2nd International Symposium on
Agricultural Technology Global Agriculture Trends for Sustainability
July 1-3, 2015 A-One The Royal Cruise Hotel Pattaya, Thailand
Proceeding
conf.kmitl.ac.th/isat2015 Organized by Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang (KMITL)
Major Sponsors
ISAT2015
Proceedings
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July 1-3, 2015
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Advisory Committee Prof.Dr. Suchatvee Suwansawat Assoc.Prof.Dr.Chamroon Laosinwattana Prof.Dr.Julian Wiseman
Committee
Prof.Dr.Gray Williams
July 1-3, 2015
Prof.Dr.Metha Wanapat
President, King Mongkut's Institute of Technology Ladkrabang (KMITL) Executive Vice President for Research and Innovation, KMITL School of Bioscience, University of Nottingham, United Kingdom School of Biological Science, University of Hongkong, Sar China Director, Tropical Feed Resource Research and Development Center, Khon Kaen University, Thailand
Steering Committee Assoc.Prof. Sakchai Choochote Assoc.Prof.Srisakul Vorachantra Asst.Prof.Dr.Paveena Taweekijakarn Asst.Prof.Peerachai Kullachai Asst.Prof.Dr.Kanya Jirajaroenrat Asst.Prof.Dr.Nutthakorn Songkram Asst. Prof.Dr.Ammorn Insung Asst.Prof.Dr.Monthon Ganmanee Asst.Prof.Dr.Thamrong Mekhora Ms.Sumnao Pattararatnant
KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
Secretary
Organizing Committee Asst.Prof.Dr.Monthon Ganmanee Assoc.Prof.Dr.Tanimnun Jaenaksorn Assoc.Prof.Dr.Chamroon Laosinwattana Asst.Prof.Dr.Paveena Taweekijakarn Asst.Prof.Dr.Kanjana Saetiew Asst. Prof.Dr.Ammorn Insung Asst.Prof.Dr.Nutthakorn Songkram Asst.Prof.Dr.Nittaya Phakamas Ms.Sumnao Pattararatnant Asst.Prof.Dr.Kanya Jirajaroenrat
Secretary
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Committee
Scientific Committee Assoc.Prof.Dr.Tanimnun Jaenaksorn Assoc.Prof.Dr.Chamroon Laosinwattana Assoc.Prof.Dr.Ronachai Sitthigripong Assoc.Prof.Dr.Tippawan Limunggura Assoc.Prof.Dr.Suneerat Ruangsomboon Asst.Prof.Dr.Kanjana Saetiew Asst. Prof.Dr.Ammorn Insung Asst.Prof.Dr. Teerawat Sarutayophat Asst.Prof.Dr.Nittaya Phakamas Dr. Nonglak Parinthawong Asst.Prof.Dr.Lampan Khurnpoon Dr. Sukunya Yampracha
Chairman
Secretary
International Scientific Committee Prof.Dr.Julian Wiseman Prof.Dr.Gray Williams Prof.Dr.Metha Wanapat
Prof.Dr.Hideo Ishii Prof.Dr. Abdul Salam Babji
Assoc.Prof.Dr. Rajeev Bhat
Assoc.Prof.Dr. Janine Croser Dr.Thierry Tran
School of Bioscience, University of Nottingham, United Kingdom School of Biological Science, University of Hongkong, Sar China Director, Tropical Feed Resource Research and Development Center, Khon Kaen University, Thailand School of Agricultural Regional Vitalization, Kibi International University, Japan School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM) Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang, Malaysia Centre for Plant Genetics and Breeding, The University of Western Australia, Australia French Agricultural Research Centre for International Development (CIRAD), France
Ceremony and Reception Committee Asst.Prof.Dr.Kanya Jirajaroenrat Asst.Prof.Dr. Sarayut Phonpho Asst.Prof.Dr. Montinee Teerarak Dr.Rutcharin Limsupavanich Lect. Dusit Aue-umneoy Assist.Prof.Dr.Komkhae Pilasombut Assist.Prof.Dr.Kanjana Saetiew Dr.Duangkamol Panrositp Thunmatiwat Dr.Suneeporn Suwanmaneepong Miss Montha Suwanrath
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KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL
KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
Secretary
July 1-3, 2015
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2 International Symposium on Agricultural Technology (ISAT2015)
Registration and Financial Committee
Committee
Asst.Prof.Dr.Nittaya Phakamas Asst.Prof.Dr.Komkhae Pilasombut Pattrarat Teamkao Phatchara Eamkijakarn Mrs.Pacharee Keadchoom Asst.Prof.Dr.Lampan Khurnpoon
July 1-3, 2015
KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
Secretary
Public Relations and Technical Committee Asst.Prof.Dr.Paveena Taweekijakarn Asst.Prof.Dr.Kanok Lertpanich Miss Wilai Punpitanusorn Mr.Apisit Sanla Asst.Prof.Dr.Nutthakorn Songkram
KMITL KMITL KMITL KMITL KMITL
Chairman
KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
KMITL KMITL KMITL KMITL KMITL KMITL
Chairman
Secretary
Welfare Committee Asst. Prof.Dr.Ammorn Insung Asst.Prof.Peerachai Kullachai Asst.Prof.Dr. Teerawat Sarutayophat Asst.Prof.Dr.Prommart Koohakan Asst. Prof.Pornthiwa Kanyawongha Dr. Nonglak Parinthawong Pattrarat Teamkao Lect. Wanida Duangkongsan Mr.Jarongsak Pumnuan
Secretary
Coordinating Committee Ms.Sumnao Pattararatnant Mrs.Pacharee Keadchoom Mr.Chatre Vijitrotai Buppha Jongput Mr.Panurat Jantup Mrs.Lamead Sumrittisut
Secretary
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Global Agriculture Trends for Sustainability
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Contents Page
Rationale……………………………............................…………..……… Report Address……………………………..........................……..……… Welcoming & Opening Remarks……………………………............…… Short Biography of Keynote and Invited Speakers……………..……… ISAT2015 Program……………………………...........……………..…… Scientific Program……………………………....................……………… Keynote and Invited Papers…………………………..………...………… Oral Full Papers……………………………....................………………… Poster Full Papers…………………………..............…………...………… List of Participants………………………………...........................……… Authors Index……………………………............................………………
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Page Keynote and Invited Papers Optimizing the Use of Feed Resources for Healthy Rumen, Increased Production and Mitigation Methane Prof. Dr. Metha WANAPAT Food Sustainability-Emerging Challenges in the ASEAN Region (Reality Versus the Myth) Assoc. Prof. Rajeev BHAT Can Feedstuff Processing Improve Digestibility of Amino Acids for Non-ruminants? Prof. Dr. Julian WISEMAN Rapid Generation Technology for Food and Forage Legumes- Harnessing in vitro Technologies for Accelerating Genetic Gain Assoc. Professor Dr. Janine CROSER Opportunity of AEC Meat Production for Halal food Market Prof. Dr. Abdul Salam BABJI Applications of Molecular Diagnostic Methods in the Management of Fungicide Resistance Prof. Dr. Hideo ISHII Biogas Reduces the Carbon Footprint of Cassava Starch: A Comparative Assessment with Fuel Oil Dr. Thierry TRAN
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Page Oral Full Papers Adapting the CSM-Canegro-Sugarcane Model to Simulate Napier Grass O-1 in Chiang Mai Province, Thailand Tupthai NORSUWAN, Attachai JINTRAWET and Carsten MARONH Effect of Azolla (Azolla spp.) on Growth and Yield of Pathumthani 1 Rice (Oryza sativa L.) O-2 Teerawat SARUTAYOPHAT, Nattawan BUSSABA and Phanupong PHONCHAROEN Allelopathic Effects of Pisonia grandis R.Br. on the Germination and Growth O-4 of Echinochloa crus-galli (L.) Beauv. Sudteerak SAIPLUEMCHIT and Chamroon LAOSINWATTANA How Far Teak Plantations Can Contribute for Climate Change Mitigation and Landscape Restoration in Tropics: O-5 A Case Study from Central India Singam Laxmana SWAMY and Alka MISHRA Antioxidant Properties of Flower Extract of Etlingera elatior (Jack) R.M. Smith O-6 Kanokporn CHANGSAWAKE, Komkhae PILASOMBUT, Chamroon LAOSINWATTANA and Montinee TERARAK Effect of Storage Conditions on the Vase Life of Homalomena O-7 Philodendron and Monstera Nipaporn YONSAWAD and Montinee TEERARAK Micro-propagation of Dalbergia cochinchinesis Pierre O-8 Wannasiri WANNARAT, Panida WONGWEAN, Sirinaree SUPANSOMPORN, Warinee KITPRECHAWANICH and Yupa PANKAEW Standardization of Drip Irrigation and Fertigation for Improving Physiology, Yield and Quality Parameters of Mango var. Alphonso O-9 under Ultra-High Density Planting K. PRAKASH, R.M. VIJAYAKUMAR and S.D. SUNDHAR SINGH Study of Using Coop Fertilizer Ameliorant on Greenhouse Gas Emission in Some Rubber Estates Agroecosystems in Peatland O-10 Hariyadi JAMIN PRIYO MINARJO, Dedi NURSYAMSI and Adi PRADIPTA Plant Nutrient Management Strategies Enhanced Growth, Yield Traits O-11 and Mitigated Leaf Reddening in Cotton (Gossypium hirsutum L.) Allahwadhayo GANDAHI, Khalillulah PANHWAR and Rabail GANDAHI Utilization of Calcium Silicate Application on Pepper Seedling Production O-12 Eakkarin SUKKAEW, Suphachai AMKHA, Thongchai MALA and Pornpairin RUNGCHAROENTHONG Behavior of Nutrient Uptake by Pummelo Growing on Salt Marsh Soil O-13 Hien HUU NGUYEN, Somsak MANEEPONG and Potjamarn SURANINPONG
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Page Oral Full Papers Eco-Friendly Utilization of TDE and Fertilizers on Soil Properties, O-14 Yield and Quality of Seed Cane J. REVATHI and M. BASKAR Investigating the Safety of Potential Probiotic Candidates Isolated O-16 from the GI Tract of Tilapia in vitro Rungtawan YOMLA, Daniel MERRIFIELD and Simon DAVIES Studied on Using of Robo-Logger Technique Measuring In Situ Inside Shell Temperature of Rocky Shore Limpet O-17 Monthon GANMANEE, Chayanid MEEPOKA, Pokamon KOCHARIN and Gray A. WILLIAMS Impacts of the PTT GC Oil Spill on Intertidal Rocky Shore Macrobenthos in Ao Phrao, Samed Island O-18 Jindarha PREMPRAMOTE, Chayanid MEEPOKA, Sujitra SAMAKRAMAN and Monthon GANMANEE A Study on Some Production Traits and Egg Quality Characteristics O-19 of Lutein Eggs Kanda LOKAEWMANEE and Supat DUANGDEE Effect of Leucaena Silage Levels on Rumen Fermentation and O-20 Nutrients Digestibility in Dairy Steers Giang NGUYEN THIEN TRUONG and Metha WANAPAT Effect of Dried Leucaena Leaf and Leucaena Silage Supplementation O-21 on Rumen Ecology, Rumen Fermentation in Swamp Buffalo Kampanat PHESATCHA and Metha WANAPAT Supplementation of Urea on Nitrogen Balance and Microbial Protein Synthesis in Swamp Buffaloes Fed on Cassava Hay and Rice Straw O-22 Thiwakorn AMPAPON, Metha WANAPAT and Kampanat PHESATCHA Modeling the Microwave Heat Distribution of Banana at Different O-24 Ripening Stage Wisara THUTO and Kittichai BANJONG Preparation, Optimization and Characterization of Carboxymethyl O-25 Cellulose from Rice Straw Using Microwave Heating Noppadol PANCHAN and Chalida NIAMNUY O-26 Optimization of Process Parameters on the Production of Bacterial Cellulose from Rice Rinsing Waste Water (nata-de-leri) by Acetobacter xylinum Alwani HAMAD, Giswantara and Endar PUSPAWININGTYAS O-28 Sensitivity Detection of Phytophthora spp. Causing Para-Rubber Leaf Fall Disease to Some Systemic Fungicides Pornprapa KONGTRAGOUL and Panot VIRIYAEKKUL
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Assessment of the Antagonistic Activity of Trichoderma spp. from Five Different Habitats on Plant Pathogenic Fungi Suriyasit SOMNUEK, Pornprapa KONGTRAGOUL and Tanimnun JAENAKSORN Colonization of Plant Root and Punctured Surface Tissue by NonPathogenic and Pathogenic Fusarium oxysporum Titi THONGKAMNGAM and Tanimnun JAENAKSORN Integrated Management of Yellow Vein Mosaic Disease in Okra C. P. KHARE, A. KOTESTHANE, D. SHARMA, A. DIXIT, and J. SINGH In Vitro Production of Cell Wall Degrading Enzymes by Pythium Species Isolated from Asymptomatic and Symptomatic Lettuce Root Chulalak TALUBNAK, Nonglak PARINTHAWONG and Tanimnun JAENAKSORN Gene Expression Analysis in Lettuce (Lactuca sativa L.) Treated with Trichoderma spp. Malatee PRADUBYAT, Nonglak PARINTHAWONG and Tanimnun JAENAKSORN Assessment of Viability and Efficacy of Fusarium oxysporum (F221-B) as BCA and PGPF During Long Term Preservation Titi THONGKAMNGAM and Tanimnun JAENAKSORN Comparison of Trichoderma Population in the Re-Circulating Nutrient Solution With and Without Supporting Material Kanet JAIKENGKAJ, Tanimnun JAENAKSORN and Prommart KOOHAKAN Morphological Identification of Trichoderma species from Different Habitats Suriyasit SOMNUEK, Pornprapa KONGTRAGOUL and Tanimnun JAENAKSORN Resistant in Rice Cultivars against Sheath Blight Disease under Artificial Inoculated Conditions P.K. TIWARI, C.P. KHARE and A.S. KOTASTHANE
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Page Poster Full Papers Effect of Naphthalene Acetic Acid and Thidiazuron on Phytotoxicity of Phenanthrene and Fluorene to Waxy Corn P-1 Waraporn CHOUYCHAI, Jaturaporn DONRODPRI and Khanitta SOMTRATOON Effect of Fertilizers on Physiological Traits Relate to Yield of Napier Pak Chong 1 Grass P-2 Nittaya PHAKAMAS, Anupong MAKLAI, Tochchakorn PERMHIRUN, Kittiya SERTSUNGNONE and Nawarat JAMPATHONG Effect of Nanoparticles on the Relationship between Crop Growth P-3 Rate and Yield of Chainat 1 Rice (Oryza sativa L.) Sutichai SAMART, Nittaya PHAKAMAS and Sutee CHUTIPAIJIT Antioxidant Determination of Nang Dam Upland Rice Bran Oil by DPPH Assay P-4 Vanapron SAE-ANG, Chitti TAWAI, Raumjit NOKKOUL and Duangkamol RUEN-NGAM Effect of Different Media and Concentrations of Growth Regulator on Callus Induction and Growing Suspension Cell Culture of San-PahP-5 Tawng 1(Oryza sativa L.) Ranyikar PORAHA, Anurug POEAIM, Saengthong PONGJAROENKIT and Pradit PONGTHONGKAM Elite Thein Corn Inbred Lines Utilized to be Synthetic Variety P-6 Kitti BOONLERTNIRUN, Suchada BOONLERTNIRUN and Choosak JOMPUK Hypoxic Responses of 6 Commercial Waxy Corn Varieties P-7 Suchada BOONLERTNIRUN, Raweewun SUVANNASARA and Kitti BOONLERTNIRUN Development of Chitosan-Based Silver Nanoparticles Coating and P-8 Study of Its Effect on Litchi Stored at Ambient Temperature Warin PIMPA and Chakkrit PIMPA Green Synthesis of Silver Nanoparticles Loaded onto Activated P-9 Carbon Using Banana Peel Extract for Environment Applications Warin PIMPA and Chakkrit PIMPA Bioactive Compounds and Antioxidant Activities in Flesh, Leaves and Endocarp of Carissa carandas Compared to Grape Seed Extract Products P-10 Surasak SAJJABUT, Wachiraporn PEWLONG, Sirilak CHOOKAEW, Jaruratana EAMSIRI and Panchalee PRAKHONGSIL Gamma Radiation Effects on Anthocyanins, Vitamin C Content and Antioxidant Activity of Carissa carandas P-11 Wachiraporn PEWLONG, Surasak SAJJABUT, Jaruratana EAMSIRI, Sirilak CHOOKAEW and Panchalee PRAKHONGSIL Influence of Electron Beam Irradiation on Hygienic Quality and Antioxidant Activities of Ground Sea Holly (Acanthus ebracteatus) P-12 Jaruratana EAMSIRI, Surasak SAJJABUT, Wachiraporn PEWLONG and Sirilak CHOOKAEW
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Evaluation of Antioxidant Activities of Essential Oils from Peppermint and Ginger Natthakiti PHURUEN, Chamroon LAOSINWATTANA and Montinee TEERARAK Antioxidant Activity and Total Phenolic Contents of Tagetes erecta L. Leaves Extracts Pattharin WICHITTRAKARN, Montinee TEERARAK and Chamroon LAOSINWATTANA Allelopathic Effect of Fresh and Dried Leaves Aqueous Extracts of Marachra capitata L. and the Action of Allelochemicals in Different Soil Types Phawinee KAMSAN, Pattharin WICHITTRAKARN, Montinee TEERARAK, and Chamroon LAOSINWATTANA Effects of Natural Herbicide from Piper betle Linn on Seed Germination, Imbibition and α- Amylase Activity of Amaranthus gracilis Desf. Pariyaporn NETSAWANG, Pattharin WICHITTRAKARN, Montinee TEERARAK, and Chamroon LAOSINWATTANA Effect of Different Temperatures on Carotenoid Content and Antioxidant Activity in ‘Khak Dam’ Papaya Kanthee SIRIVEJABANDHU and Lampan KHURNPOON Nitric Oxide Improves Cowpea (Vigna unguiculata [L.] Walp.) Growth under Lead Stress Omid SADEGHIPOUR Nutrient Dynamics in an Aquaponic System Somsak MANEEPONG The Effects of Calcium Silicate on Density of Trichomes in Field Corn Pongsakorn NITMEE, Pornpairin RUNGCHAROENTHONG, Suphachai AMKHA and Thongchai MALA Utilization of Zeolite and Isolite as Soil Conditioner to Improve Sandy Soil Nukoon TAWINTEUNG Leaf Flushing Inhibition by Application of High Phosphorus and Potassium Fertilizers for Off-season Mango Production Kannikar KAEWSONGSANG, Apisak BAOLEE and Ravie SETHPAKDEE Sand-Size Distributions of Soils on the So-Called Aeolian Sand Splay Landform in the Lower Mun-Chi Basin, Northeast Thailand Pornthiwa KANYAWONGHA and Anongnat SRIPRACHOTE Parent Material Affecting Phosphorus Distributions in Acid Sulfate Soils of Thailand Pornthiwa KANYAWONGHA, Nutcharee BOONPLANG and Anongnat SRIPRACHOTE Screening of Ametryn Resistant Bacteria from Sugarcane Cultivation Soils Pattrarat TEAMKAO, Nipaporn ROENGANAN and Siratee PONGPOUN
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Effect of Organic Fertilizers on Soil Phosphorus Sorption Capacity Sukunya YAMPRACHA and Parichart WATHAKIATTIKUL A Relationship between Loss on Ignition and Carbon Concentration Determined by Wet Digestion to Assess C/N Ratio of Plant Materials and Organic Fertilizers Nukoon TAWINTEUNG Relationship between Total Calcium and Pectin in Asparagus (Asparagus officinalis L.) Cell Wall Nutcharee BOONPLANG Antibacterial Activity of Roselle (Hibicus sabdariffa Linn.) Flower Extract Napaporn KONGKARN, Pussadee TANGWATCHARIN, Montinee TEERARAK, Kanokporn CHANGSAWAKE and Komkhae PILASOMBUT In vitro Antagonistism of Talaromyces flavus and Neosartorya pseudoficheri Against Anthracnose Disease on Coffee Mayamor SOYTONG and Supattra POEAIM Identification of Blast Resistance Gene in Yang Mawng Variety of Thai Indigenous Rice Siriporn PRAMRIT and Nonglak PARINTHAWONG Acaricidal Activity of Paracress (Acmella oleracea (L.) R.K. Jansen) Extract on the Mushroom Mite (Dolichocybe indica Mahunka) Jarongsak PUMNUAN, Jakkrapong AREEWONG, Pornprapa KONGTRAGOUL and Ammorn INSUNG A Degree-Day Simulation Model for the Population Dynamics of the Brown Planthopper, Nilaparvata lugens Stal. (Homoptera: Delphacidae) Wirote KHLIBSUWAN, Yupa HANBOONSONG and Krirk PANNANGPETCH Preliminary Study on Used Microalga (Nostoc commune) as a Protein Supplement in Dry-Wet Mixtures Feed for Juveniles Snakehead (Channa striata) Suneerat RUANGSOMBOON Protein and Some Other Constituents of Sucker Catfish’s Fish Meal Kalkullanutch PATRARASRIPONG and Kanok LERTPANICH Genetic Diversity of Seagrasses across the Eastcoast of Thailand Based on Sequence-Related Amplified Polymorphism (SRAP) Technique Pattama SRINAMNGOEN and Kanok-on DUANGPAKDEE Aquatic Polyculture Farm in Bangsaothong District, Samutprakarn Province, Thailand Nipon JITTAMNAN1 and Panneepa SIVAPIRUNTHEP Microbiological Safety of Ready-to-Eat Semi-Dried Nham, an Innovation of Thai Fermented Meat Product Thanapa CHETAWAN, Komkhae PILASOMBUT and Supaluk SORAPUKDEE Carcass Traits Comparison between Duroc and Commercial Crossbred Pigs Numfon TAJASRI, Chanporn CHAOSAP, Ronachai SITTHIGRIPONG and Rutcharin LIMSUPAVANICH
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Optimization of Culture Conditions of Bacteria Isolated from Buffalo Rumen for Cellulase and Xylanase Production Kanya JIRAJAROENRAT, Jutatip CHALERMWANICHWONG, Tipaporn NGAMSANGA and Kanokrat SRIKIJKASEMWAT Consumption of Energy, Water and Consumables in Pon-Yang-Kham Beef Cutting Process Chalermsak SAKDAPECHSIRI, Thierry TRAN, Matana OSOTHONGS and Kanya JIRAJAROENRAT Changes in Physicochemical, Microbiological and Sensorial Quality of Jerky Processed From Spent Laying Hen Meat during Storage Chanpen UESAKULRUNGRUENG, Supaluk SORAPUKDEE and Komkhae PILASOMBUT Screening of Lactic Acid Bacteria from Traditional Fermented Meat Products Jiraroj NITHISANTAWAKHUPT, Pussadee TANGWATCHARIN and Kan SUKSUPATH Screening and Identification of Lactic Acid Bacteria with Antimicrobial Activity to Mastitis Pathogen in Dairy cows; Staphylococcus aureus Khakhanang RATANANIKOM, NantiyaSUWANPANYA, Yupaporn KHONNALAO and Sarayut SRIROD Comparison of Stomoxys calcitrans (Diptera: Muscidae) Attacked and Behavioral Responses on Genetic Blood Differences of HolsteinFriesian Dairy Cattle in Thailand Ubon TANGKAWANIT, Varangrat SENASING, Wirote KHLIBSUWAN, and Tassanee JAMJANYA Opinion Towards Agricultural Further Study of Student in Nongchok District, Bangkok Metropolis Tippawan LIMUNGGURA and Panya MANKEB Using Augmented Reality (AR) Technology for the Promotion of Agricultural Products: A Case Study of Bang Krathum Processed Banana Product Nutthakorn SONGKRAM Factors Affecting Consumer Purchasing Behavior of Toxic-Free Vegetables in Muang District, Samutprakarn Province Panya MANKEB, Pratompong MATONG, Suneeporn SUWANMANEEPONG and Prapaporn CHULILUNG Financial Cost and Benefit Analysis of Commercial Orchid Farming in Krathumban District, Samutsakorn Province, Thailand Panya MANKEB, Chalida LERTKRASEMPON, Tippawan LIMUNGGURA and Prapaporn CHULILUNG Enhance Environment of a Historic Site, Ancient Benhama Maharat Building, For Future Public Use Warong NAIVINIT, Parkpoom SUEBNUKARN, Rukkeit SANPRASERT and Taweesak WIYACHAI Biodiesel Synthesis from Used Frying Oil with the Modified Catalyst from Waste Anadara granosa Shells Sasiwimol WOOTTHIKANOKKHAN, Buppha THAMNIYOM and Supamas CHUANOI
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Rationale
The global population is expected to increase from 7 billion today to 9 or 10 billion people by 2050. This situation puts increasing pressures on agriculture to produce much more food, as well as non-food crops. Agricultural practices are currently unsustainable and generate many issues that must be addressed, including over-use of pesticides and fertilizers, land use change, loss of biodiversity, soil erosion, water management for irrigation, management and utilization of crop residues and waste, increasing yields without increasing agricultural surfaces, and so on. Sustainable agriculture which defined as "an integrated system of plant and animal production practices having a site-specific application that will last over the long term" has been accepted worldwide as a significant trend for agricultural practice. This issue offers various positive aspect, for example, satisfy human food, enhance environmental quality and the natural resource base upon which the agricultural economy depends, make the most efficient use of non-renewable resources and on-farm resources and integrate where appropriate, natural biological cycles and controls, sustain the economic viability of farm operations, enhance the quality of life for farmers and society as a whole. However, knowledge gap still remains in some area. Therefore, research is urgently needed to make agricultural practices more sustainable in order to reduce environmental impacts to all production systems, including crops, livestock, fishery and forests. As above mentioned, in this event 2nd International Symposium on Agricultural Technology is organized with aim to promote the exchange of ideas and knowledge to solve current and future problems related to sustainability of agriculture. We will focus on the topic “Global Agriculture Trends for Sustainability” in which consisted of various sessions. As for plenary session, more detailed on “Global Agriculture Trends for Sustainability” and related topics will be focused. Many of oral sessions as well as poster sessions are also organized. We hope that this event will promote the exchange of ideas and knowledge to solve current and future problems related to sustainability of agriculture as well as future and practical prospects concerning self-reliance, moderation, reasonableness, and self immunity for agriculture, and other related issues in different countries.
The 2nd
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(Assistant Professor Dr.Monthon Ganmanee) Chairman Organizing Committee International Symposium on Agricultural Technology (ISAT2015)
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Report Address On behalf of the Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), I would like to extend my warm welcome and appreciation for your participation at the 2nd International Symposium on Agricultural Technology (ISAT2015). It is the goal of Faculty of Agricultural Technology to be recognized in the region as one of the top fifth Agricultural Science and Technology universities. To fulfill this goal, ISAT was first originated in 2010 hosting by the Faculty of Agricultural Technology, KMITL, jointly with Tokai University, Japan under the theme of the conference “Sufficiency Agriculture”. It was a dual conference with the 16th Asian Agricultural Symposium. For this year, the 2nd International Symposium on Agricultural Technology was organized with the main concept of “Global Agriculture Trends for Sustainability”. This symposium will offer a dynamic platform for scientific research update and knowledge sharing on Agricultural Technology among students and researchers both from local and abroad. This symposium composed a total of 150 participants including honorary guest speakers, faculty members, researchers and students from various universities and institutes in Thailand and from abroad. There are 63 posters and 40 oral research presentations submitted. I hope this delightful opportunity will create a dynamic research network and bring out more collaborated activities between KMITL and other universities or institutes both at national and international levels.
Assoc.Prof. Sakchai Choochote Dean, Faculty of Agricultural Technology King Mongkut’s Institute of Technology Ladkrabang
July 1-3, 2015
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Welcoming & Opening Remarks As global population is expected to increase dramatically, world scarcity of foods and agricultural raw material supplies are well expected. Issues in global sustainability, due to agricultural raw material production to serve this growing population, have been long recognized. But new problems arise and keep changing. It’s necessary for agricultural technologists and scientists to keep their eyes on those changing world issues. On behalf of King Mongkut’s Institute of Technology Ladkrabang, it is an honor and my great pleasure to welcome all guests and participants to the 2nd International Symposium on Agricultural Technology (ISAT2015). The goal of this symposium is to emphasize how importance for the awareness in continuing and immerging issues of world sustainability. I certainly believe that this conference will provide great motivation for all researchers and graduate students to produce more quality research work. In addition, all activities at the meeting will create an excellent floor for research exchange and collaborations among all institutions, nationally and internationally. With the goal of this conference in mind, I believe it will lead to an advance in agricultural technology not only in Thailand, but also in our international research network. I hope all participants find the poster and oral research presentation, special topics from distinguished guest speakers as well as all conference extra activities delightful and useful for their careers.
Prof.Dr. Suchatvee Suwansawat President King Mongkut’s Institute of Technology Ladkrabang
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Short Biography of Keynote and Invited Speakers Keynote Speakers "Status of global agriculture and food security situation, and the future trend" Dr. Hiroyuki KONUMA Assistant Director-General and FAO Regional Representative for Asia and the Pacific FAO Regional Office for Asia and the Pacific Phra Athit Road, Phranakorn, Bangkok Thailand Telephone: +66-2-6974000 Fax: +66-2-6974445 E-mail: FAO-RAP@fao.org
Dr. Hiroyuki Konuma, a Japanese national, he has been with FAO for 33 years. He was appointed as the FAO Assistant Director -General and the Regional Representative for Asia and the Pacific in March 2010. During this tenure as the Regional Representative of FAO for Asia and the Pacific, he chaired the Regional UN Thematic Working Group on Poverty and Hunger and coordinated the launch of Zero Hunger Initiative for Asia and the Pacific and the Regional Save Food Campaign in 2013. He promoted the role of FAO in the region and coordinated FAO’s experts and programs in the field of agriculture, food security, nutrition, rural development and with an aim to promote regional coordination and collaboration, and promote food and nutrition security towards achievement of Millennium Development Goals and Zero Hunger targets.
July 1-3, 2015
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"Optimizing the use of feed resources for healthy rumen, increased production and mitigating methane" Professor Dr. Metha WANAPAT Director of the Tropical Feed Resources Research and Development Center (TROFREC), Khon Kaen University, Thailand Department of Animal Science, Faculty of Agriculture, Khan Kaen University, Thailand Email: metha@kku.ac.th
Prof. Dr. Metha Wanapat, has long-term experience for more than 30 years in teaching, research engagement in ruminant nutrition and feeding systems in the Tropics. His research work encompasses a wide range of aspects of feed resources, conventional and non-conventional feed resources both on farms and at industrial practicalities. His current research work have been on establishment of on farm feed resources using food-feed-system approach, strategies of supplementation and manipulation of rumen using dietary means to improve rumen fermentation efficiency, methane mitigation and enhance ruminant productivity.
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"Food Sustainability - Emerging Challenges In the ASEAN Region (Reality versus the Myth) "
Associate Professor Dr. Rajeev BHAT Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang , Malaysia rajeevbhat1304@gmail.com rajeevbhat@usm.my Tel: +60-4653-5212 (off)/
Dr. Rajeev Bhat is presently working as Associate Professor of Food Technology at Univeristi Sains Malaysia. Attaining self-sufficiency, ensuring food sustainability and application of novel techniques for preservation of food have been the major theme of his research since past fifteen years. Dr. Bhat has authored more than 150 research publications (in ISI journal and as book chapters) and has four books to his credit. He is a recipient of several prestigious international awards, such as: ‘Excellence Recognition Award’ (Association of Agricultural Technology in Southeast Asia), ‘Prosper.NetScopus Award in Sustainable Development’ (UNU), ‘IUFoST/Fi Young Scientist Excellence Award’ (International Union of Food Science and Technology, Canada), Global Achievers Award (conferred by IIOH, India), etc. As an active researcher he has worked under various capacities in India, South Korea, Malaysia and Germany.
July 1-3, 2015
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Invited Speakers "Can feedstuff processing improve digestibility of amino acids for non-ruminants?" Professor Dr. Julian WISEMAN Division of Animal Science, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leicestershire United Kingdom Phone: 0115 951 6054 Fax: 0115 951 6099 Email: julian.wiseman@nottingham.ac.uk
Prof. Dr. Julian Wiseman is a head of the Division and Senior Editor of the Journal of Agricultural Science, Cambridge. He has published 81 refereed papers and 37 review articles and has written three books. Julian's research interests fall into two main areas: non-ruminant animal nutrition and product quality. Current research areas include: nutritional value of co-products from bioethanol production, home-grown legumes in diets for non-ruminants; digestibility, performance and carcass quality, evaluating exogenous enzymes in pig nutrition and muscle growth in broilers.
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"Opportunity of AEC meat production for Halal food market" Professor Dr. Abdul Salam BABJI School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia (UKM) Selangor, Malaysia Phone: +603-89215988 Fax: +603-89213232j Email: daging@ukm.my
Prof. Dr. Abdul Salam Babji is currently working as a professor in National University of Malaysia; he is a senior ex-pert with more than 30 years of experience in the field of Meat Science and Meat Technology. One of his major areas of R&D is focused on fish, meat and poultry product development with emphasis on healthy meat products. He has been actively seeking ways into replacing synthetic food additives in meat products with phytochemical extracts of herbs and spices. These extracts are tested for their effectiveness as antioxidant and antimicrobial material, also as a tool to extend the quality and shelf life of meat products.
July 1-3, 2015
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"Applications of molecular diagnostic methods in the management of fungicide resistance"
Prof.Dr. Hideo ISHII School of Agricultural Regional Vitalization Kibi International University, Japan TEL: +81-799-42-4722 FAX: +81-799-42-4701 E-mail: h-ishii@kiui.ac.jp or hi481204@yahoo.co.jp
Honorary Researcher at National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki, Japan. His major research fields include Fungicide resistance of pathogens, Pathological specialization of fungi, Disease resistance in plants, Induction and mechanism of systemic acquired resistance in plants, Disease control by natural products.
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"Rapid generation technology for food and forage legumes - harnessing in vitro technologies for accelerating genetic gain"
Assoc.Prof.Dr. Janine CROSER Centre for Plant Genetics and Breeding The University of Western Australia , Australia Phone +61 8 6488 7951 Fax +61 8 6488 1140 Email janine.croser@uwa.edu.au
Dr Janine Croser is a Project leader within the Centre for Plant Genetics and Breeding (PGB), School of Plant Biology at The University of Western Australia. Her main research interest is innovative plant breeding technologies to accelerate genetic gain, working with pasture and grain legumes, oilseeds and specialty crops. As a chief investigator, her current projects include development of accelerated single seed descent technology for grain legumes and mining of wild Cicer relatives for identification of valuable traits for chickpea improvement. Dr Croser began her career in Cell Biology at The Victorian Department of Agriculture, undertaking research for Monsanto and VanderHave Research Netherlands, before completing her PhD with The University of Melbourne. She undertook postdoctoral training at the University of Saskatchewan before joining The University of Western Australia in 2002. Her group currently consists of two postdoctoral researchers and four technical staff.
July 1-3, 2015
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"Biogas reduces the carbon footprint of cassava starch: A comparative assessment with fuel oil"
Dr.Thierry TRAN Joint Research Unit QUALISUD, CIRAD, France, and Cassava and Starch Technology Research Unit (CSTRU) Faculty of Agro-Industry, Kasetsart University, Thailand Tel: +66-86 822 0362 fax: +66-2 940 5634 Email:Thierry.tran@cirad.fr
Dr. Thierry Tran is a researcher at CIRAD, the French International Research Centre for Agricultural Development, Montpellier, France. His work focuses on food processing & environmental footprints, using mainly Life Cycle Assessment (LCA), as part of the activities of the QUALISUD research unit. He is currently based at the Cassava and Starch Technology Research Unit (CSTRU), for a collaboration between CIRAD and Kasetsart University, Bangkok, Thailand. He is also involved with development projects for increased processing of roots and tubers, such as cassava, in developing countries in Africa and South America, as well as Southeast Asia.
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ISAT2015 Program at A-One the Royal Cruise Pattaya Hotel, Chonburi, Thailand
Day 1: Wednesday, 1 July 2015 08:00-09:00
Registration and Reception Venue: In front of Suphannahong Room, 3rd Floor, Royal Wing
Opening ceremony
09:00-09:30
Venue: Suphannahong Room
09:30-10:15
Keynote address I : Dr. Hiroyuki KONUMA
10:15-10:30
Group Photo and Coffee break
10:30-11:00
Keynote address II : Prof. Metha WANAPAT
11:00-11:30
Keynote address III : Assoc.Prof. Rajeev BHAT
11:30-12:00 12:00-13:00
Poster viewing (odd number) Venue: In front of Panerai 1 Room,3rd Floor, Royal Wing Buffet Lunch Venue: Private Room, 2nd Floor, Royal Wing
Oral Presentation I Session 1: Plant and Soil for Sustainability Venue: Suphannahong Room
Session 2: Animal Production and Fisheries for Sustainability Venue: Panerai 1 Room
13:00-14:15
O-1 to O-5
13:00-14:00
O-15-O-18
14:30-15:15
O-6 to O-9
14:00-15:00
O-19-O-22
15:15-15:30 Session 1: Plant and Soil for Sustainability Venue: Suphannahong Room 15:30-16:45 16:45-17:30 18:00-20:00
July 1-3, 2015
O-10 to O-14
Coffee break Session 3: Process and Environmental Technologies for Sustainability Venue: Panerai 1 Room 15:30-16:45
O-23 to O-27
Poster Presentation (Even Number)
Venue: In front of Panerai 1 Room,3rd Floor, Royal Wing Welcome Party (Buffet) Venue: Suphannahong Room
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Day 2 : Thursday, 2 July 2015 Invited Session Venue: Suphannahong Room 09:00-09:30
Invited speaker I : Prof. Julian WISEMAN
09:30-10:00
Invited speaker II : Assoc.Prof. Janine CROSER
10:00-10:15
Coffee break
10:15-10:45
Invited speaker III : Prof. Abdul Salam BABJI
10:45-11:15
Invited speaker IV : Prof. Hideo ISHII
Oral Presentation II Session 4: Crop Protection for Sustainability Venue: Suphannahong Room 11:15-12:00
O-28 to O-30
12:00-13:00
Buffet Lunch Venue: Private Room, 2nd Floor, Royal Wing Session 4: Crop Protection for Sustainability Venue: Suphannahong Room
13:00-14:45
O-31 to O-37
14:45-15:00
Coffee break Session 4: Crop Protection for Sustainability Venue: Suphannahong Room
15:00-16:15
O-38 to O-42
Closing Ceremony 16:15-16:45
Invited speaker V : Dr. Thierry TRAN
16:45-17:15
ISAT 2015 Awards for Oral Presentation and Poster Presentation Closing speech
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Day 3 Friday, 3 July 2015
"Excursion" at Suphattra Land, Rayong Province
09.00-10.00 10.00-12.00
Journey to Suphattra Land, fruit garden, Klaeng, Rayong Province (Pick-up point at A-One the Royal Cruise Pattaya Hotel) Visit fruit garden and taste the Exotic Fruits: rambutan, durian, mangosteen, lansat, dragon fruit, jackfruit, mango, coconut‌etc.
12.00-13.00
Lunch
13.00-14.00
Visit grape garden, orchid garden, salad hydroponics and honey production
14.00-15.00
Back to A-One the Royal Cruise Pattaya Hotel
15.00-16.00
Journey to Bangkok (Drop point at Suvarnabhumi Airport)
July 1-3, 2015
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Scientific Program
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Scientific Program
Wednesday, 1 July 2015 Plenary Session Venue: Suphannahong Room 08.00-09.00 Registration and Reception Opening ceremony
09.00-09.30
09.30-10.15
10.15-10.30
10.30-11.00
11.00-11.30
VDO Presentation: Faculty of Agricultural Technology, KMITL Report address Assoc. Prof. Sakchai CHOOCHOTE Dean, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang Welcoming and Opening Speech Prof. Dr. Suchatvee SUWANSAWAT President, King Mongkut's Institute of Technology Ladkrabang Keynote address Status of Global Agriculture and Food Security Situation, and the Future Trend Dr. Hiroyuki KONUMA Assistant Director –General and FAO Regional Representative for Asia and the Pacific Coffee break Keynote address Optimizing the Use of Feed Resources for Healthy Rumen, Increased Production and Mitigation Methane Prof. Dr. Metha WANAPAT Director of the Tropical Feed Resources Research and Development Center (TROFREC), Khon Kaen University, Thailand Keynote address Food Sustainability-Emerging Challenges in the ASEAN Region (Reality Versus the Myth) Assoc. Prof. Rajeev BHAT Food Technology Division, School of Industrial Technology, Universiti Sains Malysia (USM), Malaysia
11.30-12.00
POSTER VIEWING (Odd Number)
12.00-13.00
Lunch
July 1-3, 2015
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Wednesday, 1 July 2015 Session 1: Plant and Soil for Sustainability Venue: Suphannahong Room Chairperson: Prof. Dr. Singam Laxmana SWAMY Secretary: Asst. Prof. Dr. Teerawat SARUTAYOPHAT Adapting the CSM-Canegro-Sugarcane Model to Simulate Napier Grass 13.00-13.15 O-1 in Chiang Mai Province, Thailand Tupthai NORSUWAN, Attachai JINTRAWET and Carsten MARONH
Effect of Azolla (Azolla spp.) on Growth and Yield of Pathumthani 1 Rice (Oryza sativa L.)
13.15-13.30
O-2
13.30-13.45
O-3 Kashmir, India
Teerawat SARUTAYOPHAT, Nattawan BUSSABA and Phanupong PHONCHAROEN
Experiences with Direct Seeded Rice in Jammu and Vijay BHARTI, Ashok KUMAR RAINA and Anuradha SAHA
Allelopathic Effects of Pisonia grandis R.Br. on the Germination and Growth
13.45-14.00
O-4 of Echinochloa crus-galli (L.) Beauv.
Sudteerak SAIPLUEMCHIT and Chamroon LAOSINWATTANA
14.00-14.15
O-5
How Far Teak Plantations Can Contribute for Climate Change Mitigation and Landscape Restoration in Tropics: A Case Study from Central India Singam Laxmana SWAMY and Alka MISHRA
Chairperson: Assoc. Prof. Dr. Janine CROSER Secretary: Asst. Prof. Dr. Kanjana SAETIEW Antioxidant Properties of Flower Extract of Etlingera elatior (Jack) 14.15-14.30 O-6 R.M. Smith Kanokporn CHANGSAWAKE, Komkhae PILASOMBUT, Chamroon LAOSINWATTANA and Montinee TERARAK
Effect of Storage Conditions on the Vase Life of
14.30-14.45
O-7 Homalomena Philodendron and Monstera
Nipaporn YONSAWAD and Montinee TEERARAK
Micro-propagation of Dalbergia cochinchinesis Pierre
14.45-15.00
O-8 Wannasiri WANNARAT, Panida WONGWEAN, Sirinaree SUPANSOMPORN, Warinee KITPRECHAWANICH and Yupa PANKAEW
15.00-15.15
O-9
Standardization of Drip Irrigation and Fertigation for Improving Physiology, Yield and Quality Parameters of Mango var. Alphonso under Ultra-High Density Planting K. PRAKASH, R.M. VIJAYAKUMAR and S.D. SUNDHAR SINGH
15.15-15.30 Coffee break
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Session 1: Plant and Soil for Sustainability Venue: Suphannahong Room Chairperson: Assoc. Prof. Dr. Somsak MANEEPONG Secretary: Asst. Prof. Dr. Nukoon TAWINTEUNG Study of Using Coop Fertilizer Ameliorant on Greenhouse Gas 15.30-15.45 O-10 Emission in Some Rubber Estates Agroecosystems in Peatland Hariyadi JAMIN PRIYO MINARJO, Dedi NURSYAMSI and Adi PRADIPTA
15.45-16.00
Plant Nutrient Management Strategies Enhanced Growth, Yield Traits and Mitigated Leaf Reddening in O-11 Cotton (Gossypium hirsutum L.) Allahwadhayo GANDAHI, Khalillulah PANHWAR and Rabail GANDAHI
16.00-16.15
Utilization of Calcium Silicate Application on Pepper O-12 Seedling Production
16.15-16.30
Behavior of Nutrient Uptake by Pummelo Growing on O-13 Salt Marsh Soil
16.30-16.45
O-14 Properties, Yield and Quality of Seed Cane
Eakkarin SUKKAEW, Suphachai AMKHA, Thongchai MALA and Pornpairin RUNGCHAROENTHONG
Hien HUU NGUYEN, Somsak MANEEPONG and Potjamarn SURANINPONG
Eco-Friendly Utilization of TDE and Fertilizers on Soil J. REVATHI and M. BASKAR
16.45-17.30 POSTER VIEWING (Even Number) 18.00-20.00
July 1-3, 2015
Welcome Party (Buffet) Suphannahong Room
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Session 2: Animal Production and Fisheries for Sustainability Venue: Panerai 1 Room Chairperson: Dr. Isao TSUTSUI Secretary: Asst. Prof. Dr. Monthon GANMANEE A Co-Culture System of Giant Tiger Prawn and Unexploited Seaweed and Snail 13.00-13.15 O-15 Isao TSUTSUI, Dusit AUE-UMNEOY, Jaruwan SONGPHAT KAEW, Piyarat PINPHOO, Chonlada MEEANAN, Sirimas KLOMKLING, Halethichanok SUKCHAI, Monthon GANMANEE and Kaoru HAMANO
Investigating the Safety of Potential Probiotic
13.15-13.30
O-16 Candidates Isolated from the GI Tract of Tilapia in vitro Rungtawan YOMLA, Daniel MERRIFIELD and Simon DAVIES
13.30-13.45
Studied on Using of Robo-Logger Technique Measuring O-17 In Situ Inside Shell Temperature of Rocky Shore Limpet
13.45-14.00
Impacts of the PTT GC Oil Spill on Intertidal Rocky O-18 Shore Macrobenthos in Ao Phrao, Samed Island
Monthon GANMANEE, Chayanid MEEPOKA, Pokamon KOCHARIN and Gray A. WILLIAMS
Jindarha PREMPRAMOTE, Chayanid MEEPOKA, Sujitra SAMAKRAMAN and Monthon GANMANEE
Chairperson: Prof. Dr. Julian WISEMAN Secretary: Assoc. Prof. Dr. Ronachai SITTHIGRIPONG A Study on Some Production Traits and Egg Quality 14.00-14.15 O-19 Characteristics of Lutein Eggs Kanda LOKAEWMANEE and Supat DUANGDEE
Effect of Leucaena Silage Levels on Rumen
14.15-14.30
O-20 Fermentation and Nutrients Digestibility in Dairy Steers Giang NGUYEN THIEN TRUONG and Metha WANAPAT
14.30-14.45
O-21
Effect of Dried Leucaena Leaf and Leucaena Silage Supplementation on Rumen Ecology, Rumen Fermentation in Swamp Buffalo Kampanat PHESATCHA and Metha WANAPAT
14.45-15.00
O-22
Supplementation of Urea on Nitrogen Balance and Microbial Protein Synthesis in Swamp Buffaloes Fed on Cassava Hay and Rice Straw Thiwakorn AMPAPON, Metha WANAPAT and Kampanat PHESATCHA
15.00-15.30
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Coffee break
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Session 3: Process and Environmental Technologies for Sustainability Venue: Panerai 1 Room Chairperson: Assoc. Prof. Rajeev BHAT Secretary: Asst. Prof. Dr. Lampan KHURNPOON Effects of Drying Process on Characteristics of Gac 15.30-15.45 O-23 Fruit Aril Powder
Warangkana ANGKANANON, Mudtorlep NISOA, Phanphen ATTANARSAKIT, and Visaka ANANTAWAT
15.45-16.00 O-24
Modeling the Microwave Heat Distribution of Banana at Different Ripening Stage Wisara THUTO and Kittichai BANJONG
16.00-16.15 O-25
Preparation, Optimization and Characterization of Carboxymethyl Cellulose from Rice Straw Using Microwave Heating Noppadol PANCHAN and Chalida NIAMNUY
16.15-16.30 O-26
Optimization of Process Parameters on the Production of Bacterial Cellulose from Rice Rinsing Waste Water (nata-de-leri) by Acetobacter xylinum Alwani HAMAD, Giswantara and Endar PUSPAWININGTYAS
16.30-16.45 O-27
Orthogonal Functions for Mathematical Modeling in Problems on Agriculture Technology Mohsen RAZZAGHI
16.45-17.30 POSTER VIEWING (Even Number) 18.00-20.00
July 1-3, 2015
Welcome Party (Buffet) Suphannahong Room
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Thursday, 2 July 2015 Invited Session Venue: Suphannahong Room
Invited speaker Can Feedstuff Processing Improve Digestibility of Amino Acids for Non09.00-09.30 ruminants? Prof. Dr. Julian WISEMAN
Division of Animal Science, School of Biosciences, Nottingham University, United Kingdom
Invited speaker Rapid Generation Technology for Food and Forage Legumes- Harnessing in vitro 09.30-10.00 Technologies for Accelerating Genetic Gain Assoc. Professor Dr. Janine CROSER
Centre for Plant Genetics and Breeding, The University of Western Australia (M080), Australia 10.00-10.15 Coffee break Invited speaker Opportunity of AEC Meat Production for Halal food Market 10.15-10.45 Prof. Dr. Abdul Salam BABJI
School of Chemical Science and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia
Invited speaker Applications of Molecular Diagnostic Methods in the Management of Fungicide Resistance 10.45-11.15 Prof. Dr. Hideo ISHII
School of Agricultural Regional Vitalization Kibi International University, Japan
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Session 4: Crop Protection for Sustainability Venue: Suphannahong Room Chairperson: Prof. Dr. Hideo ISHII Secretary: Dr. Nonglak PARINTHAWONG Sensitivity Detection of Phytophthora spp. Causing Para-Rubber Leaf 11.15-11.30 O-28 Fall Disease to Some Systemic Fungicides Pornprapa KONGTRAGOUL and Panot VIRIYAEKKUL
11.30-11.45
Assessment of the Antagonistic Activity of Trichoderma spp. from Five O-29 Different Habitats on Plant Pathogenic Fungi
11.45-12.00
O-30 Non-Pathogenic and Pathogenic Fusarium oxysporum
Suriyasit SOMNUEK, Pornprapa KONGTRAGOUL and Tanimnun JAENAKSORN
Colonization of Plant Root and Punctured Surface Tissue by Titi THONGKAMNGAM and Tanimnun JAENAKSORN
12.00-13.00 Lunch
July 1-3, 2015
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Session 4: Crop Protection for Sustainability Venue: Suphannahong Room Chairperson: Prof. Dr. C. P. KHARE Secretary: Dr. Nonglak PARINTHAWONG, Dr. Pornprapa KONGTRAGOUL Integrated Management of Yellow Vein Mosaic Disease 13.00-13.15 O-31 in Okra C. P. KHARE, A. KOTESTHANE, D. SHARMA, A. DIXIT, and J. SINGH
13.15-13.30
In Vitro Production of Cell Wall Degrading Enzymes by Pythium Species Isolated from Asymptomatic and O-32 Symptomatic Lettuce Root Chulalak TALUBNAK, Nonglak PARINTHAWONG and Tanimnun JAENAKSORN
13.30-13.45
Gene Expression Analysis in Lettuce (Lactuca sativa L.) O-33 Treated with Trichoderma spp.
13.45-14.00
O-34 Rice var. RD41
14.00-14.15
Rungrat VAREEKET and Kasem SOYTONG Assessment of Viability and Efficacy of Fusarium oxysporum (F221-B) O-35 as BCA and PGPF During Long Term Preservation
Malatee PRADUBYAT, Nonglak PARINTHAWONG and Tanimnun JAENAKSORN
Evaluation of Photosynthesizing Bacteria for the Growth of
Titi THONGKAMNGAM and Tanimnun JAENAKSORN
14.15-14.30
Comparison of Trichoderma Population in the Re-Circulating O-36 Nutrient Solution With and Without Supporting Material
14.30-14.45
Morphological Identification of Trichoderma species O-37 from Different Habitats
Kanet JAIKENGKAJ, Tanimnun JAENAKSORN and Prommart KOOHAKAN
Suriyasit SOMNUEK, Pornprapa KONGTRAGOUL and Tanimnun JAENAKSORN
14.45-15.00 Coffee break
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Session 4: Crop Protection for Sustainability Venue: Suphannahong Room Chairperson: Prof. Dr. P.K. TIWARI Secretary: Asst. Prof. Dr. Ammorn INSUNG Resistant in Rice Cultivars against Sheath Blight 15.00-15.15 O-38 Disease under Artificial Inoculated Conditions P.K. TIWARI, C.P. KHARE and A.S. KOTASTHANE
15.15-15.30
Influence of Weather Parameters on the Incidence of Okra Yellow O-39 Vein Mosaic Virus Disease and Whitefly (Bemisia tabaci) Population D.R. BHENDARKAR, C.P. KHARE and S.L. SWAMY
15.30-15.45
Rhodospirillum spp. from Wastewater O-40 Rungrat VAREEKET and Kasem SOYTONG
15.45-16.00
A Crossbred High Yielding and Insect Resistant Cotton O-41 Variety Having Wider Climatic Adaptability Syed BILAL HUSSAIN, Zulfiqar ALI and Hasnain NAWAZ KHAN
16.00-16.15
The Impact of Climate Variability on Agricultural Pest O-42 Ninio A. RELOX and Sharon Juliet M. ARRUEJO
Closing Ceremonies 16.15-16.45
Invited Article Biogas Reduces the Carbon Footprint of Cassava Starch: A Comparative Assessment with Fuel Oil Dr. Thierry TRAN Researcher, CIRAD, France
16.45-17.15
ISAT 2015 Awards for Oral Presentation ISAT 2015 Awards for Poster Presentation Closing Speech
July 1-3, 2015
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Friday, 3 July 2015
Excursion to Suphattra Land, fruit garden, Klaeng, Rayong Province
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Poster Session: Global Agriculture Trends for Sustainability P-1
Effect of Naphthalene Acetic Acid and Thidiazuron on Phytotoxicity of Phenanthrene and Fluorene to Waxy Corn Waraporn CHOUYCHAI, Jaturaporn DONRODPRI and Khanitta SOMTRATOON
P-2
P-3 P-4
Effect of Fertilizers on Physiological Traits Relate to Yield of Napier Pak Chong 1 Grass
Nittaya PHAKAMAS, Anupong MAKLAI, Tochchakorn PERMHIRUN, Kittiya SERTSUNGNONE and Nawarat JAMPATHONG
Effect of Nanoparticles on the Relationship between Crop Growth Rate and Yield of Chainat 1 Rice (Oryza sativa L.) Sutichai SAMART, Nittaya PHAKAMAS and Sutee CHUTIPAIJIT Antioxidant Determination of Nang Dam Upland Rice Bran Oil by DPPH Assay Vanapron SAE-ANG, Chitti TAWAI, Raumjit NOKKOUL and Duangkamol RUEN-NGAM
P-5
Effect of Different Media and Concentrations of Growth Regulator on Callus Induction and Growing Suspension Cell Culture of San-Pah-Tawng 1(Oryza sativa L.)
P-6
Elite Thein Corn Inbred Lines Utilized to be Synthetic Variety
P-7
Hypoxic Responses of 6 Commercial Waxy Corn Varieties
P-8
Development of Chitosan-Based Silver Nanoparticles Coating and Study of Its Effect on Litchi Stored at Ambient Temperature
Ranyikar PORAHA, Anurug POEAIM, Saengthong PONGJAROENKIT and Pradit PONGTHONGKAM Kitti BOONLERTNIRUN, Suchada BOONLERTNIRUN and Choosak JOMPUK Suchada BOONLERTNIRUN, Raweewun SUVANNASARA and Kitti BOONLERTNIRUN
Warin PIMPA and Chakkrit PIMPA
P-10
Green Synthesis of Silver Nanoparticles Loaded onto Activated Carbon Using Banana Peel Extract for Environment Applications Warin PIMPA and Chakkrit PIMPA Bioactive Compounds and Antioxidant Activities in Flesh, Leaves and Endocarp of Carissa carandas Compared to Grape Seed Extract Products
P-11
Gamma Radiation Effects on Anthocyanins, Vitamin C Content and Antioxidant Activity of Carissa carandas
P-12
Influence of Electron Beam Irradiation on Hygienic Quality and Antioxidant Activities of Ground Sea Holly (Acanthus ebracteatus)
P-9
P-13 P-14 P-15
Surasak SAJJABUT, Wachiraporn PEWLONG, Sirilak CHOOKAEW, Jaruratana EAMSIRI and Panchalee PRAKHONGSIL
Wachiraporn PEWLONG, Surasak SAJJABUT, Jaruratana EAMSIRI, Sirilak CHOOKAEW and Panchalee PRAKHONGSIL
Jaruratana EAMSIRI, Surasak SAJJABUT, Wachiraporn PEWLONG and Sirilak CHOOKAEW
Effect of Alginate Edible Coating on Quality of Fresh-cut ‘Kimju’ Guava A. ONDEE and S. MANURAKCHINAKORN Evaluation of Antioxidant Activities of Essential Oils from Peppermint and Ginger Natthakiti PHURUEN, Chamroon LAOSINWATTANA and Montinee TEERARAK
Improved Efficiency of Anthocyanin Extraction from Roselle Using Acidified Solvent F. NUUDOM and S. MANURAKCHINAKORN
July 1-3, 2015
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P-16
Antioxidant Activity and Total Phenolic Contents of Tagetes erecta L. Leaves Extracts Pattharin WICHITTRAKARN, Montinee TEERARAK and Chamroon LAOSINWATTANA
P-18
Allelopathic Effect of Fresh and Dried Leaves Aqueous Extracts of Marachra capitata L. and the Action of Allelochemicals in Different Soil Types Phawinee KAMSAN, Pattharin WICHITTRAKARN, Montinee TEERARAK and Chamroon LAOSINWATTANA Effects of Natural Herbicide from Piper betle Linn on Seed Germination, Imbibition and α- Amylase Activity of Amaranthus gracilis Desf.
P-19
Effect of Different Temperatures on Carotenoid Content and Antioxidant Activity in ‘Khak Dam’ Papaya
P-17
Pariyaporn NETSAWANG, Pattharin WICHITTRAKARN, Montinee TEERARAK, and Chamroon LAOSINWATTANA
Kanthee SIRIVEJABANDHU and Lampan KHURNPOON
P-20
Nitric Oxide Improves Cowpea (Vigna unguiculata [L.] Walp.) Growth under Lead Stress
P-21
Nutrient Dynamics in an Aquaponic System
P-22 P-23
P-24
P-25
Omid SADEGHIPOUR
Somsak MANEEPONG
The Effects of Calcium Silicate on Density of Trichomes in Field Corn Pongsakorn NITMEE, Pornpairin RUNGCHAROENTHONG, Suphachai AMKHA and Thongchai MALA
Utilization of Zeolite and Isolite as Soil Conditioner to Improve Sandy Soil Nukoon TAWINTEUNG
Dendoremediation Efficiency of Forest Hardwoods to Remove Cadmium Toxicity from Contaminated Soil Syed AMIR MANZOOR, Din MUHAMMAD ZAHID KHAN, Muhammad ZUBAIR, Wasif NOUMAN, Sarwat NAZ MIRZA, Syed BILAL HUSSAIN, Feehan HASSAN and Muhammad IMRAN UMAR
Leaf Flushing Inhibition by Application of High Phosphorus and Potassium Fertilizers for Off-season Mango Production Kannikar KAEWSONGSANG, Apisak BAOLEE and Ravie SETHPAKDEE
P-26
Sand-Size Distributions of Soils on the So-Called Aeolian Sand Splay Landform in the Lower Mun-Chi Basin, Northeast Thailand Pornthiwa KANYAWONGHA and Anongnat SRIPRACHOTE
P-27
Parent Material Affecting Phosphorus Distributions in Acid Sulfate Soils of Thailand Pornthiwa KANYAWONGHA, Nutcharee BOONPLANG and Anongnat SRIPRACHOTE
P-28
Screening of Ametryn Resistant Bacteria from Sugarcane Cultivation Soils
P-29
Effect of Organic Fertilizers on Soil Phosphorus Sorption Capacity
P-30
A Relationship between Loss on Ignition and Carbon Concentration Determined by Wet Digestion to Assess C/N Ratio of Plant Materials and Organic Fertilizers
Pattrarat TEAMKAO, Nipaporn ROENGANAN and Siratee PONGPOUN Sukunya YAMPRACHA and Parichart WATHAKIATTIKUL
Nukoon TAWINTEUNG
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P-31
Relationship between Total Calcium and Pectin in Asparagus (Asparagus officinalis L.) Cell Wall Nutcharee BOONPLANG
P-32
Antibacterial Activity of Roselle (Hibicus sabdariffa Linn.) Flower Extract Napaporn KONGKARN, Pussadee TANGWATCHARIN, Montinee TEERARAK, Kanokporn CHANGSAWAKE and Komkhae PILASOMBUT
P-33
Isolation of ACC-Deaminase-Producing Endophytic Bacteria from Rice (Oryza sativa)
P-34
In vitro Antagonistism of Talaromyces flavus and Neosartorya pseudoficheri Against Anthracnose Disease on Coffee
Chokchai Kittiwongwattana
Mayamor SOYTONG and Supattra POEAIM
P-35
Identification of Blast Resistance Gene in Yang Mawng Variety of Thai Indigenous Rice Siriporn PRAMRIT and Nonglak PARINTHAWONG
P-36
Acaricidal Activity of Paracress (Acmella oleracea (L.) R.K. Jansen) Extract on the Mushroom Mite (Dolichocybe indica Mahunka)
P-37
A Degree-Day Simulation Model for the Population Dynamics of the Brown Planthopper, Nilaparvata lugens Stal. (Homoptera: Delphacidae)
Jarongsak PUMNUAN, Jakkrapong AREEWONG, Pornprapa KONGTRAGOUL and Ammorn INSUNG
Wirote KHLIBSUWAN, Yupa HANBOONSONG and Krirk PANNANGPETCH
P-38
Preliminary Study on Used Microalga (Nostoc commune) as a Protein Supplement in Dry-Wet Mixtures Feed for Juveniles Snakehead (Channa striata) Suneerat RUANGSOMBOON
P-39
Assessing Impact of Artificial Reef on Local Fisherman Household Income Using Propensity Score Matching in Lang Suan District, Chumphon Province, Thailand
P-40
Protein and Some Other Constituents of Sucker Catfish’s Fish Meal
P-41
Protein Isolation from Bigeye Snapper (Priacanthus tayenus) Head By-Product Using pH-Shift Method
Rapeepan KANTAVICHAI, Monthon GANMANEE, Thamrong MEKHORA, Maytapon PORNRATANACHOTSAKUL and Ariya THONGSAMUI Kalkullanutch PATRARASRIPONG and Kanok LERTPANICH
Worawan PANPIPAT and Manat CHAIJAN
P-42
Myoglobin Redox Instability of Protein Isolate from Bigeye Snapper (Priacanthus tayenus) Head By-product Worawan PANPIPAT and Manat CHAIJAN
P-43
Utilization of Fish Residue from Fish Sauce Fermentation for High Calcium Sweet Dipping Sauce (Nam Pla Wan) Production Manat CHAIJAN, Worawan PANPIPAT and Waraporn TUMTONG
P-44
Genetic Diversity of Seagrasses across the Eastcoast of Thailand Based on Sequence-Related Amplified Polymorphism (SRAP) Technique Pattama SRINAMNGOEN and Kanok-on DUANGPAKDEE
P-45
Genetic Variation of Halodule pinifolia Collected from Rayong Province Using SRAP Marker Siraphop ADICHANUND and Pattama SRINAMNGOEN
July 1-3, 2015
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P-46
Aquatic Polyculture Farm in Bangsaothong District, Samutprakarn Province, Thailand Nipon JITTAMNAN1 and Panneepa SIVAPIRUNTHEP
P-47
Microbiological Safety of Ready-to-Eat Semi-Dried Nham, an Innovation of Thai Fermented Meat Product Thanapa CHETAWAN, Komkhae PILASOMBUT and Supaluk SORAPUKDEE
P-48
Comparison of Carcass Traits in Duroc and Two Commercial Crossbred Pigs Numfon TAJASRI, Chanporn CHAOSAP, Ronachai SITTHIGRIPONG and Rutcharin LIMSUPAVANICH
P-49
Optimization of Culture Conditions of Bacteria Isolated from Buffalo Rumen for Cellulase and Xylanase Production
P-50
Consumption of Energy, Water and Consumables in Pon-Yang-Kham Beef Cutting Process
P-51
Gel Properties of Pork Ball Containing Mixed Precooked Carotenoid-rich Vegetables
P-52
Changes in Physicochemical, Microbiological and Sensorial Quality of Jerky Processed From Spent Laying Hen Meat during Storage
P-53
Kanya JIRAJAROENRAT, Jutatip CHALERMWANICHWONG, Tipaporn NGAMSANGA and Kanokrat SRIKIJKASEMWAT
Chalermsak SAKDAPECHSIRI, Thierry TRAN, Matana OSOTHONGS and Kanya JIRAJAROENRAT
Manat CHAIJAN, Worawan PANPIPAT, Noppawan LAOPHROM and Saowanee TRUKTRONG
Chanpen UESAKULRUNGRUENG, Supaluk SORAPUKDEE and Komkhae PILASOMBUT
Screening of Lactic Acid Bacteria from Traditional Fermented Meat Products Jiraroj NITHISANTAWAKHUPT, Pussadee TANGWATCHARIN and Kan SUKSUPATH
P-54
Screening and Identification of Lactic Acid Bacteria with Antimicrobial Activity to Mastitis Pathogen in Dairy cows; Staphylococcus aureus
P-55
Comparison of Stomoxys calcitrans (Diptera: Muscidae) Attacked and Behavioral Responses on Genetic Blood Differences of Holstein-Friesian Dairy Cattle in Thailand
Khakhanang RATANANIKOM, NantiyaSUWANPANYA, Yupaporn KHONNALAO and Sarayut SRIROD
Ubon TANGKAWANIT, Varangrat SENASING, Wirote KHLIBSUWAN, and Tassanee JAMJANYA
P-56
Opinion Towards Agricultural Further Study of Student in Nongchok District, Bangkok Metropolis Tippawan LIMUNGGURA and Panya MANKEB
P-57
Using Augmented Reality (AR) Technology for the Promotion of Agricultural Products: A Case Study of Bang Krathum Processed Banana Product Nutthakorn SONGKRAM
P-58
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Factors Affecting Consumer Purchasing Behavior of Toxic-Free Vegetables in Muang District, Samutprakarn Province Panya MANKEB, Pratompong MATONG, Suneeporn SUWANMANEEPONG and Prapaporn CHULILUNG
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P-59
Financial Cost and Benefit Analysis of Commercial Orchid Farming in Krathumban District, Samutsakorn Province, Thailand
P-60
Enhance Environment of a Historic Site, Ancient Benhama Maharat Building, For Future Public Use
P-61
Biodiesel Synthesis from Used Frying Oil with the Modified Catalyst from Waste Anadara granosa Shells
Panya MANKEB, Chalida LERTKRASEMPON, Tippawan LIMUNGGURA and Prapaporn CHULILUNG
Warong NAIVINIT, Parkpoom SUEBNUKARN, Rukkeit SANPRASERT and Taweesak WIYACHAI
Sasiwimol WOOTTHIKANOKKHAN, Buppha THAMNIYOM and Supamas CHUANOI
P-62 P-63
Scanning Electron Microscope of Alkaline Treated Spent Coffee Ground
Chayaporn WONGSIRIDETCHAI, Watcharaphan CHIANGKHAM, Narissara KHLAIHIRAN and Sudathip CHANTORN
Production of α-Amylase by Newly Isolated Thermotolerant Bacillus aerius through Solid State Fermentation by Using Some Agricultural Wastes Veysi OKUMUŞ, Sadin ÖZDEMİR and Abdurrahman DÜNDAR
July 1-3, 2015
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Keynote and Invited Papers
Keynote and Invited Papers
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Optimizing the Use of Feed Resources for Healthy Rumen, Increased Production and Mitigating Methane in Ruminants Professor Dr. Metha WANAPAT Director of the Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand Email: metha@kku.ac.th
ABSTRACT Ruminants are important livestock capable of utilizing fibrous feeds in the rumen through the fermentation of microorganisms, including bacteria, protozoa and fungi. Their fermentation end-products in the rumen are those of volatile fatty acids (VFAs), ammonia nitrogen (used for microbial protein synthesis), and methane production. Local feed resources availability in various seasons can contribute as essential sources of carbohydrate and protein and can significantly impact to the rumen fermentation and the subsequent productivity of the ruminant. Manipulation of rumen fermentation to maintain rumen ecology with normal pH, essential microbes and fermentation end products such as VFA and ammonia nitrogen concentration are essential. Furthermore, manipulation of the rumen using dietary approach to mitigate methane is promising. Development of the food-feed-system (FFS) can increase food for human and feed for ruminant as well as enrich the nitrogen for the soil. Cassava root is one of the good examples, in the form of dry cassava chips, pellets, or by-product from starch factory as energy sources, and dried cassava leaves and/or cassava hay as protein sources, have been used successfully in ruminant rations. It was found that the uses of cassava could provide year-round feed, which resulted in a high yield and good quality milk, and contributed to a more lucrative dairy and beef cattle, buffalo enterprise, especially for smallholder ruminant farming systems. Apart from producing rumen volatile fatty acid and microbial protein, greenhouse gas such as methane is also produced in the rumen. There have been a number of methods used in reducing rumen methane. However, among many approaches, nutritional manipulation using feed formulation and feeding management, especially the use of plant extracts or plants containing secondary compounds (condensed tannins and saponins), banana flower powder (BAFLOP) rich in minerals, as well as plant oil have been reported with promising results. Such results have impacted on decreasing rumen protozoa, methanogens and methane mitigation. However, at the current stage, more research concerning this burning issue with the role of livestock on global warming warrants further research undertakings regarding economical feasibility and practicality especially on the wider scope of implementation. Keywords: Rumen manipulation, Methane, Plant secondary compounds, Feeding system
July 1-3, 2015
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Food Sustainability - Emerging Challenges in the ASEAN Region (The Reality versus the Myth) Associate Professor Dr. Rajeev BHAT Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang- 11800, Malaysia Email: rajeevbhat1304@gmail.com, rajeevbhat@usm.my
ABSTRACT On a global perspective, what does ‘Food sustainability’ technically mean? Is there any appropriate definition? Otherwise, how to segregate ‘Food Sustainability’ and ‘Sustainable Foods?’ Overall, the word ‘Sustainability’ literally means “the ability to satisfy the needs of our present generation by ensuring that the needs of the future generation remains uncompromised.” According to the United Nations, food sustainability aims “to ensure nutritional security without foregoing the long-term health of the ecosystem and the vital cultural scenario that provides food.’ However, on a broader perspective, food sustainability encompasses a wide array of multi-disciplinary themes, which can have an extensive paradigm (developing and implementation of novel concepts, hypothesis, policies, theories and ideas, etc) relevant to the ‘socio-economic’ state-of-affairs up to the ‘agroecological-food sector’ scenario. The concept and theme of food sustainability covering an academic notion can differ from those of agro-foods based industries. On a broader sense, food sustainability can be linked with ensuring food security (quality and safety, overcoming hidden hunger, population explosion and poverty, food loss/wastage, food governance and food crisis, food trade, etc) as well as attaining a successful ‘sustainable food production.’ In the ASEAN context, several emerging challenges are faced concerning the production of sustainable foods or for achieving comprehensive food sustainability. The theoretical policies and framework put-forward for the developed countries might not be of total practical apprehension in the developing world, as in the case of ASEAN region. This requires to be amended based on the basic requirements of the region. Besides, the regional polices can widely differ from the proposed/practised international policies. It is worth to accentuate that some of the claims made in the developing world (as in case of ASEAN countries) might me more of a “myth” rather the ‘truth/reality.’ In the ASEAN region, individual governments have been proposing diversified trend marks relevant towards ensuring food security/food sustainability. However, we need to comprehend whether the entire proposal is a reality (practically applicable), and if yes, how many ASEAN countries are self-reliable and food secured? Further, when it comes to the societal needs, is healthy, nutritious and a wholesome food being consumed by the local populations? Is there any encouragement given to the local populations to promote and encourage consumption of local and a traditional healthy sustainable food? How about underutilized resources? What are the priorities set in this regard? On a common platform, is there any knowledge shared between the government, NGO’s, concerned industries and consumers on the importance of sustainable foods? If yes, how far is this successful? Any detailed documentation is being prepared in the ASEAN context? Further, when it comes to the agro-food industry sector, are the new proposed policies and themes in the ASEAN region, such as those concerning the effects of greenhouse gases, pollutants along the food chain, reusing/reprocessing of agro-food wastes, conservation of water, fuel and energy, bio-degradable packaging materials, impact of novel food processing technologies (e.g. nanotechnology), and other requirements are successfully implemented? Page 42
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Moreover, what are the roles that need to be played by the ‘urban’ versus ‘rural’ populations to ensure food sustainability in the region is a success? How to reconnect this diversified group of populations (intra- and inter-)? Further, when it comes to the farming community, is adequate care being taken to ensure that sharing of knowledge relevant to agro-food sustainability, new governmental policies, land use planning, impacts of weather changes and natural calamities, new ways of financial assistance (assuring economic self-sufficiency), active inclusion of farmers in the government’s decision making processes are crafted? Then how about achieving sustainability in the feed processing industry (for livestock)? Finally, can we come up with a common ‘vision’ and ‘mission’ (agenda and a frame work) for the ASEAN region relevant to attaining food sustainability? What should be our new approaches in the ASEAN region to benefit our community for sustainable development? All of the above facts comparing the emerging challenges, the ‘Reality versus the Myth’ in the ASEAN regional context of ‘Food Sustainability’ will be discussed/deliberated in this talk. Keywords: Sustainable Foods, Farming Community, Governmental Policies, Food Security, Consumer
July 1-3, 2015
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Can Feedstuff Processing Improve Digestibility of Amino Acids for Non-ruminants? Professor Dr. Julian WISEMAN Professor of Animal Production Division of Animal Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK
ABSTRACT Non-ruminant livestock diets in most regions of the world are based on cereals / cereal coproducts (for example wheat distillers dark grains with solubles – WDDGS - from the rapidly developing bioethanol industry) and plant proteins that are particularly important in view of the current ban on animal proteins within the European Union. Plant proteins invariably contain naturally-occurring anti-nutritive factors, principally trypsin inhibitors that are particularly important in soya beans but also occasionally peas. The inhibitors are heat labile and denatured by heat. There are a number of technologies available for processing plant proteins but a key message is that equipment operates under variable conditions of temperature, duration and moisture addition. Over-processing risks protein being denatured; for example a trypsin inhibitor activity of 1.5mg/g is associated with a reduction in amino acid digestibility. It is crucial that processing conditions are defined accurately rather than simply name of equipment. Extrusion is a common technology used in processing raw materials. Investigations into amino acid digestibility of soybeans following extrusion have produced data confirming that both under and over-cooking will give lower figures than when conditions are optimum. Levels of the trypsin inhibitor are lower in peas (a crop that is fairly common in many parts of the world) so the need for extrusion is not as important, in fact peas appear to be more sensitive to over-cooking than soybeans so there is a greater risks of reducing amino acid digestibility. The main objective of cereal fermentation is to convert starch to bioethanol leaving WDDGS; this is moist and therefore has to be dried as quickly as possible. Unfortunately it appears that over-cooking reduces amino acid digestibility. The conclusion is that processing can indeed improve amino acid digestibility but this needs to be carefully controlled in order to optimise nutritional value of raw materials.
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Rapid Generation Technology for Food and Forage Legumes - Harnessing in vitro Technologies for Accelerating Genetic Gain Assistant Professor Dr. Janine CROSER Centre for Plant Genetics and Breeding, The University of Western Australia, Australia
ABSTRACT Rapid development of recombinant inbred line (RIL) populations for exploitation of powerful new molecular technologies is just one benefit of new tools to accelerate generation turnover in grain legumes. We have developed accelerated single seed descent (ASSD) technology to achieve a turnover of six to eight generations per year for the food legumes field pea (Pisum sativum L.), chickpea (Cicer arietinum L.), lentil (Lens culinaris Medik.) and narrow-leaf lupin (Lupinus angustifolius L.). We have adapted this technology to the forage legumes, including subclover and Medic species. The ASSD technique is robust, cost-effective and enables the development of RIL populations in less than 12 months. We are currently working to incorporate into the ASSD methodology targeted screening for tolerance to abiotic traits of interest (viz. boron toxicity in pea and lentil; low pH and aluminium toxicity in lupin and chilling tolerance in chickpea). It is envisaged combining ASSD with abiotic stress screening will provide efficient selection of adapted germplasm for rapid incorporation into breeding programs and thus permit a timely response to emerging challenges.
July 1-3, 2015
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Opportunities of AEC Meat Production for the Halal Food Market. BABJI, A.S.*, SAHILAH, A.M., NUR‘ ALIAH, D. and NURUL NADIA, M. School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi Selangor, Malaysia Tel: 603-89215988, Fax: 603-89213232 *Email: daging@ukm.edu.my
ABSTRACT Exploring and identifying the strengths of ASEAN in the production of Halal meat and processed value added meat will be the best approach towards a win-win venture between member countries. Looking at the current population growth of Muslims which is close to two billion, the Halal market has emerged as one of the most lucrative and influential market globally. Malaysia’s strength is JAKIM ,Thailand is Processed Meat, Indonesia is the huge Muslim population and large livestock industry, Philippine, the strong tie to US market and modern processing technologies availability, Singapore, its strong trading & export link globally. Other members can enlighten their resources in relation to the Livestock industry or human resource expertise to explore the potential Halal meat market. Growing demand for Halal meat in ASEAN is also largely influenced by government policy, politics, religions, customs, traditions and other factors. In the matter of the Halal Meat industry itself AEC must come to a common stand on Quality and Standards set by world bodies on the production and processing of Halal meat and food products. For example, in the matter of ingredient technology and food additives applied in the processing of meat, discoveries of new ingredients have become more complex and complicated beyond the perception of consumers as to what are the inherent original sources and raw materials. Handling of raw materials and processing technologies should ensure that the processing steps and machinery parts in contact with foods are not contaminated with forbidden and harmful substances. The challenges faced by food analyst include unavailability of common officially recognized standard methods among member countries, resulting in different laboratories producing different results and interpretations. Traceability of meat produced at the farm and food additives used in the food industry remain as major hurdles and issues for the Muslim community seeking Halal food from farm to fork. The processes and technological advancements made in raw material processing, ingredient extractions, modifications, purification and re-synthesis, restructured and fabricated into food matrices make the question of traceability and solving the problem of Halalness of the meat and food products and processes a monumental task. There has been increasing level of awareness concerning Halal Halal including foods and beverages, pharmaceuticals, cosmetics or personal care products, which is being driven by increased consumer knowledge of the ingredients used, ways of productions, technically available matured, analytical methodologies and alternative consumer’s choice of selection. The current techniques in Halal verification for Halal meat products are molecular approaches such as PCR-based techniques. They have demonstrated and proven to be more scientifically, technologically sensitive and high accuracy on authentically of Halal. Those methods are including conventional PCR, real-time PCR and PCRsouthern hybridization on DNA chips which are compliance with Syariah laws. AEC must address these issues and compete strategically within members in the spirit of ASEAN.
Introduction It is reported that the world Muslim population in 2014 is 2.038 billion. With the growth rate of 1.84% annually, Islam is the fastest growing religion in the world and the Continent of Africa is the leading region with the highest Muslim percentage of 53.04% followed by Asia with 32.16 % (Muslim Population, 2014). Due to the fact that it is obligatory for all Muslims to consume Halal food, the demand for Halal food especially in Halal meat is expected to increase in tandem with the rise in population. Nizam (2011) reports that the world Halal Page 46
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industry is estimated to be worth to US$ 2 trillion a year, an amount which indicated a huge market potential for any countries of firms that are seriously considering to engage in producing and selling Halal products. According to a report by Xinhuanet (March 28, 2015) ten Chinese quality companies from halal food, financial and infrastructure sectors will officially enter Port Klang Free Trade Zone (FTZ) in Malaysia. Contracts were signed between the two sides during the ChinaMalaysia International Trade Summit which witnessed launching of China Area of Port Klang Free Trade Zone International Trade and Halal Industrial Center in Beijing. Meanwhile, at the opening of 2015 World Halal Summit hosted by Malaysian Ministry of International Trade and Industry, Malaysian Prime Minister Najib Razak said there are great opportunities in halal industrial development, adding that Malaysia will spare no effort to promote ASEAN to become a halal trade center. Looking at the tremendous speed in the development of Halal Industry worldwide, a uniform halal standard in AEC is vital to cater to the increased number of market participants in the industry not only from Muslim countries but non-Muslim countries as well. Requirements of Halal food production are cleanliness, free from ‘contamination’ and healthy food. Of concern among the Muslim consumers are raw materials, ingredients and additives utilized in the production of Halal food products, especially meat products. Traceability of Halal meat raw materials should start at the point of animal breeding, production to the stage of Halal slaughter, processing operations and final point of consumption. It is well known in the meat trade that Muslims consume Halal meat. However, at times questions are asked, what is Halal? In Arabic it simply means permissible or allowed. Opposite to it is Haram, which means forbidden or not allowed. Halal method of slaughtering can be referred to the Muslim Guidelines (Wahab 2004). Halal slaughtering must not be done where pig are slaughtered or in the vicinity of pigs slaughtering area. The pig was chosen by other few consumers and industries in preference to the cow or chicken, because a pig produces by far the most varied collection of ingredients. Modern technology has brought the pig number 05049 from the farm to Europe’s largest processors of meat and by-products, with a market that encompasses the entire world. From there, the pig products find themselves in the most wide-ranging places and may start having a cross contact to the Halal food production practice. While demand for halal foods and other Islamic goods are increasing, food industry globally have started looking at the ‘halal’ concept as a new tool for marketing, which in turn, must understand and appreciate the religious and scientific basis of halal requirement. Establishment of ASEAN Economic Community (AEC) which involved ASEAN countries including Malaysia, who is the leader of Halal food benchmarking (Bohari et al., 2013) Thailand who managed to be the 6th biggest exporter of Halal products in the world in 2011 (Nizam, 2011), Indonesia as the largest Muslim country with more than 210 million Muslims and large livestock industry, Philippine who establish a strong tie to US market and modern processing technologies availability, Singapore who exhibit strong trading & export link globally will promise a good future ahead in establishing AEC Halal Meat. ASEAN meat production has increased by 4.6 percent a year over the past twenty-five years. Livestock production plays an important role in providing food, employment and many other contributions to both regional and national development. Malaysia, Thailand and Indonesia have invested considerably to be one of the leading Halal hubs. Vietnam has a large and quite fragmented meat and poultry processing industry as this industry has been in existence for many years in history. Other more developed countries in ASEAN like Singapore and Malaysia as well as Philippine, there are few challenges to be dealt with in the area of distribution quality for meats and poultry (Ismail et al., 2013).
July 1-3, 2015
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Research and Development on Halal Meat Products Research and development have made a major contribution to the transformation of agriculture – both crop and animal. Most of the technological gains have been applied in the developed countries. Unfortunately, the impact of most technological progress has been limited to only developing countries. Smallholder crop-livestock systems which support the large majority of the poor have remained much more reliant on the locally available knowledge and production techniques. To address the emerging challenges posed by the rapidly growing consumer and Muslim community specifically, as well as urbanization, there is a need for the adoption and the use of advances through research and development. This will enable smallholder systems to respond to the changing social, economic and environmental challenges. Developing new and modified processing technology for livestock products requires an understanding of consumers’ perception of the product category. Processing Technologies and Ingredient Specialists have been successful in blending new techniques with functional non-meat raw materials to produce quality and healthy food products. Restructured meat products offer unlimited opportunities attracting both research and commercial sectors. Product development must be complemented by industry needs to produce value added products that meets consumers demand. In Asia, traditional meat and poultry products of high fat, cholesterol, salt and chemical additives will have to give way to ‘lite and lean’ types of products. While this change is significant in developed nations, Asia is still fully utilizing its meat resources to the fullest. Unconventional protein resources such as mechanically deboned meat, connective tissues, skin and surimi-like materials, are carefully formulated and processed using modern blending and marinating technology, pre-emulsion, gelation, and coating techniques producing what consumers want: quality meat products which are nutritious and healthy (Yackel, 1992). Science and innovation are also used by industry to add value to animal by-products to search for key active molecules in nutrition like bioactive peptides, food safety (antimicrobial peptides), medicine, cosmetics or other fields, to develop new technological applications. This type of innovation towards advanced value-addition of meat by-products are continuous, especially when it comes to replacing the non-halal sources of meat by-products which had been used previously (Toldrá et al., 2012). New Processing Technology A lot of processing technologies are involved in the production of meat products with low fat and new ingredients with functional properties aimed at producing healthy products. In addition large local processing plants have utilized efficient processing operations to minimize animal wastes and by-products, improving their functional and nutritional properties and producing economical good quality value added meat products (Babji 1996; Babji and Ong, 1993; Babji and Chempaka, 1993; Babji 1993). The private sectors are well informed on the availability of modern processing technologies combined with appropriate ingredients blended with the meat to yield meat products. One of the important protein functionalities in processed meats is thermally induced gelation of muscle proteins. Thermal gelation changes of the muscle proteins account for many textural and functional properties including gel strength, gel elasticity, water holding capacity, and encapsulation of other food constituents of the processed meat products (Fukazawa et al., 1961; Asghar et al., 1985; Autio et al., 1989). High pressure processing (HPP) has been used in a commercial environment as a non-thermal decontamination technology for processed and RTE meat products particularly in cases where heat treatment is inconvenient, with high consumer acceptance of products, in comparison to other non-thermal decontamination technologies such as ionizing radiation. Page 48
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The thermo-labile nature of protein meat systems allow the development of novel meat based products and the usage of HPP as a time and energy saving processing step for the meat industry. Functional and rheological properties of meat can be improved and novel meat based products with decreased salt content can also be developed (Bajovic et al., 2012). There is a strong demand for meat products that are low in fat, salt, cholesterol and calories. The basis of the consumer demand for such products lies in research indicating greater health risks with such diets. One needs to ponder the question of origin of raw materials and address the issues related to Halal status of such food items. Modern blending and marinating technology, pre-emulsion, gelation, coating techniques and improved nutritional, quality and shelf life produced what consumers demand: Quality meat products which are nutritious, healthy and wholesome (Halal). Halal Control Measures Halal control measures can be defined as action and activities to prevent Halal threat to consumers. With regards food and drinks, Islam has laid down three very important aspects; whether the consumption of foodstuff is prohibited by Allah; whether the foodstuff is obtained through Halal or Haram means, and; whether or not the material is harmful to health. This should be governed by Syariah Law which means Islamic Law based on Al-Quran, the Sunnah, consensus of opinions of Muslims jurists (Ijma’) and analogy (qiyas) and other modes of legal reasonings (ijtihad). In Malaysia the Syariah rulings based on ijtihad or fatwa on halal matters should be in line with the Shafie Mazhab of Ahli al- Sunnah wa al-Jama’ah. Hence, a particular food becomes Halal and non-Halal by Syari’ah Law if it is considered so by any of these sources. For meat and meat products as example, traceability of Halal meat raw materials should start at the point of animal breeding, production to the stage of Halal slaughter, processing operations and final point of consumption Malaysia is a leader in the halal food benchmarking. The United Nations has cited Malaysia as the world’s best example of benchmarking of halal food in accordance with the Codex Alimentarius Commission adopted the Codex general guidelines for the use of the term halal in Geneva in 1997. This is because a single halal standard is applied throughout the country with the result that the Malaysian standard has become the basis for the development of the world’s halal food industries (SME Annual Report 2006, 2007) Department of Islamic Development Malaysia (JAKIM) was the world’s first halal certification body responsible for monitoring the halal industry, leading to the amendment of Malaysia’s Trade Description Act in 2011 which gives JAKIM a much stronger mandate to regulate the halal industry. JAKIM has developed a Malaysian Protocol for the Halal Meat and Poultry Productions to give clear guidance in the production of halal meat and poultry. This protocol is applicable to all establishments producing halal meat, poultry and their products Besides Malaysia, Thailand also showed positive development in establishment of Halal products. According to the Regulation of the Central Islamic Committee of Thailand (CICT), regarding the Halal Affair Operation of B.E. 2552, the Central Islamic Committee of Thailand shall be responsible for determining and announcing the use of Thai Halal Product Standard to be in accordance with the Islamic Principle and international standards besides approving the use of Halal Logo on Halal Product. It also functions as a Halal Accreditation Body (HAB) to accredit Halal Certification Body (HCB); coordinate and supervise the division related to Halal Affair Operation for the effective operation of Halal Product Standard (Halal Standard Institute of Thailand, 2012). To be more proactive and efficient, Thai Department of Halal Affairs strategized that Provincial Islamic Committee shall be responsible for Halal Certification at provincial level, while for other provinces without Provincial Islamic Committee, the Central Islamic July 1-3, 2015
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Committee of Thailand shall be the responsible body. Thai Halal food industry is ensured to have abided by the Islamic law and is managed by the Halal Standard Institute of Thailand. Those two countries are the examples of successful Halal body which AEC can look for in establishing Halal control measure especially in meat and meat products. The one barrier to entry into ASEAN market is that many of the countries have their own level of certification. While as example Malaysia use Halal Jakim as a standard, another country in the region may not accept that standard and vice versa. Therefore, AEC should start to create an ‘ASEAN Standards’ like what European Union did (Jayaeelan, 2015). The Halal control measures should be parallel with 3 combination elements; Syariah Law experts, enforcement and/or based on scientific evidence. It means, the determination of Halal is not standing alone by authority body and enforcement but it also must be strengthened by scientific-based measures. Such techniques used permit the detection of accidental or deliberate adulteration which cover two aspects involving the ‘religious practices’ and ‘unfair trade practice’. Halal authentication methods based on DNA technology have been established for instances in detecting porcine DNA in hard and soft capsules gelatin using polymerase chain reaction (PCR) and southern-hybridization on the biochip analysis (Sahilah et al., 2012); and detection of porcine DNA in raw meats and fats using polymerase chain reaction (PCR) (Aida et al., 2005; Chandrika et al., 2009; Sahilah et al., 2011). Whereas, Department of Chemistry Malaysia using real-time PCR to detect the porcine DNA in raw and processed foods, born and fats (Personal communication). With regards Genetic modified organisms (GMOs), foods and drinks content or products or ingredients or food additives produced from GMOs which are contained non-Halal or Haram animals genes (such as pig and rat) considered as non-Halal or Haram. Although, the ethical dilemma in GMOs is till debating our personal opinions based on Syariah Law the GMOs is Haram if they contain prohibited animal gene such as pig and rat. In terms of traceability to identify the source of contamination of Non-Halal ingredients, the WRES system of E numbers related to ingredients specification and sources are insufficient to support auditors evaluation for certification (by JAKIM, Malaysia) It is suggested that a prototype system for detection of Halal status to support Halal certification be formulated and applied to speed up the process of auditing and certifying Halal products / ingredients and raw materials. This prototype is expected to detect the source and status of halal ingredients. Utilization of this prototype software will be convenient for the auditors involved to make decision with regards to halal certification and improve productivity (Juhana et al., 2015). Marketing of Halal Meat Products Concerns by muslims on Halal meat and meat products need to be considered in the production of Halal food from farm to mouth. These include raw meat imports, local slaughter, processing operations/equipment, packaging, storage, transportation, food ingredients, additives adulterants, biotechnology and genetically modified animals, food safety and food quality aspects of ‘Thoyyibah’. Thus the importance of traceability for all these raw and processed materials is of almost importance for the Muslim consumers. According to the Livestock Sub-Sector Report for the ASEAN Strategic Plan 2016-2020 (Jabbar 2014), between 2000 and 2012, in real terms total value of livestock outputs increased in the Asean Member States at varying extent. Production increased by 2.8 times in Myanmar, more than doubled in Vietnam, by 70-78% in Malaysia, Indonesia and Brunei, by 36-49% in Thailand, Singapore, the Philippines and Lao PDR, but it is almost stagnated in Cambodia. In case of the meat trade, only Thailand is a net exporter and its net export of meat increased reasonably. All the other countries are net importers of almost all types of meat and the extent of import increased over time. Thailand ranks first for Halal exports among the 10 member countries of the Association of Southeast Asian Nations (ASEAN). Major markets Page 50
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for Thai Halal food include Indonesia, Malaysia, Nigeria, Oman, and other countries in the ASEAN region. Another ASEAN country, Brunei is aiming to expand its halal food sales to ethical consumers beyond the spectrum of Muslims. In this regard, the Sultanate recently signed a memorandum of understanding with the Guangxi Zhuang Autonomous Region of People’s Republic of China to create a halal food processing and distribution base. The Brunei authorities wish to leverage upon their halal certification standards which ensure high food quality and not just animal welfare but also the health and goodness of the product. Bearing such considerations, Brunei aims to become a key player in the global halal food industry tapping not only a Muslim customer base but also the ethical consumers from all walks of life (MIFC, 2014) Malaysia has aspirations to become a global hub for the production and trade of Halal products and services, as outlined in the 2006 Third Industrial Master Plan (IMP3). The country is a leading exporter of various halal goods. In regards of meat and meat products, Halal Hub Industry and Standardization of Halal Certificate Asian economic community (AEC) is formed to transform ASEAN into a region with free movement of goods, services, investment, skilled labour, and free flow of capital. Each ASEAN countries have their own strength in supporting AEC Halal meat production into a single market and a competitive regional economic manufacturing Halal hub industry. Thus, a unity and harmonization of Halal standard being accepted in AEC is value added. In Malaysia context, Malaysia has a comprehensive halal ecosystem which cannot be found in any other country in the world due to its complete ecosystem in terms of raw materials, production, packaging, manufacturing, logistics, certification and Islamic banking. All these features are available and must in place as a country that aspires to be a Halal producer products and the biggest Halal hub in the world by 2020. Thus, Islamic Development Department of Malaysia’s (JAKIM) is an authorized body for any Halal certification and integrity application in all foods including meats, beverages, cosmetics and pharmaceuticals industry in Malaysia, neither local nor international companies which many of them were already invested in a long period in Malaysia. Thus, the industries are responsible in producing Halalan Toyyibban (Permissible and wholesome good, clean, purify and safe) to fulfill the demand of consumers choice. The authority and experience body such as JAKIM should be given the credit and chance to lead the AEC Halal meat industry standard. JAKIM can play an important role to assist a wider opportunity for not only Malaysia Halal industries to penetrate the global market but also AEC. The halal industry is not only relevant to Islamic nations, but also to non-Islamic countries. The industry is seen as a source of revenue that can stimulate a country’s economy. Concomitant with Halal certification by JAKIM, this body can also strengthen scientificallybased regulatory systems that verify the safety and Halalness of the meat products. The above initiative is indeed in conformity with the Halal concept (permissible) which constitutes an Islamic legal value to which Muslims are required to abide in their daily lives. Conclusion Food is only Halal if the entire food chain from farm to table, is processed, handled and stored in accordance with the Shariah and/or Halal standards or Guidelines, such as in Malaysia, JAKIM: General guidelines, Malaysia standards MS 1500: 2004 and Codex Alimantaruis (Food Labeling). Here lies the challenge and importance of traceability to verify the ‘Halalness’ of the sources of Halal raw materials and final meat based food products. Thailand and Malaysia must take the lead to enhance opportunities for AEC to formulate practical marketing strategies for a win –win situation in the Halal food industry. July 1-3, 2015
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References Aida, A. A., Y.B. Che Man, C.M.V.L. Wong, A. R. Raha and R. Son. 2005. Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. Meat Science 69: 47-52. Asghar, A., K. Somehima and T. Yasui, 1985. Functionality of Muscle Proteins in Gelation Mechanisms of Structured Meat Products. CPC Cri. Rev. Food Sci. & Nutr. 22:27. Autio, K., M. Kiesuara and Polvineu. 1989. Heat Induced Gelation of Minced Rainbow Trout (Salmo gairdneri). The effect of pH, sodium chloride and setting. J. Food Sci. 54: 805. Babji, A.S. 1996. Research and Development of Value Added Products in Asia. Proc. WPC. New Delhi. Vol. III. pg. 175-180. Babji, A.S. and M.Y. Seri Chempaka. 1995. The Nutritional Value of Some Processed Meat Products in Malaysia. Mal. J. Nutr. Vol. I. No. 1: 83-94. Babji, A.S. 1993. Development and Promotion of Value Added Food Product from Livestock. Proc. XVI MSAP, Annual Conference. pg. 1-9. MSAP Publication, Malaysia. Babji, A.S. and J.W.B. Ong. 1993. Effect of Additives on the Frozen Quality of MDCM. J. Vet. Malaysia 5(2): 27-32. Bajovic, B., T. Bolumar and V. Heinz. 2012. Quality considerations with high pressure processing of fresh and value added meat products. Meat Science, 92(3), 280–289. doi:10.1016/j.meatsci.2012.04.024. Bohari, A.M., C.W. Hin and N. Fuad. 2013. The competitiveness of halal food industry in Malaysia: A SWOT- ICT analysis. GEOGRAFIA Online Malaysia Journal of Society and Space 9: 1-9. Chandrika, M., M.N. Zainon, M. Maimunah, M.B. Lesley, S. Jinap and R. Son. 2009. Meat species identification and Halal authentication analysis using mitochondrial DNA. Meat Science 83:57-61. Fukazawa, T., Y. Hashimoto and T. Yasui. 1961. Effect of Some Proteins on the Binding Quality of Experimental Sausage. J. Food. Sci. 26: 541. Halal Standard Institute of Thailand. 2012. HSIT presents slogan “One country one logo” in Thaifex 09 Available at http://www.Halal.or.th/en/main/content. php?page=sub&category=6&id=128 retrieved on 13/3/2012. Ismail, M. M., A. M. Abdullah and B. Hassanpour. 2013. Ranking the competitiveness of the ruminant meat and meat preparation sub-sector amongst Asean countries. International Journal of Economics and Management 7(1), 1–16. Jayaeelan, R. 2015. The Asean Buzz. 14 February 2015. Jabbar, M.A. 2014. Livestock Sub-Sector Report for the ASEAN Strategic Plan 2016-2020; Prepared for the FAO RAP and the ASEAN Secretariat. June 2014. Juhana, S., M. A. Shereena, W. M. Wan Aida and H. Osman. 2015. Personal communication, Universiti Kebangsaan Malaysia, Bangi, Malaysia.. Malaysia World’s Islamic Finance Marketplace (MIFC). 2014. The Halal Economy: The huge potential for Islamic Finance. 30 September 2014. Muslim Population. 2014. Available at http://muslimpopulation.com/world/. Retrieved on 25/02/2015. Nizam, Y. 2011. Emulate the Thai Halal industry, Utusan on line 03/08/2011 Available at http://ww2.utusan.com.my/utusan/special.asp?pr=theMessenger&y=2011&dt=0803& pub=theMessenger&sec=Features&pg=fe_01.htm retrieved on 13/3/2012.22 Sahilah, A.M., Y. Norhayati, A.S. Norrakiah, A. Aminah and W. M. Wan Aida. 2011. Halal authentication of raw meats using PCR amplification of mitchondrial DNA. International Food Research Journal 18 (4): 1489-1491.
