption of spicy beet chips was high. Especially garlic flavored beet chips received a higher general acceptability score than the control sample. There is no more studies released on beet chips produced by similar method. It is thought that our results will shed light on the future studies. Production of beet chips without any pretreatment will bring a new product to the market.

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REFERENCES Bhaduri, S. A comprehensive study on physical properties of two gluten-free flour fortified muffins. Journal of Food Processing & Technology. 2013, 04(08), 4–7. Cemeroğlu, B.S. Gıda Analizleri. Ankara: Bizim Grup Basımevi; 2013. Chandran J., Nisha P., Singhai R.S., Pandit A.B. Degradation of colour in beetroot (Beta vulgaris L.): a kinetics study. 2014, 51(10), 2678-2684. Er, T. Kırmızı pancarın bazı fiziksel ve fitokimyasal özellikleri üzerine farklı kurutma sıcaklıklarının etkisi. Selçuk Üniversitesi, Fen Bilimleri Enstitüsü Gıda Mühendisliği Anabilim Dalı, Konya, 2011, 65 s. Gengatharan, A., Dykes, G.A., Choo, W.S. Betalains: Natural plant pigments with potential application in functional foods. Lwt. 2015, 64(2), 645–649. Hamouia, R. Kırmızı pancar (beta vulgaris l.) Turşusu üretimi süresince renk ve antioksidan özelliklerdeki değişim. Ondokuz Mayıs Üniversitesi, Fen Bilimleri Enstitüsü Gıda Mühendisliği Anabilim Dalı, Samsun, 2018. Janiszewska, E. Microencapsulated beetroot juice as a potential source of betalain. Powder Technology. 2014, 264, 190–196. Kaya A., Aydın O., Kolaylı S. Effect of different drying conditions on the vitamin C (ascorbic acid) content of Hayward kiwifruits (Actinidia deliciosa Planch). 2010, 88, 165173. Nemzer, B., Pietrzkowski, Z., Spórna, A., Stalica, P., Thresher, W., Michałowski, T., et al. Betalainic and nutritional profiles of pigment-enriched red beet root (Beta vulgaris L.) dried extracts. Food Chemistry. 2011, 127(1), 42–53. Olumese, F.E., Oboh, H.A. Antioxidant and antioxidant capacity of raw and processed nigerian beetroot (Beta vulgaris). Nigerian Journal of Basic and Applied Sciences. 2017, 24(1), 35. Onoğur, T.A., Elmacı, Y. Gıdalarda Duyusal Değerlendirme. 2nd ed. İzmir: Sidas Medya Ltd. Şti.; 2011. Oyedeji A.B., Sobukola O.P., Henshaw F., Adegunwa M.O., Ijabadeniyi O. A., Sanni L.O., Tomlins K.I. Effect of Frying Treatments on Texture and Colour Parameters of Deep Fat Fried Yellow Fleshed Cassava Chips. Journal of Food Quality. 2017. Peng J., Yi J., Bi J., Chen Q., Wu X., Chou M., Liu J. Freezing as pretreatment in instant controlled pressure drop (DIC) texturing of dried carrot chips: Impact of freezing temperature. LWT - Food Science and Technology. 2018, 89, 365-373. Ravichandran, K., Saw, N.M.M.T., Mohdaly, A.A.A., Gabr, A.M.M., Kastell, A., Riedel, H., et al. Impact of processing of red beet on betalain content and antioxidant activity. Food Research International. 2013, 50(2), 670–675. Salvador A., Varela P., Sanz T., Fiszman S.M. Understanding potato chips crispy texture by simultaneous fracture and acoustic measurements, and sensory analysis. LWT Food Science and Technology. 2009, 42, 763-767. SAS, Statistical Analysis Programme 2001. Tuta S., Palazoğlu K. Effect of bakıng and fryıng methods on Quality characteristics of potato chips. The Journal of Food. 2017, 42(1), 43-49.

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DETERMINATION OF QUALITY CHARACTERISTICS OF DIFFERENT SWEET POTATO GENOTYPES Yasin Bedrettin KARAN1*, Özlem Gültekin ŞANLI1, Şevket ÖZDEMİR1 1

Tokat Gaziosmanpa University, Faculty of Agriculture, Department of Field Crop *Corresponding author’s e-mail: yasinb.karan@gop.edu.tr

ABSTRACT This study was conducted in 2018 under Tokat-Kazova condition. The experiment was laid out in Randomized Complete Block Design, with three replications. The objective of this research was to test performance, dry matter content, protein content and antioxidant capacity and antioxidant content of different sweet potato genotypes. Dry matter contents of the four sweet potato genotypes differed from each other as well as from each other. In this study, the dry matter content of the sweet potato genotypes ranged from 28.11 to 33.36 %. The highest dry matter content was obtained from Hatay Yerlisi genotype. The overall average of the tubers in terms of the protein content % 3.68. When ranking in terms of antioxidant content was determined that Kalem (% 32.62), Hatay Yerlisi (% 32.35), Hatay Kırmızısı (% 28.66) and Havuç (% 26.80). Keywords: Sweet potato, Dry matter, Protein content, Antioxidant capacity

INTRODUCTION Sweetpotato [Ipomoea batatas (L.) Lam] originated from South America belongs to Convolvulaceae family and it is a storage root plant. Throughout world 107.6 million tons of sweetpotatoes are produced (Faostat, 2017). Sweetpotato takes the fifth place in production following rice, wheat, corn and cassava (Scott and Maldonado, 1999). Sweetpotato is one of the appreciated crops producing the highest root dry matter content for human consumption. 70% of the dry weight of sweet potato is constituted by the starch content and high dry matter content as a significant characteristic of a good sweet potato variety. Storage-roots of sweetpotato contain 30% dry matter that 70% of it starch, 5% sugar and 5% protein with vitamin A, C and B. Especially orange colored sweetpotatoes contain vitamin A (ßeta carotene) and vitamin C (Woolfe, 1992). Sweetpotato is one of the valuable crops producing the highest root dry matter content for human consumption. Sweetpotato’s root storage have anti-diabetic and anti-oxidant properties due to the presence of valuable nutritional and mineral components. Furthermore, Ipomoea batatas root storage, which are staple item in the Americans’ diet, appear to be very beneficial in the diet of diabetics and consumers with an insulin resistance, because they have a low glycemic index (Slafer, and Savin, 1994; Woolfe, 1992). The aim of the present study was to determine storage root dry matter, protein and antioxidant content of different genotypes. MATERIAL AND METHODS The experiment was conducted during the 2018 under field conditions in the Turkey-TokatKazova. The altitude of Kazova is 571 meters. In this study, four different local genotype were used. 364

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Table 1. Plant material used in the experiment Hatay Kırmızısı Havuç

Kalem Hatay Yerlisi

The experiment was laid out in Randomized Complete Block Design, with three replications. The planting process was carried out in early May in Kazova. Sweetpotato seedlings were planted as 90 cm row spacing and 45 cm plant spacing (Yurtsever, 1984). The trials were fertilized 12kg/da 15:15:15 (N:P:K) at the time of planting in both years. 8 kg/da N was applied 45 days after planting. The local sweetpotato genotype were irrigated as required to maintain adequate moisture levels by drip irrigation. When cultural and chemical methods are needed to combat diseases and pests, necessary applications have been made. RESULTS AND DISCUSSIONS Dry Matter Content (%) At harvest, two to three medium sized storage roots per genotypes were selected for dry matter determinations. 50g of each tuber samples was sliced into small pieces and dried in oven at 105°C for 2 days with three replications. Dry matter content was determined by weighing the initial and final weight, and calculating the percentage of dried weight. The same procedures were followed for all the replications. Dry matter (%) = Dry weight of the tuber/ Fresh weight of the tuber x 100

Dry Matter Content (%) 34 33 32 31 30 29 28 27 26 25 Havuç Patates

Hatay Kırmızısı

Kalem

Hatay Yerlisi

Mean comparison among dry matter contents indicated that Hatay Yerlisi and Kalem genotypes consisted of higher dry matter content than other genotypes. In this study, the dry matter content of the genotypes ranged from 28.1 to 33.4 %. Protein Content (%) Samples were first ground with IKA A11 Basic model blender and powdered and dried in BINDER brand oven at 65°C for 48 hours. Before starting the analysis of the dry and powdered samples, the calibration curve was drawn by using Aspartic Acid standard according to the 365

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sample category to be analyzed from Thermo Cookbook and the samples were weighed and analyzed in the specified amounts.

Protein Content as Dry Matter (%) 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 Havuç Patates

Hatay Kırmızısı

Kalem

Hatay Yerlisi

The highest protein content was obtained from Kalem genotype (4.6%). The lowest protein content was measured in Havuç genotype as 2.8%. Antioxidant Capacity (%) A total of 12 samples were taken in order to determine the antioxidant activities of the storage roots as a result of the production of four Sweet potato genotypes in the experiment area each with three replications.

% Inhibition 35 30 25 20 15 10 5 0 Havuç Patates

Hatay Kırmızısı

Kalem

Hatay Yerlisi

In this study, the inhibition content of the genotypes ranged from 26.8 to 32.6%. The highest inhibition content was obtained from Kalem genotype. As a result of the analysis of four of the sweet potato genotypes grown in Tokat conditions, the protein content of the storage roots were below 5% in the dry matter specified in the literature. The highest protein content was obtained from Kalem genotype. 366

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Dry matter contents indicated that Kalem genotype had higher content than other genotypes.As a result of storage root antioxidant analysis of sweet potato genotypes in the study area, the highest inhibition rate was found in Kalem genotype (32.621%). Therefore, the highest antioxidant capacity was obtained in Kalem genotype.

REFERENCES FAO, 2017. Food and Agriculture Organizition of the United Nations. http://faostat.fao.org/ Scott, G.J. and Maldonado, L., 1999. Sweetpotato facts. A compendium of key figures and analysis for 30 important sweetpotato-producing countries. CIP. Lima, Peru. Slafer, G.A. and Savin, R. 1994. Source sink relationships and grain mass at different positions within the spike in wheat. Field Crops Res. 37: 39-49 Woolfe, J.A. 1992. Sweet potato: An untapped food resource. Cambridge University Press. Cambridge, UK. pp 60. Yurtsever, N., 1984. Deneysel İstatistik Metotları. Toprak ve Gübre Araştırma Enstitüsü Yayınları, Genel Yayın No: 121, Teknik Yayın No:56, Ankara.

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DETERMINING THE PERFORMANCE OF SOME NEW POTATO CULTIVARS AND CANDIDATES IN CENTRAL BLACK SEA REGION Güngör YILMAZ1*, Yasin Bedrettin KARAN1, Fuat BARIŞ1 1

Tokat Gaziosmanpasa University, Faculty of Agriculture, Department of Field Crop, Tokat, Turkey *Corresponding author’s e-mail: gungor.yilmaz@gop.edu.tr ABSTRACT

The present study was conducted to determine yield and yield components of 15 potato genotypes in Tokat-Artova. The experiment was a randomized complete block design with four replications. In the study, some agronomic traits such as emergence time, emergence rate, shape of plant growth, observations of flower and fruit setting, number of main stems, plant height, days to flowering, days to maturity, number of tubers per hill, average tuber weight, tuber yield, marketable tuber yield and technological values were studied. According to the results, emergence times were between 27-38 days, emergence rates 87.9%-100%, the number of main stems, 4.4-8.5, plant height 33.9-92.3 cm, days to flowering 57.8-73.3, days to maturity 96-117, the number of tubers per hill 2.9-9.3, average tuber weight 47.90-96.82 g and tuber yield 699.96-3625.04 kg/da. It was concluded that genotype Alegria (st) with the highest tuber yield (3625.04 kg/da) seemed to be suitable for the region. Keywords: Solanum tuberosum, potato cultivar, potato candidate

INTRODUCTION Potato is the third most important food crop for human consumption after wheat and rice. It originated on high plateau of the Andes Mountains of South America. It has been cultivated for some 8000 years. Recent research reports that there are 108 wild and four cultivated species of potato. Solanum tuberosum is the most common tuber bearing species, and is grown worldwide Rowe 1993). Potatoes are grown in a wide range of environments. The highest potential and actual tuber dry matter productions and yield are obtained in areas with temperate climates in Turkey (Yılmaz, 2003). Some of the factors aaffecting high quality seed potato production are as follows; healthy beginning materials, altitude of production area and free of diseases and pests, experience level of growers, and production techniques (Haverkort et al., 1991). The objective of this study was to determine the performance of some new potato cultivars and candidates in Central Black Sea Region. MATERIAL AND METHODS The experiment was conducted during 2014 in Artova Plain of Tokat Province in Turkey. The altitude of Artova is 1193 metres. In this study, six different potato cultivar candidates and nine different commercial cultivars were used.

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Table 1. Plant material used in the experiment Number

Clones

Commercial Cultivar

PAİ-MUT-46

Agata

PAİ-07-18-64

Agria

PAİ-07-26-82

Lady Claire

PAİ-07-33-103

Alegria

GH-113

Basciftlik Beyazi

Lady Olympia Navigator

Dione

Bohemia

Soraya

PAI : Potato Research Institute, Nigde-Turkey GH: Aegean Agricultural Research Institute, İzmir-Turkey Tubers were planted on 11 May, 2014. Plants were irrigated as required to maintain adequate moisture levels by sprinkler irrigation. The experiment was laid out in Randomized Complete Block Design, with four replications (Yurtsever, 1984). Harvest was performed at the end of October. When cultural and chemical methods are needed to combat diseases and pests, necessary applications were made. The trial was fertilized with 12 kg/da 15:15:15 (N:P:K) at the time of planting. 8 kg/da N was applied 45 days after planting. RESULTS AND DISCUSSION Tuber Number/Plant

8,5

9,3

6,2 4,0

4,2

4,7

5,1 2,9 369

7,7

7,2

Tuber number/plant 6,7

7,8

7,1

8,6

10,0 8,0 6,0 4,0 2,0 0,0

8,7

The tuber number/plant was between 2.9 and 9.3. The lowest tuber number/plant was obtained from Bohemia cultivar, while the highest tuber number/plant was obtained from Soraya cultivar.

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Average Tuber Weight (g)

77,29

75,21

47,9

58,35

66,08

65,06

91,77

80,92

78,89

54,16

96,46

81,67

96,82

91,15

100 90 80 70 60 50 40 30 20 10 0

84,84

According to Figure, the average tuber weight ranged from 47.9 to 96.46 g. Based on the average of the experiment, the highest average tuber weight was obtained from Lady Olympia cultivar, while the lowest average tuber weight was obtained from Bohemia cultivar.

Total Tuber Yield (kg/da)

1000,00 0,00

370

1386,02

1091,12

1239,15

1400,90

3128,06

2903,64

3245,81 1931,55

2668,45

3015,10 699,96

2000,00

2950,49

3000,00

3181,59

3276,21

The highest total tuber yield was produced from Agata cultivar (3276.71 kg/da). The results showed that the lowest total tuber yield belonged to Bohemia (699.96 kg/da).

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REFERENCES Haverkort AJ, M Van de Waart, and J Marinas, 1991. Field performance of potato microtubers as a propagation material. Potato Res 34:353-364. Rowe C.R., 1993. Potato Health Management. Department of Plant Pathology. Ohio State University, USA. Yurtsever, N., 1984. Deneysel İstatistik Metotları. Toprak ve Gübre Araştırma Enstitüsü Yayınları, Genel Yayın No: 121, Teknik Yayın No:56, Ankara

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EMPATHY AND PRO-SOCIAL BEHAVIOR IN RATS Lalazar NAZAROVA*1, Aytach AHMADZADA1 1

Baku Engineering University, Baku, Azerbaijan

*Corresponding author’s e-mail: lalauzeyir1@gmail.com

ABSTRACT Whereas human pro-social behavior is often driven by empathic concern for another, it is unclear whether nonprimate mammals experience a similar motivational state. But it has previously been shown that rats behave in a pro-social manner in response to a conspecifics distress, and that there are biological roots of empathy. To test for empathically motivated prosocial behavior in rodents, we placed four free rats in an area with a cagemate trapped in a plastic restrainer. After several sessions, the free rats, occasionally hearing distress calls from its compatriot they learned to intentionally and quickly open the restrainer and free the cagemate. Rats did not open empty or object-containing restrainers. They released cagemates even when social contact was prevented. When liberating a cagemate was pitted against chocolate contained within a second restrainer, rats opened both restrainers and typically shared the chocolate. Thus, rats behave pro-socially in response to a conspecific’s distress, providing strong evidence for biological roots of empathically motivated helping behavior. Keywords: Rats, Plastic package

INTRODUCTION Empathy, from the German “Einfujhlung,” was first translated into English by Titchener in 1909 (Hornblow, 1980; Preston & de Waal, 2002). Titchener’s idea of empathy was that a person could project conscious awareness into another person’s environment from observing that person’s environment. Empathy was thought to involve insight into the motivations of other people’s behaviors and it is an emotional response to display of distress in others and reversedempathy is an emotional response to non-distressed others in distressed subjects. Also we define empathy as the ability to understand and perhaps even ‘feel’ the emotions of others. Prosocial behavior refers to an act that is done to benefit another. In our environment, where social interaction and collaboration are keys to success in society, empathy and prosocial behavior are essential features that a human being must possess. Up until recently, it was thought that only humans and some non-human primates exhibited empathy and pro-social behavior. When we see someone else crying or sad we get sad. When we feel pain, an area of our brain, the anterior cingulate cortex (ACC), becomes active. And interestingly, it also activates when we observe someone else's pain. While we don't literally feel their pain physically, we do feel it emotionally. It's believed that this is due to "mirror neurons" in our cingulate cortex. However, recent studies by Bartal et Al suggest that rats may also exhibit these behaviors. Bartal et Al’s research used the restrainer test to elicit empathic and prosocial behaviors from the rats. Also, for the first time researchers at the University of Chicago decided to see is these intelligent creatures not only have feelings but is they are able to feel other’s emotions too! Finding helping in rats is exciting because it tells us that this is a biological inheritance,” professor Mason told. She also added that it means that we don’t teach helping. “In fact what appears to be the case is that around 4 years of age, kids start to learn who NOT to help,” the scientist revealed. The first study was started in 20007 and published in 2011. 372

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Process focused on when rats get some difficult circumstances. Do they help to each other or feel the stress of other rats? Studies demonstrate that not only are rats empathetic, they are also pro-social. They appear to demonstrate both the inner state that identifies with another but then take the next step and act upon it. Primatologist Franz de Waals calls the study groundbreaking. “We are entering a distinctly psychological realm of emotions and reactions to the emotions of others, which is where most human altruism finds its motivation.” Other scientists remain more skeptical. Alex Kacelnik of the Department of Zoology at Oxford University says, “Empathy has been proposed as the motivation behind the sort of 'pro-social' rescue behavior in which one individual tries to free another,” said Professor Kacelnik, lead author of the article, 'however, the reproductive benefits of this kind of behavior are relatively well understood as, in nature, they are helping individuals to which they are likely to be genetically related or whose survival is otherwise beneficial to the actor. “To prove empathy any experiment must show an individual understands another's feelings and is driven by the psychological goal of improving another's wellbeing. Our view is that, so far, there is no proof of this outside of humans.” MATERIAL AND METHODS Firstly, we take a one plastic package and 5 rats. The rats should live together couple a months and get to know each other very well. After this procces we get 2 plastic package which have to be small more than first one. Also need a piece of cake and add it second one. Catch one rat and The rat placed in an area. The plastic packages mouth should be half open for breathing and chocolates smell. Set them together to bigger one. In the beginning of the procces they start to protect their friend and try to open packages mouth. When they notice chocolates smell they stop it, go another package and start to fighting for chocolate. One of them stay, uncover and survive rat. Another rats win the game and got the cholocate. And they start eating together. Picture

RESULT AND DISCUSSION Process showed that rats are “selective” about whom they empathize with. When introduced to a different, unknown strain of rats, the free rat would not free the trapped rat. The research yielded the same results if the free rat was grown in isolation and wasn’t socialized even with its own strain of rats. What it basically means, is that if a rat has never seen another rat of its own strain, it won’t open the door. CONCLUSION We are human being, we have empathy. They also concluded that this provided some biological roots for empathy. What we can safely say is that rats seem to have empathy and act prosocially. This is a good thing to know, for it shows that both these traits may well be part of the human psyche that precedes learning. It doesn’t demonstrate that humans are good by nature, 373

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but it does make a strong case that being good is part of human nature. All happens in exactly the same brain region in rats as in humans.

REFERENCES P. Vernazza, L. Jorda, P. Ševeček, M. Brož, M. Viikinkoski, J. Hanuš, B. Carry, A. Drouard, M. Ferrais, M. Marsset, F. Marchis, M. Birlan, E. Podlewska-Gaca, E. Jehin, P. Bartczak, G. Dudzinski, J. Berthier, J. Castillo-Rogez, F. Cipriani, F. Colas, F. DeMeo, C. Dumas, J. Durech, R. Fetick, T. Fusco, J. Grice, M. Kaasalainen, A. Kryszczynska, P. Lamy, H. Le Coroller, A. Marciniak, T. Michalowski, P. Michel, N. Rambaux, T. Santana-Ros, P. Tanga, F. Vachier, A. Vigan, O. Witasse, B. Yang, M. Gillon, Z. Benkhaldoun, R. Szakats, R. Hirsch, R. Duffard, A. Chapman, J. L. Maestre Bored Panda, Research Shows That Rats Show Empathy, But Are Selective Regarding Fur Color Bigthink.com, Rats 'feel the distress' of other rats, Dutch neuroscientists say Arthur Dobrin D.S.W theverge.com, Rats feel emphaty for other rats, unless they are on anti-anxiety medication.

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ORGANIC DAIRY GOAT PRODUCTION MODEL FOR THE SOUTHEASTERN REGION IN TURKEY Halit Deniz SİRELİ1, Ahmet Konuralp ELİCİN2 and Muzaffer DENLİ1* 1

Department of Animal Science, Faculty of Agriculture, Dicle University, 21280, Diyarbakır, Turkey 2

Department of Agricultural Machinery, Faculty of Agriculture, Dicle University, 21280, Diyarbakır, Turkey *Corresponding author’s e-mail: muzaffer.denli@gmail.com

ABSTRACT Organic goat farming is very poorly developed although Turkey has a sufficient natural vegetation for organic goat breeding. The aim of this study was to investigate the viability of organic goat breeding as a model in the Southeastern Anatolia. Within the scope of this research, a total of 70 dairy goat capacity organic goats farm was built. The minimum indoor area (net) on the farm was 1.5 m2 for an adult goat and 0.35 m2 for a kid. The out-door run was a minimum of 2.5 m2 for adult goats and 0.5 m2 for a kid. About 13.3 adult goats per ha were allowed graze on organic pastures. Totally 70, 12 months old Kilis goats were kept in modern stables had seasonal access to pasture. Depend on the lactation period goats were fed with 60 % of roughage and 40 % concentrated feed. Live weight changes, pregnancy rate and first mating and other adaptation values were checked. The results obtained showed that Kilis goat breed may be raising is this region for organic goat production. Keywords: Organic Farming, Dairy goats, Milk performance, Southeastern region, Turkey

INTRODUCTION Organic agriculture is a production system that sustains the health of soils, ecosystems and people. It relies on ecological processes, biodiversity and cycles adapted to local conditions, rather than the use of inputs with adverse effects. Organic farming is an agricultural production method which is providing reliable food products and raising quality of food products. Starting from early 20th century, organic agricultural production started to increase. There were 2.4 million organic producer, organic farming is practiced in 179 countries at about 50.9 million hectares of agricultural land and it was estimated the global market for organic food to have reached 81.6 billion US dollars in 2015 (Willer and Lernoud, 2017). The highest organic shares of the total agricultural land, by region, are in Oceania (6.5 percent) and in Europe (2.7 percent; European Union 6.7 percent) (Willer et al., 2018). Australia is the leading country in organic land area, with more than 12 million ha, followed by China (2.3), Argentina (2.2), US (1.6, 2005 data), Italy (1.1), Uruguay (0.9), Spain (0.9), Brazil (0.9), Germany (0.8), and UK (0.6) (Willer and Youssefi, 2007). Liechtenstein is the leading country with highest share of organic land (29%), followed by Austria (13%), Switzerland (12%), Italy (9%), Estonia (9%), Greece (8%), Portugal (7%), Sweden (7%), Latvia (7%), and Timor Leste (7%) (Lu et al., 2010). Various studies reported that small-scale plays a crucial role in food security in developing countries (Tscharntke et al., 2012, Azadi et al., 2015). 375

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According to 2018 data, there are 626’885 organic farming arable area in Turkey and there has been a steady increase since 2005 and the increase in the number of producers was 421’934 in 2005 to more than 2.4 million in 2018 (Ministry of Agriculture and Forestry, 2019). Turkey is an important goat-producing country with a goat population of 10.9 million heads and 450,000 t/year milk production (Ministry of Agriculture and Forestry, 2019). Although not included in official statistics, it is estimated that approximately 0.5 million heads of Kilis goat exist in the Kilis, Gaziantep, and Hatay provinces of Turkey. The Kilis goat is known to have higher milk yield and reproductive characteristics than the Hair goat. Organic goat farming is very poorly developed although Turkey has a sufficient natural vegetation for organic goat breeding. In the present study we aimed to to investigate the viability of organic goat breeding as a model in the Southeastern Anatolia. MATERIAL AND METHODS According to the regulation of organic agriculture, total 100 head capacity barn was built in Diyarbakir province in Turkey. The out-door run was a minimum of 2.5 m2 for adult goats and 0.5 m2 for kids according to organic agriculture law and regulation. The total area of 3.000 m2 rides area was created. The minimum indoor area (net) on the farm was 1.5 m2 for an adult goat and was 0.35 m2 for a child. Kilis goat has middle-weight and long farmed and so big pendulous ears with about 28 cm average length. Depend on the lactation period goats were fed with 60 % of roughage and 40 % concentrated feed. Live weight changes, pregnancy rate and first mating and other adaptation values were checked. At the experimental station of the department of Animal Science Organic Farming about 70 Turkish Kilis goats have been kept since March, 2018 to assess under the standards of organic farming. The goats are kept in modern stables, have seasonal access to pasture (October– November) and get only farm produced feedstuffs. Under these conditions it has been shown that high productive organic dairy milk production is possible. RESULTS Data obtained from this study showed that that the Kilis goat breed may be adapt to the Diyarbakir province region. Goats are seasonally polyestrous animals with peak sexual activity occurring in the fall when day length is decreasing. In this study, almost all kilis goats reared under organic growing conditions showed estrous cycle in October and November. Live weight changes, pregnancy rate and first mating and other adaptation values will be checked next year. In addition, yield characteristics and product quality will be compared in organic and conventional goat breeding. CONCLUSIONS The results obtained from this study showed that Kilis goat breed may be raising is this region for organic goat production. Acknowledgements: This research was supported by by the Southeastern Anatolia Project (GAP) Regional Development Administration (Project No. 2015A020020) and the Scientific Research Project Fund of Dicle University (DÜBAP) under the project number Ziraat.19.001)

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REFERENCES Castellini, C., C. Mugnai, A. Dal Bosco (2002). Effect of organic production system on broiler carcass and meat quality. Meat Sci., 60 (3), 219-225. Castromán, G., M. del Puerto, A. Ramos, M. C. Cabrera, A. Saadoun (2013). Organic and conventional chicken meat produced in Uruguay: colour, pH, fatty acids composition and oxidative status. Am. J. Food Nutr., 1 (2), 12-21. Chen, X., W. Jiang, H. Z. Tan, G. F. Xu, X. B. Zhang, S. Wei, X.Q. Wang (2013). Effects of outdoor access on growth performance, carcass composition, and meat characteristics of broiler chickens. Poultry Sci., 92, 435–443. Cho, H. P., M. Nakamura, S. D. Clarke (1999). Cloning, expression, and fatty acid regulation of the human δ-5 desaturase. J. Biol. Chem., 274, 37335-37339. Christie, W. W. (1973). Lipid analysis. Pergamon Press, Oxford. Cömert, M., Y. Şayan, F. Kırkpınar, Ö. Hakan Bayraktar, S. Mert (2016). Comparison of carcass characteristics, meat quality, and blood parameters of slow and fast grown FAO, 2007. International conference on Organic Agriculture and food security. Report of the conference held in May 2007 in Rome, OFS/2007/REP [www.fao.org/organicag/index.jsp] FAO/WHO, 1999. Codex Alimentarius Commission Guidelines for the Production, Processing, Labelling and Marketing of organically produced foods. cac/gl, 32, Rome, Italy. Lu, C.D., Gangyi, X. and Kawas, J.R., 2010. Organic goat production, processing and marketing: Opportunities, challenges and outlook. Small Ruminant Research, 89(23), pp.102-109.