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Sahilah, A. M., Mohd. Fadly, L., Norrakiah, A. S. Aminah, A., W.M. Wan Aida, A.G. Ma’aruf, and A. Mohd Khan. 2012. Halal market surveillance of soft and hard gel capsules in pharmaceutical products using PCR and southern-hybridization on the biochip analysis. International Food Research Journal 19 (1): 371-375. SME Annual Report 2006. 2007. Potential growth areas for SMEs. National SME Development Council, Kuala Lumpur. Toldrá, F., M.-C. Aristoy, L. Mora and M. Reig. 2012. Innovations in value-addition of edible meat by-products. Meat science 92(3): 290–6. Wahab, A. R. 2004. Guidelines for the Preparation of Halal Food and Goods for the Muslim Consumers. AmalMerge (M) Sdn. Bhd.,.
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Applications of Molecular Diagnostic Methods in the Management of Fungicide Resistance Professor Dr. Hideo ISHII Kibi International University, Minami-awaji, Hyogo 656-0484, Japan ABSTRACT
Chemical control is the most common measure to cope with crop diseases in practice. However, development of pathogen isolates resistant to fungicides often causes a failure of disease control. It is therefore essential to monitor the change of fungicide sensitivity in pathogen populations. Cultural methods and/or bioassays using fungicide-treated media and plants, respectively, have been conventionally employed for the monitoring. Recently, some molecular biological methods such as PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) and ASPCR (Allele-specific PCR) have been developed and widely used for the diagnosis of resistant isolates of fungal pathogens. In this paper, molecular methods being routinely used for resistance monitoring are introduced. Mode of action and mechanism of resistance have been intensively studied on major group of fungicides. QoI (strobilurin) fungicides have a wide range of spectrum to control fungal and oomycete pathogens on various crops. But, they also carry high risk for resistance development in target pathogens. A point mutation of cytochrome b gene, which encodes fungicide-target protein in mitochondria, resulting in the G143A substitution is the major cause of high QoI resistance. To monitor QoI resistance, PCR-RFLP is often used in the rice blast fungus Magnapothe oryzae and other important pathogens. Loop-mediated isothermal amplification (LAMP) offers the potential for accurate and rapid detection of resistance using field samples, even for small diagnostic laboratories. Because the “on-site� methods are still insufficient, molecular diagnostics have not yet proved useful to growers in guiding in-season of anti-resistance strategies, but further developments in LAMP may overcome this limitation.
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Biogas Reduces the Carbon Footprint of Cassava Starch: A Comparative Assessment with Fuel Oil Dr. Thierry TRAN Food processing & Life Cycle Assessment (LCA) Team Joint Research Unit QUALISUD, French Agricultural Research Centre for International Development, (CIRAD), Montpellier, France Cassava and Starch Technology Research Unit (CSTRU), Kasetsart University, Bangkok, Thailand ABSTRACT
Greenhouse gases (GHG) emissions, or carbon footprint of cassava starch production, were assessed based on three factories in Thailand. The system boundaries included farm stage (production of cassava roots), transportation to factory, processing into native starch. The functional unit (FU) was one ton of native cassava starch at 13% water content. Biogas produced from the factory wastewater was the main source of thermal energy for starch drying (180–240 m3/FU). Agricultural production contributed approximately 60% of GHG emissions, mainly from the use of nitrogen fertilizers. GHG emissions of root production varied widely due to the diversity of agricultural practices within the same region. The contribution of the factory stage to the carbon footprint depended on the use of electricity, biogas and other fuels, ranging from 200 to 460 kg CO2eq/FU. Allocation rules such as wetweight or dry-weight basis allocations affected the results markedly. In the past 10 years, 90% of cassava starch factories in Thailand switched from fuel oil to biogas, which reduced GHG emissions of the cassava starch industry as a whole between 700,000 and 1,000,000 tons CO2eq/year.
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O-1
Adapting the CSM-Canegro-Sugarcane model to Simulate Napier Grass in Chiang Mai Province, Thailand Tupthai NORSUWAN1*, Attachai JINTRAWET1 and Carsten MARONH2 1
Center for Agricultural Resource System Research, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand 2 Institute of Plant Production and Agroecology in the Tropics and Subtropics, University of Hohenheim, Stuttgart, Germany *Corresponding email: tupthai.n@cmu.ac.th
ABSTRACT Plot experiment was conducted during November, 2013 to March, 2014 at Chiang Mai province, Thailand. Drip irrigation was applied as Reference Evapotranspiration (ET0) in five-day interval and 300 kg N.ha-1 was applied at two weeks after planting. Napier grass was harvested three times. Two-week interval quantification of plant components and literature reviews were used in order to calibrate ecotype and cultivar parameters of the CSM-CanegroSugarcane model in DSSAT v4.6 Beta. Calibration results displayed that simulated tiller number was corresponded with the observed values as the index of agreement (D-stat, the values varies between zero and one by one being the best fit.) equaled to 0.97, 0.36 and 0.19 in planted crop, the 1st and 2nd ratoon crop, respectively. Maximum radiation conversion efficiency (PARCEmax) was set as 6, 9 and 12 g.MJ -1 in order to find out the best simulation result of total dry matter and partitioning of dry leaf and leaf sheath weight and dry stem weight. Setting PARCEmax as 9 g.MJ-1 gave the best for simulation of planted crop as D-stat ranking between 0.94 to 0.98. In contrast, setting PARCEmax as 12 g.MJ -1 gave the best simulation results on the 1st and 2nd ratoon crop as D-stat ranking between 0.60 to 0.72. Keywords: CSM-Canegro-Sugarcane, Drip irrigation, DSSAT, Napier grass Introduction In Thailand within 2015, Napier grass would be expected as the main staple supplied in biogas power generation (6.25% of nationally total electricity production) and compressed biogas (5% replacement of NGV) (Cheokul, 2012). This demand could promote collaboration of farmers in the production plan of napier grass (DEDE, 2013). In order to reduce cost and time for setting conventional trial of field experiment, crop models can be used as policy analysis tools for yield prediction under variety of climatic and edaphic conditions (Boote et al., 1996). Decision Support System for Agrotechnology Transfer (DSSAT), a data base management program for soil, weather, and crop management and experimental data, was developed in order to facilitate the application of crop model (IBSNAT, 1993). However, napier model is not included in the DSSAT v4.6 Beta. Therefore, adapting ecotype and cultivar parameters in the CSM-Canegro-Sugarcane model (Jones and Singels, 2008; Singels et al., 2008) were reasonable because napier grass and sugarcane are C4 perennial grasses grown in tropical regions. This paper aimed to describe the calibration process and the result of the CSM-Canegro-Sugarcane in DSSAT v4.6 Beta simulating Napier Grass planted on a representative soil profile under dry season (November, 2013 to March, 2014) of Chiang Mai province. Materials and Methods Plot experiment: Three replications of plot experiment were carried out at Mae Hia Agricultural Research, Demonstration and Training Center (18º46'N, 98°55'E, 350 m a.s.l.). The soil is a July 1-3, 2015
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loamy-skeletal, mixed, isohyperthermic Typic (Kandic) Paleustult, Mae Rim series (Potijun et al., 2003). The plot was plowed at 30 cm depth by a diesel-engine tractor with a seven-disc plough on the day before planting. Each replicated plot was prepared as 4 x 5 m. Two-node napier stalks, Pak Chong 1 (Pennisetum purpureum × Pennisetum americanum), were planted as 50 x 100 cm planting space by burying the bottom node underground and setting the top node above ground on November 16, 2013. The 1st harvest was conducted on January 11, 2014, the 2nd and 3rd harvests were on February 8 and March 8, 2014, respectively. The grass was cut closely to the ground surface at harvest. Maximum and minimum of air temperature and relative humidity, and average wind speed obtained from daily statistic reports of the Thai Meteorological Department, measured at Chiang Mai airport (TMD, 2014), were used to calculate daily ET0 from the Penman-Monteith equation on a daily time step (Allen et al., 1998). Water lost by ET0 was irrigated to the plot experiment by drip irrigation in five-day intervals after the 1st harvest. 300 kg N.ha-1 of urea fertilizer was applied into excavated holes and covered with soil at two weeks after planting. In order to quantify tiller number, dry leaf and leaf sheath weight, dry stem weight and total dry matter yield, three plants were sampled in two-week interval. The fresh plant components were separated and dried in a hot-air oven as 70oC for 24 hrs. Model calibration: Ecotype and cultivar parameters of the CSM-Canegro-Sugarcane model were calibrated according to literature review and the quantification of tiller number, dry leaf and leaf sheath weight, dry stem weight and total dry matter yield. Soil input were taken from a dataset of Land Development Department of Thailand, which in classified as a loamy-skeletal, mixed, isohyperthermic Typic (Kandic) Paleustult, Mae Rim series (Potijun, et al., 2003). Weather inputs were referred form daily statistic reports of the Thai Meteorological Department measured at Chiang Mai airport (TMD, 2014). Solar radiation was calculated by WeaData v2.0 based on air temperature adopting a new coefficient adopted from Phakamas (Phakamas et al., 2013). Maximum radiation conversion efficiency (PARCEmax) was set as 6, 9 and 12 g.MJ-1 in order to find out the best simulation result of total dry matter and partitioning of dry leaf and leaf sheath weight and dry stem weight. Management input was adjusted as real practice in plot experiment. The index of agreement (Willmott, 1981) (D-stat) was applied in order to compare between model-predicted values and the observed from the field experiment. D-stat has values between zero and one by one being the best fit. n
d 1[
(P i 1 n
i
Oi ) 2
,0≤d≤1 ]
( P O ) where: n: number of observed values, Pi: predicted values for the ith measurement, Oi: observed values for the ith measurement, O : overall mean of observed values, Pi' : Pi O and O i' : O i O . i 1
' i
' i
2
Results and Discussion Adjustment of ecotype and cultivar parameters: Ecotype parameters (Table 1) in the CSMCanegro-Sugarcane model, namely, TTBASEEM, TTBASELFEX and TTBASEPOP were adjusted according to the referenced minimum temperature for growth of napier grass is about 15°C and mean minimum temperature of the coldest month is 11.5° + 5.4°C (Russell and Webb, 1976). Table 1. Ecotype parameter of plant phenology and adjusted value
Parameter Adjusted value Description TTBASEEM 11.5 Base temperature for emergence (oC) TTBASELFEX 11.5 Base temperature for leaf phenology (oC) TTBASEPOP 15.0 Base temperature for stalk phenology (oC) The table displays only the adjusted parameters
Cultivar parameters in the model were categorized as plant phenology, tiller and leaf phenology and, biomass accumulation and partitioning (Table 2, 3 and 4). Firstly, plant phenology (Table 2), TTPLNTEM and TTRATNEM were calculated based on the referred base temperatures and the daily average temperature of the napier grass emergences. TT_POPGROWTH was calculated from the referred base temperature and the daily average temperature from 14th to 16th week in the 3rd ratoon crop. Page 58
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Secondly, tiller and leaf phenology (Table 3), Tthalfo were estimated from the observed fresh weight that was half from in 2nd ratoon crop. The referred base temperature (Russell and Webb, 1976) was used for base temperature for canopy development (Tbase). LFMAX was counted on the main tiller of the final harvest in the 2nd ratoon crop. MXLFARNO was adopted from the highest sugarcane-suggested value (Jones and Singels, 2008). PI1, PI2 and PSWITCH was estimated from the period of leaf emergence between the 7th and 9th leaf in the 2nd ratoon crop. Table 2 Cultivar parameter of plant phenology and adjusted value. Parameter TTPLNTEM TTRATNEM CHUPIBASE TT_POPGROWTH
Adjusted value 72.0 10.0 10.0 146.0
Description Thermal time to emergence for a plant crop (oC.d) Thermal time to emergence for a ratoon crop (oC.d) Thermal time from emergence to start of stalk growth (oCd) Thermal time from emergence to peak tiller population ( oC.d)
Table 3 Cultivar parameter of tiller and leaf phenology and adjusted value. Parameter Tthalfo Tbase LFMAX
Adjusted value 225 15 16
MXLFAREA MXLFARNO PI1 PI2 PSWITCH
600 16 20 70 8
Description Thermal time to half canopy (oC.d) Base temperature for canopy development (oC) Maximum number of green leaves a healthy, adequately-watered plant will have after it is old enough to lose some leaves (leaf number) Max leaf area assigned to all leaves above leaf number MXLFARNO (cm2) Leaf number above which leaf area is limited to MXLFAREA (leaf number) Phyllocron interval 1 (for leaf numbers below Pswitch, (base TTBASELFEX)) (oC.d) Phyllocron interval 2 (oC.d) Leaf number at which the phyllocron changes (leaf number)
Thirdly, biomass accumulation and partitioning (Table 4), PARCEmax expresses as assimilate produced before respiration. Therefore, observed total dry matter yield could not be applied to quantify PARCEmax manually. In order to find out the best simulation results, PARCEmax was adjusted as 6, 9 and 12 g.MJ-1. APFMX was used the highest sugarcane-suggested value. STKPFMAX was observed from the harvest of the 2nd ratoon crop. Table 4 Cultivar parameters of biomass accumulation and partitioning, and adjusted value. Parameter PARCEmax
Adjusted value 6, 9 ,12
APFMX
0.90
STKPFMAX
0.14
Description Maximum (no stress) radiation conversion efficiency expressed as assimilate produced before respiration, per unit PAR (Mg.MJ-1) Maximum fraction of dry mass increments that can be allocated to aerial dry mass (Mg.Mg-1) Fraction of daily aerial dry mass increments partitioned to stalk at high temperatures in a mature crop (Mg.Mg-1)
Comparison of observed values and simulation result: The observed tiller number increased from 11.7 to 44.64 tiller.m-2 in planted crop, and increased up to 102.0 and 191.3 tiller.m-2 in the 1st and 2nd ratoon crops, respectively. According to D-stat as 0.97, 0.36 and 0.19 in planted crop, the 1st and 2nd ratoon crop, respectively, the model could simulate increasing tiller number in planted crop properly. However, the model simulated increasing tiller number from 0 to 27.5 and 30.8 tiller.m-2 in the 1st and 2nd ratoon crops, respectively. The total dry matter yield increased up to 3,440, 3,130 and 5,800 kg DM.ha-1 at harvest of planted crop, the 1st and 2nd ratoon crop, respectively (Figure 1). Simulation results displayed, according to D-stat ranking between 0.94 to 0.99 (Table 5), setting PARCEmax at 9 g.MJ-1 was the best fit for simulation of dry leaf and leaf sheath weight, dry stem weight and dry matter yield in the planted crop. However, setting at 12 g.MJ-1 gave the best simulation result as D-stat ranking between 0.60 to 0.65 and 0.70 to 0.72 in the 1st and 2nd ratoon crop, respectively. Particularly, the CSM-Canegro-Sugarcane model could not simulate early growth of napier grass in the 1st and 2nd ratoon crop (Figure 1) due to failed simulation of the continuous emergence of tiller on the transition between the planted crop and the ratoon crop. Although thermal time to emergence for a ratoon crop July 1-3, 2015
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(TTRATNEM) and thermal time from emergence to start of stalk growth (CHUPIBASE) were adjusted as 10 and 10 oC.d lower than 203 and 1,050 oC.d in sugarcane-suggested value (Table 2).
Figure 1 Observed dry matter yield and simulated dry matter yield at PARCEmax = 6, 9 and 12 g.MJ-1 at planted crop, the 1st and 2nd ratoon crop. Table 5 Simulated result and Index of agreement of dry leaf and leaf sheath weight, dry stem weight and total dry matter yield under varied maximum radiation conversion efficiency at harvest of planted crop, the 1st and 2nd ratoon crop.
Leaf and leaf sheath2 Stem Total matter yield Simulated D Simulated D Simulated D (kg DM.ha-1) (kg DM.ha-1) (kg DM.ha-1) 6 1,869 0.89 307 0.84 2,177 0.86 planted 9 3,324 0.99 547 0.94 3,871 0.98 crop 12 4,841 0.88 797 0.87 5,638 0.89 the 1st 6 237 0.51 39 0.54 276 0.49 ratoon 9 487 0.55 78 0.58 559 0.54 crop 12 777 0.62 127 0.65 904 0.60 the 2nd 6 699 0.54 114 0.57 813 0.54 ratoon 9 1,333 0.61 217 0.63 1,550 0.62 crop 12 2,043 0.70 333 0.72 2,376 0.70 PARCEmax is maximum (no stress) radiation conversion efficiency expressed as assimilate produced before respiration, per unit PAR. In st nd planted crop, the 1 and 2 ratoon crop respectively, observed dry leaf and leaf sheath weight was 2,890, 2,660 and 4,929 kg DM.ha -1, observed dry stem weight was 550, 470 and 870 kg DM.ha-1 and observed total dry matter yield was 3,440, 3,130 and 5,800 kg DM.ha-1. D is Index of agreement. Crop
PARCEmax (g.MJ-1)
Conclusion and Suggestion The ecotype and cultivar parameters in the CSM-Canegro-Sugarcane model could be adapted to simulate development and growth of planted crop satisfactorily. In order to simulate the ratoon crop, the ordinary adjustment of parameters in the model was not deserved. The model mechanism needs to be modified for cumulative tiller emergence in the transition between planted crop, the 1st and 2nd ratoon crop. Acknowledgements The author is grateful to the Energy Research and Development Institute–Nakornping, Chiang Mai University, especially, to collaboration of Assist. Prof. Dr. Pruk Aggarangsi for financial support. The author wish to express sincere thanks to Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, for providing plot experiment.
References Allen, R., G. Pereira,S .Luis, D. Raes and M. Smith. 1998. Crop evapotranspiration - guidelines for computing crop water requirements. Rome: Food and Agriculture Organization of the United Nations. Boote, K.J., J.W. Jones and N.B. Pickering. 1996. “Potential uses and limitations of crop models.” American Society of Agronomy. 88: 704-716. Cheokul, R. 2012. The renewable and alternative energy development plan for 25 percent in 10 years during 2012-2021. Retrieved 19 Mar, 2014, from http://www.dede.go.th/dede/images/stories/pdf/dede_aedp_2012_21.pdf DEDE. 2013. Napier grass. Department of Alternative Energy Development and Efficiency, Ministry of Energy, Thailand Retrieved 19 Mar, 2014, from http://weben.dede.go.th/webmax/content/napier-grass IBSNAT. 1993. The IBSNAT decade ten years of endeavor at the frontier of science and technology. Honolulu, Hawaii: Department of Agronomy and Soil Science, College of Tropical Agriculture and Human Resources, University of Hawaii. Jones, M. and A. Singels. 2008. DSSAT v4.5 Canegro sugarcane plant module. Mount Edgecombe, South Africa: International Consortium for Sugarcane Modelling. Phakamas, N., A. Jintrawet, A. Patannothai, P. Sringam and G. Hoogenboom. 2013. “Estimation of solar radiation based on air temperature and application with the DSSAT v4.5 peanut and rice simulation model in Thailand”. Agricultural and Forestry Meteorology 180: 182-193. Potijun, A., P. Viwutvongvana, and S. Wattana. 2003. Charaterization of established soil series in the north and central highland region of Thailand Reclassification According to Soil Texonomy 2003. 171p. Russell, J.S. and Webb, H.R. 1976. Climatic range of grasses and legumes and tropical grasses. Australian Journal of Experimental Agriculture and Animal Husbandry 42: 156-163. Singels, A., M. Jones and M. van den Berg. 2008. DSSAT v4.5 CANEGRO sugarcane plant module. Mount Edecombe: South African Sugarcane Research Institute. TMD. 2014. Daily statistics reports. CHIANG MAI AIRPORT weather Observation Station. Retrieved May 1, 2014, from http://www.awsobservation.tmd.go.th/web/reports/weather_days.asp Willmott, C. J. 1981. On the validation of model. Physical Geography 2(2): 184-194.
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O-2
Effect of Azolla (Azolla spp.) on Growth and Yield of Pathumthani 1 Rice (Oryza sativa L.) Teerawat SARUTAYOPHAT1*, Nattawan BUSSABA2 and Phanupong PHONCHAROEN2 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Graduate student of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kkteeraw@kmitl.ac.th
ABSTRACT Nitrogen is the element that most often insufficient and limited rice yield product while azolla contains the nitrogen-fixing endosymbiont, Anabaena azollae, so it should be used as nitrogen source for rice production. This study was conducted at the experimental field of the Department of Plant Production Technology, Faculty of Agricutural Technology, KMITL, Bangkok in 2013 to study the effect of azolla and chemical fertilizers on growth and yield of the Pathunmthani 1 rice. Randomized Complete Block Design (RCBD) with 3 replications was used. A 22-day seedlings were transplanted in space of 20x25 cm, 2 seedlings/hill with total of 10 hills/plot. Treatments were 9 fertilizer applications as follows: 1) a recommended fertilization (40 kg N/ha and 25 kg P2O5/ha), 2) and 3) inoculated azolla of 1,250 and 2,500 kg/ha on a transplanting date (TD), 4) and 5) incorporated fresh azolla of 6,250 and 12,500 kg/ha into soil for 5 days before transplanting date (DBT), 6) inoculated azolla of 1,250 kg/ha and incorporated of 6,250 kg/ha, 7) inoculated azolla of 1,250 kg/ha with 20 kg N/ha+25 kg P2O5/ha, 8) inoculated azolla of 1,250 kg/ha with 40 kg N/ha+25 kg P2O5/ha, and 9) a non-fertilization. Result showed that inoculated azolla 1,250 kg/ha with 40 kg N/ha+25 kg P2O5/ha had a significantly higher grain yield than other treatments (p<0.01) of 6,139.0 kg/ha. A recommended fertilization and inoculated azolla 1,250 kg/ha with 20 kg N/ha+25 kg P2O5/ha had an equally grain yield of 5,525.3 and 5,525.2 kg/ha, respectively. A sole inoculated azolla of 1,250 kg/ha had grain yield of 4,691.9 kg/ha, significantly higher than a non-fertilization which yield was 3,043.8 kg/ha. The results of this study indicated that inoculation azolla on a transplanting date for 1,250 kg/ha can increase grain yield of the Pathumthani 1 rice. Keywords: Azolla, Biofertilizer, Chemical fertilizer, Pathumthani 1 rice Introduction Azolla is a small free floating fern that contains the nitrogen-fixing blue-green algae (BGA), Anabaena azollae in the dorsal lobe cavity as a symbiotic association. The azolla-BGA association fixing atmospheric-N2 and supplying N to rice field is well documented. Azolla grew either as a dual crop along with rice in a flooded field or incorporated as green manure at the beginning of the growing season. It had been proved to be an important biological source in improving N balance in soil and increased rice straw and grain yield. (Bharati and Mohanti, 2000; Bhuvaneshwari, 2012; Ladha et al., 2000; Giller, 2002; Madiama and Paul, 2003). It can fix and supply 30-60 kg N/ha (EL-Shahat et al., 2006). While, Ladha et al. (2000) estimated of fixed and added N to rice field based on a 14-yr continuous doublecrop/yr for 64 kg N/ha/crop. Besides, supplying Ncan modify physic, chemical and biological
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properties of the soil and soil-water interface that are benefit to rice crop. For example, azolla reduces water temperature, suppresses weeds, curb NH3 volatilization loss, and mobilization of fixed phosphates. While, BGA liberate an extracellular organic compounds and photosynthetic-O2 during their growth (Pabby et al., 2003; Mandal et al., 1999). Although the benefits of azolla-BGA in rice field is well documented among agronomists, the practical implement to farmers are still limited. Last 50 years, farmers in tropical and subtropical increased their rice production by increasing application inorganic fertilizers, especially nitrogenous fertilizers. Present decade, farmers are facing to high and rising cost of inorganic fertilizers, and low price of their products due to high worldâ&#x20AC;&#x2122;s cereal stock (FAO, 2015). Therefore, it is time to focus on an alternative renewable resources in supplying N for rice crop. The aim of this study was to evaluate the effects of a sole azolla amounts and application methods, and combine applications with N, P2O5 fertilizers on growth and yield of lowland rice cv. Pathumthani 1. Materials and Methods Field experiment was conducted at the Department of Plant Production Technology, Faculty of Agricutural Technology, KMITL, Bangkok during Feb 4 to May 23 in 2013. The soil at the experimental site was sampling prior to carrying an experiment, characteristics of this clay soil showed high fertility, pH (5.6-5.8), organic matter (1.4-2.0%), available P (70.7244.6 ppm), soluble K (316.7-398.8 ppm), Ca (2,354.7-2,711.4 ppm) and Mg (1,890.91,932.2 ppm) (Table 1). Azolla pinnata was collected from natural habitat near KMITL and cultured for large amount at the Facultyâ&#x20AC;&#x2122;s reservoir. RCBD with 3 replications was used. Treatments were 9 azolla and chemical fertilizers applications as follows: 1) a recommended fertilization (40 kg N/ha and 25 kg P2O5/ha; 1st applied of 20 kg N/ha and 25 kg P2O5/ha at TD, the rest was applied at 30 days after transplanting date (DAT), 2) and 3) inoculated azolla of 1,250 and 2,500 kg/ha on a transplanting date (TD) , 4) and 5) incorporated fresh azolla of 6,250 and 12,500 kg/ha into soil for 5 days before transplanting date (DBT), 6) inoculated azolla of 1,250 kg/ha and incorporated of 6,250 kg/ha, 7) inoculated azolla of 1,250 kg/ha with 20 kg N/ha+25 kg P2O5/ha, 8) inoculated azolla of 1,250 kg/ha with 40 kg N/ha+25 kg P2O5/ha, and 9) a non-fertilization. A 22-day seedlings were transplanted for 2 seedlings/hill in space of 20x25 cm, with 7 hills/row, 6 rows/plot, and data were collected from 2 middle rows for 10 hills/plot. The following data: culm height, straw and grain yield, no. of panicle/hill, no of filled-unfilled grain/panicle were collected and statistically analysed. Table 1 Soil fertility characteristics of the experimental field. Level from soil surface pH OM P K (cm) (%) (ppm) (ppm) 0-20 5.8 2.0 244.6 398.8 20-40 5.6 1.4 70.7 316.7
Ca (ppm) 2,711.4 2,354.7
Mg (ppm) 1,890.9 1,932.2
Results and Discussion The effect of fertilizers on straw and grain yield, and no. of panicle/hill of rice cv. Pathumthani 1 were significant (Table 2, 3). Inoculated azolla of 1,250 kg/ha with 40 kg N/ha+25 kg P2O5/ha had highest grain yield of 6,139.0 kg/ha, higher than a recommended chemical fertilizers which yield was 5,525.3 kg/ha for 39.9%. The non-fertilizer had lowest grain yield of 3,043.8 kg/ha, lower than inoculated azolla of 1,250 kg/ha (4,691.9 kg/ha), and incorporated of 6,250 kg/ha (3,917.1 kg/ha) for 54.1 and 28.7%, respectively. Similar result was reported by Anonymous (2015) that rice yield from inoculated azolla 300 kg/ha and recommended chemical fertilizers were non-significant difference. Page 62
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Table 2 Growth and yield of Pathumthani 1 rice as resulted of azolla and chemical fertilizers. Treatment Culm ht Yield (kg/ha) Relative App. rate grain (cm) Straws Grain yield (%) (kg/ha) Fertilizer1, N+P2O5 Azolla, inoc.2 Azolla, inoc.2 Azolla, incorp.3 Azolla, incorp.3 Azolla, inoc.2+ incorp.3 Azolla, inoc.2+N+P2O5 Azolla, inoc.2+N+P2O5 Non-fertilizer F-test C.V.(%)
40+25 1,250 2,500 6,250 12,500 1,250+6,250 1,250+20+25 1,250+40+25 0+0
78.6 77.0 74.1 75.9 78.7 76.8 74.5 81.6 77.2 NS 4.6
11,040ab 8,290cd 8,690bed 7,760cd 11,600a 8,430cd 9,680abc 10,000abc 6,290d ** 15.8
5,525.3b 4,691.9cd 4,788.6c 3,917.1e 4,140.5de 4,593.0cd 5,525.2b 6,139.0a 3,043.8f ** 9.9
181.5 154.1 157.3 128.7 136.0 150.9 181.5 221.4 100.0
1
Ammonium phosphate at transplanting date and urea at 30 DAT, 2 inoculated azolla to flooded field plot in transplanting date Incorporated fresh azolla into soil for 5 days before transplanting date, NS. non-significant difference , ** significant difference at p <0.01 Means within the same column followed by different letter indicate significant difference between treatments by LSD. 3
Table 3 Yield components of Pathumthani 1 rice as resulted of azolla and chemical fertilizers Treatment No.of No.of filled No. of Percentage App. rate Panicle/ grain/panicle unfilled of filled (kg/ha) hill grain/panicle grain (%) Fertilizer1,N+P2O5 Azolla, inoc.2 Azolla, inoc.2 Azolla, incorp.3 Azolla,incorp.3 Azolla,inoc.2+ incorp.3 Azolla,inoc.2+N+P2O5 Azolla,inoc.2+N+P2O5 Non-fertilizer F-test C.V.(%)
40+25 1,250 2,500 6,250 12,500 1,250+6,250 1,250+20+25 1,250+40+25 0+0
21.50ab 21.63ab 22.60ab 23.30a 27.83a 21.95ab 25.37a 23.70a 15.87b
96.80 95.83 97.40 84.30 84.47 96.50 102.57 104.57 94.47
35.13 26.93 23.33 32.47 33.23 37.20 31.97 29.86 27.20
* 18.6
NS 15.0
NS 25.3
73.4 78.1 80.7 72.2 71.8 72.2 76.2 77.8 77.6
1
Ammonium phosphate at transplanting date and urea at 30 DAT, 2 Inoculated azolla to flooded field plot in transplanting date Incorporated fresh azolla into soil for 5 days before transplanting date, NS. non-significant difference, * significant difference at p <0.05 Means within the same column followed by different letter indicates significant difference between treatments by LSD. 3
Straw yields of inoculated and incorporated with a low rate methods were non-significantly higher than a non-fertilization, indicated well N balancing in soil of an azolla application treatments. Along the crop, some azolla renewed generations while some died, decomposed and released N gradually. Ventura et al. (1987) reported that 70% of N contents in dead azolla became available to rice within 20 days. However, culm height, no. of filled and unfilled grain/panicle were non-significant difference. The non-promoting culm height was other benefit of azolla concerning to lodging percentage.
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Conclusion Azolla application either inoculated to flooded field or incorporated into soil enhanced growth and yield of lowland rice cv. Pathumthani 1. Inoculation at transplanting period and cultured along with rice in a flooded field showed higher efficiency in increasing yield of Pathumthani 1 rice than incorporated application method. These results confirmed the benefits of azolla application in lowland rice production. Acknowledgments The authors are grateful to the Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang for financial support to this research. Also, thanks to Mr. Teerawat Riengruksa, Miss Donruthai Ditsapong and Miss Thisawan Moonpradap for their practical assistance in the field works and collecting data.
References Anonymous. 2015. The use of azolla instead of urea in rice production. Online, Available; www.ubonratchathani.doae.go.th/.../n_deang.pdf (5 June, 2015). Bharati, K. and S.R. Mohanty. 2000. Influence of incorporation or dual cropping of Azolla on methane emission from a flooded alluvial soil planted to rice Eastern India. Agric. F.co. and Environ. 79:73-83 Bhuvaneshwari K. 2012. Beneficial effects of blue-green algae and Azolla in rice culture. Journal of Environment Conservation 13(1&2) 1-5. EL-Shahat R.M., M.G.Z. Girgis, Al-ShaymaaI Ahmed and E.A. Saleh. 2006. Response of three rice varieties to conjugated applications of Azolla pinnataand different rates of urea. Journal of Annals Agric. Sci. 51(2), 373-381. FAO. 2015. World food situation; cereal supply and demand brief. Online, Available; http://www.fao.org/worldfoodsituation/csdb/en/ (5 June, 2015). Gillcr K.E. 2002. Nitrogen Fixation in Tropical Cropping Systems. pp. 63-84 Commonwealth As sociation of Biologists International Pub. Wallingford England. Ladha J.K., D. Dawa, T.S. Ventura, U. Singh, W. Ventura and I. Watanabe. 2000. Long-term effects of urea and green manure on rice yields and nitrogen balance. Journal of Soil Sci. Soc. Am. 64: 1993-2001. Madiama, C. and L.G.V. Paul. 2003. Influence of urea on biological N2-fixation and N Transfer from Azolla intercropped with rice. Plant and Soil 263:311-321. Mandal B., P.L.G. Vlek and L.N. Mandel. 1999. Beneficial effects of blue-green algae and Azolla, excluding supplying nitrogen, on wetland rice fields: a review. Journal of Biol. Fert. Soils 28: 329-342. Mishra A. K. and K.N. Mishra. 2007. Use of Azolla pinnataas biofertilizer for the production of rice pant-4 in Jaunpur District (U.P.), India. Journal of Plant Archives. Vol.7 No.1, pp.313-316. Pabby A., P. Radha and P.K. Singh. 2003. Azolla-anabaena symbiosis from traditional agriculture to biotechnology. Journal of Indian Journal of Biotechnology Vol. 2, pp.26-37. Ventura W., G.B. Mascarina, R.E. Furoc and I. Watanabe. 1987. Azolla and Sesbania as biofertilizers for lowland rice. Journal of Philipp J. Crop Sci. 12(2): 61-69.
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O-4
Allelopathic Effects of Pisonia grandis R.Br. on the Germination and Growth of Echinochloa crus-galli (L.) Beauv. Sudteerak SAIPLUEMCHIT1 and Chamroon LAOSINWATTANA1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: klchamro@kmitl.ac.th
ABSTRACT Pisonia grandis R.Br. (lettuce tree) is a famous landscaping plant in tropical garden because of a beautiful color and rough texture of leaves. In landscape design, there are various species of plants growing in the same ecology. This study verified the allelopathic effect of lettuce tree dry leaves on germination and seedling growth of Echinochloa crus-galli (L.) Beauv. (barnyard grass) with three experiments conducted in laboratory and greenhouse. First experiment was compared between dry leaf powder and aqueous extract, dry leaf power (125, 250, 375 and 500 mg/plate) was more inhibitory than aqueous extracts at equal concentration. Next experiment was conducted in greenhouse, the comparison of dried leaf powder at different rate of 0, 1, 2, 4 and 6 ton/ ha mixed with sandy loam. It was found that 6 ton/ha gave the significant inhibitory effect on plant height compared with the other concentrations. Last experiment, the dry leaf powder was incorporated into the soil at a rate of 5, 10, 15 and 20 gDW/100g Soil for 0, 1, 2, 3 and 4 weeks. Inhibitory effect on emergence of barnyard grass various depended on the amount of dry leaf powder incorporated and incubating periods. In corporation of dry leaf powder at 15 and 20 gDW/100g soil of 2 weeks showed the most inhibitory effect on emergence of barnyard grass. The results here are providing more knowledge about the allelopathic potential of lettuce tree and can be a useful tip for selecting and grouping the right plants for landscape. Keywords: Allelopathy, Lettuce tree, Germination, Seedling growth, Landscaping plant Introduction Pisonia grandis R.Br. (lettuce tree) belongs to the Nyctaginaceae family and it is well known for ornamental plant and medicinal values (Sripathi et al., 2011). In Thailand, lettuce tree is a one of a famous landscaping plant in many garden styles such as tropical garden. Allelopathy is the ability of plant to release some chemical compounds that have an impact on the development and/or growth of other plants or species (Weston, 1996). There are many studies of allelopathic from plants residues such as allelopathic of Helianthus tuberosus L. on the germination and growth of Echinochloa crus-galli (L.) Beauv. (Tesio et al., 2012), potential of dried powder of Tagetes minuta as a natural herbicide on rice weeds (Batish et al., 2007) and effects of Ambrosia artemisiifolia L. in the invasive process (Vidotto et al., 2013). The purpose of this study was to investigate the allelopathic effect of lettuce tree in terms of weed control and effect of plant growth on another landscape plants under their canopy. Materials and Methods
Plant Materials Lettuce tree leaves were collected around KMITL Ladkrabang, Thailand. The mature and healthy leaves were rinsed with tap water, dried in hot-air oven at 45Ë&#x161;C and ground to powder (100 mesh) in electrical blender. Barnyard grass (Echinochloa crus-galli (L.) Beauv.) was selected as representative of bioassay species. Their seeds were collected from paddy rice July 1-3, 2015
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field at Ladkrabang, Thailand. Barnyard grass seeds were placed at room temperature for 3 months and incubated at 45Ë&#x161;C in hot-air oven for 3 days to break their dormancy. Aqueous extract and dry leaf powder bioassay in laboratory Aqueous extract of lettuce tree was prepared by soaking 10 g of dried leaf powder in 100 mL distilled water for 4 day at 8Ë&#x161;C, and then filtration to remove any residues. The lettuce tree aqueous extract was diluted with distilled water to obtain 2.5, 5, 7.5 and 10 mg/mL (125, 250, 375 and 500 mg/Petri dish). Five milliliters of each concentration (treatment) was added into each 9 cm diameter of Petri dish, placed with 2 layers of filter paper. Another set of Petri dish was placed with dry leaf powder of lettuce tree at the rate of 125, 250, 375 and 500 mg/Petri dish then 5 mL of distilled water was added. Only distilled water was added into the Petri dish and used as control treatment. Twenty uniform seeds of barnyard grass were placed in each Petri dish. Four replicates were performed for each treatment in a completely randomized design in a growth chamber. Germination and seedling growth (shoot and root lengths) were measured at seven days after treatment. Dry leaf powder bioassay in pots This experiment was conducted in greenhouse of Faculty of Agricultural Technology KMITL Ladkrabang, Thailand, using plastic pots (10 cm diameter) filled with sandy loam. Dry leaf powder at the rates of 1, 2, 4 and 6 ton/ha were incorporated into top soil of each pots. The leaf residue extracted with ethanol was used as check and sandy loam alone was used as control. Twenty uniform seeds of barnyard grass were placed in each pot. Four replicates were performed for each treatment in a completely randomized design. Emergent plant was counted at seven days, plant height was measured at 7, 14, 21 and 28 days after treatment. Decomposition period of dry leaf powder in soil Dry leaf powder of lettuce tree at rate of 5, 10, 15 and 20 g DW/100g Soil was incorporated into the soil and placed at room temperature for 0, 1, 2, 3 and 4 weeks. After incubation periods, the soil was taken out from bottles to 9 cm diameter petri dishes and twenty uniform seeds of barnyard grass were placed in each experimental unit. Soil alone was served as control. Germination and seedling growth (shoot and root lengths) were measured at seven days after sowing. Results and Discussion Aqueous extract and dry leaf powder bioassay in laboratory The dry leaf powder of lettuce tree was more effect on seed survival and seedling growth than aqueous extract at 250 and 375 mg/plate. However, at 500 mg/plate of dry leaf powder and aqueous extract were completed inhibitory on seed survival and seedling growth (Figure 1a1c). Similarly, an inhibitory effect of aqueous extract from Tagetes minuta at concentration of 4% w/v on germination and growth of barnyard grass has been reported (Batish et al., 2007). Dry leaf powder bioassay in pots The result of dry leaf powder in pots showed that the inhibition effect on seedling emergence and plant height was increased as the increasing concentrations (Figure 2a-2b). Plant height were reduced to 36.38, 35.45, 34.27, 31.34, 29.39 and 29.1 cm by the treatments of control, check, 1, 2, 4 and 6 t/ha were, respectively (Figure 2b). In the similar to Batish et al., (2007) reported at 0, 1, 2, and 4 t/ha of Tagetes minuta dried leaf powder in field pots were effected to plant height of barnyard grass reduced to 66.5, 52.1, 44.8 and 27.5 cm, respectively.
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Survival %
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Figure 2 Allelopathic potential of dry leaf powder of lettuce tree on seed emergence and plants height of barnyard grass in pots. Decomposition period of dry leaf powder in soil The germination and seedling growth of barnyard grass were affected by the amount of dry leaf powder and decomposition periods (Figure 3a). The results indicated that germination and seedling growth of barnyard grass decreased with increasing the dry leaf powder rates. But the degree of inhibition decreased as the composition periods progressively increased from 0 to 4 weeks. This finding is in agreement with Teerarak et al. (2010) reported allelopathic effect of Spanish jasmine decreased according to the decomposition period length.
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Figure 3 Allelopathic potential of dry leaf powder of lettuce tree incorporated into the soil on seed germination and seedling growth of barnyard grass. Conclusion The results of this study had increased information of Pisonia grandis R.Br. (lettuce tree) on the allelopathic potential in terms of weed control and ability of landscaping plant. Dry leaf powder had more effect on seedling survival and seedling growth than aqueous extract in equal concentration. At 6 ton/ha gave the significant inhibitory effect on plant height compared with the other concentrations. Additionally, inhibitory effect on emergence of barnyard grass varied depending on the amount of dry leaf powder and incubating periods. Dry leaf powder at 15 and 20 g DW/100g incorporated with 2 week-incubated soil showed the highest inhibition effect on emergence of barnyard grass. Acknowledgements The authors wish to thank King Mongkut â&#x20AC;&#x2122;s Institute of Technology Ladkrabang for providing financial support for this research. References Batish, D. R., K. Arora, H. P. Singh and R.K. Kohli. 2007. Potential utilization of dried powder of Tagetes minuta as natural herbicide for managing rice weeds. Crop Prot. 26:566-571. Jung, W.S., K.H. Kim, J.K. Ahn, S.J. Hahn and I.M. Chung. 2004. Allelopathic potential of rice (Oryza sativa L.) residues against Echinochloa crus-galli. Crop Prot. 23:211-218. Sripathi, S.K. et al.2011. HPTLC fingerprinting of extracts of Pisonia grandis (R.Br.). IJPSR. 2 (9):180-183. Teerarak, M., Laosinwattana C., Charoenying P. 2010. Evaluation of allelopathic, decomposition and cytogenetic activities of Jasminum officinale L. f. var. grandiflorum (L.) Kob. on bioassay plants. Bioresour. Technol. 101, 5677-5684. Vidotto, F., Tesio F. and A. Ferrero. 2013. Allelopathic effects of Ambrosia artemisiifolia L. in the invasive process. Crop Prot. 54:161-167. Weston, L.A. 1996. Utilization of allelopathy for weed management in agroecosystems. Agronomy Journal 88(6):860-866. Page 68
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How Far Teak Plantations Can Contribute for Climate Change Mitigation and Landscape Restoration in Tropics: A Case Study from Central India Singam Laxmana SWAMY1* and Alka MISHRA1 Guru Ghasidas Vishwavidyalaya (A Central University), Bilaspur, Chhattisgarh, 495 009 India *Corresponding email: swamy_101@ yahoo.com
ABSTRACT One of the major challenges facing by the scientific community of the world today is how to stabilize the increasing levels of greenhouse gases in the atmosphere and reduce the negative impacts of climate change. Intergovernmental Panel on Climate Change (IPCC, 2014) advocated a viable strategy of mitigation and adaptation to climate change through enhancing the carbon (C) pools through via afforestation, reforestation, and agroforestry practices. Tree plantations have great scope and opportunity not only for C sequestration but also restore the degraded landscapes and developing resilient ecosystems. Tectona grandis popularly known as teak, a member of Lamiaceae is grown under commercial plantations in Central and Southern India owing to its excellent quality timber and high economic returns. A study was conducted in age sequence plantations of Teak grown as monoculture and agroforestry in Chhattisgarh state, Central India with an aim to investigate its potential for C sequestration and ameliorating the degraded landscapes. The findings revealed that biomass, C storage and nutrient status (N, P and K) were significantly higher in plantations than agroforestry and further increased by the age. The C storage ranged from 4.5 Mg ha-1 to 62.6 Mg ha- in plantations, and from 2.2 Mg ha-1 to 31.5 Mg ha-1 under agroforestry in 10 year old and 40 year old stands, respectively. Litter fall and N, P and K status were higher in soils under plantations compared to agroforestry. In conclusion, the sole teak plantation should be more beneficial for C sequestration and soil amelioration than agroforestry practices. Keywords: Agroforestry, Biomass, C sequestration, Litter fall, Nutrient storage Introduction One of the major challenges facing by the scientific community of the world today is how to stabilize the increasing levels of greenhouse gases in the atmosphere and reduce the negative impacts of climate change. Intergovernmental Panel on Climate Change (IPCC, 2014) advocated a viable strategy of mitigation and adaptation to climate change through enhancing the carbon (C) pools through via afforestation, reforestation, and agroforestry practices. There are several studies conducted in the past identified the promising species for plantations in tropics to secure ecological and economic benefits (Swamy and Puri, 2005; Sreejesh et al., 2013, Jha, 2014). Teak (Tectona grandis Linn f.) is one of the most promising timber species ranks among top five tropical hardwoods and being promoted for use in tropical plantations in Asia, Africa, and Latin America. Due to rising demand of teak wood products, the plantations are rapidly expanding as pure and agroforestry systems thus creating an opportunity for huge C sequestration in future. Since teak is a long rotation species and retention of C in teak plantations and its products for lasts for longer periods, therefore it has an advantage for C sequestration. There are few studies made on monoculture young plantation of Teak to assess the C sequestration potential but comparative studies are lacking. Therefore, the present investigation was aimed to comparatively evaluate the biomass, C storage and soil nutrients (N, P and K) under sole and agroforestry stands.
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Materials and Methods
Study area The study was conducted in Shivtarai, Kota area of Bilaspur district, Chhattisgarh, India. The study area fall in part of buffer zone of Amarkantak- Achanakmar biosphere reserve located between 220 15' to 220 58' North latitude and 810 25' to 820 5' East longitude. The climate is sub-humid tropical with an average rainfall of 1400Âą55 mm. The soils are degraded in nature with low fertility and belong to Alfisols and Inceptisols. Biomass estimation The study was conducted in three age sequences viz., 0-10 yr old, 11-20 yr old and 21-30 yr stands of Teak plantations grown by state Forest Development Corporation, Chhattisgarh and by farmers under agroforestry (on rice field bunds). The quadratic sampling procedure was adopted to enumerate the trees by randomly laying five plots of 20 * 20 m in sole plantations, while 100 m line transect was laid in agroforestry stands. Diameter at breast height (DBH) and total height of the trees were measured. Allometric regression equations developed by Buvaneswaran et al., 2006 was used for the estimation of component wise biomass of different age sequence of Teak plantations. The component wise biomass i.e. leaf, branches, bark, bole and roots were added/summed to derive total biomass in each age sequence plantations. Standing litter crop was measured by randomly laying three 1 x 1 m quadrates on forest floor in each marked sample plot (20 x 20 m) of age sequence plantation. C storage in Teak plantations and soil nutrients Carbon content of Teak biomass was estimated using the C concentration values in different components of Teak as reported by Sreejesh et al. 2013. The organic C, available N, P and K in soil were determined at 0-20cm and 20-40 cm soil depths by standard analytical methods suggested by Jackson 1967. Data on biomass, C and nutrient storage was analyzed by ANOVA methods suggested by Steel and Torrie (1983). Results and Discussion The biomass estimates of Teak revealed that all tree components viz., leaf, stem, branch, root and total biomass varied significantly (p < 0.05) among age sequences in different types (Table 1). In 20-30 year old stands, the total biomass was 142.6 Mg ha-1 and 72.6 Mg ha-1, in sole and agroforestry plantation, respectively. The biomass accumulation was more in plantation, which was 43.1%-83.8% higher than stands raised under agroforestry system. The difference was declined with an increase in age of plantation. In 20-30 year old stands, the leaves, stem, branches, bark, roots and litter contributed to total biomass between 3.6-3.9%, 26.9-52.3%, 10.6-13%, 4.2-8.3%, 11.1-18.7% and 3-3.9%, in sole and agroforestry stands, respectively. The higher root:shoot ratios were recorded in stands under agroforestry than sole plantations. The present findings on biomass are well within the range and consistent with reports of earlier workers (Buvaneswaran et al. 2006 and Jha, 2015). The higher rootshoot ratios in Teak stands under agroforestry might attributed tree-crop competition as it was also demonstrated by Swamy and Puri (2005) in Gmelina arborea stands. Carbon storage in different components of Teak showed significantly (P < 0.05) variation due to differences in age of the stands under sole and agroforestry (Table 2). The C storage increased with age of the stand and relative increase was higher in sole than agroforestry stands. The C storage was almost 2-5 times higher in sole than agroforestry stands. In 20-30 yr-old stands, the total C storage varied from 31.5 to 62.5 Mg ha-1. Plantation had highest C, which was almost 50% higher than agroforestry system. Carbon storage in different components followed the order: Stem>Root>Branches>Bark>Litter> Foliage (Table 2). C storage is relatively higher in above ground components than below ground components in sole compared to agroforestry stands. The findings on C storage are comparable and consistent with the findings of Sreejesh et al., 2013 and Jha, 2015. The higher C storage in Page 70
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sole plantations is mainly attributed to higher stand density, which was almost 5-15 times higher than agroforestry stands. The C storage in Teak plantation was lower to those reported by Sreejesh et al., (2013). The degraded site conditions coupled with low rainfall were mainly responsible for poor growth of Teak stands thus resulted in lower C storage in plantations. Available N, P, K and organic C contents in soil increased with age of the plantation (Table 3). The nutrients decreased with an increase in soil depth. In 20-30 year old stands, the levels of organic C ranged from 18.3 to 33.2 Mg ha-1, N from 290.3 to 314.5 Kg ha-1, P from 14.6 to 16.4 Kg ha-1 and K from 434.1 to 482.8 Kg ha-1. The higher soil nutrients in sole compared to agroforestry stands might be resulted as a consequence of higher litter accumulation in former than later stands. The possible reason for higher nutrient enrichment is due to more amount and better quality in sole Teak stands. Tree-crop competition might cause poor litter crop in agroforestry stands and also repeated tillage could be responsible for loss of soil nutrients. The present findings are in agreement and very well supported with the reports of Swamy and Puri, (2005) and Jha (2014). Conclusion The present study demonstrated that Teak plantation grown as sole system accumulated significant carbon in living biomass, as well as soil carbon, indicating the potential to offer the environmental service of carbon sequestration. Besides, the Teak plantations contributed significantly for soil amelioration by enriching the status of nutrients especially N, P, K and C, and thus suggested to exploit its potential for restoration degraded landscapes of tropics. Acknowledgements This work was financially supported by NRSA, Hyderabad, India. Authors are grateful to Forest Department of Chhattisgarh, India for providing necessary support during the field work. References Buvaneswaran, C., D. Perez and M. Kanninen. 2006. Biomass of Teak plantations in Tamilnadu, India and Costarica compared. Journal of Tropical Forest Science. 18(3): 195â&#x20AC;&#x201C;197. Jackson, M.L. 1967. Soil chemical analysis. Prentice Hall Inc. Englewood cliffs. N.J., U.S.A. Jha, K.K. 2014. Temporal patterns of storage and flux of N and P in young Teak plantations of tropical moist deciduous forests. India. J. For. Res. 25:75-86. Jha, K.K. 2015. Carbon storage and sequestration rate assessment and allometric model development in young teak plantations of tropical moist deciduous forest. India. J. For. Res. DOI 10.1007/s11676-015-0053-9. Sreejesh, K.K., T.P. Thomas, P. Rugmini, K.M. Prashant and P.K. Kripa. 2013. C sequestration potential of Teak (Tectona grandis) plantations in Kerala. Res. J. Recent Sci. 2:167-170. Steel, R.G.D. and J. H. Torrie. 1980. Principles and procedures of statistics. 2nd ed. McGraw Hill, New York. Swamy, S.L. and S. Puri. 2005. Biomass production and C-sequestration of Gmelina arborea in plantation and agroforestry system in India. Agroforestry Systems 64:181-195.
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AFP 0.11 1.09 2.62 0.14
Foliage SP 0.46 2.55 5.51 0.24
AFP 0.24 1.67 7.67 0.87
Branch SP 0.97 7.71 18.5 2.71
AFP 0.34 1.92 5.96 0.41
Bark SP 1.52 7.08 11.81 1.89
Bole SP 3.61 37.44 74.63 2.34
AFP 0.65 17.5 38.4 3.68
Branch SP 0.37 3.24 7.77 2.34
Bark SP 0.61 2.83 4.77 1.46
AFP 0.13 0.77 1.91 0.54
Bole SP 1.62 17.22 33.58 6.28
AFP 0.41 7.75 17.28 4.52
Root SP 3.02 15.4 26.63 1.82
Root SP 1.36 6.93 11.98 3.83
AFP2 15.3 17.6 18.9 2.89
AFP 13.3 16.5 17.8 2.89
C (Mg ha-1) 20-40 cm SP 19.8 29.2 32.2 4.62
AFP 221.8 262.2 294.5 11.6
N (Kg ha-1) 0-20 cm SP 248.5
271.8 321.3 12.8
AFP 208.2 223.4 286.2 14.2
N (Kg ha-1) 20-40 cm SP 211.1 234.5 307.8 15.7
AFP 11.1 12.4 15.4 1.8
P (Kg ha-1) 0-20 cm SP 11.36 17.2
13.5 2.1
SP 10.5 12.6 15.7 1.5
AFP 0.14 0.89 2.21 0.52
Litter
SP 1.28 2.86 5.56 1.76
AFP 0.04 0.36 0.88 0.19
SP 421.2 457.4 484.5 24.7
Total
SP 406.7 446.4 481.2 22.3
AFP 1.92 28.69 72.64 4.6
AFP 0.92 13.1 31.5 8.18
AFP 389.7 429.7 421.5 17.8
K (Kg ha-1) 20-40 cm
SP 4.64 32.3 62.55 11.93
Total
SP 11.86 73.04 142.64 6.22
AFP 411.4 432.1 446.7 19.3
K (Kg ha-1) 0-20 cm
Litter
SP 0.51 1.14 2.32 0.94
AFP 10.1 11.7 13.8 1.4
P (Kg ha-1) 20-40 cm
AFP 0.18 3.12 6.75 1.89
AFP 0.44 5.62 15.78 2.89
Table 1 Component wise biomass in sole and agroforestry plantations of Tectona grandis (Mg ha-1). Age sequence (Yr) 0-10 10-20 20-30 LSD at 5%
AFP 0.05 0.41 0.99 0.22
Foliage SP 0.17 0.97 2.09 0.36
AFP 0.11 0.71 3.46 0.56
Table 2 C stocks in sole and agroforestry plantations of Tectona grandis (Mg ha-1). Age sequence (Yr) 0-10 10-20 20-30 LSD at 5%
SP1 22.5 31.2 34.2 4.62
C (Mg ha-1) 0-20 cm
Table 3 N, P, K and C storage in under sole and agroforestry plantations of Tectona grandis. Age sequence (Yr) 0-10 10-20 20-30
= Sole plantation (SP), 2 = Agroforestry plantation (AFP)
LSD at 5% 1
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O-6
Antioxidant properties of flower extract of Etlingera elatior (Jack) R.M. Smith Kanokporn CHANGSAWAKE1, Komkhae PILASOMBUT2, Chamroon LAOSINWATTANA1 and Montinee TEERARAK1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: ktmontin@kmitl.ac.th
ABSTRACT Assessment of antioxidant properties of aqueous and ethanol extract from unopened flower of Etlingera elatior (Jack) R.M. Smith (torch ginger) was assayed by using DPPH free radical scavenging assay, metal chelating activity, reducing power, inhibition of lipid peroxidation and total phenolic contents. The results found that the values of half maximal inhibitory concentration (IC50) of DPPH radical were 603.04 ppm in aqueous extract and 2,520.61 ppm in ethanol extract while the IC50 values of the reference standard ascorbic acid (vitamin C) and butylated hydroxyl toluene (BHT) were 3.16 ppm and 21.82 ppm, respectively. In metal chelating activity, the IC50 values were 3,689.57 ppm in aqueous extract and 6,127.20 ppm in ethanol extract while the IC50 values of the reference standard ethylene diamine tetraacetic acid (EDTA) was 18.32 ppm. Moreover, the ability of reducing power and inhibition of lipid peroxidation increased as the concentrations of aqueous and ethanol extract from Torch Ginger increased. The total phenolic contents recorded were 723.43 and 236.61 mg GAE/100g DW in aqueous and ethanol extract, respectively. The results concluded that aqueous torch ginger flower extract was more potent in DPPH radical scavenger and metal chelating activity than ethanol flower extract. The aqueous and ethanol extract might not be directly involved in reducing power and lipid peroxidation. Keywords: Antioxidant properties, Etlingera elatior, Extract, Total phenolic contents Introduction Natural antioxidants are mainly found in fruits, vegetables and marine plants. It’s a substance capable of preventing or slowing the oxidation in the body and a molecule which stabilizes a free radical either donating an electron or by transferring a hydrogen atom (Cosentino and Fernandez, 2011). Torch ginger flower (Etlingera elation (Jack) R.M. Smith) belongs to the family Zingiberaceae and is widely used as an ornamental plant (Chainok, 2013). The showy pink flowers are used in decorative arrangements, while the flower buds are an important ingredient in foods. In Thailand, it is eaten in a kind of Thai salad preparation. The objective of this study was to evaluate the antioxidant properties of aqueous and ethanol flower extract of Torch ginger. Materials and Methods Preparation of the extracts Plant samples were collected in Bangkok, Thailand. Dried Torch ginger flower was broken into small pieces using a cylindrical crusher, and extracted with aqueous solution or ethanol. Extracts were then filtered through a filter paper (Whatman No.1) and stored in refrigerator for further analysis. July 1-3, 2015
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DPPH radical scavenging activity
DPPH free radical scavenging capacity of flower extract of Torch ginger was determined by the slightly modified method of Brand Williams et al. (1995) and expressed as IC50 value, which is the extract concentration providing 50% radical scavenging capacity. Ascorbic acid (vitamin C) and butylated hydroxytoluene (BHT) were used as standards. Iron chelating activity The iron chelating activity assay was determined by the slightly modified method of Benzie (1996) and expressed as IC50 which is the extract concentration providing 50% chelating ferrous ions rate. Ethylenediaminetetraacetic acid (EDTA) was used as reference standards. Lipid peroxidation inhibitory activity Inhibition of lipid peroxidation in the egg yolk was determined using a modified method from Kuppusamy et al. (2002) and expressed as IC50 value, which is the extract concentration providing 50% inhibition. Vitamin C was used as a standard. Reducing power activity The reducing power was determined using the method described previously by Oyaizu et al.(1986) and expressed as EC50 value which is the extract concentration providing absorbance of 0.5. Vitamin C was used as a standard. Total phenolic contents The total phenolic contents were determined using the spectrophotometric method a modified by Meda et al. (2005) and expressed as mg GAE/100 g DW. Statistical analysis All data of antioxidant activity tests are the average of triplicate analysis. DPPH radical scavenging activity
Results and Discussion
100 80 60 40 20 0
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The DPPH radical scavenging capacity of aqueous and ethanol extract from torch ginger had the IC50 values of 603.04 and 2,520.61 ppm, respectively. The IC50 value of vitamin C and BHT (standard) was found to be 3.16 ppm and 21.82 ppm, respectively (Figure 1). The lower IC50 gave the higher the antioxidant capacity of the extract. Similarly, determination of the antioxidant properties of Teucrium parviflorum Schreber flower showed higher scavenging capacity of ethanol extract than water extract (Türkoğlu et al., 2010). 100 80 60 40 20 0
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Figure 1 DPPH radical scavenging activity of aqueous and ethanol extract from torch ginger, and standards vitamin C and BHT. Iron chelating activity The chelating of Fe2+ of torch ginger flower had the IC50 values of 3,689.57 ppm in aqueous extract and 6127.20 ppm in ethanol extract which is lower than EDTA (IC50 = 18.32 μg/ml) (Figure 2). In contrast, the ethanol extracts of seeds of Monodora myristica greatly chelated Fe2+ than the aqueous extract (Ogunmoyole et al., 2013).
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Ferrous chelating activity (%)
80 60 40 Aqueous extract Ethanol extract
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Figure 2 Metal chelating ability of aqueous and ethanol extract from torch ginger and standards EDTA. Inhibition of lipid peroxidation Inhibition of lipid peroxidation increased as the concentrations of aqueous and ethanol extract increased. The IC50 value of vitamin C was found to be 623.72 ppm (Figure 3). Similarly, inhibition of lipid peroxidation of alcoholic extract of Medicago sativa increased as extract concentrations increased (Rana et al., 2010). 60
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Figure 3 Effect of aqueous and ethanol extract from torch ginger inhibition of lipid peroxidation and standards vitamin C. Reducing power The ability of reducing power increased as the concentrations of aqueous and ethanol extract from torch ginger increased. The IC50 value of vitamin C was found to be 45.53 ppm (Figure 4). Similar evaluation of reducing power of extract was reported by Padmanabhan and Jangle (2012), the alcoholic extract from leaves of Aloe vera, Bacopa monniera, Moringa oleifera and rhizome of Zingiber officinale showed increase as the concentrations of extract increased. 0.8
Absorbance at 700 nm
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Figure 4 Reducing power (as absorbance at 700 nm) of aqueous and ethanol extract from torch ginger and standards vitamin C. Total phenolic contents The total phenolic content of aqueous and ethanol extract from torch ginger were found to be 723.43 and 236.61 mg GAE/100g DW, respectively. The aqueous extract was found to contain the higher amount of phenol content as compared to aqueous extract (Table 1). Phenolic compounds are a class of antioxidant compounds which act as free radical terminators (Shahidi and Wanasundara, 1992).
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Table 1 The amount of total phenolic compound of aqueous and ethanol extract from torch ginger. Total phenolic contents Extracts (mg GAE/100g DW) Aqueous extract 723.43 Ethanol extract 236.61 GAE = Gallic acid equivalents. Each experiment was performed in triplicate and the results were mean ± SD. Conclusion The results concluded that aqueous torch ginger flower extract was more potent in DPPH radical scavenger and metal chelating activity than ethanol flower extract. The aqueous and ethanol extract might not be directly involved in reducing power and lipid peroxidation. Acknowledgments The authors gratefully acknowledge funding for this research from Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Thailand in the year of 2015 (B.E. 2558). Reference Benzie, I.F.F. and J.T. Strain. 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power, the FRAP assay. Anal Biochem. 239:70-76. Brand-Williams, W, M.E Cuvelier and C. Berset. 1995. Use of free radical method to evaluate antioxidant activity. Lebensm.-Wiss. u.-Techno. 28:25-30. Chainok, K. 2013. Torch ginger flower. Faculty of Pharmacy, Mahidol University. http://www.pharmacy.mahidol.ac.th. Cosentino, G.D. and J.R.M. Fernandes. 2011. Ecto-phosphatases in protozoan parasites: possible roles in nutrition, growth and ROS sensing. J. Bioenerg Biomembr. 43:89-92. Kuppusamy, U.R., M. Indran and B.R.S. Balraj. 2002. Antioxidant effects of local fruits and vegetable extracts. J. Trop. Med. Plants. 3(1):47-53. Meda, A., C.E. Lamien, M. Romito, J. Millogo and O.G. Nacoulma. 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Faso honeys as well as their radical scavenging activity. Food Chem. 91:571-577. Ogunmoyole, T, S. Inaboya. J.O. Makun and I.J. Kade. 2013. Differential antioxidant properties of ethanol and water soluble phytochemicals of false nutmeg (Monodora myristica) Seeds. Int. J. Biochem. Biotechnol. 2(1): 253-262. Oyaizu, M. 1986. Studies on product of browning reaction prepared from glucose amine. Jpn. J. Nutr. 44: 307-315. Padmanabhan, P. and S. N. Jangle. 2012. Evaluation of DPPH Radical Scavenging Activity and Reducing Power of Four Selected Medicinal Plants and Their Combinations. IJPSDR. 4(2): 143-146. Rana, M.g. R.V. katbamna, A.A. Padhya, A.D. Dudhrejiya, N.P. Jivani and N.R. Sheth. 2010. In vitro antioxidant and free radical scavenging Studies of alcoholic extract of medicago sativa L. Rom. J. Biol.- Plant biol. 55(1): 15-22. Shahidi, F, Wanasundara PKJPD. 1992. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32: 67-103. Türkoğlu, S., S. Çelik, Đ. Türkoğlu, U. Çakılcıoğlu and M. Bahsi. 2010. Determination of the antioxidant properties of ethanol and water extracts from different parts of Teucrium parviflorum Schreber. Afr. J. Biotechnol. 9(40): 6797-6805. Page 76
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Effect of Storage Conditions on the Vase Life of Homalomena Philodendron and Monstera Nipaporn YONSAWAD1, and Montinee TEERARAK1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. *Corresponding email: ktmontin@kmitl.ac.th
ABSTRACT The objective of this investigation was to study the effect of storage conditions on the vase life of Philodendron (Philodendron spp.), Homalomena (Homalomena spp.) and Monstera (Monstera deliciosa). These cut leaves were placed under three different storage conditions: (i) room temperature (33ºC) with 65% relative humidity (ii) temperature at 20ºC and 88% relative humidity in the dark and (iii) temperature at 20ºC, 88% relative humidity and 12 h photoperiod with light intensity of 10. 46 mol m-2s-1. Among the three cut leaf plants studied, Philodendron stored at temperature 20ºC with light had the longest vase life of 27.6 days. Inversely, it had the shortest vase life of 15.6 days under storage of temperature at 20ºC in the dark. Homalomena and Monstera cut leaves had the vase life in the range of 20.4225.20 days under all storage conditions. Rates of water uptake rapidly declined in all cut leaves stored at 20ºC in the dark. The results of this study suggest that Philodendron cut leaf was more sensitive to postharvest deterioration during storage and a marked reduction of vase life was observed in samples stored at 20ºC in the dark. Keywords: Homalomena, Monstera, Philodendron, Storage condition Introduction As cut leaves are commonly used in flower arrangements for decoration of the vase or bouquet, the longevity of cut leaves is an important commercial consideration in horticultural practices. Homalomena, Philodendron and Monstera genera in the Araceae family. Homalomenas are tropical to subtropical native plants found in such humid climes of Columbia, Costa Rica, Sumatra, Malaysia and the Philippines. Philodendrons are among the most popular, tolerant, and durable of all house plants. Most Philodendrons are native to the jungles of tropical America. Monstera is a genus of about 50 species. It has native to tropical regions of the Americas. Demand for cut flowers, potted foliage plants for indoor decoration as well as cut leaves for flower arrangements, is increasing dramatically around the world. During the postharvest stage the shortened shelf life of green leaf may be attributed to no optimal storage temperatures, mechanical damages, hormone unbalances or presence of ethylene (Able 2002; Ella et al., 2003). Philodendron (Philodendron spp.), Homalomena (Homalomena spp.) and Monstera (Monstera deliciosa) are now also widely traded as cut floral greens. The globalization of the cut flower market means that cut leaves have to be transported over long distances or long storage without light which accelerated senescence. The objective of this investigation was to study the effect of light or dark storage conditions on the vase life of Philodendron, Homalomena and Monstera. Materials and Methods Cut leaves of Philodendron (Philodendron spp.), Homalomena (Homalomena spp.) and Monstera (Monstera deliciosa) were purchased from a local commercial Udom garden in Ratchaburi province, Thailand and were taken immediately (within about 2 h after harvest) to the laboratory at Faculty of Agricultural Technology, King Mongkut’s Institute of July 1-3, 2015
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Technology Ladkrabang, Bangkok Thailand. Undamaged leaves were graded for uniformity and placed in containers with distilled water. These cut leaves were stored under three different storage conditions: (i) room temperature (33ºC) with relative humidity 65% (ii) temperature at 20ºC and relative humidity 88% in the dark and (iii) temperature at 20ºC, relative humidity 88% and 12 h photoperiod under light intensity of 10 mol m-2s-1. Each storage condition contained five replicates. The data were taken 2 days intervals till the end of the experiment. The characters studied were vase life, leaf water loss and reduction rate of leaf weight. Vase life of cut leaf was ended when color change or wilted leaf occurred. Results and Discussion
Vase life Data in Table 1 showed that Philodendron stored at temperature 20ºC under light had the longest vase life of 27.60 days. Inversely, it had the shortest vase life of 15.60 days under storage in the dark at 20ºC. Cut leaves of Homalomena and Monstera had the vase life ranged from 20.42 to 25.20 days under all storage conditions. Water uptake Rate of water uptake was increased at all storage conditions during 3 days after storage and rapidly declined in all cut leaves stored at temperature 20ºC in the dark (Figure1). Leaf weight loss Percentage of leaf water loss of all cut leaves increased during 4 days after storage (Figure 2). Cut leaves of Homalomena stored at room temperature and temperature of 20ºC in the dark were much more rapidly increasing leaf weight loss than others. Reduction rate of leaf weight loss of Philodendron slightly declined at room temperature and temperature of 20ºC with the light except for temperature of 20ºC in the dark. The quality of cut leaf may be observed with external quality which is represented by the visual appearances (colour, defects and mechanical damages) of produce, is necessarily required to induce the consumers to purchase the products. Temperature is the most important factor affecting post-harvest quality and shelf life of cut plant organs and their marketability. Higher temperatures accelerate senescence. Inversely, lower temperatures without any symptoms of chilling injury retard senescence due to suppression of respiratory activity, retardation of water loss and inhibition of growth of microorganisms (Wilson et al., 1999). Decay-producing microorganisms which can clog the stem result in the reduction of ability to take up water. This blockage can easily shorten the life of a cut leaf. Light significantly affected shelf life of cut Philodendron leaf and lack of light during prolonged storage significantly shortened its vase life and accelerated yellowing of leaf. In cut spinach leaves (Spinacia oleracea), the negative effect of lack of light during storage has been also reported by Toledo et al. (2003). Table 1 Effect of different storage conditions on the vase life of Philodendron, Homalomena and Monstera. Plant Storage conditions Vase life (day) Homalomena Room temp. 20.40 cd temp. 20°C in dark 21.60 bc temp. 20°C with light 22.80 abc Philodendron Room temp. 27.00 ab temp. 20°C in dark 15.60 d temp. 20°C with light 27.60 a Monstera Room temp. 20.40 cd temp. 20°C in dark 21.60 bc temp. 20°C with light 25.20 abc
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Water uptake rate (%)
1.60
Homalomena+Room temp. Homalomena+temp. 20°C in dark
1.40
Homalomena+temp. 20°C in light
1.20
Plilodendron+Room temp Plilodendron+temp. 20°C in dark
1.00
Plilodendron+temp. 20°C in light
0.80
Monstera+Room temp. Monstera+temp. 20°C in dark
0.60
Monstera+temp. 20°C in light
0.40 0.20 0.00 0
3
6
9
12
15
18
21
24
27
30
Figure 1 Effect of different storage conditions on water uptake rate of Philodendron, Homalomena and Monstera.
110.00
Homalomena+Room temp. Homalomena+temp. 20°C in dark
105.00 Leaf weight loss (%)
Homalomena+temp. 20°C in light
100.00
Plilodendron+Room temp Plilodendron+temp. 20°C in dark
95.00
Plilodendron+temp. 20°C in light
Monstera+Room temp.
90.00
Monstera+temp. 20°C in dark Monstera+temp. 20°C in light
85.00 80.00
75.00 0
3
6
9
12
15
18
21
24
27
30
Figure 2 Effect of different storage conditions on leaf weight loss (%) of Philodendron, Homalomena and Monstera. Conclusion Philodendron stored at temperature 20ºC under light had the longest vase life of 27.6 days but it had the shortest vase life of 15.6 days under storage of temperature at 20ºC in the dark. Homalomena and Monstera cut leaves had the vase life ranged 20.42-25.20 days under all storage conditions. Rates of water uptake rapidly declined in all cut leaves stored at 20ºC in the dark. The results of this study suggest that Philodendron cut leaf was more sensitive to postharvest deterioration during storage and a marked reduction of vase life was observed in samples stored at 20ºC in the dark. In the future study, effective techniques are needed for preserving postharvest quality in cut leaf of Philodendron during long distance transportation or prolonged storage without light. References Able, A.J., L.S. Wong, A. Prasad and T.J. O’Hare. 2002. 1-MCP is more effective on a floral brassica (Brassica oleracea var. Italica L.) than a leafy brassica (Brassica rapa var. chinensis). Postharvest Biol. Technol. 26:147-155.
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Ella, L., A. Zion, A. Nehemia, L. Ammon. 2003. Effect of the ethylene action inhibitor 1methylcyclopropene on parsley leaf senescence and ethylene biosynthesis. Postharvest Biol. Technol. 30, 67-74. Toledo, M.E.A., Y. Ueda, Y. Imahori and M. Ayaki. 2003. L-ascorbic acid metabolism in spinach (Spinacia oleracea L.) during postharvest storage in light and dark. Postharv. Biol. Techn. 28: 47-57. Wilson, L.G., M.D. Boyette and E.S. Estes. 1999. Postharvest Handling and Cooling of Fresh Fruits, Vegetables, and Flowers for Small Farms. Part II: Cooling. North Carolina State University.
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O-8
Micro-propagation of Dalbergia cochinchinesis Pierre 1
Wannasiri WANNARAT1*, Panida WONGWEAN , Sirinaree SUPANSOMPORN1, Warinee KITPRECHAWANICH1 and Yupa PANKAEW1 1
Kasetsart Agricultural and Agro-industrial Product Improvement Institute, Kasetsart University, Bangkok, Thailand *Corresponding email: aapwnsr@ku.ac.th
ABSTRACT Dalbergia cochinchinesis Pierre, Siamese Rosewood, is native in Thailand, Lao, Cambodia, and Vietnam. In the past, these large evergreen trees were found abundantly in the northeast of Thailand. Today, D. cochinchinesis Pierre is listed as vulnerable species because the tree populations in the natural forest have been declining dramatically due to high demand of limbers in the market. To establish in vitro conservation, the micro-propagation of D. cochinchinesis Pierre was investigated in this study. The in vitro shoot clumps were successfully induced from cotyledonary node segments excised from 14-day-old seedlings. The proliferation media supplemented with BA (3 mg/l), GA (1 mg/l), and NAA (1 mg/l) alone or in combination were tested. The highest shoot multiplication was seen when the shoots were cultured on MS containing BA (3.0 mg/l) for 60 days. The shoots rooted on MS medium supplemented with kinetin and IAA after 30-45 days of culture. The plantlets vigorously grew in the soils mixed with sand (1:1) after 30 days of acclimatization. Keywords: Dalbergia cochinchinesis Pierre, Micropropagation, Tissue culture Introduction Dalbergia cochinchinensis Pierr or Siamese rosewood (Phayung) is an economically valued leguminous tree found in mixed deciduous forest or dry evergreen forest of Southeast Asia. Siamese rosewood timbers were highly used to make furniture, wooden product and musical instruments, this leads to the gradual loss of these natural trees in Thailand. Since the tree is threatened with extinction in the near future, the development of long-term conservation program should be in a consideration. To save forest tree species, in general, it involved two strategies: in situ and ex situ conservation (Haggman et al., 2008). Ex situ strategies were seed banks, field gene collection and in vitro culture (Englemann, 1991; Engels and Visser, 2003). Establishment of in vitro plant propagation protocol is the initial step for setting up in vitro conservations including slow growth storage (Cordeiro et al., 2014) and cryopreservation (Thakur and Karnosky, 2007). The in vitro conservation has been established in many woody plant species (Padaychee et al., 2008; Bandupriya et al., 2010) but not D. cochinchinensis because the micropropagation system was available only for Dalbergia sissoo Roxb (Swamy et al., 1992). Addition, Gulati and Jaiwal (1996) reported a profound protocol to proliferate of D. sissoo Roxb microshoots by inoculating single nodal -6 segments onto MS medium supplemented with 4.4x10 M 6-benzylaminopurine (BA) and 4.4 x10-7 M β-naphthoxy acetic acid (NOA). The shoots pretreated with 10-5 M indole-3butyric acid (IBA) rooted well in the half strength MS liquid medium without plant growth regulator. Pradhan et al (1998) discovered that cotyledonary nodes of D. sissoo Roxb seedling cultured on MS 8.9 ¾M BA also proliferate shoots. The micropropagation system generated for D. cochinchinensis Pierre would be useful for an in vitro conservation, as well as, a mass propagation. To develop a protocol of micro-propagation of D. cochinchinensis Pierre, the separate experiments were carried out to examine a role of plant growth regulators on shoot proliferation and rooting in this study. July 1-3, 2015
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Materials and Methods MS (Murashige and Skoong, 1962) basal salts used in all experiments contained 100 mg/l of myo-inositol, 3% sucrose (w/v) and 0.5% agar (w/v). The pH was adjusted to 5.7 before adding agar and autoclaving (20 min, 121°C, 1.03 kg/cm2). The cultures were maintained on a 16/8 light/dark cycle under florescent light (60 µmol/m2/s) at 25+2 °C. D. cochinchinensis Pierre seeds collected from the forest at Pitsanulook Silvicultural Research Station, Royal Forestry Department of Thailand, were used in this study. For initiation culture, seeds were dipped in 90%alcohol for 1 minute and washed in running water for 30 minutes. Next, they were sterilized in 0.07% HgCl solution for 5 minutes and rinsed in sterilized autoclaved water 3 times. After rinsing, the sterilized seeds were germinated on plant growth regulator-free half strength MS medium. Seeds germinated after 7 days of culture. Once germinating, seedlings developed cotyledonary leaves with apical shoots, the excised cotyledon segments were inoculated on MS medium supplemented with BA at different concentrations (0, 0.5, 1, 2, 3 mg/l) alone or in combination with activated charcoal for 30 days. To determine the optimum plant growth regulator required for shoot multiplication, the initiated shoot clumps were cultured on MS medium contained 3 mg/l BA and/or in combination with 1 mg/l α- napthaleneacetic acid (NAA) or 1 mg/l Gibberellic acid (GA3 ) for 30 and 60 days. The obtained shoot cultures subcultured to MS medium supplemented with 3 mg/l Kinetin (Kin) at 4-week intervals for a period of 6 months were further used in rooting experiment. To investigate the effect of Indole acetic acid (IAA) and Kin on the rooting of produced shoots, elongated shoots were cultured on IAA (0.5, 1) and Kin (0.5, 1, 2, 3) alone and/or in combination. The numbers of explants used in each independent treatment were ranged from 5-10. The experiments were performed using complete randomized design with three replications. The data collected were analyzed statistically. Results and Discussion Previously, we attempted to induce shoots from axillary nodes of D. cochinchinensis. But the internodal segments cultured on MS containing with BA and GA3 at low concentration failed to form new shoots, ceased their growth and eventually died (data not shown). In the present study, the cotyledonary node excised from germinating seedling was used as explant.Similar findings were also reported earlier in the studies in other leguminous trees; D. sisso (Pradhan et al., 1998) and Pterocarpus marsupium Roxb (Chand and Singh, 2004). Our results showed that two shoot buds were induced in the cotyledonary nodes on MS medium supplemented with BA (1 mg/l) within 30 days. The proliferating shoots were subsequently transferred to shoot proliferation medium for repeating subcultures. BA alone at 3 mg/l was found to be sufficient for multiplying shoots, the 2-3 newly formed shoots were obtained from each stem (Table1). Following this procedure, 6 shoots were produced from one cotelydonary nodes after 60 days (Figure 1a). BA concentration used for shoot multiplication can be increased up to 5 mg/l without malformed shoot condition (data was not shown). Callus formed at base of shoot clump did not seem to interfere the shoot growth. Phenolic released from proliferating shoot clumps adjacent with calli turned medium to be brownish after 30 days in culture. The addition of activated charcoal to shoot induction medium could prolong a cycle of subculture. The combination of IAA and Kinetin was found the most effective for induction of root formation. The best rooting occurred at IAA (0.5 mg/l) and Kinetin (0.5 mg/l) (86.36%) after 30-45 days of culture (Figure 1c). Unlike the study reported by Gulati and Jaiwal (1996), we found that IBA-induced roots were swollen and brittle as compared with those IAA-induced roots (data not shown). Survival of the mature plantlets transferred to mixed soil was 50%.
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Table 1 Effect of BA, NAA and GA on shoot proliferation in D. cochinchinensis after 30 days and 60 days in culture. Treatments Shoot number per explant + S.E. Shoot length (cm) at 30 days of culture 60 days of culture 60 days of culture MS 2.45b+0.18 3.29b+0.25 2.907 a a MS+BA(3mg/l) 4.175 +0.19 6.66 +0.39 2.908 MS+BA(3mg/l) +GA (1 mg/l) 3.951a+0.26 5.91a+0.38 2.894 a a MS+BA(3mg/l)+NAA (1mg/l) 3.9 +0.29 5.46 +0.40 2.707 Table 2
Effect of IAA and Kinetin on root induction in D. cochinchinensis after 45 day of culture.
MS medium supplements (mg/l) IAA (0.5) IAA (1.0) Kin (1.0) Kin (2.0) Kin (3.0) IAA (0.5)+Kin (0.5) IAA (0.5)+Kin (2.0) IAA (0.5)+Kin (3.0) IAA (1.0)+Kin (1.0) IAA (1.0)+Kin (2.0) IAA (1.0)+Kin (3.0)
Rooting frequency (%) 60.00ab 42.86ab 55.00ab 53.85ab 36.36a 86.36c 59.09bc 65.00bc 57.89bc 51.85bc 47.06bc
Means within a column with the same letters are not statistically significant at Pâ&#x2030;¤0.05 (ANOVA)
a b c d Figure 1 Micro-propagation steps of D.cochinchinensis: a) Multiple shoots developed from cotyledonary nodes on MS+BA (3 mg/l) b) Shoot clumps maintained on MS+Kin (3 mg/l) c) Rooted shoots on MS+IAA (0.5 mg/l)+Kin (0.5mg/l) d) Survived plantlet transferred to soil. Conclusion This paper described the in vitro propagation of D.cochinchinensis. Shoots were initiated from coledonary nodes of very young seedlings. BA was used to induce shoot multiplication. Rooting was induced on MS medium supplemented with a combination of IAA and Kinetin.
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Acknowledgments We would like to thank Ms Onuma Sornsri, Mr Sombat Khokratiam and Ms Wichain Thaijarean for the technical assistances. This work was fully supported by KURDI, Kasetsert University, Thailand. References Bandupriya, H.D.D, S.C. Fernando, J-L Verdeil, and B. Malaurie. 2010. Cryopreservation of encapsulated plumules of coconut: effect of transport/store conditions. Asia Pacific Journal of Molecular Biology and Biotechnology 18 (1): 135-137. Chand, S. and A. K. Singh. 2004. In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, Pterocarpus marsupium Roxb. In vitro Cellular Developmental Biology-Plant 40:454-466. Cordeiro, S.Z., N.K. Simas, A.N. Henriques and A. Sato. 2014. In vitro conservation of Mandevilla moricandiana (Apocynaceae): short-term storage and encapsulationdehydration of nodal segments. In vitro Cellular Developmental Biology-Plant 50: 326336. Engles, J. M. M. and L. Visser. 2003. A guide to effective management of germplasm collections. IPGRI Handbooks for Genebanks No.6. IPGRI, Rome, Italy. Englemann, F. 1991. In vitro conservation of tropical plant germplasm. Euphytica 57: 227243. Gulati A. and P.K. Jaiwan. 1996. Micropropagation of Dalbergia sissoo from nodal explants of mature tree. Biologia Plantarum 38(2): 169-175. Haggman, H., Rusanen, M., and Jokipii, S. 2008. Cryopreseravation of in vitro tissues of deciduous forest trees. B.M. (ed.), Plant cryopreservation: A practical guide. Springer. USA. Murashige, T. and F. Skoog. 1962. A revised medium for rapid grow and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497. Padayachee, K., M.P. Watt, N. Edwards, and D.J. Mycock. 2008. Physiological responses of Eucalyptus in vitro axillary buds to cryopreparative desiccation and osmotic preculture: effects of abscisic acid. South African Journal of Botany 74 (2008): 639-646. Pradhan, C., S. Kar, S. Pattnaik and P.K. Chand. 1998. Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes. Plant Cell Reports 18: 122126. Swamy, B.V., K. Himabindu and G.L. Sita. 1992. In vitro micropropagation of elite rosewood (Dalbergia sissoo Roxb.) Plant Cell Reports. 11: 126-131. Thakur, R.C and Karnosky. 2007. Micropropagation and germplasm conservation of Central Park Splendor Chinese elm (Ulmus parvifolia Jacq. â&#x20AC;&#x2DC;A/Ross Central Parkâ&#x20AC;&#x2122;) trees. Plant Cell Report 26: 1171-1177.
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O-9
Standardization of Drip Irrigation and Fertigation for Improving Physiology, Yield and Quality Parameters of Mango var. Alphonso under Ultra-High Density Planting K. PRAKASH1*, R.M. VIJAYAKUMAR1 and S.D. SUNDHAR SINGH2 1
Department of Fruits, Horticultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India 2 Jain Irrigation Systems Ltd, Tamil Nadu, India *Corresponding email: hortidoctorpks@gmail.com
ABSTRACT An experiment was conducted with an objective of assessing the water and nutrient requirement (NPK) through fertigation on physiology, yield and quality of mango cv. Alphonso under ultra-high density planting. There were three irrigation regimes in main plots namely I1 (16 litres per day per plant; lpdp), I2 (20 lpdp) and I3 (24 lpdp) and four fertigation levels in subplots viz., F1 (50% recommended dose of fertilizer; RDF), F2 (75 % RDF), F3 (100 % RDF) and F4 (125 % RDF) replicated three times in a split plot design. The results revealed that among the irrigation treatments I3- 24 lpdp recorded the highest values for physiological traits like chlorophyll a, b and total chlorophyll content, soluble protein, nitrate reductase activity, carbohydrate content, yield characters like percentage of fruit set, fruit weight, number of fruits per tree, fruit yield per tree and quality characters like TSS, ascorbic acid, sugar, carotenoids. On comparison of sub plot treatments, the physiological parameters were at the highest by application of 125% RDF (F4) through fertigation. The yield contributing factors like percentage of fruit set, mean fruit weight, number of fruits per tree, fruit yield per tree were found to be higher by application of 100 % RDF (F3) through fertigation. In the interaction effect, application of 100% recommended dose of fertilizers (120:75:100 g NPK/tree/year) along with 24 lpdp has resulted in the improvement of all beneficial parameters leading to enhanced physiology, yield and quality of mango var. Alphonso under ultrahigh density planting. Keywords: Fertigation, Irrigation, Physiology, Quality, Yield Introduction Although India is the largest producer of mango, its productivity is only 5.5 MT tonnes /ha as against 30 tonnes/ha in Israel. Small size orchards, low yielding traditional varieties, poor orchard management like existence of wider spacing, poor or nil nutrient and canopy management practices and inadequate technological up gradation are the major constrains for lower productivity of mango orchards. Among the various factors listed above, poor water and nutrient management is an important factor, which has totally been ignored or neglected in mango, despite the fact that scientific recommendations are available in our country than at regional level (Anon., 2004). Since it is a perennial crop, attention towards meeting its water and nutritional requirements is of paramount importance. Optimum vegetative growth, better yields, competent fruit quality and greater storage life of fruits depend upon proper irrigation and nutrition to the trees. The water requirement for mango is not systematically studied in the recent time which is taken as the first objective of the present experiment. The right combination of water and nutrients is the key for high yield and quality of produce. Recently, fertigation, the most efficient method of fertilizer application, which ensures application of fertilizers directly to plant roots (Patel and Rajput, 2001) is gaining popularity in all fruit July 1-3, 2015
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crops including mango. As reiterated earlier, another reason for low productivity is wider spacing and poor land utilization. To alleviate this, adopting high density planting has been recommended (Balasubramanyan et al., 2009). Of late, the high density planting systems are becoming popular in mango, people adopting different spacing namely 5m × 5m, 10m × 5 m etc., according to their own wishes. Under such system, when more population per hectare is being planted, the demand for water and nutrients may be also varying as compared to conventional systems. One step further in this direction is the use of Ultra High Density Planting (UHDP) system with a spacing of 3m × 2m, to explore the maximum utilization of land. With this background, an experiment was undertaken to study the effect of irrigation regimes and fertigation levels on physiology, yield and quality of mango var. Alphonso under UHDP. Materials and Methods A field experiment was conducted during 2009-2010 at JISL Farm, Elayamuthur, Udumalpet, in Mango var. Alphonso by Department of Fruit Crops, Horticultural College and Research Institute, TNAU, Coimbatore to study the water and nutrient requirement and influence of different levels of NPK through fertigation on physiology and yield of mango variety Alphonso under UHDP (3m × 2m). There were three irrigation regimes in main plots namely I1 (16 lpdp), I2 (20 lpdp) and I3 (24 lpdp) and four fertigation levels in subplots viz., F1 (50 per cent RDF), F2 (75 per cent RDF), F3 (100 per cent RDF i.e. 120:75:100) and F4 (125 percent RDF) replicated three times in a split plot design. In main plots, irrigation treatments were given on daily basis and in sub plots, fertigation treatments were given at weekly interval. Thus, there were twelve treatment combinations with irrigation and fertigation. Irrigation regimes were applied through drip irrigation system as per treatment schedule at daily interval excluding December month to induce stress for flowering. Results and Discussion A comparison of three irrigation regimes indicated that application of 24 lpdp (13) resulted in higher chlorophyll content. Such increase may be due to more water absorption and more uptakes of nutrients which have close association with chlorophyll biosynthesis as reported by Raghavendra (2000). Fertigation with 125% RDF showed a significant influence on chlorophyll content of leaves. The phenomenon of increased chlorophyll content with increased nutrition, as observed in the present study, was also reported earlier by several workers viz., Ngugen et al. (2004) in mango and Sadarunnisa et al. (2010) in papaya. In the present investigation, application of 24 lpdp through drip registered the highest soluble protein content at all stages of crop growth. This might be due to high soil moisture status, thus maintaining normal cell integrity, cell elongation and functioning of biopolymers apart from enhanced nutrient uptake. On analyzing the performance of sub plot treatments, application of 125%RDF registered higher soluble protein content. Naik and Asana (1961), Del Rio et al. (1978) were of the opinion that higher nitrogen level could enhance the protein synthesis throughout the growth phase by direct participation as an essential constituent of protein. Total carbohydrates, the product of photosynthesis, were greatly influenced by exposure of plants to sunlight. The carbohydrate production is naturally extensive in leaves that are exposed to sunlight because of greater availability of light source for the production of assimilatory powers for subsequent usage in CO2 reduction process (Lythleton and Tso, 1958). In the present investigation, application of 24 lpdp through drip registered the highest carbohydrate content at all stages of crop growth. According to Klein et al. (2001), irrigation deprivation reduces carbohydrate accumulation and hence, higher levels of irrigation might have contributed for higher carbohydrate and C/N ratio. The present investigation also revealed that application of 125% RDF through drip produced higher level of carbohydrate in Page 86
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leaves than by other treatments. This might be due to higher plant vigour and chlorophyll content. Higher uptake of water and nutrients enhanced the photosynthetic efficiency thus resulting in higher accumulation of photosynthates in leaves. Among the treatments, application of 24 lpdp resulted in the highest fruit yield than trees receiving 16 lpdp (Table.1). This might be due to the percentage of fruit set, fruit weight and number of fruits per tree observed with the same treatment. Yan and Chan (1980) found that vegetative growth and photosynthesis of mango trees were reduced when soil moisture content was low. Fruit bud differentiation, panicle development, fruit set and fruit growth of mango trees increase with adequate soil moisture (Larson, 1989). When irrigation water was applied through drip irrigation system, it enhanced the water use efficiency and maintained adequate soil moisture status that resulted in higher fruit set. The present investigation is in line with findings of Wolfram Spreer et al. (2009). Among the irrigation regimes, application of 24 lpdp showed higher fruit weight. Pavel and Villers (2004) observed increase in number of fruits per tree in mango due to drip irrigation.The increased fruit weight under drip irrigation might be ascribed to better water utilization, minimum losses of water through percolation and evaporation, and excellent soil-water-air relationship with higher oxygen concentration in the root zone and higher uptake of nutrients. Among the treatments, application of 24 lpdp resulted in the highest fruit yield than trees receiving 16 lpdp. This might be due to the fruit set, fruit weight and number of fruits per tree observed with the same treatment. In the present study, application of 100% RDF recorded significantly higher fruit yield. Zekri and Koo (1991) observed in 'Valencia' orange that fertigation with higher rates of fertilizers resulted in higher availability of nutrients in soil solution which obviously led to increased growth; higher uptake of nutrients, better photo assimilation and translocation of assimilates from source to sink, thus increasing fruit yields. In this investigation, the highest TSS, total sugars, carotenoids and ascorbic acid contents were registered by application of 24 lpdp (I3) whereas the lowest levels of these traits were noticed in the treatment providing 16 lpdp (I1). Optimum moisture supply by I3 might have enhanced the enzymatic activities thus resulting in translocation and accumulation of assimilates in fruits. Moreover, the availability of adequate soil moisture with required quantity of nutrients might have resulted in more uptakes of nutrients favoured by a profused rooting system of plants. This has led to a balanced water and nutrition supply to the trees and thereby increased the quality of mango fruits. These are in line with the findings of Kadam and Mayar (1992). With regard to NPK levels, the highest TSS, total sugars, carotenoids and ascorbic acid contents were registered by application of 125 and 100% RDF whereas the lowest levels of these traits were noticed in the treatment providing 50% RDF. So, it is self-explanatory that to attain a higher fruit quality of mango, the major nutrients like NPK should be given at adequate doses. Similar observations were made by Koo (1984) in orange, Colapietra (1987) in grapes and Orphanos and Eliades (1994) in Valencia orange and Sadarunnisa et al. (2010) in papaya. Higher fruit quality especially, the high sugar content, noticed in fruits of trees provided with 125 and 100% RDF, can be explained by the role of potassium in carbohydrate synthesis, breakdown and translocation, Higher level of K available throughout the growing stages of tree favours the conversion of starch into simple sugars during ripening by activating â&#x20AC;&#x2DC;sucrose synthaseâ&#x20AC;&#x2122; enzyme, resulting in higher sugar content in fruits. The higher level of TSS observed in treatments, receiving higher levels of nutrients through fertigation, could be correlated to the enhanced photosynthetic rates. Increased dose of nitrogen, phosphorus and potassium enhanced the photosynthetic and metabolic activities, which resulted in higher amounts of acid metabolites and glucose. These products might have contributed to the synthesis of ascorbic acid and TSS. Usherwood (1985) postulated that adequate levels of major nutrients were required for proper synthesis of carotenoids. Accordingly, in the present study also, the carotenoids were at their best level in the treatments where higher doses of nutrients July 1-3, 2015
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were given. With proper K nutrition, the mango fruit is generally higher in total solids, sugars, acids and carotenoids. Ascorbic acid (Vitamin C) and beta-carotene (vitamin-A) were generally higher in fruits obtained from trees receiving 125 and 100% K and N through fertigation than in fruits from 50% RDF. The beneficial effects of K probably resulted from the combination of improved leaf photosynthetic CO2 assimilation, assimilate translocation from leaves to fruit, and improved leaf and fruit water relations, increased enzyme activation and substrate availability for ascorbic acid and beta-carotene biosynthesis (Gross, 1991). Lester et al. (2007) had expressed similar views regarding carotenoids. Table 1 Effects of irrigation regimes and fertigation levels on Number of fruits per tree and fruit yield per tree (kg). I F F1 F2 F3 F4 Mean
Percentage of fruit set
Number of fruits per tree
Fruit yield per tree (kg)
I1
I2
I3
Mean
I1
I2
I3
Mean
I1
I2
I3
Mean
0.29 0.31 0.35 0.37 0.33
0.35 0.39 0.43 0.45 0.41
0.39 0.41 0.48 0.46 0.44
0.34 0.37 0.42 0.43
25.57 26.67 29.17 30.43 27.96
28.16 30.96 35.67 32.83 31.92
32.47 34.83 39.67 37.33 36.08
28.73 30.82 34.83 33.53
5.76 6.35 7.08 7.32 6.63
6.86 7.69 9.46 8.91 8.23
8.28 9.35 11.37 10.11 9.78
6.96 7.80 9.30 8.77
I 0.005 0.015
F 0.003 0.006
IXF 0.007 0.017
I 0.298 0.828
F 0.287 0.603
IXF 0.524 1.215
I 0.148 0.410
F 0.092 0.192
IXF 0.202 0.497
SEd CD (0.05)
Conclusion Thus from this study, it is evident that application of 100% recommended dose of fertilizers (120:75:100 g NPK/tree/year) through fertigation along with 24 lpdp has resulted in the improvement of all beneficial parameters leading to enhanced physiology, yield and quality of mango cv. Alphonso under ultra-high density planting. References Del Rio, F. L. A., M. Gomez, J.A. Leal and J. Lopez George. 1978. Iron deficiency in pea plantseffect on catalase, peroxidase, chlorophyll and protein of leaves. Plant and Soil 49: 343-353. Lester, G.E., J.L. Jifon, and W.M. Stewart. 2007. Foliar potassium improves cantaloupe marketable and nutritional quality. Better Crops 91 (1): 24-25. Naik, M.A. and R. D. Asana. 1961. Effect of zinc deficiency on the synthesis of protein, mineral uptake and ribonuclease activity in cotton plant. Indian J. Plant Physiol., 4: 103-111. Ngugen, H., P. Hofman, R. Holmes, I. Bally, B. Stubbings and R. McConchie. 2004. Effect of N on skin color and other quality attributes of ripe Kensington Pride mango fruit. J. Hort. Sci. and Biotech. 79(2): 204-210. Pavel, E.W. and A.J. Villiers. 2004. Responses of mango trees to reduced irrigation regimes. Acta Hort. 646: 63-68. Raghavendra, A.S. 2000. Photosynthesis. A comprehensive treatise 1st Edn., Cambridge University Press, London, ISBN-10. Sadarunnisa, S., C. Madhumathi, K. Hari Babu, B. Sreenivasulu and M. Rama Krishna. 2010. Effect of fertigation on growth and yield of papaya cv. Red Lady. Acta Hort. (ISHS) 851: 395-400. Usherwood, N.R. 1985. The role of potassium in crop quality. In: Potassium in Agriculture (Ed: R.S. Munson). ASA-CSSA-SSSA, Madison, WI. pp. 489-513.
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O-10
Study of Using Coop Fertilizer Ameliorant on Greenhouse Gas Emission in Some Rubber Estates Agroecosystems in Peatland Hariyadi JAMIN PRIYO MINARJO1*, Dedi NURSYAMSI2 and Adi PRADIPTA3 1
Department of Agronomy and Horticulture, Faculty of Agriculture, Bogor Agricultural University (IPB), Jln. Meranti-Dramaga Campuss of IPB, Bogor 16680, West Java province, Indonesia 2 Indonesian Center for Agricultural Land Resources Research and Development (ICALRRD), Jln. Tentara Pelajar 12, Bogor 16114, West Java province, Indonesia 3 Study Program of Environmental and Natural Resources Management, Bogor Agricultural University (IPB), Baranangsiang Campus of IPB, Jln. Raya Pajajaran Bogor, 16144, West Java province, Indonesia *Corresponding email: hariyadiipb@rocketmail.com
ABSTRACT Amelioration is one of effort to increase the peatland productivity that can be applied with due to the environmental aspects. The purpose of this research was to determine the emissions from some rubber agroecosystem in peatlands and their influence on the ameliorant distributions. The research had used PVC pipe that filled with peat, which had previously taken from Jabiren, Central Kalimantan. The research was conducted at the AERC station on June to August 2012. This research consisted of two factors: the type of land use and dose of chicken manure. Types of peatland were consisting of L1 (rubber and shrubs), L2 (rubber and pineapple) and L3 (shrubs). Chicken manure dosage consisted of 0 and 4 tonnes/ha). Gas sampling was weekly taken with close chamber method adopted from IAEA. Sampling of CH4, CO2, and N2O has done manually. Analysis of CO2 and N2O using a GC equipped with a TCD and ECD detector, while CH4 was analyzed using a GC equipped with FID detector. The results showed that ameliorant distribution on land use and the rubber shrubs (L1A2) combined for the highest emissions of CO2 and N2O, which amounted to 2634.66 kg/ha/year and 67.13 kg/ha/year, while CH4 emissions were higher in the treatment without ameliorant. In the land-use of rubber and pineapple (ICCTF), the highest emissions for CH4 and N2O produced by treatment without ameliorant, respectively by 4.41 kg/ha/year and 66.53 kg/ha/year, while the highest CO2 emissions on the same land produced by treatment ameliorant distribution respectively by 2083.32 kg/ha/year. In the shrubs land, CH4 and N2O emissions were also produced at the highest treatment without giving ameliorant at 4.34 kg/ha/year and 9.96 kg/ha/year, respectively. The highest decreased emission in the land-use of rubber and pineapple (ICCTF) amounted to 12.82%. The ameliorant distribution in peatland having been overgrown of shrubs decreased emission to 7.50%. Keywords: Peat, Ameliorant, Methane, Carbon dioxide, Nitrous oxide Introduction Peat land is now a marginal land potential for expansion of agriculture (agricultural extension), at least for planting rubber. According to Rieley et al. (1996), the majority of tropical peat lands in South-East Asia Region (26.216 million ha), and Indonesia is the largest owner of peat lands. Indonesia has peat land around 14.91 million ha or 12% from the total land area of Indonesia (ICALRRD, 2011). Peat lands are scattered mainly in the islands of Sumatra, Kalimantan and Papua. There are only about 6 million hectares for agriculture. Proper management of peat lands will contribute to fulfilling the needs and national food
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security. But any exploitation of peat can cause damage to the ecosystem and will increase greenhouse gas emissions. The carbon stored in peat lands easily oxidized into CO2 gas and peat lands were prone to subsidence (subsidence) if the felled forests and drained. Subsidence is the resultant of the process of oxidation and compaction that will accelerate the process of decomposition of organic matter reserves causing CO2 and N2O emissions are likely to increase, despite a decrease in CH4 emission (Inubushi et al., 2003). Given the large carbon stocks in peat while the ecosystem is very fragile, if we do not manage properly will cause a lot of carbon, mainly in the form of methane (CH4) and carbon dioxide (CO2) into the atmosphere to increase the emission of greenhouse gases. Therefore, to minimize the impact of tropical peat land management activities by providing ameliorant that can lead to a reduction or loss rate of carbon emissions from peat lands, it is important to investigate the impact of granting ameliorant on greenhouse gas emissions in several types of land use. Materials and Methods This research was conducted at the Laboratory of Greenhouse Gases and Integrated Laboratory of ICALRRD, peatland Jabiren village, Central Kalimantan province. The material used consisted of peat soil samples with three types of land use. Other materials that will be used in this study include N2 and H2 carrier gas, standard gas CO2, CH4 and N2O, ameliorant (chicken manure). Tools used include peat drill, syringe, gas chromatography, scales, meter, electrode, pH/EH meter, 250 ml beaker, gas chamber, GPS and a digital camera. Experiment was performed using Split Plot with two treatment factors. Type of rubber agroecosystem consisted of L1 (peat land planted with rubber and shrubs), L2 (peat land planted with rubber and pineapple) = ICCTF L3 (peat land with shrubs). The chicken manure consisted of A0 (0) and A1 (4 tonnes/ha). Greenhouse gas production rate was measured using the following equation (Latin et al., 1995): E = (C24-C0) x
Vh W tan
Description: E : Emissions CO2/CH4/N2O (mg / kg soil / day) C24 : Conc.CO2/CH4/N2O at 24 hai (ppm) W tan : Weight of soil used in the incubation (g) mV : CO2/CH4/N2O molecular volume
C0 Vh mW Q
x
mW mV
x
273.2 (273.2+T)
: Concentration CO2/CH4/N2O at t0 (ppm) : Volume 'headspace' incubation in a glass cup (ml) : CO2/CH4/N2O molecular weight (g) : The average temperature incubator (0C)
Results and Discussion Treatment of Ameliorant significantly increased CO2 emission (Table 1). Table 1 CO2 Emission by Ameliorant treatment. CO2 emission Treatment of ameliorant (kg/ha/tahun) A-0 (control) 1950.1b A-1 (4 t/ha) 2497.9a Noted: values followed by the same letter, in the same of column are not significantly different by DMRT test level 5%.
CO2 emissions from peat soils of the three land use (peat land planted with rubber and shrubs, peat land planted with rubber and pineapple, and peat land with shrubs ranged from 2,115.7 kg/ha/year to 2.287 kg/ha/year. it was not significantly different. In the initial phase of incubation (8 HSI) chicken manure applied to the soil began to reshuffle in large numbers, so there was a difference in flux between the two treatment groups. CO2 emissions between treatments were not significantly different (Figure 1).
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Figure 1 CO2 flux in many provision of ameliorant materials in peat soil CH4 emissions CH4 per day for 50 days gave a value of 0.67-1.88 mg CH4/m2/day. There was no significant effect between the factors of land use and ameliorant distribution (Figure 2). But the difference was in the pattern of CH4 emission ameliorant treatment given to the three types of land use.
Figure 2 CH4 flux on any provision ameliorant materials in different land use. N2O emissions N2O flux patterns for each treatment based on the use of peat land and the provision of materials ameliorant (Figure 3). Daily N2O fluxes during the incubation period fluctuated quite large, ranging from 0.96-24.13 mg/m2/days. Based on the graph, it showed that giving ameliorant on peat soil from land use rubber people emitted the highest N2O compared with other treatments. Meanwhile granting ameliorant peat soils derived from scrub land use produces the lowest emissions.
Figure 3 N2O flux on any provision ameliorant materials in the different of land use.
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All diversity factors (land use and ameliorant) and the effects of land use and ameliorant interaction were highly significant on N2O emissions. The peat soil from land shrubs produced the low N2O emissions (9.96 kg/ha/year). While on peat soil from land ICCTF produce the highest emissions were 66.53 kg/ha/year (Table 2). Table 2 Mean values of N2O emissions (kg/ha/year). Treatment Smallholder Rubber ICCTF Shrubs Grove
Ameliorant Without Ameliorant 4 ton/ha a 24.85 ± 3.82 67.13 ± 8.47d b 66.53 ± 13.55 56.49 ± 12.65e c 9.96 ± 3.22 8.46 ± 9.36c
Noted: values followed by the same letter, in the same of column are not significantly different by DMRT test level 5%.
Conclusion During the incubation period of 50 days, it showed that the peat soil from land use smallholder rubber (rubber and shrubs) produced the highest GHG emissions for CO2 and CH4 amounted to 2,444.38 kg/ha/year and 4.70 kg/ha/year, while the highest N2O emissions generated on the land of rubber and pineapple (ICCTF) of 66.53 kg/ha/year. Giving ameliorant form of chicken manure 4 tons / ha of peat land in smallholder rubber (rubber and shrubs) resulted in total emissions of CO2 and N2O highest, amounting to 2,634.66 kg/ha/year and 67.13 kg/ha/year, however, it tended to reduce CH4 emissions in the three types of land use. Acknowledgement We thank Indonesian Center for Agricultural Land Resources Research and Development (ICALRRD) for funding this research. References Agus, F. 2009. Carbon stocks, emissions of greenhouse gases and conservation of peatlands. Anniversary Seminar Proceedings Brawidjaya University to 46. January 31, 2009. Malang. Inubushi K, Y. Furukawa, A. Hadi, E. Purnomo, H. Tsuruta. 2003. Seasonal changes of CO2, CH4, and N2O fluxes in relation to land-use change in tropical peatlands located in coastal area of South Kalimantan. Chemosphere 52: 603-608. Latin, R. S., M. J. B. Aduna, A. M. J. Javellana. 1995. Methane measurements in rice fields. Manila, International Rice Research Institute. Parish, F., A. Sirin, D. Charman, H. Joosten, T. Minayeva, M. Silvius and L. Stringer. 2007. Assessment on Peatlands, Biodiversity and Climate Change: Main Report. Global Environment Centre, Kuala Lumpur and Wetlands International, Wageningen. Raihan, H. S. and H. M. Z. Arifin. Role of organic materials against chemical properties land for corn crops Lebak dry season. Proceedings. National Seminar on Agricultural Environmental Management. Surakarta, October 21, 2003. Rieley, J. O., A.A. Ahmad-Shah, M. A. Brady. 1996. The extent and nature of tropical peat Swamps. In: Maltby E. et al., (Eds). Tropical Lowland Peatlands of Southeast Asia, Proceedings of a Workshop on Integrated Planning and Management of Tropical Lowland Peatlands held at Cisarua, Indonesia, 3-8 July 1992. IUCN, Gland, Switzerland. 294p. ISBN 2-8317-0310-7.
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O-11
Plant Nutrient Management Strategies Enhanced Growth, Yield Traits and Mitigated Leaf Reddening in Cotton (Gossypium Hirsutum L.) Allahwadhayo GANDAHI1*, Khalillulah PANHWAR1 and Rabail GANDAHI2 1
Department of Soil Science, Sindh Agriculture University Tandojam, Pakistan Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia *Corresponding email: gandahi@yahoo.com
2
ABSTRACT Cotton leaf reddening is a physiological syndrome which affects the plant growth and crop yields. A field experiment was conducted to study the effect of nutrient management on growth and yield characters of cotton (Gossypium hirsutum L.) at Soil Fertility Section, Agriculture Research Institute Tandojam, Sindh Pakistan (25°25'32"N 68°32'15"E) in a randomized complete block design. Cotton variety Haridost was studied under 4 different rates of NPK (0-0-0- NPK kg ha-1(control), 120-0-0 kg N ha-1, 120-70-0 kg N ha-1, 120-70-40 kg NPK ha-1 and 5 different secondary and micronutrients (Control, 50, 3, 4 and 1.5 kg ha-1 Mg, Fe, Zn and B, respectively) treatments. Results of study revealed that an application of NPK @ 120-70-40 kg ha-1 resulted maximum plant height, highest number of green leaves plant-1, greater number of opened bolls plant-1, more total bolls plant-1, seed index, and seed cotton yield ha-1. The treatments without incorporation of K and P (120-70-0 kg ha-1 N-P and 120-0-0 kg N ha-1) resulted in lowest values of all the above traits. Red leaves reduced to 8.43 per plant when soil was fertilized with N, P and K @ 120-70-40 kg ha-1; and plots without application of K (120-70-0 kg ha-1) and PK (120-0-0 kg N ha-1) increased number of red leaves. Present investigations further showed that application of micronutrients such as Zn @ 4 kg and B @ 1.5 kg ha-1 were highly effective to control reddening of cotton leaves with 2.43 and 3.88 red leaves plant-1, respectively. Interactive effect of NP @ 120-70 kg ha-1 × Zn @ 4 kg ha-1 resulted in maximum reduction of red leaves plant-1. Regardless the application of N, P or K and their control, reddening of leaves was linearly affected by Zn and B application. The results suggested that leaf reddening problem in cotton will be more severe in soils deficient of Zn and B. It is concluded that there was no direct effect of N, P, K, Mg and Fe on the reddening of leaves in cotton. Plant growth improved markedly when essentially needed NPK was applied @ 120-70-40 NPK in combination with Zn and B @ 4 and 1.5 kg ha-1, respectively. Keywords: Cotton, Growth, Leaf reddening, Nutrient management Introduction Innumerable abiotic and biotic factors affect cotton, thus limiting productivity. Physiological disorders appear as a consequence of nutritional/hormonal imbalances and vagaries in environmental conditions. Their effect on productivity depends upon the crop growth stage, intensity of incidence and loss of reproductive parts during ontogeny. Some of the commonly occurring physiological disorders in cotton include leaf reddening. Mineral nutrients deficiency/toxicity though not a disorder in strict terms, but it is worthwhile to know various symptoms because they affect the plant metabolism and cause various physiological disorders (Dhopte, 2001; Khandagale et al., 2011). Leaf reddening in cotton is an outcome of interaction of location, variety, environmental condition and nitrogen supply. In general, some of the hirsutum varieties and a few inter and intra specific tetraploid hybrids are July 1-3, 2015
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sensitive and vulnerable to this malady (Saini et al., 2011). Leaf reddening may occur at any growth stage of the crop. The factors affecting ideal source-sink relationship promote leaf reddening and symptoms are prolific in nature under extreme stress situations (Prakash et al., 2011). Keeping in view the economic significance of cotton and the damage caused by reddening of cotton, a field experiment was carried out to observe the individual effect of macro and micro-nutrients on the growth yield and cotton reddening. Materials and Methods A field experiment was conducted under three replicated randomized complete block design (Factorial). Cotton variety Haridost was tested against different levels of macro secondary and micronutrient. Nitrogen was applied in the form of Urea (46% N), P as DAP (18% N, 46% P2O5), K in the form of SOP (50% K2O), Mg as MgSO4 (20% Mg), Fe in the form of FeSO4 (15% Fe), Zn as ZnSO4 (21% Zn) and B (Boron) in the form of Borax (14.6% B). Agronomic observations was recorded at harvesting including plant height (cm), red leaves plant-1, green leaves-1, opened bolls plant-1, total bolls plant-1, seed index and seed cotton yield (kg ha-1). Sowing time, methods, irrigation and picking: The sowing was done on furrows in the month of July by dibbling. The distance between rows was maintained as 75 cm (2.5”). The fuzzy seed was treated with sulphuric acid (H2SO4). Seed rate was 5-6 kg acre-1. Irrigations were applied as per requirement of the crop and soil moisture. The picking was started in the first week of October when more than 50% bolls were observed to open. Statistical analysis: Data collected were analyzed for Analysis of Variance (ANOVA) of individual and interactive effects of the experimental treatments. For treatment mean discrimination, Duncan’s Multiple Range Test (DMRT) was applied. Results and Discussion The results showed that application of NPK @ 120-70-40 kg ha-1 resulted maximum plant height (128.60 cm); and discontinuation of K and P (120+70 kg ha-1 NP and 120 kg N ha-1) resulted in a reduced cotton plant height of 118.50 and 109.87 cm, respectively; while Zn @ 4 kg and B @ 1.5 kg ha-1 resulted in greater plant height of 111.47 and 110.75 cm (Table 1). Interactive effect of NPK @ 120-70-40 kg ha-1 × Zn @ 4 kg ha-1 resulted in maximum plant height (132.67 cm) and lowest (64 cm) in control. Reddening of leaves indicated that red leaves reduced to 8.43 plant-1 when soil was fertilized with N, P and K @ 120-70-40 kg ha-1; and without application of K (120+70 kg ha-1 NP) and P (120 kg N ha-1) displayed a little increase in the number of red leaves i.e. 8.60 and 8.667 plant-1, respectively as compared to 8.53 plant-1 in control. In case of micronutrients, Zn @ 4 kg and B @ 1.5 kg ha-1 were highly effective to control reddening of leaves with 2.427 and 3.883 red leaves plant-1, respectively against 12.167, 12.417 and 12.667 red leaves plant-1 in plots supplemented with 3 kg Fe, 50 kg Mg ha-1 and control, respectively. Interactive effect of NP @ 120-70 kg ha-1 × Zn @ 4 kg ha-1 resulted in maximum reduction of red leaves (2.00 plant-1) and red leaves were in maximum number (14.00 plant-1) in the interaction of 120 kg N × 50 kg Mg ha-1. Regardless the application of N, P or K and no application, reddening of leaves was linearly affected by Zn and B application. The green leaves increased markedly to 80.267 plant-1 when soil was fertilized with N, P and K @ 120-70-40 kg ha-1; and discontinuation of K (120+70 kg ha-1 NP) and P (120 kg N ha-1) considerably; and application of Zn @ 4 kg and B @ 1.5 kg ha-1 increased green leaves (73.50 and 72.833 plant-1), respectively. Interactive effect of NPK @ 120-70-40 kg ha-1 × B @ 1.5 kg ha-1 or NPK @ 120-70-40 kg ha-1 × Zn @ 4 kg ha-1 resulted in more green leaves (86.00 and 85.333 plant-1); and minimum (38.00 plant-1) in interaction of 120 kg N × 0 micronutrients.
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Table 1 Cotton growth and yield traits as affected by plant nutrient management
Macronutrients
Plant height (cm)
Control 71.60 d 120 kg N ha-1 109.9 c 120-70 kg NP ha-1 118.6b -1 120-70-40 kg NPK ha 128.6 a LSD (0.05) 1.3002 Secondary and micronutrients Control 102.5 c
Number of red leaves plant-1
Number of green leaves plant-1
Number of total bolls plant-1
42.4 d 71.2 c 77.4 b 80.3 a 1.173
Number of opened bolls plant-1 11.6d 21.4c 26.6b 34.2a 1.0966
8.60 8.70 8.60 8.50 1.1454 12.7 a
Seed cotton yield (kg ha-1)
19.3d 30.7c 34.8b 42.1a 0.955
Seed index (1000 seed weight, g) 67.3d 84.7c 91.1b 100.9a 0.908
63.9b
19.9d
30.3b
82.0c
2301.2c
105.0b 12.4a 64.3b 21.25c 50 kg ha-1 Mg 31.3b 84.7b 105.1b 12.2 a 64.6b 21.33c 3 kg ha-1 Fe 31.3b 85.1b -1 111.5a 2.4b 73.5a 28.0a 4 kg ha Zn 33.2a 89.3 a 110.8a 3.9b 72.9 a 26.6b 1.5 kgha-1 B 32.4a 88.9 a LSD (0.05) 1.4537 1.2856 1.9155 1.226 1.0678 1.0154 Means followed by common letter in each column are similar at 5% probability level.
1346.2d 2118.3c 2734.0b 3427.2a 23.930
2369.9b 2382.1b 2495.2a 2484.4a 26.754
Opening of bolls under NPK @ 120-70-40 kg ha-1 was highest, 34.20 plant-1, and the opened bolls reduced with termination of up to 26.53 and 21.40 plant-1, respectively as compared to control. Zn application @ 4 kg and B @ 1.5 kg ha-1 resulted higher number of opened bolls, 28.083 and 26.667 plant-1, respectively against 21.333, 21.250 and 19.833 opened bolls plant-1 under 3 kg Fe, 50 kg Mg ha-1 and control, respectively. Interactive effect of macro and micronutrients indicated that NPK @ 120-70-40 kg ha-1 Ă&#x2014; Zn @ 4 kg ha-1 resulted in maximum opened bolls. The application of NPK @ 120-70-40 kg ha-1 resulted in significantly greater number of total bolls (42.13) plant-1 as compared to 19.26 in control. Zn application @ 4 kg and B @ 1.5 kg ha-1 resulted in markedly higher number of total bolls, 32.41 and 33.16 plant-1, respectively against total bolls plant-1 in plots supplemented with 3 kg Fe, 50 kg Mg ha-1 and control, respectively. Seed index was highest in NPK @ 120-70-40 kg, while it reduced on termination of K application. Zinc application @ 4 kg and B @ 1.5 kg ha-1 resulted higher seed index against seed index in plots given 3 kg Fe, 50 kg Mg ha-1 and control, respectively. Seed cotton yield was highest under NPK @ 120-70-40 kg (3427.20 kg ha-1), while yield decreased to 2734.00 and 2118.30 kg ha-1 with no application of K. Zn application @ 4 kg and B @ 1.5 kg ha-1 resulted higher seed cotton yield of 2495.20 and 2484.4 kg ha-1, respectively against those plots which were given 3 kg Fe, 50 kg Mg ha-1 and control, respectively. Interactive effect of NPK @ 120-70-40 kg ha-1 Ă&#x2014; Zn @ 4 kg ha-1 resulted in maximum seed cotton yield and minimum in controls (Table 1). Leaf reddening is a physiological disorder in cotton that appears in cotton as a reflex of plant response to environmental stresses, nutritional imbalances and chemical factors. (Perumal et al., 2006) and symptoms of these disorders affect the plant metabolism and adversely affect the plant growth and crop yields (Khandagale et al., 2011). Present study showed that NPK application @ 12070-40 kg ha-1 resulted maximum plant height, slightly reduced red leaves plant-1, maximum green leaves plant-1, maximum open bolls plant-1, highest total bolls plant-1, maximum seed index and highest seed cotton yield ha-1. Without application of K and P resulted in adverse effects on all the above characters. Red leaves reduced when soil was fertilized with N, P and K @ 120-70-40 kg ha-1. Application of N, P K is essential for optimum seed cotton yield and while omitting any of these elements will result an adverse effect on this character. Hosmath et al. (2012) reported that balancing the application NPK and other micronutrients including Zn and B
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in genotype Neeraja recorded significantly lower red leaf index than others. The results clearly suggested that the leaf reddening problem in cotton will be more severe in soils deficient of Zn and B. Reddening of leaves is a physiological disorder in cotton induced by different abiotic stresses. The lipid peroxide content indicative of membrane fragmentation was decreased. In this way a multicomponent system encompassing anthocyanins, proline, and peroxidase may act coordinately to overcome abiotic stress in cotton (Endreva et al., 2002). Soil fertility, N, P and K fertility altered leaf reddening in cotton (Percy et al., 2002). Conclusion Cotton plant growth and yield traits improved markedly when macro- and micronutrients (Zn and B) were applied together. There was no direct effect of N, P, K, Mg and Fe on the reddening of leaves in cotton. However, reddening of leaves was linearly affected by Zn and B application. Plant growth improved markedly when essentially needed NPK was applied @ 120-70-40 NPK in combination with Zn and B @ 4 and 1.5 kg ha-1, respectively.
References Dhopte, A. M. 2001. Leaf reddening in cotton. Kalyani Publishers, New Delhi. Edreva, A., A. G端rel, E. Gesheva and H. Hakerlerler. 2002. Reddening of cotton (Gossypium Hirsutum L.) leaves of cotton (Gossypium hirsutum L.) leaves, Biologia Plantarum 45(2): 303-306. Hosmath, J.A., D.P. Biradar, V.C. Patil, A.R. Alagawadi and S.S. Patil. 2012. Variations in biochemical and yield parameters of Bt and non- Bt cotton genotypes. Cotton Archives 2(4): 23-29. Khandagale, G., M. Bhosale, V. Bhamare and V. Khandagale. 2011. Effect of nutrients on reddening of Bt and non Bt cotton hybrids. World Cotton Research Conference-5, Renaissance Convention Centre, Mumbai 7-11 November 2011; Abstract No. 120, Pp. 157. Percy, R., H. Moser, R. Hutmacher and S. Wright. 2002. Heritability of tolerance to early foliar decline in three pima cotton populations. The J. Cotton Sci. 6:104-114. Perumal, N.K., K.B. Hebbar, M.R.K. Rao and P. Singh. 2006. Physiological disorders in cotton. Technical Bulletin No: 28 of the CICR (Central Institute of Cotton Research), Nagpur, India. Prakash, A.H., K.R. Kranthi, V. Nagrare, S. Kranthi and V. Gotmare. 2011. Do foliar application of nutrients, PGRS and insecticides control leaf reddening in cotton. World Cotton Research Conference-5, Renaissance Convention Centre, Mumbai 7-11 November 2011; Abstract No. 53, Pp. 36. Saini, D.P., R.K. Kalyan, P.K.P. Meena and Urmila. 2011. Evaluation of Bt cotton hybrids for yield and quality components. World Cotton Research Conference-5, Renaissance Convention Centre, Mumbai 7-11 November 2011; Abstract No. 65, Pp. 131.
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O-12
Utilization of Calcium Silicate Application on Pepper Seedling Production Eakkarin SUKKAEW1, Suphachai AMKHA1,* Thongchai MALA1 and Pornpairin RUNGCHAROENTHONG2 1
Department of Soil Science, Faculty of Agriculture at KamphaengSaen, Kasetsart University, KamphaengSaen Campus, Nakorn pathom 73140, Thailand 2 Botany, Department of Science, Faculty of Liberal Arts and Science, Kasetsart University, KamphaengSaen Campus, Nakorn pathom 73140, Thailand *Corresponding email: agrscak@ku.ac.th
ABSTRACT Silicon can be classified as beneficial element. Many plants show the better growth as the silicon from available (H4SiO4 or SiO42-). The pepper production to silicon in soil Thailand is not cleared. Thus purpose of this research is to study the rate and application method of silicon fertilizer appropriated for pepper seedling production. This study was divided into two experiments. Experiment 1 was the effect of calcium silicon (Ca2SiO4) fertilizer application in pepper by soil drench method using 2x6 Factorials in Completely Randomized Design (CRD) with 4 replications. Factor A was seed preparation methods (seed priming with Ca2SiO4 fertilizer at a rate 2 g L-1 and non-seed priming) and factor B was application rates of Ca2SiO4 fertilizer at 0, 30, 60, 120, 240 and 480 kg ha-1. Experiment 2, the effect of Ca2SiO4 fertilizer application in pepper by foliar method was conducted 2x6 Factorials in Completely Randomized Design CRD with 4 replications. Factor A was seed preparation methods (seed priming with Ca2SiO4 fertilizer at a rate 2 g L-1 and non-seed priming) and factor B was application rates of Ca2SiO4 fertilizer at 0, 2, 4, 6, 8 and 10 g L-1. All experiment data were collected such as plant growth (plant height, leaf number, fresh weight, dry weight) and total silicon content in plant at 28 days after sowing (DAS). From the 1st experiment, the results showed that utilization of Ca2SiO4 fertilizer application by seed priming method at a rate 2 g L-1 with Ca2SiO4 fertilizer application by soil drench at a rate 120 kg ha-1 gave the highest plant growth and total silicon in plant. Regard to the 2nd experiment, the utilization of Ca2SiO4 fertilizer application by seed priming method at a rate 2 g L-1 with Ca2SiO4 fertilizer application by foliar at a rate 2 g L-1 gave the highest plant growth and total silicon in plant. To conclude, Ca2SiO4 fertilizer application can be employed for enhancing plant growth of pepper seedling and increasing silicon content in plant. However, the utilization of Ca2SiO4 fertilizer application at a high dose in the pepper seedling could decrease plant growth. Keywords: Foliar, Fertilizer, Pepper seedling, Silicon, Soil drench Introduction Pepper (Capsicum sp.) is vegetable popular consumption in Thailand. In 2013, pepper area plantation was 348,539 rai and tended to decreasing from 2010 (474,000 rai) (Department of Agriculture, 2013). The reason for the decline of area was the farmers changed plant for cultivation. Besides, pepper yield and quality were affected by disease and insects, also low seed quality and expensive seed. Total pepper yield was 1.3 tons rai1 and crop removal in pepper amount of 6.5 kg Si rai-1 (13.91 kg SiO2 rai-1) (Department of Agriculture, 2013). Then, silicon (Si) is necessary for pepper, Si can be classified as beneficial element (Epstein, 2005). Many plants show the better growth as the silicon form available (H4SiO4 or SiO42-). Si is reported to the component of cell (Ma et al., 2001), improves plant resistance to a range of biotic and abiotic stresses (Ma and Yamaji, 2006), stimulates growth and yield (Ma and July 1-3, 2015
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Takahashi, 2002), leaf erectness, haulm stability and lodging while increases photosynthesis (Epstein and Bloom, 2005). Now, utilization of silicon fertilizer has many variants. Kunlinda et al., (2014) reported that sweet pepper â&#x20AC;&#x153;vega 1288â&#x20AC;? seeds primed with Ca2SiO4 at 2g L-1 gave the highest of germination index (GI) and germination percentage. While, seed-primed with Ca2SiO4 at 4 g/l by foliar application gave the good of sweet pepper seedling. However, pepper production to silicon in Thailand is not cleared such as seedling production. Therefore, Si fertilizer from Ca2SiO4 can be used for benefiting pepper. Thus, purpose of this research is to study the rate and application method of Ca2SiO4 fertilizer appropriated for pepper seedling production. Materials and Methods The experiment was conduct in greenhouse at Soil Science experimental field, Department of Soil Science, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Thailand. This study was divided into two experiments. Experiment 1 was the effect of Ca2SiO4 fertilizer application in pepper by soil drench method using 2x6 Factorials in Completely Randomized Design (CRD) with 4 replications. Factor A was seed preparation methods (seed priming with Ca2SiO4 fertilizer at a rate 2 g L-1 and non-seed priming) and factor B was application rates of Ca2SiO4 fertilizer at 0, 30, 60, 120, 240 and 480 kg ha-1. Experiment 2, the effect of Ca2SiO4 fertilizer application in pepper by foliar method was conducted 2x6 Factorials in CRD with 4 replications. Factor A was seed preparation methods (seed priming with Ca2SiO4 fertilizer at a rate 2 g L-1 and non-seed priming) and factor B was application rates of Ca2SiO4 fertilizer at 0, 2, 4, 6, 8 and 10 g L-1. All experiment data were collected such as plant growth (plant height, leaf number, fresh weight, dry weight) and total silicon content in plant (Nayer et al., 1975) at 28 days after sowing (DAS). Calcium silicate (Ca2SiO4) used in this experiment was component as silica (25% SiO2), calcium (40% CaO) and magnesium (2%MgO). Statistical analyses were carried out using the R program. Differences between treatments and data variance were determined by ANOVA. Comparisons of the means among treatments were done using LSD at a significance level of P < 0.01. Results and Discussion Experiment 1 was the effect of Ca2SiO4 fertilizer application in pepper by soil drench method using 2x6 Factorials in CRD. The results showed that seed-primed with Ca2SiO4 fertilizer application at a rate 2 g L-1 was significantly different and gave the plant height, fresh weight and dry weight higher than non-seed-primed (Table1). In addition, Ca2SiO4 fertilizer application in soil was significantly different plant height, fresh and dry weight and Si concentration in plant (Table 1). By the time, Ca2SiO4 fertilizer application at a rate 120 kg ha-1 gave to tent the high of plant height, fresh and dry weight and Si concentration in plant. While, relationship between seed preparation methods with application rate of Ca2SiO4 fertilizer were significantly different that seed-primed with application rate of Ca2SiO4 fertilizer at 120 ka ha-1 gave to tent the high of plant height, fresh and dry weight and Si concentration in plant (Table 1).
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Table 1 Plant height, leaf number, fresh weight, dry weight and total concentration of Si in pepper seedling at 28 DAS (experiment 1). Factors
Plant height (cm)
Leaf number
Fresh weight (g)
Dry weight (g)
Total Si concentration in plant (%)
Seed preparation methods (A) Non-seed priming 10.31B 8.0 2.66B 0.24B 0.18 Seed priming 11.62A 8.0 2.89A 0.27A 0.19 F-test (A) ** ns ** ** ns Ca2SiO4 fertilizer application (B) (kg ha-1) 0 9.80d 8.0 2.39b 0.21c 0.01b 30 10.75 8.0 2.56a 0.24b 0.20a 60 11.63a-b 8.0 2.83a 0.26a-b 0.20a 120 11.87a 8.0 2.97a 0.28a 0.20a 240 11.06b 8.0 2.94a 0.27a-b 0.22a 480 10.74c 8.0 2.94a 0.25a-b 0.25a F-test (B) ** ns ** ** ** AxB ** ns ** ** ** CV.(%) 6.39 2.44 8.72 11.31 17.27 Remark ns= non-significantly different at P>0.01,**= significantly different at P<0.01 Means followed by common letter in each column are similar at 5% probability level.
Experiment 2, the effect of Ca2SiO4 fertilizer application in pepper by foliar method was conducted 2x6 Factorials in CRD. The results showed that seed-primed by Ca2SiO4 fertilizer application at a rate 2 g L-1 was significantly different and gave the plant height, and fresh weight higher than non-seed-primed. In addition, seed-priming and non-seed priming was not significantly different of plant leaf number, dry weight and total Si concentration in plant (Table 2). Ca2SiO4 fertilizer application by foliar method was significant different of seedling fresh weight, seedling dry weight and total Si concentration in plant (Table 2). Ca2SiO4 fertilizer application by foliar method at a rate 2 g L-1 gave to tent the seedling fresh weight, seedling dry weight and total Si concentration in plant. However, relationship between seed preparation methods with application rate of Ca2SiO4 fertilizer application by foliar method were not significantly different at P<0.01 (Table 2). Seed-primed with Ca2SiO4 fertilizer application at a rate 2 g L-1 by foliar method gave the high plant height, leaf number, seedling fresh weight, seedling dry weight and total Si concentration in plant. Conclusion The utilization of Ca2SiO4 fertilizer application by seed priming method at a rate 2 g L-1 with Ca2SiO4 fertilizer application by soil drench at a rate 120 kg ha-1 gave the highest plant growth and total silicon in plant. While, the utilization of Ca2SiO4 fertilizer application by seed priming method at a rate 2 g L-1 with Ca2SiO4 fertilizer application by foliar at a rate 2 g L-1 gave the highest plant growth and total silicon in plant. By the time, Ca2SiO4 fertilizer application can be employed for enhancing plant growth of pepper seedling and increasing silicon content in plant. However, the utilization of Ca2SiO4 fertilizer application at a high dose in the pepper seedling could decrease plant growth.
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Table 2 Plant height, leaf number, fresh weight, dry weight and total concentration of Si in pepper seedling at 28 DAS (experiment 2). Factors
Plant height (cm)
Leaf number
Fresh weight (g)
Dry weight (g)
Total Si concentration in plant (%)
Seed preparation methods (A) Non-seed priming 12.10B 8.0 3.32B 0.30 0.069 Seed priming 13.80A 8.0 3.54A 0.31 0.089 F-test (A) ?? ns ** ns ns Ca2SiO4 fertilizer application (B) (g L-1) 0 11.83 8.0 2.67c 0.25c 0.018c 2 12.43 8.0 3.69a 0.34a 0.120a 4 14.33 8.0 3.70a 0.31a 0.078b 6 13.16 8.0 3.62a 0.32a 0.072b 8 13.00 8.0 3.56b 0.32a 0.084b 10 12.96 8.0 3.37b 0.29b 0.089b F-test (B) ns ns ** ** ** AxB ns ns ns ns ns CV.(%) 12.98 2.44 6.69 10.74 18.62 Remark ns= non-significantly different at P>0.01,**= significantly different at P<0.01 Means followed by common letter in a column are similar at 5% probability level
References Department of Agriculture. 2013. The reported data of crop production annually 2013/2014. Available; http://production.doae.go.th/report/report_main2.php?report_type=1. Epstein, E. and A.J. Bloom. 2005. Minerals nutrition of plants: Principles and perspective, 2nd edition. Sinauer Associated, Inc. 400 p. Korkmaz, A. 2005. Inclusion of acetyl salicylic acid and methyl jasmonate into the priming solution improves low-temperature germination and emergence of sweet pepper. Horticultural Science.40:197-200. Kunlinda T., Thongchai M., Pornpairin R. and A. Suphachai. 2014. Utilization of calcium silicate on growth and yield quality of sweet pepper in hydroponics system. Khon Kaen Agr. J. 42 Suppl. 3. Ma, J. and N. Yamaji. 2006. Silicon uptake and accumulation on higher plants. Trends Plant Sci. 11: 392-397. Ma, J. F., Y. Miyake and E. Takahashi. 2001. Silicon as a beneficial element for crop plants. In: Silicon in Agriculture, L. E. Datnoff, G. H. Snyder and G.H. Korndorfer (eds). 17â&#x20AC;&#x201C; 39.
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O-13
Behavior of Nutrient Uptake by Pummelo Growing on Salt Marsh Soil Hien HUU NGUYEN1*, Somsak MANEEPONG1 and Potjamarn SURANINPONG1 1
School of Agricultural Technology, Walailak University, Nakhon Si Thammarat, Thailand *Corresponding email: huuhiendhv@gmail.com
ABSTRACT Pummelo growing on salt marsh soil is more superior taste than the same variety growing on other soils. This study aimed to examine soil chemical properties and their contribution to nutrient concentrations in leaves. Soil samples were taken under pummelo canopies at depth of 0-20 cm. Leaf samples of 3-5 months old were taken from beneath canopies of the same trees. Soil chemical properties and nutrient concentrations in the leaves were determined. Soil pHs were slightly alkaline (7.10.3) and their electrical conductivities were slightly saline (2.70.9 mS/cm). Concentrations of P in the soils (17794 mg/kg) were much higher than its optimum range (15-25 mg/kg), whereas the concentrations in leaves (1.40.1 g/kg) were at lower marginal of its optimum range (1.5-2.0 g/kg). Excess P in soils did not cause luxury consumption. A mole ratio of Ca/K in soils (3.61.7) was higher than that of in leaves (2.30.9). K can be uptaken better than Ca, although it had lower concentration than Ca in the soils. K also can be uptaken better than Mg, since K/Mg in the soils (0.40.2) was lower than that of in the leaves (1.50.3). A mole ratio of Ca/Mg in soils was lower than that of in leaves, indicating that Ca is able to be uptaken better than Mg. Concentrations of K, Ca and Mg in soils were higher than the optimum ranges, but only Mg concentration in leaves was higher than the optimum ranges. Antagonism effect of Mg was shown to inhibit the uptake of K and Ca, and cause excess consumption of Mg. Keywords: Nutrient uptake, Plant nutrition, Pummelo, Salt marsh Introduction Nutrient balance in soil is important for plant growth and fruit quality. Ability of nutrient uptake does not depend only on its concentration in the soil, because mobility of each nutrient into plant root is different. The nutrient uptake processes of K, Mg, and Ca are strongly antagonistic resulting in a deficiency of the depressed nutrient (Voogt, 1998; Jakobsen, 1993). A deficiency of one element could imply a relative or absolute excess of the others resulting in an imbalance for the plants (Bergmann, 1992). A sufficient Ca concentration in soil or nutrient solution is important; however, major cations frequently interfere with Ca uptake (Barber, 1995). Magnesium may strongly modify the uptake of Ca and K, whereas K and Ca restrict the uptake and translocation of Mg from the roots to the upper plant parts (Schimanski, 1981). Standard recommendations for pummelo growing soil and nutrient concentrations in pummelo leaves were established in our previous study, and similar results were published in another study (Maneepong, 2008; Zhuang et al., 1991). However, nutrient response based on the recommendations was ambiguous. Hence, the present study aims to examine the soil chemical properties and their contribution to nutrient uptake. Materials and Methods A representative pummelo plantation in Pakpanang district, Nakhon Si Thammarat province, Thailand (latitude 80 31’ 0749’’ N longitude 1000 12’ 05516’’ E) was selected for this study. The plot was arranged in ridge and trench alternatively to prevent flooding during rainy
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season. Double rows of pummelo were planted on 9 m width ridges, using 5.2 m equal spacing between rows and trees. Tuptim Sayam cultivar was grown dominantly with a small number of Khao Thong Dee cultivar. Only the Tuptim Sayam was selected for this study. Most of the pummelo trees were planted using air-layering stocks in 1997. Some trees that were replanted later were not included in this study. Soil samples were collected from 4 positions directly beneath the canopy of each tree between 0 and 20 cm depth by a sampling tube. The samples were mixed, air-dried, ground and then gravel and debris were removed by sieving through a 2 mm screen. Eighteen composite samples were selected from 6 pummelo groups. Soil pH and electrical conductivity (EC) were measured using 1:2.5 and 1:5 soil:water ratios, respectively. EC at the saturation point (ECe) was estimated by multiplying the EC by 6 (Shaw, 1999). Available P was extracted by 0.03 M NH4F in 0.10 M HCl (Bray II solution), and its concentration was analyzed by molybdenum blue method. Exchangeable K, Ca and Mg were extracted with 1 M NH4OAc at pH 7.0. Concentration of K was analyzed by a flame photometer. Concentrations of Ca and Mg were analyzed by an atomic absorption spectrophotometer (AAS) (Jones, 2001; Jones, 2003). Three- to five-month-old pummelo leaves were sampled from 3rd or 4th position of newly flush and non-fruiting twig on the outer canopy. Thirty pummelo trees were selected, and 12 to 16 leaves from each tree were collected. The samples were dried at 65 Ë&#x161;C, ground, passed through 1 mm sieve. N was analyzed by the Kjeldahl method. The samples were digested with 2:1 mixed of HNO3: HClO4 for P, K, Ca and Mg analysis. The concentration of P was analyzed using vanadomolybdate method. Concentration of K was analyzed by a flame photometer. Concentrations of Ca and Mg were analyzed by AAS (Soil and Plant Analysis Council, 1998). Results and Discussion The soil chemical properties and their optimum ranges are listed in Table 1. The soil pH was neutral and higher than its optimum range. Soil ECe varied greatly in a range of 1.3 to 5.2 mS/cm; however, most of these values fell in the optimum range. Slightly saline soil is recommended for pummelo growing. Although the soil tends to retard growth rate, but better fruit quality can be obtained (Maneepong, 2008; Samarankoon et al., 2006). Available P in the soils varied in a wide range of 75 mg/kg to 383 mg/kg, and the values are higher than its optimum range. Therefore, pummelo growing in this area should not require additional P fertilizer. The exchangeable of K, Ca and Mg were higher than their optimum ranges. If nutrient assimilation depends only on the nutrient concentration, the amounts of K, Ca and Mg should be sufficient for pummelo. However, these nutrients are strongly antagonism to each other. High Mg concentration either in soil or plant often causes poor K status in plant (Kirkby and Mengel, 1976). Zamaniyan et al. (2012) found that the K uptake by chicory cultured in nutrient solution depended on K:Ca ratio. Increasing the K:Ca ratio also increased K concentrations both in leaves and root. A K:Ca ratio higher than 6:4 decreased the yield and caused morphological damage related to Ca deficiency, such as pith hole and tip burn. The molar concentrations of K in the study soils were lower than those of Ca and Mg. Therefore, the K:Ca and K:Mg ratios opposed to those of nutrient solution for soilless culture. The nutrient concentrations in pummelo leaves and their optimum ranges are listed in Table 2. The N concentration was lower than its optimum range according to Maneepong (2008), and fell at lower margin according to Zhuang et al. (1991). N fertilizer may not be applied sufficiently, or may cause from a high N loss in NH3 form. The P concentration in the leaves was also low, despite its high concentration in the soil. Restriction in P uptake may be ascribed to a high Ca concentration in the soil together with a neutral pH. Jakobsen (1993) demonstrated that Ca can both support and inhibit P uptake. The inhibition effect results from Page 102
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the precipitation of less soluble calcium phosphate in the rhizosphere. The K concentration in the leaves was lower than its optimum range, whereas the Mg concentration was higher. Pummelo cannot uptake K to a sufficient level despite the excessive K concentration in the soil. An antagonism between K and Mg was previously described (Jones, 1999; Kirkby and Mengel, 1976). The K:Mg mole ratio in the leaves was higher than that in the soil, indicating that pummelo prefered K over Mg. The K:Ca and Ca:Mg mole ratios in the leaves were also higher than those in the soil. These results indicated that the preference order of the pummelo over these nutrients was K > Ca > Mg. However, this preference order does not agree with the concentration order in the leaves (Ca > K > Mg). Generally, farmers apply a large amount of K but ignore Ca and Mg. This practice induces Ca and Mg deficiency (Voogt, 1998; Bartal and Pressman, 1996; Jakobsen, 1993). Table 1 Chemical properties of pummelo growing soils. Soil samples (0 cm to 20 cm) were collected from 18 subplots in a salt marsh growing area. Optimum range Soil properties Unit Values (Mean ± SD) (Maneepong, 2008) pH 7.1 ± 0. 3 5.5 – 6.5 ECe mS/cm 2.7 ± 0.9 2.0 – 3.0 Available P mg/kg 177 ± 94 15 – 25 Exchangeable K mg/kg 1,013 ± 407 100 – 150 Exchangeable Ca mg/kg 3,224 ± 1,030 1,000 – 2,000 Exchangeable Mg mg/kg 1,568 ± 178 120 – 240 K:Ca mole ratio 0.35 ± 0.16 K:Mg mole ratio 0.42 ± 0.20 Ca:Mg mole ratio 1.30 ± 0.55 Table 2 Nutrient concentrations in pummelo leaves. Three- to five-month-old leaf samples were collected from 30 pummelo trees. Optimum range Nutrients Unit Values (mean ± SD) (Maneepong, 2008) N g/kg 26.1 ± 1.2 27 – 30 P g/kg 1.4 ± 0.1 1.5 – 2.0 K g/kg 13.9 ± 2.0 15 – 20 Ca g/kg 31.4 ± 9.3 30 – 40 Mg g/kg 6.0 ± 0.8 3–5 K:Ca mole ratio 0.50 ± 0.16 K:Mg mole ratio 1.48 ± 0.34 Ca:Mg mole ratio 3.16 ± 0.78 The K:Ca, K:Mg and Ca:Mg mole ratios in pummelo leaves according to the optimum ranges suggested by Maneepong (2008) were 0.5, 2.8 and 5.4, respectively. Similar ratios suggested by Zhuang et al. (1991) were 0.6, 2.8 and 4.3, respectively. The results showed that K was reached the optimum ratio compared with Ca, but less than the optimum compared with Mg. Ca also exhibited an uptake level less than the optimum compared with Mg. Excessive Mg in the soil inhibited K and Ca uptake. This problem may be solved by applying K fertilizers. Conclusion Soil in the study plantation was affected by seawater, therefore the pH was neutral and the ECe was slightly saline. The concentrations of P, K, Ca and Mg in the soil were higher than their optimum ranges. The P uptake was restricted by a high concentration of Ca in the soil. July 1-3, 2015
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The antagonism effect of Mg inhibited the uptake of K and Ca, thereby causing an excessive consumption of Mg. Acknowledgments This work was supported by the Thailand Research Fund and Walailak University. References Barbaer, S.A. 1995. Soil nutrient bioavailability: A mechanistic approach. 2nd ed. John Wiley and Sons, New York. Bartal, A. and E. Pressaman. 1996. Root restriction and potassium and calcium solutions concentration affect dry-matter production, cation uptake and blossom-end rot in greenhouse tomato. J. Am. Soc. Hort. Sci. 121: 649-655. Bergmann, W. 1992. Nutritional disorders of plants: Development, visual and analytical diagnosis. Gustav Fisher Verlang, Jena, Germany. Jakoben, S.T. 1993. Interaction between Calcium and Phosphate. Acta Agric. Scand. Sect. Soil and plant Sci. 43: 6-10. Jones, J.B. 1999. Tomato plant culture: in the field, greenhouse, and home garden. CRC Press, Florida. Jones, J.B. 2001. Laboratory Guide for Conducting Soil Tests and Plant Analysis. CRC Press, New York. Jones, J.B. 2003. Agronomic Handbook: Management of crops, soils and their fertility. CRC Press, New York. Kirkby, E.A. and K. Mengel. 1976. The role of magnesium in plant nutrition. Journal of plant nutrition and soil science. 139 (2): 209-222. Maneepong, S. 2008. A nutrient survey for establishment of standard recommendation of soil and plant analysis for pummelo. Agricultural Science Journal. 39:62-65. Samarakoon, U.C., P.A. Weerasinghe and W.A.P. Weerakkody. 2006. Effect of electrical conductivity (EC) of nutrient solution on nutrient uptake, growth and yield of leaf lettuce. Tropical Agricultural Research. 18: 13 – 21. Schimanski, C. 1981. The influence of certain experimental parameters on the flux characteristics of Mg-28 on the case of barley seedlings grown in hydroculture. Landw. Forsch. 34: 154 – 156. Shaw, R.J. 1999. Soil salinity – electrical conductivity and chloride, pp. 129-144. In: K.I. Peverill, L.A. Sparrow and D.J. Reuter (eds.). Soil analysis: An interpretation manual. CSIRO Publishing, Melboune. Soil and Plant Analysis Council. 1998. Handbook of reference methods for plant analysis. CRC Press, Boca Raton, Florida. Voogt, W. (1998). The growth of beefsteak tomato as affected by K/Ca ratios in the nutrient solution. Glasshouse Crop Research Station. Naaldwijk, the Netherlands. Zamaniyan, M., J. Panahandeh., S.J. Tabatabaei and A. Motallebie-Azar. 2012. Effects of different ratios of K:Ca in nutrient solution on growth, yield and chicon quality of wifloof chicory (Cichorium intybus L.). International Journal of Agricultural Science. 2 (12): 1137-1142. Zhuang, Y., W. Renji, C. Lixuan, X. Zhian, X. Wenbao, H. Yuzong and Z. Zhenlong. 1991. Optimum range of mineral element contents in the leaves of Guanxi honey pomelo (Citrus Grandis). Journal of Fujian Academy of Agricultural Sciences. 6 (2): 52-58.
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Eco-Friendly Utilization of TDE and Fertilizers on Soil Properties, Yield and Quality of Seed Cane J. REVATHI1* and M. BASKAR1 1
Research Scholar, Department of Soil Science and Agricultural Chemistry, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India *Corresponding email: revagrichem@gmail.com
ABSTRACT An experiment was conducted with an objective of assessing the utilization of TDE and fertilizers on soil properties, yield and quality of seed cane. There were four main plots viz., M1 (control), M2 (TDE at a dilution 1:10 on 75th days after planting), M3 (TDE at a dilution 1:10 on 75th and 120th DAP) and M4 (TDE at a dilution 1:10 on 75th, 120th and 165th DAP) and five fertilizer treatments in subplots viz, S1 (control), S2 (100 % NPK), S3 (100 % NP), S4 (75 % N and 100 % P) and S5 (75 % NP) replicated three times in a split plot design. Results revealed that among the main plot treatments application of TDE at a dilution 1:10 on 75th, 120th and 165th DAP (M4) recorded highest soil chemical properties like available N, P, K, exchangeable cations, micronutrients, yield (33.7 % over control) and quality. Among sub plot treatments, same results were recorded by application 100 % RDF (S2). On interaction, application of three times TDE along with 100 % recommended dose of fertilizers (225:62.5:112 kg NPK/ ha /year) has resulted in the improvement of soil properties leading to enhancement of the seed cane yield, quality and it was on par with three times application of diluted TDE with 75 % NP fertilizer. It is noteworthy that, by omitting 100 % K and 25 % NP, in combination with three times of diluted TDE improved yield and quality of seed cane. Keywords: Fertilizers, Seed Cane, Soil Properties, TDE, Yield Introduction Sugarcane, Saccharum officinarum L., an old energy source for human beings and India is a major producer and consumer of sugar in the world. There is a sizeable increase in acreage and production of sugarcane in India and returns from sugarcane cultivation form an important part of the Indian agricultural economy. In recent years, wastewater reuse has increased significantly worldwide. In India, the abundance of soils with low organic matter content, favours the use of industrial wastewaters containing organic matter as an organic amendment and nutrient supply to soil. Industrial waste is non-toxic, biodegradable, purely of plant origin and contains large quantities of soluble organic matter and application of distillery industrial waste water to agricultural field instead of disposing off in lakes and rivers can make crops grow better due to presence of various nutrients like N, P, Ca, Mg etc. The treated distillery effluent used in the study was dark brown in colour with unpleasant odour of burnt sugar. The TDE was found to be neutral in reaction (pH 7.5). It carries large amount of dissolved solids (85300 mg L-1), and soluble salts (EC 28.8 dSm-1). The TDE had exceptionally high BOD (4250 mg L-1), COD (48250 mg L-1) and other organic components. TDE contains all plant nutrients whose concentration is in the order of K>S>Ca>Mg>N>P (macronutrients) and Fe>Zn>Mn>Cu (micronutrients). Keeping in view the TDE and role of fertilizers in crop production, an experiment was undertaken to assess the eco-friendly utilization TDE and fertilizers on soil properties, yield and quality of seed cane
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Materials and Methods A field experiment was initiated on 30th July, 2012 in the village Karamanikuppam, Cuddalore district, Tamil Nadu by Department of Soil Science and Agricultural Chemistry ADAC&RI, TNAU, Coimbatore and EID Parry (I) Ltd, to assess the eco-friendly utilization TDE and fertilizers on soil chemical, biological properties and seed cane yield. There were four main plots viz., M1 ( control ), M2 (TDE at a dilution 1:10 on 75th day after planting), M3 (TDE at a dilution 1:10 on 75th and 120th day after planting and M4 (TDE at a dilution 1:10 on 75th , 120th and 165th days after planting) and five different combinations of fertilizer treatments viz., S1 (control), S2 (100 per cent NPK), S3 (100 per cent NP), S4 (75 per cent N and 100 per cent P) and S2 (75 per cent NP) were imposed as subplot treatments in a split plot design. Thus, there were twenty treatment combinations with TDE and fertilizers. Results and Discussions
Soil chemical properties Application of TDE had a favourable influence on the available N content in soil at each growth stage. The organic form of N supplied by the application of TDE might have been slowly released into the available pool and thereby increased the available N content. This is in line with the findings of Devarajan and Oblisami (1995) and Satisha (2000). Available phosphorus in soil was significantly increased with increasing number of TDE application. The contribution of P from TDE, the HCO3 of TDE and organic acid produced during decomposition of organic matter might have solubilized the native insoluble soil P and thus helped to increase the available P content of soil. Ghugare et al. (1988) also observed higher available P in TDE applied plots. Similar results were obtained by Murugaragavan (2002) and Anandakrishnan et al. (2008). Increase in the available K content of the surface soil was sustained even after harvest of sugarcane crop. It is quite expected that the K fertilizers have increased the soil available K. These results also agreed with the findings of Murugaragavan (2002). Chang and Li (1988) found that the application of TDE to the main crop of sugarcane increased the available K content of surface soil and remained high even after the harvest of the main crop. The application of 100 per cent RDF had high nutrient content in all the three stages of growth. In all the three stages, the highest available N status was recorded in 100 per cent RD of NP and NPK along with three times of diluted TDE might be due to combined nitrogen supply from both the inorganic source and organic source in to the available pool. Application of TDE significantly increased the exchangeable cation contents of the postharvest soil especially the beneficial cations viz., Ca, Mg and K. Since the distillery effluent contained appreciable amount of Ca (2543 mg L-1), Mg (2350 mg L-1) and K (9800 mg L-1), its application significantly increased the exchangeable cation contents of the soil. Taluk et al. (1990) observed that application of vinasse increased the CEC, K, Ca, Mg contents in soil. The population of soil bacteria, fungi and actinomycetes were significantly increased with increased number of TDE application. An increase of 52.90, 36 and 20.5 per cent bacteria, fungi and actinomycetes respectively were observed due to three times TDE received plot over control. Since, it contains highly bio-degradable organic matter with high content of essential plant nutrients; its application improves the soil biological health. Tisdall and Oades (1982) observed the stimulation of microbial activity and secretion of microbial polysaccharides due to application of TDE. The fertilizer treatments and their interaction also significantly influenced the microbial population of the post-harvest soil over control. The increase in nutrient and soil organic carbon supplied by TDE and fertilizer application might have increased the microbial population of the post-harvest soil.
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Cane yield and quality attributes The results of the field experiment revealed that the application of different levels of diluted TDE significantly increased the yield of seed cane (Figure 1). An increased cane yield of 16.5, 28.3 and 33.7 per cent were recorded for the application of one time (M2), two times (M3) and three times (M4) diluted TDE respectively over control (M1). The higher response of sugarcane for diluted treated distillery effluent application might be attributed to the enhanced availability of plant nutrients. Further, the solubilising effect of decomposing organics in TDE and the retention of more plant nutrients in the soil for longer period resulting in higher cane yield as reported by Kanwar and Kapur (1987). Maintenance of higher concentration of nutrient in soil throughout the growth period especially in three times TDE applied plots might probably be the reason for the superiority of TDE application along with the fertilizers. Similar results were reported by Devarajan et al. (1993) who observed a significant increase in the cane yield with diluted distillery effluent irrigation. Increasing doses of TDE application increased the yield attributes and cane yield due to an increase in organic carbon, enhanced supply of nutrients and improvement in soil physical properties as reported by Previna (2012). The nutrients found in the treated distillery effluent are in the organic form. The nutrients released gradually over a period of time due to fact that organic matter is being degraded slowly. In TDE applied plots, the cane yield differences among fertilizer levels viz., applications of 100 per cent NPK (S2), 100 per cent NP (S3), 75 per cent N and 100 per cent P (S4) and 75 per cent NP (S5) were not significant which indicated the supply of nutrients through TDE, especially the K. The interaction effect revealed that three times application of diluted TDE together with 75 per cent NP fertilizer (M4S5) was the most suitable combination of nutrient management for good seed cane. It is noteworthy that the application of inorganic fertilizers omitting K and 25 per cent N and P, in combination with three times of diluted TDE gave high yield as that of 100 per cent NPK combination leading to a saving of 100 per cent K and 25 per cent NP (Table1). The juice quality generally depends upon variety and genetic makeup of the cane. There was no significant change in the juice quality (Brix %) with TDE application and NPK fertilizers which is in line with the findings of Pushpavalli et al. (1999) and Baskar et al. (2004). However, increase in reducing sugar percentage was observed in sugarcane juice at 7th month of seed cane. The supply of enough quantity of N through TDE might have increased the reducing sugar per cent of seed cane (Fig.1). This might have increased the germination percentage of seed cane. The positive correlation between N fertilization, reducing sugar and germination percentage was also reported by Feyissa et al. (2009). Table 1 Effect of TDE and fertilizers on cane yield (t/ha). Treatments M1 M2 M3 M4 Mean SE d CD (0.05)
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S1 32 41 48 51 43 M 1.00 2.5
S2 S3 S4 54.6 49.8 44.8 58.6 58.1 54.9 62.8 62.4 61.5 65.5 65.3 64.1 60.3 58.9 59.3 S M Ă&#x2014;S S Ă&#x2014;M 1.09 2.21 2.19 2.2 4.7 4.5
S5 Mean 40 44.2 52.4 52.9 59.8 58.9 61.9 61.5 53.4
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Reducing sugar %
3 2.5
C
2 100 % NPK
1.5 1
100 % NP
0.5 0 Control
one time
Two times
TDE
Three times
Figure 1 Influence of TDE and fertilizers on reducing sugar percentage of sugarcane Conclusion In a nutshell, three times application of TDE at 1:10 dilution on 75, 120 & 165th days after planting along with S5 (75 per cent NP) has resulted in the improvement of soil chemical properties and quality of sugarcane nursery crop in medium textured soil (sandy loam). References Anandakrishnan, B., M. S. Dawood, M. Soundarrajan, S. Jebaraj and M. Murugesan. 2008. Conjunctive use of post methanated distillery effluent and inorganic fertilizers for sustainable sugarcane cultivation. Res. crops 9: 311-316. Devarajan, L and G. Oblisami. 1995. Effect of distillery effluent on soil properties, yield and quality of sugarcane. Madras Agric. J. 82: 397-399. Kanwar, R. S and K. Kapur.1987. Direct and residual effects of sulphitation pressmud cakes on yield, quality and nutrient of sugarcane. Indian Sugar Crops. J. 13: 1-5. Murugaragavan, R. 2002. Distillery spentwash on crop production in dryland soils. M.Sc. Thesis., TNAU, Coimbatore. Previna, S. 2012. Ecofriendly utilization of treated distillery effluent in sugarcane (Saccharum officinarum L.). Ph.D. Thesis., TNAU, Coimbatore. Pushpavalli, R., P. Kotteeswaran, M. Krishnamurthi and P. Parameswaran. 1999. Second International Conference on contaminants in the Soil Environment in the AustraliaPacific region, 12-17 December, New Delhi. Satisha. G. C. 2000. Bioconversion of sugar industrial effluent wastes into enriched compost and its effect on soils and crops. Ph.D. Thesis. TNAU, Coimbatore. Tisdall, J. M. and J. M. Oades. 1982. Organic matter and water-stable aggregates in soil. J. Soil Sci., 33:141-163.
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Investigating the Safety of Potential Probiotic Candidates Isolated from the GI Tract of Tilapia in vitro Rungtawan YOMLA1/2, Daniel MERRIFIELD1, and Simon DAVIE1 1
Aquatic Animal Nutrition and Health Research Group, Portland Square A425, Plymouth University, Devon, PL4 8AA, UK 2 Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: rungtawan.yomla@plymouth.ac.uk; kprungta@kmitl.ac.th
ABSTRACT Thrity-four bacterial colonies were isolated from the intestinal tract of nineteen tilapia collected in different cultures in Thailand. Fifteen intestinal bacteria were able to inhibit the growth of pathogenic bacteria: Aeromonas hydrophila and Streptococcus iniae. Bacterial isolates were evaluated for safety as probiotic candidates by testing antibiotic resistance and blood hemolytic activity. A number of bacterial isolates, when concentrated at more than 1014 CFU mL-1, displayed resistance to antibiotics. Macrococcus caseolyticus (isolate 2) was susceptible to all antibiotics tested. Three isolates [Staphylococcus arlettae (isolate1), Enterobacter sakazakii (isolate 7), and En. asburiae (isolate 33)] displayed resistance to erythromycin. Four bacterial isolates (Bacillus sp. (isolate 14), and Ba. cereus (isolates 15, 18, & 23) displayed resistance to sulphamethoxazole/thrimethoprim. Multi-antibiotic resistance was observed by Bacillus sp. (isolate 18), which displayed resistance to ampicillin, cephalothin, and sulphamethoxazole/thrimethoprim. A number of Bacillus isolates [Bacillus sp. (isolate 14), Bacillus sp. (isolate 18), and Ba. cereus (isolates 15, 17, 23)] displayed positive blood hemolysis. The isolates, which do not display antibiotic resistance or hemolytic activity, will be tested in in vivo trials by using adhesion properties, and resistance to gastric conditions. And then, potential probiotic candidates will be selected using multiparameters in vivo trials to quantitative data with weight score assumptions. Then, high potential probiotic candidates will be studied in with expectation of elucidating a novel potential probiotic for tilapia application. Keywords: Tilapia, Intestinal bacteria, Potential bacterial probiotic, Antibiotic resistances, Blood hemolysis Introduction Probiotics used in aquaculture support growth performance and diseases resistance, while increase immune enhancement, healthy improvements, balancing function mechanisms of fishes as well assustain gut microbes, and improve water quality (Gatesoupe, 1999; GomezGil et al., 2000; Verschuere et al., 2000; Wang et al., 2008; Merrifield et al., 2010; HachĂŠ and Plante, 2011). Probiotics are investigated following the global guideline for evaluation of probiotics in food (FAO/WTO, 2006). In vitro trials, the safety assessment as an antibiotic resistance, and hemolysis activity are important testing to demonstrate without harmful to the host. Antibiotic resistance is a stress condition, which antibiotic drugs cannot kill/treat microbial pathogens. Currently, antibiotic residues distribute in water resources and this causes aquatic microbes display to resist antibiotic (Petersen and Dalsgaard, 2003; Michel et al., 2007). They can transfer between aquatic systems to human and concern pathogenic resistance in human (Benbrook, 2002). Potential probiotic can lead to antimicrobial resistance gene. As the same of blood hemolysis, potential probiotic must safe for life with differences July 1-3, 2015
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from pathogens. For these reasons, a study of antibiotic resistance and blood hemolytic activity as safety uses of probiotic candidates were evaluated. These will be supported probiotic selection based on antibiotic susceptibility and negative result on blood hemolysis. Materials and Methods Bacterial preparation Fifteen bacterial isolates from the GI tract of tilapia were grown in TSB overnight at 320C. These bacteria were harvested by centrifugation at 5000 g for 5 minutes and washed twice with sterile 0.85% NaCl and re-suspended in this sterile solution. Finally, bacterial cell concentration was adjusted concentration at 10 (1A), 3 (1B), and 0.5 (1C) McFarland standard (9×108 cells.mL-1 of approximated cell density). Antibiotic resistance Potential probiotic preparation (1A) was screened on twelve antibiotic discs (Oxiod, UK) included ampicillin 10μg, cephalothin 30μg, enrofloxacin 5μg, erythromycin 15μg, gentamycin 10μg, kanamycline 30μg, neomycin 30μg, nitrofurantoin 300μg, oxolinic acid 2μg, oxytetracycline 30μg, sulphamethoxazole/thrimethoprim 25μg, and tetracycline 30μg, which isolate no.2 (M. caseolyticus) were displayed antibiotic susceptibility on twelve disc types. Then, 100μl of bacterial preparation (1B) was used spreading on TSA plates and incubated for 45-60 minutes tested with antibiotic disc types using duplicates of disc types. After incubation at 32 0C for 18-24 hours, the expression of bacterial isolates on antibiotic resistance was recorded as the diameter of clear zone with duplicated antibiotic disc. Results were defined following susceptible (S), intermediate (I), and resistant (R). Hemolytic testing with sheep blood agar and tilapia blood Agar plates of sheep blood agar plates (MDX1407077) and tilapia blood agar plates (2%v/v) were made of six wells (0.6 cm of a diameter). Then, 20 l of bacterial preparation (1C) was transferred into wells in these plates and incubated overnight at 320C. The appearance of clear zone diameters was recorded. Hemolytic testing was assessed by the two-way analyses of variance (ANOVA). Statistical significance was accepted at p≤0.05. These data were analyzed using the Systat software version 5.02 (Systat, Inc., 1990). Results and Discussion Antibiotic susceptibility was displayed in six isolates (Mac. caseolyticus (2), Ba. megaterium (31), Stap. sciuri (4), Bacillus spp. (12, 21, and 29). Antibiotic resistances on sulphamethoxazole/thrimethoprim were found in isolate 15, 17, and 23 (Ba. cereus), and 14 and 18 (Bacillus sp.), on erythromycin in three isolates (Stap. arlettae (1), Ent. sakazakii (7), and Enterobacter sp. (33)). In addition, Bacillus sp. (18) showed multi-antibiotic resistances on sulphamethoxazole/thrimethoprim, ampicillin, and cephalothin of this investigation. Different efficiency of potential probiotic has been resisted on different antibiotic types (Mourad and Nour-Eddine, 2006; Liasi et al., 2009; Nayak and Mukherjee, 2011; MuñozAtienza et al., 2013). Tilapia farm in Thailand has been a reported popular use antibiotic such as enrofloxacin, sulfadiazine, and trimethoprim (Rico et al., 2014). The multiple antibiotic resistance may be possible transferring an antibiotic resistance gene to other aquatic bacteria. Hemolytic testing on sheep blood and tilapia blood was shown differently significant (p≤0.05). The positive blood hemolysis on sheep and tilapia blood was displayed in Bacillus sp. (14, and 18), and Ba. cereus (15, 17, and 23) (Figure 1), which differently shown harmful to the host (Figure 2). Bacterial pathogenicity produce enterotoxins, aerolysin and hemolysin, which can cause effect on fish diseases and mortalities (Daskalov, 2006; Deen et al, 2014). According to these probiotic selection, these can be based on safety uses without antibiotic gene and negative blood hemolysis in vitro trials.
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* *
*
* * *
Figure 1 In vitro hemolytic activity on sheep and tilapia blood hemolysis of bacteria isolates and bacterial pathogens (A: back plate; B: front plate).
*
Figure 2 Diameter of clear zones (mean (mm) ± standard deviation) of fifteen bacterial isolates and two pathogenic bacteria. * significant differences between blood types and isolates
Conclusion Five isolates (Ba. megaterium (31), Stap. sciuri (4), and Bacillus sp. (12, 21, and 29) displayed antibiotic susceptibility. Ten (Stap. arlettae (1), Mac. caseolyticus (2), Stap. sciuri (4), Ent. sakazakii (7), Bacillus spp. (12, 13, 21, and 29), Ba. megaterium (31), Enterobacter sp. (33)) of fifteen isolates were shown negative blood hemolysis, except five isolates (Bacillus sp. (14, and 18), and Ba. cereus (15, 17, and 23)). Acknowledgements This project is supported by King Mongkut’s Institute of Technology Ladkrabang, Thailand. References Benbrook, C.M. 2002. Antibiotic Drug Use in U.S. Aquaculture, IATP Report. Available at: http://iatp.org/files/421_2_37397.pdf (Accessed: 25 February 2015). Daskalov, H. 2006. The importance of Aeromonas hydrophila in food safety. Food Control 17: 474–483. Deen, A.E.N.E., S.M. Dorgham, A.H.M. Hassan, and A.S. Hakim. 2014. Studies on Aeromonas hydrophila in cultured Oreochromis niloticus at Kafr El Sheikh Governorate, Egypt with reference to histopathological alterations in some vital organs. World Journal of Fish and Marine Sciences 6 (3): 233-240. FAO/WTO. 2006. Probiotics in food Health and nutritional properties and guidelines for evaluation. FAO Food and Nutrition Paper 85, Rome. Gatesoupe, F.J. 1999. The use of probiotics in aquaculture. Aquaculture, 180, pp. 147-165. Gomez-Gil, B., Roque, A. and Turnbull, J.F. 2000. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture 191: 259–270 p. Haché, R. and S. Plante. 2011. The relationship between enrichment, fatty acid profiles and bacterial load in cultured rotifers (Brachionus plicatilis L-strain) and Artemia (Artemia salinastrain Franciscana). Aquaculture 311: 201–208.
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Liasi, S. A., T. I. Azmi, M.D. Hassan, M. Shuhaimi, M. Rosfarizan and A. B. Ariff. 2009. Antimicrobial activity and antibiotic sensitivity of three isolates of lactic acid bacteria from fermented fish product, Budu. Malaysian Journal of Microbiology 5(1): 33-37. Merrifield, D. L., A. Dimitroglou, A. Foey, S. J. Davies, R.T.M. Baker, J. Bøgwald, M. Castex and E. Ringø. 2010. The current status and future focus of probiotic and probiotic applications for salmonids. Aquaculture 302: 1-18. Michel, C., C. Pelletier, M. Boussaha, D.G. Douet, A. Lautraite, and P. Tailliez. 2007. Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis. Applied and Environmental Microbiology 73 (9): 2947–2955. Mourad, K. and K. Nour-Eddine. 2006. In vitro preselection criteria for probiotic Lactobacillus plantarun strains of fermented olives origin. International Journal of Probiotics and Prebiotics 1(1): 27-32. Muñoz-Atienza, E., B. Gómez-Sala, C. Araújo, C. Campanero, R. Del Campo, P.E. Hernández, C. Herranz, and L.M. Cintas. 2013. Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture. BMC Microbiology 13 (15). Nayak, S.K. and S.C. Mukherjee. 2011. Screening of gastrointestinal bacteria of Indian major carps for a candidate probiotic species for aquaculture practices. Aquaculture Research, 42: 1034-1041. Petersen, A. and Dalsgaard, A. 2003. Species composition and antimicrobial resistance genes of Enterococcus spp., isolated from integrated and traditional fish farms in Thailand. Environmental Microbiology 5(5): 395–402. Rico, A., Phu, T.M., Satapornvanit, K., Min J., Shahabuddin, A.M., Henriksson, P.J.G., Murray, F.J., Little, D.C., Dalsgaard, A. and den Brink, PJ.V. 2014. Use of veterinary medicines, feed additives and probiotics in four major internationally traded aquaculture species farmed in Asia. Aquaculture 412–413:231–243. Systat Inc. 1995. Systat 5.02 for Windows. Copyright 1990-1993, Inc., Evancton, IL USA. Verschuere, L., G. Rombaut, P. Sorgeloos and W. Verstraete. 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev. 64 (4): 655–671. Wang, Y-B., Z-Q. Tian, J-T. Yao, and W. Li. 2008. Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune response. Aquaculture 277: 203-207.
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Studied on Using of Robo-Logger Technique Measuring In Situ Inside Shell Temperature of Rocky Shore Limpet Monthon GANMANEE1*, Chayanid MEEPOKA1, Pokamon KOCHARIN1 and Gray A. WILLIAMS2 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Swire Institute of Marine Science, University of Hong Kong, Pokfulam Road, SAR China *Corresponding email: kkmontho@kmitl.ac.th
ABSTRACT Temperature is one of the most important environmental factor driven distribution pattern and physiological traits of intertidal rocky shore organisms. However, it is difficult to understand their body temperatures under in situ conditions. In this study, the robo-logger technique was used by putting thermo-logger (waterproof device model Thermochron Maxim DS1922) inside limpet shell (Cellana toreuma) filled with Scotchcast® 2131. The robo-loggers were deployed at 3 tidal levels (high, mid and low shore) in 2 sites (intertidal rocky shore near Asadang Bay, Sichang Island, Chonburi province and Muang district, Chumporn province). Each tidal level, a pair of robo-logger was deployed in order to determine effect on temperature variation in different degree of rock surface. One of robo-logger was put on vertical rock surface or crevice and the other was put on flat rock surface. Temperature inside was recorded every 30 minutes for 9 months (August 2014 to April 2015). The data will be compared with hourly air temperature data obtained from local meteorological station. Spatial and temporal variation of in-shell temperature will be determined. Keywords: Climate change, Limpet, Robo-Logger, Rocky shore, Temperature Introduction The dramatic changes of environmental factors resulting from ongoing climate change have significant impacts on species composition and abundance. Consequently, local extinction, species distribution shift and community succession occur ubiquitously in various ecosystems, and then it becomes important and urgent to understand species distribution shift using mechanistic approach and to predict future biogeographic patterns in the scenario of climate change for future biodiversity conservation and management (Mislan et al., 2014). Because most organisms are ectotherms, therefore, temperature is one of the major factors controlling their species distribution and abundance. Global warming is also the primary effect of climate change (Cox et al., 2000, Lima and Wethey, 2012), and organisms have to face changing thermal environments (Root et al., 2003). To cope with the increasing temperature, organisms have to develop various strategies in behavior, physiological and evolutionary (Somero, 2012). Through determining which species currently live closest to their upper thermal tolerance limits that were mainly depended on the physiological systems and their physiological plasticity, it is feasible to predict the “winners” and “losers” facing the future climate change (Somero, 2012). The rocky intertidal is an excellent model to study and predict the effect of global warming in marine systems. Intertidal rocky shore species commonly live close to their upper thermal limits and are vulnerable to climate change. Extensive studies showed that temperature is the most important factor determining the zonation and biogeography of intertidal species (Hawkins et al., 2008). In the preset study, an integrative study of in situ operative body temperature monitoring, using biomimetic loggers July 1-3, 2015
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(Robo-logger) was carried out in two geographical region of Thailand (Sichang island and Chumporn) to investigate patterns of thermal stress in tropical coast and the possible impacts on the distribution of rocky shore intertidal species. Materials and Methods Limpet, which is a common species living in rocky intertidal, was chosen as a representative organism in this study. The operative body temperatures of limpets were measured in the upper distribution limits of limpet using Robo-logger technique as a previous study described by Lima and Wethey (2009). Thermo-logger (waterproof device model Thermochron Maxim DS1922) was put inside dead limpet shell (Cellana toreuma), then, filled with Scotchcast ® 2131. In the present study, the operative body temperatures of limpets were monitored on Sichang island, Chonburi province (13°08´N; 100°48´E) and rocky shore at Muang district, Chumporn province. In each site, eighteen Robo-limpets were deployed on two positions of rock surface, horizontal (sun-exposed) and vertical (shade) at three tidal levels (high, mid and low intertidal) with three replicates per tidal level. The high shore was identified at the littorinid level whereas the low shore was at the coralline algae. The middle level is approximately at the point between these two levels. It also corresponds to the mean tide level as obtained from a tide table. Operative temperature recordings were made every 30 min. Data from each Robo-logger in Sichang island was downloaded bi-monthly from September 2014 to February 2015 and October 2014 to March 2015 for Chumporn, with One Wire Viewer software. The data on operative for temperature both two sites were compared with daily air temperature data reported by Department of Meteorological of Thailand. Difference in recorded temperature in robo-logger at each site, tidal level and deployed position were analyzed using ANOVA. Homogeneity of variance was assessed using Cochran's test. LSD test were used for multiple comparisons of the mean (at α= 0.05). Results and Discussion There was few losses (a couple of shells were eroded and an electrode missing) in some robologger caused data missing in mid shore of exposed surface in Sichang island. Generally, body temperature of robo-limpet varied between season and their daily maximum temperatures at high shore were considerably higher than air temperatures, with differences as great as 12C in Sichang island and 15C in Chumporn for sun-exposed and 10C for shaded surface, respectively. Data from the present work also showed that even low-shore organisms are periodically exposed to temperatures exceeding 30°C (Table 1 and Table 2). During hot days with daytime low tides (September to mid-November in Sichang island, October to mid-November in Chumporn and from mid-January afterward for both sites), robo-logger temperatures were often > 40oC. Position of robo-limpet on rock surface and tidal height showed clear influences on temperature, with high shore sun-exposed robo-limpet experiencing hotter temperatures than their lower counterparts (Figure 1 and Figure 2).