Willer, H., Lernoud, J. and Kemper, L., 2018. The world of organic agriculture 2018: Summary. In The World of Organic Agriculture. Statistics and Emerging Trends 2018 (pp. 2231). Research Institute of Organic Agriculture FiBL and IFOAM-Organics International. Willer, H. and Lernoud, J., 2017. The world of organic agriculture. Statistics and emerging trends 2017 (pp. 1-336). Research Institute of Organic Agriculture FiBL and IFOAM-Organics International.

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CONSERVATIONAL NOTES ON THE ACANTHOLIMON AVANOSICUM DOĞAN & AKAYDIN (AVANOS: NEVŞEHIR) IN TURKEY Hüseyin Kürşad İLDENİZ1*, Ferhat CELEP2, Özal GÜNER3, Mehmet GÜL4, Evren CABİ1 1

Faculty of Arts and Sciences, Department of Biology, Tekirdağ Namık Kemal University, Tekirdağ, Turkey

Faculty of Arts and Sciences, Department of Biology, Kırıkkale University, Kırıkkale, Turkey 3 Faculty 4 Enpark:

of Sciences, Department of Biology, Gazi University, Ankara

Environment and Energy Consultant Company, Ankara, Turkey

*Corresponding author’s e-mail: kursad.ildeniz@gmail.com

ABSTRACT The genus Acantholimon Boiss. (Plumbaginaceae Juss.) includes approximately 295 species worldwide. This genus is represented by 50 species of which 32 are endemic for Turkey. Acantholimon avanosicum Doğan & Akaydın, a rare endemic taxon, grows only argillaceous slopes, between Avanos and Hacibektas in Nevsehir Province. With this study, number of individuals, and size of meta-populations, and habitat features of A. avanosicum have been investigated. According to field observations, IUCN conservation threat categories and status besides potential threats to its habitat have also been identified. Field studies were carried out between July and August in 2018. During the surveys, georeferenced occurrence data, number of individuals of A. avanosicum in metapopulations and other plant species growing together in the localities were noted. Based on extensive field surveys 3 different occurrence sites which were not far from each other were recorded. These occurrence cites can be accepted as one fragmented population. Totally 750-800 mature individuals have been recorded, however, there is no clear evidence that the number of individuals decreasing or stable. Extent of occurrence (EOO) of A. avanosicum is not more than 1.204 km2. The most appropriate rating is proposed Critically Endangered according to all findings. It grows together with Astragalus microcephalus Willd, Thymus sipyleus Boiss., Gundelia tournefortii L., Echinophora tournefortii Jaub. & Spach, Gypsophila sp., Hedysarium sp., Euphorbia sp., Scabiosa sp., Verbascum sp., Nonea sp., Rhamnus sp., Helichrysium sp., Stipa sp., Cousinia sp., Dianthus sp., Aspedoline sp., Globularia orientalis L., Eryngium campestre L., Taeniatherum caputmedusae (L.) Nevski, Peganum harmala L., Onosma sp., Acantholimon acerosum (Willd.) Boiss., Alhagi sp., Marrubium sp., Salvia hypargeia Fisch. & C.A.Mey., and S. absconditiflora (Montbret & Aucher ex Benth.) Greuter & Burdet. There are tile and brick factories around the distribution range of the species. These factories use soil as raw material for their brick process so soil taking is the main threat over the sustainability of A. avanosicum in the area. Other threats are road and field expansion studies, urbanization, storing construction waste. Keywords: Acantholimon avanosicum, endemic, Conservation biology, Nevşehir, Cappadocia

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INTRODUCTION Turkey has a very rich flora and vegetation types with 11.707 plant taxa. The main reason of this rich plant diversity is the geographic location of Turkey. Turkey intersects with EuroSiberian, Iran-Turanian and Mediterranean phytogeographic regions. Plant endemism ratio in Turkey is 32% and majority of endemic taxa have narrow distribution area or evaluated as endangered (Davis & Hedge, 1975; Güner at al., 2012; Kaya & Aksakal, 2005). The family Plumbaginaceae Juss., the only family belonging to Plumbaginales is represented by 24 genera and 775 species worldwide. The family includes 6 genera namely Limonium Miller., Goniolimon Boiss., Armeria Willd., Plumbago L., Acantholimon Boiss. and Limoniopsis Lincz. in Turkey (Jussieu, 1789; Heywood, 1978; Davis, 1982). The genus Acantholimon includes 67 taxa in Turkey of which 32 species are known just only from Turkey. (Doğan & Akaydın, 2007; Davis, 1982, 1988; Doğan & Akaydın, 2003; Boissier, 1879). A. avanosicum Doğan & Akaydın was first collected from Nevşehir (Avanos) in Turkey by Doğan & Akaydın and introduced to the scientific community. It is spiny perennial shrubs in the form of cushion and begins to bloom in early July and stays in bloom until the end of August. It has pink flowers and in the first week of September, the flowers were completely shed and only the calyx structures are remained (Doğan & Akaydın, 2002, 2007). The species grows with Astragalus microcephalus Willd. and various grasses of the Central Anatolian steppe. This rare endemic taxon, grows only argillaceous slopes, between Avanos and Hacibektas in Nevsehir province (Figure 1). Doğan & Akaydın proposed Critically Endangered (CR) (IUCN, 2001) for the taxon because of its local distribution and small population size. This study includes the preliminary results of Species Conservation Action Plan (SAP) of the A. avanosicum and provides background information about the species and an overview of the 'threats' considered to be causal in the species' population decline.

Figure 1. Argillaceous slopes, between Avanos and Hacıbektaş in Nevşehir Province

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MATERIAL AND METHODS Field studies were carried out between July and August 2018 in Avanos district of NevĹ&#x;ehir. While conducting field studies, potential habitats of A. avanosicum species were investigated. Number of individuals, and size of meta-populations, and habitat features of A. avanosicum have been determined. Other plant species were also identified growing together with A. avanosicum. The elevations and coordinates of the geo-locations of the populations using GPS device were determined and noted. With the help of Arcgis sotware aspect, slope, elevation and hillshade maps were also created based on the presence data. EOO (Extent of occurrence) value was calculated by using Geocat application of KEW herbarium. According to field observations, IUCN conservation threat category was reevaluated and potential threats to its sustaniablity have also been determined. RESULTS AND DISCUSSION Based on extensive field surveys 3 different occurrence sites which were not far from each other were recorded. These occurrence cites can be accepted as one fragmented population. Totally 750-800 mature individuals have been recorded, however, there is no clear evidence that the number of individuals decreasing or stable. Extent of occurrence (EOO) and Area of Occupancy (AOO) of A. avanosicum is not more than 1,204 km2 and Area of Occupancy is 8 km2 (Figure 2). The most appropriate threat category rating is re-evaluated and proposed Critically Endangered (CR) B1ab(i,ii)+B2ab(i,ii) according to all findings.

Figure 2. Extent of occurrence (EOO) and Area of Occupancy (AOO) of A. avanosicum A. avanosicum, between 905-1000 m, on clayey-stony slopes, Astragalus microcephalus from typical Central Anatolian Steppe vegetation. And it has been observed to distribute with various herbaceous plants belonging to this vegetation. A. avanosicum, between 905-1000 m, on clayey-stony slopes, Astragalus microcephalus from typical Central Anatolian Steppe vegetation. And it has been observed to distribute with various herbaceous plants belonging to this vegetation. It grows together with Astragalus microcephalus Willd, Thymus sipyleus 380

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Boiss., Gundelia tournefortii L., Echinophora tournefortii Jaub. & Spach, Gypsophila sp., Hedysarium sp., Euphorbia sp., Scabiosa sp., Verbascum sp., Nonea sp., Rhamnus sp., Helichrysium sp., Stipa sp., Cousinia sp., Dianthus sp., Aspedoline sp., Globularia orientalis L., Eryngium campestre L., Taeniatherum caput-medusae (L.) Nevski, Peganum harmala L., Onosma sp., Acantholimon acerosum (Willd.) Boiss., Alhagi sp., Marrubium sp., Salvia hypargeia Fisch. & C.A.Mey., and Salvia absconditiflora (Montbret & Aucher ex Benth.) Greuter & Burdet. A. avanosicum grows in shadowless and open areas. It prefers the southern and southwest of the slopes of 2 to 10 degrees (Figure 3).

Figure 3. a) Elevation map b) Hillshade map c) Aspect map d) Slope map There are tile and brick factories around the distribution range of the species. These factories use soil as raw material for their brick process so soil taking is the main threat over the sustainability of A. avanosicum in the area. Other threats are road and field expansion studies, urbanization, storing construction waste (Figure 4).

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Figure 4. a) Soil taking b) Storing construction waste c) Urbanization d) Field expansion studies. Acknowledgements: This study was supported by the General Directorate of Nature Conservation and National Parks of the Ministry of Agriculture and Forestry Affairs. REFERENCES Boissier, E. (1879). Flora Orientalis (4) (pp.823-854). Basel: Reg. Acad. Scient. Davis, P.H., Hedge, I.C. (1975). The Flora of Turkey: Past, Present and Future, Candollea, 30, 331-351. Davis, P.H. (ed.) (1982). Flora of Turkey and The East Aegean Islands. (7) pp. (478-502.) Edinburgh Univ. Press. Edinburgh. Davis, P.H., Mill, R.R., & Tan, K. (eds.). (1988). Flora of Turkey and The East Aegean Islands (10). Edinburgh: Edinburgh Univ. Press. Doğan, M., Akaydın, G. (2002). A new species of Acantholimon Boiss. (Plumbaginaceae) from Central Anatolia. Turkey. Botanical Journal of the Linnean Society, 138, 365–368. Doğan, M., & Akaydın, G. (2003). Türkiye’nin Acantholimon Boiss. (Plumbaginaceae) Türlerinin Revizyonu. TBAG 1781 (199T011). TÜBİTAK Doğan, M., & Akaydın G. (2007). Synopsis of Turkish Acantholimon Boiss. (Plumbaginaceae). Botanical Journal of the Linnean Society, 154, 397-419. Güner, A., Aslan, S., Ekim, T., Vural, M., & Babaç, M.T., (eds.). (2012). Türkiye Bitkileri Listesi (Damarlı Bitkiler). Istanbul: Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayınları. Heywood, V.H. (1978). Flowering plants of the World. Oxford: Oxford University Press. Jussieu, A.L. (1789). Paris: Genera Plantarum secundum ordines naturales disposita, juxta methodum in horto region parisiensi exaratam. Herissant. Kaya, Y., & Aksakal, Ö. (2005). Endemik Bitkilerin Dünya ve Türkiye’deki Dağılımı, Erzincan Eğitim Fakültesi Dergisi. 7(1), 85 – 99.

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DETERMINATION OF CONSUMERS’ BEHAVIORS TOWARDS IRRADIATED FOODS Bülent ERGÖNÜL*1, Kıvılcım ÇELİK1, Pelin Günç ERGÖNÜL1 1

Manisa Celal Bayar University, Food Engineering Department, Manisa, Turkey *Corresponding author’s e-mail: bulent.ergonul@hotmail.com

ABSTRACT In this study, it was aimed to determine the consumers’ knowledge and behaviors towards irradiated foods in Manisa city center. A questionnaire was prepared and a face to face interview was performed with 1200 respondents living in Manisa. According to results, it can be concluded that consumers living in Manisa are not familiar with irradiated foods and there is significantly important lack of knowledge towards irradiated foods. Keywords: Irradiation, Food, Questionnaire, Manisa, Consumer

INTRODUCTION Irradiation is known as “exposure of foods to ionizing radiation in form of gamma radiation, X-rays or electron beams to destroy pathogenic microorganisms” (FDA, 2019; Diehl, 2002). Food irradiation has about a hundred years of history (Roberts, 2014; Grolichova et al., 2004; Diehl, 2002; Tauxe, 2001). It is a scientifically established technology and a safe food process. On the other hand consumers' perceptions prevented widespread acceptance of food irradiation but Safety and efficiency of food irradiation has been approved by several authorities (FDA, USDA, WHO, FAO, etc.) and scientific societies based on extensive research (FDA, 2019; Mahami et al., 2019; Roberts, 2014; Bruhn, 1995). Many studies indicated that consumers were unfamiliar with food irradiation. Researchers recommended that consumers receive information about the process and be offered a choice of irradiated and nonirradiated foods in the marketplace. The term “irradiation” is sometimes confusing or alarming to consumers because of its perceived association with radioactivity and the causes, incidence, and prevention of foodborne disease are poorly understood by the general public (Galati et al., 2019; Bearth and Siergrist, 2018, Tauxe, 2001). So in this study, it was aimed to determine the consumers’ knowledge levels and behaviors towards the irradited foods. MATERIAL AND METHODS A face to face conversation was performed with a total of 1200 randomly selected consumers. A three-minute questionnaire was designed to obtain information on demographics of respondents, theirperceptions, and awareness of food irradiation and health concerns. The questionnaire was pilot tested on 8 respondents for clarity and validity and necessary adjustments were done. RESULTS AND DISCUSSION Demographical attributes of respondents were given as Table 1. As seen, 57% of the respondents were at ages among 21 and 60 and the percentages of female and male respondents are nearly the same. 383

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Table 1. Demographical attributes of respondents Characterisic

Age

18-20 21-40 41-60 <60

21 29 28 22

Gender

Female Male

48 52

Marital Status

Married Single

66 34

Education

Elementary High University

19 51 30

Monthly Income*

< 3000 TL 3001 – 5000 >5001

44 40 16

* Per person “Which of the following poses greater health risks in food industry?” was asked to consumers as the first question of the interview and the results obtained was given as Table 2. As seen from Table 2, food additives are of a great concern among the consumers which is followed by pesticides, fertilizers and drug residues. According to 12% of the consumers irradiated foods poses great health risks in food industry. Table 2. Which of the following poses greater health risks in food industry? Criteria Irradiated foods Frozen foods Toxins Bacteria / Microorganisms Hormones Pesticides, fertilizers and drug residues Food additives

% 12 5 17 9 5 22 30

And then the second question “Do you know how foods are irradiated?” was asked to consumers to learn about their knowledge in terms of food irradiation process and the results of the question is given as Figure 1. 72% of the consumers stated that they had enough knowledge about the food irradiation process.

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Yes

No 28 72

Figure 1. The results of the question “Do you know how foods are irradiated?”

The third question of the questionnaire was “Do you want to consume an irradiated food?” Only 20% of the respondents reported that they tend to buy irradiated foods, whereas 41% of the consumers have said that they would certainly not consume irradiated foods (Figure 2).

I would like to buy Undecided I certainly don't want to buy Figure 2. The results of the question “Do you want to consume an irradiated food?”

After the third question a brief information regarding the safeness of irradiation process was given to respondents as below and then the same question was asked again to determine how respondents’ answers changed after that brief explanation. “Irradiation is a safe process like pasteurization or sterilization, and shelf life of irradiated foods are longer. Irradiation is harmless to human health” The results of the question was given as Figure 3. As seen, the ratio of the consumers who stated that they tend to buy irradiated foods increased to 41%.But the ratio of undecided respondents did not change (%39).

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After fourth question another brief information about irradiation process was given to respondents and the same question was asked as fifth question of the interview. The second brief information was as below; “The FDA has evaluated the safety of irradiated food for more than 30 years and has found the process to be safe. The World Health Organization (WHO), the Centers for Disease Control and Prevention (CDC) and the U.S. Department of Agriculture (USDA) have also endorsed the safety of irradiated food. (FDA, 2019)”

I would like to buy Undecided I certainly don't want to buy Figure 3. The results of the question “Do you want to consume an irradiated food?” (second question) As seen from Figure 4, the ratio of the consumers who stated that they tend to buy irradiated foods increased to 70%, and the ratio of undecided consumers decreased to 10%. But 20% of the respondents were still remaining who believed the irradiation process is harmful to human health (Figure 4).

20 10 70

I would like to buy Undecided I certainly don't want to buy Figure 4. The results of the question “Do you want to consume an irradiated food?”

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As the sixth question of the questionnaire, the question “Which information is more convincing?” was asked to respondents and the results were given as Figure 5. As seen, according to consumers’ answers, the fact that the approval food irradiation by FDA and WHO was the most convincing effecting their idea according to irradiated foods.

17 15

29 21

Irradiated foods are not radioactive

Food irradiation is approved by FDA and WHO safety of irradiated foods is evaluated for more than 30 years and has found the process to be safe None of them

Figure 5. The results of the question “Which information is more convincing?” A question about Radura – the symbol of the irradiated foods – was also added into questionnaire as below (Figure 6)

Figure 6. The results of the question “Which one is the official symbol of the irradiated foods?” 387

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According to the answers of the respondents, only nearly half of the (49%) consumers were familiar with the Radura the symbol of irradiation. Last question of the interview was regarding the determine the consumers’ tendency to buy irradiated foods under different price levels compared to unirradiated foods (Figure 7). 43% of the consumers stated that they would like to buy irradiated foods if their prices was cheaper. But the 25% of the respondents were still remained undecided.

I would like to buy Undecided I certainly don't want to buy Figure 7. Consumers’ tendency to buy irradiated foods under different price levels compared to unirradiated foods CONCLUSIONS Consumer acknowledgement of irradiated foods to a great extend relies upon the awareness and knowledge of the benefits and use of irradiation process. Turkish consumer’s awareness and acknowledgement of irradiation process are low. This is related with the absence of knowledge about the process. When consumer hears a benefit statement, it increases the acceptance of irradiated foods in a considerable amount. If irradiated foods’ price is same as that of unirradiated foods acceptance of irradiated foods will increase. The only way to achieve increasement is to educate consumers about the benefit and uses of food irradiation. To advance market achievement, public education about the process can be provided at schools, media, conferences and educational practices. REFERENCES Bearth, A., Siegrist, M. (2018). “As long as ıt is not irradiated” – influencing factors of us consumers’ acceptance of food irradiation. Food Quality and Preference. Doi: https://doi.org/10.1016/ j.foodqual.2018.06.015 Bruhn, C. M., H. G. Schutz, and R. Sommer. (1986). Attitude change toward food irradiation among conventional and alternative consumers. Food Technology. 40(1):86-91. Diehl, J.F. (2002). Food irradiation – past, present and future. Radiation Physics and Chemistry. 63: 211-215. FDA. (2019). https://www.fda.gov/food/buy-store-serve-safe-food/food-irradiation-what-youneed-know (1.9.2018). 388

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Galati, A., Tulone, A., Moevero, P., Crescimanno, M. (2019). Consumer interest in information regarding novel food technologies in Italy : the case of irradiated foods. Food Research International. 119 : 291-296. Grolichova, M., Dvorak, P., Musilova, H. (2004). Employing ionizing radiation to enhance food safety â&#x20AC;&#x201C; a review. Acta Veterinaria Brno. 73: 143-149. Mahami, T., Togby-Tetteh, W., Kottoh, D.I., Amoakoah-Twum, L., Gasu, E., Annan, S.N.Y., Larbi, D., Adjei, I., Adu-Gyamfi, A. (2019). Microbial food safety risk to humans associated with poultry feed: the role of irradiation. International Journal of Food Science. Doi: https://doi.org/10.1155/2019/6915736 Roberts, P.B. (2014). Food irradiation is safe: half a century of studies, radiation physics and chemistry. Doi: http://dx.doi.org/10.1016/j.rad- physchem.2014.05.016 Tauxe, R.V. (2001). Food safety and irradiation: protecting the public from foodborne infections. Emerging Infections Diseases. 7(3): 516-521.

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CALCULATION OF IBMR SCORE VALUES BY USING PYTHON PROGRAMMING LANGUAGE Ogün DEMİR1*, Evren CABİ2 1

Department of Biology, Institute of Natural and Applied Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey

Department of Biology, Faculty of Arts and Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey *Corresponding author’s e-mail: ogundemir8@gmail.com

ABSTRACT There are many indexes developed in rivers to assess water quality and to create typologies based on flora or phytosociological communities. One of them, the Macrophyte Biological Index for Rivers (IBMR or MBIR), is used to determine the water quality of rivers. IBMR, which assesses trophic distortion as well as heavy organic pollution, becomes a difficult and time-consuming index to be calculated by classical methods as the number of studied rivers and the number of taxa evaluated increase. In this study, an application with graphical user interface has been created for calculating IBMR using Python programming language. The algorithm of the mathematical models in the IBMR index was generated in Python version 3.7.0. It was also used as a file database with xlsx extension. The database includes the taxa from the French IBMR method and the trophic level (CSi) and stenosis factor (Ei) reference values of these taxa. In the calculation phase, sample CSi and Ei values of taxa and sample taxa were used. Taxa were entered into the program with sample abundance values and the IBMR value was calculated. Accuracy of the obtained results was compared with the manual calculations. With the software we have generated, we have achieved much faster and more accurate results than the conventional calculation methods (including Microsoft Excel). We have also provided a simple interface to users, avoiding the confusion in the calculation methods in Microsoft Excel. No matter how many rivers and taxa in the determination of ecological quality status of rivers, it provides great convenience to the researcher with easy of use and performance in calculations. Keywords: IBMR, Python, Macrophyte, Quality, River. INTRODUCTION Macrophytes are any group of macro organisms (vascular plants, stoneworths (Characeae), bryophytes, pteridophytes aquatic algae even some macrofungi) which have relationship with water. They grow usually in water or edge of water bodies. They are accepted as reliable indicators for wetland ecosystems and are commonly used to determine the water quality of rivers (Lacoul et al., 2006; Franklin, 2008; WFD, 2014). There are many indexes developed for rivers to assess water quality and to create typologies based on macrophyte diversity (Ali et al., 1999; Schneider and Melzer, 2003; Dodkins et al., 2005; Willby et al., 2000; Willby et al., 2009). One of them, the Macrophyte Biological Index for Rivers (IBMR or MBIR), assesses trophic distortion as well as heavy organic pollution in rivers by using the indicator macrophyte taxa (AFNOR, 2003; Haury et al., 2006). Calculation with this index is getting a difficult and time-consuming process especially 390

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when the number of studied rivers and the number of taxa evaluated are too much. In this study, to overcome the difficulties related with calculations an application with graphical user interface has been created for IBMR using Python programming language. MATERIAL AND METHODS The algorithm of the mathematical models of IBMR index was generated in Python version 3.7.0 (Van Rossum, 1995; Van Rossum & Drake, 2009; Python Software Foundation, 2019). Calculation algorithms have been generated with math and random libraries as numeric and mathematical modules in Python according to mathematical operation in the IBMR index. Also os library was used for file and directory access. A file database with xlsx extension was also used. The database includes the indicator taxa from IBMR method and the trophic level (CSi) and stenosis factor (Ei) reference values of these taxa. Database of the species is being imported with the openpyxl (version 3.0.0) library (Gazoni & Clark, 2019) to software. In order to create the graphical user interface (GUI), the Tkinter library (Shipman, 2013) was used in Python. The results of sampling station or location were written to the excel file by using openpyxl when the calculation process is over. In the calculation phase, 10 randomly generated river macrophyte data were used. Taxa were entered into the program with sample abundance values and the IBMR value was calculated. Accuracy of the obtained results was compared with the manual calculations. RESULTS AND DISCUSSION IBMR calculations were performed with “Formulaire saisie flor IBMR” (version 4.3.1) (Hydrobio DCE, 2019), manually by using calculator, user-created spreadsheets and our software for 10 randomly generated river macrophyte data. In our software, calculations were faster and more accurate than other tools. Also calculations results are consistent with other tools. There is a GUI (Figure 1) in our software which provides convenience to users however other tools are only based on Microsoft Office Excel. Data input such as trophic level and stenosis factor is automatically carried out in our software and Hydrobio La directive cadre européenne sur l’eau (Hydrobio DCE)’s calculator. Database editing and adapting IBMR to other countries are simple in our software and Hydrobio DCE’s calculator while very difficult in other tools. The disadvantage of the Hydrobio DCE’s calculator is that it is complicated and written in French. We have achieved much faster and accurate results than the conventional calculation methods (including Microsoft Excel) with the software we generated. We have also provided a simple interface to users, avoiding the confusion in the calculation methods in Microsoft Excel. No matter how many rivers and taxa in the determination of ecological quality status of rivers, it provides great convenience to the researcher with easy of use and performance in calculations.