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Figure 1 Daily maximum body temperature profile obtained by robo-limpet deployed in difference position of rock surface compared with air temperature data at Sichang island (A) and Chumporn (B).
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Figure 2 Daily maximum body temperature profile obtained by robo-limpet deployed in difference tidal height compared with air temperature data at Sichang island (A) and Chumporn (B). This work provided the first long-term series of intertidal rocky shore animals' body temperatures in coastal region of Thailand, obtained by means of standardized methods (Lima and Wethey, 2009; Seabra et al, 2011; Lathlean et al, 2014). Overall, at each site (Sichang island and Chumporn), maximum daily body temperatures were frequently higher than air temperature for over 10ď&#x201A;°C. Body temperature of animals living on sun-exposed versus shaded associated tidal height was consistently different. Previous studied by Dong et al. (2015) showed that Maximum Habitat Temperature (MHT) exceed Median Lethal Temperature (LT50) of limpet (Cellana toreuma) in Sichang island. It means that the limpet currently suffer from intensive heat stress. The effect of global warming might generate distinction or poleward shift of the species to avoid increased heat stress. Table 1 Maximum and minimum body temperature in sun-exposed surface robo-limpet in difference tidal height obtained from Sichang island and Chumporn. Sichang island Low shore Mid shore High shore Air Temp.
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Max. 42.5 ns 47.0 35.0
Chumporn Min. 22.5 ns 19.5 16.5
Max. 43.3 47.0 51.3 36.5
Min. 19.3 19.8 21.4 18.0
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Table 2 Maximum and minimum body temperature in shaded surface robo-limpet in difference tidal height obtained from Sichang island and Chumporn. Sichang island Low shore Mid shore High shore Air
Max. 35.5 37.0 46.5 35.0
Chumporn Min. 21.5 22.0 18.7 16.5
Max. 36.5 45.5 46.9 36.5
Min. 18.0 27.5 21.3 18.0
References Cox, P.M., R.A. Betts, C.D. Jones, S.A. Spall and I.J. Totterdell. 2000. Acceleration of global warming due to carbon-cycle feed backs in a coupled climate model. Nature 408: 184−187. Dong, Y.W., G.D. Han, M. Ganmanee and J.Wang. 2015. Latitudinal variability of physiological responses to heat stress of the intertidal limpet Cellana toreuma along the Asian coast. Marine Ecology Progress Series 529:107-119. Hawkins, S.J., P. Moore, M.T. Burrows and E. Poloczanska. 2008. Complex interactions in a rapidly changing world: responses of rocky shore communities to recent climate change. Climate Research 37:123−133. Lathlean, J.A., D.J. Ayre, R.A. Coleman and T. E. Minchinton. 2014. Using biomimetic loggers to measure interspecific and microhabitat variation in body temperatures of rocky intertidal invertebrates. Marine and Freshwater Research 66(1): 86-94 Lima, F.P. and D.S. Wethey. 2009. Robolimpets: measuring intertidal body temperatures using biomimetic loggers. Limnology and Oceanography Methods 7:347−353. Mislan, K.S., B. Helmuth and D.S. Wethey. 2014. Geographical variation in climatic sensitivity of intertidal mussel zonation. Global Ecology and Biogeography 23:744−756. Seabra, R., D.S. Wethey, A.M. Santos and F. P. Lima. 2011. Side matters: Microhabitat influence on intertidal heat stress over a large geographical scale. Journal of Experimental Marine Biology and Ecology 400:200–208. Somero, G.N. 2012. The physiology of global change: linking patterns to mechanisms. Annual Review on Marine Science 4:39−61.
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Impacts of the PTT GC Oil Spill on Intertidal Rocky Shore Macrobenthos in Ao Prao, Samed Island Jindarha PREMPRAMOTE1*, Chayanid MEEPOKA1, Sujitra SAMAKRAMAN1 and Monthon GANMANEE1 1
Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: j_aoei@hotmail.com
ABSTRACT On 28 July 2013, 50,000 litres of the crude oil leaked from a pipeline owned by PTTGC Plc, and burst while oil was being transferred from an undersea well to a tanker at Rayong Province, Thailand. The oil spill moved eastward and reached rocky shore and beach at Koh Samed's Ao Phrao in the following day resulted in serious impact to the shore. Abundance (in density), diversity and community structure of macrobenthos at rocky intertidal of Ao Phrao were monitored 3, 6 and 9 months compared with unaffected reference sites. Transects were positioned at five different heights from low to mid shore (1.5 to 2.5 m above MTL). At each height, abundance of rocky shore macrobenthos was recorded within 30 replicates quadrats (25 x 25 cm). Decreases in abundance and diversity indices were observed for rocky shore macrobenthos at Ao Phrao whilst these parameters were not significantly different in reference site (p<0.05). Results in MDS (multidimentional scaling) revealed zonation pattern of rocky shore macrobenthos in Ao Phrao did not change or move up to higher shore. This finding suggested that intertidal rocky shore macrobenthos were impacted by the oil spill. Long term monitoring program should be carried out to clarify abundance and community structure of these organisms to restore to its original level before oil spill. Keywords: Community structure, Intertidal rocky shore, Macrobenthos, Oil spill, Samed island Introduction Crude oil is a complex compound containing many chemicals mainly alphatic, aromatic and polar compounds. If crude oil spilled to the sea, it spreads on the sea surface and forms a thin film, called an oil slick (Maki et al., 2001). Once the oil spreads and reaches to the shore by wind and water current and covers on the rock surface. It might have seriously affected to the coastal organisms both short-term and long-term (Lim and Shin, 2013). Latest oil spill accident in Thailand occurred on July 28, 2013, the crude oils leaked from a pipeline by PTTGC Plc., Oil company. About 50 tons of the crude oils were spilled, reached the Ao Phrao beach Koh Samed,Thailand where is one of the most popular beach for tourists. Crude oil covered sandy beach and rocky shore in this area for more than 1 week. It is necessary to evaluate the impact of the oil to intertidal marine organism in this area. The objective of this study was to determine whether intertidal rocky shore macrobenthos were impacted by the oil spill. Materials and Methods Study area Because there was no baseline data available on abundance and diversity of rocky shore macrobenthos in Ao Prao, the survey in the present study, therefore, was carried out in impact
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site (Ao Prao) comparing with rocky shore in Laem Yaa where was not be impacted by the oil spill accident. The site is located on Rayong mainland, ca.4 km from Ao Prao (Figure 1).
Figure 1 Studied sites (Ao Prao, Samed island and Laem Yaa rocky shore). Sampling protocol Samplings were done in October, December, 2013 and March 2014 (3, 5 and 8 months following the oil spill accident). Line transects (100 m) were positioned at five different heights from low to high shore (1.5, 1.75, 2.0, 2.25 and 2.5 m above Mean Tidal Level; MTL, respectively). Total of 30 replication quadrats (25×25 cm) were placed randomly at each height. Photographs of each quadrat were taken with a digital camera (Cannon G9 with autofocus). In the laboratory, abundance of macrobenthos within each quadrat was assessed by Image J®. Data analysis Sample similarity was calculated with the Bray-Curtis coefficient, after log (x+1) data transformation. Non-metric multidimentional scaling (nMDS) was used to produce twodimensional ordination plots using PRIMER v5 software (Clarke and Gorley, 2001). Spatial and temporal distribution of specific taxa of bivalves and gastropods were analysed using 3factors ANOVA (tidal level: L; sampling site: S and Sampling time: T). LSD test was used for multiple comparisons of the mean (at α= 0.05). Results and Discussion A total of 11 species of intertidal rocky shore macrobenthos were identified in the present studies both Ao Prao (impact site) and Laem Yaa (non-impact site). Most of them belong to Phylum Mollusca which were dominated by coil snail (Planaxis sulcatus), limpet (Cellana toreuma), periwinkle (Monodonta labrio and Peasiella repsotroffiana), littorinids (Echinolittorina malaccana and E. radiata), Morula sp., chiton (Acanthopleurs japonica) and oysters (Sacosstrea cucullata and Isognomon nucleus). Unfortunately, mobile species such as rock crab and amphipod (Ligia sp.) were not able to be taken photograph by this technique. Figure 2 (MDS) showed community structure of intertidal rocky shore macrobenthos for both two sites in this study. Clear vertical differences in species distributions could be distinguished between tidal levels for both two sites which could be categorized into 3 vertical groups: (1) 1.5 m to 2.0 m above MTL (2) 2.0 m to 2.25 m above MTL and (3) 2.25 to 2.5m above MTL. The first group (low shore) was characterized by limpets and oyster (S. cucullata). Most of periwinkles, coil snail and bivalves (I. nucleus) were distributed in mid shore. In the highest shore of group 3, the littorinid E. malaccana dominated of their total abundance being found at 2.5 m above MTL.
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A
B
Figure 2 Multidimentional scaling (MDS) of intertidal macrobenthos community structure in Ao Prao (A) and Laem Yaa (B). Abundance of intertidal rocky shore macrobenthos in Ao Prao was significantly different through sampling time (Figure3), being decreased from 182.2 individuals/m2 in October, 2013 to 132.0 individuals/m2 in December, 2013 and 132.0 individuals/m2, respectively. Such trend was not observed in Lam Yaa macrobenthos which their abundance was highest in December, 2013. In March, 2014 the abundance of dominant taxa, E. radiate, was rare but not for limpet (C. toreuma).
Planaxis sulcatus Peasiella roepstorffiana Morula sp. Echinolittorina malaccana E. radiata Saccostrea cucullata Isognomon nucleus limpet Monodonta labrio Acanthopleura japonica
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Figure 3 Abundance of intertidal rocky shore macrobenthos in Ao Prao and Laem Yaa. Resulted in diversity indices (Simpson Diversity Index; d, Shannon Wiener Diversity Index; H and Equitability Index; J) revealed that H and J in impact site had decreased gradually from October, 2013 to March, 2014 whereas diversity indices in non impact site was not significantly different between sampling time (Table 1). Table 1 Diversity indices (Simpson Diversity Index; d, Shannon Wiener Diversity Index; H and Equitability Index; J) of intertidal rocky shore macrobenthos in Ao Prao and Lam Yaa Sampling time Ao Prao Lam Yaa d H J d H J October 2013 1.26 1.34 0.58 1.59 1.70 0.77 December 2013 1.35 0.89 0.37 1.65 1.84 0.80 March 2014 1.13 0.55 0.25 1.09 0.85 0.44 July 1-3, 2015
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Conclusion In conclusion, this finding suggested that intertidal rocky shore macrobenthos were impacted by the oil spill. There are several studies indicated that crude oil spill accident impacted to abundances and changes in macrobenthos community structure (MacFarlane and Burchett, 2003) and affected to a decrease in the species richness in Polychaetes, insects, semiterrestrial crustaceans (Huz et al., 2005). Nagelkerken and Debrot (1995) reported the abundance of mollusc on the rocky shore of the Curacao island in the southeast of the Caribbean, is less affected by the oil spill, especially mollusc at low and mid shore because of the influence of oil-covered area but the crude oil did not affect to genetic macrobenthos communities (Pineira et al., 2008). The area affected by the oil can be recovery in the least 23 years, depending on the amount of oil covering the surface, the affected period, life cycle of macrobenthos species and the ability to move (Yamamoto et al., 2003). However, long term monitoring program should be carried out in Ao Prao to clarify abundance and community structure of these organisms to restore to its original level before oil spill. Acknowledgments Authors wish to thank staffs of Koh-Samed, Laem Yaa Marine Park for their kindly accommodation and logistic supports throughout field work sampling. This project was financial supported by Nation Council Research of Thailand and Thailand Environmental Institute. References Huz, R. de la., M. Lastra, J. Junoy, C. Castellanos and J.M. Vie´itez. 2005. Biological impacts of oil pollution and cleaning in the intertidal zone of exposed sandy beaches: Preliminary study of the “Prestige” oil spill. Estuarine, Coastal and Shelf Science 65: 19-29. Jongeneelen, F. and J. Benchmark. 2001. Guideline for urinary 1-Hydroxypyreneas Biomarker of occupational exposure to polycyclic aromatic hydrocarbons. The Annals of Occupational Hygiene 45: 3-13. Lim H. H. and H. S. Shin. 2013. Simultaneous determination of 2-naphthol and 1hydroxypyrene in fish and shellfish contaminated with crude oil by gas chromatography–mass spectrometry. 2013. Food Chemistry 138: 791-796. Maki H., T. Sasaki and S. Harayama. 2001. Photo-oxidation of biodegraded crude oil and toxicity of the photo-oxidized products. 2001. Chemosphere 44: 1145-1151. Nagelkerken I. A. and A. O. Debrot. 1995. Mollusc communities of tropical rubble shores of Curaçao: Long-term (7+ years) impacts of oil pollution. Marine Pollution Bulletin 30: 592–598. Piñeira J., H. Quesada, A.E. Rolán and E. Caballero. 2008. Genetic discontinuity associated with an environmentally induced barrier to gene exchange in the marine snail Littorina saxatilis. Marine Ecology Progress Series 357: 175–184. Samakraman S., G. A. Williams and M. Ganmanee. 2009. Spatial and Temporal Variability of Intertidal Rocky Shore Bivalves and Gastropodsin Sichang Island, East Coast of Thailand. The nagisa world congress 35-46 Yamamoto T., M. Nakaoka, T. Komatsu, H. Kawai and K. Ohwada. 2003. Impacts by heavyoil spill from the Russian tanker Nakhodka on intertidal ecosystems: recovery of animal community. Marine Pollution Bulletin 47: 91-98.
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A Study on Some Production Traits and Egg Quality Characteristics of Lutein Eggs Kanda LOKAEWMANEE1* and Supat DUANGDEE1 1
Department of Agriculture and Resources, Faculty of Natural Resources and Agro-Industry, Kasetsart University, Chalermphakiat Sakon Nakhon Province Campus, Sakon Nakon, 4700, Thailand *Corresponding author: e-mail address: csnkdp@ku.ac.th
ABSTRACT This investigation was carried out to demonstrate some production traits and egg quality characteristics of lutein eggs. One hundred twenty laying hens were used in the study. The birds were fed a basal mash diet supplemented with lutein at 0 (CON) and 50 mg/kg (LUT) for three weeks. Birds had free access to feed and water throughout the trial, and egg production were recorded weekly. Thirty eggs per group were evaluated weekly for egg quality characteristics such as yolk weight, albumen weight, shell weight, shell thickness, Haugh unit, shape index and albumen index. The egg yolk color was visually examined using the Roche Yolk Color Fan. The egg yolk color was further examined for chroma using a spectrophotometer, an objective method. The deposition of lutein into egg yolk was indentified at the end of the experiment. There was no difference in final body weight, the relative live weight, feed consumption, hen-day egg production, egg mass, feed efficiency, egg weight, yolk weight, albumen weight, shell weight, shell thickness, Haugh unit, shape index, albumen index and yolk index among dietary groups. The values of yolk color, chroma and concentration of lutein increased in raw and boiled egg yolk in the LUT group compared with the CON group (P<0.05). In conclusion, the present study might suggest that lutein at 50 mg/kg of feed enhanced raw and boiled egg yolk color by increasing chroma value and improving concentration of lutein in egg yolk compared with the lutein at 0 mg/kg of feed. Keywords: Production traits, Egg quality characteristics, Lutein egg Introduction Carotenoid is associated with a reduced incidence of several chronic diseases, including certain cancers, cardiovascular disease and age-related macular degeneration (AMD) (Tapiero et al., 2004). Lutein is one of carotenoid in the human macula and retina (Sommerburg et al., 1998. It is able to absorb blue light striking the retina, which is thought to initiate degeneration of the delicate surface membrane (Landrum and Bone, 2001). Rapp et al. (2000) indicated that lutein may be able to play a role as an antioxidant in macular surface membrane. Human trials with lutein supplements indicate that these dietary carotenoids, in addition to fruits and vegetables, can be used to elevate macular pigment concentration (Thurmann et al., 2005). Moreover, dietary lutein provides evidence of significant efficacy against light-induced skin damage (Robert et al., 2009). As the designer, egg can be considered as a new type of functional food such as eggs with vitamin E, selenium, docosahexaenoic acid (DHA) and polyunsaturated fatty acids (PUFA) (Surai and Spark, 2001). Therefore, the objective of this study was to demonstrate some production traits and egg quality characteristics of lutein eggs.
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Materials and Methods One hundred-twenty, 51-weeks-old Charoen Pokphand laying hens were divided into two groups of 3 chickens with 20 replications. Lutein was supplemented as the basal diet (Table 1) in qualities of 0 (CON) and 0.5 mg/kg (LUT). All birds were maintained in laying cages (3 chickens/cage) in an environmentally controlled room (at a temperature of around 27 ºC with a photoperiod of 16L:8D). Feed and water were available ad libitum. Until 54 weeks of age, feed consumption, hen-day egg production, egg mass, feed efficiency and livability were recorded weekly. Thirty eggs from each group were collected weekly to measure egg quality, for criteria such as egg weight, yolk weight, albumen weight, shell thickness, Haugh unit, shape index and albumen index. At first, egg weight was recorded by using an electronic digital balance. The eggs were broken onto a metal plate and the height of the albumen was measured by the distance between the metal plate and the electrode placed on top of the thick egg weight of the broken egg. Then, the weights of albumen, egg yolk and shell were measured using an electronic digital balance. Shell thickness was a mean value of measurements at three locations on the egg (air cell, equator and sharp end) by a digital caliper. Yolk color was measured visually by using the Roche Yolk Color Fan. Chroma was measured by using spectrophotometer. The values of the shape index, albumen index, yolk index and Haugh unit were calculated for each individual egg using the formula according to Tilki and Saatci (2004). At the end of experiment, lutein in the eggs was analyzed by the Insitute of Food Research and Product Development, Kasetsart University, Bangkok, Thailand. The results were reported as mean±SD and data on production traits and egg quality were statistically analyzed by one-way analysis of variance (ANOVA) supported by the Statistical Analysis System (SAS, 1997). Differences among treatment means were tested using Duncan’s New Multiple Rang Test at P<0.05 (Steel and Torrie, 1980). Results and Discussion Some production traits and egg quality did not have statistically significant differences in both CON and LUT groups (Table 2-4). Consideration of the current parameters suggests that lutein would have no detrimental effect on some production traits and egg quality, with 50 mg/kg dietary lutein. Moreover, all birds were healthy and there were no mortality in this experiment. In raw and boiled egg yolk, yolk color, chroma and concentration of lutein were higher in LUT group than the CON group (P<0.05). As the nutrition composition of the diets in both groups was almost the same, the present better yolk color, chroma and concentration of lutein seemed to be induced by lutein. Our finding are in agreement with those of Jang et al. (2014), who reported that lutein is not synthesized in the human body. Dietary lutein is the only source to meet the requirements of lutein. Leeson and Caston (2004) reported that the egg yolk color increase with adding the dietary lutein. The results from these experiments indicate that lutein is transferred into the yolk. Then, the present study might suggest that lutein at 50 mg/kg of feed enhanced raw and boiled egg yolk color by increasing chroma value and improving concentration of lutein in egg yolk compared with the lutein at 0 mg/kg of feed. Conclusion These results suggest that lutein 50 mg/kg of feed enhanced raw and boiled egg yolk color by increasing chroma value and improving concentration of lutein in egg yolk compared with the lutein at 0 mg/kg of feed.
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Table 1 Formula and composition of the basal diet fed to laying hens. Item % Corn 55.67 Defatted rice bran oil 6.00 Soybean meal 22.98 Fish meal 5.00 Rice bran oil 1.05 Oyster shell 7.85 Dicalcium phosphate 0.45 Salt 0.35 DL-methionine 0.15 Premix1 0.50 Total 100 Chemical composition (%) Crude protein 17.50 Crude fiber 3.76 Crude fat 8.20 Calcium 3.51 Available phosphorus 0.35 Lysine 0.96 Methionine 0.75 2 Metabolizable energy (kcal/kg) 2750 1 Concentrate mixture including (per kg of diet): vitamin A 10000 IU; cholecalciferol 2000 IU; vitamin E 0.25 IU; vitamin K3 2 mg; vitamin B12 10 µg; choline 250 mg; folacin 1 mg; niacin 30 mg; pantothenic acid 10 mg; pyridoxine 3 mg; riboflavin 6 mg; thiamin 2 mg; ethoxyquin 125 mg; choline 1500 mg; copper 10 mg; iodine 0.5 mg; manganese 40 mg; zinc 50 mg; selenium 0.2 mg; presernative 6.54 mg and feed supplement 26 mg. 2 Calculaed values Table 2 Body weight and the relative live weight of laying hens fed basal diet (CON) and diets supplemented with the commercial lutein (LUT) (mean±SD; n=60). Item Dietary group* CON LUT Initial BW (g), 51 wks 1900.20±128.8 1905.11±124.00 Final BW (g), 54 wks 2000±142.6 2001.20±120.45 Live weight (g), g/100g BW 1.02±0.48 1.01±0.58 *CON (the basal diet), LUT (a commercial lutein, 50 mg lutein/kg diet) Table 3 Some production of laying hens fed basal diet (CON) and diets supplemented with the commercial lutein (LUT) (mean±SD; n=60). Item Dietary group CON LUT Feed consumption (g/hen/day) 118.73±0.06 118.70±0.05 Hen-day egg production (%) 89.59±5.44 54.58±3.94 Egg mass (g/hen/day) 54.95±3.58 0.52±0.01 Feed efficiency 0.51±0.01 100 Livability (%) 100
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Table 4 Some egg quality characteristics and concentration of lutein of laying hens fed basal diet (CON) and diets supplemented with the commercial lutein (LUT) (mean±SD; n=30). Item Dietary group CON LUT Egg weight (g) 62.42±3.32 63.76±3.24 Yolk weight (g) 16.95±1.23 17.40±1.00 Albumen weight (g) 39.06±2.37 39.76±2.57 Shell weight (g) 6.49±0.42 6.63±0.25 Shell thickness (mm) 0.43±0.02 0.42±0.01 Haugh unit 79.84±5.82 79.04±5.07 Shape index 75.66±1.83 75.18±2.34 Albumen index 4.35±0.61 4.49±0.36 Yolk index 37.03±1.89 35.97±1.90 Raw egg yolk Yolk color 8.33±0.45b 12.71±0.27a Chroma 13.00±0.00b 14.00±0.00a b Concentration of lutein (µg/100g) 155.96±0.28 2944.90±0.59a Boiled egg yolk Yolk color 1.50±0.27b 7.09±0.21a Chroma 7.30±9.78b 12.00±0.00a b Concentration of lutein (µg/100g) 316.12±0.74 2812.63±0.65a a,b Means in the same row with different superscripts differ significantly (P<0.05). Acknowledgments The work was funded by the Faculty of Natural Resources and Agro-Industry, Kasetsart University Chalermphrakiat Sakonnakhon Province Campus, Thailand. References Jang, I., Y. Ko, S. Kang, S. Kim, M. Song, K. Cho, J. Ham and S. Sohn. 2014. Effects of dietary lutein sources on lutein-enriched egg production and hepatic antioxidant system in laying hens. J. Poult. Sci. 51:58-65. Landrum, J.T. and R.A. Bone. 2001. Lutein, zeaxanthin and the macular pigment. Arch. Biochem. Biophys. 385:28-40. Leeson, S. and L.Caston. 2004. Enrichment of eggs with lutein. Poult. Sci. 83:1709-1712. Rapp, L.M., S.S. Maple and J.H. Choi. 2000. Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and periphera; human retina. Invest. Opthamol. Vis. Sci. 41:1200-1209. Roberts, R.L., J. Green and B. Lewis. 2009. Lutein and zeaxanthin in eye and skin health. Clin. Dermatol. 27:195-201. SAS Institute. 1997. SAS User’s Guide: Statistics. Version 6.12 ed. SAS Institute Inc., Cary, NC. Sommerberg, O., J.E. Keunen, A.C. Bird and F.J. van Kuijk. 1998. Fruits and vegetables that are resources for lutein and zeaxanthin: the macular pigment in human eyes. Br. J. Opthalmol. 82:907-910. Steel, R.G.D. and J.H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd Ed. McGraw-Hill Inc, New York. Surai, P.F. and N.H.C. Sparks. 2001. Designers eggs: from improvement of egg composition to function food. Trends Food Sci. Tech. 12:7-16. Tapiero, H., D.M. Townsend and K.D., Tew. 2004. The role of carotenoids in the prevention of human pathologies. Biomed Pharmacother. 58:100-110. Thurmann, P.A., W. Schalch, J.C. Aebischer, U. Tenter and W. Cohn. 2005. Plasma of lutein, zeaxanthin, and 3-dehydro-lutein after multiple oral doses of a lutein supplement. Am. J. Clin. Nutr. 82:88-97. Tilki, M. and M. Sattci. 2004. Effects of storage time on external and internal characteristic in partridge (Alectoris graeca) eggs. Revue. Med. Vet. 11:561-564.
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Effect of Leucaena Silage Levels on Rumen Fermentation and Nutrients Digestibility in Dairy Steers Giang NGUYEN THIEN TRUONG1 and Metha WANAPAT1* 1
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: metha@kku.ac.th
ABSTRACT The objective of this experiment was to determine effects of Leucaena silage (LS) levels on rumen fermentation and nutrients digestibility in dairy steers. Four, rumen fistulated dairy steers with initial weight of 167 Âą 12 kg were randomly assigned to receive diets according to 4x4 Latin square design. Treatments were: T1 = untreated rice straw (Control), T2 = 70% untreated rice straw + 30% LS, T3 = 40% untreated rice straw + 60% LS and T4 = 100% LS. All treatments were fed ad libitum intake. Results revealed that dry matter intake and digestibility were linearly increased together with increasing levels of LS (P<0.05). Ruminal pH was not changed among treatments while ruminal NH3-N concentration and bacterial population were enhanced with increasing levels of LS (P < 0.05). The blood urea nitrogen was in normal ranges. The acetic acid concentration was decreased (P < 0.05) while propionic acid concentration was increased (P < 0.05) in all LS levels, especially with full LS feeding. Based on this study, it could be concluded that LS has improved digestibility of nutrients, rumen microbial population and rumen fermentation, and the highest improvement was at 100% LS level. Keywords: Dairy steer, Leucaena silage, Rumen fermentation, Rice straw Introduction Feed resources for ruminant are mainly agricultural by-products, especially in tropical countries where the roughage resources are shortage in dry season (Wanapat, 2000). These resources have low nutritive values, low crude protein and high level of ligno-cellulosic which extremely leading low performance (Wanapat et al., 2013). Therefore, protein supplement from concentrate resources make balance ration to improving animal performance is dispensable. However, the price of concentrate is quite high and not always available. For these reasons, finding locally available protein supplement resources to replace concentrate especially fodder tree and shrub legumes such as Leucaena leucocephala and Flemingia macrophylla have been exploited (Wanapat, 2009). Leucaena (Leucaena leucocephala) can be an locally excellent choice for alternative protein resources for ruminants due to its high protein, palatability, digestibility and availability. According to Garcia et al. (1996), Leucaena contain protein at high level of 29.2% crude protein in leaf and 22.03% in forage. However, it contains mimosine which is toxic for ruminants and non-ruminants (Hegarty et al., 1964), resulting in production of goiters, loss of hair, and reduced fertility. In order to overcome these disadvantages of Leucaena only if mimosine is decreased its content. There are many studies have been done to find out the effective methods of Leucaena utilization such as chemical treatment, heat treatment, molasses supplement and ensilage. Leucaena silage can improve feed intake and nutrients digestibility in ruminant and reduce 90% mimosine (Sunagawa et al., 1989). However, due to limitation of data on using Leucaena silage and its effect on dairy steer performance, therefore, the investigation on effect of Leucaena silage as an alternative protein resource on dairy steers should be conducted. July 1-3, 2015
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Materials and methods Animals, diets and experimental design: Four, rumen fistulated dairy steers with initial weight of 167 ± 12 kg were randomly assigned to receive four diets according to a 4x4 Latin square design. All animals were supplemented concentrate at 0.2% of BW/d. The treatments were as follows: T1 = untreated rice straw (Control), T2 = 70% untreated rice straw + 30% Leucaena silage, T3 = 40% untreated rice straw + 60% Leucaena silage and T4 = 100% Leucaena silage. Data collection, sampling and chemical composition analysis: The experiment was conducted for four periods, each lasting 21 days. The feed was sampled daily during period for DM intake measurement. During the last seven days of each period, feed and fecal samples was collected every day and was dried at 60°C and ground (1 mm screen using Cyclotech Mill, Tecator, Sweden) and then analysed for DM, ash and crude protein (CP) content (AOAC, 1985), neutral detergent fiber (NDF), acid detergent fiber (ADF) (Goering and Van Soest, 1991). Rumen fluid samples were collected at 0, 2, 4, 6 h post-feeding. Approximately 100 ml of rumen fluid was taken from the middle part of the rumen at each time at the end of each period. Rumen fluid was immediately measured for pH and temperature using a portable pH and temperature meter (HANNA instrument HI 8424 microcomputer, Singapore). Rumen fluid samples were then filtered through four layers of cheesecloth. After that, 45ml of rumen fluid sample was collected and kept in a plastic bottle to which 5 ml of 1 M H2SO was added and then centrifuged at 3,000 x g for 10 minutes. The supernatant was collected to analyze for NH3-N by Kjeltech Auto 1030 Analyzer (AOAC, 1995). Statistical analysis All obtained data were subjected to ANOVA according to a 4x4 Latin square design using the General Linear Models (GLM) procedures of the Statistical Analysis System Institute (SAS, 1998). Treatment means were compared by Duncan’s New Multiple Range Test (Steel and Torrie, 1980). Results and discussion Chemical composition of feeds The chemical composition of concentrate, rice straw and Leucaena silage are presented in Table 1. The rice straw was low in CP (2.4) and high in NDF (78.9) and ADF (62.5), while Leucaena silage was high in CP (25.1) and low in NDF (36.2) and ADF (21.4). Table 1. Chemical composition of dietary treatment used in the experiment. Concentrate
Rice straw
Leucaena silage
Chemical composition (%) Dry matter
89.5
30.3
Organic matter Crude protein Neural detergent fiber Acid detergent fiber
93.6 13.9 19.3 9.5
89.7 % DM 88.6 2.4 78.9 62.5
Items
90.9 25.1 36.2 21.4
Energy is usually the limiting factor for growth of anaerobic microbes and provision of urea and molasses might have increased the microbial mass that leads to increased CP (Staples et al. 1981). Thus, provision of carbon skeleton and energy for microbial growth in Leucaena silage might have synchronized with ammonia released from urea hydrolysis, consequently increasing the CP content of forages ensiled. These results showed that Leucaena silage is a good alternative for improving feed quality and preservation. Characteristics of ruminal fermentation The table 2 shows that the effects of rice straw replaced with three levels of Leucaena silage on characteristics of ruminal fermentation and blood metabolites. The findings revealed that
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the ruminal pH in 30% and 60% Leucaena silage rations was not significantly different (p<0.05) but higher in other ones, while there was not different between rice straw and 100% Leucaena silage. The result also showed that the NH3-N increased together with increasing the proportion of Leucaena silage in the diets while temperature was not different among treatments. Table 2 Effect of Leucaena silage levels on blood urea nitrogen, ruminal pH, temperature, NH3-N concentration and volatile fatty acids (VFA) concentration in dairy steers.
RST RLS30 LS SEM Significance RLS60 38.9 39.3 38.6 0.93 ns Temperature, C 39.1 a b a b 6.79 6.62 6.8 0.08 * Ruminal pH 6.57 a b d c 7.4 16.2 27.9 0.27 * NH3-N, mg/dl 22.4 RST: Rice straw, RLS30: 70% rice straw + 30% Leucaena silage, RLS60: 40% rice straw + 60% Leucaena silage, LS: Leucaena silage, a,b,c,d Means in the same row with different superscripts differ (P<0.05)*, ns = Nonsignificantly different, SEM = Standard error of the means. Items
0
The reduction in pH with increasing level of Leucaena in the diet indicates increased production of volatile fatty acids from the increased intake and breakdown of the forage. These findings support the results reported by Osakwe and Steingass (2006) that, as Leucaena proportion in the diet increases, rumen function improves. In contrast, replacement with 100% Leucaena silage did not decrease pH showing that the inefficient volatile fatty acids production. However, in this study the pH (6.57-6.8) and temperature (38.6-39.3oC) were in normal range, similar to those reported of Wanapat (1990). Many researchers found that ruminal NH3-N was increased when protein supplementation increased (Nikkhah et al., 2010). Therefore, increasing dietary protein increases ruminal NH3-N concentration. Feed intake, nutrients intake and apparent digestibility Effects of rice straw replacement with Leucaena silage levels on feed intake and nutrient digestibility are presented in Table 3. The results showed the dry matter intake were increased with Leucaena silage levels as compared to only rice straw (p<0.05). However, there was not significantly different among Leucaena silage levels. The digestibility of DM, OM, CP NDF was significantly higher in all Leucaena silage diets and highest in 60% Leucaena silage, in turn 30% and 100% Leucaena silage. The result also showed the digestibility of ADF was significantly in T2 and T3 as compared to T1 and T4. Table 3 Effect of Leucaena silage levels on voluntary feed intake and nutrient digestibility in dairy steers
RST RLS30 RLS60 LS SEM Significance level Items Total Dry matter intake 3a 3.4b 3.4b 3.5b 0.14 * kg/day a b b b 1.7 2.0 2.1 2.1 0.17 * % of BW a b b b 0.75 62.8 73.3 75.3 74.7 2.68 * g/kg BW Nutrient digestibility, % 55.4a 64.0b 71.7c 62.7d 1.02 * Dry mater a b c d 59.0 64.3 72.7 62.3 0.64 * Organic matter 47.9a 61.9b 70.8c 60.3d 1.67 * Crude protein a b c d 53.5 58.5 62.7 55.6 1.30 * Neutral detergent fiber a b c a 50.1 54.9 59.0 51.0 1.38 * Acid detergent fiber RST: Rice straw, RLS30: 70% rice straw + 30% Leucaena silage, RLS60: 40% rice straw + 60% Leucaena silage, LS: Leucaena silage, a,b,c,d Means in the same row with different superscripts differ (P<0.05)*, ns = Nonsignificantly different, SEM = Standard error of the means.
The reduction of DM intake in rice straw diet may be due to the effect of NDF high level which is considered to be the negative index of feed intake. According to Mertens (1987) who reported that feed intake can be limited by the bulkiness (fill effect) of the feed in July 1-3, 2015
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relation to the voluntary intake of the reticulo-rumen. The nutrient digestibility in 30% and 60% Leucaena silage levels had improved and highest in 60% Leucaena silage. In contrast, Jetana et al. (2012) reported that digestibility of nutrients was declined when fed diet containing 50%-84% of Leucaena in swamp buffalo and murah buffalo. This can be explained that Leucaena silage with molasses has provided a balance in protein-energy ratio for microbial growth resulting in increased nutrient digestibility. However, the 100% Leucaena silage diet probably had a high protein to carbohydrate ratio in the diet could lead to a relatively low microbial protein to VFA ratio in the end-products of fermentative (Leng, 1993) which means low digestibility. Besides, high concentration of condensed tannin reduced the DM potentially available for digestion due to not digested by the microbe (McSweeney et al., 2011).
Conclusions In conclusion, feeding of 60% Leucaena silage level to dairy steers improved DM intake, nutrient digestibility and rumen environment. Acknowledgements
The authors would like to express their most sincere gratitude and appreciation to Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, KKU, Thailand and for providing financial support for research and for the use of the research facilities and to the Vietnam International Education Department, Ministry of Education and Training for providing scholarship for the author. References
AOAC. 1995. Official method of analysis. 16th ed. Animal feeds: Association of Official Analytical Chemists, VA, USA. Garcia, G.W., T.U. Ferguson, F.A. Neckles and K.A.E. Archibald. 1996. The nutritive value and forage productivity of Leucaena leucocephala. Anim. Feed Sci. Technol. 60(1â&#x20AC;&#x201C;2), 29-41. Hegarty, M., R. Court and P.Thorne. 1964. The determination of mimosine and 3,4-dihydroxypyridine in biological material. Aust. J. Agric. Res. 15: 168-179. Jetana, T., S. Thongruay, S. Uswang and R. Hengtrakulsin. 2012. A comparative study on mimosine, 3,4dihydroxy pyridine (3,4-DHP) and 2,3-dihydroxy pyrodone (2,3-DHP), purine derivatives (PD) excretion in the urine, thyroid hormone and blood metabolites profiles of Thai swamp buffalo (Bubalus bubalis) and Murrah buffalo (Bubalus bubalis). Trop. Anim. Health Prod. 44:887-897. Leng, R. A. 1993. Quantitative ruminant nutritionâ&#x20AC;&#x201D;a green science. Aust. J. Agric. Res. 44: 363-380. McSweeney, C.S., B. Palmer, D.M. Mcneill, and D.O. Krause. 2011. Microbial interactions with tannins: nutritional consequences for ruminants. Anim. Feed Sci. Technol. 9:38-93. Mertens D. R. 1987.Predicting intake and digestibility using mathematical-models of ruminal function. J. Anim. Sci. 64: 1548-1558. Nikkhah, A., M. Kazemi-Bonchenari, K. Rezayazdi, H. Kohram, and M. Dehaghan-Banadaky. 2010. The effects of different levels of sodium caseinate on rumen fermentation pattern, digestibility and microbial protein synthsis of Holstein dairy cows. Afr. J. Biotechno. 9: 1990-1998. Osakwe, I. and H. Steingass. 2006. Ruminal fermentation and nutrient digestion in West African Dwarf (WAD) sheep fed Leucaena leucocephala supplemental diets. Agroforestry Systems 67:129-133. S.A.S. (1998). Guide for personal computers. 6 th eds. S.A.S. Institute Inc., Cary, NC, USA. Staples, C. R., G. C. Fahey Jr, L. L. Berger and R. B. Rindsig. 1981. Evaluation of dairy waste fiber as a roughage source for ruminants. J. Dairy Sci. 64:662-671. Steel, R.G.D. and J.H. Torrier. 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill Book Company, New York. Sunagawa, L., F. Hongo, Y. Kawashima, and S. Tawatana. 1989. The effect of mimosine reduced Leucaena feed on sheep. Japanese J. Zootech Sci. 60:133-140. Van Soest, P. J., J. B. Robertson and B. A. Lewis. 1991. Methods for dietary fiber neutral detergent fiber, and non starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597. Wanapat, M. 1990. Nutrition Aspects of Ruminant Production in Southeast Asia with Special Reference to Thailand. Funny Press, Bangkok, Thailand, pp 217. Wanapat, M. 2000. Rumen manipulation to increase the efficient use of local feed resources and productivity of ruminants in the tropics. Asian-Aust. J. Anim. Sci. 13 (Suppl.):59-67. Wanapat, M. 2009. Potential uses of local feed resources for ruminants. Trop. Anim. Health Prod. 41:1035-1049. Wanapat, M., Kang, S. and S. Polyorach. 2013. Development of feeding systems and strategies of supplementation to enhance rumen fermentation and ruminant production in the tropics. J. Anim. Sci. Biotechnol. 4:32.
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O-21
Effect of Dried Leucaena Leaf and Leucaena Silage Supplementation on Nutrients digestibility, Rumen Fermentation in Swamp Buffalo 1
Kampanat PHESATCHA1and Metha WANAPAT1*
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: metha@kku.ac.th
ABSTRACT The experiment was conducted to determine the effect of dried Leucaena leaf (DLL) and Leucaena silage (LS) supplementation on rumen fermentation and microbial protein synthesis in swamp buffalo fed rice straw. Four, rumen-fistulated swamp buffaloes were randomly assigned according to a 4Ă&#x2014;4 Latin square design under four dietary treatments; unsupplementation (control), supplementation with DLL at 4 kg/hd/d, supplementation with DLL at 8 kg/hd/d and supplementation with LS at 8 kgDM/hd/d. Untreated rice straw was fed ad libitum, while concentrate was fed at 0.1% BW. The results revealed that total intake was significantly improved when levels of DLL and LS supplementation increased. Ruminal ammonia nitrogen (NH3-N) concentration was significantly increased by DLL and LS at high level of supplementation. There was an increase (P<0.05) in total volatile fatty acid (TVFA), propionic acid (C3), while the highest were found in LS supplemented group. Total urinary dihydroxy pyridine (DHP) corresponded with the mimosine intake. In addition, hormonal profile in this study revealed that, the level of thyroxine (T4) was increased by DLL and LS supplementation while triiodothyroxine (T3) were not changed by treatments. Based on this study, it could be concluded high level of DLL supplementation at 8 kg/hd/d and LS at 8 kgDM/hd/d could enhance the nutrients digestibility, rumen fermentation in swamp buffaloes fed rice straw without adverse effect. Keywords: Leucaena leucocephala, Rumen ecology, Ruminant, Silage Introduction Leucaena has been reported for feeding to ruminants up to 30% of total DM intake (Shelton and Dalzell, 2007). However, the potential attributes of Leucaena are limited by the presence of the toxic amino acid, mimosine. In many tropical regions of the world, consumption of Leucaena by ruminants results in poor growth, alopecia, mouth and esophageal lesions, depressed thyroxine levels and goiter (Ram et al., 1994). During the rainy season, silage is widely used in farms and has a substantial role in animal production systems. High silage quality is a key factor to minimize cost of production and sustained animal health. Increasing use of silage has resulted in continuing efforts to minimize the quality losses (Bartzanas et al., 2013). The target of making silage is to maximize the preservation of original nutrients in the forage crop for feeding, usually a difficult task, since the fermentation process usually leads to less than optimal preservation of nutrients. Ensilaging with additional nitrogen and energy might have increased microbial mass that leads to increased crude protein and nutrient utilization in ruminants (Salem et al., 2013). Therefore, the objective of this study was to investigate the high level of dried Leucaena leaf (DLL) supplementation compare with Leucaena silage (LS) supplementation on feed intake, nutrient digestibility, rumen fermentation and urinary excretion of dihydroxypyridine (DHP) in swamp buffalo fed on rice straw.
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Materials and Methods Leucaena leucocephala was harvested and sundried. Only the leaf of Leucaena was sundried and used as dried Leucaena leaf (DLL) for the experiment. Moreover, Leucaena was harvested and immediately chopped to 2-3 cm length and ensiled with urea and molasses at 1.0 and 2.0% DM, respectively, packed well by pressing to get rid of air inside plastic bags and ensiled for 28 days at ambient temperature of 25-30 °C and used as Leucaena silage (LS). Four, rumen fistulated swamp buffaloes were randomly assigned according to a 4×4 Latin square design. The four dietary treatments were un-supplementation (control), supplementation with DLL at 4 kg/hd/d, supplementation with DLL at 8 kg/hd/d and supplementation with LS at 8 kgDM/hd/d. Untreated rice straw was fed ad libitum, while concentrate was fed at 0.1% BW. Animals received a concentrate diet at 0.1% of BW, while rice straw was offered ad libitum. The experiment was conducted for four periods and each of the four periods lasted for 21 days in length, the last 7 days were for sample collection as animals were moved to metabolism crates for total collection. At the last day of each sampling period, rumen fluid was collected at 0, 2, 4 and 6 h post morning feeding. Approximate 200 ml of rumen fluid were taken from the middle part of rumen through the rumen fistula at each sampling hour. Rumen fluid was immediately measured for pH and temperature using a portable pH meter (HANNA, instruments HI 8424 microcomputer, Singapore). Rumen fluid samples were then filtered through 4 layers of cheese-cloth. The first 45 ml of rumen fluid sample were collected and kept in a plastic bottle to which 5 ml of 1 M H2SO4 and then centrifuged at 3,000 x g for 10 min. About 20-30 ml of supernatant were collected and analyzed for volatile fatty acids (VFA) by High Performance Liquid Chromatography according to Samuel et al. (1997). Blood sample (about 10 ml) was drawn from the jugular vein at the same time as rumen fluid. Blood samples were immediately kept into tubes containing 12 mg of EDTA and plasma was separated by centrifugation at 3,500 x g for 20 min. Plasma triiodothyronine (T3) and thyroxine (T4) concentrations were determined by radioimmunoassay, according to the recommended procedures supplied by the kit manufacturer (KT1CT-T3 RIA CT and KT2CT-T4 RIA CT, RADIMS, P.A via del Mare, 125-0040, POMEZIA (ROMA)-ITALIA). Urine samples were collected at the same day of fecal sampling by total collection method (over 24 h) of each individual buffalo during the last 7 days. Mimosine, 3, 4-DHP and 2, 3-DHP in urine was determined by high performance liquid chromatography using μBondapak C18 Column (3.9×300 mm 10μ, Water Corporation, USA) (Dalzell et al., 2012). All obtained data were subjected to ANOVA according to a 4 × 4 Latin square design using the General Linear Models (GLM) procedures of the Statistical Analysis System Institute (SAS, 1998). Result and Discussion Crude protein (CP) content of concentrate, DLL, LS and rice straw were 122, 204, 258 and 22 %, respectively. DLL and LS contained OM 93.6 and 92.3%, aNDF 32.6 and 36.3%, ADF 17.3 and 20.7 % and mimosine 5.6 and 2.2%, respectively. The results of rice straw, concentrate, total DM and nutrient intakes as well as apparent nutrient digestibility as influenced by DLL and LS supplementation are shown in Table 1. High level of DLL and LS supplementation have affected on decrease rice straw intake (P<0.05) meanwhile, DM, OM, CP and aNDF digestibilities were significantly affected by DLL and LS supplementation (P<0.05) while ADF digestibility was similar among treatments (P>0.05).
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Table 1 Effect of dried Leucaena leaf and Leucaena silage supplementation on voluntary feed intake and nutrient digestibility in swamp buffaloes. Items Dry matter intake Rice straw intake kg/day g/kg BW 0.75 Concentrate intake kg/day g/kg BW 0.75 Total intake kg/day g/kg BW 0.75 Nutrient digestibility, % Dry matter Organic matter Crude protein aNeutral detergent fiber Acid detergent fiber
Control
Supplementation (kg/hd/d) DLL1 at 4 DLL1 at LS2 at 8 kg SEM kg/hd/d 8 kg/hd/d DM/hd/d
P-value
6.5 a 81.1 a
5.3 b 65.8 b
3.4 c 42.3 c
2.9 c 35.8 d
0.33 0.14
0.03 0.04
0.35 4.32
0.35 4.31
0.36 4.35
0.36 4.36
0.52 0.43
0.14 0.31
6.8 a 85.4 a
9.6 b 120.1 b
11.8 c 143.7 c
11.3 c 136.9 c
0.36 0.41
0.04 0.04
58.1 a 59.7 a 40.1a 48.6a 44.1
64.9 b 65.4 b 60.3b 54.1b 44.9
68.2 c 70.2b 69.4c 61.3c 45.2
70.1 c 71.4b 71.2 d 62.2c 45.4
0.54 0.31 0.72 0.61 0.43
0.04 0.03 0.02 0.03 1.23
1
Dried Luecaena leaf, 2 Luecaena silage, SEM = standard error of the mean, a, b, c,d Means in the same row with different superscripts differ (P<0.05) The results of ruminal pH, temperature affected by DLL and LS supplementation are presented in Table 2. The results showed that ruminal pH and temperature were similar among the four treatments value at 38.7-39.4째C and 6.7- 6.9 for temperature and pH, respectively. In addition, supplementation of DLL and LS increased total VFA and propionic acid (C3) concentration (P<0.05). The highest value (total VFA and C3) was found in the treatment LS supplementation and the lowest was in the non-supplemented group. In contrast, there was no change among treatments on acetic acid (C2) while butyric acid (C4) concentration was decreased by DLL and LS supplementation. The urinary mimosine and DHP excretion was (P<0.01) increased in buffaloes fed with DLL. DLL and LS had a mimosine concentration 5.6 and 2.2%, respectively. Total excretion of DHP in the urine over the whole experimental period was related to both the DLL consumed and total mimosine intake. DHP toxicity is commonly disrupting the endocrine system by suppressing blood thyroids and assay of serum T4 and T3 can be used as an indicator of DHP toxicity (Jones and Hegarty, 1984). The present study has shown that the levels of T4 (101-143 ng/ml) was increased by DLL and LS supplementation while T3 (4.60-5.07 ng/ml) was not changed by treatments. Conclusions Based on this study, it could be concluded that supplementation of DLL at 8 kg/hd/d or LS at 8 kg DM/hd/d resulted in improved nutrient digestibility, rumen fermentation indicating high potential use of DLL or LS in swamp buffaloes. However, DLL and LS may be simply prepared and stored for lengthy period of feeding. Presence of urinary DHP indicated relationship between mimosine in Leucaena leaf and its degradation in the rumen of swamp buffaloes. No toxic sign of mimosine was observed in this present study.
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Table 2 Effect of dried Leucaena leaf and Leucaena silage supplementation on ruminal pH, temperature, hormonal profile, volatile fatty acids (VFA) and urinary dihyroxy pyridine excretion in swamp buffaloes. Supplementation (kg/hd/d) Items Ruminal pH Temperature, oC Hormonal profile T3 (ng/mL) 3 T4 (ng/mL) 4 Total VFA, mmol/l VFA, mol/100mol Acetic acid (C2) Propionic acid (C3) Butyric acid (C4) C2:C3 Urinary DHP excretion Mimosine 3,4-DHP, ug/ml 2,3-DHP, ug/ml Mimosine+DHP, Âľg/ml
Control
DLL1 at 4 kg/hd/d
DLL1 at 8 kg/hd/d
LS2 at 8 kg DM/hd/d
SEM
P-value
6.8 38.7
6.9 39.1
6.7 39.2
6.7 39.4
0.05 0.03
0.74 0.71
4.60 101.0 a 83.1a
4.72 120.7 b 96.2b
4.81 138.9 c 102.4c
5.07 143.1 c 106.7c
0.12 0.54 0.33
0.13 0.04 0.04
74.7 13.3a 12.0 a 2.80a
68.75 22.9b 8.3b 2.60a
66.7 27.5c 5.8c 2.38b
66.1 28.3c 5.7 c 2.35b
1.22 0.47 0.38 0.35
0.67 0.04 0.03 0.04
0a 0a 0a 0a
4.2 b 21.6 b 50.7 b 76.5 b
6.1c 52.4c 70.4 c 129.2 c
4.9 c 30.7b 56.6 b 92.2 b
3.23 5.61 4.32 8.41
0.04 0.04 0.03 0.02
1
Dried Luecaena leaf, 2 Luecaena silage, 3T3 = Triiodothyroxine, 4T4 = Thyroxine, SEM = standard error of the mean, a, b, c, d Means in the same row with different superscripts differ (P<0.05) Acknowledgments The authors would like to express their most sincere thanks to all who have assisted and supported the research in this study, particularly the Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand, the Royal Golden Jubilee Ph. D. Scholarship Program for their kind financial support and provision of research facilities. References Bartzanas, T., D.D. Bochtis, O. Green, C.G. Sørensen and D. Fidaros. 2013. Prediction of quality parameters for biomass silage: A CFD approach. Computers and Electronics in Agriculture. 93:209-216. Dalzell, S.A., D.J. Burnett, J.E. Dowsett, V.E. Forbes and H.M. Shelton. 2012. Prevalence of mimosine/DHP toxicity in cattle grazing Leucaena leucocephala pastures in Queensland, Australia. Anim. Prod. Sci. 52(5): 365-372. Jones, R.J. and M.P. Hegarty. 1984. The effect of different proportions of Leucaena leucocephala in the diet of cattle on growth, feed-intake, thyroid-function and urinary excretion of 3-hydroxy4(1h)-pyridone. Aust. J. Agric. Res.35: 317-325. Ram, J.J., P.P. Atreja, A. Chhabra and R.C. Chopra. 1994. Mimosine degradation in calves fed sole diet of Leucaena leucocephala in India. Trop. Anim. Health Prod. 26:199-206. Salem, A.Z.M., Z. Chuan-She, T. Zhi-Liang, M. Mellado, M.C. Salazar, M.M.M.Y. Elghandopur and N.E. Odongo. 2013. In vitro ruminal gas production kinetics of four fodder trees ensiled with or without molasses and urea. J. Integr. Agric. 12(7), 1234-1242. Samuel, M., S. Sagathewan, J. Thomus and G. Mathen. 1997. An HPLC method for estimation of volatile fatty acids of rumen fluid. Indian J. Anim. Sci. 67: 805-807. SAS. 1998. Guide for personal computers. 6th eds. S.A.S. Institute Inc., Cary, NC, USA. Shelton, H.M. and S.A. Dalzell. 2007. Production economic and environmental benefit of Leucaena pastures. Trop Grassl. 41:174-190.
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O-22
Supplementation of Urea on Nitrogen Balance and Microbial Protein Synthesis in Swamp Buffaloes Fed on Cassava Hay and Rice Straw Thiwakorn AMPAPON1, Metha WANAPAT1*and Kampanat PHESATCHA1 1
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding author: metha@kku.ac.th
ABSTRACT The experiment was conducted, using four rumen-fistulated swamp buffaloes with initial average liveweight 355±15.0 kg, to study urea (NPN) and cassava hay supplementation on feed intake, digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes. Treatments were as follows: T1= supplementation with cassava hay at 400 g/hd/day without urea (Control), T2= supplementation with cassava hay at 400 g/hd/day + urea 30 g/hd/day, T3= supplementation with cassava hay at 400 g/hd/day + urea 60 g/hd/day, T4= supplementation with cassava hay at 400 g/hd/day + urea 90 g/hd/day. All buffaloes were randomly allocated to receive diets according to a 4 × 4 Latin square design. The present results revealed that Total dry meter intake were significantly increased level of urea (P<0.05). While, purine derivative (PD) excretion, PD absorption, N absorption, MNS and EMPS were significantly increased at 60 and 90 g/hd/d level of urea (P<0.05). Moreover, bacterial population particularly those of proteolytic, cellulolytic and amylolytic bacteria were linearly increased with increasing level of urea (P<0.05). Based on this experiment, the results revealed that supplementation of urea between 60 to 90 g/hd/d with cassava hay at 400 g/hd/d resulted in improved microbial protein synthesis in swamp buffaloes fed on rice straw. This result implies the beneficial feeding of non-protein nitrogen along with low-quality roughages especially in the tropics. Keywords: microbial protein synthesis, rice straw, swamp buffaloes, urea Introduction The availability of local feed resources in various seasons can contribute as essential sources of carbohydrate and protein which significantly impact rumen fermentation and the subsequent productivity of the ruminants (Wanapat et al., 2013). Cassava hay can be processed from whole crop of about 4 months, containing high level of crude protein (25%) and condensed tannin (3-4%) (Wanapat, 1995). Urea is commonly hydrolyzed by bacterial enzyme in the rumento ammonia and carbon dioxide then ammonia was used further with available C- skeletal to synthesize into microbial protein (Wohlt et al., 1978; McGuire et al., 2013; Wanapat et al., 2013). Piao et al. (2012) also reported the synchronization of the hourly rate of carbohydrate and nitrogen (N) released in the rumen would increase the amount of retained nitrogen for growth and thus improve the efficiency of microbial protein synthesis (EMPS) in holstein steers. However, little information is available on combination of cassava hay and urea as sources of protein in feeding buffaloes. Therefore, the objective was to study supplementation of urea on nitrogen balance and microbial protein synthesis in swamp buffaloes fed on cassava hay and rice straw. Materials and Methods Four rumen-fistulated swamp buffaloes with initial average liveweight of 355±15.0 kg, were used to study urea (NPN) and cassava hay supplementation on feed intake, digestibility, July 1-3, 2015
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rumen fermentation and microbial protein synthesis in swamp buffaloes fed on rice straw based diet. Treatments were: T1= supplementation with cassava hay at 400 g/hd/day without urea (Control), T2= supplementation with cassava hay at 400 g/hd/day + urea 30 g/hd/day, T3= supplementation with cassava hay at 400 g/hd/day + urea 60 g/hd/day, T4= supplementation with cassava hay at 400 g/hd/day + urea 90 g/hd/day, respectively. Untreated rice straw was offered ad libitum. The experiment was conducted for four periods and each period lasted for 21 days in length. Feeds offered and refusals were recorded daily throughout the experimental period for dry matter (DM) intake measurement. Feeds and fecal samples were collected by total collection of each individual buffalo during the last 7 days at morning and afternoon feeding. On day 21 of each period, rumen fluid samples were collected at 0, 2, 4 and 6 hour after the morning feeding through rumen fistula and measured immediately for pH and temperature (HANNA Instrument HI 8424 microcomputer, Singapore). Rumen fluid was separated into measure microbial populations by total direct counts of bacteria (Galyean, 1989) and cultured for groups of bacteria using the roll-tube technique (Hungate, 1969) to identify bacterial groups (i.e., cellulolytic, proteolytic, amylolytic, total viable bacterial counts). Feeds, refusals and fecal samples were dried at 60oC and ground and analyzed using standard methods of AOAC (1995) for DM, crude protein (CP) and ash. Urine samples were analyzed for total N (AOAC, 1990) and urinary allantoin, determined by HPLC as described by Chen and Gomes (1995). The amount of microbial purines absorbed was calculated from purine derivative excretion based on the relationship derived by Chen and Gomes (1995). All data were subjected to ANOVA according to a 4 x 4 Latin square design using the General Linear Models (GLM) procedures of the Statistical Analysis System Institute (SAS, 1996). Results and Discussion The present results revealed that total dry matter intake were significantly increased by level of urea (P<0.05) (Table 1). While, purine derivative (PD) excretion, PD absorption, N absorption, MNS and EMPS were significantly increased at 60 and 90 g/hd/d level of urea (P<0.05). This study revealed that PD excretion, PD absorption, microbial nitrogen supply (MNS), and efficiency of microbial protein synthesis (EMPS) were increased with increasing level of urea supplementation which using urinary PD can estimate microbial N supply in sheep and cattle with reasonable confidence (Chen and Orskov, 2003). Microbial N synthesis increased linearly with increasing urea supplementation, which is reflected in increased PD in the urine (Khattab et al., 2013). Kang et al. (2015) reported that fecal and urinary N excretions were decreased in buffaloes consumed concentrate containing higher CP level especially with urea while purine derivatives increased which resulted in a higher N balance as compared to lower CP level and SBM source treatments. Table 1 Effects of supplementation of urea and cassava hay* on feed intake in swamp buffaloes. Item 0
Urea, g/hd/d 30 60
SEM
P-Value
90
Total DM intake kg/hd/d 6.9 6.9 6.9 7.1 0.53 0.91 %BW/hd/d 2.0 1.9 2.0 2.0 0.32 0.53 0.75 a b bc c g/kg /hd/d 95.8 103.6 107.6 111.3 1.75 0.01 a,b,c,d Means in the same row with different superscripts differ (P<0.05), SEM=standard error of the mean, * Supplementation with cassava hay at 400 g/hd/day. Higher urinary purine derivative was found when level of urea increased especially between 60-90 g/hd/d. Regard to determination of rumen microorganism populations, total viable bacteria, amylolytic, proteolytic and cellulolytic bacteria were increased (P<0.05) at 60 and Page 134
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90 g/hd/d of urea (Table 2). Bacterial population particularly those of proteolytic, cellulolytic and amylolytic bacteria were increased with increasing level of urea supplementation. McSweeney et al. (1999), (2000) reported that condensed tannins altered rumen ecology and increased microbial protein synthesis. Moreover, higher bacterial counts were found at higher mass in cows fed UTRS which had higher levels of ruminal NH3-N than cows fed with untreated-rice straw (Wanapat, 2003). Table 2 Effects of supplementation of urea and cassava hay on rumen bacteria populations Item Urea, g/hd/d SEM P-Value 0 Roll-tube technique, CFU/ml 7 Amylolytic bacteria, ×10 Proteolytic bacteria, ×10
7
Cellulolytic bacteria, ×10 Total viable bacteria ,×10
8
8
30
60
90
2.5a
4.5b
4.5b
4.9b
1.43
0.03
3.6a
3.7a
4.6b
5.9b
1.01
0.02
3.7a
3.8a
5.4b
5.4b
0.76
0.02
5.5
5.6
5.9
6.8
1.37
0.09
a,b,c
Means in the same row with different superscripts differ (P<0.05), SEM=standard error of the mean, * Supplementation with cassava hay at 400 g/hd/day.
Table 3 Effects of supplementation of urea and cassava hay on excretion of urinary purine derivatives (PD) and microbial crude protein supply in swamp buffaloes Item Urea, g/hd/d SEM P-Value 0 30 60 90 PD excretion, mmol/d 26.7a 27.1a 29.8ab 32.0b 1.36 0.01 a ab b c PD absorption, mmol/d 87.4 97.2 116.8 132.4 3.93 0.04 MNS 1,gN/d 63.6a 70.6ab 84.9c 96.3c 2.31 0.02 MP,g/d 397.5a 441.3ab 530.6b 601.9c 10.2 0.01 2 a ab c c EMNS ,g/kg OMDR 28.8 31.5 38.9 43.9 2.10 0.02 a,b,c Means in the same row with different superscripts differ (P<0.05). SEM=standard error of the mean. * Supplementation with cassava hay at 400 g/hd/day. Conclusion and Recommendation Based on this experiment, the results revealed that supplementation of urea between 60 to 90 g/hd/d with cassava hay at 400 g/h/d resulted in improved microbial protein synthesis in swamp buffaloes fed on rice straw. This result implies the beneficial feeding of non-protein nitrogen along with low-quality roughages especially in the tropics. Acknowledgments Authors would like to express their most sincere thanks to Tropical Feed Resources Research and Development Centre (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University (KKU) for their kind financial support and provision of research facilities. References AOAC. 1995. Official Method of Analysis, 16th ed. Animal Feeds: Association of Official Analytical Chemists, VA, USA. July 1-3, 2015
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AOAC. 1990. Official methods of analyses, 15th ed. Association of Official Analytical Chemists. Chen, X.B. and E.R. Ă&#x2DC;rskov. 2003. Research on urinary excretion of purine derivatives in ruminants: Past, present and future. Aberdeen, UK: International Feed Resources Unit, Macaulay Land Use Research Institute, Craigiebuckler. 34p. Galyean, M. 1989. Laboratory procedure in animal nutrition research. Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM, USA. Hungate, R.E. 1969. A roll tube method for cultivation of strict anaerobes. In:Norris, J.R., Ribbons, D.W. (Eds.), Methods in Microbiology. Academic, New York, p. 117-313. Kang, S., M. Wanapat, K. Phesatcha and T. Norrapoke. 2015. Effect of protein level and urea in concentrate mixture on feed intake and rumen fermentation in swamp buffaloes fed rice straw-based diet. Trop. Anim. Health Prod. 47,671-679. Khattab, I.M., A.Z.M. Salem, A.M. Abdel-Wahed and K.Z. Kewan. 2013. Effects of urea supplementation on nutrient digestibility, nitrogen utilization and rumen fermentation in sheep fed diets containing dates. Livest. Sci. 155: 223-229. McSweeney, C.S., B. Palmer, R. Bunch and D.O. Krause. 1999. Isolation and characterization of Proteolytic ruminal bacteria from sheep and goats fed the tannincontaining shrub legume Calliandra calothyrsus. Appl. Environ. Microbiol. 62: 30753083. McSweeney, C.S., B. Palmer and D.O. Krause. 2000. Rumen microbial ecology and physiology in sheep and goats fed a tannin-containing diet. In: Proc. International Workshop on Tannins in Livestock and Human Nutrition (Ed. J.D. Brooker), ACIAR Proceedings No. 92. 171 p. Ă&#x2DC;rskov, E.R., 1999. Supplement strategies for ruminants and management of feeding to maximize utilization of roughages. Preventive Veterinary Medicine 38: 179-185. Piao, M.Y., H. J. Kim, J. K. Seo, T.S. Park, J. S. Yoon, K. H. Kim and J.K. Ha. 2012. Effects of synchronization of carbohydrate and protein supply in total mixed ration with Korean rice wine residue on ruminal fermentation, nitrogen metabolism and microbial protein synthesis in Holstein steers. SAS. 1996. SAS/STAT User's Guide: Statistics, Ver. 6.12. Edition. SAS Inc, Cary, NC, USA. Wanapat, M. 2003. Manipulation of cassava cultivation and utilization to improve protein to energy biomass for livestock feeding in the tropics. Asian-Aust. J. Anim. Sci. 16(3): 463-472. Wanapat, M., S. Kang and S. Polyorach. 2013. Development of feeding systems and strategies of supplementation to enhance rumen fermentation and ruminant production in the tropics. J. Anim. Sci. Biotechnol. 4(1) 27-32. Wanapat, M., K. Sommart and S. Uriypongson. 1995. Effect of cassava pellet leaves in beef cattle fattening rations. Final Report. Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand. Wohlt, J.E., J.H. Clark and F.S. Blaisdell. 1978. Nutritional value of urea versus preformed protein for ruminants. II. Nitrogen utilization by dairy cows fed corn based diets containing supplemental nitrogen from urea and/or soybean meal. J. Dairy Sci. 61 902905.
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Modeling the Microwave Heat Distribution of Banana at Different Ripening Stages Wisara THUTO and Kittichai BANJONG* Faculty of Agro-Industry, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kbkittic@kmitl.ac.th
ABSTRACT Microwave heating may be a viable alternative to maintain fruit quality, as a new thermal treatment method for postharvest insect control or reduce postharvest disease in agricultural commodities. Banana is one of the important high sugar containing tropical fruit crops grown commercially in many countries. This study focused on the influence of sugar content in banana (Musa (ABB group) "Kluai Nam Wa") on heat distribution during microwave heating. In this regard, COMSOL Multiphysics was used to simulate the three-dimensional computer model based on FEM and it satisfactorily predicted the temperature distributions inside the banana slices. This model was validated by comparing with temperature data obtained by an infrared thermometer. Three different ripening stages of banana were investigated and the banana samples were heated by microwave at 800 W for 1 minute. The results showed that sugar content as the effect of the response variable. Initially, the hot spot was generated in the core of the banana sample and subsequently the temperature was distributed radially outward. The banana samples with high sugar content showed higher temperature increases due to their greater dielectric loss factor and the temperature gradually increased to higher levels over time compared to those with low sugar content. The predicted temperature well agreed with the experimental data obtained from an infrared thermometer. Keywords: Heat distribution, Microwave heating, Modeling, Banana, Multiphysics Introduction Microwave heating may be available alternative to maintain fruit quality, as a new thermal treatment method for postharvest insect control or reduce postharvest disease in agricultural commodities. Sugar is an important microwave absorbing food ingredient as compared to other hydrocolloids. Sugars modify the dielectric behavior of water (Sahin and Sumnu, 2006). Banana is one of the important high sugar containing tropical fruit crops grown commercially in many countries (Maskan, 2000). About 82% of total banana production in Thailand is Kluai Namwa (Musa (ABB group)) (Boonyanuphap et al., 2004). Over the last two decades, computer simulation is becoming a promising tool to understand complex microwave heating with powerful computational techniques and development of efficient numerical methods. A computer-based simulation of microwave heating of foods can assist in optimizing design of food systems and packages to improve food quality and safety (Pitchai et al., 2014). Knowledge of the 3D temperature profile of heated products is essential when optimizing microwave heating processes (Salvi et al., 2011). This study focused on the influence of sugar content in Kluai Nam Wa at three different stages of ripeness (unripe, ripe and overripe) on temperature profile during microwave heating, COMSOL Multiphysics was used to simulate the three-dimensional computer model based on FEM and it satisfactorily predicted the heat distribution inside the banana slices. Such discussions aim at improving the understanding of temperature profiles and heat distribution within the banana slices on three different stages of ripeness during microwave heating. July 1-3, 2015
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Materials and Methods
Governing equations The electromagnetic energy distribution inside an oven cavity is governed by Maxwellâ&#x20AC;&#x2122;s wave form equation. đ?&#x2018;&#x2014;đ?&#x153;&#x17D; â&#x2C6;&#x2021; Ă&#x2014; đ?&#x153;&#x2021;đ?&#x2018;&#x;â&#x2C6;&#x2019;1 (â&#x2C6;&#x2021; Ă&#x2014;đ??¸â&#x192;&#x2014; ) â&#x2C6;&#x2019; đ?&#x2018;&#x2DC;02 (â&#x2C6;&#x2C6;đ?&#x2018;&#x; â&#x2C6;&#x2019; đ?&#x153;&#x201D; ) đ??¸â&#x192;&#x2014; = 0 (1) 0
An electromagnetic wave loses its energy while travelling through a losses dielectric medium such as food. Part of the electromagnetic power is converted into thermal energy within the food. Conversion of electromagnetic energy into thermal energy is proportional to the dielectric loss factor (đ?&#x153;&#x20AC; " ) and square of electric field strength: đ?&#x2018;&#x192;đ?&#x2018;Ł = đ?&#x153;&#x2039;đ?&#x2018;&#x201C;đ?&#x153;&#x20AC;0 đ?&#x153;&#x20AC; " |đ??¸â&#x192;&#x2014; |
2
(2)
The dissipated power term (đ?&#x2018;&#x192;đ?&#x2018;Ł , W/m3) is a heat source term in transient heat transfer. đ?&#x153;&#x2022;đ?&#x2018;&#x2021;
đ?&#x153;&#x152;đ??śđ?&#x2018;? đ?&#x153;&#x2022;đ?&#x2018;Ą = đ?&#x2018;&#x2DC;â&#x2C6;&#x2021;2 đ?&#x2018;&#x2021; + đ?&#x2018;&#x192;đ?&#x2018;Ł
(3)
Computational details A commercial finite method based software package, COMSOL Multiphisicsď&#x192;&#x2019; 4.3 was used for geometry model building and temperature prediction. All these calculations were done on a workstation with a Intelď&#x192;&#x2019; Coreď&#x192;&#x201D; i7 (CPU speed 2.8 GHz) and RAM of 16 GB running a 64-bit Windows 7. Geometric model was developed for a 800W domestic microwave oven (Model: ME711K, Samsung Electronics Co., Ltd.). The loads (banana samples) were trim cut to a uniform estimation size with transversal geometry by original banana samples, each load placed on the center of a glass plate (turntable). The size of the tetrahedral mesh elements was calculated in such a way that there would be at least 2 elements for every wavelength of microwaves. Meshing scheme implemented for oven cavity and the load as shown in Figure 1.
Figure 1 Meshing scheme implemented for oven cavity and the load. The parameters used in model were found from literature are mentioned in Table 1. Table 1 Summary of material properties and initial conditions used in the model. Parameters Values 980 kg/m3 Density (đ?&#x153;&#x152;) 3.35 kJ/kg oC Specific heat capacity (đ??śđ?&#x2018;? ) 0.49 W/m oC Thermal conductivity (đ?&#x2018;&#x2DC;) 59.1 Dielectric constant (đ?&#x153;&#x20AC; , ) " 18.9 Dielectric loss factor (đ?&#x153;&#x20AC; )
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Model validation Three different stages of ripeness (unripe, ripe and overripe) of Kluai Nam Wa were obtained from a local supermarket. To determine the total soluble solid (TSS) of the bananas, fruit were juiced and measured by using a refractometer. Prior to heating, samples were peeled manually and transversal cut. The transverse slices were obtained by cuts over the widest part of the sample. These slices had 3.0 cm and 0.6 cm for average diameter and height, respectively. The heating was done in 800W domestic microwave oven (Model: ME711K, Samsung Electronics Co., Ltd.) for 1 minute. One sample for each treatment was used and it was placed on the center of a glass plate. Every 20 seconds, the temperature of sample after heating was quickly measured using an infrared thermal thermometer, five selected points at different radial distances from the center of the samples at which temperature were measured. Each experiment was performed in triplicate and data were collected as average temperature. Results and Discussion The simulated results in terms of the temperature profiles were presented in Figure 2, it showed how microwave heating was non-uniform when applying microwaves to the samples located in the center of a microwave oven. Faster heating of one part of the samples was evident and even hot spots could be identified. The temperature distribution increased all the time from the center to outer regions of the sample. Hot spots were found to be at the center, high temperature was observed at the center as well as at the surface. In another study, hot spots were found to be at the center for spherical shaped products whereas for cylindrical products the hot spots generally occurred at the center than at other regions for slab shaped (Chandrasekaran et al., 2013).
Figure 2
Predicted results on temperature profiles of three different banana slices geometries
Increase of sugar content is another characteristic of banana ripening. The starch is converted to soluble solids as the banana fruit starts to ripen (Blankenship et al., 1993). The overripe of banana samples with the highest sugar content showed higher temperature increases due to their greater dielectric loss factor and the temperature gradually increased to higher levels over time compared to those with low sugar content.
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The prediction of temperature from the simulations were also compared with digital photograph of actual banana samples used in experiments after 1 minute of microwave heating as shown in Figure 3. It can be seen that the agreement between two final of heating patterns was qualitatively consistent, particularly in the hot spot region and it was also indicated that simulated results were quite accurately.
Figure 3 Comparison of predicted results (a) digital photograph (b)of actual banana samples used in experiments after 1 minute of microwave heating. A fairly good agreement was observed between the average experimental and simulated values, the R-squared values were 0.9832, 0.9771 and 0.9704 for unripe, ripe and overripe of banana, respectively. Conclusion To predict the temperature distributions of food sample during microwave heating, a computer model with a new simulation approach based on the finite element method was developed. The simulated results for the temperature profiles in three different stages of ripeness of banana samples are in agreement with experimental results. The quality of the predicted results illustrates that the 3D model of the heat distribution in the banana samples are satisfactory. In addition, this model provides a better understanding of the heat generation phenomenon inside the banana samples and can be used in the future for optimizing the microwave heating process. References Blankenship, S.M., D.D. Ellsworth and R.L. Powell. 1993. A ripening index for banana fruit based on starch content. Hort Tech. 3(3): 338-339. Boonyanuphap, J., D. Wattanachaiyingcharoen and K. Sakurai. 2004. GIS-based land suitability assessment for Musa (ABB group) plantation. J. Appl. Horticulture 6: 3-10. Chandrasekaran, S., S. Ramanathan and T. Basak. 2013. Microwave food processing—A review. Food Research International 52: 243–261. Maskan, M. 2000. Microwave/air and microwave finish drying of banana. J. Food Eng. 44: 71-78. Pitchai, K., J. Chen, S. Birla, R. Gonzalez, D. Jones and J. Subbiah. 2014. A microwave heat transfer model for a rotating multi-component meal in a domestic oven: Development and validation. J. Food Eng. 128: 60–71. Sahin, S. and S.G. Sumnu. 2006. Physical Properties of Foods. Springer. New York. 257 p. Salvi, D., D. Boldor, G.M. Aita and C.M. Sabliov. 2011. COMSOL Multiphysics model for continuous flow microwave heating of liquids. J. Food Eng. 104: 422–429.
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Preparation, Optimization and Characterization of Carboxymethyl Cellulose from Rice Straw Using Microwave Heating Noppadol PANCHAN1 and Chalida NIAMNUY1* Department of Chemical Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand *Corresponding email: fengcdni@ku.ac.th
ABSTRACT Rice straw is an abundant agricultural waste, which can be useful resource as it contains approximately 35% cellulose. Sodium carboxymethyl cellulose (CMC) is the most important water-soluble derivative form of cellulose, which is used in several industrial fields. Therefore, the utilization of rice straw for preparation of CMC is of interest. Due to the preparation processes of CMC requiring high temperature for a long reaction time for the conventional heating, the microwave heating is a high performance alternative technique because it can shorten the process time and environmental friendly. The aims of this work are to isolate Îą-cellulose from rice straw, which is the raw material for CMC preparation and to investigate the effect of various parameters such as sodium hydroxide (NaOH) concentrations (5, 20 and 35 g/100 ml), sodium chloroacetate (SCA) doses (2, 8 and 14 g), microwave power (160, 320 and 480 W) and etherification times (1, 5 and 9 min) on degree of substitute (DS) of prepared CMC. In addition, the experimental condition was designed using Box-Benhken experimental design. Response surface methodology (RSM) is applied to optimize the CMC preparation process, with the best condition for preparation by using 5.505 g/100 ml of NaOH concentration and 12.607 g of SCA dose with etherification at 320 W of microwave power for 7.97 min. Under this condition, CMC with DS 0.697 was produced. The Fourier transform infrared (FT-IR) spectroscopy and X-Ray diffraction (XRD) spectroscopy are also used to characterize the CMC product. Keywords: Box-Benhken experimental design, Carboxymethyl cellulose, Etherification, Microwave heating, Response surface methodology, Rice straw Introduction Rice straw is an abundant agricultural waste. Even though, it has been utilized for animal feed, fertilizer and biofuel but it still remains in large amount. Most of farmers often eliminate it by open field burning due to its most convenient, cheapest and fastest way to clear the land for the next cultivation. On the other hand, this releases pollutants to the atmosphere and causes of soil fertility degradation (Kanokkanjana and Garivait, 2013). According to the composition analysis, rice straw contains 35-40% cellulose that is able to use as raw material for produce carboxymethyl cellulose (CMC), the water soluble ionic derivative of cellulose which widely used in pharmaceuticals, detergents, cosmetics, foods, paper and textile industries, etc (Sadeghi et al., 2012). The preparation processes of CMC require high temperature for a long reaction time in the conventional heating. Microwave heating is a potential technique which converts electromagnetic energy into thermal energy, as it provides a volumetric heat that improves heating efficiencies when compared with conventional techniques (Kappe, 2004). Therefore, it is an interesting heating technique used for shorten the processing time.
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Materials and Methods Rice straw collected from a local paddy field in Ayutthaya province, Thailand was pretreated by following the method of Lu and Hsieh (2013). The first step was alkalization. 4.0 g of isolated cellulose was put into a three necks round bottom flask that was set in a modified microwave oven (LG MS2343DAR, Korea) with condenser and glass agitator. Then 200 ml of isopropyl alcohol (IPA) and 40 ml of 5, 20 and 35 g/100 ml aqueous sodium hydroxide (NaOH) solution were added. After that it was agitated at 500 rpm for 30 min. The second step was etherification. The variable parameter namely, sodium chloroacetate (SCA) dose (2, 8 and 14 g), microwave power (160, 320 and 480W) and etherification time (1, 5 and 9 min) were used in this step. After finishing the reaction, the CMC product was suspended in 80% methanol and neutralized using acetic acid, washed three times with 70% methanol and dried at 60°C for 6 h. All of 29 experiments were designed using the Box-Benhken method and response surface methodology (RSM) was employed to analyze the experimental data to determine the optimal conditions of four parameters using Design Expert® 9.0 (Stat-Ease Inc., USA) software. The prepared CMC was determined for the degree of substitution (DS) using the standard method ASTM D1439-03. The Fourier transform infrared (FTIR) spectroscopy and X-Ray diffraction (XRD) spectroscopy were also used to characterize the CMC product. Results and Discussion Optimization of CMC preparation conditions: According to the multiple regression analysis, a second-order polynomial equation was obtained: DS = -0.38497 + 0.00316A + 0.02539B + 0.00164C + 0.03902D - 0.00077AB - 3.03906×10-5AC - 3.27083×10-5AD + 2.99349×105 BC + 0.00428BD -1.53711×10-5CD + 0.00022A2- 0.00106B2 - 1.47440×10-6 C2 - 0.00285D2 (R2 = 0.9232) where A,B,C and D were the independent variables of NaOH concentration, SCA dose, microwave power and etherification time, respectively. According to the results from ANOVA, it revealed that the significance of regression model, p-value less than 0.005 inferred that A, B, D, AC and BD terms were significant independent variables of DS. Table 1 Variance analysis of regression equation Source model A B C D AB AC AD BC BD CD A2 B2 C2 D2 *
Df* 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Degree of freedom
MS** 0.053 0.042 0.25 0.019 0.29 0.019 0.021 1.541×10-5 3.303×10-3 0.042 3.871×10-4 0.016 9.358×10-3 9.241×10-3 0.013
F-value 12.03 9.52 56.28 4.23 66.46 4.36 4.80 3.474×10-3 0.74 9.52 0.087 3.71 2.11 2.08 3.04
P-value <0.0001 0.0081 < 0.0001 0.0590 < 0.0001 0.0555 0.0459 0.9538 0.4027 0.0081 0.7720 0.0745 0.1684 0.1709 0.1030
**Mean square
In addition, the results clearly revealed that the model term was also significant at 95% confidence interval. The influence of each independent variables on the DS of the prepared Page 142
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CMC was described using response surface plot as shown in figure 1(a-f). Due to degree of substitution (DS) which is a major factor in the water solubility of CMC, it was chosen to denote the CMC quality. The important role of NaOH is to swell the cellulose structure and to enhance reactivity of hydroxyl group of the cellulose chain (Zhang et al., 2011). According to the results of NaOH concentration (Figure1 (a, b and c)), the DS value was the highest at 5 g/100ml of aqueous NaOH solution corresponding to 2.02 mol NaOH/mol AGU. The DS value decreased when the NaOH concentration was increased. Due to, at 20 and 35 g/100 ml of aqueous NaOH solution gave 8.16 and 14.29 mol NaOH/ mol AGU, indicated that the alkali level to cellulose ratio was too high. Consequently, sodium glycolate formation as byproduct simultaneously occurred from a side reaction. In addition, the degradation of cellulose chain at high NaOH conditions led to decrease of the DS of the obtain CMC (Barai et al., 1997). The effect of SCA dose and etherification time were the in same trends, the DS value increased with increasing of SCA dose and etherification time (fig. 1(a, c, d, e and f)). Prolonging the etherification time was consequence of the favorable effect of time on diffusion and absorption of SCA with the ultimate effect of inducing better contacts between SCA and cellulose (Pushpamalar et al., 2006). The model predicted the maximum DS of 0.697 under the optimal conditions of 5.505 g/100 ml of NaOH concentration and 12.607 g of SCA dose at 320 W of microwave power for 7.97 min. According to confirmation of the reliability of the model, five duplications were performed and the mean DS was 0.696. (a)
(b)
(c) (d)
(e)
(f)
Figure 1 3D response surface analysis of effect of variables on DS: (a) combined effect of NaOH concentration and SCA dose (b) combined effect of NaOH concentration and microwave power (c) combined effect of NaOH concentration and time (d) combined effect of SAC dose and microwave power (e) combined effect of SCA dose and time (f) combined effect of microwave power and time on the DS of CMC
Figure 2 FTIR spectra of cellulose and prepared CMC (DS =0.697)
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Figure 3 XRD pattern of cellulose and prepared CMC (DS = 0.697)
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Characterization of prepared CMC: The FTIR spectra of cellulose and CMC (DS 0.697) from rice straw were shown in Figure 2. It presented the same functional group such as hydroxyl group (–OH stretching) at 3437 cm-1, methyl group (–CH2 stretching vibrations) at 2914 cm-1, carbonyl group (C=O stretching) at 1637 cm-1, –CH2 scissoring and –OH bending at 1435 cm-1 and 1336 cm-1, respectively. The band at 1066 cm-1 was due to ether group (>CH-O-CH2 stretching). The additional peak at 2364 cm-1might be due to the combination band from water. Carboxyl groups as their salts have frequency about 1600-1640 cm-1 and 1400-1450 cm-1. Hence, the strong peaks at 1637 and 1435 cm-1indicated the presence of carboxymethyl substituents (Zhang et al., 2011). According to Figure 3, the XRD spectra of cellulose and prepared CMC (DS = 0.697) from rice straw revealed their crystallinity of 59.7% and 45.1%, respectively. The crystallinity of prepared CMC was less than that of cellulose due to the cleavage of hydrogen bonds when it was treated with NaOH. The increased distance between cellulose polymer molecules induced the substitution of SCA into the hydroxyl groups of cellulose (Adinugraha et al., 2005). Synthesized CMC from rice straw using microwave heating was successfully prepared and characterized. The highest DS was obtained under the optimal conditions of 5.505 g/100 ml of NaOH concentration and 12.607 g of SCA by etherification using microwave power at 320 W for 7.97 min. Acknowledgements The authors are gratefully acknowledged the funding from the Kasetsart University Research and Development Institute (KURDI), Kasetsart University. References Adinugraha, M. P., D. W. Djagal and Haryadi. 2005. Synthesis and characterization of sodium carboxymethylcellulose from cavendish banana psudo stem (Musa cavendishii LAMBERT). Carbohydrate Polymers. 62: 164-169 American Society for Testing and Materials (ASTM) D1439-03. 2004. Standard test methods for sodium carboxymethylcellulose. Barai, B.K., R.S. Singhal and P.R. Kulkarni. 1997. Optimization of a process for preparing carboxymethyl cellulose from water hyacinth (Eichornia crassipes). Carbohydrate Polymers. 32: 229-231. Kanokkanjana, K. and S. Garivait. 2013. Alternative rice straw management practices to reduce field open burning in Thailand. International Journal of Environmental Science & Development. 4(2): 119-123. Kappe, C. O. 2004. Controlled microwave heating in modern organic synthesis. Angewandte Chemie. 43: 6250-6284 Pushpamalar, P., S. J. Langford, M. Ahmad and Y. Y. Lim. 2006. Optimization of reaction conditions for preparing carboxymethyl cellulose from sago waste. Carbohydrate Polymers. 64: 312-318. Lu, P.and Y. L. Hsieh. 2013. Preparation and characterization of cellulose nanocrystals from rice straw. Carbohydrate Polymer. 87: 564-573. Sadeghi, M., N. Ghasemi and F. Soliemani. 2012. Graft copolymerization methacrylamide monomer onto carboxymethyl cellulose in homogeneous solution and optimization of effective parameters. World Applied Sciences Journal. 16(1): 119-125. Zhang, G.L., L. Zhang, H. Deng and P. Sun. 2011. Preparation and charaterization of sodium carboxymethyl cellulose from cotton stalk using microwave heating. Journal of Chemical Technology & Biotechnology. 86: 584-589.
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Optimization of Process Parameters on the Production of Bacterial Cellulose from Rice Rinsing Waste Water (nata-de-leri) by Acetobacter xylinum Alwani HAMAD1*, Giswantara and Endar PUSPAWININGTYAS1 Department of Chemical Engineering, Faculty of Engineering, Muhammadiyah University of Purwokerto, Dukuh Waluh Rd, PO BOX 202 Purwokerto, Central Java 53182, Indonesia *Corresponding email :hamadalwani@yahoo.co.id
ABSTRACT Nata is a bacterial cellulose that derived from the fermentation of Acetobacter xylinum. It is often found in desserts as healthy functional foods because of a lot of fiber. Not only it is produced from coconut water known as nata-de-coco, but also it could be produced from other sources such as waste-water from rice rinsing as nata-de-leri. The waste-water is produced from rice rinsing still contains carbohydrate source that can be used as substrate of Acetobacter xylinum fermentation. This study aims to optimize paramaters of the fermentation of bacterial cellulose (nata-de-leri) using statistical method (Placket Burman Method and Response Surface Method) in order to enhance production. The optimization strategy in this study through statistically designed experiments as an effective tool for fermentation engineering. First, Plackett–Burman screening design was applied to address the most significant parameter components affecting nata-de-leri production. A large of continous factors (ratio between rice and water, source of carbon using sucrose, source of nitrogen using CO(NH2)2, source of phosphate using K2HPO4, pH, vitamin, fermentation time, concentration of starter) were screened and insignificant ones were eliminated in order to obtain a smaller, more manageable set of factors. The two-level Plackett–Burman design was used under our experiment at static culture condition at 28 – 30oC. Second, Response surface method using Central Composite Design was employed to find out the optimal levels of fermentation parameters resulted from the first step using response of yield. The result of this study showed that concentration of sucrose, concentration of CO(NH2)2, concentration of starter and fermentation time were found to be the major factors of the nata production. Estimated optimum values for the production of nata-de-leri were as follows concentration of sucrose were 17.5 g/ml; concentration of CO(NH2)2 were 12 g/ml; concentration of starter were 20%; fermentation time for 12 days. It resulted yield of nata at 97.20%. From this study, we can conclude that the optimum nata-de-leri yield was resulted from optimization which was 20 % bigger than the basal parameters. Keywords: Bacterial cellulose, Nata De Leri, Acetobacter xylinum, Rinsing rice waste water, Fermentation Introduction Bacterial cellulose (BC) produced by Acetobacter xylinum at the air-liquid interface of coconut water is popularly known as nata-de-coco. Not only coconut water is used as substrate as nata, rice rinsing waste water can also be used as nata-de-leri. A. xylinum uses the nutrients in the coconut water medium, forms a thin slimy, transparent layer of cellulose on the surface of the medium, which thickens with age forming a thick with sheet after 7–20 days (Iguchi et al., 2000). Although the attributes of bacterial cellulose have been recognized on several field applications, productivity of cellulose production needs to be addressed so as July 1-3, 2015
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to make it economically compatible. Hence it becomes necessary to optimize the yields of cellulose production by the use of process improvement strategies. Various workers have optimized medium constituents and process parameters for increased bacterial cellulose (BC) yield (Embuscado et al., 1994, Bae and Shoda, 2005, Jagannath, 2008, Zenga et al., 2011) The objectives of the present investigation include optimization of nata-de-leri production from A. xylinum RSM. Medium ingredients and process parameters were screened by Plackett-Burman (PB) Design and optimization of significant factors was done applying a Central Composite Design (CCD). Materials and Methods Microorganisms and preparation of Nata â&#x20AC;&#x201C; de - leri starter Acetobacter xylinum (wild type) culture obtained from Food and Bioprocess Research Centre, University of Muhammadiyah Purwokerto, Indonesia was maintained on liquid medium. A. xylinum grown on 500 ml coconut waters was inoculated into sterilized media containing 17.5 g fructuse, 3 g CO(NH2)2 in 500 ml of cocout water, Then pH was adjusted to 4.5 with glacial acetic acid. The inoculated media was incubated in sterilized jar statically at 30 oC for 7 days. Culture media and growth conditions A volume (500 ml) of medium (Rice Rinsing Waste Water) in sterilized container with dimensional 30 x 20 x 5 cm was inoculated with bacterium and incubated at room temperature for 8-16 days under stationary conditions to observe the cellulose pellicles that would form at air-liquid interface. The sterilized of medium is added in fermentation like fructuse, CO(NH2)2, K2HPO4 and Biotin. Screening of the Independent Variables-Plackett Burman Design The concentrations of medium components like fructuse, CO(NH2)2, K2HPO4 and Biotin and also parameters process like ratio between rice and rinsing water, pH, concentration of starter that added in culture and fermentation times were varied from high to low concentrations (Table 1) and 27 experimental trials were conducted in two batches and their effects on BC yield production was determined. Table 1 Component of factors and their high, basal and low level of Plackett Burman method. Variables Rice/rinsing water Sucrose (%w/v) CO(NH2)2 (%w/v) K2HPO4 (%w/v) pH Fermentation time (days) Starter (%v/v) Biotin (drops)
Low level (-1) 1/1 1 0 0 6 8 2 0
Basal level (0) 1/2 3.5 0.6 0.6 4,5 12 12 3
High Level (+1) 1/3 6 1.2 1.2 3 16 22 6
Table 2 Variables and their levels for CCD. Range level in 500 ml culture Variables
-Star point
Low level
Center level
High level
+Star point
Sucrose (g) CO(NH2)2 (g) Fermentation time (days) Starter (ml)
(-Îą) 0.5 0 6 1
(-1) 5 0.5 8 10
0 17.5 3 12 60
(+1) 30 6 16 110
(+Îą) 50 12 20 150
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Optimization of the independent variables-response surface methodology To optimize carbon source (sucrose), nitrogen source (CO(NH2)2), fermentation time and volume of starter, a Central Composite Design (CCD), consisting of a set of 29 experiments with replicates at central point was conducted. A four factor of 5 level CCD with 29 experiments was used (Table 2). Results and Discussion Screening of the independent variables: Plackett-Burman Design From the Main effects plot (Figure 1) and analysis of yield using PB design, it can be seen that carbon source using sucrose, nitrogen sources using CO(NH2)2, fermentation times and volume of starter have major significant effect on nata-de-leri production when compared to another variables (ratio betwen rice and rinsing water, source of phosphate using K2HPO4, pH and source of vitamin like biotin. This is because of source of carbon and nitrogen useful for Acetobacter xylinum to grow in order to make pelicles of nata. The growth of the nata pelicles is also affected by amount of microorganisms itself and time of incubation (Yang et al., 1995, Iguchi et al., 2000, Budhiono et al., 1999). Hence to optimize nata production it is necessary to optimize the levels of significant variables using different concentrations or levels whereas other components of the medium can be kept constant.
Figure 1 Main effect plot for the screening of medium components using PB design. Optimization of the independent variables: Response surface methodology To optimize carbon (sucrose) and nitrogen (CO(NH2)2 ) sources, fermentation times and volume of starter, a Central Composite Design consisting of a set of 29 experiments with four independent at central point was conducted. The results showed the yields from experiments was at the range of 0.00 to 95.53%. The experiment also showed variables and their levels for central composite design (CCD). All the experiments were performed in sterilized container containing 500 ml of media (rinsing rice water). The quadratic model expressed by equation represents nata yield (Y) as a function of sucrose (X1), CO(NH2)2 (X2) , fermentation times (X3) and volume of starter (X4). The response equation from the above set of experiments can be written as: Y = - 4.330 + 2.570 (X1)+ 4.370 (X2)+ 2.324 (X3) + 0.929 (X4) – 0.053 (X1)2 – 0.093 (X2)2 – 0.261 (X3)2 – 0.008 (X4)2 – 0.113 (X1) (X2) + 0.056 (X1) (X3) + 0.001 (X1) (X3) – 0.299 (X2) (X3) + 0.050 (X2) (X3) + 0.037 (X3) (X4) Table 3 Analysis of Variances. Source Regression Linear Square Interaction Residual Error Lack-of-Fit Pure Error Total
DF 14 4 4 6 14 11 3 28
Seq SS 25093.5 14858.7 8026.1 2208.6 5874.7 5866.3 8.3 30968.1
Adj SS 25093.48 2377.47 7996.08 2208.64 5874.65 5866.34 8.32
Adj MS 1792.39 594.37 1999.02 368.11 419.62 533.1 2.77
F 4.27 1.42 4.76 0.88
P 0.005 0.28 0.012 0.536
192.39
0.001
S = 20.48, R-Sq = 81.0%, R-Sq(adj) = 62.1%
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The estimated coefficients for nata yield are presented in Table 3. The significance of each coefficient was determined by student’s T-test and P values. The larger the magnitude of Tvalue and smaller P value, the more significant is the corresponding coefficient. The response obtained under different combinations of variables and defined experimental design. Table 3 was analyzed using the Analysis of Variance (ANOVA) appropriate to the experimental design. Table 3 which indicated that the sum of squares due to regression (first and secondorder terms) was found to be significant (p < 0.05) and lack of fit was not significant. The value of coefficient of determination (R2 = 0.810) suggested that the model is a good fit though accurate. The 3D plot (Figure 2) provided a visual interpretation of the interaction between two factors. In the response surface plot, we can see interaction among variables to show interaction effects. Estimated optimum values for the production of nata-de-leri are as follows: concentration of sucrose are 17.5 g/ml; concentration of CO(NH2)2 are 12 g/ml; concentration of starter are 20%; fermentation time for 12 days. It resulted in yield of nata at 97.20%.
Figure 2 Surface plot of showing the interaction effects Conclusion This work has demonstrated the use of central composite design by determining the conditions which are required to get optimum yield of nata-de-leri production from Acetobacter xylinum. From this study we can conclude that the optimum nata-de-leri yield was resulted from optimization which is 20 % bigger than the basal parameters. References Bae, S. and M. Shoda. 2005. Statistical Optimization of Culture Conditions for Bacterial Cellulose Production using Box-Behnken Design. Biotechnol and Bioengineering 90: 20-28. Budhiono, A., B. Rosidi, H. Taher and M. Iguchi. 1999. Kinetic aspects of bacterial BC formation in nata-de-coco culture system. Carbohy Polym. 40: 137-143. Embuscado, M.E., J.S. Marks and N.B. BeMiller, 1994. Bacterial Cellulose. II. Optimization of Cellulose Production by Acetobacter xylinum through Response Surface Methodology. Food Hydrocolloid 8: 419-430. Iguchi, M., S. Yamanaka and A. Budhiono. 2000. Bacterial cellulose—a masterpiece of nature's arts. Journal of Materials Science 35(2): 261–270. Jagannath, A., A. Kalaiselvan, S.S. Manjunatha, P.S. Raju and A.S. Bawa. 2008. The Effect of pH, sucrose and ammonium sulphate concentrations on the sroduction of bacterial cellulose (Nata-De-Coco) by Acetobacter xylinum. World J. Microbiol. Biotechnol. 24: 2593-2599. Yang, S.H.P., Z. Jung Wook Hwang, Q. Yu Ryang Pyun and Y. Sam Kimi. 1998. Cellulose production by Acetobacter xylinum BRCS under agitated condition. Journal of Fermentation And Bioengineering 85(3): 312-317. Zenga, X., D.P. Smallb and W. Wanb. 2011. Statistical optimization of culture conditions for bacterial cellulose production by Acetobacter xylinum BPR 2001 from maple syrup. Carbohydrate Polymers 85:506–513. Page 148
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Sensitivity Detection of Phytophthora spp. Causing Para-Rubber Leaf Fall Disease to Some Systemic Fungicides Pornprapa KONGTRAGOULl*and Panot VIRIYAEKKUL1 1
Program in Horticulture, Department of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Chumphon, Thailand *Corresponding email: kkpornpr@kmitl.ac.th
ABSTRACT Leaf fall disease of para-rubber was diagnosed by tissue transplanting technique. The 12 isolates of oomycetes were obtained and then observed for morphological characteristics by growing on V8 agar for 5 days. All isolates showed white colony and the growth rate of the colonies was 14.54Âą1.46 mm/day. The mycelia showed hyaline branching, non-septate hyphae, and formed sporangium. These morphological characteristics were identified as Phytophthora sp. The resistibility to some systemic fungicides including dimethomorph, fosetyl-Al, and metalaxyl were detected from the growth of colony by poisoned food technique. The level of resistance to each fungicide was evaluated and grouped into 2 representative phenotype reactions where fungicide resistance (FgR) was for the isolate that be able to grow on each fungicide at more than, or equal to the field-use recommended rate and fungicide sensitivity (FgS) for the isolate that could grow on at less than the field-use recommended rate. Nine isolates were classified as the FgR to metalaxyl. However, all isolates were classified as the FgS to dimethomorph and fosetyl-Al. Keywords: dimethomorph, fosetyl-Al, metalaxyl, para-rubber, Phytophthora sp. Introduction Para-rubber (Hevea brasiliensis) is a major economic crop in Thailand. It produces latex for the manufacturing of a wide range of products. The more than 90% of rubber products and natural rubber in Thailand are exported to overseas markets. However, leaf fall disease caused by Phytophthora sp. is an important disease on para-rubber in tropical wet areas such as southern Thailand. An outbreak of this pathogen from infected leaves to the trapping panel can cause a 40% drop in yield (Drenth and Sendall, 2004). Therefore, application of systemic and contact fungicide is recommended to control this pathogen. However, the effective of systemic fungicide has declined over time because the new resistant strains have appeared in the field. It is a significant limiting factor for disease control since systemic fungicide is having site-specific inhibitors which high risk of resistant development in pathogen (Ishii, 2006). In this study, three fungicides used are from each of the systemic groups that have distinct performance action: carboxylic acid amides (i.e., dimethomorph), phosphonates (i.e., fosetylAl), and phenylamides (i.e., metalaxyl). Dimethomorph inhibits cell wall biosynthesis with low to medium risk of fungicide-resistance problems. Fosetyl-Al, having unknown mode of action, is not appearing in the list derived from reclassified fungicides with low risk of resistance. Metalaxyl inhibits nucleic acids synthesis with high risk (Fungicide Resistance Action Committee, 2013). The objective of this study was to evaluate the sensitivity of Phytophthora sp. causing para-rubber leaf fall to dimethomorph, fosetyl-Al and metalaxyl.
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Materials and Methods
Collection and isolation Naturally infected para-rubber leaves that showed leaf fall symptoms were collected from orchards in Chumphon province, southern Thailand. Tissue transplanting technique was performed to isolate the Phytophthora sp. on PAR(PH)-V8 selective medium. Each isolate was cultured by transferring onto clarified V8 medium (V8) (Campbellâ&#x20AC;&#x2122;s V8 juice, 100 ml; CaCO3, 1.5 g; agar, 15 g; and distilled water, 900 ml). The morphological characteristics of colony and sporangium structures were observed to confirm identification of Phytophthora sp. Fungicide sensitivity test The following systemic fungicides were used in this study: dimethomorph {(E,Z) 4-[3-(4chlorophenyl)-3-(3,4-dimethoxyphenyl) acryloyl] morpholine 50% WP} at 30 g/20 l of fielduse recommended rate, fosetyl-Al (aluminium tris-o-ethlphosphonate 80% WG) at 50 g/20 l of field-use recommended rate, and metalaxyl [methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DLalaninate 25% WP] at 40 g/20 l of field-use recommended rate. The reaction of all isolated Phytophthora sp. to systemic fungicide sensitivity was determined through in vitro comparison of mycelial growth by poisoned food technique. Five-mm diameter mycelial disc was cut from the margin of 4-day-old cultures and transferred onto V8 amended with each fungicide at the final concentration of 0.5x, 1x, and 2x of field-use recommended rate and control (without fungicide). All tested fungicides were added to V8 after autoclaving and the plates were incubated at room temperature for 3-4 days. After inoculation, diameter of each colony was measured and the percentages of growth were calculated. Values obtained were categorized as fungicide sensitivity was evaluated and grouped into 2 representative phenotype reactions; resistant (FgR), and able to grow on each fungicide at more than, or equal field-use recommended rate and sensitive (FgS) at less than field-use recommended rate. Results and Discussion In our sampling of para-rubber, leaf fall disease was detected in Chumphon province, southern Thailand. This is the major para-rubber production zone and rainfall is often very conducive for disease development. Twelves fungal isolates were isolated successfully from para-rubber leaf samples showing typical leaf fall symptom (Figure 1a).
(a) (b) (c) (d) Figure 1 Symptom of para-rubber leaf fall disease (a) caused by Phytophthora sp., colony characteristic on V8 agar (b), hyaline branching and non-septate hyphae (c), and sporangium (d).
All isolates were observed for morphological characteristics by growing on V8 agar for 5 days. The results showed that the colony characteristic of all isolates was white colony and the growth rate of the colonies was 14.54Âą1.46 mm/day (Figure 1b). The mycelia of all isolates showed hyaline branching, non-septate hyphae, and formed sporangium (Fig 1c, d). These morphological characteristics were identified as Phytophthora sp. (Erwin and Ribeiro 1996; Zheng 1997). Page 150
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Figure 2 Fungicide-resistant assay of Phytophthora spp. causing para-rubber leaf fall diseases on V8 agar amended with dimethomorph, fosetyl-Al, and metalaxyl. Control
0.5X
1X
2X
Control
0.5X
1X
2X
Control
0.5X
1X
2X
Isolate code
dimethomorph
fosetyl-Al
metalaxyl
Isolate code
dimethomorph
Fosetyl-Al
metalaxyl
Figure 3 Fungicide-resistant assay of Phytophthora spp. causing para-rubber leaf fall diseases on V8 agar amended with dimethomorph, fosetyl-Al, and metalaxyl at concentrations of 0.5x, 1x, and 2x of the field-use recommended rate.
The resistibility of all isolates to dimethomorph, fosetyl-Al, and metalaxyl were tested by mycelial growth assays on V8 amended with each fungicide at the final concentration of 0.5x, 1x, and 2x of field-use recommended rate and control (without fungicide). After incubation at room temperature, the diameter of each colony was measured and each isolate was classified July 1-3, 2015
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into two representative reactions of each fungicide as follows; fungicide resistance (FgR) for the isolate that be able to grow on each fungicide at more than, or equal to the field-use recommended rate (1x and 2x) and fungicide sensitivity (FgS) for the isolate that could grow on at less than the field-use recommended rate (0.5x). The result showed that nine isolates were classified as the FgR and three isolates as the FgS to metalaxyl. However, all isolates were classified as the FgS to dimethomorph and fosetyl-Al (Figure 2, 3). This showed that Phytophthora sp. treatment with metalaxyl failed to control resistant isolates, but dimethomorph and fosetyl-Al were usually effective to control. As many other studies indicated that repeated applications of metalaxyl for controlling Phytophthora sp. enhances development of resistant strain (Timmer et al., 1998; Judelson and Roberts. 1999; Fontem et al., 2005; Grünwald et al., 2006; Yuan et al., 2006; Earnshaw and Shattock, 2012). Conclusion Twelve isolates of Phytophthora sp. were isolated from para-rubber fall leaf. The nine isolates were resistant to only metalaxyl. However, all tested isolates were sensitive to dimethomorph and fosetyl-Al. Acknowledgments We express our sincere thanks to King Mongkut’s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Chumphon, Thailand for financial support. I am grateful to Prof. Dr. Hideo Ishii, Assoc. Prof. Dr. Tanimnun Jaenaksorn, and Dr. Sarunya Valyasevi for encouragement and valuable advice.
References Drenth, A. and B. Sendall. 2004. Isolation of Phytophthora from infected plant tissue and soil, and principles of species identification. In ‘Diversity and Management of Phytophthora in Southeast Asia’. (Eds A Drenth, DI Guest) pp. 94–102. (Australian Centre for International Agricultural Research:Canberra, Australia). Earnshaw, D. M. and R. C. Shattock. 2012. Sensitivity of progeny of Phytophthora infestans to fungicides. Asian Journal of Agriculture Sciences, 4 (3): 213-224. Erwin, D. C. and Ribeiro, O. K. 1996: Phytophthora Diseases Worldwide St Paul, MN: APS Press. Fontem, D. A., O. M. Olanya, G. R. Tsopmbeng and M. A. P. Owona. 2005. Pathogenicity and metalaxyl sensitivity of Phytophthora infestans isolates obtained from garden huckleberry, potato and tomato in Cameroon. Crop Protection 24: 449-456. Fungicide Resistance Action Committee. 2013. Fungicides sorted by FRAC Code. Available: http://ipm.ifas.ufl.edu/resources/success_stories/T&PGuide/pdfs/Appendices/Appendix6-FRAC.pdf Grünwald, N. J., A. K. Sturbaum, G. R. Montes, E. G. Serrano, H. Lozoya-Saldana, and W. E. Fry. 2006. Selection for fungicide resistance within a growing season in field populations of Phytophthora infestans at the center of origin. Ecology and Epidemiology. 96 (12): 1397-1403. Hammi, A., Y. Msatef, A. Bennani, A. E. L. Ismaili and M. N. Serrhini. 2002. Mating type, Metalaxyl resistance and aggressiveness of Phytophthora infestans (Mont.) de Bary in Morocco. Journal Phytopathology 150: 289-291. Ishii, H. 2006. Impact of fungicide resistance in plant pathogens on crop disease control and agriculture environment. JARQ 40 (3): 205-211. Judelson, H. S. and S. Roberts. 1999. Multiple loci determining insensitivity to Phenylamide fungicides in Phytophthora infestans. Phytopathology 89 (9): 754-760. Timmer, L. W. J. H. Graham, and S. E. Zitko. 1998. Metalaxyl-resistant isolates of Phytophthora nicotianae: Occurrence, sensitivity, and competitive ability on citrus. Plant Disease 82: 254- 261. Zheng, X. B. 1997: Phytophthora and its Research Techniques. Beijing: China Agricultural Press. Page 152
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Assessment of the Antagonistic Activity of Trichoderma spp. from Five Different Habitats on Plant Pathogenic Fungi Suriyasit SOMNUEK1* Pornprapa KONGTRAGOUL2 and Tanimnun JAENAKSORN1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, Thailand 2 Program in Horticulture, Department of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Chumphon, Thailand *Corresponding email: suriyasitsom@gmail.com
ABSTRACT The antagonistic efficacy of Trichoderma spp. from five different habitats (T121Kh, T114Kb, T114So, T112Sc and commercial Trichoderma) were determined on mycelial growth of six plant pathogenic fungi, namely Alternaria sp., Curvularia sp., Fusarium sp., Helminthosporium sp., Pestalotia sp., and Rhizoctonia sp. by dual culture test. The results showed that all tested Trichoderma spp. had potential to inhibit the mycelial growth of all tested pathogens. T114Kb showed the highest growth inhibition on Alternaria sp., Curvularia sp., Fusarium sp., Helminthosporium sp. and Rhizoctonia sp. in the range of 67.40-77.77 percent and followed by commercial Trichoderma with 64.81-77.40 percent inhibition against Alternaria sp., Curvularia sp., Helminthosporium sp. and Rhizoctonia sp. regarding antagonistic mechanism, antibiosis and competition mechanism were shown. Therefore, these tested Trichoderma spp. can be used for further studies on in vivo to confirm the feasibility of using in plant disease control. Keywords: Trichoderma spp., Antagonists, Plant pathogenic fungi Introduction Fungi are responsible for approximately more than 70% of all major crop diseases (Agrios, 2005). Plant diseases that are caused by fungi reduce the crops, create markings, affect the flowers and fruits, finally causing death of the plant. In recent years, there has been a growing trend towards the search for alternatives to chemical pesticides in many crops (Walter, 2009) due to the harmful effect of chemical to human and environment. In regard to the control of such plant diseases, biological control is considered as one of the more reliable, sustainable, eco-friendly approaches to the management of plant disease. Trichoderma fungi are the most popular agents used in a biological control (Monte, 2001; Gveroska and Ziberoski, 2012). Before the application of biocontrol agent, it is however necessary to first do various in vitro studies such as impact of each species of biocontrol agent on each pathogen. Therefore, the purpose of this study was to determine the antagonistic effect of indigenous Trichoderma spp. from different habitats in Thailand on 6 genera of plant pathogenic fungi in laboratory condition. Materials and Methods Antagonistic fungi. The tested Trichoderma isolates were obtained from different habitats in Thailand. These include T121Kh and T114Kb from nutrient solution in hydroponics farm and forest soil at Nakhon-Ratchasima province, T114So and T112Sc from clay soil in organic and chemical rice field in Suphanburi province, respectively. While, T. com was obtained from commercial product.
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Plant pathogenic fungi. Six genera of plant pathogenic fungi (Alternaria sp., Curvularia sp., Helminthosporium sp., Pestalotia sp., Fusarium sp. and Rhizoctonia sp.) were provided by Plant Pathology Laboratory, Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok Thailand. Antagonism assessment. The antagonistic activities of all Trichoderma spp. isolates were tested determined by dual culture technique. The experimental design used was a completely randomized with five replications for each isolates. A mycelial plug (5 mm in diameter) from the margin of 3 day-old culture of Trichoderma spp. and 7 day-old culture of plant pathogenic fungi were placed opposite to each other equidistant from the periphery (2 cm). In control plates, an agar plug of only Trichoderma (without pathogen) or only plant pathogenic fungi (without Trichoderma) was placed on the PDA plates. All plates were incubated at room temperature. Diametrical growth of pathogen isolates was daily measured and the percentage of growth inhibition (GI) was calculated in relation to growth of the controls without Trichoderma as follows: Growth inhibition percentage (GI) (%) = [(CP-T)/CP] Ă&#x2014; 100; where GI is inhibition of diametrical mycelial growth; CP is the diameter of colony measurement of each plant pathogenic fungi in the control; T is the diameter of colony of the pathogen the presence of Trichoderma isolates. Results and Discussion Antagonistic activities of Trichoderma spp. from 5 different habitats were determined against mycelia growth of 6 plant pathogenic fungi by dual culture test. The results showed that all tested Trichoderma spp. presented antagonistic potential against all tested fungi from the early stage of incubation (3 DAI) and their potentialities on inhibiting fungal growth varied among tested fungi in the range of 8.5-49 % through mostly antibiosis mechanism (Figure 1). At the end of incubation (9 DAI), all 5 isolates of Trichoderma spp. were capable of influencing the growth of all tested pathogens (64.44-77.77 % growth inhibition) and their antagonistic activities were significantly different in all tested pathogens except in Alternaria sp. Overall, T114Kb from forest soil was found to be more efficient in influencing the growth of tested pathogens through competition mechanism with marked inhibitory effect on mycelial growth of all test fungi (in the range of 67.4-77.77 %) except for Pestalotia sp., while T114So from clay soil of organic rice field was shown to induce the maximum inhibition of growth of Pestalotia sp. (76.66%) as well as Alternaria sp. (77.77%) and Curvularia sp. (73.33%) (Figure 1, 2). Besides, no deformations of pathogen hyphae were observed under microscopic observations implying that test Trichoderma spp. having no exploitation mechanism. Fungi of the genus Trichoderma have long been recognized for their ability to act as biocontrol agents against plant pathogens (Harman, 2006; Gveroska and Ziberoski, 2012; Patil et al., 2012; Jat and Agalave, 2013). In present study, we found Trichoderma from forest soil (T114Kb) and from clay soil of organic rice field (T114So) were having quite high antagonistic activity (64.44-77.77 %) against test pathogens which were in line with the other researches being revealed that Trichoderma spp. gave the highest inhibition on growth of Alternaria sp. (73.56%), Curvularia sp. (68.22%) (Pradubyart et al., 2013) and F. oxysporum (73%) (Thongkamngam et al., 2013).
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80 60
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Figure 1 Antagonistic activity of Trichoderma spp. on plant pathogenic fungi by dual culture test at 3 DAI and 9 DAI. ; Alt. = Alternaria sp., Cur. = Curvularia sp., Fus. = Fusarium sp., Hel. = Helminthosporuim sp., Pes. = Pestalotia sp. and Rhi. = Rhizoctonia sp.; Values are means of five replicates. The same letter on column bars with in each pathogen are not different significantly according to Duncanâ&#x20AC;&#x2122;s Multiple Range Test (DMRT) (Pâ&#x2030;¤ 0.05); A = Antibiosis, C = Competition mechanism
Figure 2 Dual-culture of Trichoderma spp. against plant pathogenic fungi at 3 and 9 DAI. CP = Control pathogen plate; DC = Dual culture plate; CT = Control Trichoderma plate
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Conclusion This study confirms in vitro biological activity of all tested Trichoderma spp. from 5 different habitats towards 6 genera of plant pathogenic fungi. T114Kb from forest soil was found to be more efficient in giving marked inhibitory effect on mycelial growth of all test fungi in the range of 67.4-77.77 %, while T114So from clay soil of organic rice field also offered significant inhibition of growth (73.33-77.77 %) of 3 genera of tested fungi. To conclude, the strong antagonistic effect of T114Kb and T114So towards Alternaria sp., Curvularia sp., Fusarium sp., Helminthosporium sp., Pestalotia sp. and Rhizoctonia sp. can be applied in biological control of these pathogens and should be further studied in order to be incorporated into other eco-friendly control measures such as cultural and physical control. Acknowledgment The authors are sincerely thankful to Plant Pathology Laboratory, Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology for facilities support and providing the plant pathogenic fungi. References Agrios, G.N. 2005. Plant pathology, Elsevier Academic Press, California, 952 p. Gverroska, B. and J. Ziberoski. 2012. Trichoderma harzianum as a biocontrol agent against Alternaria alternata on tobacco. Applied Technologies and Innovation 7(2): 67-76. Harman, G.E. 2006. Overview of mechanism and uses of Trichoderma spp. Phytopathology 96:190-194. Jat, J.G. and H.R. Agalave. 2013. Antagonistic properties of Trichoderma species against oilseedborne fungi. Science Research Reporter 3(2): 171-174. Monte, E. 2001. Understanding Trichoderma: Between biotechnology and microbial ecology. International. Microbiology 4:1-4. Patil, A., A. Laddha, A. Lunge, H. Paikrao and S. Mahure. 2012. In vitro antagonistic properties of selected Trichoderma species against tomato root rot causing Pythium species. International Journal of Science, Environment 1 (4): 302-315. Pradubyart, M., N. Parinthawong and T. Jaenaksorn. 2013. In vitro screening of antagonistic Trichoderma sp. against pathogenic fungi causing leaf spot of vegetable crop grown in hydroponics. Proceedings of 6th Rajamangala University of Technology Tawan-ok Research Conference: 52-57. Thongkamngam, T., P. Koohakan, and T. Jaenaksorn. 2013. In-Vitro assessment of antagonistic efficacy of Trichoderma isolates against Fusarium oxysporum f. sp. lactucae incitant of wilt on hydroponically grown lettuce. King Mongkutâ&#x20AC;&#x2122;s Agricultural Journal 31 (3): 57-67. Walters, D. 2009. Disease control in crops: Biological and environmentally-friendly approaches. Wiley-Blackwell, West Sussex, 280 p.
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Colonization of Plant Root and Punctured Surface Tissue by Non-pathogenic and Pathogenic Fusarium oxysporum Titi THONGKAMNGAM* and Tanimnun JAENAKSORN *Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding authors: Titi29950@hotmail.com
ABSTRACT Purpose of this research was to assess the colonization of non-pathogenic Fusarium oxysporum strain F221-B and pathogenic F. oxysporum strain F221-R and F422-G on roots of lettuce (Lactuca sativa L.) grown in hydroponics and on punctured surface tissue of pakchoi (Brassica chinensis var. chinensis). The colonization was assessed in terms of epiphytic and endophytic colonization. Besides, survival of the tested fungi in nutrient solution was also determined. The results showed that non pathogenic F221-B could colonize on root surface (as epiphytic colonization with 28.5 and 54 CFU/ml in the 1st and 3rd week after inoculation, respectively) as well as inside the root (endophytic colonization was about 60 CFU/ml in the 1st wk. and 76 CFU/ml in the 3rd wk after inoculation) without producing any disease symptom. Unexpectedly, the pathogenic F221-R and F422-G were found to colonize only on root surface as epiphytic colonization. Regard to the colonization on punctured surface tissue, the top-view as well as cross-sectional view of colonization of plant surface tissue and top-view of epidermal peel of a plant leaf were visualized and recorded. Keywords: Non-pathogenic Fusarium oxysporum F221-B, Epiphytic colonization, Endophytic colonization, Lactuca sativa L., Brassica chinensis var. chinensis Introduction Plant disease management through the use of biocontrol agents (BCAs) could offer the prospect of an environmentally safe alternative (Sikora, 1997). Successful colonization in plant tissue is an important step for their suitability as BCA. In some instances, endophytes, normally live asymptomatically inside the plant, could develop mutualistic relationships with plants, acting as antagonists to diseases (Sikora, 1992; Sikora, et al., 2003; Paparu et al., 2006). When endophytes are artificially reintroduced into the host plant, biological control of disease can be greatly enhanced (Clay, 1989). Hence, the potential of endophytes as BCA (such as protective strains of Fusarium oxysporum) has currently drawn attention to many researchers (Eparvier and Albouvette, 1994; Olivain and Albouvette, 1997; Bao and Lazarovits, 2001; Olivain et al., 2006; Nahalkova et al., 2008). Non-pathogenic F. oxysporum (F221-B), originally isolated from root of lettuce grown in hydroponics, has been proved so far as plant growth promoting fungus (Thongkamngam et al., 2013) and BCA (unpublished). In order to maximize its biological control potential, it is essential that high initial colonization and subsequent persistence could occur. However, nothing has been known about the pattern of lettuce root colonization by this non pathogenic F221-B, such information is important in understanding its mechanism of protecting plant against disease. Therefore, the aim of this study was to assess the colonization (in terms of epiphytic and endophytic colonization) of non pathogenic F221-B compared to pathogenic F. oxysporum (F221-R and F422-G) on roots of lettuce grown in hydroponics and on punctured surface tissue of pakchoi. Besides, their patterns of surface tissue colonization were described. Materials and Methods Colonization of non-pathogenic F. oxysporum strain F221-B and pathogenic F. oxysporum strain F221-R and F422-G on roots of lettuce grown in hydroponics To assess the colonization of non-pathogenic F221-B and 2 strains of pathogenic F. oxysporum, Completely Randomized Design (CRD) with 3 replications of 3 plants was employed. Before transplanting into the DRFT, each 14 day-old-lettuce seedling root was July 1-3, 2015
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dipped into 5 ml of spore suspension of each tested F. oxysporum (1x108 spore/ml for F221-B and 1x106 spore/ml for F221-R and F422-G) while sterile water was used for healthy control. All the treated plants were grown in mini DFT under outdoor condition. Root colonization was assessed in terms of epiphytic and endophytic colonization at 1, 2 and 3 weeks after inoculation. Besides, survival of tested fungi in nutrient solution was also monitored accordingly. Epiphytic colonization assessment (Osono, 2008) Treated roots were washed under running tap water for 10 minutes and then were surfacesterilized by briefly in series with 70 percent ethanol for 30 seconds, 10 % sodium hypochlorite for 1-2 minutes and further cleaned by passing through two sets sterile water. Rinse water from the second rinse was kept and poured onto plate containing Trichoderma selective media (TSM). Culture plates were incubated at room temperature for 3 days then fungal colonies were counted. Endophytic colonization (Elizabeth and Herre, 2003) Treated roots were washed under running tap water for 10 minutes and then were surfacesterilized by briefly in series with 70 percent ethanol for 30 seconds, 10 % sodium hypochlorite for 10 minutes and finally rinsed 3 times with sterile water (3 minutes each). The sterile root was crushed in 1 ml sterile water with mortar. Sample solution was poured onto TSM plate and cultures were kept at room temperature for 3 days then fungal colonies were counted. Colonization of non-pathogenic F. oxysporum (F221-B) and two strains of pathogenic F. oxysporum (F221-R and F422-G) on punctured surface tissue of pakchoi To study the colonization of non-pathogenic F221-B and 2 strains of pathogenic F. oxysporum on punctured surface tissue of pakchoi sheath leaf, two agar plugs from 7 day old culture of each tested strain of F. oxysporum were inoculated on 2 wounds per sheath leaf (5 leaves per tested strain). Sterile PDA plug was used as non-inoculated control. All inoculated sheath leaves were incubated at room temperature. The colonization of plant tissue on top-view, cross-sectional view and top-view of epidermal peel of a plant leaf were observed under stereomicroscopy and light microscopy and then photographed. Besides, disease lesion was measured and disease severity was calculated. Results and discussion Colonization of non-pathogenic F. oxysporum (F221-B) and pathogenic F. oxysporum (F221-R and F422-G) on root of lettuce (Lactuca sativa L.) grown in hydroponics Our result revealed that non pathogenic F221-B could successfully colonize both on surface and inside the root without producing any disease symptom (Figure 1 and 2). However, the amount of colonization was pretty low compared to those from original inoculated amount (epiphytic colonization was only 0.29x 102 and 0.54x102 CFU/ml while endophytic colonization was about 0.6x102 CFU/ml in the 1stand 3rd week after inoculation, respectively,). Contrary to expectations, both isolates of pathogenic F. oxysporum were found to colonize only on root surface with lower amount than that of non-pathogenic F221-B from beginning till the 3rd week after inoculation (0.23x102 CFU/ml for F221-R and 0.16x102 CFU/ml for F422). Furthermore, it was demonstrated that survivals of the three tested fungi in nutrient solution were much higher than those in plant tissue, this probably due to fungal inoculation method onto plant root and into nutrient solution at the same time. However, their survivals were decreased along with increasing incubation times. At the 3rd week after inoculation, survival amount of tested fungi in nutrient solution were in the range of 1.74-2.2x102 CFU/ml (Figure 1). Our study confirming colonization ability of non-pathogenic F221-B as epiphyte and endophyte is in agreement with those described by Paparu et al. (2005); Olivain et al. (2006); Nahalkova et al. (2008) who stated that many strains of non-pathogenic F. oxysporum could colonize root surface and penetrate intact host tissues. Regard to our result on colonization ability of two isolates of pathogenic F. oxysporum as epiphyte, this contradicts to Scott et al. (2014) who described the intensity of pathogen colonization of the vascular stele of lettuce cultivars. However, endophytic colonization of the 2 strains of pathogenic F. oxysporum from this study could probably be detected if incubation period is set longer.
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A
B
Figure 1 Colonization of non-pathogenic F. oxysporum (F221-B) and pathogenic F. oxysporum (F221-R and F422-G) including on root of lettuce (Lactuca sativa L.) grown in hydroponics their survival in nutrient solution (A) and colony on PDA at 21 DAI (B).
Figure 2 Colonization of non-pathogenic Fusarium oxysporum (F221-B) and pathogenic F. oxysporum (F221-R and F422-G) on root of lettuce grown in hydroponics (at 7 and 21 DAI). Colonization of non-pathogenic Fusarium oxysporum (F221-B) and two strains of pathogenic F. oxysporum (F221-R and F422-G) on punctured surface tissue of pakchoi. Regard to the colonization on punctured surface tissue of pakchoi leaf by non-pathogenic F. oxysporum (F221-B) and two strains of pathogenic F. oxysporum (F221-R and F422-G), the result showed that pattern of leaf surface tissue colonization was similar for both strains of pathogenic F. oxysporum with 82.5-100 % disease severity (DS) while no obvious disease symptom was noted with colonization of non-pathogenic F221-B (top-view and crosssectional view of colonization) (Table 1). Besides, severe disease lesion (its size about 3-4 cm) was observed with the depression of the leaf surface at 5 DAI. Abundant white hyphae of three tested strains of F. oxysporum were found spreading on the epidermis while only hyphae of two pathogenic strains could be observed from internal tissues. Based on epidermal peel view, normal tissue was illustrated on non-inoculated tissue while hyphae of 2 strains of pathogenic F. oxysporum were visible in intercellular and intracellular spaces. Interestingly, only macroconidia of non-pathogenic F. oxysporum (F221-B) were observed on epidermal peel view. These results are in line with Paparu et al., (2006) who provided the first in situ observation and quantification of endophyte colonization and also demonstrated the ability of F. oxysporum to penetrate intact host tissue. To the extent that such differences between colonization on lettuce root and on punctured surface tissue of pakchoi leaf by pathogenic F. oxysporum are probably due to rather short incubation period in lettuce experiment. Conclusion Our study reveals the first investigation of the endophytic and epiphytic colonization ability of non-pathogenic F. oxysporum (F221-B) on roots of lettuce grown in hydroponics. This information of its successful colonization internally would be worthwhile for further research in terms of potential development for exploiting an endophytic F221-B as a promise BCA and PGPF in the near future. Acknowledgement The authors would like to thank the Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang for providing all the research facilities. Thanks also go to KMITL for financial support on presenting this research at the Symposium.
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Table 2 Colonization of non-pathogenic Fusarium oxysporum (F221-B) and pathogenic F. oxysporum (F221-R and F422-G) on punctured surface tissue of pakchoi.
References Bao, J.R. and G. Lazarovits. 2001. Differential colonization of tomato roots by nonpathogenic Fusarium oxysporum strains may influence Fusarium wilt control. Phytopathology 91: 449-456. Clay, K. 1989. Clavicipitaceous endophytes of grasse: their potential as biocontrol agents. Mycological Research 92: 1-12. Elizabeth, A.A. and A.E. Herre. 2003. Canopy cover and leaf age affect colonization by tropical fungal endophytes: Ecological pattern and process in Theobroma cacao (Malvaceae). Mycological 95(3): 388-398. Eparvier, A. and C. Alabouvette. 1994. Use of ELISA and GUS transformed strains to study competition between pathogenic and non-pathogenic Fusarium oxysporum for root colonization. Biocontrol Science Technology 4: 35–47. Nahalkova, J., J. Fatehi, C. Olivain and C. Alabouvette. 2008. Tomato root colonization by fluorescent-tagged pathogenic and protective strains of Fusarium oxysporum in hydroponic culture differs from root colonization in soil. Federation of European Microbiological Societies 286: 152-157. Olivain, C. and C. Alabouvette. 1997. Colonization of tomato root by a non-pathogenic strain of Fusarium oxysporum. New Phytologist 137: 481–494. Olivain, C., C. Humbert, J. Nahalkova, J., F. FatehiHaridon and C. Alabouvette. 2006. Colonization of tomato root by pathogenic and nonpathogenic Fusarium oxysporum strains inoculated together and separately into the soil. Applied and Environmental Microbiology 72: 1523–1531. Osono, T. 2008. Endophytic and epiphytic phyllosphere fungi of Camellia japonica: seasonal and leaf age-dependent variations. Mycological 100(3): 387-391. Paparu, P., T. Dubois, C.S. Gold, B. Niere, E. Adipala and D. Coyne. Colonization pattern of nonpathogenic Fusarium oxysporum, a potential biological control agent, in root and rhizomes of tissue cultured Musa plantlets. Annals of Applied Biology 149: 1-8. Pavithra, N., L. Sathish and K. Ananda. 2012. Antimicrobial and enzyme activity of endophytic fungi isolate from Tulsi. Journal of Pharmaceutical and Biomedical Sciences (JPBMS) 16(12): 1-6. Scott, J.C., D.N. Mcroberts and T.R. Gordon. 2014. Colonization of lettuce cultivars and rotation crops by Fusarium oxsporum f.sp. lactucae, the cause of fusarium wilt of lettuce. Plant Pathology 63: 548-553. Sikora, R.A. 1992. Management of antagonistic potential in agricultural ecosystems for the control of plantlet parasitic nematodes. Annual Review of Phytopathology 12: 245-270. Sikora, R.A. 1997. Biological system management in the rhizosphere: an inside-out/outside-in perspective. Mededelingen van de Faculteit Landbouwwetenschappen Rijksuniversiteit Gent 62: 105-112. Sikora, R.A., B. Niere and J. Kimenju. 2003. Endophytic microbial biodiversity and plantlet nematode management. In Biological control in IPM Systems in Africa 2nd ed, pp. 179-192. Thongkamngam, T., P. Koohakan and T. Jaenaksorn. 2013 Fusarium oxysporum F221-B as plant growth-promoting fungus PGPF on six plants in hydroponics and its growth characteristics on different media. In Proceedings of 6th Rajamangala University of Technology Tawan-ok Research Conference 46-51.(In Thai)
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Integrated Management Yellow Vein Mosaic Disease of Okra C. P. KHARE1*, A. KOTESTHANE, D. SHARMA2, A. DIXIT2 and J. SINGH2 1*
Department of Plant Pathology, Department of Vegetable Science, Indira Gandhi Agricultural University Raipur â&#x20AC;&#x201C; 492006 (Chhattisgarh), India *Corresponding email: dr.cpkhare@rediffmail.com 2
ABSTRACT Okra is a multipurpose crop due its diversified uses. Diseases are the key factors affecting its cultivation in India and yellow vein mosaic disease is major among them. The disease causes enormous losses both qualitative and quantitative. Considering the importance of disease, its proper management, the present study was undertaken in two parts at Indira Gandhi Agricultural University, Raipur (Chhatisgarh) India in Kharif/rabi season of 2012-13. In the first part, a trial was conducted in RBD with three replications with the plot size 4.8 x 2.7 m2 and the spacing 45 x 30 cm. The crop was raised with good agronomic practices and insecticides alongwith neem oil and neem oil (alone) were evaluated against the disease. They were sprayed four times in the crop at 10 days interval and observations of disease were recorded in 15 days interval after the appearance. In the second part, resistant seventeen entries (AVT-I & AVT-II group) were evaluated against the disease. The trial was conducted in RBD with three replications and planting was done. The plot size used was 4.8 x 2.7 m2 with the spacing 45 x 30 cm. the crop was raised with good agronomic practices and observations of disease incidence were recorded in 15 days interval. The lowest disease incidence was recorded by spraying of Acephate 75% SP @ 1.5 gm/lit+ Neem oil 0.15% EC @ 2.0 ml/lit followed by Imdacloprid 17.80% SL @ 0.5 ml/lit+ Neem oil 0.15% EC @ 2.0 ml/lit followed by Admire @ 2 g/l+ Neem oil and Hostothion + Neem Oil at 10 days interval. Out of 17 entries evaluated under various groups two (2012/OK YVRES 5and 2012/OKYVRES-6), were free from the disease from each group i.e. AVT-I& AVT-II. One entry (2012/OK YVRES 2) from AVT-I group was highly resistant to the disease. The insecticides play a significant role in the management of the disease by reducing whitefly population and incidence of the disease. The spraying of Acephate 75% SP @ 1.5 gm/lit+ Neem oil 0.15% EC @ 2.0 ml/lit. Spray of Imdacloprid 17.80% SL @ 0.5 ml/lit+ Neem oil 0.15% EC @ 2.0 ml/lit or spray of Admire @ 2 g/l+ Neem oil and Hostothion + Neem Oil at 10 days interval can be applied for management of the disease (YVMV). Likewise, development of virus resistant varieties has been considered the most effective, economical and resistant entries (2012/OK YVRES 5 and 2012/OKYVRES-6, 2012/OK YVRES 2) can be used for cultivation as well as for generating breeding material as well. Keywords: Disease incidence, Insecticides, Resistant entries
Introduction Okra [Abelmoschus esculentus (L.) Moench] is an important vegetable crop in the tropics. Young fruit are consumed fresh or cooked. It is a good source of vitamin A, B, C and protein, carbohydrates, fats, minerals, iron and iodine. Fresh fruit are harvested when 3 - 7 days old. In India, it is mainly grown for its tender green fruits and ranks first in the world (Dhankhar and Mishra, 2004). It is grown throughout the year except in heavy winter month in northern India however summer crop fetches more income. Among its growing constraints, diseases are the key factors affecting its cultivation in whole area of the country and Yellow vein mosaic disease caused by YVMV is major among them. In the world, Yellow vein mosaic July 1-3, 2015
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disease of okra was reported for the first time from India by Kulkarni (1924). It was named as yellow vein mosaic of okra by Uppal et al., (1940). Capoor and Verma (1950) established viral nature of the pathogen causing the disease. Considering the importance of the disease for its proper management, the present study was undertaken to deal with its management by pesticides application and utilization of resistance to combat with the problem. Material and Methods The present investigation, was conducted in two parts in first, resistant entries were evaluated against the disease in Chhatisgarh plain condition and were grown in Kharif season of 201213 at horticultural research farm, IGKV Raipur with seventeen entries of Advance Varital Trial 窶的 year (AVT-I) and Advance Varital Trial 窶的I year (AVT-II) group received from Indian Institute of Vegetable Research Varanasi under the AICRP on vegetable crop project for Raipur condition. The resistant evaluation trial was conducted in RBD with three replications and planting was done in Kharif seasons. The plot size used was 3.0 x 2.7 m2 with the spacing of 45 x 30 cm. The crop was raised with good agronomic practices and observations of disease incidence were recorded in 15 days interval after sowing of the crop seed. In the second trial, maize crop sown as border crop 15 days earlier to the main test crop (okra). The okra cv. Parbhani kranti, susceptible to disease was raised with good agronomic practices for the trial in RBD with three replications at horticultural research farm, IGKV Raipur in Kharif season of 2012-13. The plot size was 4.8 x 2.7 m2 and the spacing was 45 x 30 cm, where insecticides along with neem oil and neem oil alone were evaluated against the disease by spraying them four times in the crop at 10 days interval. The observations of disease incidence were recorded in 15 days interval after sowing of the crop seed. Results and Discussion Out of 10 (AVT-I) entries, one (2012/OK YVRES 5) was free from the disease or highly resistant category. While only 2% incidences were recorded in entry (2012/OK YVRES 2), the remaining were susceptible to highly susceptible. Out of 7 entries (AVT-II), one (2012/OKYVRES-6) was free from the disease while remaining were susceptible to highly susceptible. The similar resistance against yellow vein mosaic disease was also found in okra entries by Raghupati et al., 2000; Singh et al., 2002 and Vijaya, 2004. In the second trial, disease management by application of pesticide, where insecticides along with neem oil and neem oil alone were evaluated against the disease by spraying. The data on disease incidence of YVMV reveals that the lowest disease incidence (30.94%) of YVMV was recorded in sequential sprays of Acephate 75% SP @ 1.5 gm/lit+ Neem oil 0.15% EC @ 2.0 ml/litre followed by Imdacloprid 17.80% SL @ 0.5 ml/litre+ Neem oil 0.15% EC @ 2.0 ml/litre followed by Admire @ 2 g/l+ Neem oil and Hostothion + Neem Oil at 10 days interval and the highest disease incidence was observed in T7 (control). The highest marketable yield was recorded in the same with 42.10Q/ hac-1while the lowest marketable yield was recorded in T7 (control). As regard, the cost of the treatment for spray was the highest in T 3- (Imdacloprid 70% WG @ 2 gm/15 litre+ Neem oil 0.15% EC @ 2.0 ml/litre) and lowest in T 6-(Neem oil 0.15% EC @ 2.0 ml/litre) while the highest CB ratio was calculated in T4 (1: 4.83) followed by T6 (1: 2.58). The findings were similar to the result of the AICRP group meeting. (Anonymous, 2014)
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Table 1 Percent disease incidence of YVMV. S N 1 2 3 4 5 6 7 8 9 10
Entries (AVT-I)
1 2 3 4 5 6 7
2012/OKYVRES-4 2012/OKYVRES-5 2012/OKYVRES-6 Arka Abhaya Pusa Sawani Arka Anamika VRO-6
2012/OK YVRES 1 2012/OK YVRES 2 2012/OK YVRES 3 2012/OK YVRES 4 2012/OK YVRES 5 2012/OK YVRES 6 Arka Abhaya (C) Pusa Sawni (SC) Arka Anamika VRO 6 Entries (AVT-II)
15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Percent disease incidence (DAS) 30 45 60 75 90 0.00 0.00 0.61 1.32 7.22 0.00 0.00 0.57 0.57 2.27 0.00 0.00 2.69 17.03 48.03 0.00 0.00 3.88 9.56 44.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9.24 20.92 38.98 0.00 0.00 3.23 10.79 51.51 0.00 0.00 5.48 15.11 41.70 0.00 0.00 9.22 28.73 47.48 0.00 0.00 4.70 14.08 37.12
105 11.66 2.27 67.41 50.56 0.00 40.81 67.53 60.31 54.38 53.37
Marketable yield Q/ha. 62.97 40.00 25.30 50.37 28.02 32.96 27.65 34.93 31.11 22.71
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00 0.00 0.00
54.68 57.95 0.00 53.39 57.18 61.99 31.68
48.89 36.91 34.19 29.25 32.83 28.02 28.27
0.00 0.00 0.00 0.00 0.00 0.00 0.00
5.82 2.67 0.00 5.58 7.53 4.29 4.29
10.69 12.26 0.00 8.38 18.88 7.57 6.90
33.24 37.42 0.00 23.78 48.05 38.17 24.74
Table 2 Percent disease incidence of YVMV (cv. Parbhani kranti). S N
4
Application of insecticides (Four times at10 days interval) T1 -Acephate @1.5g/litre + Neem oil T2 窶的midacloropid 0.5 ml. + Neem oil T3-Admire @ 2 g/l+ Neem oil T4-Hostothion + Neem Oil
5
T5-T1 T2, T3 T4
6
T6-Neem Oil
7
T7-Control
1 2 3
CD CV
Percent disease incidence (DAS) 15 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 -
30 0.0
45 0.0
60 2.04
0.0
0.0
4.69
0.0
0.0
0.00
75 10.9 5 12.8 1 7.50
0.0
0.0
4.77
3.70
0.0
0.0
0.58
4.95
0.0
0.0
1.59
8.68
0.0
0.0
2.06
8.14
-
-
-
-
90 15.7 3 14.9 9 17.9 2 14.8 8 12.7 3 13.7 8 26.6 7 -
Marketabl e yield Q/ha
Cost Benefit Ratio
32.24
1:1.10
33.63
1:1.49
34.62
1:1.77
32.49
1:1.39
42.10
1:4.83
34.10
1:2.58 -
29.44 4.94 8.65
Conclusion Out of 17 entries of various groups, two (2012/OK YVRES 5and 2012/OKYVRES-6) were free from the disease from each group i.e. AVT-I& AVT-II. One entry (2012/OK YVRES 2) from AVT-I group was highly resistant to the disease and can be used for further cultivation as well as for generating breeding material as well. July 1-3, 2015
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While moderately resistant and commercial popular varieties/entries can be used for cultivation along with control measures. In the disease management by application of pesticide, the lowest disease incidence of YVMV was recorded by spraying of Acephate 75% SP @ 1.5 gm/litre+ Neem oil 0.15% EC @ 2.0 ml/litre followed by Imdacloprid 17.80% SL @ 0.5 ml/litre+ Neem oil 0.15% EC @ 2.0 ml/litre followed by Admire @ 2 g/litre+ Neem oil and Hostothion + Neem Oil at 10 days interval. Acknowledgement The authors wish to acknowledge the IGKV, Raipur and PC AICRP on vegetable crops for their all kind support for this study. References Capoor, S.P. and P.M. Verma. 1950. Yellow vein mosaic of H. esculentus (L.). Indian J. Agril. Sci. 20: 217-230. Dhankhar,-B-S; -J-P Mishra. 2004 Objectives of okra breeding. Journal-of-New-Seeds. 6(2/3): 195-209. Kulkarni, G.S. 1924. Mosaic of other related diseases of crops in the Bombay Presidency. Poona Agric. College Magazine. XVI: 6-12. Ragupathi, N., D. Veergavathatham, and S. Thamburaj. 2000. Reaction of okra cultivars on bhendi yellow vein mosaic virus (BYMV) disease. South Ind. Hort. 48(16): 103-104. Singh, A.K., R.B.S. Sanger and C.R. Gupta. 2002. Performance of different verities of okra to yellow vein mosaic virus under field conditions in Chattisgarh. Progressive Horticulture 34(1):113-116. M. 2004. Vijaya, M. 2004. Screening of okra entries to yellow vein mosaic virus (YVMV) disease under field conditions. The Orissa J. Horticulture 32(1):75-77. Uppal, B.N., P.M. Verma and S.P. Capoor. 1940. Yellow vein mosaic of bhendi. Current Sci. 9: 227-228.
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O-32
In Vitro Production of Cell Wall Degrading Enzymes by Pythium Species Isolated from Asymptomatic and Symptomatic Lettuce Root Chulalak TALUBNAK1, 2*, Nonglak PARINTHAWONG2 and Tanimnun JAENAKSORN2 1
Department of Biotechnology in Plant Pathology, International College, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Plant Production, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: chulalakkmitl@gmail.com
ABSTRACT This research investigated the ability of six isolates of Pythium species isolated from asymptomatic and symptomatic lettuce roots in producing cell wall degrading enzymes, eg. cellulase, pectinase and chitinase on solid media. The test pathogens were grouped as the non to weakly virulent and the moderately virulent groups. Activities of cellulase and pectinase enzymes were determined by hydrolysis capacity (HC) value which was the ratio of the hydrolysis zone and colony diameter. For chitinase, its activity was categorized based on relative increase in diameter as well as intensity of purple color on colloidal chitin medium. The results revealed that all tested isolates of Pythium species showed low potential to produce cellulase with HC value of less than 1.00 while absent cellulase was detected in Phytophthora IS40. Regard to pectinase enzyme production, absence as well as presence of activity with low HC value (<1.00) were observed in the non to weakly virulent group of Pythium. Meanwhile, only low HC value (<1.00) of pectinase was detected in moderately virulent group. For chitinolytic enzyme, two isolates in the non to weakly virulent group presented moderate activity and the rest showed absent activity. Keywords: Cellulase, Chitinase, Pectinase, Pythium Introduction Plant cell wall degrading enzymes produced and secreted by many plant pathogenic fungi are considered of major importance in the intercellular invasion and maceration of host tissue and therefore play significant roles in plant pathogenesis. For example, Fusarium oxysporum strain SUF 850 was found to be the producer for cellulose and xylan degrading enzymes (Yoshida et al., 1989). Production of pectolytic and cellulolytic enzymes has been identified in several plant pathogenic Pythium species, including P. aphanidermatum (Janardhanan and Husain, 1974; Sutton et al. 2006). P. myriotylum, responsible for root rot disease of cocoyam, was reported to cause a significant loss of pectin in epidermal cells of root apex during early infection probably via degradation by hydrolytic enzymes (Boudjeko et al., 2006). Three Pythium species namely P. violae, P. sulcatum and P. ultimum principally responsible for cavity spot on carrot roots could secrete pectate lyase, cellulase, polygalacturonase and β-1, 4-glucanase but in different amount and time (Campion et al., 1997). Generally, the greater level of pathogenicity of plant pathogenic fungus is associated with its ability to produce a wide range of these enzymes. Differences in cell wall degrading enzymes levels and mycelial growth rates of isolates of a pathogen are highly correlated with differences in virulence (Chen et al. 1998; Owen-Going et al., 2004; Onuh and Ohazurike. 2008). For chitinolytic enzymes, they are receiving attention in regard to their development as microbial biocontrol agents. In this sense, biological control of some soil-borne fungal diseases has been correlated with chitinase production (Herrera-Estrella and Chet, 1999). Therefore, aim of this July 1-3, 2015
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research was to investigate the ability of Pythium species isolated from asymptomatic and symptomatic root of hydroponically grown lettuce in producing of cell wall degrading enzymes (viz. cellulase, pectinase and chitinase) on solid media. Materials and Methods Pythium culture Thirty isolates of Pythium spp. were isolated from asymptomatic and symptomatic roots of hydroponically grown lettuce and being determined for their pathogenicity (Talubnak et al., 2014). Of these, 4 isolates of none to weakly virulent group (namely, ASR23, SR33, SR36 and IS39) and 3 of moderately virulent group (namely, ASR9, ASR26 and SR31) were selected for qualitative enzyme assay on solid media while standard enzymes were used as control. Moreover, Phytophthora IS40 isolated from infested soil of durian was represented as one of the non-virulent oomycete. The selected Pythium were cultured on potato dextrose agar (PDA) at 25°C for 3 days and ready for being used for cellulase, pectinase and chitinase assay. Enzymatic production Cellulase: Screening for cellulolytic activity of Pythium was done on carboxymethylcellulose (CMC) agar medium (0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.2% carboxymethylcellulose (CMC) sodium salt, 0.02% peptone and 1.7% agar) and determined qualitatively by iodine plate assay (Kasana et al., 2008). Pectinase: Screening for pectinolytic activity of Pythium was done on Czapek agar medium (2 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4.7H2O, 0.5 g KCl, 1 ml of 1% ZnSO4, 1 ml of 0.5% CuSO4, 30 g sucrose, 20 g agar and 1 L of water) containing 1% of pectin and determined qualitatively by iodine plate assay (Priya and Sashi, 2014). Chitinase: Screening for chitinolytic activity of Pythium was done on colloidal chitin medium (0.3 g of MgSO4.7H2O, 3 g of (NH4)2SO4, 2 g of KH2PO4, 1 g of citric acid monohydrate, 20 g of agar, 200 Οl of Tween-80, 4.5 g of colloidal chitin, 0.15 g of bromocresol purple and 1 L of water) (Agrawal and Kotasthane, 2012). The pH value was adjusted to 4.7. Calculation of enzymatic activity assay Cellulase, pectinase and chitinase produced by Pythium were examined for their potential to degrade cellulose, pectin and chitin, respectively. Activities of cellulase and pectinase enzymes were determined by hydrolysis capacity (HC) value which was the ratio of the hydrolysis zone and colony diameter (Taechapoempol et al., 2011). For chitinase, its activity was categorized based on relative increase in diameter as well as intensity of purple color on colloidal chitin medium (Agrawal and Kotasthane, 2012). Results and Discussion Pythium species, isolated from asymptomatic and symptomatic lettuce root, were grouped into non to weakly virulent (ASR23, SR33, SR36 and IS39) and moderately virulent (ASR9, ASR26 and SR31) groups (Talubnak et al., 2014) and then were determined their abilities in producing cell wall degrading enzymes on solid media. The results showed that all tested isolates of both groups showed low potential to produce cellulase with HC value of less than 1.00 while absent cellulase was detected in Phytophthora IS40 (a reference for non virulent oomycete) compared to standard enzyme having HC value of more than 3 (Table 1, Figure 1). In regard to pectinase production, standard pectinase showed the HC value of more than 3 while all 3 isolates of Pythium from moderately virulent group and IS39 from non to weakly virulent group could produce small amount of pectinase with HC value of less than 1.
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Table 1 Enzymes production of cellulase, pectinase and chitinase on solid media. Isolates Standard enzyme (Control) The non to weakly virulent group Pythium ASR23 PythiumSR33 Pythium SR36 Pythium IS39 The moderately virulent group Pythium ASR9 Pythium ASR26 Pythium SR31 Reference as non-virulent oomycete Phytophthora IS40
Cellulase1/ ++++
Pectinase1/ ++++
Chitinase2/ high
+ + + +
+
medium absence medium low
+ + +
+ + +
absence absence absence
-
-
absence
1/
b
c
d
e
f
g
h
i
j
(2)Pectinolyticenzyme at 2 d.
a
(3)Chitinolytic enzyme at 5 d.
(1)Cellulolytic enzyme at 2 d.
HC value as follow: - = absence; + = HC value is < 1.00; ++ = HC value is 1.01-2; +++ = HC value is 2.01-3 and ++++ = HC value is > 3. 2/ Absence; low, medium, high activity was categorized based on relative increase in diameter as well as intensity of purple color on colloidal chitin medium.
a
b
c
d
e
f
g
h
i
j
a
b
c
d
e
f
g
h
i
j
Figure 1 The hydrolyzed zone of (1) cellulolytic, (2) pectinolytic and (3) chitinolytic enzymes by Pythium isolates on medium supplemented with CMC, pectin and colloidal chitin, respectively. a = uninoculated (negative control), b = inoculated with standard (positive control), c = Pythium ASR23, d = Pythium SR33, e = Pythium SR36, f =Pythium IS39, g =Pythium ASR9, h =Pythium ASR26, i = Pythium SR31, and j = Phytophthora IS40
Besides, the rest of non to weakly virulent group did not produce pectinase. For chitnase, only 3 isolates (ASR23, SR36 and IS39) from the non to weakly virulent group could produce it with low to medium activity while high activity was observed from standard chitinase enzyme. Altogether, our results indicated that most of Pythium species, isolated from asymptomatic and symptomatic lettuce root, could produce cellulolytic and pectinase enzymes but only in a small amount. This observation was in agreement with Janardhanan and Husain (1974), and Sutton et al. (2006) who reported the ability of P. aphanidermatum to produce cellulolytic and pectolytic enzymes and also mentioned that the ages of the culture had influence in the quantity and activity of these enzymes. The small amount of cellulolytic and pectinase enzymes detected from our study probably due to the fact that Pythium species was regarded as weak parasite. This was in harmony with the work of Chen et al. (1998), and Owen-Going et al. (2004) who reported that differences in cell wall degrading enzymes levels were highly correlated with differences in virulence. Interestingly, chitinase activity was detected only from Pythium species of non to weakly virulent group. In this sense, such data led to the suggestion that we could probably try to July 1-3, 2015
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employ these chitinase producing isolates causing no disease as microbial biocontrol agent. However, before adopting, further research on this regard is still needed. Conclusion Most of Pythium species, isolated from asymptomatic and symptomatic lettuce root, could produce cellulolytic and pectinase enzymes on agar medium but only in a small amount with HC value of less than 1. In regard to chitinase, its low to medium activity was detected only from Pythium species of non to weakly virulent group. Acknowledgements This research was supported by the Royal Golden Jubilee Ph.D. Program, the Thailand Research Fund (Grant No. PHD/0286/2551). We are thankful to the Faculty of Agricultural Technology, KMITL for providing all laboratory facilities. Thank also goes to Asst. Prof. Kanjana Saeteaw, Department of Plant Production, Faculty of Agricultural Technology, KMITL for providing us the standard pectinase and cellulase enzymes.
References Agrawal, T. and A.S. Kotasthane. 2012. Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India. SpringerPlus 1(73): 1-10. Boudjeko, T., C. Andéme-Onzighi, M. Vicré, A.P. Balangé, D.O. Ndoumou and A. Driouich. 2006. Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum. Planta 223: 271-282. Campion, C., P. Massiot and F. Rouxel. 1997. Aggressiveness and production of cell-wall degrading enzymes by Pythium violae, Pythium sulcatum and Pythium ultimum, responsible for cavity spot on carrots. European Journal of Plant Pathology 103: 725-735. Chen, W.C., H.J. Hsieh and T.C. Tseng. 1998. Purification and characterization of a pectin lyase from Pythium splendens infected cucumber fruits. Botanical Bulletin of Academia Sinica 39: 181-186. Herrera-Estrella, A. and I. Chet. 1999. Chitinases in biological control. Experientia Supplementum. 87: 171-184. Janardhanan, K.K. and A. Husain. 1974. Production of toxic metabolite and pectolytic enzyme by Pythium butleri. Mycopathologia et Mycologia Applicata 52: 305-330. Kasana, R.C., R. Salwan, H. Dhar, S. Dutt and A. Gulati. 2008. A rapid and easy method for the detection of microbial cellulases on agar plates using gram’s iodine. Current Microbiology 57: 503-507. Onuh, O.M. and N.C. Ohazurike. 2008. Effects of culture ages on the production and activities of polygalacturonase and cellulase (cx) enzymes produced by pythium aphanidermatum (edsonfitzpat.) isolated from soft stem rot disease of cowpea. Science World Journal 3(2): 5-9. Owen-Going, T.N., C.W. Beninger, B. Christie, J.C. Sutton and J.C. Hall. 2004. Role of phenolic compounds in epidemics of Pythium root rot (Pythium aphanidermatum) of hydroponic pepper. Canadian Journal of Plant Pathology 26(1): 410-419. Priya, V. and V. Sashi. 2014. Pectinase enzyme producing microorganisms. International Journal of Scientific and Research Publications 4(3): 1-4. Sutton, J.C., C.R. Sopher, T.N. Owen-Going, W. Liu, B. Grodzinski, J.C. Hall and R.L. Benchimol. 2006. Etiology and epidemiology of Pythium root rot in hydroponic crops: current knowledge and perspectives. Summa Phytopathologica 32(4): 307-321. Taechapoempol, K., T. Sreethawong, P. Rangsunvigit, W. Namprohm, B. Thamprajamchit, S. Rengpipat and S. Chavadej. 2011. Cellulase-producing bacteria from Thai higher termites, Microcerotermes sp.: enzymatic activities and ionic liquid tolerance. Applied Biochemistry and Biotechnology 164: 204-219. Talubnak, C., N. Parinthawong and T. Jaenaksorn. 2014. In-vitro screening for non-pathogenic isolates of Pythium spp. from asymptomatic and symptomatic lettuce in hydroponics. In: The 12th International Symposium on Biocontrol and Biotechnology, 11-13 December 2014 at KMITL Chumphon campus, Chumphon Thailand. Yoshida, N., T. Fukushima, H. Saito, M. Shimosaka and M. Okazaki. 1989. Cellulose and xylan degrading enzymes of the plant pathogenic fungus, Fusarium oxysporum SUF850. Agricultural and Biological Chemistry 53(7): 1829-1836.
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O-33
Gene Expression Analysis in Lettuce (Lactuca sativa L.) Treated with Trichoderma spp. Malatee PRADUBYAT1, 2* Nonglak PARINTHAWONG1 and Tanimnun JAENAKSORN1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand. 2 Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand *Corresponding email: mee_yard@msn.com
ABSTRACT This study was aimed at gene expression in hydroponically grown lettuce treated with 4 isolates of Trichoderma. The experiment was arranged into Completely Randomized Design having 9 treatments with 3 replications of 6 plants. Pathogenesis related protein (PR) 1 and phytoalexin (LTC1, LTC2) were analyzed from lettuce leaf of each treatment at 0, 24, 48 and 72 hrs. Besides, plant growth as well as yield was recorded at harvest. Results revealed the total RNA extract and the presence of 18s bands were clearly observed. PR1 was clearly observed while no LTC1 and LTC2 were detected. The expression level of PR1 gene was variable linked to the isolation of Trichoderma and incubation period. The tested Trichoderma had positive as well as negative effect but insignificant on yield of lettuce compared to control. Keywords: Trichoderma spp., Induced resistance, PR protein, Lettuce, Hydroponics technique Introduction Pathogenesis related protein (PRs) have been defined as plant proteins that are induced in pathological or related situation (Van Loon et al., 1994). PR-1 protein could play important role in disease resistance since it is having antifungal activity at the micromolar level against a number of plant pathogenic fungi, including Uromyces fabae, Phytophthora infestans and Erysiphe graminis (Niderman et al., 1995). Along with PR proteins, complementary DNA (cDNA) products of LTC1 and LTC2 were normally recovered from plants which accumulate phytoalexins (Bennett et al. 2002) suggesting that they are also importance of plant defense. Recent discoveries show that Trichoderma spp. are opportunistic, avirulent plant symbionts, as well as bio-control agents against fungal phytopathogens (Chet and Inber, 1994, Harman et al., 2004 and Gajera et al., 2013) with direct or indirect mechanisms. Direct mechanism comprises competition for nutrients and space, modification of environmental conditions, antibiosis and induced of plant defensive mechanism (Gajera et al., 2013). Therefore, this research was conducted to study an induction of plant defensive mechanism by Trichoderma in lettuce (Lactuca sativa L.) being grown in hydroponics. Gene expression such as LTC1, LTC2 and PR1 were analyzed accordingly. Materials and Methods Induction of resistance in lettuces by Trichoderma Three isolates of Trichoderma spp. (LK019, BR002 and G003) were obtained from Malatee et al. (2013) and the other one was from commercial product and set as positive control. All antagonistic Trichoderma were cultured on Potato dextrose agar (PDA) medium and then seven days old Trichoderma spp. culture were used for preparing the conidia suspension and being adjusted to 1x106 cfu/ml. July 1-3, 2015
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Two-week-old lettuce seedlings were transplanted in hydroponic system. The experiment was arranged into Completely Randomized Design having 9 treatments with 3 replications of 6 plants. Treatment was set for control, treatment 2-5 were treated with test Trichoderma on root while treatment 6-9 were treated on leaf of 3-week-old lettuce. Leaves were collected at 0, 24, 48 and 72 hrs for LTC1, LTC2 and PR1 analysis. Besides, plant growth included leaf number and leaf area were measured every week and fresh yield was recorded at harvest. Plant RNA extraction and polymerase chain reaction (PCR) analysis (LTC1, LTC2 and PR1) RNA was extracted with TRIzol reagent (Invitrogen, USA) according to the manufacture’s instruction. Thereafter, total RNA was resuspended with RNA free water. Genomic DNA contamination was removed using DNaseI (Thermo scientific, EU). The cDNA was synthesized from 1 µg of total RNA using the RevertAid reverse transcriptase (Thermo scientific, EU). Concentrations of cDNA were adjusted with distilled water based on 18s ribosomal primer band. The cDNA obtained from all samples was used in PCR assay with specific primers, the oligonucleotide sequences of LTC1, LTC2 and PR1 were designed. The program PCR was carried out as following: pre-denaturation 95°C for 5 min, 35 cycles of denaturation 95°C for 45 sec, annealing 54°C (LTC1) or 56°C (LTC1 and PR1) for 45 sec and extension 72°C for 50 sec, and final extension 72°C for ?. PCR product was separated by 1% agarose gel electrophoresis, stained with ethidium bromide and photographed by Gene genius bio imaging system (Syngene, Genegenius, Japan). Result and Discussion Result revealed the total RNA extract and the presence of 18s bands were clearly observed (Figure 1a). Surprisingly, LTC1 and LTC2 were not detected from lettuce either treated with or without Trichoderma spp. which is on the contrary to research of Harman et al.(2004) reporting that T. virens has ability to induce phytoalexin production and localized resistance in cotton. Regarding PR1 expression, it was observed mostly from treated lettuce but not from control. Besides, its expression level was varied among Trichoderma isolates and incubation time (Figure 1b). Interestingly, PR1 seemed to be systemic induced resistance since its expression was detected from leaf of lettuce in which its root was treated with Trichoderma spp. Level of gene expression could be estimated from band intensity. Our result was in line with the work of Alfano et al. (2006) revealing that T. hamatum 382 actively induced systemic changes in plant physiology and disease resistance through systemic modulation of the expression of stress and metabolism genes. T. harmatum (T-22) was reported to have ability to induce systemic resistance to pathogens in maize (Harman et al., 2004). In terms of plant growth, leaf numbers from all treatments were not statistically different from control while lettuce treated with LK019 and G003 tended to give larger leaf area than control and the rest (Table 1). Regard to yield, the result was in line with those of leaf area (Figure 2). On this regard, Trichoderma spp. was also reported to be plant growth promoting fungi on wheat (Salehpour et al., 2005), bitter gourd, loofah and cucumber (Lo and Lin, 2002). Taken together, tested Trichoderma spp. were shown to induce systemic resistance and promote growth of lettuce.
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a
0 hr 24 hrs 48 hrs 72 hrs 1Kb T1
T2
T3
T4
T5
T6 T6.T T7 T7.T T8 T8.T T9 T9.T
b
48 hrs 48 hrs 1Kb T1
T2
T3
T4
T5
T6 T6.T T7 T7.T T8 T8.T T9 T9.T
Figure 1 Photograph of agaroes gel of the amplified 18s band (a) and the bands associated with the PR1 gene (b). Lane :1 Kb = molecular weight marker, 1 Kb + DNA ladder; T1= control, T2= root treated with Trichoderma LK019 , T3 = root treated with Trichoderma BR002, T4 = root treated with Trichoderma G003, T5 = root tread with commercial product, T6 = leaf untreated with Trichoderma LK019, T6.T = leaf treated with Trichoderma LK019, T7 = leaf untreated with Trichoderma BR002, T7.T = leaf treated with Trichoderma BR002, T8 = leaf untreated with Trichoderma G003, T8.T = leaf treated with Trichoderma G003, T9 = leaf untreated with commercial product, T9.T = leaf treated with commercial product. The 18s bands clearly showed that the same amount of cDNA were presented in the different sample. What two panels of 48 hrs (b) standed for? Table 1 Effect of 4 isolates of Trichoderma spp. 4 on growth of lettuce (at harvest, 45 days) Treatments Control-water treated Root treated - LK019 Root treated - BR002 Root treated - G003 Root treated - product Leaf treated - LK019 Leaf treated - BR002 Leaf treated - G003 Leaf treated - product
Plant growth Leaf no. Leaf area (cm) 17.2 a1/ 54.0 ab 18.1 a 58.4 a 15.9 a 44.9 b 18.0 a 50.6 ab 17.0 a 47.7 ab 16.7 a 48.7 ab 16.8 a 54.6 ab 18.2 a 57.5 a 18.0 a 54.8 ab
Yield (g) 44.7 abc 52.1 a 37.8 bc 47.2 abc 36.3 c 43.1 abc 42.3 abc 50.5 ab 43.4 abc
1/
Means follow by the same letter in each column do not differ significantly according to Duncanâ&#x20AC;&#x2122;s Multiple Range Test at P=0.05.
Conclusions This study was aimed at gene expression in hydroponically grown lettuce treated with 4 isolates of Trichoderma. Result revealed the total RNA extract and the presence of 18s bands were clearly observed. LTC1 and LTC2 were not detected from lettuce. Regarding PR1, it was observed mostly from treated lettuce but not from control. In terms of plant growth, leaf numbers from all treatments were not statistically different from control. Taken together, tested Trichoderma spp. were shown to induce systemic resistance and promote growth of lettuce. July 1-3, 2015
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Figure 2 Effect of Trichoderma spp. 4 isolates treated on hydroponically grow lettuce (45 days post inoculation). Acknowledgments This research is partially supported by the Center of Excellence on agricultural Biotechnology Science and Technology Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of Education (AG-BIO/PERDOCHE). Thanks also go to King Mongkut’s Institute of Technology Ladkrabang for financial support for presenting this research at the symposium. References Alfano, G., M.L. Lewis Ivey, C. Cakir, J.I.B. Bos, S.A. Miller, L.V. Madden, S. Kamoun and H.A.J. Hoitink. 2007. Systemic modulation of gene expression in tomato by Trichoderma hamatum 382. Biological Control 97: 429-437. Bennett, M.H., J.W. Manssfield, M.J. Lewis and M.H. Beale. 2002. Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.). Phytochemistry. Vol. 60, pp. 255-261. Chet, I. and J. Inbar. 1994. Biological control of fungus pathogens. Applied Biochemistry and Biotechnology 48: 37-43. Gajera, H., R. Domadiya, S. Patel, M. Kapopara and B. Golakiya. 2013. Molecular mechanism of Trichoderma as bio-control agents against phytopathogen system-A review. Current Research in Microbiology and Biotechnology 1:133-142. Harman, G.H., Howell C.R., Viterbo A., Chet I. and Lorito M. 2004. Trichoderma speciesopportunistic, avirulent plant symbionts. Microbiology 2:43-56. Lo, C.T. and Lin C.Y. 2002. Screening strains of Trichoderma spp. for plant growth enhancement in Taiwan. Plant Pathology Bulletin. 11:215-220. Niderman, T., I. Genetet, T. Bruyère, R. Gees, A. Stintzi, M. Legrand, B. Fritig and E. Mösinger. 1995. Pathogenesis-related PR-1 proteins are antifungal; isolation and characterization of three 14kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans. Plant Physiology. 108:17-27. Pradubyart, M., N. Parinthawong and T. Jaenaksorn. 2013. In vitro screening of antagonistic Trichoderma spp. against pathogenic fungi causing leaf spot of vegetable crops grown in hydroponics. Proceedings of 6th Rajamangala University of Technology Tawan-ok Research Conference 52-57. Salehpour, M., H.R. Etebarian, A. Poustaei, G. Khodakaramian and H. Aminian. 2005. Biological control of common root rot of wheat (Bipolaris sorokiniana) by Trichoderma isolates. Plant Pathology Journal 85-90. Van Loon, L.C., W.S. Pierpoint, T. Boller and V. Conejero. 1994. Recommendations for naming plant pathogenesis-related proteins. Plant Molacular Biology Reporter 245-264. Page 172
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Assessment of Viability and Efficacy of Fusarium oxysporum (F221-B) as BCA and PGPF during Long Term Preservation Titi THONGKAMNGAM1* and Tanimnun JAENAKSORN1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: Titi29950@hotmail.com
ABSTRACT Long term preservation of Fusarium oxysporum (F221-B) using modified filter paper technique was monthly assessed on the basis of its viability, morphological characteristics and in vitro efficacy as biocontrol agent (BCA) and plant growth promoting fungus (PGPF). The results showed that the 6-month storage of F221-B was very satisfactory since its viability of cultures was still 100 percent until the end. Remarkable consistency in retaining its morphological characteristics was noted during preservation. Main hypha was in the range of 110-121x2.2-2.6 µm, hyphal tip was 82.04-94.04x3.2-3.5 µm whereas macro conidia were 28.23-29.85x3.0-3.5 µm. The biocontrol efficacy of F221-B against Colletotrichum sp., Curvularia sp. and Fusarium sp. remained relatively constant and unchanged during 6 months which was in the range of 36.1-74.8 percent from dual culture test and 31.6-48.2 percent from agar well diffusion assay when using 100 percent of culture filtrate concentration. Regarding its effectiveness as PGPF, the treated seeds with spore suspension of F221-B at 103 and 106 spores/ml could germinate 2-day faster than non-treated seeds resulting in higher percentage of seed germination and seedling length at a given day. To conclude, 6-month preservation of F221-B using modified filter paper technique was successful with 100 percent viability and its unchanged efficacy as BCA and PGPF. Keywords: Fusarium oxysporum (F221-B), Modified filter paper technique, Long term preservation, Biocontrol agent, Plant growth promoting fungus Introduction Fusarium oxysporum (F221-B) has shown its ability as PGPF on various plants such as Butterhead, Cos, Green oak, Red oak lettuce, kale and mung bean (Titi et al., 2012) and as BCA against plant pathogenic fungi (unpublished). Therefore, it is necessary to employ the proper method for keeping the culture of F221-B as long as possible without any changes on its morphological characteristics and potentialities. Modified filter paper culture method was shown to be effective in preserving the F221-B viable without any contamination at the early stage of preservation (Thongkamngam and Jaenaksorn, 2014). However, its viability, morphological characteristics and in vitro efficacy as BCA and PGPF have not yet been assessed for long-term period. Hence, the research was conducted to preserve the culture of F221-B using modified filter paper and kept at 4° C while assessment of its viability and efficacy as BCA and PGPF was monthly monitored during 6 months preservation. Materials and methods Assessment of viability of F221-B during 6-month storage Viability, purity, mycelial growth, spore production and morphological characteristics of F221-B were monitored immediately after storage, at 1, 2, 3, 4, 5, and 6 months after storage. One piece of the inoculated filter paper was randomly removed from eppendorf tube from 4° C storage under aseptic conditions and then inoculated onto PDA (5 pieces per plate of 10 plates) for 3 days at room temperature. All parameters were observed accordingly. July 1-3, 2015
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Assessment of biocontrol efficacy of F221-B during 6-month storage against plant pathogenic fungi Biocontrol efficacy of F221-B against 3 plant pathogenic fungi, namely Colletotrichum sp. Curvularia sp. and Fusarium sp. was monthly assessed during 6 months preservation by dual culture test and agar well diffusion assay. Dual culture test (Dennis and Webster, 1971) A mycelial disc (9 mm diameter) obtained from the 7-day-old cultures of tested pathogenic fungi and F221-B was placed simultaneously on the periphery, about 1 cm from the edges of the Petri dishes (9 cm diameter) at opposite sides. Petri dishes containing PDA inoculated either with the tested pathogens or F221-B alone served as control. All plates were incubated at 28 °C. Radial growth of pathogen in dual plate was daily measured until the culture of control pathogen was full. The percentage growth inhibition of tested pathogens in presence of BCA was calculated over control using the formula: C- T / C x100, where C= pathogen growth in control and T = pathogen growth in dual plate. Agar well diffusion assay (Fiddman and Rossall, 1993) To prepare culture filtrate, 5 discs (5 mm diameter) of F221-B were cut from vigorously growing margin of 7-day-old cultures and inoculated into flask containing 50 ml of sterile Potato dextrose broth (pH 5.6). Flasks were incubated at 28°C for 10 days. After incubation, the cultures were filtered first through Whatman filter paper No.1 and then through microfilter (0.2 μm ?) to obtain sterile culture filtrate. The culture filtrate was adjusted to different concentrations 0, 20, 40, 60, 80 and 100% by mixing with sterile water and ready to be used. Each pathogen was inoculated on PDA plate and incubated for 3 days. Then, six wells (5 mm diameter) were punched in the pathogen-inoculated plates and filled with 30 µl of each concentration of culture filtrate of F221-B. The medium devoid of culture filtrate served as control. All plates were kept in room temperature. The radial growth of tested pathogens was daily measured and the percentage of growth inhibition of tested pathogens was calculated. Assessment of plant growth promoting ability of F221-B during 6-month storage Plant growth promoting ability of F221-B was evaluated with Cos and Red oak lettuce seeds using completely randomized design. Fifty seeds of each were soaked into 5 ml of 2 concentrations (103 and 106 spore/ml) of F221-B spore suspension for 5 minutes before transplanting the inoculated-lettuce seeds onto Petri plate containing moistened-Whatman filter paper (No.1). Data collected as percentage of germinated seed and seedling length were statistically analyzed. Results and discussions Six month preservation of F221-B at 4° C by modified filter paper method was shown successfully. Regard to its viability, the result showed 100 percent viability since all pieces of inoculated paper were recovered each time from storage and showed the initial colony characteristics, and the same growth rate as the original culture (Table 1). Besides, remarkable consistency in retaining its morphological characteristics was noted throughout 6 month period. Main hypha was in the range of 110-121x2.2-2.6 µm, hyphal tip was 82.0494.04x3.2-3.5 µm whereas macro conidia were 28.23-29.85x3.0-3.5 µm which were matched the original fungal identification documented for F221-B. These results are in agreement with that described by Fong et al. (2000) who could preserve Fusarium oxysporum for 4 years, Marasmiellus inoderma for 2 years and Ganoderma isolates for 5 months using a modified filter paper. In regard to this, another report by Hu et al. (2014) who described a success long –term preservation and fast revival of filamentous fungi using a novel, highly efficient method based simply on sterile cellophane squares (CPS) which is similar to modified filter paper method.The biocontrol efficacy of F221-B during 6 month preservation period against Colletotrichum sp., Curvularia sp. and Fusarium sp. remained relatively constant and unchanged which was in the range of 36.1-74.8 percent from dual culture test (Figure 1, 2) and 31.6-48.2 percent in agar well diffusion assay (at the 100 percent of culture filtrate concentration) (Figure 1, 3). Since our research has focused on preservation of only a nonpathogenic F. oxysporum (F221-B), newly isolate BCA and PGPF, no related observation has been made by others. However, the results of the current study provided evidence for the antagonistic ability of F221-B which was still in line with those decribed by Park et al. (1988) and Abeysinghe (2006) who revealed the ability of selected non-pathogenic F. oxysporum . Page 174
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Regarding its potential as plant growth promoting fungus, the treated seeds with spore suspension of F221-B at 103 and 106 spores / ml could germinate 2 days faster than nontreated seeds resulting in higher percentage of seed germination and seedling length. Taking into account, our present research confirmed that the potential of F221-B as BCA and PGPF remained unchanged during 6 month storage. Table 1 Viability and morphological characteristics of Fusarium oxysporum (F221-B) during 6 month preservation Month
80 60 40 20 0
50 40 30 20 10 0 50 40 30 20 10 0 50 40 30 20 10 0
a
Colony color
1 Day
3 Day
100 100 100 100 100 100 100
yellow ” ” ” ” ” ”
a
a
a
a
Morphology of Fusarium oxysporium F221-B (at 3 day) Length x Width (µm) Macroconidia Amount Main hypha Hyphal tip Size(µm) (spore/ml) 117×2.6a 86.7×3.4a 29.7×3.5 3.14×104 116×2.2a 82.04×3.5a 29.85×3.2 3.26×104 110×2.4a 87.42×3.2a 28.56×3.5 2.43×104 112×2.6a 92.04×3.2a 28.85×3 3.26×104 121×2.6a 90.04×3.4a 28.28×3.2 3.16×104 118×2.4a 94.04×3.4a 29.03×3.4 2.88×104 116×2.4a 85.13×3.4a 28.23×3.2 2.86×104
a
Dual-culture Colletotrichum sp. Curvularia sp. Fusarium sp.
1
2
3 4 5 Month of preservation
6
Colletotrichum sp.
1
2
3 4 Curvularia sp.
Agar well
5
6
Figure 2 In vitro effectiveness of Fusarium oxysporum F221-B against 3 plant pathogen fungi by Dual culture test at 6 month of preservation. 20% 40% 60% 80%
1
1
2
2
3 4 Fusarium sp.
5
3 4 5 Month of preservation
6
Concentration
% Growth inhibition % Growth inhibition % Growth inhibition
% Growth inhibition
0 (before storage) 1 2 3 4 5 6
Viability (%)
100%
6
Figure 1 In vitro effectiveness of Fusarium oxysporum (F 221 -B) against 3 plant pathogenic (check also in Fig 2) fungi during 6 month preservation.
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Figure 3 Effectiveness of Fusarium oxysporum F221-B concentration at 0, 20, 40, 60, 80 and 100 percent by Agar well diffusion assay at 2, 4 and 6 month of preservation.
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Table 2 Assessment of plant growth promoting ability of F221-B during 6 month storage Seed germination Seedling length (cm) Cos Red oak Cos Red oak 2 day 3 day 2 day 3 day 2 day 3 day 2 day 3 day Control 0 80 0 65 0c1 1.7c 0c 1.3c 1 103 spore/ml 100 100 80 100 2.3ab 3.1ab 1.8ab 2.8ab 106 spore/ml 100 100 100 100 2.41a 3.74a 2.1a 3a Control 0 80 0 60 0c 1.8c 0c 1.4c 2 103 spore/ml 100 100 85 100 2.1ab 3.2ab 1.9ab 2.6ab 106 spore/ml 100 100 100 100 2.56a 3.51a 2.3a 3.1a Control 0 85 0 67 0c 1.65c 0c 1.1c 3 3 10 spore/ml 100 100 80 100 2.1ab 3.1ab 1.7ab 2.5ab 106 spore/ml 100 100 100 100 2.41a 3.63a 2.4a 2.9a Control 0 85 0 62 0c 1.7c 0c 1.3c 3 4 10 spore/ml 100 100 80 100 2.3ab 3.4ab 1.8ab 2.8ab 106 spore/ml 100 100 100 100 2.46a 3.9a 2.1a 3a Control 0 82 0 65 0c 1.7c 0c 1.4c 5 103 spore/ml 100 100 80 100 2.1ab 3.1ab 1.6ab 2.7ab 6 10 spore/ml 100 100 100 100 2.5a 3.74a 2.5a 3a Control 0 85 0 65 0c 1.7c 0c 1.3c 6 103 spore/ml 100 100 80 100 2.2ab 3.5ab 1.8ab 2.9ab 6 10 spore/ml 100 100 100 100 2.41a 3.84a 2.6a 3.4a 1/ Mean of 50 replications in a column followed by same letters are not significantly different according to Duncan’s Multiple Range Test (DMRT) at P=0.05 Month
Treatment
Conclusion In conclusion, our current study provides evidence that 6 month storage of F. oxysporum (F 221-B) at 4° C using modified filter paper method was very satisfactory since its viability, stability of living cells, and morphological characteristics were remarkable consistency as same as that of the original culture. Besides, the potential of F221-B as antagonist and plant growth promoting fungi remained unchanged. Therefore, it would be worthwhile to extend this research for longer monitoring in order to validate the reliability of this preservation method and ensure the stability of living cells of F 221-B during longer period of preservation. Furthermore, the stability of F 221-B living cells should be assessed by molecular parameters. Acknowledgements The authors would like to thank the Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang for providing all the facilities both in laboratory and in hydroponic greenhouse. Thanks also go to KMITL for financial support on presenting this research at the Symposium. References Abeysinghe, S. 2006. Biological control of Fusarium oxysporum f.sp. radicis-cucumerinum the casual agent of root and stem rot of Cucumis sativus by non-pathogenic Fusarium oxysporum. Ruhuna Journal of Science 1: 24-31. Dennis, C. and J. Webester. 1971. Antagonist properties of species group of Trichoderma; Production of volatile antibiotics. Transactions British Mycological Society 57: 41-78. Fiddman, P.J. and S. Rossall, 1993. The production of antifungal volatile by Bacillus subtilis. Journal of Applied Bacteriology 74: 119-126. Fong, Y.K., S. Anuar, H.P. Lim, F.Y. Tham and F.R. Asnderson 2000. A modified filter paper technique for long-term preservation of some fungal cultures. Mycologist 14(3): 127-130. Hu, X., G. Webster, L. Xie, C. Yu, Y. Li and X. Liao 2014. A new method for the preservartion of axenic fungal cultures. Jounal of Microbiological Methods 99: 81-83. Park, C.S., T.C. Paulitz and R. Baker. 1988. Biocontrol of Fusarium wilt of cucumber resulting from interactions Pseudomonas putida and non-pathogenic isolates of Fusarium oxysporum. Phytopathology 78: 190-194. Thongkamngan, T. and T. Jaenaksorn (2014). Evaluation of the modified filter paper culture method for maintenance the culture of Fusarium oxysporum (F221-B) The 12th Naresuan Research Conference. Naresuan University, 28 - 29 October 2014. (In Thai) Thongkamngan, T., P. Koohakan and T. Jaenaksorn 2013. Fusarium oxysporum F221-B as plant growth-promoting fungus PGPF on six plants in hydroponics and its growth characteristics on different media. In Proceedings of 6th Rajamangala University of Technology Tawan-ok Research Conference 46-51. (In Thai)
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Comparison of Trichoderma Population in the Re-Circulating Nutrient Solution With and Without Supporting Material Kanet JAIKENGKAJ1 Tanimnun JAENAKSORN1 and Prommart KOOHAKAN1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand. *Corresponding author: E-mail: kkpromma@kmitl.ac.th
ABSTRACT Effects of supporting material on Trichoderma population in the re-circulating nutrient solution were studied. The experiment was done in closed re-circulating system with or without supporting material. In the system with supporting material, vermiculite (1 L) mixed with 50 g of Trichoderma bioproduct was filled into the plastic bottle, while control was only bottle without supporting material. Nutrient solution passed through the container and ran off to the nutrient solution tank. Population of Trichoderma in the nutrient solution with and without supporting material was weekly detected. The result showed that Trichoderma population in the system with supporting material changed slightly by 4.4-5.3 log cfu/ml during seven weeks. Though in control, Trichoderma population reduced rapidly from 5.3 to 3.3 log cfu/ml within 2 weeks, then decreased to 2.6-2.7 log cfu/ml until the end of experiment. For Trichoderma population in the nutrient solution, it was lower than that with supporting material. Interestingly, the population in the nutrient solution with supporting material was 10 times higher than that of without supporting material. Keywords: Hydroponics, Trichoderma sp., Biological control Introduction Hydroponics for plant production is nowadays accepted by many growers. It is a cultivation without soil and mostly re-circulated the nutrient solution for economic and environmental purpose. This system is really less of disease, except for root disease that the pathogens can transmit via the nutrient solution (Stanghellini and Rasmussen, 1994; Koohakan et al., 2004). The concerning on disease development in this system is that nutrient solution flow to every plants. Therefore, the incidence and severity of certain plant disease is more than that in soilbased system. Many countermeasures to eliminate the pathogen in the nutrient solution are proposed, including physical methods, chemical methods and biological methods (Ikeda et al., 2002). However, biological method is extensively studied. Biological control uses antagonistic fungi for example Chaetomium sp., Gliocladium sp. and Trichoderma sp. or beneficial bacteria such as Bacillus sp. and Pseudomonas sp. (Paulitz and Belanger, 2001; Kanjanamaneesathian et al., 2013). They can suppress the pathogens, promote plant growth and induce resistance for plant disease (Paulitz and Belanger, 2001; Gul et al., 2008; Vinale et al., 2009; Cai et al., 2013; Nawrocka and Malolepzsa, 2013). Some biological control agent is developed to bio-product. Though, it is convenient for using but several problems for application in the system are encountered. If bio-product such as Trichoderma sp. added to the system, its population might be reduced until it disappeared. Thus, that is useless in a long term. For that reason, we need to develop the strategies for maintaining the population of Trichoderma in the re-circulating nutrient solution of hydroponics. In this experiment, we firstly studied the effect of supporting material on Trichoderma population and contributed the results to further study.