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Figure 1. GUI of IBMR method

REFERENCES AFNOR (2003) Détermination de l’Indice Biologique Macrophytique en Rivière (IBMR). NF T 90‐395. Association Française de Normalisation, La Plaine Saint‐Denis, France. Ali M. M., Murphy K. J. & Abernethy V. J. (1999). Macrophyte functional variables versus species assemblages as predictors of trophic status in flowing waters. Hydrobiologia, 415, 131–138. Dodkins I., Rippey B. & Hale P. (2005). An application of canonical correspondence analysis for developing ecological quality assessment metrics for river macrophytes. Freshwater Biology, 50, 891–904. Franklin, P., Dunbar, M., & Whitehead, P. (2008). Flow controls on lowland river macrophytes: a review. Science of the total environment, 400(1-3), 369-378. Gazoni, E., & Charlie, C. (2019). A Python library to read/write Excel 2010 xlsx/xlsm files, version 3.0.0. Available at https://openpyxl.readthedocs.io/en/stable/ Haury, J., Peltre, M. C., Trémolières, M., Barbe, J., Thiébaut, G., Bernez, I., ... & Dutartre, A. (2006). A new method to assess water trophy and organic pollution—the Macrophyte Biological Index for Rivers (IBMR): its application to different types of river and pollution. In Macrophytes in aquatic ecosystems: From biology to management (pp. 153158). Springer, Dordrecht. Hydrobio DCE (2019). Macrophytes cours d’eau. Retrieved 25 November 2019, from https://hydrobio-dce.irstea.fr/telecharger/macrophytes-rivieres-2/ Lacoul, P., & Freedman, B. (2006). Environmental influences on aquatic plants in freshwater ecosystems. Environmental Reviews, 14(2), 89-136. Python Software Foundation (2019). Python Language Reference, version 3.7.0. Available at http://www.python.org Schneider S. & Melzer A. (2003). The trophic index of macrophytes (TIM) – a new tool for indicating the trophic state of running waters. International Review of Hydrobiology, 88, 49–67. Shipman, J. W. (2013). Tkinter reference: a GUI for Python. New Mexico Tech Computer Center. Van Rossum, G. (1995). Python tutorial, Technical Report CS-R9526, Centrum voor Wiskunde en Informatica (CWI), Amsterdam. 392

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Van Rossum, G., & Drake, F. L. (2009). Introduction To Python 3: Python Documentation Manual Part 1. CreateSpace. Willby N. J., Abernethy V. J. & Demars B. O. L. (2000). An attribute‐based classification of European hydrophytes and its relationship to habitat utilisation. Freshwater Biology, 43, 43–74. Willby N., Pitt J. A. & Phillips G. (2009) The Ecological Classification of UK Rivers Using Aquatic Macrophytes. UK Environment Agency Science Reports. Project SC010080/SR1. Environmental Agency, Bristol. WFD (2014). UKTAG Lake Assessment Method Macrophytes and Phytobenthos, Macrophytes (Lake LEAFPACS2). Scotland: Water Framework Directive – United Kingdom Advisory Group (WFD-UKTAG). ISBN: 978-1-906934-45-3.

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DEVELOPING A SOFTWARE BASED ON LEAFPACS2 CALCULATIONS BY USING PYTHON PROGRAMMING LANGUAGE Ogün DEMİR1*, Aybüke KIZILIRMAKLI1, Evren CABİ2 1

Department of Biology, Institute of Natural and Applied Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey

Department of Biology, Faculty of Arts and Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey *Corresponding author’s e-mail: ogundemir8@gmail.com

ABSTRACT Macrophytes are any group of macro organisms (vascular plants, stoneworths (Characeae), bryophytes, pteridophytes aquatic algae even some macrofungi) which have relationship with water. They grow usually in water or edge of water bodies. They are accepted as reliable indicators for wetland ecosystems and commonly used to determine the water quality of lakes. Lake LEAFPACS2, one of the common classification methods, enables the evaluation of macrophytes in lakes according to the requirements of the Water Framework Directive (WFD). This method mostly used by Environment Agency, SEPA and Natural Resources Wales for detect the impact on lake macrophytes of nutrient enrichment. Normally the macrophyte metric EQRs, the entire EQR and the Confidence of Class can calculate by manually using a calculator or spreadsheets such as MS Excel but when the number of stations and the number of species increase, it is difficult to calculate Lake LEAFPACS2 by manually or with spreadsheet Microsoft Excel and the rate of error increases. So as to minimize the calculation errors we developed a user friend software by using Python, version 3.7.0. Keywords: LEAFPACS2, Python, Ecology, Quality, Lake

INTRODUCTION Macrophytes are any group of macro organisms (vascular plants, stoneworts (Characeae), bryophytes, pteridophytes aquatic algae even some macrofungi) which have relationship with water. They grow usually in water or edge of water bodies. They are accepted as reliable indicators for wetland ecosystems and are commonly used to determine the water quality of lakes (Coops et al., 2007; Penning et al., 2008; Poikane et al., 2018). There are many ecological evaluation indices based on macrophytes. These indices have been developed specifically for countries or regions (Schaumburg et al., 2004; Søndergaard et al., 2013; Ciecierska et al., 2014; Portielje et al., 2014). Lake LEAFPACS2 which is one of the common classification method, enables the evaluation of macrophytes in lakes according to the requirements of the Water Framework Directive (2014a). Lake LEAFPACS2 includes five metrics which describes different aspects of the macrophyte communities: “Lake Macrophyte Nutrient Index (LMNI) – a taxon-specific nutrient response score. Number of functional groups of macrophyte taxa (NFG) - a diversity metric, individual taxa are allocated to one of 18 “functional groups”* Number of macrophyte taxa (NTAXA) - a diversity metric, the number of scoring taxa recorded in the field survey. Mean percent cover of hydrophytes (COV) – derived from lake macrophyte survey data Relative percent cover of filamentous 394

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algae (ALG) – derived from lake macrophyte survey data”. This method mostly used by Environment Agency, SEPA, Natural Resources Wales and General Directorate of Water Management of the Ministry of Agriculture and Forestry to determine ecological quality of lakes based on macrophytes communities (Willby, 2008; WFD, 2014a; Bakır, 2015). While using this index, Ecological Quality Ratios (EQRs) of each above mentioned metrics has to be calculated and then the obtained scores are used to obtain the final EQR. These calculations are usually carried out manually by using a calculator or spreadsheets such as MS Excel. If number of stations and recorded species numbers in the stations will increase, it will be difficult to calculate Lake LEAFPACS2 by manually or with spreadsheet Microsoft Excel and also the possibility of making mistakes will be increased. So as to minimize the calculation errors we developed a user friend, practical software by using Python, version 3.7.0 (Van Rossum, 1995; Van Rossum & Drake, 2009; Python Software Foundation, 2019). Python, a programming language, has quickly become the focus of interest in the scientific world, although it is not designed specifically for the computing needs of scientists. It provides various libraries and simple syntax to scientists for make computations easier (Oliphant, 2007; Millman & Aivazis, 2011). MATERIAL AND METHODS Math and random libraries in Phyton have been used as numeric and mathematical modules while forming calculation algorithm of LEAFPACS2. We also used os library for file and directory access.¶ The species information given in the LEAFPACS2 method has been transferred to the Microsoft Excel file and is being imported with the openpyxl (version 3.0.0) library (Gazoni & Clark, 2019) to the software. In order to create the graphical user interface (GUI), the Tkinter library (Shipman, 2013) was also used in Python. When the calculation process is over, the results of station are written to the excel file with openpyxl. RESULTS AND DISCUSSION For the purpose of comparison of efficiency of our software, we tested other classical Lake LEAFPCAS2 calculation tools such as Water Framework Directive’s calculator (WFD, 2014b), user-created spreadsheets and finally by using calculator. 10 randomly generated lake values and macrophyte diversity data used for testing. As a result of comparison we obtained the same results with the other classical tools. Our calculations were much faster and accurate than other classical tools. The GUI (Figure 1) in our software also provides convenience to users otherwise other tools are only based on Microsoft Office Excel. Data input such as LMNI, NFG and NTAXA is automatically calculated in our software and WFD’s calculator. Database editing and adapting Lake LEAFPACS2 to other countries are simple in our software while very difficult in WDF’s calculator. As a result of this study, we generated a software which is faster, more accurate results, easily editable and user-friend.

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Figure 2. GUI of our software

REFERENCES Bakır, N. (2015). Su çerçeve direktifine göre biyolojik kalite unsuru: Makrofit. TC. Orman ve Su İşleri Bakanlığı Su Yönetimi Genel Müdürlüğü, Ankara (Expert Thesis). Ciecierska, H., & Kolada, A. (2014). ESMI: a macrophyte index for assessing the ecological status of lakes. Environmental monitoring and assessment, 186(9), 5501-5517. Coops, H., Kerkum, F. C. M., Van den Berg, M. S., & Van Splunder, I. (2007). Submerged macrophyte vegetation and the European Water Framework Directive: assessment of status and trends in shallow, alkaline lakes in the Netherlands. Hydrobiologia, 584(1), 395-402. Gazoni, E., & Charlie, C. (2019). A Python library to read/write Excel 2010 xlsx/xlsm files, version 3.0.0. Available at: https://openpyxl.readthedocs.io/en/stable/ Millman, K. J., & Aivazis, M. (2011). Python for scientists and engineers. Computing in Science & Engineering, 13(2), 9-12. Oliphant, T. E. (2007). Python for scientific computing. Computing in Science & Engineering, 9(3), 10-20. Penning, W. E., Mjelde, M., Dudley, B., Hellsten, S., Hanganu, J., Kolada, A., ... & Ecke, F. (2008). Classifying aquatic macrophytes as indicators of eutrophication in European lakes. Aquatic ecology, 42(2), 237-251. Poikane, S., Portielje, R., Denys, L., Elferts, D., Kelly, M., Kolada, A., ... & van den Berg, M. S. (2018). Macrophyte assessment in European lakes: Diverse approaches but convergent views of ‘good’ecological status. Ecological indicators, 94, 185-197. Portielje, R., Bertrin, V., Denys, L., Grinberga, L., Karottki, I., Kolada, A., ... & Phillips, G. (2014). Water Framework Directive Intercalibration Technical Report Central Baltic Lake Macrophyte Ecological Assessment Methods. Python Software Foundation (2019). Python Language Reference, version 3.7.0. Available at http://www.python.org

Schaumburg, J., Schranz, C., Hofmann, G., Stelzer, D., Schneider, S., & Schmedtje, U. (2004). Macrophytes and phytobenthos as indicators of ecological status in German lakes—a 396

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contribution to the implementation of the Water Framework Directive. Limnologica, 34(4), 302-314. Shipman, J. W. (2013). Tkinter reference: a GUI for Python. New Mexico Tech Computer Center. Søndergaard, M., Phillips, G., Hellsten, S., Kolada, A., Ecke, F., Mäemets, H., ... & Oggioni, A. (2013). Maximum growing depth of submerged macrophytes in European lakes. Hydrobiologia, 704(1), 165-177. Van Rossum, G. (1995). Python tutorial, Technical Report CS-R9526, Centrum voor Wiskunde en Informatica (CWI), Amsterdam. Van Rossum, G., & Drake, F. L. (2009). Introduction To Python 3: Python Documentation Manual Part 1. CreateSpace. Willby, N. J. (2008). LEAFPACS: Development of a system for the classification of rivers and lakes in the UK using aquatic macrophytes. WFD (2014a). UKTAG Lake Assessment Method Macrophytes and Phytobenthos, Macrophytes (Lake LEAFPACS2). Scotland: Water Framework Directive – United Kingdom Advisory Group (WFD-UKTAG). ISBN: 978-1-906934-45-3. WFD (2014b). Lake LEAFPACS2 class and metric calculator. Available at: https://www.wfduk.org/resources/lakes-macrophytes

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ASSESSMENT OF ECOLOGICAL QUALITY BY USING AQUATIC MACROPHYTES IN LAKE AVLAN Aybüke KIZILIRMAKLI1*, Evren CABİ2, Nesibe TURAN3 1

Department of Biology, Institute of Natural and Applied Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey

Department of Biology, Faculty of Arts and Sciences, Tekirdağ Namık Kemal University, Tekirdağ, Turkey 3

Republic of Turkey, Ministry of Agriculture and Forestry, Turkey *Corresponding author’s e-mail: kizilirmakliaybike@gmail.com

ABSTRACT In this study, it was aimed to estimate the ecological quality according to aquatic macrophytes in Lake Avlan, Antalya. Diversity and coverage of macrophytes were determined throughout 4 different vegetation periods in 2014 and 2015. Six submerged taxa (Polygonum amphibium L., Myriophyllum spicatum L., Schoenoplectus lacustris subsp. lacustris (L.) Palla, Potamogeton crispus L., Ranunculus trichophyllus Chaix ex Vill. and Potamogeton nodosus Poir.), three emergent taxa (Bolboschoenus maritimus subsp. maritimus (L.) Palla, Butomus umbellatus L. and Nasturtium officinale R.Br.) and two algae (Cladophora sp. and Zygnema sp.) were recorded throughout 4 vegetation periods. Ecological quality calculation is measured based on LEAFPACS2 Lake index methodology. Calculation were performed according to optimum vegetation period which Polygonum amphibium L., Myriophyllum spicatum L., Butomus umbellatus L., Bolboschoenus maritimus subsp. maritimus (L.) Palla, Potamogeton crispus L., Potamogeton nodosus Poir., Ranunculus trichophyllus Chaix ex Vill. and Nasturtium officinale R.Br. recorded. Bolboschoenus maritimus subsp. maritimus (L.) Palla and Nasturtium officinale R.Br. which are not exist in LEAFPACS2 Lake macrophyte taxa list, were not included in the calculation. As a result of calculation, we measured ecological quality of Lake Avlan as moderate with 0,52 index score. This study was supported by the Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Water Management. Keywords: Macrophytes, Lake, LEAFPACS2, Avlan

INTRODUCTION Macrophytes are a group of ecological organisms that spend the whole year or at least a few months in the water, live freely on the surface of the water or some of which develop upward from the water (Cook et al.,1974; Westlake, 1981). Macrophytes react to changes in physical or chemical conditions in water such as water level, nutrient content and heavy metal content. The response of macrophytes to physical and chemical changes affects development of macrophytes, which informs us about the conditions of lakes and rivers. For this reason, it is important to investigate the development of macrophytes in wetland and shallow lakes and to know their indicative properties in order to understand these ecosystems. (Westlake, 1981; Melzer, 1999; Wersal et al., 2006; Rejmankova, 2011; Doğan, 2018). Macrophytes in the 398

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littoral regions of the lakes are prominent as limnological indicators and unlike bacteria and microalgae, react slowly and gradually to changes in nutrient conditions for several years. Also aquatic macrophytes accumulate nutrients and form biomass to a large extent using their nutrients. For these reasons, they can be used effectively as biological indicators (Reddy & Busk, 1985; Melzer, 1999). Lake Avlan is a shallow lake which has rich biodiversity with 54 endemic plant taxa and important for migration route of birds . Although the lake, which was dried in the 1970s, was recovered 1997, the main problems of the lake still persist. Main problem of Lake Avlan is the road passing through the middle of lake which disrupts the integrity. In addition exhaust gases from vehicles passing by the road pose a threat to the environment. Therefore, determination of the EQR (Ecological Quality Ratio) of the Lake Avlan is important for the sustainability and protection of the lake (Keske & Ketenoğlu, 2010; Ünlüsoy & Ketenoğlu, 2011). Various researches have been carried out so far about Lake Avlan such as flora and fauna studies (Deniz & Sümbül, 2004; Tezcan et al., 2007; Van Neer et al., 2008; Keske & Ketenoğlu, 2010), vegetation studies (Ünlüsoy & Ketenoğlu, 2011) and reasons and environmental effects of drying of the Lake Avlan (Kum & Sönmez, 2016). Also EQR of Lake Avlan was determined based on phytoplankton (Toudjani et al. 2017). However macrophytes have not been used to determine the EQR of Lake Avlan. In this study, it was aimed to estimate the ecological quality according to aquatic macrophytes in Lake Avlan. MATERIALS AND METHODS As a study area, Lake Avlan covers an approximate area of 850 ha which is located on southern end of Elmalı polje around 1016 meters above sea level. Also Lake Avlan surrounded by Bey Mountains (Southeast), Susuz Mountain (Southwest) and Elmalı district (North) in Antalya province. Diversity and coverage of macrophytes were determined throughout 4 different vegetation periods in 2014 and 2015 (Table 1) according to the TS EN 15460 standard, which is also accepted by the General Directorate of Water Management of the Ministry of Agriculture and Forestry. Sampling sites were chosen according to the size of the lake to reflect the diversity of the lake and to surround the lake as evenly as possible. A 100 meters length area parallel to the shore from the sampling point was determined. Macrophytes were sampled in 4 different transects perpendicular to an area of 100 meters. Also coverages of macrophytes were recorded during sampling. Table 1. Detailed dates of vegetation periods. Vegetation Period

Month

Year

First

September

2014

Second

November

2014

Third

April

2015

Fourth

August

2015

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Ecological quality calculation is measured based on Lake LEAFPACS2 index methodology which is part of WFD (Water Framework Directive) (WFD, 2014). In addition, Calculations was performed by using Lake LEAFPACS2 Software (Demir et al., 2019). Data of Lake Avlan such as physicochemical, altitude, lake area, average alkalinity and lake depth were obtained from General Directorate of Water Management of the Ministry of Agriculture and Forestry. RESULTS AND DISCUSSION As a result of sampling, Six submerged taxa, three emergent taxa and two algae (Table 2) were recorded throughout 4 vegetation periods in Lake Avlan between 2014-2015. Coverage of species were were also determined (Table 3). Polygonum amphibium L. has the highest coverage during all vegetation periods except third vegetation period. Also Polygonum amphibium L. is the most recorded macrophyte together with Myriophyllum spicatum L. Table 2. Species were recorded throughout 4 vegetation periods in Lake Avlan between 2014-2015. Taxon Polygonum amphibium L. Myriophyllum spicatum L. Schoenoplectus lacustris subsp. lacustris (L.) Palla Potamogeton crispus L. Ranunculus trichophyllus Chaix ex Vill. Potamogeton nodosus Poir. Bolboschoenus maritimus subsp. maritimus (L.) Palla Butomus umbellatus L. Nasturtium officinale R.Br. Cladophora sp. Zygnema sp.

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Life Forms Submerged Submerged Submerged Submerged Submerged Submerged Emergent Emergent Emergent Algae Algae

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Table 3. Coverages of Macrophytes each vegetation period No

Taxon

First Vegetation Period T1 (%) T2 (%)

T3 (%)

T4 (%)

Total Coverage T (%) (%) 90 22,5 45 11,25 25 6,25

P. amphibium L. 30 10 30 20 M. spicatum L. 30 5 10 0 S. lacustris subsp. lacustris (L.) 0 5 0 20 Palla Second Vegetation Period 2 0 2 0 4 1 1 Cladophora sp. 10 5 15 10 40 10 2 P. amphibium L. 15 5 5 0 20 5 3 M. spicatum L. 0 5 0 20 25 6,25 4 S. lacustris subsp. lacustris (L.) Palla Third Vegetation Period 1 0 0 0 1 0,25 1 P. crispus L. R. trichophyllus Chaix ex Vill. 0 0 0 2 2 0,5 2 0 0 5 0 5 1,25 3 Zygnema sp. Fourth Vegetation Period 5 10 5 15 35 8,75 1 P. amphibium L. 5 5 5 5 20 5 2 M. spicatum L. 10 5 5 5 25 6,25 3 B. maritimus subsp. maritimus (L.) Palla 1 5 5 5 16 4 4 B. umbellatus L. 2 2 5 2 11 2,75 5 P. crispus L. 5 10 10 5 30 7,5 6 P. nodosus Poir. 0 4 2 2 8 2 7 R. trichophyllus Chaix ex Vill. 0 5 5 0 10 2,5 8 N. officinale R.Br T: Transect, T1: First Transect, T2: Second Transect, T3: Third Transect, T4: Fourth Transect. 1 2 3

Dataset of Lake Avlan such as physicochemical (nutrients, Secchi depth, alkalinity, chlorophyll-a) and descriptive data (surface area, altitude, mean alkalinity and depth) were given in the Table 4.

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Table 4. Physicochemical and descriptive data of Lake Avlan Parameter

Fourth Vegetation Period

Mean

0,5

Third Vegetation Period 1016 850 0,2

0,25

0,3625

Parameter Quality Class -

9,22 23

9,6 16,2

8,51 21

8,66 24,9

8,99 21,82

High -

507 9,63 122 11,1

308 8,55 18,8 18

278 9,17 19,20 8,00

348 7,42 34,40 8,90

360,25 8,69 48,60 11,50

COD (mg/L)

73,15

32,80

44,70

55,95

TOC (mg/L) AN (mg/L) Nitrite Nitrogen (mg/L) Nitrate Nitrogen (mg/L) TKN (mg/L) OPP (mg/L) Salinity (‰) Alkalinity (mgCaCO3/L) Total Nitrogen (mg/L) Total Phosphorus (mg/L) Secchi depth (m) Chlorophyll-a (µg/L)

5,6 <0,10 0,01

5,7 0,313 0,102

<1,0 <0,1 0,002

9,01 0,132 0,004

5,20 0,14 0,03

High High Moderate (III) Moderate (III) High -

<0,10

0,18

0,11

<0,1

0,10

High

1,52 <0,20 0,24 15,00

0,46 <0,2 0,18 15,00

0,41 <0,01 0,14 15,00

0,34 <0,01 0,16 15,00

0,68 0,05 0,18 15,00

Good Good -

1,66

1,06

0,58

0,48

0,94

High

0,44

0,95

0,04

0,03

0,37

Moderate (III)

0,5

0,23

0,2

0,25

0,295

Hypertrophic

<3,10

<0,1

1,95

0,80

Oligotrophic

Altitude (m) Lake area (ha) Lake Depth (m) pH Temperature (oC) EC (µs/cm) DO (mg/L) SS (mg/L) BOD (mg/L)

First Vegetation Period

Second Vegetation Period

0,5

AN: Ammonium Nitrogen, BOD: Biological Oxygen Demand, COD: Chemical Oxygen Demand, DO: Dissolved Oxygen, EC: Electrical conductivity, OPP: Ortho-Phosphate Phosphorus, SS: Suspended Solids, TKN: Total Kjeldahl Nitrogen, TOC: Total Organic Carbon. As a result of calculations, EQR of three vegetation period were achieved as poor status. Only the EQR of fourth vegetation period which is optimum vegetation period, was found as moderate status (Table 5). Calculation were performed according to optimum vegetation period which Polygonum amphibium L., Myriophyllum spicatum L., Butomus umbellatus L., Bolboschoenus maritimus subsp. maritimus (L.) Palla, Potamogeton crispus L., Potamogeton nodosus Poir., Ranunculus trichophyllus Chaix ex Vill. and Nasturtium officinale R.Br. were recorded. Bolboschoenus maritimus subsp. maritimus (L.) Palla and Nasturtium officinale R.Br. 402

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are not existing in LEAFPACS2 Lake macrophyte taxa list. Therefore, taxa recorded in optimum vegetation period were not included in the calculation. As a result of calculations, we measured ecological quality of Lake Avlan as moderate with 0,52 index score. Detailed calculation tables of vegetation periods are given in the Appendix 1. Table 5. Calculation results of EQR Vegetation Period

EQR and Status

First

0,30 (Poor)

Second

0,38 (Poor)

Third

0,36 (Poor)

Fourth

0,52 (Moderate)

P. amphibium L., P. crispus L., Cladophora sp. and Zygnema sp. taxa are known to have a relatively high tolerance. Also these taxa are mostly characterise in moderate-poor status lakes. In addition, it has been reported that the P. crispus L. commonly exists in the eutrophic shallow lakes (Penning et al., 2008; Poikane, 2018). Also M. spicatum L. is effectively used as a bioindicator for monitoring water in terms of heavy metals and it characterises lakes with nutrient-rich conditions (Yabanli et al., 2014; Søndergaard et al., 2010). The taxa observed during the four vegetation periods are mostly indicators of moderate-poor water bodies. This confirms the calculation results of EQR in Lake Avlan. In addition, Toudjani et al. (2017) were determined EQR of Lake Avlan based on phytoplankton and as a result of their study they found EQR of Lake Avlan as moderate status in two of three different indexes and good in one. This research generally confirms that our findings are consistent with EQR of Lake Avlan based on phytoplankton. The response of macrophytes to the environmental effects of eutrophication and assessment of EQR based on macrophytes has been demonstrated in several studies (Coops et al., 2007; Penning et al., 2008; Søndergaard et al., 2010; Poikane et al., 2018). In this context, our study shows that the macrophytes are an important quality element in the assessment of the lakes. Especially they provide information about eutrophication in lakes. Acknowledgement: This study was supported by the Directorate General for Water Management of the Ministry of Agriculture and Forestry (Republic of Turkey) (Project no: 188.02.01) and DOKAY-ÇED Company.

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Appendix 1 First vegetation period Lake LEAFPACS2 calculation. Species No 1 2 3

LEAFPACS2 Observed Values Reference Values EkoLEAFPACS Status

LMN I FG 8,25 10 6,23 7

Taxon Polygonum amphibium Myriophyllum spicatum Schoenoplectus lacustris subsp. lacustris

NTAX A

NFG

7,24

4,933

5,321

9,265

LMNI

NF NF

%Coverag e 22,5 11,25

COV ALG 16,87 5 0 8,2

0,05

NF NF -

%Coverage 22,5 11,25 -

0,3 Poor

Second vegetation period Lake LEAFPACS2 calculation. Species No 1 2 3

LEAFPACS2 Observed Values Reference Values EkoLEAFPACS Status

Taxon Polygonum amphibium Myriophyllum spicatum Schoenoplectus lacustris subsp. lacustris LMNI 7,24 4,933

LMNI 8,25 6,23 -

NFG 2 5,321

NTAXA COV ALG 2 16,875 0 9,265 8,2 0,05

10 7 -

0,3 Poor

Third vegetation period Lake LEAFPACS2 calculation. Species No 1 2 3

LEAFPACS2 Observed Values Reference Values EkoLEAFPACS Status

Taxon Polygonum amphibium Myriophyllum spicatum Schoenoplectus lacustris subsp. lacustris LMNI 7,24 4,933

LMNI 8,25 6,23 -

F 10 7 -

NF NF -

%Coverage 22,5 11,25 -

NFG NTAXA COV ALG 2 2 16,875 0 5,321 9,265 8,2 0,05 0,3 Poor 404

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Fourth vegetation period period of Lake Avlan. Species No 1 2 3 4 5 6 7 8 LEAFPACS2 Observed Values Reference Values EkoLEAFPACS Status

Taxon Polygonum amphibium Myriophyllum spicatum Bolboschoenus maritimus Butomus umbellatus Potamogeton crispus Potamogeton nodosus Ranunculus trichophyllus Nasturtium officinale

LMNI 8,25 6,23 7,97 7,5 6,25 5,31 -

LMNI 6,918 4,933

FG 10 7 13 17 8 18 -

NFG NTAXA 6 6 5,321 9,265 0,52 Moderate

F NF NF NF NF NF NF -

%Coverage 8,75 5 4 2,75 7,5 2 -

COV 5 8,2

ALG 0 0,05

REFERENCES Cook, C.D., Gut, B.J., Rix, E.M., & Schneller, J. (1974). Water plants of the world: a manual for the identification of the genera of freshwater macrophytes. Springer Science & Business Media, 561s, England. Coops, H., Kerkum, F. C. M., Van den Berg, M. S., & Van Splunder, I. (2007). Submerged macrophyte vegetation and the European Water Framework Directive: assessment of status and trends in shallow, alkaline lakes in the Netherlands. Hydrobiologia, 584(1), 395-402. Demir, O., Kızılırmaklı, A., & Cabi, E. (2019). Developing a Software Based on Lake LEAFPACS2 Calculations by Using Python Programming Language. International Biological, Agricultural And Life Science Congress, Lviv, Ukraine. Deniz, I. G., & Sümbül, H. (2004). Flora of the Elmalı cedar research forest (Antalya/Turkey). Turkish Journal of Botany, 28(6), 529-555. Doğan, M., Karataş, M., & Aasım, M. (2018) In vitro Koşullarda Ceratophyllum demersum L.’un Krom (III) Akümülasyonunun Araştırılması. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, 21(3): 277-285. WFD (2014). UKTAG Lake Assessment Method Macrophytes and Phytobenthos. Kum, G., & Sönmez, M. E. (2016). The relatıonshıp between spatıal change of avlan lake and surroundıng clımate. Marmara coğrafya dergisi, 34, 300-311. Melzer, A. (1999). Aquatic macrophytes as tools for lake management. In The Ecological Bases for Lake and Reservoir Management, 181-190. Reddy, K.R., & De Busk, W.F. (1985). Nutrient Removal Potential of Selected Aquatic Macrophytes 1. Journal of Environmental Quality, 14(4): 459-462. Rejmankova, E. (2011). The role of macrophytes in wetland ecosystems. Journal of Ecology and Environment, 34(4): 333-345. Søndergaard, M., Johansson, L. S., Lauridsen, T. L., JØRGENSEN, T. B., Liboriussen, L., & Jeppesen, E. (2010). Submerged macrophytes as indicators of the ecological quality of lakes. Freshwater Biology, 55(4), 893-908.