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Materials and Methods
Antagonistic fungus The antagonistic fungus used in this experiment was bio-product of Trichoderma sp. Its survival in product-formulated was checked as follows; 1 g of bio-product was mixed with 9 ml sterile water. Then it was diluted in various concentrations to 10-1, 10-2, 10-3, 10-4 and 10-5. After that, the diluents were poured on Trichoderma selective media: TSM (Elad et al., 1981) by pour plate technique and the colony was detected and calculated to the population by Trichoderma population =
number colony on TSM concentration of stock sample
Ă&#x2014; dilution factor
The systems and experimental condition The re-circulating nutrient solution systems were set up. In the system with supporting material, vermiculite (1 L) mixed with 50 g of Trichoderma bio-product was filled into the plastic bottle (approximately 2.5x105 cfu/container by calculation), while control was only bottle without supporting material. Three replications of each treatment were used in this experiment. Nutrient solution (NS) was passed through the container and run off to the NS tank for 14 weeks throughout the experiment. In the first period, NS was circulated in the system without plant for 7 weeks. The second period, green oak lettuce (3-week-old) was transplanted to the system and grown in the system for 4 weeks. The last period, all plants were moved out and the NS was still circulated in the system without plant for 3 weeks. Data collection Population of Trichoderma in the supporting material and NS was weekly detected. Supporting material (1 gram) or NS in tank (1 ml) were sampling. Solid samples were blended or shaken in sterile water and 10-fold diluted from 10-1-10-4. Population of Trichoderma in diluents of each sample was detected by pour plate technique in TSM in triplicate. The population was calculated and log transformed before plotting to determine their population dynamics. Results and Discussion Population dynamic in the supporting material At the beginning of experiment, Trichoderma population in the system with supporting material (vermiculite) changed slightly by 5.3 to 5.1 log cfu/ml within three weeks, while in control the population reduced rapidly from 5.3 to 2.7 log cfu/ml. In week seven, Trichoderma population in the system with supporting material decreased to 4.9 log cfu/ml but it decreased to 2.6 log cfu/ml in control. In the last period (week 11), Trichoderma population in the system with supporting material reduced to 4.7 log cfu/ml. Though in control, it reduced to 2.0 log cfu/ml (Figure 1). These results indicated the effect of supporting material to maintain the population of Trichoderma sp. in re-circulating system. Similar result to Singh et al. (2007) who studied on shelf life of T. harzianum on different substrate such as tea leaves, maize cob, rice husk, saw dust and wheat barn, they found that all substrates could maintain the population of T. harzianum for 30 days. Rajput et al. (2014) also reported that organic substrate such as sorghum grain could increase Trichoderma population. According to antagonistic activity of Trichoderma sp., Dantnoft et al. (1995) reported that T. harzianum at the concentration of 108 cfu/ml applied to soil could control root rot of tomato caused by Fusarium sp. In addition, Koohakan and Nantagij (2005) tested on bio-products of T. harzianum at 103-106 cfu/ml andhe results showed that every concentrations could control Pythium myriotylum, causative of root rot of lettuce grown in hydroponics, however at the concentration of 106 cfu/ml of some product could absolutely inhibit mycelial growth of the pathogen. Others studies have reported the potential concentration of Trichoderma sp. as seed treatment to control peanut brown root rot. It was found that at concentration of 102-106 cfu/seed was effective in disease reduction Page 178
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(Rojo et al., 2007). Therefore, the survival of Trichoderma at the suitable population directly affects on disease suppression, and substrate or other supporting material is one factor to assist its biological control activities.
Figure 1 Trichoderma sp. population in the supporting material. Dynamic population in the nutrient solution tank
Figure 2
Trichoderma sp. population in the nutrient solution tank.
According to Figure 2, Trichoderma population in the NS tank of system with supporting material was detected at 3.0-3.5 log cfu/ml, while it was detected at 2.0-2.5 log cfu/ml in control. Comparison to the dynamics pattern of Trichoderma population, it was not different between 2 systems except for its amount. In this study, the amount of Trichoderma in NS tank of system with supporting material was 10 times higher than control throughout the experiment. As previously discussed, the antagonistic activity of biological control agents depended on their population. Thus, higher population might be higher efficiency. However, further study should be done on the other supporting material. Conclusion In the system with supporting material, Trichoderma population was higher than 4.4 log cfu/ml along the experiment. Whereas, the population in system without supporting material reduced rapidly to 2.6 log cfu/ml and drop to 2.0 log cfu/ml at the end of experiment, resulted
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in the population in the nutrient solution with supporting material was 10 times higher than that of without supporting material. Acknowledgement The author would like to thank the Faculty of Agricultural Technology, KMITL for providing all the facilities both in laboratory and in hydroponics greenhouse as well as for financial support on presenting this research at the symposium. Thank you Mr. Chitipan Thongcharoensukchai for writing assistance. Importantly, the author would like to thank Assoc. Prof. Dr. Tanimnun Jaenaksorn and my advisor Asst. Prof. Dr. Prommart Koohakan for providing counsel in this research.
References Cai, F., G. Yu, P. Wang, Z. Wei, L. Fu, Q. Shen and W. Chen. 2013. Harzianolide, a novel plant growth regulator and systemic resistance elicitor form Trichoderma harzianum. Plant Physiology and Biochemistry 73: 106-113. Dantnoft, L.E., S. Nemec and K. Pernezny. 1995. Biological control of Fusarium crown and root rot of tomato in Florida using Trichoderma harzianum and Glomus intraradicces Biological Control 5: 427-431. Elad, Y., I. Chet and Y. Henis. 1981. Selective media for improving quantitative isolation of Trichoderma spp. from soil. Phytoparasitica 9(1): 59-67. Gul, A., Kidogin F., Y. Tuzel and H.I. Tuzel 2008. Effect of Bacillus amyloliquefaciens on tomato (Solanum lycopersicum L.) growing in perlite. Spainish Journal of Agricultural Research 6(3): 422-429. Ikeda, H., P. Koohakan and T. Jeanaksorn. 2002. Problems and countermeasures in the reused of the nutrient solution in soilless production. Acta Horticulturae 578: 213-219. Kanjanamaneesathian, M., Wiwattanapatapee R., Rothiam, W., Pengnoo A., Wongpetkhiew W. and Tanmala V. 2013. Application of concentration formulation of Bacillus velezensis to control root rot of hydroponically-grown vegetable. New Zealand Plant Protection 66: 229-234. Koohakan, P. and I. Nantagij. 2005. Efficiency of some biological control products and rhizobacteria isolated from lettuce root hydroponically grown for inhibition the growth of Pythium myriotylum. Agricultural Science Journal 36 (5-6): 1195-1198. Koohakan, P., H. Ikeda, T. Jeanaksorn, M. Tojo, S.I. Kusakari, K. Okada and S. Sato. 2004. Evaluation of the indigenous microorganisms in soilless culture: occurrence and quantitative characteristics in the different growing systems. Scientia Horticulturae 101: 179-188. Nawrocka, J. and U. Malolepsza. 2013. Diversity in plant systemic resistance induced by Trichoderma. Biological Control 67: 149-156. Paulitz, T.C. and R.R. Belanger. 2001. Biological control in greenhouse system. Annual Review Phytopathology 39: 103-133. Rajput, A.Q., M.A. Khanzada and S. Shahzad. 2014. Effect of different organic substrates and carbon and nitrogen soruces on growth and shelf life of Trichoderma harzianum. Journal of Science Technology 16: 731-745. Rojo, F.G., M.M. Reynoso, M. Ferez, S.N. Chulze and A.M. Torres. 2007. Biological control by Trichoderma species of Fusarium solani causing peanut brown root rot under field conditions. Crop Protection 26: 549-555. Singh, A., S. Srivastava and S.B. Singh. 2007. Effect of substrate on growth and shelf life of Trichoderma harzianum and it use biocontrol of disease. Bioresource Technology 98: 470-473. Stanghellini, M.E. and S.L. Rasmussen. 1994. Hydroponics: A solution for zoosporic pathogens. Plant Disease 78: 1129-1138. Vinale, F., G. Flematti, K. Sivasithamparum, M. Lorito, R. Marra, B.W. Skelton and E.L. Ghisalberti. 2009. Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum. Journal of Natural Products 72: 2032-2035.
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Morphological Identification of Trichoderma species from Different Habitats Suriyasit SOMNUEK*1, Pornprapa KONGTRAGOUL2 and Tanimnun JAENAKSORN1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang 2 Program in Horticulture, Division of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Chumphon *Corresponding author: suriyasitsom@gmail.com
ABSTRACT Trichoderma species were collected from four different habitats, eg. organic and non-organic rice field in Suphanburi province, forest at Sakaerat Environment Research Station in Nakhon Ratchasima province and nutrient solution sample from hydroponic farm. Nine isolates were successfully isolated on the modified Trichoderma-selective media by soil plate and dilution plate techniques. The result of morphological identification showed all isolates grew completely on PDA (9 cm diameter) in 3 days at room temperature. The colony color was initially white and turned bright green to dark green later. Growth rate of colonies ranged between 2.68-4.07 cm/day. Mycelia were branched, septate, and hyaline. Single-celled light green, mostly globose conidia were formed and developed on phialide in the range of 2.6-3.5 ×2.5-2.9 μm. The phialides were in the range of 4.9-8.4 × 2.5-3.4 μm. These morphological characteristics were identical to the T. harzianum as reported in Chaveri and Samuels (2003). Keywords: Morphological identification, T. harzianum, Organic rice field, Non organic rice field Introduction Plant pathogens are one of the causes of economical losses in crop production (Agrios, 2005). Fungicides have been successfully used to control pathogens. However, fungicide residues are serious problems leading to health and environmental pollution risk (John et al., 2010; Hajieghrari et al., 2008). Therefore, biological control is a non-hazardous method for managing plant diseases. Trichoderma species are one of the most widely studied biocontrol agents for controlling plant pathogenic fungi owing to its various antagonistic mechanisms (Gveroska and Ziberoski, 2012). The mechanisms include antibiosis, competition, mycoparasitism, induced resistance and growth promoting action on plant (Hajieghrari et al., 2008; Shaigan et al., 2008; John et al., 2010; Kalaiselvi et al., 2011; Sreedevi et al.; 2011; Lone et al., 2012; Patil et al., 2012). However, their ecological adaptability makes them ideal candidates for biocontrol application in a variety of habitats, but the sensitivity of different isolates to abiotic environmental factors must be considered (Hjeljord and Tronsmo, 2005). It is worthwhile to try to discover the new indigenous potent Trichoderma species. Therefore, the present investigation was undertaken to isolate and identify Trichoderma species from different habitats such as organic and non-organic rice field in Suphanburi province, forest at Sakaerat Environment Research Station in Nakhon Ratchasima province, from hydroponics farm in Nakhon Ratchasima province, Thailand. Materials and Methods Sample collection and Isolation Soil samples were collected from different habitats such as organic and non-organic rice field in Suphanburi province, forest at Sakaerat Environment Research Station in Nakhon July 1-3, 2015
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Ratchasima province while nutrient solution sample from hydroponics farm in Nakhon Ratchasima province. For isolation of Trichoderma species, a serial dilution technique was performed and a 103 dilution of each sample was prepared. One millilitre of each solution was used for dilution plate techniques while one gram for soil plate techniques onto the modified Trichoderma-selective media plate. All culture plates were incubated at room temperature for 3 days. Then, individual colonies were isolated from the same plates onto PDA plate. Each isolate was transferred onto PDA slants and stored at 4 °C for identification study. Identification Two techniques, namely visual observation on Petri dishes as macro-characteristics and micro-morphological studies on slide culture, were adopted for identification of Trichoderma species. For visual observation on distinct morphological characteristics, the isolates were grown on PDA medium for 3 days and mode of mycelial growth, colony color, and changes of medium color for each isolate were examined daily. For micro-morphological studies, tentative identification characteristics such as shape, size, color, arrangement and development of conidiophores or phialides and conidia were examined by slide culture technique. Isolates were compared to taxonomic key for the species of Trichoderma (Kubicek and Harman, 2002; Chaveri and Samuels, 2003). Results and Discussion In this study, a total of 9 isolates of Trichoderma spp. were isolated from four different habitats. On the basis of visual observation on Petri dishes cultures as macro-characteristics, the colony color was initially white and turned bright green (T111So from organic rice field soil) to dark green later (T112Sc-non organic rice field; T114Kb and T415-2 from forest soil; T121Kh, T515-1 and T515-2 from hydroponics farm) while T114So and T111Sc from organic and non organic rice field showed dark green at the center at final stage. Besides, colony color at the backside of T111So and T111Sc changed to yellow color. Interestingly, colony of T114Kb was quite different from other, that is, only few mycelia mat was exhibited at the central part of PDA plate at the later stage (Table 1). On the basis micro-morphological studies on slide culture, tentative identification characteristics such as shape, size, color, arrangement and development of conidiophores or phialides and conidia of all isolates were similar. Hyphae were septate, branched and hyaline. Conidia, light green, single-celled, globose to sub globose (in the range of 2.6-3.5 Ă&#x2014;2.5-2.9 Îźm) were formed and developed on phialides. These fore-mentioned morphological characteristics of all isolates were identical to the T. harzianum as reported in Kubicek and Harman (2002); Chaveri and Samuels (2003). From our study, it revealed that T. harzianum was the only species recovered from all isolates from 4 different habitats in Thailand such as organic and non-organic rice field soil, forest soil and hydroponics farm. Based on recent researches worldwide, T. harzianum was reported to be the most frequently isolated species such as from rhizosphere of rubber trees in Malaysia (Zakaria, 1989), from 60 soil samples from different places (including from indigenous and planted forests) in Kenya (Okoth et al., 2007), from rice fields in 4 provinces in the Philippines (Cumagun et al., 2000) and in Iran (Khalili et al., 2012), from 4 different bioclimatic zones of Tunisia (Sadfi-Zouaoui et al., 2009), and from hydroponics farm in Thailand (Pradubyart et al., 2013). Our present study was in accordance with the above findings.
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Table 1 Characterisation of Trichoderma species isolated from four different habitats.
2.6-3× 2.6-2.9 2.8-3.2× 2.5-2.9 2.6-3.1× 2.5-2.7
globose globose
2-3
5.3-7.2× 2.8-3.4
globose
3-3.4× 2.7-2.8
T515-2 HF
2-3
5.7-6.4× 2.9-3.2
globose
2.7-3.3× 2.6-2.9
2.7-3.4× 2.6-2.8
2.9-3.5× 2.8-2.9
Sub-to globose 2.7-2.8× 2.7-2.9
3.6
globose
globose
Sub-to globose 2.6-3.2× 2.8-2.9
globose
5-8.4× 2.6-3.3 4.9-7× 3-3.4 7.2-8.0× 2.5-2.9 5.3-6.7× 3.1-3.4 6.7-7× 2.7-3.0 7-8× 2.6 -2.8
3-4 2-3 3-4 3-4 2-3
3.4 3.3 3.5
T515-1 HF
3.7
T121Kh HF
5.2-6× 2.5-2.6
T415-2 FR
2-3
T114Kb FR
2-3
T112Sc NRF
4.0
T111Sc NRF
4.1
T114So ORF
micro-characteristics phialides conidia photograph GR NV size shape size phialides conidia (µm) (µm)
3.4
T111So ORF
macro-characteristics photograph of colony front backside
3.5
isolate source
ORF= organic rice field, NRF= non-organic rice field, FR= Forest, HF= hydroponics farm; GR= growth rate (cm/day); NV= number on verticils; bars = 10 µm
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Conclusion Nine isolates of Trichoderma species were collected from 4 different habitats in Thailand. Interestingly, morphological characteristics of all isolates were identical to T. harzianum. From the above findings, it can be concluded that T. harzianum is the only predominated species recovered from all isolates and can occupy different habitats. Acknowledgment The authors would like to thank Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology for research facilities support. References Agrios, G.N. 2005. Plant pathology, Elsevier Academic Press, California, 952 p. Chaverri, P. and G. J. Samuels. 2003. Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): species with green ascospores. Studies in Mycology 48:1â&#x20AC;&#x201C;116 Cumagun, C.J.R., J. Hockenhull and M. Lubeck. 2000. Characterization of Trichoderma isolate from Philippine rice fields by UP-PCR and rDNA-ITS1 analysis: Identification of UP-PCR markers. Journal of Phytopathology 148: 109-115. Gverroska, B. and J. Ziberoski. 2012. Trichoderma harzianum as a biocontrol agent against Alternaria alternata on tobacco. Applied Technologies and Innovation 7(2): 67-76. Hajieghrari, B., M. Torabi-Giglou, M. R. Mohammadi, and M. Davari. 2008. Biological potential of some Iranian Trichoderma isolates in the control of soil borne plant pathogenic fungi. African Journal of Biotechnology 7(8): 967-972. Hjeljord, L. and A. Tronsmo. 2005. Trichoderma and Gliocladium in biological control: an overview. in Harman, G. E. and C. P. Kubicek. Trichoderma and Gliocladium, Volume 2: Enzymes, Biological Control and Commercial Applications 393 p, 115-127. John, R. P., R. D.Tyagi, D. Prevost, S. K. Brar, S. Pouleur and R. Y. Surampalli. 2010. Mycoparasitic Trichoderma viride as a biocontrol agent against Fusarium oxysporum f. sp. adzuki and Pythium arrhenomanes. Crop Protection 29: 1452-1459. Kalaiselvi, S. and Panneerselvam. 2011. In vitro assessment of antagonistic activity of Trichoderma sp. Againt Sarocladium oryzae causing sheath rot disease in paddy. International Journal of Applied Biology and Pharmaceutical Technology 2 (1): 179-183. Khalili, E., M. Sadravi, S. Naeimi and V. Khosravi. 2012. Biological control of rice brown spot with native isolates of three Trichoderma species. Braziliian Journal of Microbiology: 297-305. Kubicek, C.P. and G.E. Harman. 2002. Trichoderma and Gliocladium: Basic Biology, Taxonomy and Genetics, Volume1. Taylor and Francis Ltd, 278 p. Lone, M. A., M. R. Wani, S. A. Sheikh, S. Sahay and M. S. Dar. 2012. Antagonistic potentiality of Trichoderma harzianum againt Cladosporium spherospermum, Aspergillus niger and Fusarium oxysporium. Journal of Biology, Agriculture and Healthcare 2 (8): 2224-3208. Okoth, S. A., H. Roimen, B. Mutsotso, E. Muya and P. Okoth. 2007. Land use systems and distribution of Trichoderma species in Embu region, Kenya. Trop. and Sub Trop. Agroeco. Sys. 7: 105-112. Patil, A., A. Laddha, A. Lunge, H. Paikrao and S. Mahure. 2012. In vitro antagonistic properties of selected Trichoderma species against tomato root rot causing Pythium species. International Journal of Science, Environment 1(4): 302-315. Pradubyart, M., N. Parinthawong and T. Jaenaksorn. 2013. In vitro screening of antagonistic Trichoderma sp. against pathogenic fungi causing leaf spot of vegetable crop grown in hydroponics. Proceedings of 6th Rajamangala University of Technology Tawan-ok Research Conference: 52-57. Sadfi-Zouaoui, N., I. Hanachi, M. Rouaisi, M.R. Hajilaoui, M.B. Rubio, E. Monte, A. Boudabous and M.R. Hermasa. 2009. Biodiversity of Trichoderma strains in Tunisia. Cannadian Journal of Microbiology 55: 154-162. Shaigan, S., A. Seraji and S. A. M. Moghaddam. 2008. Identification and investigation on antagonistic effect of Trichoderma spp. on tea seedlings white foot and root rot (Sclerotium rolfsiiSacc.) in vitro condition. Pakistan Journal of Biological Sciences 11: 2346-2350. Sreedevi, B., C.M. Devi and D.V.R. Saigopal. 2011. Isolation and screening of effective Trichoderma spp. against the root rot pathogen Macrophominaphaseolina. Journal of Agricultural Technology 7(3): 623-635. Zakaria M. H. 1989. Some aspects of the biology and chemically assisted biological control of Ganoderma species in Malaysia, PhD, University of Putra, Malaysia.
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O-38
Resistant in Rice Cultivars against Sheath Blight Disease under Artificial Inoculated Conditions P.K. TIWARI1*, C.P. KHARE1 and A.S. KOTASTHANE1 1
Department of Plant Pathology, IGKV, Raipur-492012 (C.G.) India *Corresponding email: pradeep.tiwari95@gmail.com
ABSTRACT Sheath blight of rice caused by Rhizoctinia solani is one of the most serious diseases of irrigated rice. It is the major disease in particular Chhattisgarh state of India and Raipur district is considered as a hot spot location for the disease. In nature, the varieties sustained for sheath blight resistant are required and the process of selecting resistant varieties is continued. An, attempt therefore was made to identify the sources of resistance in available rice germplasms against the sheath blight disease. The two thousands and five hundred of rice cultivars were screened in different trails and a total number of 11 rice germplasms were collected on the basis of resistant reactions. The selected resistant germplams were rescreened by sowing in simple three row plots under artificial inoculated conditions during the consecutive seasons from 2011-2013. The seeds were sown in row of 2 m., spaced at 20X10 cm. in the field. The disease severity was recorded at 21 days after inoculation followed by SES scale (1996). Among the 11 cultivars tested against the sheath blight of rice under artificial inoculated conditions, none was found immune while 11 entries viz, IET22240, IET22250, IET21665, CBO9-153, Dhan Prasad, GP.no.545191, GP.no.461231, GP.no.545206, G.P no. 450284, GP no. 450296 & G.P.no 463893 proved to be resistant and locally adopted variety Swana produced highly susceptible reaction. Keywords: Sheath blight, Artificial inoculated condition, Resistant varieties, Entries, Disease severity Introduction Rice is the most important crop in India, which plays pivotal role in food security. More importantly, it is a choice crop of the millions of poor and small farmers not only for income but also for house hold food security. India ranks first in area (44 m ha) and second in the world in production of rice (102.7 mt). About 34% of the total area of the nation is under rice cultivation. Chhattisgarh is being considered as â&#x20AC;&#x153;bowl of riceâ&#x20AC;?, where about 82 percent populations depend on rice cultivation for their livelihood. Rice occupies an area of 3.57m ha with the average productivity of 1571kg/ha in the state (FAO, 2009). Sheath blight is a most important disease of rice incited by Rhizoctonia solani Kuhn, first reported by Paracer and Chahal (1963) from Gurdaspur in Punjab state. It is a major production constraint in Punjab, Haryyana, Eastern UP, Bihar, West Bangal, Orrsa, Assam, Tripura, Coastal Andhra Pradesh, Coastal Tamilnadu, Kerla, parts of Karnataka and Chhattisgarh. It can reduce the yield losses 25- 30%. Sheath blight of rice has become more prevalent on many improved varieties currently grown in India. In India, host plant resistant is the most important tool for rice disease management and has played a key role in sustaining rice productivity. There is a need to discover genes confirming resistant to sheath blight where no host resistant is known .The breeding strategies for development of sheath blight resistant cultivar as suggested by Khus (1977) were: Continuous evaluation of germplasm to identify cultivars with higher levels of resistant. Screening of all advanced breeding lines and elimination of all susceptible materials. July 1-3, 2015
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An organized breeding program using selected linens with moderate levels resistant. The program aims at pyramiding minor genes for resistant from several parents in to the same line by a method of recurrent selection. Materials and Methods Layout Transplant 25-day-old seedlings. Each test entry was planted in three rows each of 1 meter wide and two meter length, adopting a plant to plant spacing of 20x20 cm. Fertilizers Adequate nitrogen (150 kgN/ha). One- half nitrogen was applied as basal and the remaining in two equal splits at tillering and panicle initiation stages. P, K and Zn were applied as per the recommendation practices. Inoculation Multiply pure culture of the pathogen on autoclaved rice culm bits (5-7 cm). Inoculated the test entries at tillering stage by placing the inoculums between the tillers just above the water lines (Anonymous, 2014). The observation on disease severity was recorded on 21 days after inoculation by adopting SES scale (1996). Rice bits or Typha bits inoculation method Developed by Bhaktavatsalm (1978) is a simple, rapid and mass inoculation technique to induce sheath blight disease in rice to evaluate germplasm and breeding lines in field and glasshouse. Many research workers follow this method of artificial inoculation. For screening of sheath blight under AICRP program, this inoculation method is followed most of the cases (Prakasm et.al, 2013). Results and Discussion Raipur district of Chhattisgrh is a traditional rice growing area and is recognized as a hot spot for sheath blight of rice (shb). Two thousand and five hundred rice entries were screened with Rhizoctinia solani isolate collected from Raipur. The rice entries which were showed resistant to highly resistant reactions were re-screened for the consecutive years (2011-2013) and the variety Swarna was used as susceptible check (Table 1). The eleven rice entries viz, IET22240, IET22250, IET21665, CBO9-153, Dhan Prasad, GP.no.545191, GP.no.461231, GP.no.545206, G.P no. 450284, GP no. 450296 and G.P.no 463893 were reconfirmed as resistant to sheath blight and locally adopted variety Swarna produced highly susceptible reaction for the disease under Raipur conditions. Similar findings were also confirmed by Reddy et al., 1981b that the rice cultivars KRC346, KRC355, KRC356, ZANITH (ck) and RP 1057-393-1, CR280-5, JR49 and MLG14 were recorded as highly resistant for sheath blight. Rani and Styanarayna, 1992 also reported that eight thousand F2 plants from three crosses, RP1821 (OS4/RPW6-17), RP1819 (Pankaj/RP1821) and RP 1822 (Pankaj/IET5656) were field tested under favorable conditions and RP 1821 crosses were found most promising. Though, a number of varieties has been released through AICRP with moderately level of resistance to sheath blight (Rani et al., 2011).
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Table 1 List of rice entries indicating sheath blight score as per standard evaluation system of rice (SES1988) during three consecutive years. S.No. 1 2 3 4 5 6 7 8 9 10 11 12
Entries/ Variety IET22250 IET22240 IET21665 IETCB09-153 Dhan Prasad IET545191 IET461231 IET545206 IET450284 IET450296 IET463893 Swarna LSI
2011 3 3 3 3 3 3 3 3 5 3 3 9 2.5
2012 3 3 3 1 3 3 3 3 3 3 3 9
2013 1 1 1 1 3 3 1 1 1 1 1 9
Conclusion The eleven cultivars proved to be promising resistance against the sheath blight of rice under artificial inoculated conditions at Raipur are IET22240, IET22250, IET21665, CBO9-153, Dhan Prasad, GP.no.545191, GP.no.461231, GP.no.545206, G.P no. 450284, GP no. 450296 and G.P.no 463893 and locally adopted variety Swarna produced highly susceptible reaction. Acknowledgements The All India Coordinated Rice Pathology Program of the Indian Institute of Rice research for provide the germplasm for screening and financial support. References Bhaktavatsalam G., K. Satyanarayana, P.K. Reddy and V.T. John. 1978. Evaluation of sheath blight resistance in rice. Int Rice Res Newsl 3:9-10 p. Khus, G.S. 1977. Disease and insect resistance in rice. .Adv. Agron. 29: pp 268-341. Paracer, C.S. and D.S. Chahal. 1963. Sheath blight of rice caused by Rhizoctonia solai Khhn -A new record in India. Curr. Sci. 32: pp.328-329. Prakasam V., D. Ladalakshmi, G.S. Laha, D. Krishnaveni, M.S. Madhav, J. Badri, M.S. Prasad and B.C. Viraktamath. 2013. Sheath blight disease of rice and its management. Tech Bull 72. Directorate of Rice Research Hyderabad India. 58p. Reddy C.S., Gosh, A., Satyanarayna, K., Reddy, A.P.K .and John, V.T. 1981b. Central Rice Research Institute, Cuttuk. Annual Report: pp 247-248.IARI, New Delhi. Rani, N.S. and Satyanarayna, K. 1992. Testing for sheath blight resistant in rice. Int. Rice Res. News 7(5): pp 7 Rani N.S., G.S. Varaprasad, L.V.S Rao, I. Sudharshan, A.S. Hariprasad, A.S.R. Prasad, T. Ram, V.R. Babi, G. Padmavati, V. Bhadana, K. Suneetha and B.C. Viraktamath. 2011. High-yielding rice varieties in India. Tech Bull 55. Directorate of Rice Research Hyderabad India 192p.
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P-1
Effect of Naphthalene Acetic Acid and Thidiazuron on Phytotoxicity of Phenanthrene and Fluorene to Waxy Corn Waraporn CHOUYCHAI1*, Jaturaporn DONRODPRI1, and Khanitta SOMTRATOON2 1
Biology Programme, Faculty of Science and Technology, Nakhonsawan Rajabhat University, Nakhonsawan, Thailand 2 Department of Biology, Faculty of Science, Mahasarakham University, Mahasarakham, Thailand *Corresponding email: chouychai@yahoo.com
ABSTRACT Phenanthrene and fluorene are polycyclic aromatic hydrocarbons (PAHs) which are toxic to plant growth. This phytotoxicity is a limiting factor for a success of PAH phytoremediation. Thus, plant growth regulators were used to alleviate PAH toxicity. The effect of 2 plant growth regulators, naphthalene acetic acid (NAA) and thidiazuron (TDZ) to alleviate PAH phytotoxicity was studied. Waxy corn seeds were immersed in 10 mg/l NAA, or TDZ for 1 or 8 h, and then sown in 400 mg/kg phenanthrene and 400 mg/kg fluorene contaminated sand. Seeds immersed in distilled water for 1, and 8 h were done as control. Seedlings from NAA immersed seeds had higher root length and shoot fresh weight than control. On the other hand, seed immersion in TDZ decreased length and root fresh weight of waxy corn seedling compared to control. The immersion periods of 1 and 8 h affected only on root length and root dry weight of waxy corn seedling. For NAA, the 8 h immersed seed produced higher root length and root dried weight than that of 1h. This result indicated that seed immersion in 10 mg/l NAA for 1 h could enhance waxy corn seedling growth in phenanthrene and fluorene-contaminated sand and will be useful for enhancing the plant growth in phytoremediation process. Keywords: Naphthalene acetic acid, Thidiazuron, Polycyclic Aromatic Hydrocarbons, Waxy corn, Toxicity testing Introduction Polycyclic aromatic hydrocarbons (PAHs) were the pollutants that have to rapidly remove from the environment. There are many routes of PAHs to enter the environment, such as incomplete combustion of fossil fuel, transportation, and rice straw open field burning (Oanh et al., 2000; Gadde et al., 2009).Phytoremediation is an effective method-to remove PAHs from soil. Various plant species, such as corn, wing bean (Somtrakoon et al., 2014a) and ridge gourd (Somtrakoon et al., 2014b) have been used to remove PAHs from contaminated soil, but their efficiency of these plants were limited by phytotoxicity of PAHs. Use of plant growth regulators to induce plant growth in PAH-contaminated soil will be an alternative choice to increase phytoremediation efficiency. Some plant growth regulators were reported to increase plant tolerant to organic pollutant. For example, Gibberellic acid was reported to enhance 4% of benzo[a]pyrene biodegradationin cadmium + benzo[a]pyrene co-contaminated soil planted with Tagates patula (Sun et al., 2013). Synthetic auxin, such as NAA, and phenylurea cytokinin, such as TDZ, were interested to induce waxy corn growth in PAH-contaminated soil. Watering with 2.4 mg/l IAA increased fluoranthene removal in soil planted with ryegrass (Li et al., 2015). Also, watering with 0.01 mg/l TDZ increased waxy corn growth in hexachlorocyclohexane-contaminated soil (Chouychai et al. 2015). In this July 1-3, 2015
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study, the capacity of NAA and TDZ, to alleviate phenanthrene and fluorenephytotoxicty to waxy corn was studied and their possibility to use in phytoremediation proposes was assessed. Materials and Methods The phytotoxicity assay described by Somtrakoon et al. (2014c). For each experiment, 50 g of autoclaved sand were added to a glass Petri dish in triplicate. Mixture of phenanthrene (Sigma-Aldrich, purity 98%) and fluorene (Sigma-Aldrich, Germany, purity 98%) was weighed and dissolved in acetone, then transferred to a glass sprayer and spiked to soil to final concentrations of 800 mg/kg dried soil (1:1). As a control, acetone without any PAHs was sprayed into sand. Sand in each dish was thoroughly mixed with a metal digger. The spiked sand was air-dried at 28-300C for more than 24 h or until the smell of acetone had disappeared. Seeds of waxy corn (commercial seeds of Thailand) were used in this study. Plant growth regulators used in this experiment were naphthalene acetic acid (Fluka, purity 99%) and Thidiazuron (Fluka, purity 99%). Seeds of waxy corn were immersed in the following solutions: (a) 10 mg/l NAA for 1 h; (b) 10 mg/l NAA for 8 h; (c) 10 mg/l TDZ for 1 h; (d) 10 mg/l TDZ for 8 h; (e) distilled water for 1 h; and (f) distilled water for 8 h. Ten seeds were inoculated into each Petri dish containing 50 g of sand spiked with phenanthrene and fluorene. These were done in triplicate. The dishes were kept at 29Âą20C in a room under natural sunlight. Each plate received 10 to 20 ml of water daily. After 10 days, all seedlings were randomly removed for measurement their shoot and root length, as well as the fresh and dry weight of the shoots. One-way ANOVA was used to test for significant differences between treatments followed by LSDâ&#x20AC;&#x2122;s test. Results and Discussion Seed immersion in NAA could induce root length, shoot fresh weight, and root dried weight of waxy corn seedling grown in PAH-contaminated sand obviously. Root length of waxy corn seedlings from seed immersed in NAA for 8 h was 13.2 cm that was longer than root length of seedling from seed immersed in distilled water for 8 h (4.1 cm) (Figure 1). The immersion period of NAA as 8 h was more pronounced to induce root length and root dry weight of waxy corn seedlings when compared with 1 h period. NAA was the effective auxin for enhance plant growth in PAH-contaminated soil. For example, seed immersion in NAA for 3 h could enhance shoot fresh weight of rice cv. RD47 seedling grown in 100 mg/kg fluoranthene-contaminated soil (Chouychai et al., 2014a). However, indolebutyric acid (IBA), another auxin, could not enhance waxy corn and longbean seedling grown in 400 mg/kg phenanthrene contaminated soil (Chouychai et al., 2014b). On the other hand, seed immersion in TDZ inhibited root growth and shoot length of waxy corn seedlings. Root fresh weight of waxy corn seedlings from seed immersed in TDZ for 1 h was 34.4 mg that was less than root fresh weight of seedling from seed immersed in distilled water for 1 h (127.4 mg) (Figure 2). Even though TDZ could induce diverse plant response, including callus formation and activation of cotyledon growth, TDZ often reported to suppress root growth of many plant species, such as corn, wheat, and radish (Murthy et al., 1998). The inhibition to root growth of TDZ was also reported in Brassica chinensis seedling grown in endosulfan sulfate-contaminated sand. The root of 10 day-old B.chinensis seedling from seed immersed in 10 mg/l TDZ for 3 h were not developed (Somtrakoon et al., 2013).
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Figure 1 Shoot length (A), and root length (B) of waxy corn seedling from seed immersed in plant growth regulators for 1 or 8 h and then grown in 400 mg/kg phenanthrene and fluorene contaminated sand for 10 days. Different lower case letter showed significant difference (P<0.05) between plant growth regulators and * represented significant difference (P<0.05) between immersed time.
Figure 2 Shoot fresh weight, root fresh weight, shoot dried weight and root dried weight of waxy corn seedling from seed immersed in plant growth regulators for 1 or 8 h and then grown in 400 mg/kg phenanthrene and fluorene contaminated sand for 10 days. Different lower case letter showed significant difference (P<0.05) between plant growth regulators and * showed significant difference (P<0.05) between immersed time.
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Conclusion Seed immersion in 10 mg/l NAA for 1 h was effective to enhance waxy corn seedling growth in 400 mg/kg phenanthrene and fluorene contaminated sand. Seed immersion in 10 mg/l TDZ decreased root growth of waxy corn. Acknowledgement Plant growth regulators used in this experiment was supported by the Thailand Research Fund (Grant No. MRG5480043) and the Commission on Higher Education, Ministry of Education, Thailand and Nakhonsawan Rajabhat University. References Chouychai, W., P. Chartchai, and K. Somtrakoon. 2014a. Effect of naphthalene acetic acid and benzyladenine on fluorene and fluoranthene toxicity in rice cv. RD47. Journal of Agricultural Research and Extension 31 (1): 13 - 22 (In Thai with English abstract). Chouychai, W., W. Kongtoom, and K. Somtrakoon. 2014b. Effect of Plant growth regulators on Phenanthrene Toxicity in Waxy Corn and Long bean. Journal of Agricultural Research and Extension. 31 (3):11-23. (In Thai with English abstract). Chouychai, W., M. Kruatrachue and H. Lee. 2015. Effect of plant growth regulators on phytoremediation of hexachlorocyclohexane-contaminated soil. International Journal of Phytoremediation (in press). Gadde, B., S. Bonnet, C. Menke, and S. Garivait. 2009. Air pollutant emissions from rice straw open field burning in India, Thailand, and the Philippines. Environmental Pollution 157: 1554 – 1558. Li, W., L. Xu, J. Wu, L. Ma, M. Liu, J. Jiao, H. Li, and F. Hu. 2015. Effects of indole-3acetic acid, a plant hormone, on the ryegrass yield and the removal of fluoranthene from soil. International Journal of Phytoremediation 17: 422 – 428. Murthy, B.N.S., S.J. Murch, and P.K. Saxena. 1998. Thidiazuron: A potent regulator of in vitro plant morphogenesis. In Vitro Cell Developmental Biology- Plant 34:267 – 275. Oanh, N.T.K., L. B. Reutergardh, N. Tr. Dung, M. H. Yu, W.X. Yao, and H.X. Co. 2000. Polycyclic aromatic hydrocarbons in the airborne particulate matter at a location 40 km north of Bangkok, Thailand. Atmospheric Environment. 34: 4557 – 4563. Somtrakoon, K., and M. Kruatrachue. 2013. Effects of alpha-naphthalene acetic acid and thidiazuron on Brassica chinensisseedling growth in sand contaminated with endosulfan-sulfate.Science and Technology Ubonratchthanee University Journal 15 (1): 1-11 (In Thai with English abstract). Somtrakoon, K., W. Chouychai, and H. Lee.2014a. Comparing Anthracene and FluoreneDegradation in Anthracene and Fluorene-Contaminated Soil by Single and Mixed Plant Cultivation.International Journal of Phytoremediation.16: 415-428. Somtrakoon, K., W. Chouychai, and H. Lee.2014b. Phytoremediation of anthracene- and fluoranthene-contaminated soil by Luffa acutangula.Maejo International Journal of Science and Technology 8: 221-231. Somtrakoon, K., and M. Kruatrachue. 2014c. Effect of alpha-naphthalene acetic acid and thidiazuron on seedling growth of economic crops growing on endosulfan sulfate-spiked sand. Journal of Environmental Biology 35: 1021 – 1030. Sun, Y., Y. Xu, Q. Zhou, L. Wang, D. Lin, and X. Liang. 2013. The potential of gibberellic acid 3 (GA3) and Tween-80 induced phytoremediation of co-contaminationof Cd and benzo[a]pyrene (B[a]P) using Tagates patula. Journal of Environmental Management 114: 202 – 208.
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P-2
Effect of Fertilizers on Physiological Traits Relate to Yield of Napier Pak Chong 1 Grass Nittaya PHAKAMAS1*, Anupong MAKLAI1, Tochchakorn PERMHIRUN1, Kittiya SERTSUNGNONE1 and Nawarat JAMPATHONG1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kpnittay@kmitl.ac.th
ABSTRACT Napier Pak Chong 1 (Pennisetum purpurem x Pennisetum americanum cv. Pak Chong 1) grass becomes one of the important economic forage crops in Thailand. The objective of this study was to determine the effect of chemical and organic fertilizers on the physiological traits relate to dry matter yield of Napier Pak Chong 1 grass. The experiment was conducted in the field of the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, during 15 December 2013 to 18 December 2014. The experiment was laid out in a randomized complete block design (RCBD) with 4 replications. Plot size was 1 x 2 m, planted with spacing of 1.2 m between rows and 0.8 m between plants, using stem-cutting materials. Five types of fertilizer were used i.e., no fertilizer (control), cattle manure (12,500 kg ha-1), urea (93.75 kg ha-1), cattle manure (6,250 kg ha-1) + urea (46.87 kg ha-1) and 15-15-15 chemical fertilizer (312.5 kg ha-1). Plant height, tiller per plant, leaf area (LA), leaf area index (LAI), crop growth rate (CGR), chlorophyll A, chlorophyll B, fresh yield and dry matter yield were obtained for 4 times in every 60 days-interval, and then analysis of variance and multiple regression were done for all those traits. The result found that, there was significant effect of fertilizers for chlorophyll A, chlorophyll B, LA, LAI, CGR, fresh yield and dry matter yield. The result indicated that CGR was the dominant physiological traits that contributed to yield of Napier Pak Chong 1 grass. Keywords: Forage crop, Energy crop, Physiological traits, Crop growth rate (CGR) Introduction Presently, Napier Pak Chong1 (Pennisetum purpurem x Pennisetum americanum cv. Pak Chong 1) becomes one of important economic forage crops in Thailand. It has high potential for forage crop production as it has high yield, high protein and good response to irrigation and fertilizers. Moreover, it is tolerant to drought. This grass is propagated through stemcutting and could be maintained as a perennial crop up to 6-7 years. It can be harvested at 4560 day intervals. Under field conditions and good water management, Napier Pak Chong1 had biomass yield of 49.13 t ha-1 year-1and crop growth rate (CGR) of 36.12 kg m-2 d-1 at 60day intervals and had high leaf area index (LAI) at 45 or 60 day intervals (Pongtip et al., 2014). In Sri Langka, proper management practices together with the correct application of fertilizers, irrigation in drought spells and cut at the suitable height and intervals were important for maximum profit and persistence of hybrid Napier (Pennisetum purpurem x Pennisetum americanum cv. CO-3) (Premaratne and Premalal, 2006). Application of chemical fertilizers plus organic amendment gave significantly greater growth of forage sorghum (Sorghum bicolor L. Moench) than adding chemical fertilizers (Pholsen and Suksri, 2004). The research done so far has not shown the clear results on the effect of fertilizers on physiological traits especially for dominant physiological trait related to yield. Therefore, the July 1-3, 2015
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objective of this study was to determine the effect of different fertilizers on the physiological traits related to yield of Napier Pak Chong1 grass. Materials and Methods The experiment was conducted in the field at the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, during 15 December 2013 to 18 December 2014. The treatments consisting of five types of fertilizers (no fertilizer; control, cattle manure at the rate of 12,500 kg ha-1, urea at the rate of 93.75 kg ha-1, cattle manure at the rate of 6,250 kg ha-1 + urea at the rate of 46.87 kg ha-1 and 15-15-15 chemical fertilizers at the rate of 312.5 kg ha-1) were arranged in a randomized complete block design (RCBD) with 4 replications. Plot size was 1 x 2 m with spacing of 1.2 m between rows and 0.8 m between plants, and stem cuts were used as planting materials. Irrigation water was supplied to the crop at 7-day intervals to avoid drought stress during the dry spell. Fertilizers of all types were applied to the crop as basal dose prior to planting and at 30-day intervals for 10 times. Leaf area (LA) was measured from 20 leaves of each plot using a leaf area meter (LI-3100 Area Meter) and then, the leaf area index (LAI) was calculated. All plants in each plot were oven-dried at 60 oC for 72 hours and weighed to determine dry matter yield. Dry matter yield was used to calculate crop growth rate (CGR). Chlorophyll A and Chlorophyll B were measured according to the methods described previously (Lichtenthaler, 1987; Shabala et al., 1998). Plant height and tiller number per plant were also obtained, and then analysis of variance and multiple regression were done for all traits. Results and Discussion Types of fertilizers were significantly different (P≤0.05 and P≤0.01) for fresh yield of Napier Pak Chong 1 grass at 1st, 2nd, 3rd and 4th cuttings (Table 1). All types of fertilizers were significantly higher than control at all cuttings. At the first cutting, all types of fertilizers ranging from 58.3 to 66.6 t ha-1 were similar but significantly higher than control (37.9 t ha-1). At second cutting, urea gave the highest (50.1 t ha-1) but it was not significantly higher than cattle manure (43.7 t ha-1). At third cutting, cattle manure (48.0 t ha-1) gave the highest but it was not significantly higher than urea (44.9 t ha-1) and cattle manure plus urea (32.7 t ha-1). At fourth cutting, cattle manure (13.6 t ha-1), urea (13.6 t ha-1) and cattle manure plus urea (11.9 t ha-1) gave significantly higher than 15-15-15 chemical fertilizer (6.9 t ha-1). Significant differences (P≤0.01) among treatments were observed for dry matter yield of Napier Pak Chong 1 grass at 1st and 2nd cuttings, whereas the differences at 3rd and 4th cuttings were not significant (Table 2). At 1st cutting, all fertilizer treatments ranging from 12.5 to 16.9 t ha-1 were significantly higher than control (8.1 t ha-1) and urea was highest (16.9 t ha-1). At 2nd cutting, all fertilizer treatments ranging from 9.4 to 17.9 t ha-1, only for cattle manure (17.9 t ha-1) and urea (16.3 t ha-1) were significantly higher than control (5.8 t ha-1). Yields of the grass tended to reduce at the latter cuttings. In previous study, yield of well-irrigated Napier was recorded at 49.13 t ha-1 year-1 when the crop was harvested at 60 days intervals (Pongtip et al., 2014).
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Table 1 Effect of different types of fertilizers on fresh yield of Napier Pak Chong 1 grass Fresh yield (t ha-1) Treatment 1st cutting 2ndcutting 3thcutting 4thcutting Control 37.9b 18.9d 22.0c 6.7b Cattle manure 66.6a 43.7ab 48.0a 13.6a Urea 64.5a 50.1a 44.9ab 13.6a Cattle manure + Urea 65.6a 37.8bc 32.7abc 11.9a 15-15-15 chemical fertilizer 58.3a 30.0cd 25.2bc 6.9b F-test ** ** ** * C.V. % 15.4 15.1 28.3 28.7 *,** = significantly different at P≤ 0.05and P≤0.01, respectively Means in the same column followed by the same letter are not significantly different by DMRT
Table 2 Effect of different types of fertilizer on dry matter yield of Napier Pak Chong 1 grass Dry matter yield (t ha-1) Treatment 1st cutting 2ndcutting 3thcutting 4thcutting Control 8.1c 5.8b 4.1 3.8 Cattle manure 13.5ab 17.9a 8.5 5.9 Urea 16.9a 16.3a 8.6 5.6 Cattle manure + Urea 14.1ab 8.7ab 6.1 6.7 15-15-15 chemical fertilizer 12.5b 9.4ab 3.8 3.1 F-test ** ** ns ns C.V. % 12.8 37.1 61.4 47.3 ns = non significantly different ** = significantly different at P ≤ 0.01 Means in the same column followed by the same letter are not significantly different by DMRT
The treatments were significantly different (P≤0.01) for crop growth rate at 1st and 2nd cutting, whereas the differences were not significant at 3rd and 4th cuttings (Table 3). Similar to dry matter yield, all fertilizer treatments ranging from 20.9 to 28.1 g m-2 d-1 were significantly higher than control (13.6 g m-2 d-1) at 1st cutting. At 2nd cutting, only cattle manure (29.8 g m-2 d-1) and urea (27.1 g m-2 d-1) were significantly higher than control (9.6 g m-2 d-1). Table 3 Effect of different types of fertilizer on crop growth rate of Napier Pak Chong 1 grass Crop growth rate (g m-2 d-1) Treatment 1st cutting 2ndcutting 3rdcutting 4thcutting Control 13.6c 9.6b 6.9 7.6 Cattle manure 22.6ab 29.8a 14.3 11.9 Urea 28.1a 27.1a 14.3 11.2 Cattle manure + Urea 23.6ab 14.5ab 10.2 13.4 15-15-15 chemical fertilizer 20.9b 15.7ab 6.2 6.2 F-test ** ** ns ns C.V. % 12.8 37.1 61.3 47.3 ns = non significantly different ** = significantly different at P ≤ 0.01 Means in the same column followed by the same letter are not significantly different by DMRT
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Reduction in crop growth rate was observed at latter cuttings. In forage sorghum, application of chemical fertilizer plus organic amendment gave greater growth than adding chemical fertilizer alone (Pholsen and Suksri, 2004). Correlation coefficients between fresh yield and CGR (R2=0.93; Pâ&#x2030;¤0.01), and dry matter yield and CGR (R2=0.91; Pâ&#x2030;¤0.01) were significant and positive (Figure 1). Types of fertilizers were also significantly different for chlorophyll A, chlorophyll B, LA and LAI (data not shown). Multiple regression analysis identified that CGR was the dominant physiological trait contributing to yield of Napier Pak Chong 1 grass.
Figure 1 Relationships between CGR and fresh yield (a), and CGR and dry matter yield (b) Conclusion Types of fertilizers significantly affect physiological traits and yield of Napier Pak Chong 1 grass.The correlation coefficients between crop growth rate and fresh yield, and crop growth rate and dry matter yield were positive and significant. Crop growth rate was an important physiological trait that contributed to yield of Napier Pak Chong 1 grass. Acknowledgments The authors gratefully acknowledge Nakhonratchasima Animal Nutrition Research and Development Center for their supporting for stem-cutting materials of Napier Pak Chong 1 grass. References Linchtenthaler, H.K. 1987. Method Enzymol. 148:350-380. Pholsen, S. and A. Suksri. 2004. Effect of organic amendment and chemical fertilizer on growth yield and fodder quality of a forage sorghum (Sorghum bicolor L. Moench). Pakistan Journal of Biological Sciences 7(4):651-657. Pongtip, A., P. Pitipong, N. Tonmukayakul, P. Mani-in, J. Muangpan and J. Changkaewmanee. 2014. Physiological biomass yield and energy productivity of grasses under wetland and water management areas. Agricultural Science Journal 45(2) (Suppi.): 301-304. Premaratne, S. and G.G.C. Premalal. 2006. Hybrid Napier (Pennisetumpurpurem x Pennisetumamenicanum) var CO-3: A resourceful fodder grass for dairy development in Sri Lanka. The Journal of Agricultural Sciences 2(1): 22-33. Shabala, S.N., S.I. Shabala, A.I. Martynenko, O. Babourina, andI.A. Newman. 1998. Salinity effect on bioelectric activity, growth, Na+ accumulation and chlorophyll fluorescence of maize leaves: a comparative survey and prospects. J. Plant Physiol. 25: 609-616. Page 196
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P-3
Effect of Nanoparticles on the Relationship between Crop Growth Rate and Yield of Chainat 1 Rice (Oryza sativa L.) Sutichai SAMART1*, Nittaya PHAKAMAS2 and Sutee CHUTIPAIJIT1 1
College of Nanotechnology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand * Corresponding email: samarteen_oxiy@hotmail.com
ABSTRACT Nanoparticle materials can be used in agriculture but knowledge is still limited. The objective of this study was to determine the effect of zinc oxide (ZnO) and titanium dioxide (TiO2) nanoparticles on physiological traits relate to rice yield. The experiment was conducted in the field of the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang during February to June, 2013. Treatments were arranged in 2×5 factorials in RCBD with 3 replications. Zinc oxide and titanium dioxide nanoparticles were used as the factor A. Factor B was the concentrations of nanoparticles i.e., 100, 200, 400, 600 and 800 mgL-1. ZnO and TiO2, both types nanoparticle were used for every 14-days interval until 7 days before harvesting. Crop growth rate (CGR) was recorded at two stages; during planting to flowering (0 to 58 days) and flowering to harvest (58 days to harvest). Spikelet per m2, 1,000 grain weight, total dry matter, harvest index (HI) and grain yield were also collected. Analysis of variance and multiple regressions were done for all those traits. The results were found that there were significant effects of ZnO and TiO2 nanoparticles on growth and yield of Chainat 1 rice. TiO2 at a rate of 200-600 mg.L-1 promoted greater growth and yield of Chainat 1 rice than utilizing of ZnO. CGR was significantly correlated to yield of Chainat 1 rice (R2 =0.20*). Nanoparticles can promote CGR, especially, during flowering to harvest stage (58 days to harvest). Keywords: Chainat 1 rice, Physiological traits, Nanoparticle, Crop growth rate (CGR) Introduction Presently, nanoparticle materials have been widely applied in agriculture in the world. Zinc oxide (ZnO) and titanium dioxide (TiO2) are metal oxide nanoparticles that have been used in agricultural research and only few studies have focused on their effects on plant growth. Boonyanitipong et al. (2011) reported that application of nano-ZnO stunted root length and reduced number of roots in rice (Oryza sativa L.), whereas nano-TiO2 did not have significant effect. Monica and Cremonini (2009) explained that the effects of nanoparticles on the inhibition of root growth varied greatly among nanoparticles and plants, and concentration of nanoparticles was an important factor affecting the effects of nanoparticles. Zhang et al. (2005) indicated that the application of nano-TiO2 could promoted seed germination and seed vigor of spinach (Spinacia oleracea) plant. Moreover, nano-TiO2 could increase chlorophyll formation and photosynthetic rate, and the authors demonstrated that these physiological traits were related to dry matter accumulation during growth stage of spinach plant (Zhang et al., 2005). The previous studies have revealed that nanoparticle materials may play some important role in plant growth. However, the knowledge of the effect and mechanisms of nanomaterial on plant growth and yield is still limited. In non-photoperiod sensitive rice, Somjit and Phakamas (2012) reported that crop growth rate (CGR) during flowering to July 1-3, 2015
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harvest stage was an important physiological trait related to rice yield as CGR is a function of photosynthetic rate, respiration losses by existing tissue and leaf area interception light, and these physiological traits affected plant growth. Therefore, the objective of this study was to determine the effects of ZnO and TiO2 nanoparticles on physiological traits relate to yield of Chainat 1 rice cultivar. Materials and Methods The experiment was carried out at the experimental field of the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang during February to June 2013. Treatments were arranged in 2×5 factorials in RCBD with 3 replications. ZnO and TiO2 nanoparticles were used as the factor A. Factor B included the concentrations of nanoparticles i.e., 100, 200, 400, 600 and 800 mg.L-1. ZnO and TiO2 were applied to the crop at 14-day intervals until 7 days before harvest. Chainat 1 rice cultivar was planted in the cement plots with diameter of 1 m and height of 0.35 m. Each plot contained 250 kg of mixed soil of loamy clay and sand in a proportion of 2:1 (v/v). The spacing was 0.2x0.2 m between rows and plants with 16 hills per a cement plot. Chemical fertilizer formula 16-20-0 of N-P-K was applied at 30 days after planting at the rate of 312.5 kg.ha -1, and urea was applied at 75 days after planting at the rate of 93.8 kg.ha-1. Irrigation water was monitored daily and supplied at the controlled level of 15 mm within a cement plot. CGR was recorded at two stages; during planting to flowering (0 to 58 days) and flowering to harvest (58 days to harvest). Spikelet per m 2, 1,000 grain weight, total dry matter, harvest index (HI) and grain yield were also collected. Analysis of variance and multiple regressions were done for all traits. Results and Discussion ZnO and TiO2 nanoparticles were not significantly different for CGR during planting to flowering stage (Table 1). However, they were significantly different (P≤0.01) for CGR during flowering to harvest stage, and CGR of application TiO2 (10.73 g m-2 d-1) were significantly higher than ZnO (9.34 g m-2 d-1). Concentrations of nanoparticles were significantly different (P≤0.01) for CGR during planting to flowering stage, whereas they were not significantly different at flowering to harvest. Application at a rate of 800 mg L-1 gave the highest CGR (3.30 g m-2 d-1) followed by 100 mg L-1 (2.58 g m-2 d-1), 600 mg L-1 (2.26 g m-2 d-1), 200 mg L-1 (1.72 g m-2 d-1) and 400 mg L-1 (1.69 g m-2 d-1), respectively. However, only the rate of 800 mg L-1 was significantly higher than the others. Nanoparticles at all concentrations, when they were separately applied to the crop, were not significantly different for CGR during flowering to harvest stage. The interactions between nanoparticles and concentrations were not significant for CGR at both growth stages. ZnO and TiO2 were significantly different (P≤0.01) for total dry matter, but they were not significantly different for grain yield. TiO2 produced 136.85 g.plant-1 of average total dry matter, which was significantly higher than ZnO (124.07 g.plant-1). The concentrations of nanoparticles when considered as main effect and combination were not significantly different for total dry matter, and the interactions between type of nanoparticles and concentration were not significant for total dry matter. Types and concentrations of nanoparticles were not significantly different for grain yield, and the interaction between type and concentration was not significant for this trait. However, there was a tendency that application of TiO2 at the rate of 200-600 mg.L-1 promoted greater grain yield of Chainat 1 rice than ZnO.
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Table 1 Effect of nanoparticles on crop growth rate (CGR) at different growth stages, total dry matter and grain yield of Chainat 1 rice cultivar. CGR (g m-2 d-1) at each stage Planting to flowering
Flowering to harvest
Total dry matter (g.plant-1)
Grain yield (g.plant-1)
2.39 2.23
9.34b 10.73a
124.07b 136.85a
40.00 45.34
2.58ab 1.72b
9.18 10.18
124.40 125.55
37.78 41.64
1.69b
10.37
127.37
45.81
2.26b 3.30a
10.95 9.48
139.57 135.40
45.95 42.17
2.61 1.94
9.09 9.12
123.78 116.86
37.52 38.81
ZnO x 400 mg L-1
1.86
9.04
115.21
40.72
-1
2.05
11.03
138.06
40.77
ZnO x 800 mg L-1
3.51
8.41
126.45
42.18
-1
2.55
9.27
125.02
38.04
-1
1.50
11.24
134.24
44.48
-1
1.52
11.71
139.53
50.91
-1
2.48 3.09
10.87 10.54
141.09 144.35
51.13 42.16
A
ns
**
*
ns
B
**
ns
ns
ns
AxB
ns
ns
ns
ns
23.68
12.78
11.50
16.74
Treatments Nanoparticles (A) ZnO TiO2 Concentrations (B) 100 mg L-1 200 mg L-1 400 mg L-1 600 mg L-1 800 mg.L-1 AxB ZnO x 100 mg L-1 ZnO x 200 mg L-1 ZnO x 600 mg L
TiO2 x 100 mg L TiO2 x 200 mg L TiO2 x 400 mg L
TiO2 x 600 mg L TiO2 x 800 mg L-1 F-test
C.V. (%)
ns = non significantly different *, ** = significantly different at P ≤ 0.01 and P ≤ 0.05, respectively Means in the same column followed by the same letter are not significantly different by DMRT
CGR during flowering to harvest stage was significantly correlated with grain yield of Chainat 1 rice (R2 = 0.20*; P≤0.05) (Figure 1). Multiple regression analysis identified that CGR was important physiological trait related to grain yield of Chainat 1 rice cultivar. Similarly results were reported by Somjit and Phakamas (2012) who indicated that CGR during flowering to harvest stage was important and related to rice yield of non-photoperiod sensitive rice.
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Figure 1 Relationship between CGR during flowering to harvest stage and yield of Chainat 1 rice cultivar. Conclusion The results of this study can be concluded that there were significant effects of ZnO and TiO2 nanoparticles on CGR during flowering to harvest stage and total dry matter of Chainat 1 rice. TiO2 at a rate of 200-600 mg L-1 promoted greater growth and yield of Chainat 1 rice than utilizing of ZnO. CGR was substantially correlated to yield of Chainat 1 rice (R2 =0.20*). Nanoparticles can promote CGR especially during flowering to harvest stage (58 days to harvest), and CGR is an important physiological trait that contributed yield of Chainat 1 rice cultivar. Acknowledgments The authors gratefully acknowledge College of Nanotechnology, King Mongkut’s Institute of Technology Ladkrabang for financial support, and also thank the Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang for experimental field and field equipments supporting. References Boonyanitipong, P., B. Kositsup, P. Kumar, S. Baruah, and J. Dutta. 2011. Toxicity of ZnO and TiO2 nanoparticles on germinating rice seed Oryza sativa L. International Journal of Bioscience, Biochemistry and Bioinformatics 4: 282-285. Monica, R. C. and R. Cremonini. 2009. Nanoparticles and higher plants. Caryologia 62: 161165. Somjit, P. and N. Phakamas. 2012. Relationship between crop growth rate at different stages and yield in non-photoperiod sensitive rice variety (in Thai). King Mongkut’s Agricultural Journal 29: 51-57. Zhang, L., Hong F., Lu S., and C. Liu. 2005. Effect of nano-TiO2 on strength of naturally aged seeds and growth of Spinach. Biological Trace Element Research 105: 83-91.
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P-4
Antioxidant Determination of Nang Dam Upland Rice Bran Oil by DPPH Assay Vanapron SAE-ANG, Chitti TAWAI, Raumjit NOKKOUL and Duangkamol RUEN-NGAM1* 1
Department of Biology, Faculty of Science, King Mongkut’s Institute of Technogy Ladkrabang, Bangkok 10520, Thailand *Corresponding email: krduangk@kmitl.ac.th, modeliebe@gmail.com
ABSTRACT Upland rice is one type of Thai rice grown in area where ecosystem is different to Thai jasmine rice. The upland rice is grown on the dry land without flooding while only dew, rainfall and moisture in soil are enough to grow the upland rice. It can be grown in all parts of Thailand, especially in southern, Chumpon provinces is one of an important growing area. The upland rice normally has good smell and may be a source of nutrition such as protein and iron. Rice bran, by product from milling process, composes of many types of antioxidant such as vitamin E, -oryzanol, phenolics compounds and tocopherols. This research was carried out to evaluate the antioxidant activity of rice bran oil of the Nang Dam upland rice. Antioxidant activity was evaluated using the DPPH assay. DPPH radical inhibition percentage of the Nang Dam rice bran oil was compared to antioxidants standard such as BHT, Trolox and Ascorbic acid (Vitamin C), including with commercial rice bran oil which is available in the local market. Results found that DPPH radical inhibition of the Nang Dam rice bran oil is in the range of such upper standard. The research has been done on two range sizes; 150 and 850 m. The extraction process has been done by shaking method with circulation rate of 200 rpm in 30 minutes using ethanol as solvent in dark condition. Amount of the obtained Nang Dam rice bran oil extracted were 0.1010 and 0.0598 g/g dry weight for both range sizes of the rice bran and antioxidant activity for 50% DPPH radicals inhibition (IC50) at 4.14 mg/ml and 3.78 mg/ml for 850 and 150 m, respectively while the BHT, Trolox and Vit C IC50 were 0.1857, 0.0139 and 0.0110 mg/ml. However, IC50 of the commercial rice bran oil has less than 50% which has antioxidant inhibition around 34% at concentration of 10 mg/ml. This research has demonstrated the Nang Dam upland rice bran oil has potential to be an alternative antioxidant for consumers and entrepreneurs. Keywords: Rice bran oil, -oryzanol, % DPPH radical inhibition Introduction Rice (Oryza sativa L.) as cereal grain for staple food for a large part of the world’s human population has rich source of many bioactive compounds. Especially upland rice can grow in water restrictions, dry land without flooding. The upland rice namely Nang dam grows in Chumpron provinces (Nokkoul et al., 2010). Rice bran is a by-product obtained from the outer layer of the brown (husked) rice kernel during milling to produce white rice. It is rich in nutrients with 14%-16% protein, 12%-23% fat and 8%-10% crude fiber. Rice bran oil (RBO) offers significant potential as an alternative low-cost and low valued co-product of rice milling process. Rice bran is a well-known source of tocopherols, tocotrienols and phenolic compounds. Crude rice bran oil contains high levels of components with antioxidant properties: tocopherols/tocotrienols (up to 300 mg/kg of Vitamin E) and -oryzanol (up to 3,000 mg/kg) (Xu and Godber, 1999). Such antioxidants can reduce the risk of coronary heart disease and cancer, prevent oxidative damage of lipid and low-density lipoproteins, prevent of oxidative stress-related diseases (Willcox et al., 2004). The α-tocopherol has been found to July 1-3, 2015
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show a DPPH radical scavenging capability high up to about 7-folds of those does -oryzanol and often used in cosmetic formulations. The main issue is an efficiency of extraction of different sizes of material as follows Zhu et al., 2010’s research that DPPH decreased with increased surface area to mass and DPPH inhibition also did not increase with particle size reduction. The information of upland rice, which is referred as conventional rice in Thailand and well known of antioxidant rich sources, is scarcely and added value of waste product from rice milling process is necessary therefore the efficiency of antioxidant according to DPPH assay is done by different rice bran sizes. Materials and methods Material: Rice bran of the Nang dam upland rice from Chumpon province, Thailand and the γ-oryzanol analytical grade standard purchased from Wako pure chemical industries Ltd, Japan, were materials used in this experiment. Sample preparation and extraction upland rice bran: Rice bran sample was sieved pass through mesh screen size of 150 and 850 m. The sample was then kept in the dark at -20°C until use. A 5 g of rice bran was extracted with 95% ethanol with the ratio of rice bran and solvent of 1:5 w/v at 150 rpm for 30 min and kept away from light. The solvent was removed under vacuum by evaporator at 40°C. Determination antioxidant activity on DPPH radicals: Yield of the crude extract was determined in percentage of antioxidant inhibition on DPPH assay. DPPH radical scavenging activity assay was modified from Ruen-Ngam et al. (2014). The 0.2 mM DPPH ethanolic solution, which was prepared the concentration in range of 0.25-20 mg/ml, was added to the extracted rice bran oil with ratio of 1:1 (v/v). The well-mixed mixture was kept in dark for 30 minutes. The mixure was measured at 517 nm with Microplate reader (Labsystems: EMS Reader MF). BHT, Vit C and Trolox in range of 3.125-500µg/ml were used as references. Antioxidant inhibition (%) was derived by the following equation, Antioxidant inhibition, AI (%) = [(AB – AA) /AB] × 100 when AA absorbance value of testing solution, AB absorbance value of control solution. The concentration of the extract inhibited DPPH radicals by 50% is expressed as IC50. The lower value of IC50 indicates a higher antioxidant activity. Analysis amount of -oryzanol with HPLC: The content of -oryzanol was determined according to Ruen-Ngam et al. (2014)’s research with RP-HPLC system (Alliance 2690) with photodiode-array detector was used. The crude extracts were dissolved in the mobile phase, methanol isopropanol and ethyl acetate in ratio 47.5/40/12.5 (v/v/v), and detected at 330 nm. Statistical analysis: Analysis means of antioxidant activity on DPPH assay were reported in triplicate for both 150 and 850 m. Statistical analysis was performed using one-way analysis of variance (ANOVA). Mean comparison was carried out using Duncan’s multiple range test. The statistical analysis was performed by SPSS 22.0 at significant level of 95 % (p<0.05). Results and discussion Antioxidant activity percentage: The rice bran was sieved and had wide range size in range of 150-850 m, coarse size was polished whole grain or unmilled bran and fine bran was obtained from whitening and polishing processes. The antioxidant efficiency would be reported in two forms; % antioxidant inhibition (from now on referred as %AI) and IC50. The rice bran oil can decolorized dark blue color of the DPPH solution and the %AI trend along with rice bran oil concentration was shown in Figure 1. Remark: The suitable DPPH concentration for Nang Dam is 0.2 mM whereas generally uses 0.1 mM (Butsat et al., 2010). Comparison of %AI between two rice bran sizes; 150 and 850 µm found at the same concentration 20 mg/ml, was able to fade the colors of DPPH as %AI up to 88.69 and 89.04%, respectively, as occurring the same in range with Wanyo et al., 2010 and Butsat et al., 2010’s research that analysis 500 µm coarse Thai rice bran size (Khao Dawk Mali 105) had %AI around Page 202
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87.93%. Increasing the rice bran oil concentration can enhance exponentially antioxidant ability as equation in Figure 1. The polynomial correlation between %AI and rice bran oil concentration were found in both rice bran sizes and determined as y = 0.2157x2 + 8.9185x - 6.8967 (R2= 0.9963) and y = 0.4112x2 + 6.7542x - 2.0593 (R2= 0.9957) that was contrasted with Vorarat et al. (2010) which investigated in linear form Y = 1.4404 X + 18.485 (R2 = 0.9844). Above both equations is useful for %AI determination by DPPH assay of Nang Dam rice bran oil without doing experiment that can save the chemical expense and the time. The first hypothesis of this research is because of -oryzanol appearance in rice bran oil results to decolorize DPPH solution that might be true, however, the %IA don’t relate on the amount of -oryzanol as demonstrated the amount of -oryzanol for both rice bran sizes in the third column in Table 1. The oil from 150 µm rice bran size has amount of -oryzanol around 49.14% higherthan from 850 µm that correlated as previously discussed about the size effect. This might be that other antioxidant compound than -oryzanol was extracted. In actually the rice bran oil also has been found several types of antioxidants such as α-tocopherol, γ- tocopherol, δ-tocopherol, α-tocotrienoland anthocyanin (Butsat et al., 2010). The use of %AI by DPPH assay represents the amount of -oryzanol content in rice bran oil is not available, however, there are other better way for testing antioxidant activity of -oryzanol as in previous researches used FRAP, ABTS and ORAC assay. IC50 value: IC50values can be found out from the %AI and discovered that fine (150 m) and coarse (850 m) rice bran size were the same range, 3.64 ± 0.24 and3.89 ± 0.38 mg/ml, respectively, and no significantly different in both sizes as shown the same statistic symbol on the second column in Table 1 and contradicts with Zhu et al. (2010).The IC50 value contrasted with Vorarat et al. (2010) which got IC50 around 21.8793 mg/ml and Brewer et al. (2014) investigated the effect of three range sizes of wheat bran, fine (100-200 µm), medium (240-500 µm) and coarse (900 µm), found that total antioxidant capacity from the coarse got significant lower than the medium and fine particle (equivalent Vit C per 1 g of defatted bran were 146.20 mg, 46.38 mg of ascorbic acid, and 43.13 mg of ascorbic acid). Actually the theory of extraction, the higher surface area to mass might give higher amount of extracted compounds or get higher antioxidant activity but in this research is in contrast, however, it is necessary to be judged more about both sizes. To insist by assuming the rice bran particle has sphere shape in which one particle surface area per extracting solvent are 5.90×10-7 and2.06×10-7 m2/ml for 150 and 850 m, respectively, or the size of 150 µm has 2.86-folds higher of surface area than the size of 850 µm or otherwise when considering 1 ml solvent, the rice bran with 150 µm particle size can be extracted 112.5folds higher than the 850 µm size (Remark: 0.18×1012 particles for 150 µm size and 0.16×1010 particles for 850 µm size and bulk density of 150 μm size is 0.63 g / ml whereas 850 μm is 0.38 g / ml). Therefore, it can be concluded that the rice bran size of 150 μm is the better choice for extraction in form of higher opportunity contact of solvent results to minimize the solvent expense.
Figure 1 Antioxidant inhibition (%) in different rice bran oil concentration. Table 1 IC50 values of 150 µm and 850 µm rice bran size. Material sizes Concentrate at IC50 (mg/ml) -oryzanol content (µg/ml ) a 150 µm 3.64 ± 0.24 0.8671+0.02a 850 µm 3.89 ± 0.38 a 0.5814+0.00b Note: Different letters in the same row are statistical difference at significant level of 95%. July 1-3, 2015
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Comparison antioxidant activity in other researches The value of %AI and IC50 of well known standard and commercial rice bran oil demonstrate in Table 2. IC50 of all standards were in the same range as shown in Table 2. Nang Dam rice bran oil had more antioxidant significantly than commercial rice bran, however, significant lower than standard significance level of 95%. The antioxidant inhibition activity of rice bran oil was higher than from wheat bran oil in which got %AI around 14.58% at 14.25 mg/100 g wheat bran oil (wheat bran size is 900 m) (Brewer et. al., 2014). Comparison to other type rice, Fajr and Tarem from Iran (Araba et al., 2011), had lower %AI than Nang dam (70% at 50 mg/ml Iranian rice bran oil). Butsat et al., 2010 investigated the %AI of KDML 105 rice from different growing site found that rice bran oil from Pone-Sai district can get higher %AI than BHT (0.2 mg/ml) at around 86.7%. Such extracted rice bran oil contains -oryzanol content around 5.38 mg/g and occurring of other compounds such as -tocopherol and -tocopherol around 82.8 and 4.74 g/g, respectively. Table 2 IC50 values from different sources.
Materials IC50 (mg/ml) Vitamin C 5.99 ×10-3 a Trolox 14.47 ×10-3 a BHT 0.18 a The rice bran oil (150 µg) 3.64 b Commercial rice bran oil 10.28 c Note: Different superscript letters are statistical difference at significant level of 95%.
Conclusion Scavenging DPPH radicals of Nang Dam upland rice bran oil were no significant difference in both 150 and 850 µm sizes at significant level of 95%, however, this research demonstrated the advantage in the use of 150 µm rice bran size. This research could provide useful information for further studies on antioxidant activity by DPPH assay. Nang Dam upland rice bran oil has potential sources of antioxidant such as -oryzanol. Acknowledgments The author would like to thank research department of King Mongkut’s Institute of Technology Ladkrabang (KMITL) for financial support. References Araba, F., Alemzadehb, I. and Maghsoudi, V. 2011. Determination of antioxidant component and activity of rice bran extract. ScientiaIranica C. 18 (6): 1402 –1406. Butsat, S., Siriamornpun, S. 2010. Antioxidant capacities and phenolic compounds of the husk, bran and endosperm of Thai rice. Food Chemistry 119: 606–613. Brewer, L. R., Kubola, J., Siriamornpun S., Herald, T. J. and Shi, Y. C. 2014. Wheat bran particle size influence on phytochemical extractability and antioxidant properties. Food Chemistry 152: 483–490. Nokkoul, R., Wichitparp, T. and Hussanant, S. 2010. Selection on weed tolerance upland rice varieties. Agricultural Science 41(3/1): 57-60. Ruen-Ngam, D., Thawai, C., Raumjit, N. and Sukonthamut, S. 2014. Gamma-Oryzanol Extraction from Upland Rice Bran. International Journal of Bioscience, Biochemistry and Bioinformatics 4(4): 252-255. Vorarat1, S., Managit1, C., Iamthanakul1, L., Soparat1, W. and Kamkaen, N. 2010. Examination of antioxidant activity and development of rice bran oil and gamma-oryzanol microemulsion. Journal of Health Research 24(2): 67-72. Wanyo, P., Siriamornpuna, S. and Meeso, N. 2011. Improvement of quality and antioxidant properties of dried mulberry leaves with combined far-infrared radiation and air convection in Thai tea process. Food and Bioproducts Processing 89: 22–30 Willcox, J. K., Ash, S. L. and Catignani, G. L. 2004. Antioxidants and prevention of chronic Disease. Critical Reviews in Food Science and Nutrition 44: 275–295. Xu, Z., Hua, N. and Godber, J. S. 1999. Antioxidant activity of tocopherols, tocotrienols and gamma -oryzanol components from rice bran against cholesterol oxidation accelerated by 2,20 azobis(2-methylpropionamidine) dihydrochloride. Journal of Agricultural and Food Chemistry 49: 2077–2081. Zhu, K. X., Huang, S., Peng, W., Qian, H. F. and Zhou, H. M. 2010. Effect of ultrafinegrinding on hydration and antioxidant properties of wheat bran dietary fiber, pp. 943–948. Food Research International, 43(4). Page 204
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P-5
Effect of Different Media and Concentrations of Growth Regulator on Callus Induction and Growing Suspension Cell Culture of San-pah-tawng 1 (Oryza sativa L.) Ranyikar PORAHA1, Anurug POEAIM1*, Saengthong PONGJAROENKIT2 and Pradit PONGTHONGKAM3 1
Department of Biology, Faculty of Science, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, 10520, Thailand 2 Department of Genetics, Faculty of Science, Maejo University, Chiang Mai, 50290, Thailand 3 Thepstri Rajabhat University, 321 Naraimaharat Road Tambon Talaychubsorn Amphur Muang, Lopburi, 15000, Thailand *Corresponding email: kpanurug@kmitl.ac.th
ABSTRACT The aim of this study was to find out enhancement on callus induction and growing suspension cell culture in San-pah-tawng 1 rice. The mature seeds of rice were cultured on solid MS (Murashige and Skoog, 1962) and NB (Nitsch and Nitsch, 1969) media containing with 30 g/l sucrose combined with 1 g/l L-proline, 100 mg/l casein hydrolysate, 0.5, 1, 2, 3 and 5 mg/l 2,4-D (2,4-Dichlorophenoxyacetic acid) and 2.6 g/l phytagel, Cultured in the dark condition for 4 weeks. NB medium was found better than MS medium for callus induction. The suitable media for callus induction of San-pah-tawng 1 were NB medium supplemented with 2 mg/l 2,4-D, found to be effective for callus induction which induced the biggest size and the maximum weight of calli. After that, transferred the calli to liquid NB medium containing 1 mg/l 2,4-D, 30 g/l sucrose, 1 g/l proline and 100 mg/l casein hydrolysate. Study the growth phases at 0, 3, 6, 9, 12, 15, 18 and 21 days, respectively, were determined by measuring fresh and dry weight of the cell. Keywords: San-pah-tawng 1, Callus induction, Cell suspension culture, Growth curve, 2, 4Dichlorophenoxyacetic acid Introduction Rice (Oryza sativa L.) belongs to grass family Poaceae and it is one of the most important crops and major staple food in the worldwide. Moreover, in the world 80% rice production are based on indica (Wanichananan et al., 2010), including San-pah-tawng 1 which is glutinous rice. The main characters of San-pah-tawng 1 is resistant to both blast and bacterial blight, high yield and growing all season. Nowadays, world population is increasing rapidly and to meet its demands rice production needs to be increased (Tariq et al., 2008; Bashir et al., 2010). Therefore, plant tissue culture has also become an important tool for breeding improvement in rice (Ge et al., 2006). A basic protocol is required for the callus induction and cell suspension culture for the studies related to In vitro culture of plants. Objective of the present study was to determine the most suitable basal medium, concentration of growth regulators (2,4-D) for improvement in calli induction and growth curve of cell suspension culture of San-pah-tawng 1. Materials and Methods Mature seeds of San-pah-tawng 1 rice were dehusked and sterilized by 70% ethanol for 1 min. Followed by 30 minutes shaking in 20% Sodium hypochloride and finally rinsing three times in sterile distilled water. Seeds were then kept on a sterile filter paper in a sterile PetriJuly 1-3, 2015
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dish to soak water from the seeds. After that, culture seeds on MS and NB media supplemented with 30 g/l sucrose, 1 g/l L-proline, 100 mg/l casein hydrolysate and 2.6 g/l phytagel, and different concentrations 0.5, 1, 2, 3 and 5 mg/l of 2,4-D. Cultured seeds maintained in the dark condition at 25±1 ºC. After 4 weeks, the frequency of callus induction (%), mean size and mean weight of callus formation were calculated according to the following formula: Callus induction frequency (%)
=
Mean size of callus formation (mm3) = Mean weight of callus formation (g)
=
Number of calli Number of incubated seeds
x100
Ʃ(width x length x height)of calli Number of seeds that produced calli Ʃ(weight of calli) Number of seeds that produced calli
Suspension cultures were initiated by inoculating 0.15 g of callus formed from in the best induction into 10 ml liquid medium at the same compositions as medium that induced the largest size of callus induction. The cultures were incubated on rotary shaker at 120 rpm and maintained at 25±1 ºC. Study growth phases at 0, 3, 6, 9, 12, 15, 18 and 21 days, respectively. The cells were separated from the culture medium by filtration through filter paper (Whatman no. 1) to evaluate fresh weight. Dry weight was determined after drying the cells at 110°C for 1 h, when a constant weight was obtained. Results and Discussion MS and NB media were used for callus induction in mature seeds. Different concentrations (0.5, 1, 2, 3 and 5 mg/l) of 2,4-D were used to induce callus in the mature seeds. The characteristic of callus was yellowish or bright brown, compact and nodular (Figure 1). The response of the seeds to different concentrations of 2,4-D in terms of callus induction and percentage of seeds producing calli were calculated as shown in Table 1. Table 1 The frequency of callus induction, mean size and mean weight of callus formation of San-pah-tawng 1 rice on MS and NB media containing different concentrations of 2,4-D Mean size of Mean weight of Concentration Frequency of callus formation Media of 2,4-D callus induction callus formation (mm3) (g) (mg/l) (%) b b 0.5 28.33 28.3528 0.0285c 1 61.67a 57.5049a 0.0768a a a MS 2 65.00 57.8359 0.0557ab 3 46.67a 47.0961ab 0.0447bc a ab 5 55.00 48.3055 0.0555ab 0.5 0.00d 0.0000c 0.0000b c ab 1 60.00 76.1272 0.0870a NB 2 100.00a 94.1915a 0.1175a 3 91.67a 86.8563ab 0.1158a b b 5 80.00 63.9036 0.0890a The values followed by different letters indicating significant differences according to Duncan’s Multiple Range Test (p≤0.05)
The overall frequency (%) of callus induction on NB medium was found better than that on MS medium. The difference in the composition of culture media can result in variation in Page 206
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callus induction (Torbet et al., 1998). The callus induction frequency of San-pah-tawng 1 was maximum 100% when cultured on NB medium. The calli were also compared for their mean size and mean weight on MS and NB media containing different concentrations of 2,4-D as shown in Table 1. The mean sizes and mean weight of callus on NB medium supplemented with 2 mg/l 2,4-D gave the biggest mean size of callus 94.19 mm3 and its gave the highest mean weight of callus 0.1175 g. Result of the present study were in agreement with those of Poeaim et al., (2001) who reported cultured embryo of Suphanburi 60 rice cultivar on solid NB medium containing 2 mg/l 2,4-D, 1 g/l L-proline, 100 mg/l casein hydrolysate, 20 g/l sucrose and 2.6 g/l phytagel showed the highest frequency of embryogenic callus formation. Many research stressed the role of 2,4-D in rice tissue culture. Thus Pandey et al., (1994) worked on dehusked rice seeds, using different level of 2,4-D in nutrient medium and they concluded that 2,4-D at the concentration of 2 mg/l gave the best response for callus formation.
Figure 1 Callus induction of San-pah-tawng 1 rice cultivar with NB medium (A-E) and MS medium (F-J) with different concentration of 2,4-D; (A,F) 0.5 mg/l, (B,G) 1 mg/l, (C,H) 2 mg/l, (D,I) 3 mg/l and (E,J) 5 mg/l at 4 weeks.
Figure 2 Growth rate of fresh and dry weight of cell suspension culture for San-pah- tawng 1 rice in liquid NB medium contained with 2 mg/l 2,4-D for 21 days. Growth of callus on NB liquid medium supplemented with 2 mg/l 2,4-D showed on 15th days the maximum of fresh weight and dry weight were 0.6227 g/10 ml and 0.0933 g/10 ml of medium, like a report of Poraha et al., (2014) found the maximum of fresh and dry weight of Jow Haw rice in NB medium at 15th days. Initially of San-pah-tawng 1 rice there was a lag July 1-3, 2015
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phase (0 to 3 d), followed by a log phase (3 to 15 d), stationary phase (15 to 18 d) and finally a death phase (after 18 d) (Figure 2). Callus taken from the late log phase, transfer to fresh medium for increase callus or bring callus used for conducting various tests and other experiments. Conclusion From this experiment, it could be concluded that the suitable for callus induction of San-pahtawng 1 was NB medium supplemented with 2 mg/l 2, 4-D. The maximum of fresh weight and dry weight of cell suspension culture was found on 15th days. The system of callus induction and cell suspension culture were preliminarily established and can be used for plant breeding purposes to produce high yield and quality of San-pah-tawng 1 or new variety rice in the future. Acknowledgements This research paper was supported by the Office of the Higher Education Commission Thailand and Department of Biology, Faculty of Science, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, Thailand. References Bashir, M.U., N. Akbar, A. Iqbal and H. Zaman. 2010. Effect of different sowing dates on yield and yield components of direct seeded coarse rice (Oryza sativa L). Pak. J. Agri. Sci. 47: 361-365. Ge, X., Z. Chu, Y. Lin and S. Wang. 2006. A tissue culture system for different germplasms of indica rice. Plant Cell Rep. 25:392-402. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum15:392-402. Nitch, J.P. and C. Nitch. 1969. Haploid plants from pollen grains. Science 163: 85-87. Pandey, S. K., B. Ramesh and P. K. Gupta. 1994. Study on the effect of genotype and culture medium on callus formation and plant regeneration in rice (Oryza sativa L.). Indian J. Genet. Plant Breed. 54: 293-299. Poeaim, A. and N. Saengduan. 2001. Plant regeneration from suspension culture of Suphanburi 60 rice (Oryza sativa L.). Proceedings of the 39th Kasetsart University Annual Conference: Science, Natural Resources and Environmental Economics 174180. Poraha, R., A. Poeaim., S. Pongjaroenkit and P. Pongthongkam. 2014. Callus induction and growing cell suspension culture of Jow Haw rice (Oryza sativa L.). Proceedings of the 3rd ICIST, Pakse, Laos, PDR. November 27-28:78-84. Tariq, M., G. Ali, F. Hadi, S. Ahmad, N. Ali and A.A. Shah. 2008. Callus induction and In vitro plant regeneration of rice (Oryza sativa L.) under various conditions. Pak. J. Biol. Sci., 11(2): 255-259. Torbet, K.A., H.W. Rinse and D.A. Somers. 1998. Transformation of oat using mature embryo-derived tissue cultures. Crop Sci., 38: 226-231. Wanichananan, P., T. Teerakathiti and S. Roytrakul. 2010. A highly efficient method for Agrobacterium mediated transformation in elite rice varieties Oryza sativa L. spp. indica. Afr. J. Biotechnol. 9: 5488-5495.
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P-6
Elite Thein Corn Inbred Lines Utilized to be Synthetic Variety Kitti BOONLERTNIRUN1*, Suchada BOONLERTNIRUN1 and Choosak JOMPUK2 1
Faculty of Agricultural Technology and Agro-industry, Rajamangala University of Technology Suvarnabhumi, Phranakhon Si Ayutthaya, Thailand 13000 2 Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand
*Corresponding email: kitti.b@rmutsb.ac.th ABSTRACT
Thein corn synthetic variety is alternative technology for farmers who need to improve their Thein corn production to be high yield and quality. Good synthetic variety was actually produced from good general combining ability (GCA) of inbred parents. In this study, inbred lines were extracted by standard method from three open pollinated Thein corn populations and 4 hybrid populations were also extracted. Such S 4 inbred lines were selected to be 101 lines and then crossed with 3 testers, TBK-17-1-1-1 (T1), TKKU1-7-2-1-1 (T2), and TSW23-2-1-1-1 (T3), to produce 259 top crosses. Yield trials were done using augmented design. Line x tester was analyzed and GCA in terms of yield was also evaluated and found that top cross line of tester named T1, T2 and T3 had averaged ear numbers 95,188, 100,950 and 102,388 ears ha-1, respectively. Dehusked yield of such lines was 7.63, 7.25 and 7.75 ton ha -1, respectively. Ten selected prolific S4 inbred lines were L3, L5, L22, L23, L28, L29, L47, L51, L59 and L65. The ear numbers of such selected lines ranged 112,225 to 124,444 ears ha -1 and GCA value in terms of ear numbers were between 12,494 to 24,719 ears ha-1. Keywords: Thien corn, Synthetic variety, Inbred line Introduction Thein corn is one of local waxy corn. It is popular to consume as fresh corn. Currently, marketing demand is continuously increasing. Thein corn production technology is various depending on farmer experience in each location. Synthetic variety having high yield and quality is an optimized technology for transferring to the local farmers. Farmers can use 2 to 3 generation seed multiplied by themselves. Broad genetic base of synthetic variety can adapt to various environments, especially low input environment. Kutka and Smith (2007) reported that synthetic variety was comprised of 5-12 inbred lines having high combining ability. Line cross tester was an efficiency method for inbred line selection to high combining ability. (Hallaure et al., 1988). Specific combining ability between inbred lines and testers can separate inbred lines into heterotic groups. (Fan et al., 2009). The inbred lines showed high performance in both line per se and line cross tester, indicating that frequency of additive gene was more important than dominant gene effect in traits (Bekavac et al., 2008). Thien corn had small ear. Standard ear size determined by Department of Agriculture was 10-15 cm of dehusked-ear length, 2.5-3.5 cm of ear diameter and 8-12 seed rows. Prolificacy is an important trait for line selection to improve yield. (Ketthaisong et al., 2011). This research aimed to select prolific inbred lines and then separated into different heterotic groups for developing 2 new synthetic varieties. Materials and Method Plant materials S4 Thein corn inbred lines were extracted by standard method from three open pollinated July 1-3, 2015
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populations namely TKKU1, TBK and TSW and 4 hybrid populations namely THT/Gi2, THT/CS, TSK/E1 and INS/TBK//INS were also extracted. Agronomic traits of S4 inbred lines were record and 101 inbred lines were selected and then crossed with 3 testers, name TBK17-1-1-1 (T1), TKKU1-7-2-1-1 (T2), and TSW-23-2-1-1-1 (T3), to produce 259 top cross lines. Experimental design Two hundred and fifty-nine top cross lines were randomly arranged to be 10 sets in augmented design. Twenty-six inbred lines in each set were planted with 4 check varieties (Thein ban Koh (TBK), Thein Sawan (TSW), Thein Numwang (TNW) and Thein INS/TBK Comp#1 (TIS). Twenty-five corn plants (one experimental unit) were grown with one row of 5 m long and 0.20 m x0.75 m of plant spacing. The experiment was conducted at field plot of Plant Science Section, Faculty of Agricultural Technology and Agro-industry, Rajamangala University of Technology Suvarnabhumi, Phranakhon Si Ayutthaya, Thailand, in rainy season (August-September, 2014). Data analysis Analysis of variance for augmented design and least significant difference (LSD) for mean comparison were performed by R Stat software and box plot was also plotted. Combining ability parameters were evaluated by line cross tester analysis method (Singh and Chaudhary, 1977). Results and Discussion Top cross lines of tester name T1, T2 and T3 had averaged dehusked yield of 7.63, 7.25 and 7.75 ton.ha-1 respectively (Figure 1) which was significantly (p<0.05) higher than check varieties; TBK, TSW, TNW and TIS which gave 5.58, 4.55, 6.14 and 6.11 ton ha-1 respectively (data not shown). Average ear numbers of these top cross lines were 95,188, 100,950 and 102,388 ears ha-1, respectively (Figure 1), which was higher than check varieties; TBK, TSW and TNW which gave 84,000, 70,330 and 84,000 ears ha-1, respectively, but not significantly different from TIS which gave 101,670 ears ha-1 (data not shown). Top cross lines of T2 and T3 tester showed more ear number distribution than T1 tester (Figure 1), it was used to clearly classify combining ability of inbred line. ton.ha-1
104 ears ha-1
T1
T2
T3 (a)
T1
T2
T3 (b)
Figure 1 Boxplot for ear number (a) and unhusked yield (b) of 3 groups of top cross line. The correlation coefficient between ear number and yield of T1, T2, and T3 top cross line groups was 0.56, 0.51 and 0.74, respectively (Table 1), and showed highly significant Page 210
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(p<0.01). It indicated that lines were prolific resulted in getting high yield. Prolificacy is an important trait for line selection to improve yield (Ketthaisong et al., 2011). The correlation coefficient between ear number and ear width showed no significance (p>0.05) in all groups of top cross line (Table 1). Ear width was positively correlated with yield; direct selection for increasing yield also resulted in bigger ear size. Prolificacy traits and ear size were suitable criteria to improve high yield and quality in Thein corn breeding. Table 1 Correlation between ear numbers (EN), unhusked yield (UHY), ear width (EW) and ear length (EL) of 3 groups of top cross lines. T1 top cross lines
T3 top cross lines
Traits
UHY
EW
EL
UHY
EW
EL
UHY
EW
EL
EN
0.56** 1
-0.22
-0.18
0.51**
-0.05
-0.18
0.74**
0.13
0.07
0.37*
0.08
0.46**
0.15
UHY EW 1
T2 top cross lines
-0.1
0.51** 0.49**
-0.14
0.26
*,** = significant at the probability 0.05 and 0.01, respectively.
Ten elite inbred lines namely L65, L22, L3, L29, L51, L47, L5, L28, L23, and L59 showed highly significant positive GCA of 24,900, 19,300, 17,100, 17,100, 16,000, 16,000, 13,800, 13,800, 12,600 and 12,600 ears.ha-1, respectively (Figure 2). It could be explained that elite inbred lines had high combining ability for prolificacy. Murray et al. (2003) reported that the variation in GCA components was the results of both additive and dominant genetic effects. Large GCA effect of the parents was mainly due to additive and additive x additive gene actions (Griffing, 1956). Elite inbred lines showed different heterotic groups. The first group comprised L22, L23, L28, L29, and L65 showed high SCA with T2 tester. The second group comprised L3, L5, L47, L51, and L59 showed high SCA with T3 tester (Figure 2). Synthetic variety comprised 10 selected inbred lines having high GCA and SCA in different heterotic groups should have great proportion of favorable alleles and be diverse in allele frequency effect to heterosis. The expression of heterosis depended on the difference in allele frequency of the parents and dominant effect at various loci (Falconer and Mackay, 1996).
4
-1
Grand mean = 9.94 x 10 ears.ha 4
-1
4
-1
LSD0.05 = 1.30 x 10 ears.ha
LSD0.01 = 1.76 x 10 ears.ha
Figure 2
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SCA and GCA for ear number trait (104 ears ha-1) of 10 elite inbred lines.
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Conclusion Combining ability of inbred line evaluated by Line cross tester achieved 10 inbred lines with high general combining ability. These were classified to be 2 groups as high SCA with T2 tester and high SCA with T3 tester. They were taken to recombine within group to produce 2 synthetic varieties for two separated population improvement through reciprocal recurrent selection method. Acknowledgments This study was granted research budget by National Research Council of Thailand (NRCT). The authors would like to thank NRCT and Rajamangala University of Technology Suvarnabhumi, Thailand for providing facilities in this research. References Bekavac G., B. Purar and D. Jockovic. 2008. Relationships between line per se and testcross performance for agronomic traits in two broad-based populations of maize Euphytica 162(3): 363-369. Fan, X.M., Y.M. Zhang, W.H. Yao, H.M. Chen, J. Tan, C.X. Xu, X.L. Han, L.M. Luo and M.S. Kang. 2009. Classifying maize inbred lines into heterotic groups using a factorial mating design. Agron. J. 101:106-112. Hallauer, A.R. and J.B. Miranda Fo. 1988. Quantitative genetics in maize breeding. 2nd ed, Iowa State Univ. Press. Amers. Ketthaisong, D., Suriharn, B. and K. Lertrat. 2011. Selection for Prolificacy in â&#x20AC;&#x2DC;Khon Kaen Compositeâ&#x20AC;&#x2122; Small Ear Waxy Corn Population. Agricultural Sci. J. 42 3/1 (Suppl): 207210. Kutka, F.J. and M.E. Smith. 2007. How many parents give the highest yield in predicted synthetic and composite populations of maize? Crop Sci. 47:1905-1913. Singh, R. K., and B. D. Chaudhary. 1977. Biometrical Methods in Quantitative Genetic Analysis. Kalyani publishers, New Delhi. Falconer, D.S. and T.F.C. Mackay. 1996. Introduction to quantitative genetics. 4thed. Longman, London, UK. Griffing, B. 1956. Concept of general and specific combining ability in relation to diallel crossing systems. Aust. J. Biol. Sci. 9: 463-493. Murray, L.W., I.M. Ray, H. Dong, and A. Segovia-Lerma. 2003. Clarification and reevaluation of population based diallel analyses: Gardner and Eberhart analyses II and III revisited. Crop Sci. 43: 1930-1937.
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P-7
Hypoxic Responses of 6 Commercial Waxy Corn Varieties Suchada BOONLERTNIRUN1* Raweewun SUVANNASARA1 and Kitti BOONLERTNIRUN 1 1
Faculty of Agricultural Technology and Agro-industry, Rajamangala University of Technology Suvarnabhumi, Phranakhon Si Ayutthaya province, Thailand 13000 *Corresponding email: suchada.b@rmutsb.ac.th
ABSTRACT Hypoxic soil condition is defined as oxygen deficiency in soil which negatively affects growth and yield potential of many crop species. The objective was to evaluate hypoxic tolerance at V7 growth stage of 6 commercial waxy corn varieties. Plot experiment was performed using a split plot in Randomized complete block (RCBD) with 4 replications. Hypoxic and normal conditions were used as main plot whereas waxy corn varieties named Big white 852, Sweet white 25, Violet white 926, Neaw simoung 111, Tender 58 and Neaw wan 4985 were used as subplot. The results indicated that there was a significant difference between normal and hypoxic conditions on leaf greenness, plant height, leaf area, dry weight and relative growth rate of all waxy corn varieties, negative effect was remarkably observed under hypoxic condition. Considering variety effect, it indicated that each waxy corn variety differently responded to hypoxia. Big white 852 and Sweet white 25 varieties showed high waterlogging index on dry weight, leaf greenness, plant height and leaf area, this may be explained that these two varieties tended to well adapt under hypoxic condition. Keywords: Hypoxic condition, Waxy corn, Waterlogging index Introduction Waxy corn, local economic crop in Thailand, is popular to consume as green corn because of being gentle and lightly sweet. Furthermore, short harvesting time of 60-65 days is suitable to be cash crop after rice. However, one problem in waxy corn growing after rice is heavy soil texture (clay soil) which exhibits poor drainage resulted in occurring waterlogged condition after heavy rain. Under this condition, the root capacity to supply water and nutrients into plant shoot is interrupted (De Simone et al., 2002). The endogenous levels of nutrient are reduced in different parts of plants (Ashraf et al., 2011). Waterlogging stress is also known to affect morphological and anatomical changes in plants (Ashraf, 2012), therefore, it seriously affects growth and yield potential of many crop species. Besides waterlogging also influences biochemical processes in plants, i.e. photosynthesis and respiration, relating to growth and yield potential (Setter et al., 2003). Waxy corn waterlogged at V7 growth stage for 12 days shows the most negative effect on growth and yield. (Boonlertnirun et al., 2013). The response to waterlogging is varied among varieties, growth stage of diverse crop species such as maize (Zaidi et al., 2004) cotton (Kuai et al., 2014) and wheat (Li et al., 2011). This present study aimed to screen hypoxic tolerance at V7 growth stage of 6 commercial waxy corn varieties. Materials and method A split plot in RCBD with 2 main plots and 6 sub plots was performed and replicated 4 times. Main plot was normal and hypoxic conditions and subplot was 6 waxy corn varieties namely Big white 852, Sweet white 25, Violet white 926, Neaw simoung 111, Tender 58 and Neaw wan 4985. Each waxy corn variety was separately planted in 60 cm-diameter cement tank. One cement tank (one experimental unit) contained 4 plants. All required cultural practices were performed as recommendation. When corn age was 28 days (V7 growth stage) after planting, Hypoxic condition was imposed by flooding into the cement
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tank for 12 days. After hypoxic period (12 days) was completely finished, leaf greenness plant height, dry weight and leaf area were recorded. Relative growth rate, waterlogged index and differential values were also recorded as the following equations:
Waterlogging index = (X2/X1)/(Y2/Y1) X1= characters of each genotype measured in N, X2= characters of each genotype measured in H Y1= character average of all genotypes measured in N, Y2= character average of all genotypes measured in H Differential value (D) = (X1-X2)/X1 X1 = character measured in N, X2 = character measured in H Relative growth rate = lnW2- lnW1 (T2 - T1) W1 = dry matter at the beginning period (before subjecting to waterlogging) W2 = dry matter at the end period (after subjecting to waterlogging) T1 = timing at the beginning period, T2 = timing at the end period
All collected data were subjected to analysis of variance according to experimental design used and LSD (Least significant difference) was utilized to compare treatment means. Results and discussion Leaf greenness: Leaf greenness of all varieties was significantly decreased under hypoxic condition. Sweet white 25 and Big white 852 showed darker green than the other varieties, consequently low differential value was found whereas Tender 58 was the most sensitive under hypoxic condition. Kuai et al. (2014) reported that total chlorophyll in the leaves subtending the cotton boll was remarkably reduced under water logging stress. Waterlogging occurring at any growth stage usually caused degradation of chlorophyll in leaves (Li et al., 2011). Table1 Hypoxic responses of 6 commercial waxy corn varieties on leaf greenness (spad unit). Corn variety (M)
Condition (S)
Differential value (D)
Normal condition (N) Hypoxic condition (H) Big white 852 45.47 25.71 0.43 Sweet white 25 46.48 26.57 0.43 Violet white 926 42.17 17.43 0.59 Neaw si moung 111 41.33 18.25 0.56 Tender 58 46.69 18.85 0.6 Neaw Wan xc 4985 45.92 18.90 0.59 Condition mean 44.68 A 20.95 B LSD 0.5 (M) * LSD 0.5 (S) * LSD 0.5 (M) * (S) ns CV (%) 12.62 Means followed by the different lower case letter in the same column significance at P <0.05 \ Means followed by the different letter in the same row significance at P <0.05
Corn variety mean 35.59 a 36.53 a 29.80 b 29.79 b 32.77 ab 32.41 ab
Plant height: Under hypoxic condition, Sweet white 25 height remarkably declined over than the other ones, however its variety mean was still good as Big white 852 and Tender 58. Height of Neaw wan xc 4985 rather tolerated to hypoxia over than the others due to showing lower differential value. Changdee et al. (2009) found that height of cotton under waterlogging for was reduced by 38 % when compared to the control. Leaf area: Leaf areas of all corn varieties were strongly inhibited under hypoxic condition. Sweet white 25 variety showed low differential value, so it could maintain leaf area the best under hypoxia. Neaw wan xc 4985 was the most sensitivity due to being shown the highest differential value. Grassini et al. (2007) found that waterlogging during grain filling period had negative effect on leaf areas of sunflower (Helianthus annuus L.) and pigeon pea (Cajanus cajan L.) (Kumutha et. al., 2008). Relative growth rate (RGR): RGR of all corn varieties under hypoxic condition was significantly low when compared to that under normal condition. RGR of Sweet white 25 was the highest and its differential value was also the lowest, it indicated that this variety was well adaptive. RGR of Neaw simoung 111 was dramatically reduced under hypoxic condition, so this one was classified as the poorest tolerance. Ye et. al. (2003) reported that relative growth rate of Bruguiera gymnorrhiza decreased significantly under 12 weeks of waterlogging. Dry weight: Significantly different dry weight of all varieties was found between normal and hypoxic Page 214
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conditions. Dry weight of all varieties was higher under normal condition. Big white 852 having low differential value could retain dry weight better than the others whereas Neaw si moung 111 poorly accumulated dry weight under hypoxia. Waterlogging significantly decreased dry matter of maize (Zea mays L.) and pigeon pea (Cajanus cajan L.) (Yong-zhonget. al., 2010; Kumutha et. al., 2008). Table 2 Hypoxic responses of 6 commercial waxy corn varieties on plant height (cm). Corn variety (M)
Condition (S) Differential value (D) Normal condition (N) Hypoxic condition (H) Big white 852 128.40 49.00 0.62 Sweet white 25 127.80 46.25 0.64 Violet white 926 98.06 40.16 0.59 Neaw si moung 111 101.83 37.83 0.63 Tender 58 129.06 52.41 0.59 Neaw Wan xc 4985 107.73 47.00 0.56 Condition mean 115.48A 45.44B LSD 0.5 (M) * LSD 0.5 (S) * LSD 0.5 (M) * (S) ns CV (%) 10.98 Means followed by the different lower case letter in the same column significance at P <0.05 Means followed by the different letter in the same row significance at P <0.05
Corn variety mean 88.70a 87.20ab 69.11c 69.83c 90.74a 77.36bc
Table 3
Hypoxic responses of 6 commercial waxy corn varieties on leaf area (cm2/plant).
Table 4
Hypoxic responses of 6 commercial waxy corn varieties on relative growth rate (g/day/)
Corn variety (M)
Condition (S) Differential value (D) Normal condition (N) Hypoxic condition (H) Big white 852 235.56 140.62 0.4 Sweet white 25 249.95 157.21 0.37 Violet white 926 255.11 137.50 0.46 Neaw si moung 111 273.30 133.83 0.51 Tender 58 199.71 100.00 0.5 Neaw Wan xc 4985 225.65 100.00 0.56 Condition mean 239.88 A 128.19 B LSD 0.5 (M) * LSD 0.5 (S) * LSD 0.5 (M) * (S) ns CV (%) 12.08 Means followed by the different lower case letter in the same column significance at P <0.05 Means followed by the different letter in the same row significance at P <0.05
Corn variety mean 188.09ab 203.58a 196.30a 203.56a 149.85c 162.82bc
Corn variety (M)
Condition (S) Differential value (D) Corn variety mean Normalcondition (N) Hypoxic condition (H) Big white 852 0.334 0.260 0.22 0.297 Sweet white 25 0.361 0.290 0.2 0.325 Violet white 926 0.359 0.267 0.26 0.313 Neaw si moung 111 0.355 0.209 0.41 0.282 Tender 58 0.353 0.237 0.33 0.295 Neaw Wan xc 4985 0.336 0.220 0.35 0.278 Condition mean 0.350A 0.247B LSD 0.5 (M) * LSD 0.5 (S) ns LSD 0.5 (M) * (S) ns CV(%) 15.98 Means followed by the different letter in the same row significance at P <0.05
Waterlogging index: Each character of each variety differently responded to hypoxia. Generally, Sweet white 25 showed the highest waterlogging index in several characters, i.e. leaf greenness, relative growth rate and leaf area whereas the highest waterlogging index in terms of plant height and dry weight was achieved from Neaw Wan xc 4985 and Big white 852 varieties respectively. It could be explained that Sweet white 25 variety tended to tolerate to hypoxia greater than the other varieties. On the other hand, the most sensitive variety was observed in Neaw si moung 111. July 1-3, 2015
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Table 5
Hypoxic responses of 6 commercial waxy corn varieties on dry weight (g/plant).
Corn variety (M)
Condition (S) Differential (D) Normal condition Hypoxic condition value Big white 852 44.50 35.76 0.2 Sweet white 25 49.24 36.24 0.26 Violet white 926 48.00 35.14 0.27 Neaw si moung 111 49.86 33.24 0.33 Tender 58 47.61 34.12 0.28 Neaw Wan xc 4985 42.00 32.51 0.22 Condition mean 47.02A 34.50 B LSD 0.5 (M) * LSD 0.5 (S) ns LSD 0.5 (M) * (S) ns CV(%) 14.07 Means followed by the different letter in the same row significance at P <0.05
Table 6
Corn variety mean 40.18 42.74 41.97 41.55 40.86 37.26
Waterlogging index of plant height, dry weight, leaf greenness, relative growth rate and leaf area of 6 commercial waxy corn varieties.
Corn variety Big white 852 Sweet white 25 Violet white 926 Neaw si moung 111 Tender 58 Neaw Wan xc 4985
Waterlogging index Plant height
Dry weight
Leaf greenness
Relative growth rate
Leaf area
0.97 0.92 1.04 0.94 1.03 1.11
1.09 1 0.99 0.9 0.97 1.05
1.38 1.39 1.01 1.07 0.98 1
1.1 1.14 1.05 0.83 0.95 0.93
1.12 1.18 1.01 0.92 0.94 0.83
Conclusion From this study, Sweet white 25 varieties tended to well tolerate to hypoxia at V7 growth stage because of high waterlogging index value in many characters. Therefore, it should be classified as tolerant variety whereas the sensitive variety was observed in Neaw si moung 111 due to low waterlogging index value in many characters. Acknowledgement Special thank and appreciation were extended to Rajamangala Univesity of Technology Suvarnnabhumi, for being granted research budget for this study. References
Ashraf. M. A., M.S.A. Ahmad, M. Ashraf, F. Al-Qurainy and M.Y. Ashraf. 2011. Alleviation of waterlogging stress in upland cotton (Gossypium hirsutum L.) by exogenous application of potassium in soil and as a foliar spray. Crop Pasture Science 62: 25-38. Ashraf. M. A. 2012. Waterlogging stress in plants: A review. African Journal of Agricultural Research 7(13): 1976-1981. Boonlertnirun. S., R. Suvannasara and K. Boonlertnirun 2013. Effects of Hypoxic Duration at Different Growth Stages on Yield Potential of Waxy Corn (Zea mays L.) International Journal of Agricultural, Biosystems Science and Engineering 7:651-653. Changdee. T., A. Polthanee, C. Akkasaeng and S. Morita. 2009. Effect of different waterlogging regimes on growth, some yield and roots development parameters in three fiber crops (Hibiscus cannabinus L., Hibiscus sabdarifa L. and Corchorusolitorius L.). Asian Journal of Plant Sciences 8: 515-525. De Simone. O., K. Haase, E. Müller, W.J. Junk, G. Gonsior and W. Schmidt. 2002. Impact of root morphology on metabolism and oxygen distribution in roots and rhizosphere from two Central Amazon floodplain tree species. Functional Plant Biology 29: 1025-1035. Grassini. P., G. V. Indaco , M. L. Pereira, A.J. Hall and N.Trapani. 2007. Responses to short-term waterlogging during grain filling in sunflower. Field Crops Research 101: 352–363. Kumutha. D., R.K. Sairam, K. Ezhilmathi , V. Chinnusamy and R.C. Meena. 2008. Effect of waterlogging on carbohydrate metabolism in pigeon pea (Cajanus cajan L.): Upregulation of sucrose synthase and alcohol dehydrogenase. Plant Science 175: 706–716. Kuai. J., Z. Liua, Y. Wanga, Y. Menga, B. Chena, W. Zhaoa, Z. Zhoua and D. M. Oosterhuisb. 2014. Waterlogging during flowering and boll forming stages affects sucrose metabolism in the leaves subtending the cotton boll and its relationship with boll weight. Plant Science 223: 79–98. Li. C., D. Jiang, B. Wollenweber, Y. Li , T. Dai and W. Cao. 2011. Waterlogging pretreatment during vegetative growth improves tolerance to waterlogging after anthesis in wheat. Plant Science 180: 672–678 Setter, T. L. and I. Waters. 2003. Review of prospects for germplasm improvement for waterlogging tolerance in wheat, barley and oats. Plant Soil 253: 1-34. Ye.Y., N. F.Y. Tam, Y.S. Wong and C.Y. Lu. 2003. Growth and physiological responses of two mangrove species (Bruguiera gymnorrhiza and Kandelia candel) to waterlogging. Environmental and Experimental Botany 49:209-221. Yong-zhong. L., T. Bin, Z. Yong-lian, M. Ke-jun, X. Shang-zhong and Q. Fa-zhan. 2010. Screening Methods for Waterlogging Tolerance at Maize (Zea mays L.) Seedling Stage. Agricultural Sciences in China 9 (3): 362-369. Zaidi. P. H., S. Rafiquea, P.K. Raia, N.N. Singha and G. Srinivasan. 2004. Tolerance to excess moisture in maize (Zea mays L.): susceptible crop stages and identification of tolerant genotypes. Field Crops Research 90: 189–202.
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P-8
Development of Chitosan-Based Silver Nanoparticles Coating and Study of Its Effect on Litchi Stored at Ambient Temperature Warin PIMPA1* and Chakkrit PIMPA2 1
Department of Agro-Industry, Faculty of Agriculture, Natural Resource and Environment, Naresuan University, Phitsanulok 65000, Thailand 2 Northern Region Operation Division, Electricity Generating Authority of Thailand, Phitsanulok 65000, Thailand *Corresponding e-mail: warin@nu.ac.th
ABSTRACT The fabrication of silver nanoparticles was accomplished by reducing silver nitrate with nontoxic and biodegradable chitosan. The chitosan-based silver nanoparticles (CS-AgNPs) presented a spherical shape with a wide range of diameters (<20 nm), as observed by transmission electron microscope (TEM). The CS-AgNPs showed both fast and long-lasting antibacterial effectiveness against both Gram-positive and Gram-negative bacteria. The application of CS-AgNPs coating into litchi delayed the decrease in anthocyanin content, the increase in PPO activity and the changes in colour index and moisture loss. It also more effectively delayed the decay of stored litchi as compared to chitosan coating itself. Hence, it could conclude that treatment with CS-AgNPs coating exhibited a potential for shelf life extension of litchi at ambient temperature. Keywords: Silver nanoparticles, chitosan, litchi, shelf life Introduction Litchi (Litchi chinensis Sonn.), a tropical fruit with high commercial value, is an extremely perishable fruit. Freshly harvested litchi rapidly loses its qualities, such as sweet and juicy fresh and attractive bright red pericarp, within a couple of days under ambient temperature. The short shelf life of litchi not only greatly limits the marketing of the fruit, but also constraints in litchi industry due to its short season production from mid-May to early July. The high rate of senescence of litchi has been attributed to the moisture loss and browning due to the heat of respiration (Marie et al., 2008). Respiratory behavior of litchi is related to the fruitâ&#x20AC;&#x2122;s character, ambient temperature and property of coating (Lin and Liang, 2003). The previous studies indicated that chitosan, a unique polysaccharide derived from deacetylation of chitin, has been successfully used as an edible coating to prolong storage life of litchi. Litchi coated with chitosan had produced less bio-heat than that of uncoated (Lin et al., 2011). As far as we known, there are no details available about the specially formulated chitosan-based silver nanoparticles (CS-AgNPs) coating which may provide additional protection against contamination of microorganisms affected the fruit deterioration. Consequently, the aim of this work was to provide a new angle of view to study the effect of CS-AgNPs coating on the quality changes and the shelf life extension of litchi stored at ambient temperature. Materials and Methods Preparation, characterization and antibacterial property of the CS-AgNPs Chitosan flakes from crab shells (practical grade >85% deacylated: Sigma-Aldrich) was dissolved in 1% acetic acid solution. CS-AgNPs was prepared according to the method of Wei et al. (2009) with slight modification. The freshly prepared 52.0 mM AgNO3 solution (4
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ml) and 10 ml of a solution of chitosan (6.92 mg/ml) were mixed with constant stirring at 95oC. The color of the solution was progressed from colorless to light yellow, and finally to yellowish brown within an hour after the reaction was initiated, indicating the formation of AgNPs. UV-vis spectrum of the synthesized CS-AgNPs solution was recorded as a function of wavelength on a UV/Vis spectrophotometer (UV-260 Shimadzu) from 325-850 nm. The size and morphology of the CS-AgNPs were evaluated by using a transmission electron microscope (TEM, JEOL, JEM-1011; Japan). The disc diffusion assay, modified from the method by Case and Johnson (1984), was carried out to assess the antibacterial potential of the CS-AgNPs with both gram positive (S. aureus) and gram negative (E. coli) bacteria. The bacterial suspension (100 l of 1x107 CFU/ml) was applied uniformly on the surface of a nutrient agar plate and wells were punched at required places. 50 l of the prepared colloidal CS-AgNPs was then loaded into the well followed by incubating at 37oC for 24 h. The clear zone around the well reflects the bacterial sensitivity towards CS-AgNPs as compared to the control. To prepare fruit coating solution, the CS-AgNPs at an appropriate amount of AgNPs was blended with 1% (w/v) chitosan and 1.5% (w/v) glycerol was used as plasticizer. The coating solution was stirred for 15 min at ambient temperature. The final practical silver concentration 0.06 mg/l for coating, which is safe for consumer according to United State Environmental Protection Agency (USEPA), was used. Plant material, coating procedure and fruit quality measurements Fresh litchi (Litchi chinensis Sonn.) fruits were obtained from a local orchard in Northern area of Thailand. Fruits of uniform size, free of physical damage, injury caused by insects and fungal infection were dipped in CS-AgNPs coating solution, allowed to dry for 1 h and store at ambient temperature (302oC, 60±5%RH). Non-coated litchi served as the control for this study. The samples were analyzed weight loss, polyphenol oxidase (PPO) activity (Tan and Li, 1984) and the contents of anthocyanin (Pirie and Mullins, 1976). The subjective evaluation of skin browning index was well correlated with the objective determination of the value of absorbance at 410 nm of the skin extract (Jaing, 2000). The good quality fruit percentage (GFP) was measured and calculated as described by Lin et al. (2011). The storage life means that the GFP would be greater than 60%. To assess the effectiveness of CS-AgNPs coating on the control of decay expressed as percentage of fruit infected, samples were examined for mould regularly and considered infected when a visible lesion was observed. Results and Discussion Preparation, characterization and antibacterial activity of CS-AgNPs The UV-visible absorption spectrum of the CS-AgNPs showed a single, narrow and strong peak at 410 nm (Figure 1a), which is characteristic of surface Plasmon resonance of AgNPs (Mulvaney, 1996). TEM micrograph revealed the well-dispersed and spherical AgNPs with distributed particles of various diameters (<20 nm) (Figure 1b). The CS-AgNPs showed effective antibacterial activity against both S. aureus and E. coli as illustrated by their obvious clear zone inhibition in Figure 1c. It has been shown that chitosan itself has antimicrobial activity due to its cationic properties that cause a membrane-disrupting effect (Pavinatto et al., 2007). The increase in antimicrobial activity of CS-AgNPs coating might be expected as compared to chitosan itself because of a high infiltration of the silver component by its interaction with sulfur containing functional groups from membrane-bound enzymes with a resulting high bactericidal effect (Rhim et al., 2006).
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(a)
(b)
(c)
Figure 1 (a) UV-visible absorption spectra of CS-AgNPs (the inset shows the color of the synthesized CS-AgNPs; (b) TEM image of CS-AgNPs (the scale bar corresponds to 20 nm) at an acceleration voltage of 360 kV; (c) Antibacterial activity of CSAgNPs against S. aureus (left) and E. coli (right), corresponding with their clear zone inhibition Effect of CS-AgNPs coating on the qualities and shelf life of litchi fruit The increase in weight loss was observed with longer storage life of litchi (Figure 2a). However, it was significantly slower than that of non-coated control. The decrease of moisture loss suggested that the outside surface of CS-AgNPs coating on litchi pericarp had characteristics of air-dried film that chitosan molecular chains had arranged in order and closely packed like a barrier to prevent the passage of heat from surrounding into litchi, the transfer of moisture, gases or flavor, and thus to maintain or improve the quality and increase the shelf life of litchi. Because CS-AgNPs coating could avoid the water in litchi vaporized too fast and restrain the respiration of litchi to produce bio-heat, it indirectly delayed the increase of PPO activity (Dong et al., 2004). Under this condition, the storage life of the control and CS-AgNPs coated groups was 3 and 10 days, respectively. After storage for 3 days, the GFP of the control and the treatment groups were around 60% and 100%, respectively. It meant that CS-AgNPs coating was beneficial for storage life of the litchi at ambient temperature. The CS-AgNPs coating also delayed the decrease in anthocyanin content, the increase in PPO activity (Figure 2b) and the changes in colour index. Postharvest browning of litchi pericarp is a major problem, resulting in accelerated shelf life and reduced commercial value of the fruit. The browning was generally thought to be a rapid degradation of red pigment caused by polyphenol oxidase (PPO) and peroxidase (POD). It was found that increase in browning index of litchi fruit pericarp was associated with the reduction of anthocyanin concentration. Although PPO cannot directly oxidize litchi anthocyanin, the anthocyanin is degraded rapidly in an anthocyanin-PPO-phenol system (Jaing, 2000). Furthermore, since attack by pathogens is also a major factor causing browning of the fruit (Chen, 1984), inhibiting decay partially could be beneficial in delaying browning. The growth of microorganisms were hindered by the coating and inhibited by the antimicrobial characteristics of AgNPs located within the film network, leading to significantly decrease in decay incidence of litchi, as successively compared to chitosan itself and the non-coated control.
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(a)
(b)
Figure 2 Relative changes of (a) browning index and weight loss and (b) anthocyanin content and PPO activity of CS-AgNPs coated litchi stored at ambient temperature. Conclusion The integrated approach of AgNPs and chitosan prepared as Cs-AgNPs coating is found to be an important research for extending shelf life of litchi at ambient temperature by both delaying the browning of pericarp and the decay incidence of litchi. Since the storage is a perplexing process, the coating extended litchi shelf life was the result of integrative function such as the structure of coating, transpiration, respiration and so on. We recommend the application of CS-AgNPs coating to control browning and decay in litchi fruit in combination with other methods such as low temperature and suitable packaging. Acknowledgment This study is financially supported by the research grant 2015 from Naresuan University. References Case, C.L. and Johnson, T.P. 1984. Laboratory experiments in microbiology, Benjamin Cummings Pub Inc., California. pp. 126-129. Chen, W.S., 1984. A brief profile on studies of postharvest storage of litchi fruit. Litchi Sci. Bull. 34, 47–51. Dong H.Q., L.Y. Cheng, J.H. Tan, K.W. Zheng and Y.M. Jiang. 2004. Effects of chitosan coating on quality and shelf life of peeled litchi fruit. J. Food Eng. 64: 355–358. Jiang, Y.M. 2000. Role of anthocyanins, polyphenol oxidase and phenols in litchi pericarp browning. J. Sci. Food Agri. 80:305–310. Lin, B.F. and Liang, X.Q. 2003. Guangxi Sci., 10, 68-71. Lin, B., Y. Du, X. Liang, X. Wang, X. Wang and J. Yang. 2011. Effect of chitosan coating on respiratory behavior and quality of stored litchi under ambient temperature. Journal of Food Engineering 102: 94–99. Marie, N., Ducamp, C., Ramason, H., et al. 2008. Posth. Biol. Technol., 49, 241-246. Mulvaney P; 1996. Surface Plasmon Spectroscopy of Nanosized Metal. Particles. Langmuir 12: 788800. Pavinatto F.J., A. Pavinatto, L. Caseli, D.S. Jr. Santos, T.M. Nobre, M.E. Zaniquelli and O.N. Jr. Oliveira. 2007. Interaction of chitosan with cell membrane models at the air-water interface. Biomacromolecules 8:1633–1640. Pirie, A.; and M.G. Mullins. 1976. Changes in anthocyanin and phenolics content of grapevine leaf and fruit tissues treated with sucrose, nitrate, and abscisic acid. Plant Physiol 58: 468–472. Rhim, J.W., S.I. Hong, H.M. Park and P.K.W. Ng. 2006. Preparation and characterization of chitosan-based nanocomposite films with antimicrobial activity. Journal of Agricultural and Food Chemistry 54: 5814–5822. Tan, X.J. and Y.B. Li. 1984. Partial purification and properties of polyphenol oxidase in litchi fruit peel. Acta Phytophys. Sin. 10: 339–346. Wei D., W. Sun, W. Qian, Y. Ye and X. Ma. 2009 The synthesis of chitosan-based silver nanoparticles and their antibacterial activity. Carbohyd Res 344: 2375–2382.
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P-9
Green Synthesis of Silver Nanoparticles Loaded onto Activated Carbon Using Banana Peel Extract for Environment Applications Warin PIMPA1 and Chakkrit PIMPA2 1
Department of Agro-Industry, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand 2 Northern Region Operation Division, Electricity Generating Authority of Thailand, Phitsanulok 65000, Thailand. Corresponding e-mail: warin@nu.ac.th
ABSTRACT
Silver nanoparticles (AgNPs) were successfully synthesized from aqueous silver nitrate through a simple green route using the extract of banana peel as a reducing as well as capping agents. The results obtained from UV-visible spectroscopy and transmission electron microscope (TEM) supported the biosynthesis and characterization of AgNPs. The synthesized AgNPs presented a relative spherical shape with a wide range of size diameters around <15 nm. The AgNPs indicated a good antibacterial activity against E. coli with very low minimal inhibitory concentration of 15 ď g/ml. The presence of AgNPs loaded on to activated carbon did not change significantly morphology of the activated catbon itself, examined by scanning electron microscope (SEM), and methylene blue adsorption ability. Thus, the AgNPs-loaded activated carbon might be potential use for prevention and treatment of microbial inflection and cationic dye contamination for environmental application. Keywords: Silver nanoparticles, Banana peel, Activated carbon Introduction The bio-inspired synthesis of silver nanoparticles (AgNPs) has become significant in the recent years. AgNPs prepared using biological materials have the properties of a high surface area, smaller in size and high dispersion (Kaviya et al., 2011). It is well known that silver is an effective antibacterial agent and possess a strong antibacterial activity against bacteria, viruses and fungi, although the mechanism and the manner of action are still not well known (Sharma et al., 2009). The high antibacterial activity of AgNPs is a result of well-developed surface, providing maximum contact with the environment (Krutyakov et al., 2008). In the present study, biological synthesis was adopted by using banana peel extract. On the basis of the available literature, we hypothesized that banana peel which is rich in polymers (such as lignin, hemicelluloses and pectin) (Emaga et al., 2007) and antioxidant components (especially catichin and epicatichin) (Pimpa and Nuangchamnong, 2009) could be used in the synthesis of AgNPs. The synthesized AgNPs were then loaded onto activated carbon. The experiments on the antibacterial activity against E. coli and the methylene blue adsorption of the AgNPs-loaded activated carbon were investigated. We expected that AgNPs might bring an antibacterial property for activated carbon giving AgNPs-loaded activated carbon with effective inhibition of bacterial growth and good adsorption ability of the hazardous dyes for wastewater treatment applications.
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Materials and Methods Preparation of banana peel extract Banana peel (Musa sapientum L) was obtained from local dried banana processing in Phitsanulok, washed thoroughly with distilled water. The banana peel extract was prepared according to the procedures described by Yang and Li (2013) with slight modification. Such peels (100 g) were added to 250 ml distilled water and crushed by a juicer. The extract was filtered through a filter cloth and stored at -4oC for the further use for AgNPs synthesis. Preparation and characterization of AgNPs and AgNPs loaed activated carbon With the modified procedure of Yang and Li (2003), the banana peel extract (3ml) was mixed with 1 x10-3 M AgNO3 solution (27 ml) and heated at 80oC in a water bath for 15 min. The appearance of yellowish brown solution indicated the formation of AgNPs. The stability of silver nanoparticles was examined by exposing them to ambient condition for 3 months. It was observed that no sign of aggregation even at the end of this period. To characterize the synthesized AgNPs, UV-vis spectroscopy (UV-260 Shimadzu) and transmission electron microscope (TEM, JEOL, JEM-1011; Japan) were performed. AgNPs were directly loaded onto activated carbon by mixing AgNPs solution with activated carbon under vigorous stirring for 1 h. The product of AgNPs-loaded activated carbon powder was obtained by drying at 75oC. The preparation factors were adjusted to have 1% AgNPs (w/w activated carbon). This product was characterized by scanning electron microscope (SEM) and further used in the adsorption and antibacterial tests. Adsorption experiment The batch adsorption experiments of activated carbon and AgNPs-loaded activated carbon were carried out according to the methodology described by Pimpa and Pimpa (2011). Thus, 0.01 g of activated carbon or AgNPs-loaded activated carbon was placed in a vial and contacted with 10 ml of aqueous solution of the dye with a concentration of 2 x 10-5 M, with adjusted pH 2, 3, 5 and 7. After shaking at 30oC for certain contact duration, the adsorbent was filtered off and the residual concentration of the MB remained in the filtrate was measured by UV-visible spectroscopy at 665 nm. The amount of the MB adsorbed on the adsorbent was reported as adsorption capacity (mg/g). Antibacterial experiments For the qualitative antibacterial assay, the nutrient agar medium was mixed with 0.15 g of AgNPs-loaded activated carbon. After solidification of medium, E. coli was streaked uniformly and then incubated at 37oC for 24 h. The growth of the bacteria was observed by white colonies appeared on the medium as compared to the controls that of the nutrient agar medium itself and the nutrient agar medium-mixed activated carbon. For the qualitative antibacterial assay of AgNPs-loaded activated carbon (modified from the method by Tuan et al., 2011), the amount of AgNPs was adjusted from 2 to 200 ď g/ml and the final concentration of E. coli was 1 x 106 cell/ml. Then the mixture was incubated in a shaker at 37oC. The bacterial concentration was determined by measuring the optical density at 595 nm at 24 h. Results and Discussion Preparation and characterization of AgNPs using banana peel extract as a reducing and capping agents The initial pH value of the aqueous AgNO3 solution was an important parameter in the synthesis of AgNPs using banana peel extract. It was observed that the color of AgNO 3 solution changed to yellowish brown at initial pH 3-7. Control silver nitrate solution (without banana extract) neither developed the brown colors nor did they display the characteristic peaks. The color change would be an indication of silver bioreduction mediate by banana peel extract and the subsequent formation of AgNPs after 15 min of the reaction. The Page 222
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absorption spectrum of metal nanoparticles is dominated by the surface Plasmon resonance depending upon the particle size, shape, state of aggregation and the surrounding dielectric medium (Mulvaney, 1996). The UV-visible spectra obtained from solutions at different initial pH after reaction equilibrium showed characteristic absorption bands of AgNPs at around 410-420 nm (Figure 1a). Under an extreme acidic condition (PH 2.0) such biomolecules are likely to inactivated, thus there was neither a change in color nor a characteristic. Furthermore, pH values greater than 7.0, the white-grey precipitate was observed. The differences in the reaction products obtained over the range of pH could be due to a variation in the dissociation constants (pKa) of functional groups on the active components in banana peel extract that are involved in the reduction process. A variety of biomolecules are postulated to be involved in biological nanoparticle synthesis (Pimprikar et al., 2009). TEM measurements were carried out to observe the size and morphology of the AgNPs as prepare under pH 3.0, 5.0 and 7.0. As shown in Figure 1b, all the synthesized AgNPs were relatively spherical in shape. The shape of the AgNPs prepared under lower pH was less regular and more aggregate. The sizes of AgNPs were varied with a wide range of diameters (< 15 nm) under condition at pH 3-7.
Figure 1 (a) UV-visible absorption spectra of AgNPs synthesized at different pH values (2.0, 3.0, 5.0 and 7.0) (the inset shows the color of the synthesized AgNPs at equilibrium state); (b) TEM image of AgNPs (the scale bar corresponds to 10 nm) at an acceleration voltage of 500 kV. Preparation, characterization and properties of AgNPs-loaded activated carbon Activated carbon consisted of many particles with random form and size. The AgNPs attached on the surface of activated carbon or they could be loaded in the appropriate pores and spaces between particles. Although they could not vividly showed the different morphology between activated carbon and AgNPs-loaded activated carbon as measured by SEM (Figure 2a), the existence of AgNPs in AgNPs-loaded activated carbon may be recognized by the dark spots appeared in the TEM image (Figure 2b). The amount of MB adsorbed on AgNPs-loaded activated carbon (q; mg/g) depended on the initial concentration of MB (c; mg/ml). The value of q and c is a function of time. The removal capacity at equilibrium state depended strongly on the surface area of AgNPs-loaded activated carbon, the pH of the MB solution, the temperature and the mechanism of the adsorption process. A value of 210 mg/g was obtained for the complete monolayer adsorption capacity in Langmuir model. No significant different MB adsorption capacity between activated carbon and AgNPs-loaded activated carbon was observed. The qualitative antibacterial study of AgNPsloaded activated carbon on nutrient broth medium confirmed a strong inhibition of AgNPsloaded activated carbon against the growth of E. coli as compared to that of activated carbon which did not inhibit the growth. Furthermore, the MIC of AgNPs against the growth of E. coli is 15 ď g/ml which is close to the MIC, normally ranging from 1 to 16 ď g/ml, of antibitotics used for the treatment of E. coli (Kim et al., 2003). July 1-3, 2015
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Figure 2 (a) SEM images (magnified 2000x) and the appearances of (left) activated carbon and (right) AgNPs-loaded activated carbon and (b) TEM image of AgNPs-loaded activated carbon. Conclusion The green synthesis of AgNPs was accomplished by reducing silver nitrate with banana peel extract. The stability of the synthesized AgNPs was examined by exposing them to ambient condition for 3 months. It was observed that no sign of aggregation even at the end of this period. The AgNPs-loaded activated carbon indicated good antibacterial activity and dye adsorption ability, which could be potential use for wastewater treatment application. Acknowledgment This study is financially supported by the research grant 2015 from Naresuan University. References Emaga, T. H., R. H. Andrianaivo, B.Wathelet, J. T. Tchango, and M. Paquot. 2007. Effects of the stage of maturation and varieties on the chemical composition of banana and plantain peels. Food Chem., 103(2):590–600. Kaviya, S., J. Santhanalakshmi, B. Viswanathan, J. Muthumary, and K. Srinivasan, 2011. Biosynthesis of silver nanoparticles using citrus sinensis peel extract and its antibacterial activity. Spectrochimica Acta Part A, 79(3): 594–598.. Kim S., S.S. Kim, Y.J. Bang, S.J. Kim and B.J. Lee. 2003. In vitro activities of native and designed peptide antibiotics against drug sensitive and resistant tumor cell lines. Peptides 24:945–953. Krutyakov, Y. A., A. A. Kudrinskiy, A. Y. Olenin and G. V. Lisichkin. 2008. Synthesis and properties of silver nanoparticles:advances and prospects. Russ. Chem. Rev. 77: 233–257 Mulvaney, P. 1996. Surface Plasmon Spectroscopy of Nanosized Metal. Particles. Langmuir 12: 788-800. Pimpa, W. and N. Nuengchamnong. 2009. Agri. Sci. J. 40, 588-592. Pimpa W. and Pimpa W. 2014. Adv. Mater. Res., 931, 286-290. Pimprikar PS, S.S. Joshi, A.R. Kumar, S.S. Zinjarde and S.K. Kulkarni. 2009. Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589. Colloids Surf B Biointerfaces 74(1):309-316. Sharma, V.K.; R.A. Yngard and Y. Lin, Silver nanoparticles. 2009. Green synthesis and their antimicrobial activities. Adv. Colloid Interface Sci. 145: 83–96. Tuan, T.Q., N.V. Son, H.T.K. Dung, N.H. Luong, B.T. Thuy, N.T.V. Anh, N.D. Hoa and N.H. Hai. 2011. Preparation and properties of silver nanoparticles loaded in activated carbon forPreparation and properties of silver nanoparticles loaded in activated carbon forbiological and environmental applications, J. Hazard. Mater. 192:1321–1329. Yang N. and W. H. Li. 2013 Mango peel extract mediated novel route for synthesis of silver nanoparticles and antibacterial application of silver nanoparticles loaded onto non-woven fabrics. Ind. Crop. Prod. 48: 81-88.
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P-10
Bioactive Compounds and Antioxidant Activities in Flesh, Leaves and Endocarp of Carissa carandas Compared to Grape Seed Extract Products Surasak SAJJABUT1*, Wachiraporn PEWLONG1, Sirilak CHOOKAEW1, Jaruratana EAMSIRI1 and Panchalee PRAKHONGSIL1 1
Thailand Institute of Nuclear Technology (Public Organization), Ongkharuk, Nakhon Nayok 26120, Thailand *Corresponding author: saksajja@yahoo.com
ABSTRACT Carissa carandas (Nhamdaeng in Thai) is an herbal plant commonly found in Thailand. The purposes of this study were to analyse for total phenolics, free radical scavenging power (DPPH assay), ferricâ&#x20AC;&#x201C;ion reducing power (FRAP) and proanthocyanidins (vanillin-HCL assay) in flesh, endocarp and leaves of C. carandas comparing with two brands of grape seed extract products (BM and VT). The results showed that the total phenolics and free radical scavenging power in grape seed extract products (BM and VT) were higher than those in C. carandas samples, while the highest FRAP value was found in the endocarp and BM product. Surprisingly, this study indicated that proanthocyanidin content (an important bioactive compound in grape seeds) in C. carandas endocarp was higher than in BM and VT grape seed products. Keywords: Carissa carandas, Grape seed extract, Antioxidant, Proanthocyanidins Introduction Carissa carandas L. is a plant commonly known as karanda (India and Malaysia), nhamdaeng (Thailand), caramba (Philippines) and ci huangguo (Chinese). The plant is native to India and cultivated in Taiwan, Indonesia, Malaysia, Burma, Sri Lanka, Thailand and the Pacific Islands (Wiart, 2006). Whole plant and its parts are used in traditional medicine for treatments of ailments. C. carandas leaves can be used as an easily accessible source of natural antioxidants. Unripe fruits can be used to treat liver dysfunction, break fever and counteract putrefaction of blood (Wiart, 2006). In addition, fully-ripe fruits are a good source of anthocyanin, whereas unripe fruits are a good source of vitamin C (Pewlong et al., 2014). Other plant products that are considered a good source of natural anthocyanin are grape seeds. Grape seeds, the by-product in wine and grape juice industries, have gained more attention in the last decade because of their potential antioxidant properties. After grapes are pressed and the juice is collected, the remaining materials are mainly grape seeds. Grape seeds can be separated, extracted, and purified into the grape seed extract (GSE). GSE is recommended for use as antioxidant. The component of interest is polyphenols, mainly proanthocyanidins which are condensed tannins (Nuttall et al., 1998). Nowadays, the GSE product is expensive for consumers in Thailand and other countries. Thus, the aim of this study was to investigate whether C. carandas would have a potential to be developed as a commercial alternative to other natural antioxidants such as the grape seed extract. Product development would create an added value to this plant and provide an alternative to the consumers who prefer the health benefits of natural antioxidants.
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Materials and Methods
Sample preparation The experimental materials consisted of dried flesh and endocarps (Figure 1) (dried at 50๐c for 48 h in a hot-air oven), fresh leaves of C. carandas and commercial grape seed extract products (abbreviated BM and VT to protect their identities). The flesh, the endocarp and the leaves were ground in a blender and the samples were extracted with 40% ethanol. seed
endocarp
Figure 1 Seeds and endocarp showing the morphology and the size in comparison to Thai 1 Baht coin (1.9 cm diameter). Sample extraction One gram of each sample was extracted with 100 mL of 40% ethanol in an ultrasonic bath at room temperature for 1 hour. The sample suspensions were centrifuged, and the supernatant was filtered through a filter paper No.2 and the filtrate stored at -20°c until the analysis. Determination of total phenolics The total phenolic content was estimated using the Folin-Ciocateau assay according to the method developed by Velioglu et al. (1998). Determination of free radical scavenging power Determination of free radical scavenging power was performed as previously described by Khattate et al. (2008). Determination of ferric-ion reducing power FRAP assay was performed according to the method described by Benzei and Strain (1996). Determination of proanthocyanidins Proanthocyanidins in the samples were determined by vanillin-HCl assay (Sun et al., 1998). Statistical analysis Data were reported as the means ± SD of three replicate determinations. Where appropriate, the data were tested by one-way ANOVA using IBM SPSS v.19, followed by Duncan post hoc test. Differences of p < 0.05 were considered significant. Results and Discussion The total phenolic content of the samples ranged from 16.17±0.80 mg GAE/g for C. carandas flesh to 72.15±6.51 mg GAE/g for BM commercial grape seed extract product (Figure 2). The total phenolic contents of the grape seed extract products (both BM and VT) were higher than those of the flesh, the endocarp and the leaves of C. carandas. Among the samples of C.carandas, total phenolics in the endocarp was the highest and only 12 – 13% less than that of the grape seed extract products. DPPH is a stable organic free radical with an absorption band at 517 nm. It loses this absorption on accepting an electron or a free radical species, which results in a visually noticeable discolouration from purple to yellow colour. In this study, VT showed the highest DPPH radical scavenging power among all samples. However, the radical scavenging powers of BM and VT were not statistically different. When only C. carandas samples were considered, the endocarp demonstrated the highest DPPH radical scavenging power (Figure 3).
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mgGAE/g
mgAAE/g
Figure 2 Total phenolic contents in the flesh, the endocarp and the Figure 3 Free radical (DPPH) scavenging power of the flesh, the leaves of C. carandas, and two grape seed extract products: endocarp, and the leaves of C. carandas, and two grape seed extract products: BM and VT. Means±SD are shown at the BM and VT. Means±SD are shown at the top of the data top of the data bars. Values followed by the same letters are bars. Values followed by the same letters are not not significantly different (p < 0.05). AAE = ascorbic acid significantly different (p < 0.05). GAE = gallic acid equivalent. equivalent.
The ferric-ion reducing power of the samples, an indication of the antioxidant activity, was determined using a modified Fe3+ to Fe2+ reduction assay, whereby the yellow colour of the test solution changed to various shades of blue, depending on the reducing power of the samples. The presence of antioxidants in the samples would cause the reduction of the Fe3+/ferricyanide complex to the Fe2+ form, where Fe2+ was monitored by measuring the absorbance at 596 nm. In Figure 4, this study showed the reducing power following the order of BM = endocarp > VT > leaves = flesh. mgCTE/g molFeSO4/g
Figure 4 Ferric-ion reducing power of flesh, endocarp, leaves and two grape seed extract products. Means followed by the same letter are not significantly different (p < 0.05).
Figure 5 Proanthocyanidins in flesh, endocarp, leaves and two grape seed extract products. Means followed by the same letter are not significantly different (p < 0.05). (CTE = catechin equivalent)
The proanthocyanidins contents of various samples, which were analyzed by the vanillin-HCl assay, were shown in figure 5. Surprisingly, the endocarp of C. carandas contained much higher proanthocyanidins content than the two grape seed extract products. In this experiment, the leaves exhibited non-detectable proanthocyanidins contents, but proanthocyanidins had previously been reported in the leaves of other plants such as tea (Savitri kumar et al., 2008) and lantana (Bhakta and Ganjewala, 2009).
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Conclusion The results showed that the total phenolic content and the free radical scavenging power of grape seed extract products (BM and VT) were higher than those of C. carandas samples, while the highest FRAP value was found in the endocarp of C. carandas and BM product. Surprisingly, this study indicated that proanthocyanidin content (an important bioactive compound in grape seeds) in C. carandas endocarp was higher than in BM and VT grape seed extract products. However, proanthocyanidins was not detectable in C. carandas leaves. Thus, the endocarp of C. carandas appeared to be an interesting material with a potential for development as a health product in the future. Acknowledgements We gratefully acknowledge Dr. Kanokporn Boonsirichai for reviewing the manuscript. References Bhakta, D. and D. Ganjewala. 2009. Effect of leaf positions on total phenolics, flavonoids and proanthocyanidins content and antioxidant activities in Lantana camara (L). Journal of Scientific and Research 1(2): 363 - 369. Benzie, L.F.F. and J.J. Strain. 1996. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power the FRAP Assay. Analytical Biochemistry 239(1): 70 - 76. Khattak, K.F., T.J. Simson, and Ihasnullah. 2008. Effect of gamma irradiation on the extraction yield, total phenolic content and free radical-scavenging activity of Nigella staiva seed. Food Chemistry 110(4): 967 - 972. Nuttall, S.L., M.J. Kendall, E. Bombardelli and P. Morazzoni. 1998. An Evaluation of the antioxidant activity of a standardized grape seeds extract, leucoselect. Journal of Clinical Pharmacy and Therapeutics 23(5): 385-389. Savitri Kumar, N., W.M.A. Maduwantha, V. Kumar, P.A. Nimal Punyasiri and I.B.A. Sarath. 2008. Separation of proanthocyanidins isolated from tea leaves using high-speed counter current Chromatography. Journal of Chromatography A 1216(19): 4295 - 4302. Sun, B., J.M. Ricardo-da-Silva and I. Spranger, 1998. Critical factors of vanillin assay for catechins and proanthocyanidins. Journal of Agricultural and Food Chemistry 46(10): 4267 - 4274. Velioglu, Y.S., G. Mazza, L. Gao and B.D. Oomah. 1998. Antioxidant capacity and total phenolics in selected fruit, vegetable and grain products. Journal of Agricultural and Food Chemistry 46(10): 4113 - 4117. Pewlong, w., S. Sajjabut, J. Eamsiri and S. Chowkaew. 2014. Evaluation on antioxidant activities, anthocyanins, total phenolic content, vitamin content and cytotoxicity of Carissa carandas Linn. ChiangMai University Journal of Natural Science Special Issue on Food and Applied Bioscience 13(1): 509 - 517. Wiart, C. 2006. Medicinal plants classified in the family Apocynaceae. In plants of Asia and the Pacific pp: 245-260. Taylor and Francis Group, LLC. USA.
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P-11
Gamma Radiation Effects on Anthocyanins, Vitamin C Content and Antioxidant Activity of Carissa carandas Wachiraporn PEWLONG1*, Surasak SAJJABUT1, Jaruratana EAMSIRI1, Sirilak CHOOKAEW1 and Panchalee PRAKHONGSIL1 1
Thailand Institute of Nuclear Technology (Public Organization), Ongkharuk, Nakhon Nayok 26120, Thailand *Corresponding email: wachiraporn03@yahoo.com
ABSTRACT Food and agricultural products can be preserved for long term storage by gamma radiation. A low dose of gamma radiation has been reported to improve the antioxidant activity of some fruit extracts. The aim of this study was to investigate the effect of gamma radiation on antioxidant activity, anthocyanins and vitamin C content in Carissa carandas which is commonly known as nham-daeng in Thai. Ripe fruit samples were irradiated with gamma rays for total doses of 0.3 to 2.0 kGy at a dose rate of 5.2 kGy/h. Antioxidant activities of fruit extracts were evaluated based on total phenolic content, 1,1-diphenyl-2-picryhydrazyl (DPPH), free radical scavenging ability, and ferric-ion reducing power (FRAP). In addition, total anthocyanin content was analyzed by pH differential method, and vitamin C content was determined by High Performance Liquid Chromatography (HPLC). Results showed that radiation treatment up to 2.0 kGy did not affect the level of anthocyanin and vitamin C contents. In terms of antioxidant activities, there were negligible changes in the amounts of total phenolic and total anthocyanin content in fruit extracts following irradiation. No significant differences in the antioxidant activity and vitamin C content were observed between non irradiated and irradiated samples. These results indicated that gamma radiationup to 2.0 kGy did not affect the chemical properties of C. carandas. Keywords: Gamma radiation, Carissa carandas, Antioxidant Introduction Carissa carandas Linn. is a traditional plant in Asia with medicinal use in treating various diseases such as liver dysfunction and break fever (Wiart, 2006). The plants also provide bioactive compounds that function as antidiabetic, analgesic, and anti-inflammatory (Itankar et al., 2011, Begum et al., 2013). In addition, ripe C. carandas serves as a good source of anthocyanin and has become a new source of functional food (Pewlong et al., 2014). Radiation processing is an application of nuclear technology that can be used for food preservation (Pednekar et al., 2010). The efficiency and safety of the technique has been approved by CODEX, FDA, USDA, WHO and FAO. For postharvest treatment, gamma radiation has shown to extend the self-life of fresh mushroom (Beaulieu et al., 2002) and fruit juice (Alighourchi et al., 2008). Despite its benefits in food preservation, antioxidant activities in fruits can be altered by gamma radiation. The increase in antioxidant activity and total phenolic compounds were reported in 1.0 kGy irradiated Viola tricolor and 30.0 kGy irradiated ethanol extract from rosemary leaf (Perez et al., 2007). However, the decrease in total anthocyanin was reported in 2.0 kGy irradiated pomegranate juice (Alighourchi et al., 2008). In this research, we aimed to investigate the effect of gamma radiation on total anthocyanin content, vitamin C content and antioxidant activity of C. carandas fruit extract.
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Materials and Methods Preparation of fruit extract and gamma irradiation Ripe C. carandas (dark purple in color) were collected from Samut Songkhram province, Thailand. As a comparison to control group (non-irradiated, 0 kGy), some ripe C. carandas were packed in plastic bags and exposed to gamma rays for a total dose of 0.3, 0.5, 1.0 and 2.0 kGy in Gamma Chamber 5000 (BRIT, India). Ten grams of samples were soaked in 100 ml of 40% ethanol in water for 24 hours. Free radical-scavenging assay with DPPH and ferric reducing antioxidant potential (FRAP) The antioxidant activity was performed as previously described (Khattak et al., 2008) with slight modifications in which 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used as a radical scavenger. The free radical-scavenging activity was expressed as ascorbic acid equivalent. FRAP assay was performed according to the method described by Benzei and Strain (1996). The antioxidant potential of the samples was analyzed based on a calibration curve plotted using FeSO4.7H2O at concentrations ranged from 400 to 2,000 µM. Total phenolic content The content of reducing components was estimated using the Folin-Ciocateau assay according to the method developed by Velioglu et al. (1998). The total phenolic content was expressed as ascorbic acid equivalent (AAE) mg/g. Total anthocyanin content Total anthocyanin content was measured by the pH-differential spectrophotometric method (Wrolstad et al., 2005). Vitamin C content Vitamin C (ascorbic acid) was analyzed by HPLC (Jasco, Japan) with a UV detector. The separation was carried out using a C18, Phenomenex column. The ascorbic acid peak was detected at 205 nm. The entire chromatographic separation was performed at an isocratic mobile phase of methanol and 0.05 M potassium dihydrogen phosphate (pH 3.0) at the ratio 3:97 v/v. Statistical analysis All measurements were done in triplicate. Results were expressed as mean standard deviation (SD). Data were analyzed using the one-way ANOVA and DUNCAN tests (SPSS software) for mean differences between control and irradiated samples for all parameters. Results and Discussion Antioxidant activity Using DPPH and FRAP assays, the antioxidant activity of C. carandas were determined and shown in Table 1. No significant difference was found in the scavenging activity of the control and irradiated samples. In term of DPPH assays, the DPPH activities of control and irradiated samples were within the same range about 2.09 ± 0.48 to 2.67 ± 0.71 mgAAE/g. The DPPH activity of control was 2.58 ± 0.28 mgAAE/g while the irradiated samples varied in the range of 2.09 ± 0.48 to 2.67 ± 0.71 mgAAE/g. Similarly, gamma radiation did not affect the antioxidant activity of ferric reducing antioxidant power (FRAP). The FRAP values of non-irradiated and irradiated samples were within the same range, 25.68 ± 8.90 – 27.84 ± 6.69 µmolFeSO4/g. The FRAP value of control was 27.82 ± 8.67 µmolFeSO4/g while the irradiated samples varied in the range of 25.68 ± 8.90 to 27.84 ± 6.69 µmolFeSO4/g.
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Table 1 Analysis of antioxidant activities by DPPH and FRAP, total phenolic and vitamin C content of C. carandas extracts at varied irradiation doses. Irradiation DPPH Dose (mgAAE/g) (kGy) a
FRAP (µmol FeSO4/g)
Total phenolic content (mgGAE/g)
27.82 ± 8.67
a
4.46 ± 1.38
a
Total anthocyanin content (mg/L) 58.44 ± 5.87
Vitamin C content (mg/g)
a
1.67 ± 0.22a
0
2.58 ± 0.28
0.3
2.67 ± 0.71a
27.84 ± 6.69a
3.99 ± 0.98a
55.01 ± 4.04 a
1.66 ± 0.20a
0.5 1.0
2.12 ± 0.41a 2.09 ± 0.48a
26.60 ± 8.98a 25.68 ± 8.90a
4.03 ± 0.70a 4.00 ± 1.28a
55.07 ± 4.97 a 52.85 ± 5.52 a
1.60 ± 0.40a 1.60 ± 0.70a
2.0
2.37 ± 0.74a
27.06 ± 8.40a
4.03 ± 0.92a
50.03 ± 5.19 a
1.66 ± 0.12a
Values are expressed as mean ± SD of triplicate measurements. Different letter in the same column indicate significant differences at p<0.05.
Total phenolic content The total phenolic content of control and irradiated samples of C. carandas fruit was determined using Folin-Ciocalteau’s reagent. The results, expressed as mg equivalents of gallic acid/g of fruit, are shown in Table 1. The total phenolic content in irradiated samples was found within the same range as control about 3.99 ± 0.98 - 4.46 ± 0.92 mgGAE/g. No significant changes in phenolic contents were observed following irradiation up to 2.0 kGy. Total anthocyanin content The total anthocyanin content of C. carandas fruit extract was illustrated in Table 1. The total anthocyanin content of non-irradiated sample was 58.44 ± 5.87 mg/L and irradiated samples were 50.03 ± 5.19 – 55.01± 4.04 mg/L. It showed that gamma radiation caused a slight decrease in anthocyanin concentration compared to control. Vitamin C content The vitamin C content of C. carandas fruit extract was exhibited in Table 1. Vitamin C content of non-irradiated sample was 1.67 ± 0.22 mg/g and irradiated samples were 1.60 ± 0.40 – 1.67 ± 0.22 mg/g. No significant difference on vitamin C content was observed between non-irradiated and irradiated samples. Conclusion In this present study, we found that gamma radiation of C. carandas up to the total dose of 2 kGy did not alter the antioxidant activity, total phenolic, total anthocyanin and vitamin C content. Acknowledgements The authors would like to acknowledge Dr. Pimporn Uttayarat for her help in editing the menuscript. References Alighourchi, H., M. Barzegar, and S. Abbasi. 2008. Effect of gamma irradiation on the stability of anthocyanins and shelf-life of various pomegranate juices. Food Chemistry 110(4): 1036-1040. Beaulieu, M., B.D. Aprano and M. Lacroix. 2002. Effect of dose rate of gamma irradiation on biochemical quality and browning of mushrooms Agaricus bisporus. Radiation Physics and Chemistry 63: 311-315.
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Begum, S., A.A. Syed, B.S. Siddiqui, S.A. Sattar and M.I. Choudhary. 2013. Carandinol: First isohopane titerpene from the leaves of Carissa carandas L. and its cytotoxicity against cancer cell lines. Phytochemistry Letters 6: 91-95. Benzie, L.F.F. and J.J. Strain. 1996. The ferric reducing ability of plasma (FRAP) as a measure of â&#x20AC;&#x153;antioxidant powerâ&#x20AC;?: the FRAP assay, Analytical Biochemistry 239: 70-76. Itankar, R.A., S.J. Lokhande, P.R. Verma and S.K. Arora. 2011. Antidiabetic potential of unripe Carissa carandas Linn. fruit extract. Journal of Ethnopharmacology 135: 430433. Khattak, K.F., T.J. Simpson and Ihasnullah. 2008. Effect of gamma irradiation on the extraction yield, total phenolic content and free radical-scavenging activity of Nigella sativa seed. Food Chemistry 110(4): 967-972. Pednekar, M., A.K. Das, V. Rajalakshmi, and A. Sharma. 2010. Radiation processing and functional properties of soybean (Glycine max). Radiation Physics and Chemistry 79(4): 490-494. Perez, M.B., N.L. Calderon and C.A. Croci. 2007. Radiation-induced enhancement of antioxidant activity in extracts of rosemary (Rosemarinus officinalis L.). Food Chemistry 104(2): 585-592. Pewlong, W., S. Sajjabut, J. Eamsiri and S. Chookaew. 2014. Evaluation on antioxidant activities, anthocyanins, total phenolic content, vitamin C content and cytotoxicity of Carissa carandas Linn.ChiangMai University Journal of Natural Science special issue on Food and Applied Bioscience 13(1): 509 - . Velioglu Y.S., G. Mazza, L. Gao and B.D. Oomah. 1998. Antioxidant capacity and total phenolics in selected fruits, vegetable and grain products. Journal of Agricultural and Food Chemistry 46(10): 4113-4117. Wiart, C. 2006. Medicinal Plants Classified in the family Apocynaceae. pp. 245. In Plant of Asia and the Pacific. Taylor and Francis Group, LLC. USA. Wrolstad, R.E., R.W. Durst and J. Lee. 2005. Tracking color and pigment changes in anthocyanin products, Trends in Food Science and Technology 16(9): 423-428.
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P-12
Influence of Electron Beam Irradiation on Hygienic Quality and Antioxidant Activities of Ground Sea Holly (Acanthus ebracteatus) Jaruratana EAMSIRI 1*, Surasak SAJJABUT1, Wachiraporn PEWLONG1 and Sirilak CHOOKAEW1 1
Thailand Institute Nuclear and Technology (Public Organization), Ongkharak, NakornNayok 26120, Thailand *Corresponding email: jarurattt@gmail.com
ABSTRACT Roles of electron beam irradiation on improving hygienic quality and antioxidant activities of ground sea holly (Acanthus ebracteatus) were studied. The radiation processing was carried out at the doses of 0, 5, 10, 15 and 20 kGy using an electron beam accelerator (Mevex, MB 20/16) at the energy of 8 MeV, beam current of 10 mA and dose rate of 5 kGy/pass. The irradiated and unirradiated samples were analyzed for microbiological aspects and antioxidant properties. Results indicated that irradiation using electron beam significantly reduced microbial contamination. After irradiation at 5 kGy, total viable bacterial count (TVC) and total yeast and mold (TYM) were diminished by 2 and 1 log cycle, respectively. The dose of 5 kGy was adequate to diminish TVC and TYM to meet the Thai Community Product Standard of dry herbs (TCPS.480/2557) and to purge of pathogens such as coliform bacteria, Escherichia coli, Salmonella spp. and Clostridium perfringens. The antioxidant properties such as total phenolic content (TPC), ferric reducing antioxidant potential (FRAP) and free radical scavenging activity (DPPH) displayed insignificant changes for all doses applied in this study. Keywords: Sea holly, Electron beam, Irradiation, Microbiological quality, Antioxidant activity Introduction In Thailand Acanthus ebracteatus Vahl., andA. ilicifolius Linn. have been used as traditional medicine. They are used as a purgative and anti-inflammatory for arthritis. The whole plant is boiled in water used for bathing in order to heal rash and skin diseases. The fresh plant is crushed and applied as a poultice in boils or taken orally as depurative. However, spices and aromatic herbs are often highly contaminated by microbes. The contamination can occur from the plants themselves, soil, water, air and dust during postharvest and processing, and it can be a cause of serious food-borne illness (Seo et al., 2007). Therefore, decontamination of plant materials is necessary to increase the safety of medicinal plants. Medicinal plants can be decontaminated using heat, fumigation (ethylene oxide; ETO) and irradiation. However, heat is not suitable for heat sensitive products and ETO is prohibited in many countries such as Japan and some European countries, because it reacts with organic components leaving harmful residues (Chmielewski and Migdal, 2005) The electron irradiation for medicinal plant is produced by an electron accelerator. The major advantage of using electrons is that the source for the electrons can be turned off when not in use. Treatment with electron beams can provide the highest throughput rates and lowest unit costs. However, the major limitation of electron beam is its low penetrating power, therefore it is unsuitable for thick products (IAEA, 2008).The aim of this present research is to investigate and compare the effects of electron beam at various doses on microbiological quality and antioxidant properties of ground sea holly (Acanthus ebracteatus).
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Materials and Methods
Source of samples Ground sea holly (Acanthus ebracteatus) was purchased from local suppliers in Thailand. Samples of this herb were irradiated at Gems Irradiation Center, Thailand Institute of Nuclear Technology (Public Organization) using an electron beam accelerator (Mevex, MB 20/16) at the energy of 8 MeV, beam current of 10 mA and dose rate of 5 kGy/pass for a total dose of 5, 10, 15 and 20 kGy. Non-irradiated samples were also prepared as a reference. Microbiological analysis Microbiological evaluations such as total viable bacterial count (TVC), total yeast and mold (TYM), coliforms bacteria, Escherichia coli, Bacillus cereus, Clostridium perfringens and Salmonella spp. of irradiated and non-irradiated ground sea holly were conducted or enumerated according to the Association of Official Analytical Chemists (AOAC, 1990). Determination of free radical scavenging activity (DPPH), ferric reducing antioxidant potential (FRAP) and Total phenolic content (TPC) The antioxidant activity was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The 100 µl of each extract was added to 900 µl of DPPH in methanol solution (150 µM) in a test tube and shaken vigorously. After incubation at room temperature for 15 min in the dark, the absorbance of each solution was determined at 517 nm. The free radical-scavenging activity was expressed in the form of half maximal inhibitory concentration (IC50, mg/mlFRAP assay was performed according to the method of Benzie and Strain (1996). The content of reduced components (expressed in a form of TPC) was estimated using the FolinCiocalteu assay according to a method developed by Velioglu et al. (1998). Results and Discussion Effect of irradiation on the microbial load Total elimination of the microorganism is the principal aim of irradiation. The results of total viable bacterial counts indicated that the non-irradiated samples of ground sea holly were highly contaminated with bacteria at the level of 1.27x106 cfu/g. The values exceeded the level of 1.0x104 cfu/g reported by WHO (1998) as the maximum permissible total count level. The effects of electron beam irradiation at various doses on TVC and TYM are shown in Figure 1. The initial total bacterial counts and of non-irradiated samples were 1.27x106 cfu/g, whereas that of samples irradiated at 5 kGy were 1.02x104 cfu/g. TYM were eradicated after irradiated at 5 kGy. The irradiation at 5 kGy was sufficient to practically eliminate the total bacteria and total yeast and mold. Both microbiological analyses decreased linearly with absorbed doses. These results were in good agreement with those reported by Nemtanu et al. (2006). They found that irradiation of green coffee beans at 5 kGy by electron beam led to the desired microbial reduction, while irradiation at 10 kGy resulted in a complete absence of microorganism.
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1.8
1. 6
6
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1.6
1.2
8 0.
1 0.8
6 0.
0 kGy 5 kGy 10 kGy 15 kGy 20 kGy
6
1
0.6
3 0.
0.4 0.2
3 0.
0. 4
log cfu/g
1.4
0 0
0
0
0 TVC
TYM
Figure 1 Effect of irradiation on total viable bacterial counts and total yeast and mold counts of ground sea holly. In comparison among various doses, electron beam irradiation resulted in the same decontamination tendency. The values of pathogenic microorganisms, for unirradiated samples as well as samples irradiated by electron beam at different doses, are tabulated in Table 1. Food spoilage microorganisms such as B. cereus and C. perfringens were eliminated when irradiated at 5 kGy. Salmonella spp., coliform bacteria and E. coli were not detected in all irradiated and non-irradiated samples. Table 1 Effect of irradiations on pathogenic microorganism in ground sea holly. Microbiological 0 kGy 5 kGy 10 kGy 15 kGy Coliform Bacteria (MPN/g) <3 <3 <0.3 <0.3 E. coli (MPN/g) <3 <3 <0.3 <0.3 Salmonella spp. Absent Absent Absent Absent B. cereus (CFU/g) 1.4x102 <10 Est <1 Est <1 Est C. perfringens (MPN/g) 3.6 <3 <0.3 <0.3 = estimated value Est
20 kGy <0.3 <0.3 Absent <1 Est <0.3
Determination of Total phenolic content (TPC), ferric reducing antioxidant potential (FRAP) and free radical scavenging activity (DPPH) The effects of electron beam irradiation in terms of TPC, FRAP and DPPH of ground sea holly were summarized in Table 2. The results from electron beam irradiation at various doses indicated that the levels of TPC varied between 4.64-5.19 (mg GAE/g). The values of FRAP at various doses fluctuated between 75.69 and 83.23 Âľmol FeSO4/g. DPPH was in the form of half maximal inhibitory concentration (IC50, mg/ml). The values altered between 4.00 and 4.17 mg/ml. The results also revealed that electron beam irradiation did not significantly affect TPC, FRAP and DPPH. On the other hand Suhaj et al. (2006) reported that the DPPH scavenging activities of black pepper significantly decreased after irradiation at 5, 7.5, 10, 20 and 30 kGy.
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Table 2 Effect of electron beam irradiation on antioxidant properties of ground sea holly Dose TPC FRAP DPPH assay (kGy) (mg GAE/g) (µmol FeSO4/g) (IC50 (mg/ml) 0 4.760.52a 83.232.74 a 4.170.49 a 5 4.660.39 a 81.963.63 a 4.090.44 a 10 4.640.42 a 75.699.66 a 4.000.45 a 15 5.080.32 a 82.193.57 a 4.160.46 a a a 20 5.190.52 80.441.47 4.080.49 a Values are expressed as meanSD of triplicate measurement. Superscripts with different letters are significantly different at p<0.05 within the same column. The GAE was the abbreviation of gallic acid equivalent.
Conclusion Results from this study showed that electron beam irradiation was proved to be an effective process to improve the quality of ground sea holly. Irradiation at 5 kGy can eliminate total bacteria, total yeast and mold, Coliform bacteria, Escherichia coli, Salmonella sp. and Bacillus cereus in ground sea holly. The optimum dose of irradiation to diminish the microbial contamination was 5 kGy. On the contrary, there was no significant changes in TPC, FRAP and DPPH after irradiation up to 20 kGy. References Association of Official Analytical Chemists. 1990. Official Method of Analysis (15th ed.). Washington DC. Benzie, I.F.F. and J.J. Strain, 1996. The ferric reducing ability of plasma (FRAP) as a measurement of “antioxidant power”: the FRAP assay. Analytical Biochemistry 239: 70-76. Chmielewski, A.G. and W. Migdal. 2005. Radiation decontamination of herbs and spices. Nukleonika 50: 179-184. IAEA, 2008. Trends in radiation sterilization of health care products. Vienna, Austria: IAEA. Nemtanu, M.R., M. Brasoveanu, R. Minea, M.N. Grecu, M. Albulescu, and E. Mitru. 2006 Microbiological decontamination of Spirulina platensis and green coffee using accelerated electron beams. Romanian Journal of Biophysics 16: 141-148. Seo H., J. Kim, H. Song, D. Kim, M. Byun, J. Kwon, and K. Kim, 2007. Effects of gamma irradiation on the yields of volatile extracts of Angelica gigas Nakai. Radiation Physics and Chemistry 76: 1869-1874. Suhaj, M., J. Racova, M. Polovka and V. Brezova. 2006. Effect of gamma irradiation on antioxidant activity of black pepper (Piper nigrum L.). Food Chemistry 97: 696-704. Velioglu, Y.S., G. Mazza, L. Gao and B.D. Oomah, 1998. Antioxidant capacity and total phenolics in selected fruits, vegetables and grain products. Journal of Agricultural and Food Chemistry 46: 4113-4117. World Health Organization. 1998. Food irradiation a technique for preservation and improving the safety of food. World Health Organization of the United Nations, Geneva.
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P-14
Evaluation of Antioxidant Activities of Essential Oils from Peppermint and Ginger Natthakiti PHURUEN1, Chamroon LAOSINWATTANA1 and Montinee TEERARAK 1
1*
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, Thailand *Corresponding email: ktmontin@kmitl.ac.th
ABSTRACT The aim of this study was to evaluate antioxidant activities of essential oils of peppermint (Mentha piperita L.) and ginger (Zingiber officinale Roscoe) by using DPPH (2, 2-diphenyl1-picrylhdrazyl) free radical-scavenging, metal chelating and reducing power activities. Both essential oils exhibited a radical scavenging activity against DPPH radical with IC50 (half maximal inhibitory concentration) values of 7,870.56 and 93,005.75 mg/L for ginger and peppermint essential oils, respectively. The IC50 values of standard antioxidants were 3.16 mg/L for ascorbic acid and 21.82 mg/L for butylated hydroxyl toluene (BHT). Furthermore, the essential oils also exhibited metal chelating activity with IC50 365.68 mg/L for ginger essential oil and 989.13 mg/L for peppermint essential oil while ethylene-dinitrilo-tetraacetic acid (EDTA) as standard IC50 value was 18.32 mg/L. Moreover, reducing power ability of essential oils of peppermint and ginger increased concentration-dependently and the ability was low in essential oil of peppermint compared with essential oil of ginger. Keywords: Antioxidant activity, Essential oil, Metal chelator, Reducing power Introduction Essential oils are organic natural substances that possess a wide range of biological properties, including antimicrobial, insect repellent, herbicidal, and antioxidant activities. The essential oils have strong antimicrobial properties because the main component of essential oil is a phenolic compound such as carvacrol, thymol and eugenol (Lambert et al., 2001; MihajilovKrstev et al., 2010). Essential oils from plants have been interested and researched a lot about the properties of antioxidants (Li et al., 2010). Peppermint (Mentha piperita L.) is one of the mentha species. Menthol (29%) and menthone (20-30%) are the major components of the peppermint essential oil (Ahijevych and Garrett, 2004). Ginger (Zingiber officinale Roscoe) is consumed worldwide as a spice and flavoring agent and is attributed to have many medicinal properties. The active ingredients in ginger are thought to reside in its volatile oils, which comprise sesquiterpenes: bisapolene, zingiberene, and zingiberol (Connell and Sutherland, 1969). The antioxidant from plants has been reported to scavenge free-radical, to protect the human body from disease (Kinsella et al., 1993) and to retard lipid oxidative rancidity. Lipid oxidation, which occurs during the storage of raw materials, processing, heat treatment and subsequent storage of final products, is one of the main causes of rancidity in food products (Tepe et al., 2005). In recent years, there has been a considerable interest in seeking for potential natural and possibly economic and effective antioxidants to replace the synthetic antioxidant. In this study, free radical-scavenging, metal chelating and reducing power activities of essential oils of peppermint and ginger were analyzed.
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Materials and methods
Essential oils The essential oils of peppermint (Mentha piperita L.) and ginger (Zingiber officinale Roscoe) were purchased from Hong Huat company limited (Bangkok, Thailand). 2 ,2′-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging A volume of 2 mL of essential oils of different concentrations was added to 2 mL of DPPH (5.9 mg/100 methanol). The mixtures were well shaken in a vortex and then placed in a dark room for 30 min. The absorbance of the mixture was read at 517 nm against a blank of absolute ethanol without DPPH. Ascorbic acid and butylatedhydroxytoluene (BHT) were used as references. The percentage inhibition of the DPPH radical was calculated according to the formula of Yen and Duh (1994). % I = [(AB-AS)/AB]x100 Where I = DPPH inhibition (%), AB = absorbance of control sample and AS = absorbance of a tested sample at the end of the reaction. Sample concentration providing 50% inhibition (IC50) was calculated from the graph plotted of inhibition percentage against sample concentration in the reaction system. Ferrous Ion-chelating Ability Assay The ferrous ion-chelating (FIC) assay was carried out according to the method of Singh and Rajini (2004), with some modifications. An aliquot (1 ml) of different concentrations of essential oils was mixed with 1.5 ml ethanol and 0.05 ml FeSO4·4H2O. After 5 min incubation, the reaction was initiated by the addition of ferrozine (0.1 ml). The mixture was shaken vigorously and after a further 10 min incubation period the absorbance of the solution was measured spectrophotometrically at 562 nm. The percentage inhibition of ferrozine–Fe+2 complex formation. Ferric Reducing Antioxidant Power The ferric reducing power (FRAP) was determined by using the potassium ferricyanide– ferric chloride method. Different dilutions (1 ml aliquots) of essential oils were each added to 2.5 ml phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%). The mixtures were incubated at 50°C for 20 min, after which 2.5 ml trichloroacetic acid (10%) was added and then centrifuged at 3,000 rpm, 25°C for 10 min. An aliquot of the mixture (2.5 ml) was taken and mixed with 2.5 ml water and 0.5 ml 1% FeCl3. The absorbance at 700 nm was measured after allowing the solution to stand for 30 min. Statistical analysis For all the experiments, three samples were analyzed and all the assays were carried out in triplicate. The results were expressed as mean±standard deviation. Results and discussion The activity of antioxidants has been assigned to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging (Yildrim et al., 2001). The radical scavenging capacity of the essential oils was tested using the stable free radical, DPPH and measured in terms of hydrogen-donating or radical scavenging ability. Table 1 shows DPPH radical-scavenging activities of peppermint and ginger essential oils, as compared to BHT and ascorbic acid. The essential oils of ginger and peppermint exhibited a radical scavenging activity against DPPH radical with IC50 values of 7,870.56 and 93,005.75 mg/L, respectively. The IC50 values of standard antioxidants were 3.16 mg/L for ascorbic acid and 21.82 mg/L for butylated hydroxyl toluene (BHT). One of the possible mechanisms of the antioxidative action is the chelation of transition metals. Ferrous iron can initiate lipid peroxidation by the Fenton reaction as well as accelerating peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl Page 238
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radicals (Halliwell, 1991; Deshpande and Deshpande, 1997). The obtained results showed that the essential oil of ginger had high metal chelating activity, IC50 value was 365.68 mg/L while ethylenedinitrilo-tetraacetic acid (EDTA) as standard had IC50 value was 18.32 mg/L (Table 2). From the result it was evident that both the extracts possessed Fe2+ chelating activity and might play a protective role against oxidative damage induced by metal catalyzed decomposition reactions (Dorman et al., 2003). In addition, Viuda-Manuel et al. (2010) reported that the ability of the essential oils from five spice plants in antioxidant activity by DPPH and the ability to compete for binding to metal ions Fe2+ increased as the concentration increased. Table 1 DPPH free radical-scavenging activities of peppermint and ginger essential oils, BHT and ascorbic acid. Substances IC50 (mg/L) Peppermint essential oil 93,005.75 2.24 Ginger essential oil 7,870.56 6.53 Ascorbic acid 3.16 0.01 BHT 21.82 0.18 IC50 Concentration (mg/L) for a 50% inhibition. Table 2 Metal chelating activities of peppermint and ginger essential oils and EDTA. Substances IC50 (mg/L) Peppermint essential oil 989.13 5.09 Ginger essential oil 365.68 5.67 EDTA 18.32 2.53 IC50 Concentration (mg/L) for a 50% inhibition. The reducing power was determined by the Fe3+ being reduced to Fe2+in the presence of the essential oil (Ferreira et al., 2007). The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. Reducing power of essential oils of peppermint and ginger as well as standard compounds with increasing concentrations followed the order, ascorbic acid > ginger essential oil > peppermint essential oil (Figure 1). It indicated that ascorbic acid showed an excellent reducing power.
Figure 1 Reducing power of peppermint and ginger essential oils and standard (ascorbic acid).
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Conclusions According to the obtained results, DPPH radical-scavenging, metal chelating and reducing power activities of essential oil of ginger was superior to those of essential oil of peppermint. Acknowledgment This research was supported financially by the Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Thailand via the annual budgets allocated by the Thai Government in the year of 2015 (B.E. 2558). References Ahijevych, K and B.E. Garrett. 2004. Menthol pharmacology and its potential impact on cigarette smoking behavior. Nicotine &Tobacco Research 6: S17-S28. Connell, D. and M. Sutherland. 1969. A re-examination of gingerol, shogaol and zingerone, the pungent principles of Ginger (Zingiber officinale Roscoe). Australian Journal of Chemistry 22:1033-43. Deshpande, S.S. and U.S. Deshpande. 1997. Nutritional and health aspects of food antioxidants. In Food Antioxidants. Technological, Toxicological and Health Perspectives.D.L.Madhavi, S.S. Deshpande and D.K. Salunkhe. Marcel Dekker. New York. Pp. 361-470. Dorman, H.J.D., M. Kosar, K. Kahlos, Y. Holm and R. Hiltunen. 2003. Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties and cultivars. Journal of Agricultural and Food Chemistry 51: 4563-4569. Ferreira, C. F. R., P. Baptista, M. Vilas-Boas and L. Barros. 2007. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food Chemistry 100: 1511-1516. Halliwell, B. 1991. Reactive oxygen species in living systems: Source, biochemistry, and role in human disease. American Journal of Medicine 91: S14-S22. Kinsella, J.E., E. Frankel, B. German and J. Kanner. 1993. Possible mechanisms for the protective role of antioxidants in wine and plant foods. Food Technology 47: 85â&#x20AC;&#x201C;89. Lambert, R.J.W., P.N. Skandamis, P.J. Coote and G.J.E. Nychas. 2001. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology 91:453- 462. Li, J., S. Nie, Z. Qiu, M. Che, C. Li and M. Xie. 2010. Antimicrobial and antioxidant activities of the essential oil from Herba Moslae. Science of Food and Agriculture 90: 1347-1352. Mihajilov-Krstev, T., D. Radnovic, D. Kitic, Z. Stojanovic-Radic and B. Zlatkovic. 2010. Antimicrobial activity of Satureja hortensis L. essential oil against pathogenic microbial strains. Archives Biological Science Belgrade 62: 159-166. Singh, N. and P.S. Rajini. 2004. Free radical scavenging activity of an aqueous extract of potato peel. Food Chemistry 85: 611-616. Tepe, B., D.Daferera, A. Sokmen, M. Sokmen and M. Polissiou. 2005. Chemical composition, antioxidant and antimicrobial properties of the essential oils of three Salvia species from Turkish flora. Food Chemistry 90: 333-340. Viuda-Martos, M., Y.R. Navajas and E.S. Zapata, J. Fernandez-Lopez and J.A. PerezAlvarez. 2010. Antioxidant activity of essential oils of five spice plants widely used in a Mediterranean diet. Flavour and Fragrance Journal 25: 13-19. Yen, G.C. and P.D. Duh. 1994. Scavenging effect of methanolic extracts of Peanut Hulls on freeradical and active-oxygen species. Journal of Agricultural and Food Chemistry 42: 629-632. Yildrim, M. Oktay and V. Bilaloglu. 2001. The antioxidant activity of the leaves of Cydonia vulgaris. Turkish Journal of Medical Sciences 31: 23-27.
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P-16
Antioxidant Activity and Total Phenolic Contents of Tagetes erecta L. Leaves Extracts Pattharin WICHITTRAKARN, Montinee TEERARAK and Chamroon LAOSINWATTANA* Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: klchamro@kmitl.ac.th
ABSTRACT This study was performed to evaluate the antioxidant potential of the flower extract from Tagetes erecta L. The ethanol is a recommended solvent for extracting antioxidants from this plant. The antioxidant activity was evaluated using 2, 2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging activity, metal chelating ability and total phenolic content. The result showed that DPPH free radical-scavenging activity with the half maximal inhibitory concentration (IC50) value was 111.84 ppm compared with IC50 values of standard antioxidants were 3.16 ppm for ascorbic acid and 21.82 ppm for butylated hydroxyl toluene (BHT), respectively. In addition, the flower extract has metal chelating activity with IC50 with of 22,490.11 ppm and ethylene-diaminetetraacetic acid (EDTA) was used as standard of 18.32 ppm. Furthermore, the phenolic content was found to contain 4,203.455 mg GAE/100g of dry weight (expressed as gallic acid equivalents). The results indicate that these compounds contribute to the antioxidative activity. Keywords: Tagetes erecta L., DPPH free radical-scavenging, Metal chelating ability, Total Phenolic content Introduction An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction involving the loss of electrons or an increase in oxidation state. Oxidation reactions can produce free radicals. Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness (Baillie et al., 2009). Antioxidants also have many industrial uses, such as preservatives in food and cosmetics and to prevent the degradation of rubber and gasoline. Recently, natural antioxidants have attracted considerable interest among nutritionists, food manufacturers and consumers, due to their presumed safety and prospective therapeutic values. Tagetes erecta L. belong to the family Asteraceae. It is a small shrub, which grows up to 1-2 m. It is different shades yellow and orange flower. T. erecta has also widely used as medicine, cosmetic and perfumery (Vasudevan et al., 1997). The objectives of this study were evaluated Tagetes erecta L. as a source of natural antioxidants using DPPH radical scavenging activity and metal chelating assay to determine their antioxidant capacities. Because of important roles of the total phenolics content as antioxidants, the amount of total phenolics in the extract was also determined. Materials and Methods Preparation of plant extracts The mature flowers of Tagetes erecta L. were collected from the experiment field of KMITL, and dried up in hot-air oven at 45 °C for 3 days. Five grams of dried flower was soaked in 95 mL of ethanol and placed at room temperature for 3 days. After 3days, infusions were filtered July 1-3, 2015
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through Whatman No. 1 filter paper to obtained 50,000 ppm of stock extraction then stored in a brown bottle at 4 °C. Determination of total phenolic content The total phenolic content was quantified using Folin- Ciocalteu method according to the procedures of Chumyam et al. (2013). An aliquot, 1 mL of sample extract were mixed with 0.5 mL Folin - Ciocalteu (2N) reagent and was treated to the mixture and shaken well. After 5 min, 4 mL of aqueous sodium carbonate (7.5%) were added to the mixture. The samples were incubated for an hour at room temperature in the dark. The absorbance was measured at 765 nm using the spectrophotometer. The total phenolic contents were calculated on the basis of the calibration curve of gallic acid standard. Results were expressed as mg gallic acid equivalent per gram dry weight (mg GAE/g DW) of the plant material. DPPH radical scavenging activity DPPH free radical-scavenging of flower extracts was measured by bleaching a purple solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, was based on the measurement of the reducing ability of antioxidant toward the DPPH radical. The method described by Ebrahimzadah et al. (2008) was used with slight modification. An aliquot, 2 mL of flower extract of different concentrations was mixed with 2 mL of DPPH solution (100 µM) in ethanol. After incubation in the dark at room temperature for 30 min, changed in the absorbance of the extract was measured at 517 nm using the spectrophotometer. Ascorbic acid and butylated hydroxyl toluene (BHT) were used as standard. Samples were analyzed in triplicate. IC50 values denote the concentration of sample, which required scavenging 50% inhibition of DPPH radical. Metal chelating activity The chelating ability of the plant extract for ferrous ions (Fe2+) was quantified according to the modified method of Dinis et al. (1994). An aliquot, 1 mL of sample extract at various concentrations were mixed with 1.5 mL of ethanol and 50 µL of ferrous sulfate heptahydrate (2 mM) was added. The reaction was initiated by the addition of 100 µL of ferrozine solution (5 mM). Then, the mixture was shaken vigorously and incubated at room temperature for 10 min. Absorbance of the samples was then measured at 562 nm. Ethylene-diaminetetraacetic acid (EDTA) was used as standard. The ability in chelating ferrous ions of the sample was expressed as the percentage of metal chelating activity and was calculated using the following equation; Metal chelating effect (%) = [(Acontrol – Asample ) /Acontrol] x 100 where Acontrol is the absorbance of the control and Asample is the absorbance of the tested extract. Metal chelating activity is presented by IC50 values where concentration providing 50% inhibition of chelate ferrous ions. Results and Discussion Total phenolic content Phenolics or polyphenols are secondary plant metabolites that are ubiquitously present in plants and plant products. Many of phenolics have been shown to contain high levels of antioxidant activities (Marvibaig et al., 2014). The total phenolic content was determined using Folin-Ciocalteu reagent. The results showed that, the amount of total phenolic content of the ethanolic flower extracts of Tagetes eracta was 4,203.455 GAE/100g of dry weight.