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Wersal, R. M., Madsen, J.D., McMillan, B.R., & Gerard, P.D. (2006). Environmental factors affecting biomass and distribution of Stuckenia pectinata in the Heron Lake System, Minnesota, USA. Wetlands, 26(2): 313. Westlake, D.F. (1981). Temporal changes in aquatic macrophytes and their environment. Keske, P., & Ketenoğlu, O. (2010). The Flora of the Vicinities of Avlan Lake (Antalya-Elmalı). Journal of Applied Biological Sciences (JABS) E-ISSN: 2146-0108, 4(3), 11-20. Penning, W. E., Mjelde, M., Dudley, B., Hellsten, S., Hanganu, J., Kolada, A., ... & Ecke, F. (2008). Classifying aquatic macrophytes as indicators of eutrophication in European lakes. Aquatic ecology, 42(2), 237-251. Poikane, S., Portielje, R., Denys, L., Elferts, D., Kelly, M., Kolada, A., ... & van den Berg, M. S. (2018). Macrophyte assessment in European lakes: Diverse approaches but convergent views of ‘good’ecological status. Ecological indicators, 94, 185-197. Tezcan, S., Jeanne, C., & Keskin, B. (2007). Ground beetles (Coleoptera, Caraboidea) of the ecologically managed cherry orchards of Western Anatolia (Turkey) along with some new additional data. Toudjani, A. T., Çelekli, A., Gümüş, E. Y., Kayhan, S., Lekesiz, H. Ö., & Çetin, T. (2017). Ecological status assessment based on phytoplankton of lentic systems in West Mediterranean Basin (Turkey). Ecology 2017 International Symposium, Kayseri, Turkey. Ünlüsoy, A. Y., & Ketenoğlu, O. T. D. (2011). Avlan gölü çevresi vejetasyonunun sintaksonomik analizi (Antalya-Elmalı/Türkiye) (Doctoral dissertation, Ankara Üniversitesi Fen Bilimleri Enstitüsü Biyoloji Anabilim Dalı). Van Neer, W., Wildekamp, R. H., Küçük, F., & Ünlüsayın, M. (2008). The 1997-1999 surveys of the Anatolian fish fauna and their relevance to the interpretation of trade at Sagalassos. Geo-and Bio-Archeology at Sagalassos and in its Territory. Leuven University Press, Leuven, 299-323. Yabanli, M., Yozukmaz, A., & Sel, F. (2014). Heavy metal accumulation in the leaves, stem and root of the invasive submerged macrophyte Myriophyllum spicatum L.(Haloragaceae): an example of Kadin Creek (Mugla, Turkey). Brazilian Archives of Biology and Technology, 57(3), 434-440.

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IS SKIN PRICK TEST EFFECTIVE TO DETERMINE AEROALLERGENS? Meliha Merve ÇİÇEKLİYURT*1 1

Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Medical Biology, Çanakkale, Turkey *Corresponding author’s e-mail: mervemeliha@comu.edu.tr

ABSTRACT Skin prick test is widely used to determine allergic reactions. In contrast, the long distance atmospheric transport of pollens may be trigger allergenic reactions. The main advantage of the SP-test is easy to use and determine the allergens, especially aero-allergens. In contrast the test consists of common aero-allergens. Thus, in that paper we would like to evaluate the power of skin prick test in current literature and our laboratory based knowledge. Keywords: Aeroallergens, Skin Prick test.

INTRODUCTION The skin prick test is a pain free test which is gold standard to determine the especially aeroallergens. Skin prick test cause mini allergical reactions to identify allergens by bound IgE together with mast cell degranulation (Gupta et al., 2019). Although the test is efficient, there is some limitations during the SP-test. In this literature review, our aim to evaluate the effectivity of skin prick test with our previous knowledge. MATERIAL AND METHODS In this study, we aimed to review to effectivity of skin prick test. For this purpose, literature review was done in Pubmed. During the literatüre research the key words skin prick test, aeroallergens, sensitivity, DISSCUSSION Skin prick test is widely used as a major diagnostic tool to determine specific type of allergy. Although the test is cost effective, drugs especially oral anti-histamines, topical corticosteroids and anxiolytic drugs effects the performance of the test (Yuen et al. 2007). In addition, only healthy skin is suitable to perform skin prick test. The allergic patients with serious urticarial skin or eczema must be performed the test in unaffected skin area. The patient with flagrin mutation is also higher risk of asthma and allergic rhinitis due to the easily access of allergens because of disrupted skin barrier (van den Oord et al., 2009; Marenholz et al., 2006; McAleer and İrv,ne 2013). In contrast, there is no direct association were found between flagrin mutations and disease but trigger secondary sensitization (Hougaard et al. 2014). Skin prick test reactivity is also effected from sex and age status of the patients(Åslund et al., 2008; Barbee et al., 1987, Gergen et al., 1992). The patients with earlier age of onset of the disease also able to detect more in SPT only (Yuen et al., 2007). Immunosenescence, immune system deterioration depend on aging, is the main reason of incorrect interpretation of skin testing in geriatric. As indicated in literature, thymus enlarge during childhood period and 407

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shrinks after puberty. Thymus involution cause immune system gets weak and immunosenecense occurs in geriatric people. The most possible explanation is reduced immunity and histamine skin reactivity epend on age (Scichilonea et al., 2011; Song et al 2011). The majority of studies were shown decrease in skin prick test reactivity in geriatric age (Scichilonea et al., 2011). Skassa-Brociek et al. have been reported the histamine reactivity reduced after 50 years as well reached lower level in 60 years and harder to evaluate after 70 years. However, the relation between sex and skin prick test differs in literature (Barbee et al., 1987, Warm et al., 2012, Urik et al., 2000). In our previous studies we have found that the house dust mite allergy able to determine in skin prick test, in contrast patients with mite allergy could not determine by prick test. The main reason of the low power of skin prick test is the panel only consist of two common species D. farinae and D. Pteronyssinus, but there must be different species in Çanakkale region (Unpublished data). In addition, the pollen content of the skin prick test is also weak to determine the endemic aero-allergens (Çiçekliyurt ve Akkaya, 2019). CONCLUSION Depend on our previous studies we can conclude that skin prick test is effective to identify allergies against to pollen, mold, pet dander, dust mites and foods. In contrast, the skin prick test is weak to determine the endemic pollens or mite species. REFERENCES Aslund, N, Thomsen SF, Mølgaard E, Nolte H, Backer V. Changes in skin test reactivity among adults with atopic disease: a 3-year prospective study. Ann Allergy Asthma Immunol. 2008;101:524–528. Barbee RA, Kaltenborn W, Lebowitz MD, Burrows B. Longitudinal changes in allergen skin test reactivity in a community population sample. J Allergy Clin Immunol. 1987;79:16– 24. Çiçekliyurt MM, Akkaya M. Determination of the amount of allergic pollen in the atmosphere at Çanakkale, Turkey. Determination airborne pollen distribution in Çanakkale, Turkey. Phytologia Balcanica. Gergen PJ and Turkeltaub PC. The association of individual allergen reactivity with respiratory disease in a national sample: data from the second national health and nutrition examination survey, 1976-80 (NHANES II). J Allergy Clin Immunol. 1992;90:579–588. Gupta N, Agarwal P, Sachdev A, Gupta D Allergy Testing - An Overview. Indian Pediatr. 2019;56:951-957. Marenholz I, Nickel R, Ruschendorf F et al. Filaggrin loss-of-function mutations predispose to phenotypes involved in the atopic march. J Allergy Clin Immunol 2006; 118: 866–871 McAleer MA and Irvine AD. The multifunctional role of filaggrin in allergic skin disease. J Allergy Clin Immunol. 2013;131:280-91. Pesonen M, Kallio MJ, Siimes MA, Ranki A. Allergen skin prick testing in early childhood: reproducibility and prediction of allergic symptoms into early adulthood. J Pediatr. 2015; 166(2):401-406. Scichilonea N, Callaria A, Augugliaroa G, Marchesea M, Togiasb A, Belliaa V. The impact of age on prevalence of positive skin prick tests and specific IgE tests. Respir Med. 2011;105:651-658. van den Oord RA, Sheikh A. Filaggrin gene defects and risk of developing allergic sensitisation and allergic disorders: systematic review and meta-analysis. BMJ 2009;339: b2433.

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Warm, K, Backman H, Lindberg A, Lunbäck B, Rönmark E. Low incidence and high remission of allergic sensitization among adults. J Allergy Clin Immunol. 2012;129:136–142. Ulrik CS and Backer V. Atopy in Danish children and adolescents: results from a longitudinal population study. Ann Allergy Asthma Immunol. 2000;85: 293–297. Yuen AP, Cheung S, Tang KC, Ho WK, Wong BY, Cheung AC, Ho AC. The skin prick test results of 977 patients suffering from chronic rhinitis in Hong Kong. Hong Kong Med J. 2007; 13:131-6.

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ASSESSMENT OF GLOBAL DNA METHYLATION IN LUNG TISSUE OF RABBITS THAT EXPOSED ROCURONIUM DURING ANESTHESIA Meliha Merve ÇİÇEKLİYURT*1, Betül ALTINIŞIK2 1

Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Medical Biology, Çanakkale, Turkey 2

Çanakkale Onsekiz Mart University, Faculty of Medicine, Department of Anesthesia and Reanimation, Çanakkale, Turkey *Corresponding author’s e-mail: mervemeliha@comu.edu.tr

ABSTRACT The study was carried out to examine the epigenetic changes on lung tissue of rabbits under Rocuronium exposure. 7 New Zealand White (NZW) adult male rabbits (weight: 2-2.5 kg) were used. The control and rocuronium group consist of 3 and 4 rabbit respectively. We analyzed the Global DNA methylation status of rabbit lung tissue by using 5-mC DNA ELISA method. The percentage of genomic DNA methylation was calculated and statistical analyses were performed by SPSS 18.The results showed that the methylation patterns of rocuronium group is significantly higher 1.3 times more than controls (p<0.05). Keywords: DNA methylation, Epigenetics, Rocuronium

INTRODUCTION The long-term epigenetic effects of anesthetic molecules are still unknown but accumulating evidence from literature shown that pregnant women, neonates, infants that exposed aesthesia has higher prevalence of cognitive disorders, learning and memory disabilities depend on the anaesthesia induced apoptosis, synaptic deficiency, neuroinflammation, Tau phosphorylation in brain of the young animals (Andropoulos et al. 2017; Shen et lal. 2011; Schenning et al, 2017; Zhang et al. 2019). In addition, the effects of anaesthetic molecules differs due to the exposed time of the life. In example, rodents exposed to isoflurane in adult or elderly more prone to developed cognitive impairment (Callaway et al., 2012; Culley et al., 2004; Hovens et al., 2014; Ji et al., 2014 Kong et al., 2013; Lin and Zuo, 2011) in contrast neonatal stage isoflurane exposure able to tolerate better (Loepke et al., 2009; Sanders et al., 2009; Yang et al. 2014; Zhong et al., 2015). Furthermore, epigenetic side effects of anaesthetic molecules also differs depend on the kind of anaesthetic molecules. General anaesthetic molecules decrease global DNA methylation (Lirk et al., 2012) whist opinoids increase the global DNA methylation (Doehring et al., 2011 ve Doehring et al., 2013). Rocuronium, aminosteroid neuromuscular blocking agent, is widely used during general anesthesia to induce skeletal lung relaxation. The molecule is effective and safe for infants, children, adults and geriatric patients. In the current study, we aim to explore the effects of rocuronium on global DNA methylation status in adult rabbits. MATERIAL AND METHODS In this study 6 New New Zealand White (NZW) adult male rabbits (weight:2-2,5 kg) were were divided into control and Rocuronium group. The rabbits were housed in plastic cages, 12 h light 410

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/ 12 h dark photoperiod at 21 ± 2 ˚C. The animals were feed by standard rabbit diet (Bil-Yem Ltd. Co., Ankara, Turkey) without any limitation of water throughout the experiment. Rocuronium group were exposed 1mg/kg rocuronium This study was approved by Animal Experimental Ethics Commitee of Çannakkale Onsekiz Mart University (Approval Numnber: ). The study were done accordenca with the frame of Policy on laboratory animals and minimal number of animals were used. The rabbits in each group were fasted for 8 hours the night before the experiment. Xylazin 5 mg/kg were applied intra-muscular before general anaesthesia procedure. While the rabbits were sedated, Group 2 animals received 1 mg / kg rocuronium. This procedure were applied to the rabbits twice a day apart, and after 3rd application all rabbits were sacrificed.The lung tissue of rabbits were surgically removed from animals and then immediately transfer into DNA shield (ZymoResearch) to protected DNA from the stress caused by freeze thaw cycling and store long periods of time before purification of DNA. Genomic DNA was first extracted by Thermo DNA isolation kit (Thermo Scientific™, K0512) according to manufacturer’s guide. Global DNA methylation level was examined by 5-mC DNA ELISA kit (Zymo Research) using 5-mC monoclonal antibodies in ELISA method. Obtained data analyzed with SPSS 20.0 pack programme. Data processing was carried out with count, mean value, standard deviation, Median value, minimum and Maximum values. As a result of Normal Distribution Suitability Test, Non-parametric test Mann-Whitney U was used. P<0,05 was accepted for Statistical significance. RESULTS The changes of global levels of DNA methylation in lung tissue of adult rabbits after exposed to rocuronium of 1 mg/kg for 3 days were shown on the Figure 1.

Figure 3. Global DNA methylation status of control and rocuronium groups The authors found a substantial increase of global DNA methylation level of lung tissue under Rocuronium exposure. We observed that lower methylation levels were present in control group(p<0.005). 411

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DISCUSSION The epigenetic effects of anaesthetic molecules varies depend on the chemical structure of the molecules. The molecules that has metal or hydrocarbon group more probable to trigger epigenetic changes. Aside, the developmental stage of organism and the exposure duration of anaesthetic molecules are two other important point underlie the molecular interactions between the anaesthetic molecules and epigenetic alterations. In addition, the epigenetic effects of anaesthetic molecules could be observed not only exposed individuals also first generation and after second generation (Chanlon et al., 1981). Methylated cytosines throughout genomes (the 'methylome') is important issue for the development of the central nervous system. DNA methylation is a covalent modification and is characterized by the formation of a 5-methyl cytosine (5m-C) structure by attaching a methyl group (-CH3) to carbon 5 of the cytosine (C) base. DNA methylation takes place in regions where CpG sequences are concentrated by sequential sequencing of cytosine (C) and guanine (G) pairs in the genome. Although there is no change in genome structure by the rearrangement of chromosomes and/or alteration of gene expression, clinical results has been related to changes of epigenic patterns depends on hypo methylations. In the literature, opioids are known to increase global DNA methylation levels (Doehring et al., 2011 and Doehring et al., 2013), while local anesthetics reduce global DNA methylation (Lirk et al., 2012). In addition, there is grooving evidence of sevoflurane increase global DNA methylation and cause alteration on development of the central nervous system (Ju et al. 2016; Ju et al., 2018, Zhu et al., Zhang et al. 2019). The folate metabolism is disturbed by sevoflurane anaesthesia cause demyelination in the developmental brain and trigger cognitive disorder via epigenetic alterations on myelination-related gene (ERMN) (Zhang et al. 2019). Emerging studies have revealed that epigenetic dysregulation in neurodevelopmental effects of anaesthetic molecules. In contrast, there is no consideration related to effects on the lung or relation with respiratory disorders. CONCLUSIONS The pattern of methylated cytosine in the genome important issue to understand influence of the molecules on genome thus useful tool to understand the evidence for biochemical links between the anaesthetic molecules and health. This is the first study that evaluate the global methylation status of rocuronium on rabbit lung tissue. The results is rocuronium trigger epigenetic changes more than control group. Depend on the current data authors conclude that rocuronium may be the cause of respiratory disease and postoperative pulmonary complication. Acknowledgements: This work was supported by Ă&#x2021;anakkale Onsekiz Mart University The Scientific Research Coordination Unit, Project number: TSA-2018-2611. REFERENCES Andropoulos DB, Greene MF. Anesthesia and Developing Brains - Implications of the FDA Warning. N Engl J Med. 2017;376:905-907. Callaway, J.K., Jones, N.C., Royse, C.F. (2012). Isoflurane induces cognitive deficits in the Morris water maze task in rats. Eur J Anaesthesiol, 29, 239-245. Chalon J, Tang CK, Ramanathan S, Eisner M, Katz R, Turndorf H. Exposure to halothane and enflurane affects learning function of murine progeny. Anesth Analg. 1981;60:794â&#x20AC;&#x201C;7. Culley, D.J., Baxter, M.G., Crosby, C.A., Yukhananov, R., Crosby, G. (2004). Impaired acquisition of spatial memory 2 weeks after isoflurane and isoflurane-nitrous oxide anesthesia in aged rats. Anesth Analg, 99, 1393-1397; table of contents.

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Doehring A, Geisslinger G, Lotsch J (2011). Epigenetics in pain and analgesia: an imminent research field. Eur J Pain 15: 11–16. Doehring A, Oertel BG, Sittl R, Lotsch J (2013). Chronic opioid use is associated with increased DNA methylation correlating with increased clinical pain. Pain 154: 15–23. Hovens, I.B., Schoemaker, R.G., van der Zee, E.A., Absalom, A.R., Heineman, E., van Leeuwen, B.L. (2014). Postoperative cognitive dysfunction: Involvement of neuroinflammation and neuronal functioning. Brain Behav Immun, 38, 202-210. Ji, M., Dong, L., Jia, M., Liu, W., Zhang, M., Ju, L., Yang, J., Xie, Z., Yang, J. (2014). Epigenetic enhancement of brain-derived neurotrophic factor signaling pathway improves cognitive impairments induced by isoflurane exposure in aged rats. Mol Neurobiol, 50, 937-944. Kong, F., Chen, S., Cheng, Y., Ma, L., Lu, H., Zhang, H., Hu, W. (2013). Minocycline attenuates cognitive impairment induced by isoflurane anesthesia in aged rats. PLoS One, 8, e61385. Lin, D., Zuo, Z. (2011). Isoflurane induces hippocampal cell injury and cognitive impairments in adult rats. Neuropharmacology, 61, 1354-1359. Lirk P, Berger R, Hollmann MW, Fiegl H. Lidocaine time- and dose-dependently demethylates deoxyribonucleic acid in breast cancer cell lines in vitro. Br J Anaesth. 2012;109:200– 207. Ju L.S., Yang J.J., Morey T.E. Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next generation of male, but not female, rats. Br J Anaesth. 2018;121(2):406–416. Ju L.S., Jia M., Sun J. Hypermethylation of hippocampal synaptic plasticity-related genes is involved in neonatal Sevoflurane exposure-induced cognitive impairments in rats. Neurotox Res. 2016;29(2):243–255 Loepke, A.W., Istaphanous, G.K., McAuliffe, J.J., 3rd, Miles, L., Hughes, E.A., McCann, J.C., Harlow, K.E., Kurth, C.D., Williams, M.T., Vorhees, C.V., Danzer, S.C. (2009). The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth Analg, 108, 90-104. Sanders, R.D., Xu, J., Shu, Y., Januszewski, A., Halder, S., Fidalgo, A., Sun, P., Hossain, M., Ma, D., Maze, M. (2009). Dexmedetomidine attenuates isoflurane-induced neurocognitive impairment in neonatal rats. Anesthesiology, 110, 1077-1085. Schenning K.J., Noguchi K.K., Martin L.D. Isoflurane exposure leads to apoptosis of neurons and oligodendrocytes in 20- and 40-day old rhesus macaques. Neurotoxicol Teratol. 2017;60:63–68 Shen X., Dong Y., Xu Z. Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology. 2013;118(3):502–515 Yang, B., Liang, G., Khojasteh, S., Wu, Z., Yang, W., Joseph, D., Wei, H. (2014). Comparison of neurodegeneration and cognitive impairment in neonatal mice exposed to propofol or isoflurane. PLoS One, 9, e99171. Zhu Y., Wang Y., Yao R. Enhanced neuroinflammation mediated by DNA methylation of the glucocorticoid receptor triggers cognitive dysfunction after sevoflurane anesthesia in adult rats subjected to maternal separation during the neonatal period. J Neuroinflammation. 2017;14(1):6 Zhong, T., Guo, Q., Zou, W., Zhu, X., Song, Z., Sun, B., He, X., Yang, Y. (2015). Neonatal isoflurane exposure induces neurocognitive impairment and abnormal hippocampal histone acetylation in mice. PLoS One, 10, e0125815. Zhang L, Xue Z, Liu Q, Liu Y, Xi S, Cheng Y, Li J, Yan J, Shen Y, Xiao C, Xie Z, Qiu Z, Jiang H. Disrupted folate metabolism with anesthesia leads to myelination deficits mediated by epigenetic regulation of ERMN. EBioMedicine. 2019;43:473-486. 413

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EFFECT OF THE USE OF AUTOCHTHONOUS STARTER CULTURES ON THE COLOUR CHANGES THAT TAKE PLACE THROUGHOUT THE DRYINGRIPENING PROCESS OF GALICIAN CHORIZO SAUSAGE Miriam Rodríguez-GONZÁLEZ1,2*, Javier CARBALLO1,2 1 2

Food Technology Area, Faculty of Sciences, University of Vigo, 32004 Ourense, Spain

CITACA, Agri-Food Research and Transfer Cluster, Campus da Auga, University of Vigo, 32004 Ourense, Spain *Corresponding author’s e-mail: mirrodriguez@uvigo.es

ABSTRACT In this work, we studied the effect of the use of starter cultures made from strains previously isolated from traditional Galician sausages on the colour changes that take place during the drying ripening process of Galician chorizo, a Spanish traditional sausage. Three different batches of Galician chorizo were manufactured in triplicate: CNT batch, non-inoculated control, EQU batch, inoculated with Lactobacillus sakei LS131 and Staphylococcus equorum SB23, and SAP batch, inoculated with L. sakei LS131 and Staphylococcus saprophyticus SB12. From each of the three replicates of each batch, samples were taken at day 0 (mix before stuffing) and after 2, 5, 9, 14, 21 and 30 days of ripening. Each sample was analysed for moisture content, pH value, nitrosylheme pigments, total heme pigments, percentage conversion to cured meat pigments, and colour parameters (L*, a* and b*). At the end of the manufacturing process, the values of a * and b * were significantly higher in sausages inoculated with starter cultures than in the control batch. Similarly, the conversion to nitrosylheme pigments at the end of manufacturing were also significantly higher in the inoculated batches than in the control batch. Keywords: Colour parameters, Meat pigments, Lactobacillus sakei, Staphylococcus equorum, Staphylococcus saprophyticus, Starter cultures

INTRODUCTION

Raw-cured sausages are one of the oldest processed foods known to man. Galician chorizo (Chorizo Gallego) is the most important within the raw-cured sausages made in the northwest of Spain. Nowadays this product is still elaborated in a totally empirical way, based on traditional recipes transmitted from generation to generation, at homes or in small industries. The consequence of this is the great heterogeneity in the characteristics and quality of the products present in the markets, which often negatively influences the demand of these products by consumers and limits their diffusion to wider markets. These products are characterized, among other attributes, by a marked reddish color and a characteristic aroma (Fonseca et al., 2013b). Color deficiencies in this type of foods are likely to cause rejection even if they have a good taste and texture (Ling et al., 1996; Gómez et al., 2008). The paprika, due to its high coloring power, provides an initial redness to the fresh sausage that is maintained until the end of the shelf life of the products (Gómez et al., 2008). The maintenance of this characteristic red coloration is also due to the formation of 414

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nitrosylheme pigments such as nitrosylmioglobin and nitrosylmiochromogen (Honikel., 2008, Yong et al., 2019). These pigments are formed throughout the ripening process of the sausage as a result of the reaction of nitric oxide with myoglobin leading to nitrosoheme pigment formation (Acton et al., 1974). The decrease in pH in this type of sausages, which is mainly due to the accumulation of lactic acid as a product of the metabolism of carbohydrates by the LABs (Fonseca, 2013), provides the necessary acidic conditions for this pigment formation reaction to occur. In addition to the pH, the formation of this type of pigment is also influenced by environmental conditions during ripening (relative humidity and temperature) (FernándezFernández et al., 1998). The use of autochthonous starter cultures specifically selected and designed for this type of products allows, without sacrificing the sensory attributes, the obtaining of sausages with a marked personality and a high and constant organoleptic and sanitary quality, together with a greater control on the physico-chemical changes that take place throughout the ripening process (Fonseca et al., 2013b). The strains selected to integrate the starter cultures should present adequate technological properties, such as the ability to prevailing over the contaminant microbiota at the pH, temperature and NaCl concentration occurring in the sausage throughout the entire ripening process, acidifying, proteolytic and lipolytic capacities, good sanitary aptitude, etc. (García Fontan et al., 2007a, b; Cachaldora, 2011; Fonseca et al., 2013b). In this work, the effect of the use of starter cultures integrated by strains previously isolated from traditional Galician sausages and selected on the basis of their metabolic activities on the changes taking place in the color of the Galician chorizo sausage has been studied. MATERIALS AND METHODS Sausage manufacturing and sampling In triplicate and following traditional techniques, three different batches of Galician chorizo were manufactured, two of them with addition of different starter strains. Batches were named according to the starter culture added: (i) CNT batch, non-inoculated control, (ii) EQU batch, inoculated with Lactobacillus sakei LS131 and Staphylococcus equorum SB23, and (iii) SAP batch, inoculated with L. sakei LS131 and Staphylococcus saprophyticus SB12. L. sakei LS131 was added to the mix in an amount of 106 C.F.U./ g, while each Staphylococcus strain was added in an amount of 107 CFU/g. The starter cultures were chosen from a set of isolates after identification by 16s rRNA gene sequencing and technological characterization; they showed suitable technological properties (ability to grow at temperature and pH values of fermenting sausage, and at high NaCl concentrations, as well as nitrate reductase, proteolytic and lipolytic activities) and safety aptitude (absence of amino acid decarboxylase and enterotoxigenic activities) (Cachaldora et al., 2013). Galician chorizo formulation includes lean pork (80%), pork back fat (20%), sweet paprika (22 g/kg), NaCl (15 g/kg), garlic (4 g/kg), spicy paprika (1 g/kg) and water (40 mL/kg). The lean pork and the pork back fat were ground through a 10 mm diameter mincing plate and vacuum mixed together with the other ingredients for 3 min. The mix was maintained at 4 ºC for 24 h and then stuffed into natural porcine casings with a diameter of 36-38 mm. The sausages were fermented for 9 days at 6 ºC and 80% relative humidity and then transferred to a drying-ripening chamber where they were kept for 21 more days at 12 ºC and 75% relative humidity. From each of the three replicates of each batch, samples were taken at day 0 (mix before stuffing) and after 2, 5, 9, 14, 21 and 30 days of ripening.

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Analytical methods The moisture content of the samples was determined by dehydration at 105 ºC until constant weight according to the ISO recommended standard 1442:1997 (ISO, 1997). The pH of samples was measured using a pH metre GLP21 (Crison Instruments, Barcelona, Spain) according to the AOAC 14.022 method (AOAC, 1980) after mixing 10g of sample with 90ml of distilled water for 2min in a Sorvall Omnimixer homogeniser (Omni International, Waterbury, CT, USA). The nitrosylheme pigments, total heme pigments and percentage conversion to cured meat pigments were determined following the methods described by Zaika et al. (1976). Colour was analysed by the CIEL*a*b* system (CIE (Commission Internationale de l'Eclairage) 1978) and using a portable colorimeter (Chroma Meter CR-400, Konica Minolta Sensing, Inc., Osaka, Japan) to determine the L* (lightness), a* (balance between red and green) and b* (balance between yellow and blue) coordinates. RESULTS AND DISCUSSION Total solid contents Table 1 shows the evolution of the total solid contents along the ripening of the different sausage batches. Total solids increased in a significant way (P<0.05) throughout the ripening process reaching final values of 75.57, 71.80 and 73.06 in CNT, EQU and SAP batches, respectively. The total solid contents were not significantly affected by the addition of the starter cultures. Table 1: Evolution of the total solid contents along the ripening process of Galician chorizo sausage manufactured using different starter cultures (values are means of three batches). Drying-ripening (days)

SEM

CNT

38.02

41.96

46.88

51.55

60.45

65.53

75.57

EQU

36.98

41.49

48.10

51.53

59.10

66.25

71.80

SAP

36.52

40.81

46.93

51.14

57.65

67.42

73.06

1,14

PcxPt

n.s

***

n.s

Total solids expressed as g/100g of sausage. CNT: control batch manufactured without starter cultures; EQU: inoculated with L. sakei + S. equorum; SAP: inoculated with L.sakei + S. saprophyticus. Significance: Pc: effect of the use of starter cultures. Pt: effect of the ripening time. n.s: non significant; *(P<.0.05); ** (P<0.01); *** (P<0.001). pH values The evolution of the pH values throughout the ripening process is shown in Fig. 1. At the end of the process, the pH values showed significant differences (P <0.001) between the control batch (5.81 ± 0.22) and the inoculated batches (5.60 ± 0.14 and 5.59 ± 0.18 for EQU and SAP respectively), with no significant differences between the two inoculated batches. The higher pH value at the end of the ripening process in the control batch could be related to the less acidifying capacity of the spontaneous microbiota when compared with the Lactobacillus strain added in the inoculated batches. Also, the growth of molds and yeasts capable of consuming lactic acid could be partially responsible for the less acidity after the two days of ripening in the

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control batch; the strains used as starter cultures managed the ripening process which would prevent growth of this type of microbiota (Fonseca et al, 2013a). 6,10 6,00 5,90 5,80 5,70 5,60 5,50 5,40 5,30 5,20

CNT EQU SAP

5 9 14 Drying-ripening time (days)

Figure 1. Evolution of the pH values throughout the drying-ripening process of Galician chorizo sausage manufactured using different starter cultures (plotted values are means of three batches). EQU: inoculated with L. sakei + S. equorum; SAP: inoculated with L. sakei + S. saprophyticus. A more intense proteolysis with the release of peptides, amino acids and ammonia from proteolytic reactions could also be responsible for the higher pH values in the control batch (Gรณmez et al., 2008; Spaziani et al., 2009). Pigments (heme and nitrosyl-heme)

% Conversion of pigments

The percentage of conversion of pigments (from heme to nitrosyl-heme) had initial values of 35.16%, 39.63% and 37.56% in CNT, EQU and SAP respectively, with no differences between the three batches. As indicated in Figure 2, the pigment transformation percentages showed a significant upward trend (P <0.001) throughout the entire ripening process, reaching final values of 81,88% in the CNT batch and of 82.02% and 86.26% in the EQU and SAP batches, respectively. The final value in the SAP batch was significantly (P <0.05) higher than in the control batch. 95 80 65

CNT EQU SAP

50 35 20 0

Drying-ripening time (days)

Figure 2. Evolution of the values of the % of conversion of pigments throughout the dryingripening process of Galician chorizo sausage manufactured using different starter cultures (plotted values are means of three batches). EQU: inoculated with L. sakei + S. equorum; SAP: inoculated with L .sakei + S. saprophyticus.

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Colour Changes in the CIELAB colour coordinates (L∗, a∗ and b∗) throughout the dryingripening process of the sausages are shown in Figure 3. The use of starter cultures did not have a significant effect on the luminosity (L*) of the sausages that decreased significantly during the whole drying-ripening process (values from 46-48 to 31-32) both in the control and in the batches inoculated. The decrease of this parameter during ripening occurred as a result of weight loss (Lorenzo et al., 2013; Lorenzo et al., 2012; Olivares et al., 2010) directly related to moisture loss, thus becoming a darker product. The evolution of the a * parameter (red-yellow coloration) coincides with the data described by Gómez et al., (2008), with an increase up to 5 days of maturation, reaching a maximum value of 31.94 in the SAP batch, then decreasing to the end of the process with final values of 17.47, 20.70 and 20.25 (in CNT, EQU and SAP respectively). Significant differences were observed both during ripening (P <0.001) and due to the use of starter cultures (P <0.01). The initial increase is obviously due to the formation of nitrosylmioglobin by the reaction of nitrogen monoxide (NO) with myoglobin (Lorenzo et al 2014; Baka et al., 2011). The initial redness is also influenced by the use of paprika with a high coloring power, just as the oxidation of the carotenoids present in this ingredient contribute to the loss of coloration. (Gomez et al., 2008). Finally, the parameter b * undergoes an evolution similar to that observed for redness. The initial increase in this parameter could be related to lipid oxidation. Again, we observed significant differences both during ripening and due to the use of starter cultures.

Color parameter value

60 50 40 30

CNT

EQU

SAP

0 0

9 14 21 30 0 L

9 14 21 30 0

a* Dry-ripening time (days)

9 14 21 30 b*

Figure 3. Evolution of the values of the colour parameters throughout the drying-ripening process of Galician chorizo sausage manufactured using different starter cultures (plotted values are means of three batches). EQU: inoculated with L. sakei + S. equorum; SAP: inoculated with L. sakei + S. saprophyticus. Conclusions The use of starter cultures did not have a significant effect on the luminosity (L *) of sausages that decreased significantly during the entire drying-maturation process both in the control lot and in the inoculated lots. However, at the end of the manufacturing process, the values of a * and b * were significantly higher in sausages inoculated with starter cultures than in the control batch, thus maintaining a more intense red coloration. Similarly, the conversion to nitrosylheme 418

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pigments at the end of manufacturing were also significantly higher in the inoculated batches than in the control batch. Acknowledgements: This work was financially supported by the Xunta de Galicia (The Regional Government), Spain, through the CITACA Strategic Partnership ED431E 2018/07. REFERENCES Acton, J. C, and J. E. Keller. (1974). Effect of fermented meat pH on summer sausage properties. J. Milk Food Technol. 37, 570-576. AOAC (Association of Official Analytical Chemists) (1980). Official methods of analysis (13thed.). Washington DC. Baka, A.M., Papavergou, E.J., Pragalaki, T., Bloukas, J.G. and P. Kotzekidou (2011). Effect of selected autochthonous starter cultures on processing and quality characteristics of Greek fermented sausages. LWT – Food Sci. and Technol., 44, 54-61 Cachaldora, A. (2011). Estudio de la aptitud tecnológica de cepas microbianas aisladas de embutidos tradicionales de Galicia. Unpublished Doctoral dissertation, Universidade de Vigo, Spain. Cachaldora, A., Fonseca, S., Franco, I., and Carballo, J. (2013). Technological and safety characterization of Staphylococcaceae isolated from Spanish traditional drycured sausages. Food Microbiol., 33(1), 61-68. Fernández-Fernández, E., Vázquez-Oderíz, M.L., and Romero-Rodríguez, M.A (1998) Colour changes during manufacture of Galician chorizo sausage. Z Lebensm Unters Forsch, 207, 18–21 Fonseca, S. (2013). Efecto de la Adición de cultivos iniciadores autóctonos sobre las características físico-químicas, microbiológicas y sensoriales del Chorizo Gallego. Unpublished Doctoral dissertation, Universidade de Vigo, Spain. Fonseca, S., Cachaldora, A., Gómez, M., Franco, I. and Carballo, J. (2013a). Effect of different autochthonous starter cultures on the volatile compounds profile and sensory properties of Galician Chorizo, tradicional Sapnishs dry fermented sausage. Food Control, 33 (1), 6-14. Fonseca, S., Ouoba, L.I.I., Franco, I. and Carballo, J. (2013b). Use of molecular methods to characterize the bacterial community and to monitor different native starter cultures throughout the ripening of Galician chorizo. Food Microbiol., 34, 215-226. García Fontán, M.C. Lorenzo, J.M. Martínez, S. Franco, I. and Carballo J. (2007a) Microbiological characteristics of Botillo, a Spanish traditional pork sausage. LWT– Food Sci. and Technol., 40 (9), 1610-1622. García Fontán, M.C., Lorenzo, J.M., Parada, A., Franco, I. and Carballo, J. (2007b). Microbiological characteristics of “androlla”, a Spanish traditional pork sausage. Food Microbiol., 24 (1) 52-58 Gómez R., Alvarez-Orti, M. and Pardo J.E. (2008). Influence of the paprika type on redness loss in red line meat products. Meat Sci., 80, 823-828 Gómez, M. and Lorenzo, J.M. (2013). Effect of fat level on physicochemical, volatile compounds and sensory characteristics of dry-ripened “chorizo” from Celta pig breed. Meat Sci., 95, 658-666 Honikel, K. O. (2008). The use and control of nitrate and nitrite for the processing of meat products. Meat Sci., 78, 68–76. ISO (International Organization for Standardization) Determination of moisture content, ISO 1442:1997 standard International standards meat and meat products, International Organization for Standardization, Genève, Switzerland (1997)

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Ling, P.P., Ruzhitsky, V.N., Kapanidis, A.N. and Lee, T.-C. (1996). Measuring the colour of food. Chemical Technol., 11 (3), 46-52 Lorenzo, J.M. and Franco, D (2012). Fat effect on physico-chemical, microbial and textural changes through the manufactured of dry-cured foal sausage. Lipolysis, proteolysis and sensory properties. Meat Sci., 92 (4), 704-714. Olivares, A., Navarro, J.L. and Flores M (2011). Effect of fat content on aroma generation during processing of dry fermented sausages. Meat Sci., 87, (3) 264-273 Spaziani, M., Torre, M.D., and Stecchini, M.L. (2009). Changes of physicochemical, microbiological, and textural properties during ripening of Italian low-acid sausages. Proteolysis, sensory and volatile profiles. Meat Sci., 81 (1), 77-85 Yong, H.I., Lee, S.H., Kim, S.Y., Park, S., Park, J., Choe, W. and Jo C. (2019). Color development, physiochemical properties, and microbiological safety of pork jerky processed with atmospheric pressure plasma. Innovative Food Science & Emerging Technol., 53, 78-84 Zaika, L. L., Zell, T. E., Smith, J. L., Palumbo, S. A, and Kissinger, J. C. (1976). The role of nitrite and nitrate in Lebanon bologna, a fermented sausage. J. Food Sci., 41, 14571460.

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CARBON NANOMATERIALS PRODUCTION FROM THE GAS PRODUCT OF COMPOSITE PACKAGING WASTE PYROLYSIS Ece YAPICI1, Aysun ÖZKAN*1, Zerrin GÜNKAYA1, Göktuğ GÜNKAYA2, Müfide BANAR1 1

Eskişehir Technical University, Department of Environmental Engineering, 26470, Eskişehir, Turkey 2

Anadolu University, Department of Glass, 26555, Eskişehir, Turkey *Corresponding author’s e-mail: eceturan@eskisehir.edu.tr

ABSTRACT Carbon nanomaterials have been produced from composite low density polyethylene (C/LDPE) waste feedstock via a two stage process; pyrolysis and chemical vapor deposition (CVD) process. At the first stage, C/LDPE wastes were grinded and then pyrolyzed in a nitrogen-swept fixed bad stainless steel reactor operated at 600 ° C with 20° C/min heating rate. At the second stage, the pyrolysis gas was passed through the tubular quartz CVD reactor. The CVD reactor was operated with two different catalysts (iron and nickel) at 600 °C. To determine the optimum conditions of carbon nanomaterials growth, scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyzes were carried out. Keywords: Carbon nanomaterials, chemical vapor deposition, C/LDPE, pyrolysis.

INTRODUCTION The largest consumer of plastic materials is the packaging industry. Most of the packaging waste is composite packaging (primarily PA-PE-Al laminate, a laminated foil made from paper, polyethylene, and aluminum foil) material that is difficult to recycle because of relatively high cost and low resale value (Xia et al., 2013). C/LDPE (composite/low density polyethylene) used in this study is one of the packaging materials that is located in the plastic/aluminum class and plastic content is more than aluminum content. Plastic waste is managed by different techniques including, reducing, reusing, recycling, energy recovery and disposal at landfill sites (Ouda et al., 2016; Sadef et al., 2016). Due to the fact that carbon is the major constituent of plastics, the waste plastics can therefore provide a carbon source for carbon-based value-added products. Such products include carbon black/activated carbon, carbon fibers, fullerenes, carbon nanotubes, and graphene. Therefore converting waste plastics into such value-added products is an excellent example of upcycling (Zhou et al., 2014). The current existing methods for carbon nanomaterial production are electric arc discharge method, laser ablation method, catalytic chemical vapor deposition (CVD). Among these methods, CVD seems to currently be the most promising and preferred method for large-scale production (Bazargan and McKay, 2012). At this point, pyrolysis process is a method that can be used as a step for separating the materials in such wastes and converting them into valuable liquid oil and gases via thermal decomposition (Chen et al., 2015). Acomb et al. (2014) used a two stage process where evolved pyrolysis gases are passed directly onto the second stage, producing large amounts of carbon nanotubes. Liu et al. (2011) also used a two stage pyrolysis

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process where plastics are pyrolysed in a first stage and the gases passed to a second stage where carbon nanotubes were produced using a nickel catalyst. In this study, it is aimed to produce carbon nanomaterials from C/LDPE waste feedstock via a two stage process; pyrolysis and chemical vapor deposition (CVD) process. To characterize the nanomaterials, scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyzes were carried out. MATERIALS AND METHODS Carbon nanomaterials have been produced from C/LDPE waste feedstock via a two stage process; pyrolysis and CVD. First of all, 10 g of waste C/LDPE sample was placed in the reactor, and the pyrolysis experiments were carried out under atmospheric pressure at 600 °C with 20° C/min heating rate in a fixed bed stainless steel reactor (Figure 1). Gases were collected in a Tedlar bag and analyzed by gas chromatography-thermal conductivity detector (GC-TCD).

Figure 1. Pyrolysis system At the second stage, the generated gaseous products were then passed through to the CVD reactor and passed over 0.1 g of catalyst (NiO or Fe2O3) allowing carbon deposition to occur on the catalyst surface. The reactor consists of a horizontal quartz tube with a maximum working temperature of 1100°C, a heating zone length of 250 mm and a diameter of 70 mm (Figure 2). The temperature of this reactor held at 600 ◦C. Nitrogen was used as the carrier gas with a flow rate of 300 mL/min. When the desired temperature was reached, 30 minutes feed was made with pyrolysis gas as carbon source. Then the furnace was shifted to provide instant cooling.

Figure 2. CVD system The phase structure of the carbonaceous products were characterized by X-ray diffraction (XRD) using a Rigaku Rink 2200 operating at 40 kV and 15 mA. The morphology of the

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product was examined by scannig electron microscopy (SEM) using a Zeiss SUPRA 50VP operating at 20 kV. RESULTS The composition of pyrolysis gas product was determined by GC-TCD, and the results were given in Table 1. Table 1. Pyrolysis gas composition vol. % Hydrogen

2.03

Carbon dioxide

31.03

Carbon monoxide

25.39

Methane Ethane Ethylene

12.33 9.82 19.41

The conditions of first stage were kept constant at 20°C/min and 600°C, the pyrolysis gas was passed through the tubular quartz CVD reactor. Two sets of CVD experiments were realized with 0.1 g of NiO and Fe2O3 at 600 °C. Figure 3 shows the waste C/LDPE samples and the carbonaceuous product obtained from this material by two-stage process.

(a)

(b)

Figure 3. (a) waste C/LDPE (b) carbonaceuous product Figure 4 shows the XRD patterns of the samples obtained. It reveals that a sharp peak corresponding to highly graphitic material at 2θ around 26° for sample produced with NiO catalyst (Arnaiz et al., 2013).

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Figure 4. XRD spectra of products SEM was undertaken on the carbon deposition on the catalyst surface, with the images obtained shown in Figure 5. The SEM image of the NiO catalyst obtained at 600 ◦C, shows the presence of filamentous carbons, as well as accumulations of more amorphous carbon (Acomb et al, 2015).

Figure 5. SEM image of carbon deposition on NiO catalyst CONCLUSIONS A two-stage pyrolysis–CVD process has been used to produce carbon nanomaterials from waste C/LDPE on NiO catalyst. XRD patterns show the material is highly graphitic, and SEM image shows the presence of filamentous carbons. Transmission electron microscopy (TEM) will be held to be sure that the filamentous carbons were CNTs, and Raman spectroscopy will be used to determine the purity of CNTs in further work. Acknowledgements: This study was supported by the Turkish Scientific and Technological Research Council (TÜBİTAK) (Project No. 117Y041). REFERENCES Acomb, J. C., Wu, C., & Williams, P. T. (2014). Control of steam input to the pyrolysisgasification of waste plastics for improved production of hydrogen or carbon nanotubes. Applied Catalysis B: Environmental, 147, 571-584. Acomb, J. C., Wu, C., & Williams, P. T. (2015). Effect of growth temperature and feedstock: catalyst ratio on the production of carbon nanotubes and hydrogen from the

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pyrolysis of waste plastics. Journal of analytical and applied pyrolysis, 113, 231238. Arnaiz, N., Gomez-Rico, M. F., Martin Gullon, I., & Font, R. (2013). Production of carbon nanotubes from polyethylene pyrolysis gas and effect of temperature. Industrial & Engineering Chemistry Research, 52(42), 14847-14854. Bazargan, A., & McKay, G. (2012). A reviewâ&#x20AC;&#x201C;synthesis of carbon nanotubes from plastic wastes. Chemical engineering journal, 195, 377-391. Chen, D., Yin, L., Wang, H., & He, P. (2014). Pyrolysis technologies for municipal solid waste: a review. Waste management, 34(12), 2466-2486. Liu, J., Jiang, Z., Yu, H., & Tang, T. (2011). Catalytic pyrolysis of polypropylene to synthesize carbon nanotubes and hydrogen through a two-stage process. Polymer degradation and stability, 96(10), 1711-1719. Ouda, O. K., Raza, S. A., Nizami, A. S., Rehan, M., Al-Waked, R., & Korres, N. E. (2016). Waste to energy potential: a case study of Saudi Arabia. Renewable and Sustainable Energy Reviews, 61, 328-340. Sadef, Y., Nizami, A. S., Batool, S. A., Chaudary, M. N., Ouda, O. K. M., Asam, Z. Z., ... & Demirbas, A. (2016). Waste-to-energy and recycling value for developing integrated solid waste management plan in Lahore. Energy Sources, Part B: Economics, Planning, and Policy, 11(7), 569-579. Xie, M., Qiao, Q., Sun, Q., & Zhang, L. (2013). Life cycle assessment of composite packaging waste managementâ&#x20AC;&#x201D;a Chinese case study on aseptic packaging. The International Journal of Life Cycle Assessment, 18(3), 626-635. Zhuo, C., & Levendis, Y. A. (2014). Upcycling waste plastics into carbon nanomaterials: A review. Journal of Applied Polymer Science, 131(4).

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LIFE CYCLE ASSESSMENT OF MULTI WALLED CARBON NANOTUBES SYNTHESIS VIA CHEMICAL VAPOR DEPOSITION Ece YAPICI*1, Kübra Cebeci TOPBAŞ1, Aysun ÖZKAN1, Zerrin GÜNKAYA1, Müfide BANAR1 1

Eskişehir Technical University, Department of Environmental Engineering, 26555 Eskişehir, Turkey *Corresponding author’s e-mail: eceturan@eskisehir.edu.tr

ABSTRACT Carbon nanotubes (CNTs) have a significant role in a wide range of industrial applications and products owing to their invaluable mechanical, thermal and electrical properties. Chemical vapor deposition (CVD) is the commonest synthesis method of CNTs on account of its higher degree of control and scalability. Life cycle assessment (LCA) is a useful method to assess the environmental effects of processes and products. In this study, an LCA has been carried out to determine the environmental performance of multi-walled carbon nanotubes (MWCNT) synthesized via a laboratory scale CVD. A cradle-to-gate approach has been employed for this assessment. The functional unit was considered a typical laboratory ‘batch’ of the MWCNT grown. Inventory data was collected through onsite measurements, material/energy consumption and Ecoinvent database. The environmental impact assessment was carried out using CML-IA method for the selected impact categories (abiotic depletion, global warming, ozone depletion, toxicity, photochemical oxidation, acidification and eutrophication). The results of the study showed the hot points through which significant environmental benefits and “greener” CNTs could be achieved. Keywords: Carbon Nanotubes (CNT), Chemical Vapor Deposition (CVD), environmental impacts, Life Cycle Assessment (LCA)

INTRODUCTION The enormous and escalating volume of waste plastics causes harmful environmental impacts on the health of living beings (Pol, 2010). Efficient conversion of waste plastics into advanced materials is of conspicuous environmental, social and economic benefits (Zhou et al, 2010). Most importantly, the huge amounts of plastic waste generated can also be viewed as a potential hydrocarbon resource (Tripathi et al, 2017). Since carbon is the main constituent of plastics, the waste plastics can provide a carbon source for carbon-based value-added products, such as carbon nanotubes (Zhuo and Levendis, 2013). CNTs with different structure have a major role in engineering because of their excellent mechanical, thermal and electrical properties and very low mass/volume ratio (Borsodi et al, 2016). CNT synthesis from plastic waste could be achieved with different methods, such as arc discharge, laser ablation, pyrolysis, chemical vapor deposition (CVD) and plasma assisted deposition (Mishra et al, 2012). Indeed, the CVD method is one of the most successful methods that has been used to make MWCNTs on an industrial scale (Tripathi et al, 2017). Due to the increased awareness for the environmental protection from all the possible impacts associated with products manufactured and consumed, a demand on the development of methods for better comprehension about the reduction of all these negative impacts has arisen. One of the techniques developed in response to this demand is Life Cycle Assessment 426

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(LCA) (Trompeta et al, 2015). LCA, which is based on the concept of life cycle thinking, is a systematic method for assessing the potential environmental impacts of products, services and processes across their entire life cycles. LCA is recognized as being a powerful tool to identify potential life cycle impacts of nanotechnologies and its application in this field has proliferated in the past decade (Salieri et al., 2018). So, an LCA study has been carried out to determine the environmental impacts of a multi-walled carbon nanotubes (MWCNT) production process in this study. MATERIAL AND METHODS This study was conducted according to ISO 14040 and ISO14044 standards developed by the International Organization for Standardization (ISO). This LCA study compromises four stages according to the ISO standards: The goal and scope definition (functional unit, system description and system boundaries), life cycle inventory (data collection), life cycle impact assessment and interpretation. Goal and scope definition The goal of this study is to determine the environmental effects of carbon nanotubes synthesized by chemical vapor deposition method from gas product obtained from pyrolysis of waste C/LDPE material. For this purpose, the LCA functional unit was determined for a typical laboratory â&#x20AC;&#x2DC;batchâ&#x20AC;&#x2122; of the MWNTs grown via the CVD process, approximately 0.553 g of product. System boundaries Cradle-to-gate system boundaries, which includes laboratory scaled synthesis of CNT in CVD reactor and material/energy/emission flows, were applied. The system boundaries that were considered in this LCA analysis are shown in Figure 1, based on the system description. The total environmental impact, regarding waste C/LDPE pyrolysis, consists of 3 components: indirect impact caused by energy and the material production stage, direct impact caused by CNT synthesis, and impacts avoided by valuable products (recycled material and energy).

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Figure 1. System boundary Life cycle inventory For the life cycle inventory, the data was distinguished into two parts as primary and secondary data. Primary data on the input (electricity) and output were mainly obtained from the onsite measurements and materials usage. The summary of the primary data is given in Table 1.

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Table 1. Primary inventory data on experimental work (based on functional unit) Data Input Nitrogen (N2)

Quantities

Comments/assumptions

20.31 g

Nickel oxide (NiO) Dichloromethane Energy consumption Electricity

0.1 g 31.65 g

Reaction: 60 min x 300 mL/min Cooling: 60 min x 300 mL/min Catalyst

Output Nitrogen (N2) Hydrogen (H2) Carbon dioxide (CO2) Carbon monoxide (CO) Methane (CH4) Ethane (C2H6) Ethylene (C2H4) Aluminium (Al) Wax Residual

4.39 kWh

Pyrolysis reaction: 60 min x 600°C Evaporator: 30 min x 40°C Muffle furnace: 30 min x 900°C CVD reaction: 65 min x 600°C

20.31 g 0.098 g 1.716 g 0,896 g 0.240 g 0.360 g 0.027 g 0.514 g 3.80 g 0.066 g

Mass balance

SimaPro 8.5 software was used to develop unit process by using primary data and to link the primary to the secondary data. Secondary data was gathered from the EcoInvent (v.3.0.3) database which is embodied in SimaPro 8.5. In addition to the secondary data obtained from Ecoinvent, electricity production data was gathered from Günkaya et al. (2016). This electricity mix data was modified based on the electricity mix of Turkey in 2018 (30.5% natural gas, 22.7% hydroelectric, 36% coal (mainly coal, imported coal and lignite), 6.2% for wind, 2.3% geothermal). Nickel oxide was not found in the database; therefore, the data of nickel production was used instead. Life cycle impact assessment At the impact assessment step, the CML-IA method, embodied in SimaPro 8.5, was applied for the selected impact categories: abiotic depletion (element) potential (ADPe), abiotic depletion (fossil fuels) potential (ADPff), global warming potential (GWP), ozone depletion potential (ODP), human toxicity potential (HTP), photochemical oxidation potential (POP), acidification potential (AP) and eutrophication potential (EP). Normalization results were also calculated using the EU25+3, 2000 calculation method under the CML-IA baseline (v3.00) method. RESULTS Characterization and normalization results are given in Table 2. According to normalization results, ADPff, GWP, POP and AP are the dominant (>%5) impact indicators of the synthesis of CNT.

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Table 2. Characterization and normalization results. Impact category ADPe ADPff GWP ODP HTP POP AP EP

Unit

Characterization

Normalization

% Impact

kg Sb eq. MJ kg CO2 eq. kg CFC-11 eq. kg 1.4-DB eq. kg C2H4 eq. kg SO2 eq. kg PO4 eq.

3.86E-08 3.91E+01 4.27E+00 2.16E-06 1.84E-01 7.58E-03 4.70E-02 2.55E-03

6.41E-15 1.12E-12 8.20E-13 2.12E-13 3.67E-13 4.38E-12 2.79E-12 1.38E-13

0.07 11.35 8.34 2.16 3.74 44.57 28.39 1.40

Distribution of the characterization results to the input processes is shown in Figure 2, according to which, among all the impact categories, the environmental impact resulting from electricity consumption is observed to be overwhelming compared to other process components (CNT synthesis, nitrogen production, dichloromethane production, nickel production). 100 80 CNT Synthesis Nitrogen Dichloromethane Nickel Electricity

60 40 20 0 % ADPe % ADPff % GWP % ODP % HTP % POP

% AP

% EP

Figure 2. Distribution of characterization results to the input processes Figure 3 (a) shows that coal consumption has the highest amount in the ADP ff impact category. This effect is caused by burning coal during electricity generation. The ADPff impact can be decreased by wax recovery (Figure 3 (b)). In this context, a significant decrease will be observed, especially in the use of crude oil. 0,05

30 20

0 Coal

CNTsynthesis Nitrogen Dichloromethane Nickel Electricity

Crude oil Natural gas

-0,05 -0,1

Aluminium Wax

-0,15 0 Coal

-0,2

Crude oil Natural gas (a)

(b)

Figure 3. ADPff impact of 0.553 g of CNT synthesis: a) distribution of characterization results to the input processes, and b) avoided characterization results by valuable products The main pollutants causing the GWP effect of CNT synthesis are CO2 and CH4, and the effects are 93.5% and 5.8%, respectively (Figure 4 (a)). This significant impact of CO2 430

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emissions on GWP is due to the burning of coal in electricity generation. Figure 4 (b) shows that wax recovery was found to have a larger contribution than Al recovery to avoiding GWP effect. This contribution can be made especially by preventing the release of CO2 emissions. 0

CNT Synthesis Nitrogen Dichloromethane Nickel Electricity

3 2

Carbon dioxide

Methane

-0,0005

kg CO2 eq.

-0,001 -0,0015 Aluminium

-0,002

Wax

0 Carbon dioxide

-0,0025

Methane

(a)

(b)

Figure 4. GWP (100 a) impact of 0.553 g of CNT synthesis: a) distribution of characterization results to the input processes, and b) avoided characterization results by valuable products

5E-08 0 -5E-08 -1E-07 -1,5E-07 -2E-07 -2,5E-07 -3E-07 -3,5E-07 -4E-07

CNT Synthesis Nitrogen Dichloromethane Nickel Electricity

0,007 0,006 0,005 0,004 0,003 0,002 0,001 0

Carbon Sulfur monoxide dioxide

Methane

Ethane

Ethylene

kg C2H4 eq.

In the CNT synthesis, the main pollutant components causing POP effect are carbon monoxide and sulfur dioxide and their effects are 76.3% and 21.1%, respectively (Figure 5 (a)). On the basis of the process, the highest impact was generated by electricity production with 98.3. The POP supplement category is influenced by carbon monoxide electricity generation and sulfur dioxide from nitrogen production. It has been shown that wax recovery has a greater contribution to the recovery of POP than Al recovery (Figure 5 (b)). This contribution is thought to be made especially by preventing the release of sulfur dioxide emissions.

(a)

Aluminium Wax (b)

Figure 5. POP impact of 0.553 g of CNT synthesis: a) distribution of characterization results to the input processes, and b) avoided characterization results by valuable products Figure 6 (a) shows that the SO2 and NOx emissions released during the production of the electricity are the major pollutants that cause the acidification potential (%86.48 and %13.41, respectively). These emissions are caused by the burning of coal and natural gas used in electricity generation. According to Figure 6 (b), wax recovery will have a significant role on the reduced acidification effect due to the avoided SO2 and NOx emissions.

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0,05

Sulfur dioxide

Nitrogen

-2,00E-06

kg SO2 eq.

0,04

kg SO2 eq.

0,00E+00

CNT Synthesis Dichloromethane

0,03

Nickel

Electricity

0,02 0,01

-4,00E-06 -6,00E-06 -8,00E-06

0 Sulfur dioxide (a)

Nitrogen oxides

Aluminium Wax

-1,00E-05

Nitrogen oxides

(b)

Figure 6. AP impact of 0.553 g of CNT synthesis: a) distribution of characterization results to the input processes, and b) avoided characterization results by valuable products CONCLUSIONS In this study, environmental impacts of CNT synthesis were determined by using LCA. It is seen that the study will bring an innovative solution to the waste problem in the country with the further conversion of C/LDPE wastes and form a nanotechnological product such as carbon nanotube. In addition, natural resource utilization is prevented by wax and Al recovery. When the LCA results are analysed on the basis of all processes, it is observed that the highest impact is the result of electricity consumption. Replacement of fossil fuels by various sources of renewable energy (photovoltaic, wind power, etc.) in Turkey's electricity production is quite likely to reduce these environmental impacts. This is the first LCA study on waste C/LDPE pyrolysis in Turkey. Therefore, the findings of this study are likely to be of assistance for decision-makers and practitioners by providing a framework to better understand the major environmental effects of CNT synthesis and its conversion to useful products. Acknowledgements: This study was supported by the Turkish Scientific and Technological Research Council (TÜBİTAK) (Project No. 117Y041). REFERENCES Borsodi, N., Szentes, A., Miskolczi N., Wu C., Liu X. (2016). Carbon nanotubes synthetized from gaseous products of waste polymer pyrolysis and their application. J. Anal. Appl. Pyrolysis,120: 304–313. Günkaya, Z., Özdemir, A., Özkan, A., Banar, M (2016). Environmental Performance of Electricity Generation Based on Resources: A Life Cycle Assessment Case Study in Turkey. Sustainability, 8 (1097): 1-14. Mishra, N., Das, G., Ansaldo, A., Genovese, A., Malerba, M., Povia, M., Ricci, D., Fabrizio, E.D., Zitti, E.D., Sharon, M. (2012). Pyrolysis of waste polypropylene for the synthesis of carbon nanotubes. J. Anal. Appl. Pyrolysis,94: 91–98. Pol, V.G. (2010). Upcycling: Converting Waste Plastics into Paramagnetic, Conducting, Solid, Pure Carbon Microspheres. Environ. Sci. Technol., 44: 4753–4759. Salieri, B., Turner, D.A., Nowack, B., Hischier, R. (2018). Life cycle assessment of manufactured nanomaterials: Where are we?. NanoImpact, 10: 108–120. Tripathi, P.K., Durbach, S., Coville, N.J. (2017). Synthesis of Multi-Walled Carbon Nanotubes from Plastic Waste Using a Stainless-Steel CVD Reactor as Catalyst. Nanomaterials, 7, 284. Trompeta, A.F., Vlachou,N., Perivoliotis, D.K., Koklioti, M.A., Markakis, V., Tsiliki,G., Sarimveis, H., Charitidis, C.A. (2015). Environmental assessment of carbon 432

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nanotubes synthesis via chemical vapor deposition.10th Panhellenical Conference of Ministry of Mathematics, Patras, Greece. Zhuo, C., Levendis, Y.A. (2013). Upcycling waste plastics into carbon nanomaterials: a review. J. Appl. Polym. Sci., 4: 131. Zhuo, C., Zhang, Q., Luo, G., Huang, J.Q., Zhao, M.Q., Wei,F. (2010). Coupled process of plastics pyrolysis and chemical vapor deposition for controllable synthesis of vertically aligned carbon nanotube arrays. Appl Phys A,100: 533â&#x20AC;&#x201C;540.

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EFFECT OF THE ADDITION OF DATES ON THE PHYSICOCHEMICAL AND SENSORY CHARACTERISTICS IN RAW-CURED BEEF SAUSAGES Inmaculada FRANCO1, Sihem LATRACH1, Lucia GÓMEZ-LÍMIA1, Sidonia MARTÍNEZ1 1

Food Technology, Faculty of Science, University Campus as Lagoas s/n, 32004 Ourense, University of Vigo, Spain *Corresponding author’s e-mail: inmatec@uvigo.es

ABSTRACT The effects of dates on quality and sensory characteristics of raw-cured beef sausages manufactured with beef lean and the addition of olive oil were investigated. Four batches were manufactured without dates (Control) and with 100 g/kg (D100), 200 g/kg (D200) and 300 g/100 g/kg (D300) of dates, respectively. Samples at 0 (mix immediately before stuffing), 3, 7, 14 and 21 days, were taken for subsequent analysis. Weight losses (%) were significantly affected by days of manufacturing, increasing along the ripening process. Weight loss values were significantly lower in sausages made with more dates. Weight losses are produced basically because of the dehydration, so that its evolution will be opposite to dry matter. The final total solids content of the sausages was similar in the four batches (control and mixed sausages) and around 50%. Fat content of the four batches of sausage remain practically constant along the manufacture process, with values near to 35% of the dry matter content. The evolution of pH values were very different when the four sausages were compared. In sausages made with dates the values decrease rapidly in the first three days of maturation. This evolution could be related to the high content of fermentable sugars in this fruit. Regarding to the sensorial analysis, consumers has a clear preference for sausages made with 10% of dates fruit. Keywords: Raw-cured beef sausage, Olive oil, Dates, Sensory analysis, Biochemical parameters INTRODUCTION In the current scenario of a competitive food industry, it is essential to improve or create innovative ideas for new products (Banović et al., 2016; Polizer Rocha et al., 2019). The technology of raw-cured meats offers a wide range of possibilities and variations regarding ingredients and production processes, which has led to the development of a large number of products. Different studies have been carried out on the use of fruits and vegetables in the production of meat products, mainly in order to reduce fat and introduce specific functional properties (Wang et al., 2019; Xiang et al., 2019). Dates are recognized as nutrient-rich fruits because of their rich content of essential nutrients, which include carbohydrates, salts and minerals, dietary fiber, vitamins, fatty acids, amino acids and protein (Allaith, 2008; Hasnaoui et al. 2011). Moreover, the addition of dates could be used for making high value-added meat products and would further enhance the quality of final product above all thanks to its antioxidant capacity (Al-Turki et al., 2010). In addition to the nutritional benefits, date have been related to various biological effects mainly due to the high content of polyphenols and flavonoids (Chaira et al., 2009). Animal fat plays an important role in formulation of products due to its unique texture (Pehlivanoğlu et al., 2018). An alternative to the reduction or elimination of animal fat in raw-cured meats is replacing it with oils. Olive oil has been widely used for this purpose (Bloukas et el., 1997). 434

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The aim of this work is to study the effect of the use of dates in the elaboration of a raw-cured beef sausage. MATERIAL AND METHODS Sausage production and sampling Four batches of sausage were manufactured using lean beef (80%), olive oil (20%), paprika (26 g/kg), NaCl (15 g/kg) and garlic (4 g/kg). The lean beef was ground through a 10 mm diameter mincing plate and mixed together with the other ingredients. Next, the mixture was divided into four batches as follows: control (without dates-C-), D100 with 100 g/kg of dates, D200 with 200 g/kg and D300 with 300 g/100 g/kg of dates. The mix was maintained at 4 ºC for 24 h and then stuffed into natural sheep casings with a diameter of 40 mm. The sausages were fermented for 9 days at 6 ºC and 80% relative humidity and then transferred to a dryingripening chamber where they were kept for 21 more days at 12 ºC and 75% relative humidity. Two replicates of the experiment were carried out. Samples at day 0 (mix before stuffing) and after 3, 7, 14 and 21 days of ripening were taken. To prepare samples for analysis, after removing and discarding the outer casing of each chorizo, the edible part was ground until a homogeneous mass was obtained. After determining the pH the samples were vacuum-packed and frozen at -80ºC for no longer than a month before further analysis. Physicochemical analysis At each sampling time the units of sausage were individually weighted to calculate the weight losses, and expressed as percentage of the initial weight. The moisture content of the samples was determined by dehydration at 105 ºC until constant weight according to the ISO recommended standards 1442:1997. The pH of samples was measured using a pH meter GLP21 (Crison Instruments, Barcelona, Spain). Fat was extracted according to Folch et al. (1957) procedure. Fat extract was weighted and the fat content was expressed as percentage of fat per 100 g of dry matter. All analytical determinations were made at least in triplicate. Sensory analysis The final sausages (after 21 days of ripening) from each batch were submitted to sensory evaluation to assess the differences between control samples and samples with date fruits. For this test 50 people were questioned to predict the attitudes of the target population. The evaluated parameters were general appearance, taste and aroma. Sausage samples were threedigit coded and in order to assess the section appearance and the perceptions during mastication they were presented in oblique slices approximately 1 cm thick. Water and unsalted crackers were provided to clean palate between samples. Finally, each person was given the four coded samples to determine their preference. Statistical analysis Data was subjected to an analysis of variance (ANOVA) using the General Linear Model procedure of the computer programme Statistica 8.0 for Windows (Statsoft Inc., Tulsa, OK, USA) to determine the overall effect of date addition on physicochemical and sensory parameters.

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RESULTS AND DISCUSSION The evolution of weight losses (%), during ripening of sausages, is shown in the Figure 1. Weight losses were significantly affected by days of manufacturing (P<0.05), increasing along the ripening process. Values of weight losses were around the 50% in the final product. Weight losses are produced basically because of the dehydration (Papadima and Bloukas 1999), so that its evolution will be opposite to dry matter. Significant differences (P0.05) are observed between the different sausages with different percentages of dates. This trend could be related to several factors such as higher fiber content, the sausage diameter, the air rate in the drying room or the fat content (Papadima and Bloukas 1999). Similar weight losses values were found by various authors in different dry-cured sausages (Campagnol et al., 2011; Papadima and Bloukas, 1999). The lowest losses were evidenced in the D300 sausages after 14 days of maturation. The values found in these sausages (D300) showed significant differences (P<0.05) with those determined in sausages C, D100 and D200, respectively. This trend in these sausages, could be related to its higher fiber content, among other components, which could retain water and reduce losses during ripening.

d c

* a

Figure 1. Evolution of weight losses of sausages control and elaborated with different amounts of dates. C (Control), D100 (100 g of dates/kg), D200 (200 g of dates/kg) and D300 (300 g of dates/kg). a-d Means not followed by the same letter differ significantly (P<0.05) during ripening. *Values that differ significantly (P<0.05) when comparing the different sausages. The initial moisture content of the sausages was similar in the four batches (control and mixed sausages) and around 60%. Total solids were significantly affected by the days of manufacturing (P<0.05). Values increased progressively, reaching final mean values around 55% in the four batches (Figure 2). The evolution of this parameter, due to the same casings diameter and manufacture process (same temperature and relative humidity conditions), was very similar in the sausages from the different formulations. Although the dry matter contents were higher in D300 sausages, no significant differences were found (P>0.05) when all sausages were compared. The final values were in the range of those found in other dry-cured sausages (Díaz et al., 2009; Franco et al., 2004; Elías and Carrascosa, 2010; Fernández-López et al., 2008; Franco, et al., 2002; Salgado et al., 2006). Fat content of the four batches of sausage remain practically constant along the manufacture process, with values near to 35% of the dry 436

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matter content. The fat contents are lower to several types of dry-cured sausages (ElĂas and Carrascosa, 2010; Franco et al., 2004).

Figure 2. Total solids and fat content (21 days of ripening) of sausages control and elaborated with different amounts of dates. C (Control), D100 (100 g of dates/kg), D200 (200 g of dates/kg) and D300 (300 g of dates/kg). The evolution of pH values, during ripening of sausages, is shown in the Figure 3. In sausages elaborated with dates the pH values decrease rapidly (P<0.05) in the first three days of maturation. This evolution could be related to the high content of fermentable sugars in this fruit, containing a high sugar content, which is around 50%-75% of dry weight and based mostly of inverted form (glucose and fructose). Fresh varieties contains higher amount of inverted sugars, the semi dried varieties have equal amount of inverted sugars and sucrose, while dried varieties have higher sucrose levels (Nehdi et al. 2010; Baliga et al. 2011). a b

Figure 3. Evolution of pH values of sausages control and sausages elaborated with different amounts of dates. C (Control), D100 (100 g of dates/kg), D200 (200 g of dates/kg) and D300 (300 g of dates/kg). a-d Means not followed by the same letter differ significantly (P<0.05) during ripening. *Values that differ significantly (P<0.05) when comparing the different sausages. 437

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The pH reduction over the first days of the manufacturing process resulted low (around 0.4 pH units) compared with other studies when carbohydrates and starters cultures are included on sausage formulation (Fernández-López et al., 2008; Campagnol et al., 2011). As well, fermentation and pH decreasing is limited when ripening process is carried out at low temperatures (Fonseca et al., 2012). A pH increase at the end of the production process could be attributed to ammonia production as a consequence of proteolytic phenomena, an increase of buffer substances, and a decrease of electrolytes (Campagnol et al., 2011) and to the growth of microorganisms (especially molds and yeasts) capable of consuming lactic acid (Fonseca et al., 2012). The sensory characteristics of the final product are the result of a complex interaction of physicochemical, biochemical and microbiological processes with a role in the formation and equilibrium of the chemical compounds and in the modification of molecules responsible for the texture and appearance. Taste of foods is due to low-molecular weight compounds, such as sugars, sodium chloride, amino acids, peptides and nitrogen compounds. Sausages made with different dates contents (D100, D200 and D300) differ significantly from the control sausages in the smell quality and intensity both in the external perception and when slicing; differences were also observed in flavor intensity during mastication. The flavor perception in the sausages with higher date content is possibly responsible for the better overall acceptability in these batches when compared to the control (Figure 4). C D300 D200

D100

Figure 4. Percentages of preference of the different sausages by consumers. C (Control), D100 (100 g of dates/kg), D200 (200 g of dates/kg) and D300 (300 g of dates/kg). The consumers rated the different sausages from 1 to 5. All of them noticed the intense flavor of the sausage with the highest concentration of dates. The majority had appreciated this flavor even so a very few number of consumers rated this flavor negatively. The study of consumers' preferences showed the sausage with 10% of date’s fruit as the mostly preferable one with 50%. CONCLUSIONS In this work, some physicochemical parameters and sensory analysis of a raw-cured beef sausage elaborated with olive oil and dates fruit were described. In general no significant effect of the addition of date fruit on evolution of weight losses, total solids and fat content were found. Moreover, a significant effect was observed on pH, an earlier drop of the pH of the experimental samples. Regarding to the sensorial analysis, consumers has a clear preference for sausages made with 10% of dates fruit. 438

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Regarding to the result of the present study it can be concluded that the best manufacturing process would include a 10% of dates. Acknowledgements: This work was funded by Xunta de Galicia, Spain (CITACA Strategic Partnership, Reference: ED431E 2018/07). L. Gómez-Limia, thanks University of Vigo because of this research was supported by University of Vigo predoctoral scholarship. REFERENCES Allaith, A. A. A. (2008). Antioxidant activity of Bahraini date palm (Phoenix dactylifera L.) fruit of various cultivars. Int. J. Food Sci. Technol., 43: 1033–1040. Al-Turki, S., Shahba, M. A., Stushnoff, C. (2010). Diversity of antioxidant properties and phenolic content of date palm (Phoenix dactylifera L.) fruits as affected by cultivar and location. J. Food Agric. Environ., 8: 253–260. Baliga, M. S., Baliga, B. R. V., Kandathil, S.M., Bhat, H. P., Vayalil, P. K. (2011). A review of the chemistry and pharmacology of the date fruits (Phoenix dactylifera L.). Food Res. Int., 44: 1812–1822. Banović, M., Krystallis, A., Guerrero, L. Reinders, M. J. (2016). Consumers as cocreators of new product ideas: An application of projective and creative research techniques. Food Res. Int., 87: 211–223. Bloukas, J. G., Paneras, E. D., Fournitzis, G. C. (1997). Effect of replacing pork backfat with olive oil on processing and quality characteristics of fermented sausages. Meat Sci., 45: 133–144. Campagnol, P. C. B., Santos, B. A., Morgano, M. A., Terra, N. N., Pollonio, M. A. R. (2011). Application of lysine, taurine, disodium inosinate and disodium guanylate in fermented cooked sausages with 50% replacement of NaCl by KCl. Meat Sci., 87: 239-243. Díaz, O., Ros, C., Veiga, A., Cobos, A. (2009). Including chestnuts ans sugar beet pulp in diets for pigs, the effects on the quality of pork meat and the sensory properties of dry-cured sausage (chorizo gallego). J. Muscle Foods, 20: 449-464. Elías, M., Carrascosa, A. V. (2010). Characterisation of the paio do Alentejo, a traditional Portuguese Iberian sausage, in respect to its safety. Food Control, 21: 97-102. Fernández-López, J., Sendra, E., Sayas-Barberá, E., Navarro, C., Pérez-Álvarez, J. A. (2008). Physico-chemical and microbiological profiles of salchichón (Spanish dry-fermented sausage) enriched with orange fiber. Meat Sci., 80: 410-417. Folch, J., Lees, M., Sloane-Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Fonesca, F., Cachaldora, A., Gómez, G., Franco, I., Carballo, J. (2013). Monitoring the bacterial population dynamics during the ripening of Galician chorizo, a traditional dry fermented Spanish sausage. Food Microbiol., 33: 77-84. Franco, I., Prieto, B., Cruz, J. M., López, M., Carballo, J. (2002). Study of the biochemical changes during the processing of Androlla, a Spanish dry-cured pork sausage. Food Chem., 78: 339-345. Hasnaoui, A., Elhoumaizi, M. A., Hakkou, A., Wathelet, B., Sindic, M. (2011). Physicochemical characterization, classification and quality evaluation of date palm fruits of some Moroccan cultivars. J. Sci. Res., 3: 139-149. Nehdi, I., Omri, S., Khalil, M. I., Al-Resayes, S. I. (2010). Characteristics and chemical composition of date palm (Phoenix canariensis) seeds and seed oil. Ind. Crop. Prod., 32: 360365. Papadima, S. N., J. G. Bloukas. (1999). Effect of fat level and storage conditions on quality characteristics of traditional Greek sausages. Meat Sci., 51: 103-113. 439

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Polizer Rocha, Y. J., de Noronha R. L. F. Trindade, M. A. (2019). Relations between consumer's concern with own health and their perception about frankfurters with functional ingredients. Meat Sci., 155: 91-101. Pehlivanoğlu, H., Demirci, M., Toker, O. S., Konar, N., Karasu, S., Sagdic, O. (2018). Oleogels, a promising structured oil for decreasing saturated fatty acid concentrations: Production and food-based applications. Critical Reviews in Food Science and Nutrition, 58: 1330-1341. Salgado, A., García Fontán, M., Franco, I., López, M., Carballo, J. (2006). Effect of the type of manufacture (homemade or industrial) on the biochemical characteristics of chorizo de cebolla a Spanish traditional sausage. Food Control, 17: 213-221. Wang, X., Xu, M., Cheng, J., Zhang, W., Liu, X., and Zhou, P. (2019). Effect of Flammulina velutipes on the physicochemical and sensory characteristics of Cantonese sausages. Meat Sci., 154: 22-28. Xiang, R., Cheng, J., Zhu, M. and Liu, X. (2019). Effect of mulberry (Morus alba) polyphenols as antioxidant on physiochemical properties, oxidation and bio-safety in Cantonese sausages. LWT Food Sci. Technol., 116: 108-118.

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EFFECTS OF PROCESSING TECHNIQUES AND STORAGE ON OXIDATIVE STABILITY OF DIFFERENT OILS IN A CANNED EEL Lucía GÓMEZ-LİMİA*1,2, Inmaculada FRANCO1,2, Hiar VİLLAR1, Sidonia MARTÍNEZ1,2 1

Food Technology, Faculty of Science, University Campus as Lagoas s/n, 32004 Ourense, University of Vigo, Spain

CITACA - Clúster de Investigación y Transferencia Agroalimentaria del Campus Auga, Universidad de Vigo, 32004-Ourense, España *Corresponding author’s e-mail: lugomez@uvigo.es

ABSTRACT The European eel (Anguilla anguilla) is a very precious fish in some countries of Europe and Asia. However, its excessive capture of juveniles and the impossibility of captive breeding have made the eel a very limited product. The elaboration of a canned eel would allow it to be consumed throughout the year. Canned food products are susceptible to quality deterioration because of the different treatments suffered during its preparation. Lipid oxidation can occur during cooling of cooked fish. The aim of this study was to determine the oxidative variation in different oils used as covered liquids in a canned eel after frying and sterilizing treatment and during the storage at two months and one year. For the elaboration of eel canned three different oils were used: sunflower oil, olive oil and spicy olive oil. The frying and the sterilization treatment produced in the oil an increase of the peroxide index with respect to the crude oil. No significant differences were observed after the sterilization treatment between the three cover fluids. During storage of preserves, the peroxide index decreased. The TBA value increased after frying in the three oils under study, and decreased after sterilization treatment in olive oil and spicy olive oil. It did not show significant changes in sunflower oil, with respect to fresh oil. Sunflower oil showed no significant changes, with respect to fresh oil. During the storage, no significant changes were observed in the sunflower oil. In olive oil the index values of TBARs decreased after 2 months of storage and increase again at year of storage to values similar to those obtained after sterilization. No significant changes were obtained in the spicy olive oil after 2 months of storage, but it decreased at one year of storage in relation to the values obtained after sterilization. Keywords: Olive oil, Sunflower oil, Canned eel, Lipid oxidation INTRODUCTION Canned fish are products obtained from various marine species, packaged with different types of filled medium, in airtight containers and sterilized by heat treatment. Fish canning is one of the most popular forms of fish consumption. Since 2012, there has been an increase in the consumption of canned fish in Spain according to data obtained by the Ministry of Agriculture and Fisheries, Food and Environment (2017). Canned fish are characterized by their high nutritional value. Vegetable oils such as sunflower or olive oils are widely used as filling medium in canned fish. For canned fish, different vegetable oils are widely used. During processing and storage, interactions between filling oil and the fish muscle take place. Filling medium affect the nutrient content of the final product, and can produce dilution and extraction of some components in fish muscle. On the other hand, the different filling medium (such as brine, olive oil, sunflower 441

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oil, soybean oil and other vegetables oils) and their state can affect the quality of canned fish. Canning can provoke oxidation during fish processing, but there are also a significant effect of filling medium. Oxidative reactions during heat treatment produce a deterioration of the oil, decreasing its quality, and affecting canned fish (Gertz et al., 2000; Warner, 2002). In addition, oxidation can be minimized by using filling medium containing natural antioxidant such as tocopherols or polyphenols (Choe and Min, 2006). The rancidity of the oils has been linked to harmful health effects such as cancer and neurological disorders, because of the production of potentially toxic compounds (Kaleem, et al., 2015). The aim of this study was to determine the oxidative variation in different oils used as covered liquids in a canned eel (European eel, Anguilla anguilla) after frying and sterilizing treatment, and during the storage at two months and one year. MATERIAL AND METHODS Sampling and canned preparation European eel were caught by professional eel fishermen operating in the River Ulla (Galicia, NW Spain), during the authorized fishing season (winter). The fish were transported to tanks connected to freshwater recirculation modules, where they were held until slaughter by ice water immersion. They were then purchased and transferred to the laboratory. Eels with weights between 200 and 400 g were used for canning. The fish were eviscerated, washed, packed in vacuum bags and stored at -20 °C until use. Before canning, frozen eels were defrosted in 12% brine. The head and skin were removed, and the fish was cut in small pieces. The pieces were fried in sunflower oil, olive oil, and olive oil with garlic. Then, the portions were placed in jars. Three filling medium were tested: olive oil, sunflower oil and olive oil with pepper and chilli pepper. The jars were vacuum-sealed and sterilized at 118ºC for 30 min. Then, the cans were cooled and storage at room temperature. Samples of the different oils were sampled, raw, after the frying of the fish and after the sterilization treatment. Samples were also sampled after two months and one year of room storage. In the case of canned fish, the cans were opened and the liquid part was carefully drained off gravimetrically, filtered by means of a filter paper and collected. Oxidation analysis Peroxide is the main product that gives rise to disagreeable flavor in food products, proceeds through the free radical chain reaction. The peroxide value (POV), was performed according to the Official Methods of Analysis of Oils and Fats (BOE, 1977), with some modifications, and expressed as mEq O2/kg oil. The thiobarbituric acid index (TBA-i) (mg malondialdehyde/kg oil) was determined according to Kirk and Sawyer (1991). The malondialdehyde (MDA) is the main product resulting from the degradation of hydroperoxides generated by lipid oxidation. It is based on the reaction between MDA and TBA (thiobarbituric acid) that generates a pink-red compound measurable by spectrophotometry, with wavelengths between 532-535 nm. Statistical analysis All analyses have been done at least in triplicate. The statistical treatment of the data was carried out by analysis of variance (ANOVA). The least squares test (LSD) was applied for a 95% confidence interval (P ≤ 0.05) for the comparison of the mean values, using the statistical software Statistica version 7.1 of Statsoft © Inc. (Tulsa, OK, USA). 442

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RESULTS The peroxide values are reported in Figure 1 for sunflower oil, olive oil and spicy olive oil. 10

Sunflower oil

Olive oil

9 d

8 e

meq. de O2/kg

de de

Spicy olive oil

e f

6 f 5 g

3 2

1 0 Fresh

Fried

After sterelized

2 months

1 year

Figure 1.- Peroxide values of different oils used as filling medium (means ± standard deviation) (a-h.- values with different letters are significantly different, p < 0.05). The peroxide index values in raw oils were 1.50 ± 0.35 and 2.49 ± 0.44 mEq. O2/kg in sunflower and olive oils, respectively. Frying and sterilization treatment produced an increase in peroxide index with respect to crude oil. In the case of frying, the increase was higher in sunflower oil than in olive oil. The highest increase in the peroxide index during frying in sunflower oil can be due to its higher content in polyunsaturated fatty acids. No significant differences were observed after the sterilization treatment between the three cover liquids. Canning storage resulted in a decrease in the peroxide index. In the case of sunflower oil, the decrease in the peroxide index was significant after 2 months of storage; however, this decrease was not appreciated in olive oils until 12 months of storage. That decline was more pronounced in olive oil stored for 12 months and less in spicy olive oil. The TBA index are reported in Figure 2 for sunflower oil, olive oil and spicy olive oil.

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1,2

Spicy olive oil

mg MDA/kg

1,0

c 0,8 0,6 b

0,4

b f ef

0,2

f e

g 0,0 Fresh

Fried

After sterelized

2 months

1 year

Figure 2.- TBA index of different oils used as filling medium (means Âą standard deviation) (a-g.- values with different letters are significantly different, p < 0.05). The raw sunflower oil presented TBA index values of 0.117 mg MDA/kg, and olive oil of 0.365 mg MDA/kg. These values increased with frying, as much in sunflower oil as in olive oil and in spicy olive oil. However, the sterilization treatment resulted in a decrease in the TBA index in olive oil and spicy olive oil, and did not cause significant changes in sunflower oil, compared to raw oil. No significant changes in sunflower oil were observed during storage. In the case of olive oil, TBA index values decreased after 2 months of storage, to increase again after one year of storage to values similar to those obtained after sterilization. On the other hand, in the spicy olive oil, no changes were observed after 2 months of storage, but there was a decrease after one year of storage in relation to the values obtained after sterilization. DISCUSSION The increase in the peroxide index during frying and sterilization is due to oxidation processes. The subsequent decrease during storage occurs when peroxides are transformed, as they are very unstable, into other chemical compounds such as aliphatic carbonyls (Benzie, 1996). Because of the peroxides formed are not stable compounds, the peroxide index is not always correlated with the degree of oxidation of an oil. Alhibshi et al. (2016) reported that when peroxide values are between 30 and 40 mEq. O2/kg the rancid flavor is clearly noticeable. In the samples under study, this value was never exceeded. During secondary oxidation malonaldehyde is formed. Very high treatment temperatures or prolonged treatment times cause an increase in oxidation by-products such as malonaldehyde (Shanmugasundaram et al., 2019). Subsequent oxidation of secondary products and the formation of carboxylic acids, and other compounds that are not reactive to 2-thiobarbituric acid may cause a decrease in TBA index during storage (Tagh-Vaei et al., 2014). Valenzuela et al. (2017) also observed that in the olive oil the TBA index increased more during frying than in sunflower oil. In previous studies, it has been demonstrated that olive oil contain natural polyphenols, which have a key role in determining oxidation (Tsimidou et al., 1992). Medina et al. (1999)

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also observed that extra virgin olive oil had a higher potential antioxidant activity on canned fish lipid than other filling medium as refined olive oil, refined soybean oil or brine CONCLUSIONS In spicy olive oil, higher changes in oxidation parameters during canning were observed than in sunflower oil or olive oil. In addition, the increase of the storage time produced higher changes in oils under study. The frying treatment generally caused higher changes in the oxidation parameters than the sterilization treatment of the oils. Acknowledgements: This work was funded by Xunta de Galicia, Spain (CITACA Strategic Partnership, Reference: ED431E 2018/07). L. Gómez-Limia, thanks University of Vigo because of this research was supported by University of Vigo predoctoral scholarship.

REFERENCES Alhibshi, E. A., Ibraheim, J. A., Hadad, A. S. (2016). Effect of heat processing and storage on characteristic and stability of some edible oils. 6th International Conference on Agriculture, Environment and Biological Sciences, Kuala Lumpur, Malasia. Benzie, I. (1996). Lipid peroxidation: a review of causes, consequences, measurement, and dietary influences. Int. J. Food Sci. Nutr., 46: 233-261. Choe, E., Min, D. B. (2006). Mechanisms and factors for edible oil oxidation. Compr. Rev. Food Sci. F., 5: 169-186. Gertz C., Klostermann S., Kochhar S. P. (2000). Testing and comparing oxidative stability of vegetable oils and fats at frying temperature. Eur. J. Lipid Sci. Tech., 102: 543–551. Kaleem, A., Aziz, S., Iqtedar, M. (2015). Investigating changes and effect of peroxide values in cooking oils subject to light and heat. FUUAST J. Biol., 5: 191-196. Kirk, R. S., Sawyer, R. (1991). Pearson's composition and analysis of foods. Nº. Ed. 9. Longman Group Limited, London, United Kingdom. Medina, I., R. Sacchi, L. Biondi, S. P. Aubourg, L. Paolillo (1998). Effect of packing media on the oxidation of canned tuna lipids. Antioxidant effectiveness of extra virgin olive oil. J. Agric. Food Chem., 46: 1150−1157. Shanmugasundaram, B., Singh V. K., Shrestha, S., Sarkar, S. K., Jeevaratnam, K., Koner, B. C. (2019). Comparative evaluation of peroxidation of sesame and cottonseed oil induced by different methods of heating. J. Sci. Ind. Res. India, 178: 162-165. Tagh-Vaei, M., Jafari, S. M., Mahoonak, A. S., Nikoo, A. M., Rahmanian, N., Hajitabar, J., Meshginfar, N. (2014). The effect of natural antioxidants extracted from plant and animal resources on the oxidative stability of soybean oil. J. Food Sci. Techn. 1: 124–130. Tsimidou, M., Papadopulos, G., Boskou, D. (1992). Phenolic compounds and stability of virgin olive oils part I. Food Chem., 45: 141–144. Warner K. (2002). Chemistry of frying fats. In: Akoh C. C. and Min D. B. (eds), Food Lipids: Chemistry, Nutrition and Biotechnology. New York: Marcel Dekker, 167–180.

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MACROPHYTE BIODIVERSITY OF THE NORTH AEGEAN BASIN IN TURKEY Ezgi BÜKE*1, Evren CABİ2, Nesibe TURAN3 1

Tekirdağ Namık Kemal University, Graduate School of Natural and Applied Sciences, Department of Field Crops, Tekirdağ, Turkey

Tekirdağ Namık Kemal University, Faculty of Arts and Sciences, Department of Biology, Tekirdağ, Turkey 3

Republic of Turkey Ministry of Agriculture and Forestry, General Directorate of Water Management, Turkey *Corresponding author’s e-mail: ezgibuke@gmail.com

ABSTRACT North Aegean Basin which is located in the Aegean and Marmara geographical regions is one of the 25 hydrological basins in Turkey. This basin covers only 1.3% of the surface area of Turkey. According to the EU Water Framework Directive all member states need to the monitor and assess their surface waters using specific biological quality elements, in addition to hydrochemical data. Macrophytes are one of the important parameters used as biological indicators of organic pollution and also one the ecological quality assesments to monitor and assess the ecological status of water bodies. For this purpose abundance and macrophyte composition in total 19 water bodies in the basin were determined and assessed according to the requirements of the Water Framework Directive. Surveys carried out during the project “Establishment of a Water Quality Ecological Assessment System Specific to Our Country” which were conducted by the General Directorate of Water Management of the Ministry of Agriculture and Forestry. During the surveys between 2014 and 2015, 16 rivers and 3 dam reservoirs were visited and 73 different macrophyte taxa were recorded from these 19 water bodies. Different methodologies have been followed for sampling macrophytes from dams and rivers. Mentha longifolia subsp. typhoides (Briq.) Harley and Pulicaria dysenterica (L.) Bernh. are found the most frequent emergent species. Myriophyllum spicatum L. is common rooted submergent species in the study area. The most floating leaved macrophytes are Potamogeton spp. The most frequent algae taxa in the basin were noted as Cladophora sp. and Spirogyra sp. The highest richness was found in the Menderes Stream (Çanakkale) (23 different taxa in the first vegatation period) and the lowest number of species was recorded in Geyikli Stream (İzmir) (only 1 taxon).All macrophyte life forms are present, but floating plants are represented only by Lemna minor and not common in the area, contrary Potamogeton sp. genus is most common and represented by 5 taxa. Emergent and rooted submergent species are more frequent both in rivers and dams. Keywords: Macrophytes, Rivers, Dam Reservoirs, North Aegean Basin, Turkey

INTRODUCTION Aquatic macrophytes, in other words, hydrophytes, are important components of wetlands (Rejmánková, 2011). Macrophytes as a biological group include many diverse taxonomic groups such as macro algae (Cladophora sp., Chara sp., etc.), bryophytes, liverworts, pteridophytes and vascular plants. (Wetzel, 1975; Casper & Krausch 1981; Frahm & Frei, 446

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1992). Macrophytes, as one of the biological quality elements, can provide valuable information about the ecological status of the water bodies (Newbold & Holmes, 1987; Grasmück et al., 1995). Indicator taxa and their densities can be used to determine trophic level of water (Carbiener et al., 1990). Haury et al. (2000), have indicated the important features of the macrophytes as bioindicators. Macrophytes diversity and biomass can easily linked to many other ecosystem functions and services (e.g., provision of wildlife habitat), so loss of macrophytes taxa may lead not only to a decline in productivity, but also a decline in globally important processes like carbon storage (Hooper et al., 2012). The Water Framework Directive (WFD), which came into force in 2000, aims to provide a common approach to water management for EU member and candidate countries. The directive aims to prevent and improve further deterioration of aquatic environments in particular; longterm conservation of existing water resources. (Dalkılıç & Harmancıoğlu 2008; Water Framework Directive, 2000). Macrophytes are considered by the WFD as one of the main biological groups suitable for biological monitoring. The North Aegean Basin is located in the northwest of Anatolia between 40° - 38° northern latitudes and 26° - 28° east longitudes and includes Karamenderes River, Tuzla River, Havran River, Madra River, Güzelhisar River and Bakırçay River water collection areas that discharge the waters to the Aegean Sea. The basin border starts from the Dardanelles Strait in the north and passes through the water section line of Kayalıdağ (879 meters), Kazdağ (1,766 m) and Kocakatran Mountains; east of Havran, Bergama, Soma and Kırkağaç districts; to the south, Kılıçdağ, Dumanlıdağ (1.098 m) passes through the water section line and connects to the Aegean Sea near Foça. Freshwater biodiversity is declining at an alarming rate. This rate is much greater than has been noted for even in the most affected terrestrial systems (Millennium Ecosystem Assessment, 2005). The first step of the conservation and rehabilitation of freshwaters is to identify the species in these areas. In this study, macrophyte diversity in 16 rivers and 3 dam lakes located in the North Aegean Basin was determined and the status of water resources in the basin was interpreted based on the indicator properties of these species. MATERIAL AND METHODS Sampling were carried out in 16 rivers and 3 dam reservoirs located in the basin which includes certain parts of Çanakkale, Balıkesir, Manisa and İzmir provinces. Sampling was conducted among 4 vegetation periods, between September 2014 and August 2015. Exact study areas according to their coordinates were given in Figure 1.

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Figure 1. Location of sampling areas in the North Aegean Basin For survey in rivers we followed TS EN 14184 standards, 100 m transect was chosen for sampling of river macrophytes. When sampling in the lakes it is followed TS EN 15460, standards. Collected macrophyte samples were pressed and dried or taken into alcohol solution for further identification process. After sampling, identification procedures were carried out in the Laboratory of Plant Morphology and Anatomy, Department of Biology, Tekirdağ Namık Kemal University, Tekirdağ. Identification of macrophyte samples were carried out bu using Seçmen & Leblebici (2008)’s Wetland Vegetation and Crops of Turkey, Davis (1965-1985)’s Flora of Turkey and East Aegean Islands (Vol 1-9) and Türkiye Bitkileri Listesi (Damarlı Bitkiler) (2012) books were used. RESULTS AND DISCUSSION Determining the composition of macrophytes, which are important parameters in specifying the ecological quality of water bodies, is very important in terms of revealing the ecological quality of water bodies and planning of future conservation studies. A total of 73 different macrophyte taxa have been found in 16 rivers and 3 dam reservoirs in the North Aegean Basin (Table 1). Out of the 73 taxa, 11 (15%) were Poaceae, 9 (12%) Cyperaceae, 6 of them were Asteraceae, Lamiaceae and Potamogetonaceae (%8 for each family). The distribution of the sampled macrophyte taxa according to families is given in Figure 2.

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Table 2 Macrophyte species collected from the North Aegean Basin Taxa Name

Family

Adiantum capillus-veneris L.

Pteridaceae

Alisma plantago-aquatica L.

Alismataceae

Apium nodiflorum (L.) Lag.

Apiaceae

Aster subulatus (Michx.) Hort. ex Asteraceae Michx Bidens tripartita L. Asteraceae Calystegia sepium subsp. sepium (L.) Convolvulaceae R.Br.

Cardamine hirsuta L.

Brassicaceae

Carex sp.

Cyperaceae

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Water Body Code EDSKEN05 EDSKEN01 EDSKEN08 EDSKEN04 EDSKEN08 EDSKEN09 EDSKEN11 EDSKEN03 EDSKEN06 EDSKEN01 EDSKEN01

Vegetation Period 1 2 1 2 2,4 3 2 1 1 1 1

EDSKEN02 EDSKEN09 EDSKEN14 EDSKEN15 EDSKEN16 EDSKEN01 EDSKEN04 EDSKEN08 EDSKEN11 EDSKEN14 EDSKEN16 EDSKEG01

3 3 3 3 3 1,2,3 3 3 1,2,3,4 3,4 3,4 3

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Table 3 Macrophyte species collected from the North Aegean Basin (Continues) Taxa Name

Family

Carex flacca Schreb. subsp. erythrostachys Cyperaceae (Hoppe) Holub

Water Body Code EDSKEN04

Vegetation Period 4 1,2 1 1 1 1,2

Catabrosa aquatica (L.) P. Beauv.

Poaceae

EDSKEN02 EDSKEN04 EDSKEN05 EDSKEN11 EDSKEN12

Ceratophyllum demersum L.

Ceratophyllaceae

EDSKEN07 EDSKEG02

1,4 1,2,3,4

Chara vulgaris Thuill.

Characeae

Chenopodium botrys L.

Amaranthaceae

EDSKEN06 EDSKEN08 EDSKEN12 EDSKEN11

1,2 2 1 1 1,4 1,4 2 1,3,4 3 1,3,4 2,4 3,4 3 3,4

Cladophora sp.

Cladophoraceae

EDSKEN01 EDSKEN02 EDSKEN03 EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN10 EDSKEN12 EDSKEN13 EDSKEN15

Conyza bonariensis (L.) Cronquist

Asteraceae

EDSKEN02 EDSKEN12

1 1,2

Cynanchum acutum subsp. acutum L.

Apocynaceae

EDSKEN01 EDSKEG03

1 1

Cyperus difformis L.

Cyperaceae

EDSKEN04 EDSKEN11

1 1,4

Cyperus fuscus L.

Cyperaceae

EDSKEN02 EDSKEN07

1 1

Cyperus glaber L.

Cyperaceae

EDSKEN02 EDSKEN04

1 1

Cyperus longus L.

Cyperaceae

EDSKEN01 EDSKEN02 EDSKEN06 EDSKEN11 EDSKEN16

1,4 1 1 1,4 4

Cyperus serotinus var. serotinus Rottb.

Cyperaceae

EDSKEN08

Crypsis schoenoides (L.) Lam.

Poaceae

EDSKEN02

Poaceae

EDSKEN02 EDSKEN05 EDSKEN11

1 1 1

Digitaria sanguinalis (L.) Scop.

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Table 4 Macrophyte species collected from the North Aegean Basin (Continues)

EDSKEN06 EDSKEN08 EDSKEN01 EDSKEN02 EDSKEN03 EDSKEN04 EDSKEN11 EDSKEG03 EDSKEN04 EDSKEN05 EDSKEN04 EDSKEN08 EDSKEN11 EDSKEN12 EDSKEN05 EDSKEN12 EDSKEN07 EDSKEN04 EDSKEN12 EDSKEN02 EDSKEN02 EDSKEN07 EDSKEN08 EDSKEN11 EDSKEN03 EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN07

Vegetation Period 1 1 1 1 1 1 1 1,4 1,2 1,2 1 1 1,4 1,2 1 1 1 2 2 1 1,2 1 1,2,4 1,2 2 1,2,4 4 1,2 2

Lamiaceae

EDSKEN01 EDSKEN02 EDSKEN04 EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN10 EDSKEN11

1,2 1 1 1,4 1,2 1,2,4 2,4 1

Lythraceae

EDSKEN01 EDSKEN03 EDSKEN04 EDSKEN06 EDSKEN08 EDSKEN10 EDSKEN11 EDSKEN16

1,4 4 1 1 1 4 1 4

Lamiaceae

EDSKEN02 EDSKEN07 EDSKEN16

1 1 4

Taxa Name

Family

Echinochloa colona (L.) Link

Poaceae

Echinochloa crus-galli (L.) P. Beauv.

Poaceae

Equisetum sylvaticum L.

Equisetaceae

Epilobium hirsutum L.

Onagraceae

Eupatorium cannabinum L.

Asteraceae

Fimbristylis bisumbellata (Forsskal) Bubani

Cyperaceae

Inula viscosa (L.) Aiton

Asteraceae

Juncus bulbosus L.

Juncaceae

Juncus inflexus subsp. inflexus L.

Juncaceae

Lemna minor L.

Araceae

Lemna sp.

Araceae

Lycopus europaeus L.

Lythrum salicaria L.

Mentha aquatica L.

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Water Body Code

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Table 5 Macrophyte species collected from the North Aegean Basin (Continues) Water Body Code

Vegetation Period

Mentha longifolia L. subsp. typhoides (Briq.) Lamiaceae Harley

EDSKEN02 EDSKEN03 EDSKEN04 EDSKEN05 EDSKEN06 EDSKEN07 EDSKEN10 EDSKEN11 EDSKEN12 EDSKEN16

1 1 1 1 1,4 1 2,4 1,4 1,2 4

Mentha pulegium L.

Lamiaceae

EDSKEN03

Mentha spicata subsp. spicata L.

Lamiaceae

EDSKEN02

Haloragaceae

EDSKEN06 EDSKEN07 EDSKEN08 EDSKEG02

1,2 1,4 1,2,3 1,4

Brassicaceae

EDSKEN02 EDSKEN05 EDSKEN06 EDSKEN11

2 2 2 2

Paspalum distichum L.

Poaceae

EDSKEN01 EDSKEN02 EDSKEN03 EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN10 EDSKEG01

1,2,3,4 1 1,2,4 1,2,4 1,2,4 1,2 4 1,2,4

Phyla nodiflora (L.) Greene

Verbenaceae

EDSKEN03

Phragmites australis (Cav.) Trin. ex Steud.

Poaceae

EDSKEN01 EDSKEN10

1,2,3,4 2,3,4

Phytolacca americana L. Phytolaccaceae Plantago major L. subsp. intermedia (Gilib.) Plantaginaceae Lange Plantago major subsp. major L. Plantaginaceae

EDSKEN04

EDSKEN11

EDSKEN02

Poa annua L.

Poaceae

EDSKEN04

Polygonaceae

EDSKEN01 EDSKEN03 EDSKEN04 EDSKEN06 EDSKEN07 EDSKEN10 EDSKEN11

1,2,4 1,2,4 1 1 1 2 1

Taxa Name

Myriophyllum spicatum L.

Nasturtium officinale R.Br. Aiton

Polygonum lapathifolium L.

Family

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Table 6 Macrophyte species collected from the North Aegean Basin (Continues) Taxa Name

Family

Polygonum hydropiper L.

Polygonaceae

EDSKEN03 EDSKEN09

Vegetation Period 2 2

Polypogon monspeliensis (L.) Desf.

Poaceae

EDSKEN02 EDSKEN04

1 1

Potamogeton berchtoldii Fieber

Potamogetonaceae

EDSKEN06

1,4

Potamogeton crispus L.

Potamogetonaceae

EDSKEN06 EDSKEN08 EDSKEG02

2,4 1,2 4

Potamogetonaceae

EDSKEN06 EDSKEN08 EDSKEG02

1,2,4 1,2,4 1,4

Potamogeton perfoliatus L.

Potamogetonaceae

EDSKEN07 EDSKEN08 EDSKEG02

1,4 1,2,4 1,2,4

Potamogeton trichoides Cham. & Schltdl.

Potamogetonaceae

EDSKEN06 EDSKEN08

1 1,2

Puccinellia ciliata Bor.

Poaceae

EDSKEN06

Pulicaria dysenterica (L.) Bernh.

Asteraceae

EDSKEN01 EDSKEN02 EDSKEN03 EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN11 EDSKEN12 EDSKEN16

1,2 1 1 1 1 1,4 1 1 4

Ranunculus trichophyllus Chaix ex Vill.

Ranunculaceae

EDSKEG02

Rorippa thracica (Griseb.) Fritsch

Brassicaceae

EDSKEN08

Scirpoides holoschoenus subsp. holoschoenus Cyperaceae (L.) Sojรกk

EDSKEN06 EDSKEN07 EDSKEN08 EDSKEN11 EDSKEN16

1,4 1 1,2 1,2,4 4

Setaria viridis (L.) P.Beauv.

Poaceae

EDSKEN04 EDSKEN06 EDSKEN11

1,4 1 1

Solanum dulcamara L.

Solanaecae

EDSKEN01

Zygnemataceae

EDSKEN02 EDSKEN06 EDSKEN11 EDSKEN12 EDSKEG02

1 1 4 1 2

Potamogeton nodosus Poir.

Spirogyra sp.

453

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Table 7 Macrophyte species collected from the North Aegean Basin (Continues) Taxa Name

Family

Tamarix tetrandra Pall. ex M.Bieb.

Tamaricaceae

EDSKEN01 EDSKEN03

Vegetation Period 1,2,3,4 1,2,3,4

Typhaceae

EDSKEN06 EDSKEN07 EDSKEN08

1,4 4 1,2,3,4

Urticaceae

EDSKEN01 EDSKEN02 EDSKEN09 EDSKEN11

3 1,2,3 2 1,2

Ulotrichaceae

EDSKEN04 EDSKEN06 EDSKEN07

1,3,4 1 3

Plantaginaceae

EDSKEN02 EDSKEN04 EDSKEN07 EDSKEN08 EDSKEN09 EDSKEN11

1,2 1,3,4 1 1,2 2 1,2

Vitex agnus-castus L.

Lamiaceae

EDSKEN03 EDSKEN06 EDSKEN08 EDSKEG03

1,2,3 1,2,3 1,2,3 1,2,3

Zannichellia palustris L.

Potamogetonaceae

EDSKEN06 EDSKEG02

2,4 1,2

Zygnema sp.

Zygnemataceae

EDSKEN06 EDSKEN12

2 1,2

Typha angustifolia L.

Urtica dioica L.

Ulothrix sp.

Veronica anagallis-aquatica L.

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Asteraceae (6 taxa) Cyperaceae (9 taxa) 12%

Lamiaceae (6 taxa)

34%

Poaceae (11 taxa) Potamogetonaceae (6 taxa) 8%

Araceae (2 taxa) Plantaginaceae (2 taxa) Juncaceae (2 taxa) Polygonaceae (2 taxa)

3% 3%

15% 3%

Zygnemataceae (2 taxa) Other (1 taxa)

Figure 2 Composition by family The Poaceae family is the most commonly represented by taxa, and individuals belonging to this family are generally found in the coastal zone. Phragmites australis, which has an emergent life form, is a member of this family and has a significant effect on maintaining a certain level of methane concentration in water (Arkebauer et al., 2001). According to life forms of macrophytes the most of the macrophytes are in emergent form (56 taxa), there are 6 taxa of algea and 5 taxa of floating-leaved macrophytes are in the basin. This situation shows that emergent species are dominant in the basin and that the most frequent family of macrophytes living in water is Potamogetonaceae. The sampled macrophyte taxa according to their life forms and the results are given in Figure 3.

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1% 6%

7% Algae (6 taxa) Emergent (56 taxa) Floating Leaved (5 taxa) Submersed (4 taxa) Free-floating (1 taxa) 78%

Figure 3 Composition according to macrophyte life forms Emergent macrophytes are particularly effective in preventing sediment loss in rivers. Roots and stems stabilize small sediment particles in the water (Greenway, 2007). Emergent macrophytes also significantly reduce wind-induced sediment resuspension in shallow lakes (Dieter, 1990). Emergent macrophytes are also important because they provide shelter, feeding and reproduction areas for other living things (Cooperman & Markle, 2004; Schmidt et al., 2005). Therefore, their predominance in wetlands is important in terms of ecosystem sustainability. Floating leaved macrophytes have an active role especially in heavy metal accumulation and nutrient cycling (Denny, 1980; 1987). The presence of Potamogeton spp., one of the macrophytes in the basin, is important in this respect. Among the 73 macrophyte taxa in the basin, 25 taxa were found to be bioindicator taxa. According to Macrophyte Biological Index for Rivers (IBMR or MBIR) (AFNOR 2003, Haury et al., 2006). Most taxa are indicated for eutrophic and mesotrophic rivers (Table 2). Therefore, we can say that there are generally mesotrophic-eutrophic water bodies in the basin.

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Table 8 Indicator species for water ecological quality Taxa

Indication

Taxa

Indication

A. plantago-aquatica subsp. plantago-aquatica

Eutrophic

P. australis

Mesotrophic-Eutrophic

A. nodiflorum

Mesotrophic-Eutrop hic

P. hydropiper.

Mesotrophic-Eutrophic

C. aquatica.

Mesotrophic-Eutrop hic

P. berchtoldii

Mesotrophic-Eutrophic

C. demersum

Mesotrophic-Eutrop hic

P. crispus

Mesotrophic-Eutrophic (Oligotrophic)

C. vulgaris

Mesotrophic-Eutrop hic

P. nodosus

Mesotrophic-Eutrophic

Cladophora sp.

Mesotrophic-Eutrop hic

P. perfoliatus

Mesotrophic-Eutrophic

J. bulbosus

Oligotrophic-Mesotr ophic

P. trichoides

Mesotrophic-Eutrophic

L. minor

Mesotrophic-Eutrop hic

Spirogyra sp.

Mesotrophic-Eutrophic

L. europaeus

Mesotrophic-Eutrop hic

T. angustifolia

Mesotrophic-Eutrophic

M. aquatica

Mesotrophic-Eutrop hic

Ulothrix sp.

Mesotrophic-Eutrophic

M. spicatum

Mesotrophic-Eutrop hic

V. anagallis-aquatica

Mesotrophic-Eutrophic

N. officinale

Mesotrophic

Z. palustris.

Eutrophic

Zygnema sp.

Mesotrophic

M. longifolia subsp. typhoides (Briq.) Harley and P. dysenterica (L.) Bernh. are found the most frequent emergent species. M. spicatum is common rooted submergent species in the study area. The most floating leaved macrophytes are Potamogeton spp. The most frequent algae taxa in the basin were noted as Cladophora sp. and Spirogyra sp. All macrophyte life forms are present, but free floating plants are represented only by L. minor and not common in the area, contrary genus Potamogeton is most common and represented by 5 taxa. Most taxa (23 taxa) were found during the first period sampling of EDSKEN06 point; the poorest point in terms of macrophyte richness was identified as EDSKEN13 (1 taxon). CONCLUSIONS Macrophytes have been identified by the WFD as one of the biological quality elements involved in the determination of water quality. Rapid urbanization and agricultural activities have negative effects on wetlands. These activities are a threat to species living in these areas. Aquatic macrophytes are the primary producers in wetlands, are a source of food for animals and are bioindicators. For this reason, identification and monitoring of macrophyte taxa in wetlands is important for wetland conservation, sustainability and rehabilitation studies. This 457

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is the first study which conducted in the North Aegean Basin using macrophyte biological quality elements. In this study, the macrophyte composition in the basin was determined and the general status of the basin was obtained. Acknowledgements: This study was supported by the Directorate General for Water Management of the Ministry of Agriculture and Forestry (Republic of Turkey) (Project no: 188.02.01) and DOKAY-ÇED Company. REFERENCES AFNOR (2003). Détermination de l’Indice Biologique Macrophytique en Rivière (IBMR). NF T 90‐395. Association Française de Normalisation, La Plaine Saint‐Denis, France. Arkebauer, T.J., Chanton J.P., Verma S.B., Kim J. (2001). Field measurements of internal pressurization in Phragmites australis (Poaceae) and implications for regulation of methane emissions in a midlatitude prairie wetland. American Journal of Botany, 88(4), 653–658. Güner, A., Aslan, S., Ekim, T., Vural, M., & Babaç, M.T., (eds.). (2012). Türkiye Bitkileri Listesi (Damarlı Bitkiler). Istanbul: Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayınları. Carbiener, R., Trémolières, M., Mercier, J.L., & Ortscheit A. (1990). Aquatic macrophyte communities as bioindicators of eutrophication in calcareous oligosaprobe stream waters (Upper Rhine plain, Alsace). Vegetatio, 86, 71. Casper, S. J., & Krausch, H. D. (1981). Pteridophyta und Anthophyta 23/24 (pp. 942). Ettl, H., Gerloff, J. & Heynig, H. (Eds), Stuttgart: Die Süßwasserflora von Mitteleuropa. Gustav Fischer Verlag. Cooperman, M.S. & Markle, D.F. (2004). Abundance, size, and feeding success of larval shortnose suckers and Lost River suckers from different habitats of the littoral zone of Upper Klamath Lake. Environmental Biology of Fishes, 71, 365–377. Dalkılıç, Y & Harmancıoğlu, N. (2008). Avrupa Birliği Su Çerçeve Direktifinin Türkiye’de Uygulama Olanakları. TMMOB İnşaat Mühendisleri Odası 2. Su Politikaları Kongresi. Ankara. Davis, P.H. (1965-1985). Flora of Turkey and East Aegean Islands (Vols. 1-9). Edinburg: University Press. Denny, P. (1980). Solute movement in submerged angiosperms. Biological Review, 55, 65–92. Denny, P. (1987). Mineral cycling by wetland plants a review. Archiv fur Hydrobiologie Beith, 27, 1–25. Dieter C.D. (1990). The importance of emergent veget-ation in reducing sediment resuspension in wetlands.Journal of Freshwater Ecology,5,467-473. Frahm, J.P. & Frey, W. (1992). Moosflora. (3rd. ed., 525 pp.). Stuttgart, Germany: Ulmer. Grasmück, N., Haury, J., Léglize, L., & Muller, S. (1995). Assessment of the bio-indicator capacity of aquatic macrophytes using multivariate analysis. Hydrobiologia 300, 115. Greenway, M. (2007). Environmental Bioremediation Technologies. The Role of Macrophytes in Nutrient Removal using Constructed Wetlands. (pp. 331-351). Springer, Berlin, Heidelberg. Haury, J., Peltre, M.C., Muller, S., Thiebaut, G., Tremolieres, M., Demars, B., Barbe, J., Dutartre, A., Guerlesquin, M., & Lambert, E. (2000). Les macrophytes aquatiques bioindicateurs des systemes lotiques – Interets et limites des indices macrophytiques. Synthese bibliographique des principales approches europe´ennes pour le diagnostic biologique des cours d’eau. UMR INRA-ENSA 458

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EQHC Rennes & Phytoecologie Univ.Metz. Agence de l’Eau Artois-Picardie, Etudes sur l’Eau en France n87, Min. Ecologie Dev. Durable, 101 pp. Haury, J., Peltre, M. C., Trémolières, M., Barbe, J., Thiébaut, G., Bernez, I., & Dutartre, A. (2006). A new method to assess water trophy and organic pollution—the Macrophyte Biological Index for Rivers (IBMR): its application to different types of river and pollution. In Macrophytes in aquatic ecosystems: From biology to management (pp. 153-158). Springer, Dordrecht. Hooper, D., Adair, E., Cardinale, B., Byrnes, J., Hungate, B., Matulich, K., Gonzalez, A., Duffy, J.E., Gamfeldt, L. & O’Connor, L. (2012). A global synthesis reveals biodiversity loss as a major driver of ecosystem change. Nature, 486, 105–108 Millennium Ecosystem Assessment. (2005). Ecosystems and Human Well-being: Biodiversity Synthesis. Millennium Ecosystem Assessment. World Resources Institute, Washington DC. Newbold, E., & Holmes, N.T.H., 1987. Nature conservation: water quality criteria and plants as water quality monitors. Water Pollut. Control, 86, 345-364. Rejmánková, E. (2011). The role of macrophytes in wetland ecosystems. Journal of Ecology and Field Biology, 34(4), 333-345. Seçmen, Ö., & Leblebici, E. (2008). Türkiye Sulak Alan Bitkileri ve Bitki Örtüsü. İzmir: Ege Üniversitesi Yayınları. Schmidt, M.H., Lefebvre, G., Poulin, B. & Tscharntke, T. (2005). Reed cutting affects arthropod communities, potentially reducing food for passerines. Biological Conservation, 121, 157–166. TS EN 14184. (2014). Water quality – Guidance standard for the surveying of aquatic macrophytes in running waters. Türk Standartları Enstitüsü, Ankara. TS EN 15460. (2008). Water quality – Guidance standard for the surveying of aquatic macrophytes in lakes. Türk Standartları Enstitüsü, Ankara. Water Framework Directive (2000), Directive 2000/60/EC of European Parliament and of the Council establishing a framework for the Community action in the field of water policy. Wetzel, R.G. (1975). Limnology (pp.742). Philadelphia: W.B. Saunders Company.

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ADAPTATION OF NEW APRICOT VARIETIES FOR TURKEY Alimcan KİPÇAK1, Burhanettin İMRAK*2 2 2

Çukurova University Agricultural Faculty Horticulture Dept, Adana, Turkey

Çukurova University Pozantı Agricultural Research and Application Center, Adana, Turkey *Corresponding author’s e-mail: imrakburhan@gmail.com

ABSTRACT The study was carried out on 4 years old 13 new apricot varieties which is grafted on Myrobolan 29C rootstock, 4x1.5m planting distance. The trees were pruned as central leader system and cultivated in Çukurova University Pozantı Agriculture Research and Application Center at an altitude 1100m. The aim of the study was to determine the adaptability of new apricot varieties to high altitude regions. Pomological analyzes (Fruit weight, stone weight, flesh firmness, TSS (Total soluble solids) and yield values (kg/tree) were carried out in order to determine the fruit quality criteria. In the study where standard fertilization and irrigation programs were applied. However, the results of phenological observations (bud break, first bloom, full bloom, and harvesting date) were recorded. In addition, the chill accumulation of the research area was determined according to standard and chill unit methods. According to the pomological analysıs result the highest fruit weight and firmness was obtained from Gady Cat cultivar (49.10g 6.47kg/cm2). In terms of harvesting date Orange Ruby and Mediabel (28.06.2019) were detected the first apricot cultivars, Gady Cat and Pincot were harvested in 02.07.2019. There were not detected any problem about chilling requirement of all new apricot varieties. In terms of quality and yield values Gady cat, Mediabel, Orange Ruby and Faralia varieties was found suitable for high altitude areas to apricot growing. Keywords: Apricot, Adaptation, Chill Accumulation, High altitude

INTRODUCTION Apricot is one of the most common cultivated stone fruits (Ercisli, 2009) and Turkey is the major producer of apricots in the world, Turkey is the important producer of fresh and dry apricot in the world followed by Iran, Uzbekistan, Algeria and Italy with an annual production of 985.000tonnes in 2017 (FAOSTAT, 2017). Turkey is the leading producer 50% of the total dried apricots in world is produced in Turkey. A significant portion of fresh and 90–95% of dried apricots of Turkey is produced in Malatya region (Asma, 2011). Dried and fresh apricot production are of agronomic importance for Turkey. Almost 50% of the total dried apricots in world is produced in Turkey. Apricot production is mainly based in Malatya region which is the major ecological zone in Turkey for cultivation. Approximately 50% of the fresh apricots and 90% of the dried apricots are produced in this region (Ünal and Sener, 2015). According to FAO statistical data, 90.321 tonnes dried apricots were exported from Turkey and export value of apricots in Turkey was $152 million in 2018 (FAO, 2018). The late spring frost could have caused further flower and small fruit injuries, affected proper pollination and reduced the yield. Frost damage and freezing injury to flower buds of Prunus armeniaca L. during winter period is the main important factor of irregular yields. During the dormancy period, the flower buds are the most resistant to damage caused by low temperatures. While, for a number of days, the developmental processes in buds begin, weakening their resistance to temperatures below 0oC. 460

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According to researchers indicated that a temperature drop to -2.2oC during the full bloom can cause significant damage to flowers of apricot. Early or late ripening new fresh apricot cultivars are sold with much higher prices than midseason apricots. Apricot season is usually around 30-90 days. Harvesting dates are extended with the higher elevations. It is foreseen that increasing of new apricot production during the off-season particularly in highland region provides a great advantage for domestic fresh consumption and exportation in Turkey. MATERIAL AND METHODS Material The study was carried out in two period (2018 and 2019) at the experimental orchard of Cukurova University Pozantı Agricultural Research and Application Center (POZMER) orchards at 110 m elevations in Adana (37° 37'.32”N-35o 35° 21'54.78"E) at altitude of 110m. The four years old thirteen new apricot trees (Prunus armeniaca L.) Which is self-fertile cultivar and grafted on Myrobolan 29C rootstock, planting distance 1.5x4m and trees were pruned Central Leader system (Table 1). Table 1. New apricot cultivars for Turkey Faralia

Harvali

Narroured

Farbaly

Pricia

Pinckot

Orange Ruby

Hargunet

Fortillo

Farclo

Rubista

Mediabel

Gadycat

Phonological Observations Flowering periods of genotypes in the research areas were determined by observations. Waking up from the dormancy (the period in on which 50% of the buds show green end), first bloom (the period when 5% of the flowers blossomed), full bloom (the period when 70% of the flowers blossomed) and harvesting time period when the fruits maturating date were observed. Pomological Analyses In order to detected pomological characteristics of fruit, samples taken from the types were examined according to the International Plant Genetic Resources Apricot Identification Guide "International Board for Plant Genetic Resources (IBPGR) Revised Descriptor List for Apricot (Prunus armeniaca L.)". Fruit weight (g) was measured by using a digital scale (Shinko DJ-600E, Japan precision (0.1 g).) Total soluble solids (TSS) content was determined by digital hand refractometer (ATC1. Atago, Tokyo, Japan), fruit elasticity (shore) was determined by a penetrometer (N.O.W. FHR-5. Tokyo Japan) equipped with an 8 mm cylindrical plunger. The measurement was performed on two opposite faces of the equatorial zone, after skin removal of the equatorial zone. Titratable acidity (TA) was determined by using an automatic titration apparatus (877 Titrino plus, Metrohm, Herisau, Switzerland) with 0.1 mol/L NaOH up to pH 8.1; results were shown as g citric acid/100ml (Ozkaya et al., 2006) Data derived from observation and pomological analyses conducted on the identified types after the studies were evaluated with "Weighted Rating Method" (Yazgan, 1979).

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Chilling accumulation of the experimental area The chilling accumulation of the area were calculated as daily maximum and minimum temperatures of 24h by using a computer program which is prepared by Miller and Küden in 1989 according to the Richardson’s chill unit, by using asymcur curve model (Anderson et al 1982, Anderson 1987). Also the standard method of hours for chilling accumulation was below 45°F. Trees need to fulfill their chilling requirement for bud breaking (Campoy et al., 2011b). RESULTS Phonological Observations The apricot cultivars were found to be different in terms of phenological stages (Budbreak, first bloom, full bloom and fruit set time). However, it was found that the phenological stages changes between 5-7 days were determined between years. Dormancy breaking stage occurred 8 March in 2018 and 11 March in 2019. First bloom and Full bloom stage occurred in March for both year. Harvesting time was between 22 June and 24 July. However, the earliest cultivars were found Mediabel and Orange Ruby. The yield of cultivars ranged from 3.1 to 7.6 kg/tree. Mediabel gave the best yield (7.6 kg/tree) among the cultivars. Phenological data and yield quantity (kg/tree) of new apricot cultivars of this study are given in (Table 2). Pomological Analyses Apricot fruits, of which pomological analyses conducted, were collected during tree maturation period and when the suture went yellow or red. Fruit weight valued were observed in the study. The maximum fruit weight was found Gady Cat (49.10gr) and Faralia (43.35g) varieties. While minimum fruit weight was detected in Pincot and Fortillo varieties respectively 26.13-26.10) (Table 3) Fruit frimness is ranging from 4.36-6.46 kg/cm2. Highest value measured from Gady Cat variety with 6.46 kg/cm2 the lowest frimness value were measured from Fortillo varieties as 4.36 kg/cm2 (Table 3). In terms of TSS content, Harvali variety were found to be extremely good (> 25.00), Orange Ruby, Farbaly and Mediabel varieties were good (15.01-20.00) and the other all varieties were evaluated as moderate 10.01-15.00 (Table 3).

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Table 2. The Phenological stages dates and yield of the apricot cultivars Cultivars Orange Ruby Mediabel

Gady Cat

Harvali

Farclo

Farbaly

Faralia

Pricia

Pincot

Fortillo

Narroured

Year

Bud Break

First Bloom

Full Bloom

2018

08.03

13.03

20.03

2019

11.03

17.03

23.03

28.06

4.2

2018

08.03

13.03

20.03

22.06

6.8

2019

11.03

17.03

23.03

28.06

7.6

2018

08.03

13.03

20.03

01.07

4.9

2019

11.03

17.03

23.03

02.07

5.4

2018

08.03

13.03

20.03

04.07

3.9

2019

11.03

17.03

23.03

09.07

4.3

2018

08.03

13.03

20.03

10.07

5.4

2019

11.03

17.03

23.03

12.07

5.9

2018

08.03

13.03

20.03

10.07

4.9

2019

11.03

17.03

23.03

12.07

5.7

2018

08.03

10.03

20.03

10.07

4.6

2019

11.03

16.03

21.03

12.07

5.3

2018

08.03

14.03

20.03

05.07

3.4

2019

11.03

17.03

21.03

09.07

3.9

2018

08.03

17.03

23.03

28.06

3.1

2019

11.03

19.03

24.03

02.07

3.8

2018

08.03

15.03

24.03

20.07

4.9

2019

11.03

17.03

28.03

24.07

5.4

2018

08.03

16.03

20.03

10.07

3.0

10.07

5.8

10.07

4.2

2019 2018

Hargunet

Rubista

2019

22.06

Yield (kg/tree) 3.3

Spring Frost Damage 08.03

2019 2018

Fruit Set

16.03

20.03

Spring Frost Damage 08.03

16.03

20.03

Spring Frost Damage

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Table 3. Pomological analyses result Varieties Gady Cat Faralia Harvali Orange Ruby Farbaly Prica Mediabel Farclo Pincot Fortillo LSD: %5

Fruit Weight Fruit Frimness (gr) (kg/cm2) 49.10a 6.46a 43.35ab 4.33bc 39.99bc 3.46c 37.49bc 1.51d 36.70bc 4.50b 35.90bc 3.56bc 34.64c 2.36d 32.96cd 3.50bc 26.13d 3.96bc 26.10d 4.36bc 8.29 0,10

Brix TSS (%) 13,03b 12.01b 26.00a 18.5ab 11.26b 16.30b 17.00ab 12.10b 16.80b 12.23b 9.33

Chilling accumulation of the experimental area Chilling accumulation of the experimental area was sufficient in both winter period (20182019) to apricot growing (Table 4). In terms of low temperatures corresponding to the frost damage on flowering period significant differences was detected in March which is flowering period between the years. Almost all cultivars affected by the late spring frost in 2019 March. But some cultivars such as Mediabel, Gady Cat and Fortillo cultivars less affected by frost depend on flower density (Figure1). Table 4. Chilling accumulation of the experimental area (2018-2019)

Month

November December January February March Total

2018 (2017 - 2018 Period) Chıll Unit Hours (CU) below 45°F 90 150 107 156 118 337 197 550 179 503 691 1696

2019 (2018 - 2019 Period) Chıll Unit Hours (CU) below 45°F 98 161 112 122 124 342 196 569 182 512 712 1706

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Temperature C 0

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16 14 12 10 8 6 4 2 0 -2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 -4 -6 Days 2018

2019

Figure 1. Maximum and Minimum temperature in March. DISCUSSION Freezing injury to flower buds of Prunus armeniaca L. during winter is the main cause of irregular yields. According to Asma et al. (2017), severe damage to apricot flower buds occurs with drop in temperature to 0 C0 in March. Severity of damage, however, depends on temperature fluctuation during the whole winter. In apricot, severe damage to buds occurs with a drop in temperature to 0 to -2 C0 (Ercişli, 2009). Our results harmony with this values, especially in 2019 March in terms of frost damage on flowers. Higher fruit weight values important aspect for both dried and table apricot production, varieties were also observed in the study. Average fruit weight was shown to be ranged between 24.962.1 g by the study of Akça Özyörük et al. (1992) conducted in 1989 and 1990 in Iğdır Plain for Şalak, Tebereze, Ağerik, Ordubat ve Ağcanabat apricot varieties grown in the plain. In this research fruit weight values ranged between 26.10-49.60gr. Faralia, Gady Cat, Harvali, Orange Ruby, Farbaly, Prica, Mediabel and Farclo varieties was found sufficient by weight for fresh apricot varieties. According to Pınar et al. (2010), fresh apricot weight were found generally range between 21-69g. This result was found accordance with our research result. CONCLUSIONS There was not any problem about chilling requirement of all new apricot varieties. In terms of quality and yield values Gady Cat, Mediabel, Faralia and Orange Ruby varieties was found suitable for high altitude areas to apricot growing. Gady Cat, Mediabel, Faralia and Orange Ruby from 13 apricot varieties which are tested at 1100 m high altitude will be resistant to late spring frosts as a result these varieties can be recommended to high altitude regions in terms of yield and quality. Acknowledgements: We are thankful to financial support of the Scientific Research Projects Unit of Cukurova University (Project No: FYL -2019-11945). REFERENCES Akça Y, Askın A (1992). Clonal Selection in the Apricot Cultivar Hacihaliloglu. In X International Symposium on Apricot Culture, 384, 169-172. Anderson J.L., Rıchardson E.A. (1987). The Utah chill unit/flower bud phenology models for deciduous fruit: Their implication for production in subtropical areas. Acta Hortic, 199:45-50. 465

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Anderson, J.L., Rıchardson, E.A. And Kesner. D (1986). Validation of Chill Unit and Flower Bud Phenology Models for ‘‘Montmorency’’ Sour Cherry. Acta Hort. 184:71-78. Asma B.M (2011). Her Yönüyle Kayısı, Uyum Ajans Ankara. Asma, B.M (2012). New Apricot Selections for Dried and Table Consumption in Eastern Anatolia-Turkey. XV. IS on Apricot Breeding and Culture. Acta Hort. 966: 291294. Campoy JA, Ruiz D, Rees DJG, Celton JM, Martinez-Gomez P. (2011b). Inheritance of Flowering Time in (Prunus armeniaca L.) and Analysis of Linked Quantitative Trait Loci (QTLs) using Simple Sequence Repeat (SSR) Markers. Plant Mol. Biol. Report., 29(2): 404-410 Ercişli S. (2009). Apricot Culture in Turkey. Scientific Research and Assay, 4(8): 715-719. Faostat (2016). Food and Agricultural Organisation. www.fao.org Faostat (2017). Food and Agricultural Organisation. www.fao.org Faostat (2018). Food and Agricultural Organisation. www.fao.org M. Ü. Ünal., Sener, A (2015). Two-Year Comparison of the Biochemical Properties of Polyphenol Oxidase from Turkish Alyanak Apricot (Prunus Armenica L.). Food Chemistry, 190 (2016) 741–747. Özyörük, C. Güleryüz, M., 1992. Iğdır Ovasında Yetişen Kayısı çeşitleri Üzerine Pomolojik. Biyolojik ve Fenolojik Araştırmalar (Yüksek Lisans tezi) Atatürk Üniverstesi Fen Bilimleri enstitüsü. Pınar, H., Bircan, M., Yılmaz, C., Kargı paydaş, S., Kaşka, N.,Yıldız, A., Son, L (2010). The Performance of Some Apricot Cultivars in the Mersin Ecological Conditions. XIVth IS on Apricot Breeding and Culture. Acta Hort. 862: 109-112 Yazgan, A (1979). Bahçe Bitkileri Deneme Tekniği. Alata Bahçe Kültürleri Araştırma Enstitüsü Yayınları. Erdemli. Bee, G., G. Guex, W. Herzog (2004). Free-range rearing of pigs during the winter: adaptations in muscle fiber characteristics and effects on adipose tissue composition and meat quality traits. J. Anim. Sci., 82, 1206–1218.

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