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DPPH radical scavenging activity The results showed that flowers extract has based on the measurement of the reducing ability of antioxidants toward DPPH. The DPPH radical scavenging (%) activity is shown in the Figure 1. The IC50 values were found to be 111.838, 3.16 and 21.82 ppm for the flowers extract, ascorbic acid and BHT, respectively. It was similar results to Uppal and Nigam (2012), who reported that Euphorbia hirta at the concentration of 10-80 µg/mL showed concentration dependent reduction in absorbance and percentage inhibition of 15.18, 24.82, 55.18, 66.75 and 73.98%, respectively, and M. arundinacea extract exerted the IC50 of the extract was 293.4 μg/mL, while that of BHT was 226.7 μg/mL (Nishaa et al., 2012). 100
80
80
80
60
y = 54.992x - 62.656 IC50 = 111.84
40 20
% DPPH radical
100
% DPPH radical
% DPPH radical
BHT
Ascorbic acid
Flower extract 100
60 y = 80.223x + 9.9254 IC50 = 3.16
40 20
0
0.5
1
1.5 2 2.5 Concentrations (log)
3
3.5
40
y = 53.033x - 21.003 IC50 = 21.82
20
0
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Figures 1 DPPH free radical scavenging activity of flower extract from Tagetes eracta L., ascorbic acid and BHT Metal chelating activity The metal chelating capacity of flowers extracts from T. eracta were estimated by assessment of their ability to compete with ferrozine for ferrous ions. Figure 2 showed the IC50 values were found to be 22,490.11 and 18.32 ppm for the flowers extract and EDTA, respectively. The metal chelating activity was followed a dose dependent pattern. V. album extracts from cashew recorded greater metal chelating activity than cocoa V.album (Oluwaseun and Ganiyu, 2008). EDTA
80 60
y = 51.041x - 172.13 IC50 = 22490.11
40 20 0 0
1
2 3 Concentrations (log)
4
5
% inhibition of metal chelating
% inhibition of metal chelating
Flower extract 100
100 80 y = 69.612x - 37.923 IC50 = 18.32
60 40 20 0 0.0
0.5
1.0 Concentrations (log)
1.5
2.0
Figures 2 Metal chelating activity of flower extract from Tagetes eracta L., EDTA (ethylenediaminetetraacetic acid). Conclusion Ethanolic extract of Tagetes erecta L. was found rich in phenolic compounds content that have wide range of biological functions, including antioxidant activity. The obtained results suggested that flower of T. erecta could be potential rich source of natural antioxidant activity and total phenolic content. Acknowledgments The authors wish to thank King Mongkut’s Institute of Technology Ladkrabang for providing financial support for this research. July 1-3, 2015
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References Baillie J.K., A.A. Thompson, J.B. Irving, M.G. Bates, A.I. Sutherland, W. Macnee, S.R. Maxwell and D.J. Webb. 2009. Oral antioxidant supplementation does not prevent acute mountain sickness: double blind, randomized placebo-controlled trial. An International Journal of Medicine 102 (5): 341-348. Chumyama A., K. Whangchaia, J. Jungklanga, B. Faiyuec and K. Saengnila. 2013. Effects of heat treatments on antioxidant capacity and total phenolic content of four cultivars of purple skin eggplants. Science Asia 39: 246-251. Dinis T.C.P., V.M.C. Madeira and M.L.M. Almeida. 1994. Action of phenolic derivates (acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and Biophysics 315: 161-169. Ebrahimzadeh M.A., F. Pourmorad and A.R. Bekhradnia. 2008. Iron chelating activity, phenol and flavonoid content of some medicinal plants from Iran. African Journal of Biotechnology 7 (18): 3188-3192. Marvibaigi M., N. Amini, E. Supriyanto, S. Jamil, F.A.A. Majid and S. Khangholi. 2014. Total phenolic content, antioxidant and antibacterial properties of Scurrula ferruginea extracts 70 (5): 65-72. Nishaa S., M. Vishnupriya, J.M. Sasikumar, H.P. Christabel and V.K. Gopalakrishnan. 2012. Antioxidant activity of ethanolic extract of Maranta arundinacea L. tuberous Rhizomes. Asian Journal of Pharmaceutical and Clinical Research 5 (4): 85-88. Oluwaseun A.A. and O. Ganiyu. 2008. Antioxidant properties of methanolic extracts of Mistletoes (Viscum album) from cocoa and cashew trees in Nigeria. African Journal of Biotechnology 7 (17): 3138-3142. Umamaheswari M. and T.K. Chatterjee. 2008. In vitro antioxidant activities of the fractions of Coccinia Grandis L. leaf extract. African Journal of Traditional, Complementary and Alternative medicines 5: 61-73. Uppal, G. and V. Nigam. 2012. In vitro antioxidant activity of ethanolic extract of Euphorbia hirta Linn. International Journal of Current Research 4 (10): 155-159. Vasudevan, P., S. Kashyap and S. Sharma. 1997. Tagetes: A multipurpose plant. Bioresource Technology 62: 29-35.
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P-17
Allelopathic Effect of Fresh and Dried Leaves Aqueous Extracts of Marachra capitata L. and the Action of Allelochemicals in Different Soil Types Phawinee KAMSAN, Pattharin WICHITTRAKARN, Montinee TEERARAK and Chamroon LAOSINWATTANA* Department of Plant Production Technology, Faculty of Agricultural and Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: klchamro@kmitl.ac.th
ABSTRACT Allelopathic effects of aqueous extracts from fresh and dry leaves of Marachra capitata L. at concentrations of 1.25, 2.5, 5 and 10% were evaluated on germination and seedling growth of barnyard grass (Echinochloa crus-galli (L.) Beauv.) and wild pea (Phaseolus lathyroides L.). The distilled water was used as control. It was found that aqueous extract from dry leaves had inhibitory effect on seed germination and seedling growth of barnyard grass and wild pea was stronger than aqueous extracts from fresh leaves. In addition, the concentration of 5% aqueous extract from dry leaves had completely inhibited seed germination of wild pea. The influence of 5 various planting media (fertile soil, fertile sand and sterile soil, sterile sand and only leaves powder) on dry leaf potential was also studied at concentrations of 250 and 500 mg/ petri-dish on germination and seedling growth of barnyard grass and wild pea compared with only germination paper (control). The results indicated that at 500 mg/petri-dish had completely inhibited on seed germination and seedling growth of wild pea. Keywords: Marachra capitata L, Germination and seedling growth, Fresh and dry leaves Introduction Allelopathy refers to the beneficial or harmful effects of one plant on another plant, both crop and weed species, from the release of biochemicals, known as allelochemicals, from plant parts by leaching, root exudation, volatilization, residue decomposition, and other processes in both natural and agricultural systems. Allelochemicals are a subset of secondary metabolites not required for metabolism (growth and development) of the allelopathic organism. Allelochemicals with negative allelopathic effects are an important part of plant defense against herbivory (Fraenkel et al., 1959). Crop development is affected by allelopathy from certain weed species. Allelochemicals from allelopathic weeds can disturb the root and shoot growth of emerging crop seedlings, as well as cause several other damages. Malachra capitata L. contained about 12 species, mostly found and bleeding in America (the Encyclopedia of Plants in Thailand, 2012). and species of herbaceous plant in the family Malvaceae mostly erect, coarse, annual or perennial herb 1-2 m tall, throughout densely whitish- or yellowish-tomentose with stellate hairs and usually also moderately to copiously hispid with simple or stellate hairs to 2 mm (Dmitrovic et al., 2014). In this study aimed to evaluate the effects of aqueous extracts from fresh and dry leaves of Marachra capitata L., the influence of 5 various planting media on germination and seedling growth of tested weeds, and action of allelochemicals in different plating materials.
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Materials and Methods
Plant Materials Mature and healthy leaves of M. capitata L. plants were collected from the plant growing at the experimental field of King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand, immediately cleaned of soil with running tap water and cut into small pieces (fresh leaves) dried in a hot-air oven at 45C for 3 days, and ground to powder (100 mesh) in an electric blender (dried leaves). Wild pea (Phaseolus lathyroides L.) used as representative of broadleaf weed. Seed of wild pea collected from an upland field, hard seed coats were scrubbed with No. 0 sandpaper to break their dormancy. Seeds of barnyard grass (Echinochloa crus-galli (L.) Beauv.) used as representative of grasses weed were placed in the shade at room temperature for 3 months and then incubated at 60°C in a hot-air oven for 48 hours to break their dormancy. Effects of Aqueous Extracts Bioassay Aqueous extracts were prepared from fresh and dried leaf of M. capitata by soaked 10 g of each material in 100 ml of distilled water at 10oC for 72 hours followed by filtration through three layers of cheesecloth to remove any debris. The inhibitory activities of aqueous extracts from fresh and dry leaves was tested at the concentrations of 1.25%, 2.5%, 5% and 10% (w/v) on seed germination and seedling growth of barnyard grass and wild pea in Petri dish test. Five milliliters of each treatment was added to the germination paper placed in each 9 cm diameter glass petri dish. Then, twenty uniform seeds of barnyard grass and wild pea were placed in each petri dish. Effects of dried leaves of M. capitata L. in different soil types Various planting media used in this experiment was fertile soil, fertile sand, sterile soil and sterile sand. Soil and sand sterilized by using an autoclave at a temperature of 120°C and pressure at 15 psi for 20 minutes. Fifteen grams of each planting media was added into glass petri dish placed with germination paper, then dried leaf powder of M. capitata at the rates of 250 and 500 mg/petri-dish was added into separated petri-dish for their inhibitory test. Twenty uniform seeds of barnyard grass and wild pea placed in each petri dish. Four replicates were maintained per treatment in a completely randomized manner in a growth chamber with a temperature of 25-31oC, 12/12h dark/light photoperiod, and relative humidity of around 80%. Treatment with distilled water was used as control. Germination was deemed to occur only after the radical had protruded beyond the seed coat by at least the dimension of the seed at seven days after treatment. Seedling growth was measured as the root and shoot lengths at seven days after treatment. The percentages of growth were calculated from the following equation: Inhibition (% of control) = 100-(treatment/controlx100) Statistical analysis Each treatment consisted of four replications in completely randomized design. Analysis of variance was calculated for all data and comparisons between treatments were made at probability level p ≤ 0.05 using Tukey’s Studentized Range Test. Results and Discussion Effects of aqueous extracts from fresh and dried leaves of M. capitata L. on seed germination and seedling growth of barnyard grass and wild pea. Inhibition effects of aqueous extracts from fresh and dry leaves of M. capitata were assayed at concentrations of 1.25%, 2.5%, 5% and 10%(w/v) on seed germination and seedling growth of barnyard grass and wild pea compared with distilled water as control. The results indicated that degree of inhibitory was significantly different depending on difference source
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and concentration being tested. It was found that aqueous extract from dry leaves had inhibitory effect on seed germination and seedling growth of barnyard grass and wild pea was stronger than aqueous extracts from fresh leaves and the inhibition effect increased with increasing the concentration. At 5% aqueous extract from dry leaves completely inhibited seed germination of wild pea and barnyard grass had completely inhibited by concentration of 10% (Figure 1). barnyard grass
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Figure 1 Effects of aqueous extracts from fresh and dried leaves of Marachra capitata L. on seed germination and seedling growth of barnyard grass and wild pea. Effects of dried leaves of M. capitata L. in different planting media on seed germination and seedling growth of barnyard grass and wild pea. The influenced of 5 various planting media; fertile soil, fertile sand and sterile soil, sterile sand and dried leaves powder alone were bio-assayed on seed germination of barnyard grass and wild pea. The results indicated that dried leaves or powder alone at 500 mg/petri-dish had completely inhibited seed germination and seedling growth of barnyard grass and wild pea followed by sterile sand, fertile sand, sterile soil and fertile soil, respectively (Figure 2).
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barnyard grass
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Figure 2 Effects of dried leaves of M. capitata L. in different planting media on seed germination and seedling growth of barnyard grass and wild pea. Conclusion Aqueous extract from dry leaves had more inhibitory effect than aqueous extracts from fresh leaves on seed germination and seedling growth of barnyard grass and wild pea. The inhibitory effect of dried leaves depended on planting media; the inhibitory was low in the soil as compared to the seed indicating that the allelochemicals activity of M. capitata was interfered by the soil properties. Acknowledgements This research was supported by grants from Office of the Higher Education Commission (OHEC) pass through Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, Thailand. References Fraenkel, G.S. 1959. The Raison d'Etre of Secondary Plant Substances. Science 129: 14661470. The Encyclopedia of Plants in Thailand, 2012. Available online: http://web3.dnp.go.th/ botany/dictindex.aspx Dmitrovic, S., A. Simonovic, N. Mitic, J. Savic, A. Cingel, B. Filipovic and S. Ninkovic. 2014. Hairy root exudates of allelopathic weed Chenopodium murale L. induce oxidative stress and down-regulate core cell cycle genes in Arabidopsis and wheat seedlings. Plant Growth Regul. 1-18.
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P-18
Effects of Natural Herbicide from Piper betle Linn. on Seed Germination, Imbibition and α- Amylase Activity of Amaranthus gracilis desf. Pariyaporn NETSAWANG1, Pattharin WICHITTRAKARN1, Montinee TEERARAK1, Chamroon LAOSINWATTANA1* 1
Department of Plant Production Technology, Faculty of Agricultural and Technology, King Mongkut’s Institute of Technology Ladkrabang Bangkok 10520 *Corresponding email: klchamro@kmitl.ac.th
ABSTRACT The study on natural product herbicide from Piper betle Linn. in soluble liquid (SL) formula at concentrations of 312 625 1,250 and 2,500 ppm on seed germination, seed imbibition and α- amylase activities of Amaranthus gracilis Desf. were evaluated. The distilled water was used as control. The results showed that at 2,500 ppm completely inhibited seed germination of A. gracilis. The repeated experiment was conducted at concentrations 2,500 ppm on seed imbibition and α-amylase activities of A. gracilis. The results showed that seed germination of A. gracilis decreased, when increasing the concentration. These results indicated that the inhibitory effects of natural herbicide on seed germination of A. gracilis might be resulted from inhibiting seed imbibition and decreasing of α-amylase activities of A. gracilis. Keywords: Natural herbicide, P. betle, A. gracilis, α-amylase, Imbibition Introduction Nowadays, the residue of chemicals and pesticides in agriculture has become an important issue because of its effects on the health and the environment. However, the advantage in science and technology is growing rapidly; the development of natural herbicides that affect friendly environment has been more interested for sustainable agriculture. Depending on the plant, allelopathic substances can be released from a plant’s flowers, leaves, leaf debris and leaf mulch, stems, bark, roots, or soil surrounding the roots (Rice, 1984). Allelopathic compounds have been considered as the natural herbicide potential. The Piper betle is an evergreen and perennial creeper containing an articulate with the short roots. It can be used for medicinal properties such as anesthetic effects and stimulates blood circulation. The previous studies suggested that a high concentration of aqueous extract inhibits the growth of tested plants. However at the low concentration, it can stimulate the growth of plants. The allelopathy potential from the plant depends on each species and stage of its growth by gene regulation. The purposes of this research were to assess the potential of natural product herbicide from P. betle on the germination, seed imbibition and α-amylase activity of Amaranthus gracilis. Materials and Methods Plant materials Leaves of P. betle were collected from plants growing at the experimental field at Suphan Buri province, Thailand. The leaves were cleaned from soil immediately with running tap water, dried-up in a hot-air oven at 45°C for 3 days and ground to powder (100 mesh) in an electrical blender. Germination and seedling growth bioassay Seeds of A. gracilis were collected from upland field at Ladkrabang district, Thailand. Doseresponse study was conducted under laboratory conditions using Petri dish test. Petri dishes July 1-3, 2015
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were lined with two layers of germination paper moistened with 5 ml of 0, 312, 625, 1,250 and 2,500 ppm of tested natural product herbicide and 100 seeds of A. gracilis were placed into Petri dish. Four Petri dishes were maintained as replicates for each treatment in a completely randomized design. Petri dishes were placed in a growth chamber with a temperature of 25/32°C, 12 h/12 h dark/light photoperiod, light intensity of 100 µmol m-2s-1, and relative humidity of ~80%. The rate of germination was measured every day, and shoots and roots lengths of the seedling were measured at 7 days after treatment. Seed imbibition and α-amylase activity bioassay Measurement of seed imbibition was done by following the method of Teerarak et al., (2012). The 100 uniform seeds of A. gracilis were weighed and recorded as original seed weight (W1), and were separately germinated in 5 ml of natural product herbicide at difference concentration (312, 625, 1,250 and 2,500 ppm) and distilled water used as control. Seed weight was recorded as final seed weight (W2) for each concentration and exposure time. Sets of this experiment were the same as above experiment except the amount of seed number (100 seeds) per Petri dishes. After measuring imbibition, seeds were homogenized with a 4 ml ice-cold solution of 0.1 M CaCl2 and centrifuged at 9600 × g for 10 min. Supernatant was used as the enzyme extract. The α-amylase was then assayed by measuring the rate of generation of reducing sugars from soluble starch. The reaction medium (3 ml) contained 1 ml of 1% soluble starch in acetate buffer solution at pH 5.5 and 1 ml of the enzyme. The assay medium was incubated for 15 min at 37oC. The reaction was terminated by adding of 1 ml dinitrosalicylic acid (DNS reagent; 40 mM 3,5dinitrosalicylic acid, 0.4 N NaOH and 1M K-Na tartrate), and immediately heated in a boiling water bath for 5 min. The mixture was cooled under running tap water. A total volume was made up to 7 ml with distilled water. The intensity of color was measured as absorption at 560 nm in a spectronic GENESYS 20 spectrophotometer. A standard graph was prepared using maltose, and the amount of α -amylase present in the sample was calculated from the standard curve and expressed as µmol maltose min-1 g-1(FW) (Feo et al., 2002). Statistical analysis Data were subjected to analysis of variance and the treatment mean were separated by Tukey’s Studentized Range Test of significance at the P≤ 0.05 levels. Results and discussion Germination and seedling growth bioassay The results (Table 1) showed that inhibition on seed germination increased with increasing concentration of natural product herbicide from P. betle. At concentration 2,500 ppm caused complete inhibition on seed germination and seedling growth of A. gracilis. Besides, percentage germination at concentration of 312, 625 and 1,250 ppm were 71.25, 42.5 and 27.5%, respectively. The natural product herbicide not only reduced the percent germination but also the seedling growth of A. gracilis. There was drastic reduction in shoot and root length of germinated seeds with increasing concentration of natural product herbicide. The herbicidal action of natural product herbicide was similar results in terms of germination percentage and seedling growth were reported by Kato and Macías, (2005) and Batish et al., (2001), hence these results indicated that the highest amount of allelopathic effects was recorded in the seedling growth and seed germination.
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Table 1 Effects of natural product herbicide from Piper betle L. on germination and seedling growth of Amaranthus gracilis. Inhibition
Natural product
Germination (%)
Shoot (cm.)
Root (cm.)
Control
100.00a
2.90a
2.03a
312 ppm
71.25b
1.79b
1.81a
625 ppm
42.50c
1.56c
1.51b
1,250 ppm
27.50d
0.89d
0.69c
2,500 ppm
0.00e
0.00e
0.00d
Note: In each column, means having the same letter are not significantly different by Tukey’s Studentized Range Test of significance at the P≤ 0.05.
Effect of seed imbibition The effect of natural product herbicide on seed imbibition of A. gracilis during seed germination was investigated, as shown in (Figure 1). At soaking period 6, 12, and 24 h, the results showed that percentages of seed imbibition increased when increasing soaking period. The percentages of seed imbibition treated with natural product herbicide at the concentration of 312 to 2,500 ppm was 20.18% to 17.83% at imbibition time of 6h, 26.66% to 22.57% at imbibition time of 12h and 30.28 % to 15.37% at imbibition time of 24h, respectively. While the percentages of seed imbibition of control were 22.93, 28.23 and 32.21 % at imbibition time of 6h 12h and 24h, respectively. These results indicated that the seed imbibition increased with increasing soaking period whereas decreased with increasing concentrations. This result was similar with Singh et al., (2005) who reported that aqueous extract from Partenium hysterophorus inhibited seed imbibition to A. gracilis. Effect on α-amylase activity The effect of natural product herbicide from P. betle on α-amylase activities of A. gracilis seeds during seed germination was also investigated (Fig.1). The α-amylase activity of seeds increased by prolonging the soaking period (6–24 hours) under the same natural product herbicide concentration. The results showed that incubation of 6 h in this natural product herbicide, value of α-amylase activity was observed about 18.22 mol maltose min-1g-1(FW) at 312 ppm, but it decreased down to 10.57 mol maltose min-1g-1(FW) at 2,500 ppm. After the further incubation period (12 h), value of α-amylase activity was observed about 26.27 mol maltose min-1 g-1(FW) at 312 ppm, but it decreased down to 17.43 mol maltose min-1 g-1(FW) at 2,500 ppm. At the longest incubation (24h), value of α-amylase activity was observed about 30.57 mol maltose min-1g-1(FW) at 312 ppm, but it decreased down to 19.32 mol maltose min-1 g-1(FW) at 2,500 ppm compared with control. The α-amylase activity decreased by increasing the concentration of the natural product herbicide at increased all soaking time and similar results in terms of α-amylase activity inhibition were reported of A. gracilis.
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Figure 1 Effects of natural product herbicide from Piper betle L. on water uptake and αamylase activities of Amaranthus gracilis during seed germination. The values represent treatment means. Different letters indicate significant differences (p<0.05) between treatment times. Conclusion The natural product herbicide from P. betle in soluble liquid (SL) formulation at the concentration of 2,500 ppm completely inhibited on seed germination and seedling growth of A. gracilis. The seed imbibition and α-amylase activities decreased with increasing concentration of natural herbicide from P. betle. These results suggested that inhibition on seed germination of natural product herbicide might cause by reducing of seed imbibition and α-amylase activities of A. gracilis weed during seed germination. Hence, the use of natural product herbicide from P. betle for weed control might be possible. Acknowledment The authors wish to thank King Mongkut’s Institute of Technology Ladkrabang for providing financial support for this research. References Batish, D.R., H.P. Singh, R.K. Kohli and S. Kaur. 2001. Crop allelopathy and its role in ecological agriculture. Crop Production 4: 121-161. Feo, V.D., F.D. Simone and F. Sentore. 2002. Potential allelochemicals from the essential oil of Ruta graveolens. Phytochemistry 61: 573-578. Kato-Noguchi, H. and F.A. Macías. 2005. Effects of 6-methoxy 2-benzoxazolinone on the germination and -amylase activity in lettuce seeds. Journal of Plant Physiology 162: 1304-1307. Rice, E.L. 1984. Allelopathy Second ed. Acacemic Press, Inc, Olendo. Singh, H.P., D.R. Batish, J.K. Pandher and R.K. Kohli. 2005. Phytotoxic effects of Partenium hysterophorus residues on three brassica species. Weed Biology and Management 5: 105 -109. Teerarak, M., P. Charoenying and C. Laosinwattana. 2012. Physiological and cellular mechanisms of natural herbicide resource from Aglaia odorata Lour. on bioassay plants. Acta Physiologiae Plantarum 15:1-9.
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P-19
Effect of Different Temperature on Carotenoid Content and Antioxidant Activity in ‘Khak Dam’ Papaya Kanthee SIRIVEJABANDHU1 and Lampan KHURNPOON 1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institutes of Technology Ladkrabang, Bangkok 10520 Thailand *Corresponding email: kplampan@gmail.com
ABSTRACT The effect of different temperatures (4, 12 and 25°C) on carotenoid content and antioxidant activity in whole fruits and fresh cut ‘Khak Dam’ papaya fruits were studied. At 6 days in storage of whole fruits at 4 and 12°C showed significant difference in L*, a* and b* values in peel and pulp color from fruits stored at 25°C. Fruits stored at 4 and 12°C had lower in a* value of the peel about -10.1 and -8.6, respectively, while it was about 3.3 when stored at 25°C. Low temperature storage could delay the reduction in fruit firmness, about 126.4, 98.9 and 66.1 N after being stored at 4, 12 and 25°C, respectively, but lower in percentage of weight loss and TSS/TA ratio. The percentage of free radical inhibition in fruit stored at 4, 12 and 25°C were about 62, 64 and 70%, respectively. Highest carotenoids content was found when fruit stored at 4°C about 3.4 µg/g FW. In fresh cut sample after being stored at 4 and 12°C showed significant difference in L* and a* values of the pulp but no difference in b* value when compared to sample stored at 25°C. Fruits stored at 4 and 12°C could delay the change in a* value of the pulp and preserved the fruit firmness better than fruit stored at 25°C. Significant difference between storage temperatures was found in percentage of weight loss but not in TSS/TA ratio. Low temperature storage could maintain the change in carotenoid content but lower in the percentage of free radical inhibition than stored at 25°C. Keywords: Papaya, Weight loss, Free radical, Carotenoid, Antioxidant Introduction Papaya (Carica papaya L.) fruit is rapidly becoming and important commodity worldwide, both as a fresh fruit and as processed products. Papaya is a very healthy fruit, and it is appreciated because of its attractive pulp color, flavor, succulence, and characteristic aroma (Rivera-Lopez et al., 2005). Papaya fruit grow in tropical and sub-tropical regions and are marketed around the world. Several tropical fruits are rich in antioxidants such as polyphenols, vitamins and carotenoids (Rivera-Pastrana et al., 2010). Like other climacteric fruits, papaya undergoes a series of biochemical changes after harvest. Although low temperature may extend storage life of tropical fruit, it also causes chilling injury, characterized by browning of the skin, greater firmness and off-flavours in the fruit (Mitra, 1997). Papaya is a good source of carotenoids; natural pigments responsible for the color of the fruit and related to biological functions or action in human such as provitamin A activity, carotenoids present in red-fleshed papayas are more efficient antioxidants and have been linked reduction of the risk of cancer (Ahmed et al., 2012) Several carotenoids such a ßcarotene, lycopene, lutein, and zeaxanthine are known to exhibit antioxidant activity, but ßcarotene has been the most thoroughly studied. As a group, vitamin C, E, and ß-carotene comprise the so-called antioxidant vitamins (Kaur and Kapoor, 2001). The objective of this study was to determine the effect of temperature on the amount of carotenoid content and antioxidant activity in ‘Khak Dam’ papaya.
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Material and Methods ‘Khak Dam’ papaya fruits were cleaned with water and air-dried. Samples were separated into two groups; whole fruit and fresh cut samples. Fruits were peeled then cut into the size of 2*3 cm2, packed on foam tray and wrapped with PVC film. Both groups of samples were stored at 4, 12 and 25°C until senescence. Peel and pulp color change was measured by Color Flex spectrophotometer and reported as L*, a* and b* values. The L* value represented the lightness, a* value represented the redness (+a) or greenness (-a) and b* value represented the yellowness (+b) or blueness (-b). Pulp firmness was measured by using fruit firmness tester and reported as newton (N). Total soluble solids (TSS) content was measured from the fruit juice by using hand refractometer and report as %brix. Titratable acidity (TA) content was measured from the fruit juice titrated with a standard alkaline solution (0.1N NaOH) and report as %titratable acidity. The percentage of weight loss was obtained from the different between the initial weight and one at the end of storage. Extraction and analysis of carotenoid content were done by modified the method of Dere et al. (1998) and the percentage of free radical inhibition by modified the method of Torun et al. (2013). Result and Discussion After 6 days in storage, whole fruits stored at 4 and 12°C showed significantly difference in L*, a* and b* values in peel and pulp color from fruits stored at 25°C. Fruits stored at 4 and 12°C had lower in lightness (L* value), redness (a* value) and yellowness (b* value) of the peel when compared with fruits stored at 25°C. This result confirmed by the report from Caron et al. (2013) that high temperature increase ripeness by increasing external color papayas. In the pulp color change, fruits stored at low temperature had higher in L* value than fruits stored at 25°C but lower in a* and b* value than fruits stored at 25°C. Fruits stored at 4 and 12°C had higher in fruit firmness than fruit stored at 25°C about 65 and 37N, respectively, on 6 days and preserved at 133.9 and 20.8N at the end of storage. Fruits stored at low temperature had lower in the percentage of weight loss than fruits stored at 25°C approximately 3 and 7%, respectively. Fruits stored at 4 and 12°C had the TSS/TA ratio lower than fruits stored at 25°C. The TSS/TA ratio was about 35.2, 39.1 and 49.9 at the end of storage when stored at 4, 12 and 25°C (Table 1). Carotenoid content in fruits stored at 4, 12 and 25°C decreased during storage. Fruits stored at 25°C had rapidly decreased in carotenoid content from 3.4 to 1.8µg/g FW on days 3 then to about 1.5µg/g FW at the end of storage. Fruits stored at 4 and 12°C had lower carotenoid content at the beginning than those stored at 25°C. It was about 1.7 and 1.5µg/g FW on days 12 (Table 1). Rivera-Pastrana et al., 2010, reported the effect of low temperature on carotenoid content was observed lower carotenoid levels in papaya fruit stored at 1°C could be a result of a delayed ripening or abnormal maturation as a symptom of chilling injury. Maximum levels of carotenoids were observed in fruit stored at 25°C, which correspond to the development of orange-red mature flesh color. At 12 days after storage fruits stored at 4 and 12°C had the percentage of free radical inhibition about 61.5 and 68.6%, respectively (Table 1).
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Table1
Changes in weight loss (%), firmness (N), TSS/TA ratio, carotenoid content (µg/g FW) and Free radical inhibition (%) in whole fruit ‘Khak Dam’ papaya stored at different temperature for 12 days. Quality parameters
Storage temperatures
Percentage of weight loss
Pulp firmness
TSS/TA ratio
Carotenoid content
Percentage of free radical inhibition
4oC
10.0±0.41/
133.9±10.6
35.2±2.6
1.7±0.2
61.5±6.4
o
5.5±0.6
20.8±6.1
39.1±6.2
1.5±0.8
68.6±2.3
61.6±7.8
49.9±4.6
1.5±0.6
70.8±8.4
12 C o
1/
25 C 8.8±0.3 Data shown are mean ± standard deviation.
In fresh cut sample after stored at 4 and 12°C showed significantly difference in L* and a* values of the pulp but no difference in b* value when compared to sample stored at 25°C. At 3 days after storage, fruits stored at 4 and 12°C had higher in L* value than fruits stored at 25°C were about 64.3, 64.2 and 60.1, respectively. Fruits stored at 4 and 12°C could delay the change in a* value of the pulp color but not difference in b* value of the pulp among treatment (Table 2). Fruits stored at 4, 12 and 25°C had significantly different in fruit firmness and then decrease during storage. During 3 days in storage, fruits stored at 25°C had decreased rapidly in fruit firmness. Fruits stored at 4 and 12°C had higher in fruit firmness than fruit stored at 25°C about 124 and 80N, respectively, on days 6 and preserved at 107.5 and 35.1N at the end of storage. Fruits stored at 4 and 12°C had lower percentage of weight loss than fruits stored at 25°C approximately 7 and 6%, respectively (Table 2). The firmness loss of fresh cut papaya ‘Maradol’ was observed as a result of higher storage temperature (Rivera-Lopez et al., 2005). Fruits stored at 4 and 12°C had the TSS/TA ratio higher than fruits stored at 25°C. The TSS/TA ratio was about 49.7, 42.6 and 35.1 at the end of storage when stored at 4, 12 and 25°C (Table 2). Carotenoid content in fruits stored at 4, 12 and 25°C decreased during storage. Fruits stored at 25°C had rapidly decreased in carotenoid content from 3.4 to 1.5µg/g FW on days 3 in storage. Fruits stored at 4 and 12°C had lower carotenoid content at the beginning than those stored at 25°C. It was about 1.8 and 1.4µg/g FW at the end of storage (Table 2). Falah et al., 2015 reported carotenoid content of fresh-cut papaya was still increase during stored under different storage conditions, this indicate that the sample of the fresh-cut papaya still to be maturity and ripening processes. At the end of storage fruit stored at 4, 12 and 25°C had percentage of free radical inhibition were about 64, 60 and 77%, respectively (Table 2). The storage temperature significantly affected the ORAC value of fresh-cut papaya fruit. Fresh-cut papaya changed slightly during storage at 5°C. However, significant reduction of ORAC values were found in fresh-cut papaya at 10°C and 20°C (Rivera-Lopez et al., 2005). Table 2 Changes in pulp color, weight loss (%), firmness (N), TSS/TA ratio, carotenoid content (µg/g FW) and free radical inhibition (%) in fresh cut ‘Khak Dam’ papaya stored at different temperature for 12 days. Quality parameters Pulp color
Storage temperatures
L*
a*
b*
Percentage of weight loss
Pulp firmness
TSS/TA ratio
Carotenoid content
Percentage of free radical inhibition
4oC
64.4±7.01/
13.0±7.1
30.5±2.5
7.8±2.9
107.5±6.6
49.7±8.0
1.8±0.2
63.6±8.4
o
65.1±6.4
12.0±7.2
30.4±2.8
9.3±8.5
35.1±7.2
42.6±6.0
1.4±0.5
59.5±6.9
25oC 60.1±5.9 20.8±4.0 31.0±2.9 1/ Data shown are mean ± standard deviation.
8.9±4.6
4.3±1.3
35.1±3.5
1.5±0.5
77.0±6.1
12 C
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Conclusion ‘Khak Dam’ papaya fruits stored at 4 and 12°C showed lower significantly difference in L*, a* and b* values in peel and pulp color change than fruits stored at 25°C. Fruits stored at 4 and 12°C could maintained pulp firmness, reduced percentage of weight loss, TSS/TA ratio and percentage of free radical inhibition but higher in carotenoid content than fruits stored at 25°C. In fresh cut sample after stored at 4 and 12°C showed significantly difference in L* and a* values in pulp color change but no difference in b* value when compared to stored at 25°C. Fruits stored at 4 and 12°C had significantly lower pulp softening, percentage of weight loss and percentage of free radical inhibition than fruits stored at 25°C, but no difference in TSS/TA ratio among treatments. Fruits stored at 4, 12°C had carotenoid content higher than fruits stored at 25°C. Acknowledgements We thank the Faculty of Agricultural Technology, KMITL for financial support. References Ahmed, J., M. G. Lobo and F. Ozadali. 2012. Tropical and Subtropical Fruits: Postharvest Physiology, Processing and Packaging. John Wiley & Sons, Iowa. 648p. Caron, V. C., G. M. Chitolina, and A. P. Jacomino. 2013. Influence of Low Temperature Storage on the Postharvest Quality of Papaya Fruit (Carica papaya L.). Proceedings of the Florida State Horticultural Society 126: 200–202. Dere, S., T. Gunes and R. Sivaci. 1998. Spectrophotometric determination of chlorophyll–A, B and total carotenoid contents of some algae species using different solvents. Turkish Journal of Botany 22: 13-17. Falah M. A. F., M. D. Nadine and A. Suryandono. 2015. Effect of Storage Conditions on Quality and Shelf-life of Fresh-cut Melon (Cucumis melo L.) and Papaya (Carica papaya L.). Procedia Food Science 3: 313-322. Kaur, C. and H.C. Kapoor. 2001. Antioxidants in fruits and vegetables-the millennium’s health (Review). International Journal of Food Science & Technology 36: 703–725. Mitra, S.K., 1997. Postharvest Physiology and Storage of Tropical and Subtropical Fruits. CABI Publishing, New York. 441p. Rivera-Lopez, J., F. A. Vazquez-Ortiz, J. F. Ayala-Zavala, R. R. Sotelo-Mundo and G. A. Gonzalez-Aguilar. 2005. Cutting Shape and Storage Temperature Affect Overall Quality of Fresh-cut Papaya cv. ‘Maradol’. Journal of Food Science 70 (7): 482-489. Rivera-Pastrana, D. M., E. M. Yahiab and G. A. Gonzalez-Aguilar. 2010. Phenolic and carotenoid profiles of papaya fruit (Carica papaya L.) and their contents under low temperature storage. Journal of the Science of Food and Agriculture 90 (14): 2358- 2365. Torun, H., F. A. Ayaz, N. Colak, J. Grúz and M. Strnad. 2013. Phenolic Acid Content and Free Radical-Scavenging Activity of Two Differently Processed Carob Tree (Ceratonia siliqua L.) Pod. Food and Nutrition Sciences 4: 547-553.
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P-20
Nitric Oxide Improves Cowpea (Vigna unguiculata [L.] Walp.) Growth Under Lead Stress Omid SADEGHIPOUR1* Department of Agronomy, Yadegar-e-Imam Khomeini (RAH) Branch, Islamic Azad University, Tehran, Iran * Corresponding author: osadeghipour@yahoo.com
ABSTRACT Lead (Pb) is one of the hazardous heavy metals which its increasing levels in soil exerting a wide range of adverse effects on growth and metabolism of plants. Nitric oxide (NO) is an important signal molecule modulating responses to both biotic and abiotic stresses in plants. In the present study, the effect of exogenous NO on some traits of cowpea plants exposed to 0 and 200 mg Pb (NO3)2 kg-1 soil was investigated. Seeds soaked for 20 h with 0.5 and 1 mM sodium nitroprusside (SNP) as NO donor. Results showed that Pb toxicity significantly reduced plant height (35%), seed protein content (21%) and protein yield (65%) as compared to control. On the other hand, exogenous NO improved measured traits under lead stress. Both NO levels were effective in Pb stress tolerance although 0.5 mM was more effective. These results indicate that NO plays an important role in protecting cowpea plants against Pb toxicity. Keywords: Pb toxicity, Plant height, Protein yield, Seed protein content Introduction Lead (Pb) is one of the potentially toxic heavy metal pollutants of the environment with no known biological function and its concentrations are rapidly increased in agricultural soil. The most significant factors which can distribute lead as a pollutant in the environment are burning of fossil fuels, agricultural manufacturing, mining, pesticides and fertilizers. In plants, it has been widely reported that accumulation of Pb may cause many physiological, biochemical and structural changes like decline in photosynthetic rate and essential elements absorption, the roots and shoots growth inhibition, chlorosis and decrease in water potential and plant hormones. Also, this metal can generate different types of reactive oxygen species (ROS) which can disturb cell membrane activities (Noorani Azad et al., 2011) Nitric oxide (NO) is a small highly diffusible and ubiquitous bioactive molecule that takes part in many physiological processes in plants. Its chemical properties makes NO a versatile signal that functions through interactions with cellular targets via either redox or additive chemistry. It is involved in seed germination, growth and development, flowering, hormonal responses, mitochondrial functionality and gravitropism. NO is itself a ROS and its dual behavior (protective or toxic) depends upon both concentration and the tissue where it acts. The protective role is based on its ability to regulate the level and toxicity of ROS. NO has been suggested to be involved in defense responses to biotic and abiotic stresses and appears to be present in most of stress reactions. It has been reported to exert a protective effect in response to drought stress, osmotic stress, salt stress, heavy metal stress and oxidative stress (Kumari et al., 2010). However, experimental evidences for NO effects on Pb toxicity in cowpea are very limited. The present study, therefore was done in order to investigate the impact of exogenous application of NO on plant height, seed protein content and protein yield of cowpea under lead stress. July 1-3, 2015
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Materials and Methods This pot experiment was conducted in summer 2014 in research field of the Yadegar-e-Imam Khomeini Branch, Islamic Azad University, Tehran, Iran. Longitude, latitude and altitude are 51° 28´ E, 35° 35´ N, and 1000 m, respectively. Cowpea (Vigna unguiculata cv. Kamran) seeds without visible defect, insect damage and malformation were surface sterilized using 5% sodium hypochlorite solution for 5 min and then rinsed 3 times with sterile distilled water. For the pretreatment, seeds were soaked in different levels (0, 0.5, and 1 mM) of sodium nitroprusside (SNP) as NO donor for 20 h. Then seeds were sown in 50 cm in diameter and depth; plastic pots filled with 20 kg soil containing an equal mixture of peat, decomposed manure and farm soil. Before sowing; soil of pots was mixed with appropriate amount of Pb (NO3)2 to supply 0 and 200 mg kg-1 soil. Sowing date was 20th June 2014 and then pots were placed in farm conditions. The pots were arranged in a completely randomized design with three replicates. In each pot, 20 seeds were sown in 3 cm depth and at 3 leafy stages after thinning; 6 seedlings remained. Irrigation was carried out regularly at the plant needs using tap water. At physiological maturity; plants height was recorded. Afterwards plants were harvested and sundried. Then seed yield per plant was measured. Seed protein content was calculated by kjeldahl method (6.25 × N). Protein yield was determined by product of seed yield and seed protein percentage. Collected data were analyzed by MSTATC statistical software and the means were compared by Duncan′s Multiple Range Test (DMRT) at the 5% probability level. Results and Discussion Pb toxicity significantly reduced cowpea plant height by 35% compared with the control. Nonetheless, seeds soaking in SNP raised plant height under lead stress. 0.5 and 1 mM concentrations of SNP under Pb stress increased plant height by 30% and 23%, respectively, as compared to Pb treatment alone. In lead stress conditions, the highest plant height was observed in 0.5 mM SNP application, which was statistically at par with 1 mM (Figure 1). Similar to our results; Pb addition caused a significant reduction in plant height of cotton at both Pb levels (50 and 100 μM) compared with the control and the decrease in growth was dose-dependent (Bharwana et al., 2014). Verma and Dubey (2003) also found that with 1000 mM Pb in the medium, up to 40% reduction in root length and 31% reduction in shoot length was observed in 20-day grown rice seedlings. Decreased seedling vigor in rice due to Pb could possibly be attributed to the interference of Pb with the metabolic and biochemical processes associated with normal growth and development of the plant. Alike to our findings, exogenous NO increased shoot length of maize under boron stress (Esim and Atici, 2013) and canola under cadmium toxicity (Jhanji et al., 2012). A noticeable decrease in seed protein content was observed in Pb treatment resulted in a decline by 21% as compared to control. SNP pretreatment increased seed protein content under lead stress. At 0.5 and 1 mM concentrations of SNP under Pb stress elevated seed protein content by 17% and 10%, respectively, compared with the Pb treatment alone. In lead stress conditions, the maximum seed protein content was recorded in 0.5 mM SNP application, which was statistically at par with 1 mM (Figure 1). The effect of lead on the total concentration of protein is unclear, although high concentrations may decrease the protein pool. This quantitative decrease in total protein content is the result of several lead effects: acute oxidative stress of ROS, modification in gene expression, increased ribonuclease activity, protein utilization by plants for the purposes of lead detoxification and diminution of free amino acid content that is correlated with a disturbance in nitrogen metabolism. However, certain amino acids, like proline, increase under lead stress. Such proteins play a major role in the tolerance of the plant to lead (Pourrut et al., 2011). Kausar et
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al. (2013) reported that exogenously applied NO significantly increased soluble proteins in wheat cultivar S-24 under both saline and nonsaline regimes. As shown in Figure 1 protein yield of cowpea was affected negatively by Pb toxicity. A 65% decline was observed compared with the control. SNP treatment had a positive effect on protein yield under lead stress. 0.5 and 1 mM concentrations of SNP under Pb toxicity were increased protein yield by 108% and 58% respectively, as compared to Pb treatment alone. Both SNP levels were effective in improved protein yield of cowpea under lead stress. However, 0.5 mM was more effective. Excess Pb cause a number of toxicity symptoms in plants e.g. stunted
growth, chlorosis and blackening of root system. Pb inhibits photosynthesis, upsets mineral nutrition and water balance, changes hormonal status, and effects membrane structure and permeability (Sharma and Dubey, 2005). Similar to our results, yield of two genotypes of mung bean were declined by chromium and lead stresses (Hussain et al., 2006). Moreover, excess Pb reduced dry weight and grain yield of rice (Chatterjee et al., 2004). Kumari et al. (2010) observed that NO donor (SNP) treatments improved the chickpea yield and 50% increase in seed yield was observed with long and short term Cd stress. Application of NO caused high accumulation of K+ and low accumulation of Na+, and it increased the yield of wheat under saline conditions (Kausar et al., 2013). Jhanji et al. (2012) showed that SNP treatments resulted in enhancement of siliqua number and seed yield plant-1 in control as well as Cd-treated canola plants. The significant increase in yield with SNP treatments could be the result of enhanced supply of assimilates towards developing siliqua.
Control Pb
a
Pb+SNP 0.5 mM
Plant height (cm) Seed protein content (%) Protein yield (g plant-1)
b
Pb+SNP 1 mM
b
c
a
c
b b a
c
ab bc
Figure 1 Effect of Pb (200 mg kg-1 soil) and SNP (0.5 and 1 mM as NO donor) on plant height, seed protein content and protein yield of cowpea. Different letters in each trait indicate significant differences at P â&#x2030;¤ 0.05 level using DMRT.
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Conclusion The present results revealed that Pb caused significant decrease in cowpea stem height, seed protein content and protein yield. Exogenous NO ameliorated measured traits under Pb stress. It can be expected that NO would be a useful tool in reducing Pb toxicity. Acknowledgement The author gratefully acknowledges the Yadegar-e-Imam Khomeini (RAH) Shahre-Rey Branch, Islamic Azad University, Tehran, Iran for funding of this project. References Bharwana, S.A, S. Ali, M.A. Farooq, N. Iqbal, A. Hameed, F. Abbas and M.S.A. Ahmad. 2014. Glycine betaine-induced lead toxicity tolerance related to elevated photosynthesis, antioxidant enzymes suppressed lead uptake and oxidative stress in cotton. Turkish Journal of Botany 38: 281-292. Chatterjee, C., B.K. Dube, P. Sinha and P. Srivastava. 2004. Detrimental effects of lead phytotoxicity on growth, yield and metabolism of rice. Communications in Soil Science and Plant Analysis 35: 255-265. Esim, N. and O. Atici. 2013. Nitric oxide alleviates boron toxicity by reducing oxidative damage and growth inhibition in maize seedlings (Zea mays L.). Australian Journal of Crop Science 7: 1085-1092. Hussain, M., M.S.A. Ahmad and A. Kausar. 2006. Effect of lead and chromium on growth, photosynthetic pigments and yield components in mash bean [vigna mungo (L.) Hepper]. Pakistan Journal of Botany 38: 1389-1396. Jhanji, S., R.C. Setia, N. Kaur, P. Kaur and N. Setia. 2012. Role of nitric oxide in cadmiuminduced stress on growth, photosynthetic components and yield of Brassica napus L. Journal of Environmental Biology 33: 1027-1032. Kausar, F., M. Shahbaz and M. Ashraf. 2013. Protective role of foliar-applied nitric oxide in Triticum aestivum under saline stress. Turkish Journal of Botany 37: 1155-1165. Kumari, A., S. Sheokand and K. Swaraj. 2010. Nitric oxide induced alleviation of toxic effects of short term and long term Cd stress on growth, oxidative metabolism and Cd accumulation in chickpea. Brazilian Journal of Plant Physiology 22: 271-284. Noorani, Azad, H., A.H. Shiva and R. Malekpour. 2011. Toxic effects of lead on growth and some biochemical and ionic parameters of sunflower (Helianthus annuus L.) seedlings. Current Research Journal of Biological Sciences 3 (4): 398-403. Pourrut, B., M. Shahid, C. Dumat, P. Winterton and E. Pinelli. 2011. Lead uptake, toxicity and detoxification in plants. Reviews of Environmental Contamination and Toxicology 213: 113-136. Sharma, P. and R.S. Dubey. 2005. Lead toxicity in plants. Brazilian Journal of Plant Physiology 17 (1): 35-52. Verma, S. and R.S. Dubey. 2003. Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant Science 164: 645-655.
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P-21
Nutrient Dynamics in an Aquaponic System Somsak MANEEPONG1* 1
School of Agricultural Technology, Walailak University, Nakhon Si Thammarat, Thailand. * Corresponding email: msomsak@wu.ac.th
ABSTRACT Aquaponics is an integrated system of circulating aquaculture and soilless culture. The system mainly aims to reduce waste discharge and maximize nutrient use. An aquaponic system consisting of a 500 l fish tank, sedimentation and pH control tank, degassing tank and three vegetable growing beds was assembled and tested for 17 weeks. Fifty Nile Tilapia (Oreochromis niloticus) were reared and fed them three times daily with a complete diet containing 31.8% protein. Dead corals were installed to control pH, and maintain vegetables at different growth states in the system throughout the experiment. Water convolvulus (Ipomoea aquatica) and Tokyo Bekana (Brassica rapa) were rotationally grown. Water samples were collected once a week to analyze the pH, NH3/NH4+, NO3-, phosphate, SO42-, K, Ca, Mg and Fe. Fish weight increased from 50 g/fish at the beginning to 228 g/fish at the 15 weeks. Water pH increased from 6.0 before rearing to 7.0 at the 4th week, and varied within a small range of 6.9 - 7.0 until the end of the experiment without any additional acid or alkali. Total NH3/NH4+ increased to 10.2 mg-N/l on the 2nd week and rapidly declined to lower than 2.0 mg-N/l. Phosphate, SO42- and Mg accumulated in the system, whereas Ca gradually increased and reached equilibrium at 47 ď&#x201A;ą 2 mg/l. K and NO2-/NO3- varied significantly at low concentrations compared with the general requirement of vegetables. Both vegetables initially grew well at the first crops, but their growth rates declined greatly and showed complicated nutrient deficiency on the latter crops. Keywords: Aquaponic, Aquaculture, Nile Tilapia, Plant nutrient Introduction Aquaculture requires a large volume of water and discharges a large volume of wastewater simultaneously. An integrated system of recirculating aquaculture and soilless culture has been developed to solve this problem. Waste from aquatic animals will be converted to plant nutrients by microorganisms. The nutrients will be removed by plants in a soilless subsystem, and the clean water will be recirculated back to the aquaculture subsystem. However, the optimum nutrient concentrations for plant growth are difficult to maintain. Nutrients excreted from the aquaculture do not match plant requirements; hence, plant yield reduces over an extended culture period. The present study was conducted to examine the changing of nutrient concentrations in an aquaponic system. The growth rates of fishes and vegetables affected by the water properties were also examined. Materials and Methods An aquaponic system consisting of a 500 l fish tank, sedimentation and pH control tank, degassing tank, three floating raft growing beds and a sump tank was assembled. Dead corals were installed to control the pH in the system. Bio-balls were placed in the growing beds for microbial substrate. A submersible pump controlled by a floating switch was installed in the sump tank to recirculate the water back to the fish tank. The water in the system was totally recirculated with some compensation for evaporation loss. Fifty Nile Tilapia were reared and fed them three times daily with a complete diet containing 31.8% protein. Up to 15 fish were sampled for monthly measurement of weight gain. Water convolvulus and Tokyo Bekana July 1-3, 2015
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were seeded for a week and transplanted to the growing bed for 3 weeks. Growing area in each growing bed was 0.6 m2, and 58 vegetables were grown. The water depth in the growing beds was maintained at 15 cm. The vegetables were grown rotationally to maintain equal age in the system throughout the experiment. The vegetables were harvested once a week, their roots were removed, and their fresh weights were determined. Water samples from the fish tank were collected once a week to analyze the pH, NH3/NH4+, NO3-, phosphate, SO42-, K, Ca, Mg and Fe. Fe-DTPA (11.3% Fe) was added into the system during the second week at a rate of 15 mg/l. The pH was determined by a pH meter. The concentration of NH3/NH4+ was determined by distillation and titration method. The concentrations of NO3- and SO42- were determined using ion chromatography. The phosphate concentration was determined by a molybdenum blue method. The concentrations of K, Ca, Mg and Fe were determined using atomic absorption spectrophotometry. Results and Discussion Water pH increased from 6.0 to an equilibrium value of 7.0 Âą 0.1 from the fourth week until the end of the study without adding acid or alkali. Dissolution of CaCO3 from the dead corals provided excellent role for pH controlling. The concentration of NH3/NH4+ reached the maximum of 10.2 mg-N/l by the second week and sharply declined to a range of 0.6 mg-N/l to 3.1 mg-N/l (Figure 1). Nitrification process occurred naturally within two weeks. Ideally, NH3/NH4+ concentration should not exceed 1 mg-N/l, because it will affect fish respiration (Connolly and Trebic, 2010). This problem occurred because of occasional pipe clogging, which decreased the water flow rate during that period. The concentration of NO3- reached a first peak following NH3/NH4+, because it was transformed from NH3/NH4+ by the nitrification process. The concentration increased again after 12th week, because vegetable growth was restricted during that period. The NO3- concentration was very low compared with its toxic level to fish (300 mg-N/l to 400 mg-N/l; Connolly and Trebic, 2010) and the normal level for hydroponic solution (100 mg-N/l to 200 mg-N/l; Jones, 1997). 14 NH3/NH4+
Concentration (mg-N/L)
12
NO3 10 8 6 4 2 0 0
2
4
6
8
10
12
14
16
18
Week after starting
Figure 1 Variation of NH3/NH4+ and NO3- concentrations in the fish tank. Input source of N came from pellet feed containing 31.8% protein. The K concentration increased from 2.6 mg/l at the beginning to 11.2 Âą 1.8 mg/l. The vegetables withered under strong sun exposure, indicating that the diet did not supply K sufficiency for this system. Seawright et al. (1998) demonstrated that a large portion of K was retained by fish; furthermore, fish biomass and K showed a strong correlation. The K is generally insufficient in aquaponic systems. Thus, K may be added in the form of KOH to Page 262
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serve both as a nutrient supplement and an acid neutralizing agent (Rakocy et al., 2006). The concentrations of phosphate, SO42- and Mg gradually increased in the same trend (Figure 2), indicating that assimilation of these nutrients was less than fish excretion. Increasing rates after 12th week were faster than that the preceding weeks, because vegetable growths were highly restricted. The vegetables growing area should be enlarged to avoid excessive accumulation of these nutrients. Seawright et al. (1998) found that phosphate concentration sharply declined, due to precipitation of Ca3(PO4)2 at heater elements. The water temperature in the present study varied from 23.0 째C to 33.7 째C; therefore, heating was not required. The Ca concentration increased rapidly at the beginning from 4 mg/l to 48 mg/l and remained constant afterward (Figure 2). Releasing of Ca from dissolution of CaCO3 progressed well in acid condition, but slowed in neutral. Dead corals provided both pH control and Ca source of the system. Seawright et al. (1998) found that Ca concentration in their systems declined rapidly, and had to be supplemented with CaCl2 6H2O.
Concentration (mg/L)
50
40 Ca 30
sulfate Mg
20
phosphate 10
0 0
2
4
6
8
10
12
14
16
18
Week after starting
Figure 2 Variation of phosphate, SO42-, Mg and Ca concentrations in the fish tank. Input source of Ca came from both pellet feed and dead corals, while the other nutrients came from pellet feed alone. The Fe concentration sharply increased to 1.8 mg/l during the second week due to addition of the Fe-DTPA, and gradually decreased afterward (Figure 3). The system required approximately 206 mg-Fe/m2 of growing area each month (equivalent to 1.8 g/m2 of FeDTPA). Seawright et al. (1998) reported that quantity of Fe consumed by their aquaponic systems exceeded those provided by the diet. All fish survived throughout the 17-weeks study period. Fish weight increased from 50 g/fish to 228 g/fish within 15 weeks; having growth rate of 11.9 g/week, and feed conversion ratio (FCR) of 1.46. The system showed well performance for fish production. A combination culture of Nile tilapia and bell pepper system yielded a fish growth rate of 7.6 g/week and FCR of 1.81 (Kamal, 2006). Rakocy et al. (2006) reported Nile tilapia rearing in the UVI aquaponic system yielded a growth rate of 30.8 g/week and FCR of 1.7. Yields of water convolvulus and Tokyo Bekana at the beginning stages were better than those at the middle and latter stages (Table 1). Most nutrient concentrations at the beginning were low except Fe, but plant growth was excellent. The balance of nutrients is more important for plant growth than the concentrations.
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2.0
Fe (mg/L)
1.5
1.0
0.5
0.0 0
2
4
6
8
10
12
14
16
18
Week after starting
Figure 3 Changes in Fe concentration in the system. Fe-DTPA was added in the second week. Table 1 Yields of water convolvulus and Tokyo Bekana grown at different stages. Vegetables water convolvulus Tokyo Bekana
Vegetable yield (kg/m2) Beginning state Middle state Last state st th th th th (1 – 6 week) (7 – 12 week) (13 – 17th week) 3.74 ± 0.32 3.06 ± 0.30 2.60 ± 0.03 1.95 ± 0.04 0.73 ± 0.14 0.27 ± 0.06
Conclusion The aquaponic system achieved well performance for fish production, but it was not successful for vegetable production. The nitrification process started rapidly and progressed well. The NH3/NH4+ accumulated to higher than 2 mg-N/l, because of clogging in the recirculating pipe. Phosphate, SO42- and Mg accumulated in the system, whereas K and Fe were insufficient. Acknowledgments This study was supported by the Thailand Research Fund. References Connolly, K. and T. Trebic. 2010. Optimization of a backyard aquaponic food production system. Faculty of Agricultural and Environmental Sciences, McGill University. Jones, J.B. 1997. Hydroponics: A Practical Guide for the Soilless Grower. CRC Press. Boca Raton, Florida. Kamal, S.M. 2006. Aquaponic production of Nile tilapia (Oreochromis niloticus) and bell pepper (Capsicum annuum) in recirculating water system. Egypt J. Aquat. Biol. & Fish. 10(3):85-97. Rakocy, J.E., M.P. Masser and T.M. Losordo. 2006. Recirculating aquaculture tank production systems: Aquaponic – integrating fish and plant culture. Southern Regional Aquaculture Center Publication No. 454 Seawright, D.E., R.R.Stickney and R.B. Walker. 1998. Nutrient dynamic in integrated aquaculture-hydroponics systems. Aquaculture. 160:215-237.
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P-22
The Effects of Calcium Silicate on Density of Trichomes in Field Corn Pongsakorn NITMEE1, Pornpairin RUNGCHAROENTHONG1,* Suphachai AMKHA2 and Thongchai MALA2 1
Department of Science, Faculty of Liberal Arts and Science, Kasetsart University, KamphaengSaen Campus, Nakorn pathom 73140, Thailand 2 Department of Soil Science, Faculty of Agriculture, Kasetsart University, KamphaengSaen Campus Nakorn pathom 73140, Thailand *Corresponding email: faasprr@ku.ac.th
ABSARACT The effects of Ca2SiO4 were studied on density of trichome in field corn at Kasetsart University, KamphaengSaen Campus from March to November in 2014. Experimental design was 2x7 factorials in CRD with factor 1) non-coated seed and Ca2SiO4 coated seeds, factor 2) application rates of Ca2SiO4 in soil 0, 31.25, 37.50, 43.75, 50, 56.25 and 62.50 kg/ha. The results showed that Ca2SiO4 induced the density of trichome in leaves of field corn. Seeds coated with Ca2SiO4 gave the higher the number of trichomes (2.25 trichome/mm) than noncoated (0.75 trichome/mm). Both coated and non coated seeds combined with Ca2SiO4 31.2562.5 kg/ha in soil increased the number trichome 2-3 times higher than the control. The higher the rate of Ca2SiO4 into soil, the larger the number of trichome was. Moreover, coated seeds and combined withCa2SiO4 in soil increased more number of trichomes than noncoated seed. In addition, field corn treated with Ca2SiO4 increased in leaf thickness which was higher than the control. Keywords: Calcium silicate, Field corn, Trichome Introduction Field corn (Zea may) is used in the dairy and livestock industry. Corn is important in Thailand for both grain and silage. There is main component in feed about 39.7 % of raw material in 2009. Environmental change such as drought stress, flooding, low and height temperature or light intensity or lack of fertility at growth stage are all factors that can result in less than optimum yield of corn. Each of these factors, along with pest problems, can bring to yield potential. High demand of corn for livestock industrial and ethanol production increased dramatically. However, high quality of seed is important for corn production. Seed priming with nutrition Fe, Zn and Mn improved seedling quality (Muhammad et al., 2013). Also, seed coated with calcium silicate showed better, more uniform emergence and higher plant growth than uncoated seeds (Pongsakorn et al., 2015). Silicon can benefit plant growth through greater yields in wheat and cumber (Samules et al., 1993; Gong et al., 2003). Silicon also can be very useful especially when these plants are under abiotic or biotic stress. Silicon may enhance soil fertility, improve disease and pest resistance, regulate evapotranspiration, increase tolerance to toxic elements such as Al and heavy metal and promotes orientation of leaves toward the sun in such a way as to maximize light interception, photosynthesis, influences the absorption and transport of several mineral elements (Ma and Takahashi, 2002; Epstein and Bloom, 2005; Marschner, 2012). Trichomes are principal considered to be the mechanical against predators, pathogens and competitor (Wagner et al., 2004). Previous reported silicate application in soybean induced trichome and increased the resistance from Euschistus heros (Souza et al., 2014). This study to examine the effect of seed coated with
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Ca2SiO4 and combined with application Ca2SiO4 in soil on density of trichome and leaf thickness of field corn. Materials and Methods Seeds of field corn were non-coated and coated with Ca2SiO4 2,000 ppm. Experimental design was 2x7 factorials in CRD with factor 1) non-coated seed and Ca2SiO4 coated seeds, factor 2) application rates of Ca2SiO4 in soil 0, 31.25, 37.50, 43.75, 50, 56.25 and 62.50 kg/ha. Seeds of non-coated and coated with Ca2SiO4 were sown in polyethylene pots size 6 inch each capacity of 1 kg of planting media using 3:1 mixture of soil and coconut coir. Ca2SiO4 mixed into planting media at sowing time. The number of trichome and leaf thickness was observed in third leaf from apex at 21 days after sowing. The numbers of trichome were investigated under stereo-microscopic and photographed by MShot digital microscope camera. Leaf thickness was examined by cross-section at the middle of leaf blade and measured under compound microscopic. Results and Discussions Calcium silicate significantly increased the number of trichome and leaf thickness in field corn (Table 1). Seed coated with Ca2SiO4 induced the number of trichome was 5.11 trichomes/mm higher than non-coated at 1.93 trichomes/mm. The application rate of Ca2SiO4 31.25 to 62.5 kg/ha enhanced the number of trichome around 2.88-4.75 than control at 1.5. Apply Ca2SiO4 50 kg/ha gave the highest number of trichome at 4.75. The number of trichome seemed to be decreased when apply Ca2SiO4 at 62.5 kg/ha. The interaction between seed coated and soil drench application gave more number of trichomes around 2.25 to 6.25 than that of non-coated seed around 0.75-3.50. Both coated and non-coated seeds combined with Ca2SiO4-soil drench showed the density of trichome 2-3 times higher than the control. This can be indicated that Ca2SiO4 promoted the density of trichome in field corn. Paulo et al., (2014) reported that silicate increased the number of trichome of soybean and plants associated reduce attractiveness of E. heros. Seed coated with Ca2SiO4 showed thicker and longer trichome than that of non-coated seed (Figure 1). This characterize of trichome related with rate of Ca2SiO4. The higher the rate of Ca2SiO4, the more the density of trichome was. Silicon containing treatment increased in plant resistance due to not only the deposition of silicon in leaf tissues and trichomes but also produced phenolic compound (Ferreira et al., 2011;Valle et al., 2012). Table 1 The number of trichome and leaf thickness of non-coated and coated seed with Ca2SiO4 and combined with soil drench of Ca2SiO4 in field corn. Ca2SiO4 (kg/ha) Soil drench (B) 0 31.25 37.50 43.75 50.00 56.25 62.50 Average (A)
No. of trichome/mm Seed coating(A) Coated with NonCa2SiO4 coated 2.25 0.75 4.75 1.00 4.75 2.00 5.50 2.50 6.00 3.50 6.25 2.25 6.25 1.50 5.11 Y2/ 1.93 X
Leave thickness (mm) Aver. (B) 1.50 C1/ 2.88 BC 3.38 AB 4.00 AB 4.75 A 4.25 A 3.88 AB
Seed coating(A) Coated with NonCa2SiO4 coated
0.30 b 0.31 b 0.33 b 0.33 b 0.37 b 0.35 b 0.76 a 0.45 Y
0.19 b 0.22 b 0.28 b 0.30 b 0.32 b 0.33 b 0.37 b 0.29 X
Aver. (B) 0.25 B 0.27 B 0.31 B 0.32 B 0.34 B 0.34 B 0.56 A
1/and 2/mean within the same column and row followed by the same letter indicated no statistical difference using by DMRT.** indicated significant difference at P< 0.01
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A1
A2
B1
B2
C1
C2
D1
A1
E1
D2
E2
F1
F2
G1
G2 1 mm
Figure 1 The morphology of trichome by coated seed (left:1) with Ca2SiO4 and non-coated (right:2) and combined with different rate of Ca2SiO4 in soil at 0(A1:A2), 31.25(B1: B2), 37.5(C1:C2), 43.75(D1:D2), 50(E1:E2), 56.25(F1:F2) and 62.5 kg/ha (G1:G2) of corn.
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In the case of leaf thickness, seed coated with Ca2SiO4 promoted leaf thickness more than non-coated seed. Also, soil drench application increased leaf thickness. The interaction between seed coated with Ca2SiO4 and soil drench application stimulated higher leaf thickness than control. Leaf thickness was highest at 0.76 mm when coated seed and combined with Ca2SiO4 into soil 62.5 kg/ha. Silicon deposits 2.5 μm thick between cuticle and endodermal cell compared with general only 0.1 μm thick in rice (Ma and Takahashi, 2002). According to Gong et al. (2003), they found that 7.14 mmol Na2SiO3 per 8 kg of soil resulted in an increase in leaf thickness. Conclusion Seed coated with Ca2SiO4 and combined with Ca2SiO4 soil drench application induced number of trichome and leaf thickness in field corn. Acknowledgments We appreciated thank you Chia tai company for material support. Faculty of Liberal Arts and Science for financial support. References Epstein, E. and A.J. Bloom. 2005. Mineral nutrition of plants: principles and perspectives. 2 nd ed. Sunderland (MA): Sinauer Associates, Sunderland, MA. Gong, H., K. Chen, G. Chen, S. Wang and C. Zhang. 2003. Effects of silicon on growth of wheat under drought. J. Plant Nutr. 26:1055–1063. Ma, J.F. and E. Takahashi. 2002. Soil, fertilizer, and plant silicon research in Japan, 1 st ed. Elsevier, Amsterdam. Marschner, H. 2012. Mineral nutrition of higher plants. 3nd ed . Academic Press, New York. Muhammad, I., A. Mahmood, V. Romheld and G. Neumann. 2013. Nutrient seed priming improves seedling development of maize exposed to low root zone temperatures during early growth. Europ. J. Agronomy 49:141– 148. Pongsakorn, N., P. Rungcharoenthong, S. Amkha and T. Mala. 2015. Effects of seed coating with calcium silicate and soil drench on seedling growth of field corn. Khon Kaen Agr J. 43 Suppl : 76-82. Samuels, A.L., A.D.M. Glass, D.L. Ehret and J.G. Menzeis. 1993. The effect of silicon supplement on cumber fruit: change in surface charteristics. Ann. Bot. 72:433-440. Souza, P.V., B.R. Machado, D.C. da Silva, I P.P. Menezes, M.S. Arujo and F. G. de Jesus. 2014. Effect of resistance and trichome inducer on attraction of Euschistus heros (Hemiptera: Pentatodae) to soybeans. African J. Agr. Research. 9(10):889-894. Wagner, G.J., E. Wang, and R.W. Shepherd. 2004. New approaches for studying and exploiting an old protuberance, the plant trichome. Ann. Bot. 93: 3–11. Valle, G.E., A.L. Lourencao and J.B. Pinheiro. 2012. Adult attractiveness and oviposition preference of Bemisia tabaci biotype B in soybean genotype with different trichome density. J. Pest. Sci. 85:431-442. Ferreira, R.S., J.C. Moraes and C.S. Antunes. 2011. Silicon influence on resistance induction against Bemisia tabaci Biotype B (Genn.) (Hemiptera: Aleyrodidae) and on vegetative development in two soybean cultivars. Neotrop. Entomol. 40:495-500.
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P-23
Utilization of Zeolite and Isolite as Soil Conditioner to Improve Sandy Soil Nukoon TAWINTEUNG1* 1
Department of Plant Production Technology, Faculty of Agriculture Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: ktnukoon@kmitl.ac.th
ABSTRACT Utilization of zeolite and isolite as soil conditioner was carried out in order to improve sandy soil (Ng Series). The experiment was laid out in Completely Randomized Design (CRD) consisted of 8 treatments with 4 replications i.e. 5 and 10% (w/w) of zeolite, isolite and isolite cake, compost (2 ton/rai) as a compared treatment and chemical fertilizer as a control. The soils were collected in 3 layers (0-15, 15-50 and 50-100 cm) and 75 kg of soils were packed into each lysimeter (PVC column with 24.5 cm diameter and 120 cm long) in optimum bulk density and soil layers. All treatments were received N, P2O5 and K2O 112.5, 112.5 and 56.25 kg/ha, respectively and a sweet corn was planted in each lysimeter. Plant growth and yield were recorded. Plant samples were analyzed for nutrients concentration. Leachates were collected and analyzed for nutrients leaching. The results of this study indicated that CEC of zeolite was very high (102 cmol kg-1) resulted to an increase of CEC of sandy soil to 18.2 cmol kg-1 for 10% application. Compost and isolite cake were very high of organic matter and available phosphorus. The applications of soil conditioners affected to growth and yield of corn. The highest of fresh ear yield and nutrients uptake (macro nutrients) per lysimeter was found in zeolite 10% followed by zeolite 5% and compost, respectively. It was found only the trend that zeolite application could decrease nitrate and ammonium leaching as compared to control. After cropping, isolite cake increased soil pH from 4.75-4.92 to 7.51-7.57 whereas available phosphorus was increased to 57.1-65.0 mg kg1 .Soil organic matter was decreased in all treatments. CEC of 10% zeolite was decreased to 11.8 cmol kg-1. Keywords: Soil conditioner, Sandy soil, Zeolite, Isolite Introduction Sandy soils are very common in northeast Thailand, covering 80% of its area (Yuvaniyama, 2001). These soils are acid, poor in organic matter and macro- and micro-nutrients and low in CEC. Conceivable technologies for ameliorating, the above-mentioned infertile sandy acid soil are application of soil amendments such as liming material, chemical and organic fertilizers and minerals (Wada, 2005). Zeolite is natural mineral that has been used as soil conditioner because it has unique cation exchange properties, molecular sieving and adsorption which made this mineral attractive to agricultural utilization. Consequently, the most important reason of zeolite application is high cation exchange capacity and the capacity of zeolite to absorb ammonium ion and available nutrients (Watanabe, 1967). It is found that zeolite incorporated with chemical and organic fertilizer as slow release agent (Ming and Dixon, 1986). Isolite is produced from diatomaceous earth, formed in uniform granules with proprietary combustion, resulting in an extremely stable porous ceramic that will not break down or migrate in the soil. It is consisting of up to 74% pore space with very high of surface area (4.6 m2 g-1) that conserves water, decreases compaction, increases soil surface resiliency, and favorably affects total soil porosity and oxygen levels. Figgge et al. July 1-3, 2015
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(1995) found that isolite application could increase seed germination and improve plant establishment and growth. Furthermore, isolite has been used as a filter material on sodium glutamate industry which by-product is isolite cake. The appropriate use of this by-product was to improve soils, making them more productive for plant growth while ensuring that they do not pose a significant environmental risk. The aim of this study was to evaluate the potential of zeolite and isolite as soil conditioner to improve sandy soils. Materials and Methods The green house experiment was laid out in CRD (Completely Randomized Design) consisted of 8 treatments with 4 replications i.e. 5 and 10% (w/w) of zeolite, isolite and isolite cake, compost (12.5 ton ha-1) as a compared treatment and chemical fertilizer as a control. The sandy soils (Ng soil series, isohyperthermic Ustoxic Quartzipsamments) were collected in 3 layers (0-15, 15-50 and 50-100 cm) and 75 kg of soils were packed into each lysimeter (PVC column with 24.5 cm diameter and 120 cm long) in optimum bulk density and soil layers. All treatments were received N, P2O5 and K2O 112.5, 112.5 and 56.25 kg ha-1, respectively and a sweet corn was planted in each lysimeter. Plant growth and yield were recorded. Soil and plant samples were analyzed followed Carter (1993). Leachate was collected and analyzed for nutrients leaching followed Office of Science for Land Development (2004). Data collected were subjected to analysis of variance (ANOVA) test and Duncan Multiple Range Test at p < 0.05 was used for comparisons. Results and Discussion Physico-chemical characteristic of soil and soil conditioners The soil texture was sand with a sand content between 84-88% (Table 1). The soil was very strongly acid with very low of organic matter (OM) and cation exchange capacity (CEC). Zeolite was neutral and very high of CEC (102 cmol kg-1) resulted to an increase CEC of the sandy soil to 18.2 cmol kg-1 for 10% application. Isolite was very strongly alkaline whereas isolite cake was extremely acid resulted to decrease soil pH to 4.75-4.92. Moreover, it was very high of OM (30.1%) and available P (97.5 mg kg-1). Both isolites were not dominant on CEC (Table 2). Table 1 Physico-chemical characteristics of soil (Ng soil series). Exch. Base* Depth pH (1:1) ECe OM P (Bray II) -1 (cm) soil:water mS cm-1 % K Ca Mg Na mg kg 0-15 4.98 1.77 0.37 19.2 0.1 0.66 0.1 0.14 15-50 4.89 0.72 0.21 11.6 0.1 0.54 0.1 0.10 50-100 4.77 0.36 0.11 7.75 0.1 0.22 0.1 0.09 * = cmol kg-1, S = Sand, LS = Loamy sand
Particle (%) BS Texture % Sand Silt Clay 1.14 88.6 87 12 1.0 S 1.10 67.3 88 11 1.0 S 1.45 28.3 85 9.0 6.0 LS
CEC*
Table 2 Chemical properties of soil conditioners Soil pH (1:1) ECe conditioners soil:water mS cm-1 Zeolite 7.29 0.44 Isolite 10.03 0.54 Isolite cake 3.86 0.66 Compost 6.53 4.06 * by dry ashang
OM % 0.07 0.36 30.1 10.4
P (Bray II) mg kg-1 44.38 14.41 97.5 780*
-1
Exch. base (cmol kg ) K Ca Mg Na 5.8 35.3 0 11.2 0 0.21 0.07 3.13 0.34 1.56 0.30 1.51 4.08 21.0 0.09 0.52
CEC (cmol kg-1) 102 4.78 4.17 27.6
BS % 51.1 71.3 89.0 93.1
Growth and yield of sweet corn Soil conditionersaffected on growth and yield of the sweet corn as shown on Table 3. The highest of ear yield was found in zeolite 10% followed by zeolite 5% however, it was not significantly different (p < 0.05) as compared to those others treatment, accept 10%isolite cake which gave the lowest of ear yield. Compost gave the similar stem and total yield compared to both zeolite and isolite 10%. Ippolito et al. (2011) found that weight of corn in amended sandy soil, zeolite application at 22 Mg ha-1 seemed to increase compared to the control however, corn weight was decreased when applied 90 Mg ha-1 due to the elevated zeolite Na content (3%). Page 270
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Table 3 Growth and yield of sweet corn high dry wight (g/Lysimeter) cm ear stem total 1 Control 144 c 26.2 b 64.3 cd 90.4 bcd 2 Compost 191 a 18.9 b 118 a 137 a 3 Zeolite 5% 165 ab 27.8 b 98.7 ab 126 ab 4 Zeolite 10% 169 ab 38.3 a 99.7 ab 138 a 5 Isolite 5% 164 ab 17.3 b 61.5 cd 78.9 cd 6 Isolite 10% 185 ab 18.4 b 91.5 abc 110 abc 7 Isolite cake 5% 182 ab 13.4 bc 85.6 bcd 99.0 bcd 8 Isolite cake 10% 157 bc 3.08 c 58.6 d 61.7 d ** * * * F-Test 11.3 41.4 22.5 22.9 CV (%) Means in each column followed by the same letters are not significantly different by DMRT, * = p < 0.05, ** = p < 0.01 Treatments
Nutrients concentration and uptake of sweet corn The highest of primary minerals uptake were found in zeolite 5 and 10% which were similar to compost (Table 4). The application of both zeolite gave 1.70-1.87, 0.25-0.27 and 0.13-0.15 g/plant for N, P and K, respectively. Moreover, the highest of secondary minerals and micronutrients uptake were also found in that zeolite application. The zeolite is very effective in increasing the N in plant tissues because it has a pronounced selectivity for cations, such as ammonium (Ames, 1967). Zeolite has been used as fertilizer, being a source of N slowrelease compounds, if previously treated with ammonium ions (Ahmed et al., 2006). The high nutrients uptake of sweet corn may be explained by the fact that zeolite has a very high CEC and stability compared to other materials used (Ayan, 2007). Table 4 Uptake of macronutrients and micronutrients of corn. Treatments 1 Control 2 Compost 3 Zeolite 5% 4 Zeolite 10% 5 Isolite 5% 6 Isolite 10% 7 Isolite cake 5% 8 Isolite cake 10% F-Test CV (%)
N 1.26 b 1.78 a 1.70 a 1.87 a 1.14 b 1.30 b 1.07 b 0.91 b * 9.75
Macronutrients Uptake (g/plant) P K Ca 0.16 b 0.072 d 0.78 abc 0.30 a 0.112 bc 1.11 ab 0.25 a 0.131 ab 0.52 c 0.27 a 0.154 ab 0.91 abc 0.16 b 0.069 d 0.53 bc 0.16 b 0.086 cd 1.09 abc 0.17 b 0.061 d 1.13 a 0.17 b 0.066 d 0.93 abc ** * * 19.9 20.3 40.3
Mg 0.18 bc 0.38 a 0.24 ab 0.27 b 0.15 bc 0.24 ab 0.15 bc 0.13 c * 26.7
Mircronutrients Uptake (mg/plant) Fe Mn Zn Cu 22.3 abc 29.2 a 2.99 ab 0.113 cd 29.6 a 11.5 bc 2.70 abc 0.272 a 25.0 ab 15.5 b 3.31 a 0.216 ab 23.0 abc 13.7 bc 3.52 a 0.201 abc 26.5 ab 18.4 b 1.97 bcd 0.114 cd 25.0 ab 20.0 b 2.92 ab 0.132 bcd 24.1 ab 6.64 cd 1.71 cd 0.106 cd 13.8 c 3.03 d 1.03 d 0.061 d * * * * 27.8 36.5 30.5 40.1
Means in each column followed by the same letters are not significantly different by DMRT, * = p < 0.05, ** = p < 0.01
Nutrients leaching Soil conditioners did not affect on nutrients leaching as shown on Table 5. It was found only the trend that zeolite application could decrease nitrate and ammonium leaching as compared to the control. Huang and Petrovic (1994) found that application of zeolite to the sandy soil at a ratio of 1:9 (w/w) could reduce both NH4-N and NO3-N leaching, likely because of NH4+ retention. The small molecule size of the open-ringed structure (10-6 to 10-9 m) can physically protect NH4+ ions against microbial nitrification (Ferguson and Pepper, 2004). Chemical properties of soils after cropping After cropping, isolite cake increased soil pH from 4.75-4.92 to 7.51-7.57 whereas available phosphorus was increased to 57.1-65.0 mg kg-1 (Table 6). Soil organic matter was decreased in all treatments. CEC of zeolite 10% was decreased from 18.2 to 11.8 cmol kg-1. July 1-3, 2015
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Table 5 Nutrients leaching. Total leachate mL 1 Control 5510 2 Compost 6550 3 Zeolite 5% 3401 4 Zeolite 10% 2621 5 Isolite 5% 4430 6 Isolite 10% 2734 7 Isolite cake 5% 5796 8 Isolite cake 10% 6339 F-test ns CV (%) 58.5 Treatments
pH 5.26 5.00 5.77 4.73 6.41 6.28 6.27 4.92 -
nutrients of leachate (mg/Lysimeter) EC mS cm-1 NO3_ NH4+ P K Ca Mg 3.26 298 6.01 0.10 89.1 541 75 2.82 288 5.93 0.05 109 455 52 1.62 121 2.58 0.04 67.1 158 22 1.93 97.4 3.42 0.03 37.8 145 17 1.72 162 3.53 0.08 83.8 384 60 3.00 104 3.33 0.06 54.0 144 20 1.83 195 4.03 0.06 75.6 242 32 2.58 210 9.12 0.08 96.1 232 40 ns ns ns ns ns ns 72.2 81.9 90.5 86.9 111 117
Na 262 196 93 65 220 112 113 187 ns 91
ns = non significantly different by DMRT at p < 0.05
Table 6 Chemical properties of soils after cropping. pH (1:1) ECe -1 soil:water mS cm Control 5.59 0.21 Compost 6.34 0.53 Zeolite 5% 5.74 0.33 Zeolite 10% 6.55 0.46 Isolite 5% 5.93 0.29 Isolite 10% 6.55 0.37 Isolite cake 5% 7.51 0.30 Isolite cake 10% 7.57 0.45 n=4 Treatments
1 2 3 4 5 6 7 8
OM P (Bray II) mg kg-1 % 0.26 37.7 0.33 32.9 0.31 18.8 0.34 23.2 0.25 32.3 0.25 29.7 0.43 57.1 0.75 65.0
Exch. base K Ca 0.05 0.17 0.05 0.62 0.68 2.00 1.34 2.85 0.07 0.45 0.07 0.59 0.02 0.40 0.03 0.40
(cmol Mg 0.15 0.17 0.49 0.79 0.15 0.16 0.12 0.15
kg-1) CEC -1 Na (cmol kg ) 0.10 0.67 0.81 2.10 2.59 6.10 3.40 11.8 0.25 0.97 0.82 2.00 0.96 1.52 1.00 1.74
BS % 70.1 78.6 94.4 71.0 94.8 82.0 98.7 90.8
Conclusion Zeolite was neutral and very high of CEC (102 cmol kg-1) resulted in an increase CEC of the sandy soil to 18.2 cmol kg-1 for 10% application. Consequently, the highest of fresh ear yield and nutrients uptake were found in zeolite 10% followed by zeolite 5%. Isolite was very strongly alkaline whereas isolite cake was extremely acid resulted to decrease soil pH to 4.75 however, it was increased to 7.57 after cropping. Both isolite and isolite cake were not dominant on CEC. The application of isolite gave the stem and total yield similar to zeolite but, it was lower of nutrients uptake. Isolite cake gave the lowest of corn yield nevertheless it could increase SOM and available P. These results indicated that zeolite had a high potential as a soil conditioner to improve sandy soils. Further studies are required to assess the optimum rate and residual effect of isolite and isolite cake. References Ahmed, O. H., H. Aminuddin and M. H. A. Husni. 2006. Reducing ammonia loss from urea and improving soil-exchangeable ammonium retention through mixing triple superphosphate, humic acid, and zeolite. Soil Use and Management 22 (3): 315–319. Ames, L. L. 1967. Trivalent cerium equilibria with a synthetic heulandites-clinoptilolite series zeolite. Journal of Inorganic and Nuclear Chemistry 29:262–266. Ayan, S. 2007. Fidan Yeti¸stiriciligi ve A˘ gaçlandırma Çalı¸˘ smalarında Zeolit Mineralinin Kullanımı. http://w3.gazi.edu.tr/∼sezginay/yayinlar/zeo-kast.doc. Carer, M.R. 1993. Soil Sampling and Method of Analysis. Lewis Publishers. USA. 823 pp. Figge, D. A. H., B. A. D. Hetrick and G. W. T. Wilson. 1995. Role of expanded clay and porous ceramic amendments on plant establishment in mine spoils. Environmental Pollution 88: 161-165. Hatfield, J. L. and J. H. Prueger. 2004. Nitrogen over-use, under-use, and efficiency. ‘‘New directions for a diverse planet.’’ Proceedings of the 4th International Crop Science Congress. Brisbane, Australia, September 26 October 1. Huang, Z. T. and A. M. Petrovic. 1994. Clinoptilolite zeolite influence on nitrate leaching and nitrogen use efficiency in simulated sand based golf greens. J. Environ. Qual. 23:1190Y1194. Ippolito , J. A. , D. D. Tarkalson, and G. A. Lehrsch, 2011. Zeolite Soil Application Method Affects Inorganic Nitrogen, Moisture, and Corn Growth. Soil Science. 176: Number 3, 136-142. Ming, D.W. and J.B. Dixon. 1986. Clinoptilolite in South Texas soils. Soil Sci. Soc. Amer. J. 50:1618-1622. Office of Science for Land Development. 2004. Laboratory Manuals for Soil, Water, Fertilizer and Plant Samples Analysis to Certify Commodity Standards. Department of Land Development. Bangkok. Thailand. (in Thai). Wada, H. 2005. Managing sandy soils in Northeast Thailand. Pp. 82-92 in Management of Tropical Sandy Soils for Sustainable Agriculture. 27th November- 2nd December 2005 Khon Kaen, Thailand. Watanabe, Y. 1967. Effect of natural occurring zeolite as soil amendment in special reference to their cation exchange capacities Memoradum the 4th committee. Research Association of Zeolite December 11, 1967. Yuvaniyama, A. 2001. Management of problem soils in the northeast of Thailand. Pp. 147-156 in S.P. Kam et al. (eds.), Natural Resource Management Issues in the Korat Basin of Northeast Thailand. IRRI, Manila, Philippines.
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P-25
Leaf Flushing Inhibition by Application of High Phosphorus and Potassium Fertilizers for Off-season Mango Production Kannikar KAEWSONGSANG1*, Apisak BAOLEE1 and Ravie SETHPAKDEE2 1
Business Research and Development Division, SOTUS International CO., LTD., Pak Kret, Nonthaburi, Thailand 2 Department of Horticulture, Kasetsart University Nakhon Pathom, Thailand *Corresponding email: kannikar@sotus.co.th
ABSTRACT A key obstacle in off-season mango production is early leaf flushing. Although application of foliar 0-52-34 fertilizer can inhibit flushing. Consecutive sprays can lead to a difficulty in the flower induction. This research aims to investigate how new foliar 0-40-22 fertilizer inhibit leaf flushing and affect the subsequent flowering. The experiment was conducted at Sung Neon district, Nakhon Ratchasima province and was composed of no foliar fertilizer (control), 0-52-34 at 5.0g/L, 0-40-22 at 2.5 g/L and 5.0 g/L fertilizers. The foliar fertilizer applications commenced at 49 days after pruning at leaf pre-mature stage and two more re-applications were made at weekly intervals. Flowering of mature shoots were induced by thiourea and potassium nitrate at 70 or 90 days after pruning. The shoots were sprayed with 0-52-34 at 5.0 g/L, 0-40-22 at 2.5 g/L and 5.0 g/L fertilizers flushing at 28, 45 and 25% notably lower than the control flushing 100%. For induced flowering at 70 days after pruning, the shoots treated with 0-52-34 at 5.0 g/L, 0-40-22 at 2.5 g/L and 5.0 g/L fertilizers flowered 73, 45 and 81%, respectively, whereas there were no flowering on the control shoots. Similarly, the shoots induced at 90 days after pruning flowered at 71, 65 and 69%, respectively. The inflorescences from the shoots treated with 0-52-34 at 5 g/L, 0-40-22 at 2.5 g/L and 5.0 g/L fertilizers were 42.0, 49.2 and 49.2 cm in length, existing 22.8, 24.2, 27.7 peduncles, respectively. The results indicated that foliar application of 0-40-22 at 2.5 or 5.0 g/L can appreciably inhibit leaf flushing, resulting in simple flower induction and healthy inflorescences afterwards. Keywords: Flowering, Monopotassium phosphate, NUTAC® Extra-P, Potassium nitrate, Thiourea Introduction Pruning for off-season mango production was done after fruit harvest. Successful induction of flower is due to the amount of food accumulated in the mango tree. However there are usually rains during off-season mango production, resulting in increasing soil moisture and translocation of nitrogen from soil into the tree. Too much nitrogen in the tree causes a leaf flushing instead of flowering. Nowadays, leaf flushing can be inhibited by applying 0-52-34 (Monopotassium phosphate; MKP) fertilizer but consecutive sprays (more than 3 times) can lead to a difficulty in the flower induction and may cause toxicity because MKP is an alkaline solution. This research aimed to investigate how new 0-40-22 (NUTAC® Extra-P; NEP) foliar fertilizer inhibited leaf flushing and affected the subsequent flowerings when compared with a 0-40-22 fertilizer. Materials and Methods The cultivar ‘Nam Dokmai’ mango was selected with the same canopy size. The age of mango trees were 6 years old. Pruning was done on June 2014. Two weeks after pruning, paclobutrazol was applied for preparing to start off-season mango production. During this July 1-3, 2015
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period, some leaf flushing occured. The experiment was conducted at Nakhon Ratchasima province (14̊ 48̍ 30.37̎ N, 101̊ 52̍ 15.80̎ E), Thailand during June to December 2014. Four treatments composed of 1) no foliar fertilizer (control), 2) 0-52-34 at 5.0g/L, 3) 0-40-22 at 2.5 g/L and 4) 5.0 g/L fertilizers, were applied in Completely Randomized Design (CRD) lay out. The foliar fertilizer applications commenced at 49 days after pruning at pre-mature leaf stage and two more applications were made at weekly intervals. Flowering of mature shoots was induced 2 times by thiourea and potassium nitrate with the rates 0.25:1.25% at 70 or 90 days after pruning and applied again 15 days after the first flowering induction. Statistical analysis was done by Analysis of Variance (ANOVA). Result and Discussion Leaf flushing The shoots sprayed with 5.0 g/L of 0-52-34, 2.5 and 5.0 g/L of 0-40-22 showed leaf flushing at 28, 45 and 25% (p<0.01), respectively, notably lower than the control flushing 100% (Figure 1). During this experiment, there were some rains that consequent increased the soil moisture and influent the translocation of nitrogen from soil into the tree. Nitrogen is a plant nutrient that can promote vegetative growth, consequently leaf flushing, increasing vegetable bud and encourage large and green leaves (Sruamsiri, 2011). Therefore, there was 100% leaf flushing control. The mango tree sprayed with 0-52-34 and 0-40-22 fertilizers received phosphorus and potassium that can help in food accumulation and change in C/N ratio, caused a lower leaf flushing when compared with control. A similar result was found when applied with 0-52-34 fertilizer in ‘Nam Dokmai’ mango causing a slower flushing when compared with the unsprayed mango (Phaopadetkarn, 1992). Percentages 100 a
100 80 45 c
60 28 b
40
25 b
20 0 control
0-52-34: 100 g
0-40-22: 50 g
0-40-22: 100 g
Treatments
Figure 1 Leaf flushing percentage at 45 days after fertilizer spray Flowering The shoots sprayed with 5.0 g/L of 0-52-34 and 5.0 g/L of 0-40-22 showed non significant difference on flowering percentage at 45 days after flowering induction with the value of 73 and 81 (p<.01), respectively. The shoot sprayed with 2.5 g/L of 0-40-22 showed a lower flowering percentage at 45%. There is no significant difference between shoot sprayed with 0-52-34 and 0-40-22 fertilizer at 90 days after pruning but there was no flowering in the control (Figures 2 and 3). The difference between mango trees sprayed with fertilizer and non-sprayed control is the change from vegetative bud to reproductive bud needing more ATP (adenosine triphosphate). Therefore, applied with a fertilizer containing high phosphate portion can induce the metabolism and the change from vegetative bud to reproductive bud (Phaopadetkarn, 1992). High total non-structural carbohydrate (TNC) in the tree indicates food accumulation. Therefore, trees showing high TNC may show higher flowering percentage, observed when Page 274
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applied with 5,000 ppm of 0-52-34 fertilizer. The high TNC in ‘Rad’ and ‘Nam Dok Mai’ mango (Suton, 1994) may occur because potassium helps translocation of the starch and sugar or act as a catalyst of starch metabolism (Martin, 1984). Thus, it can increase food accumulation in the mango tree. Shoots sprayed with 2.5 g/L of 0-40-22 fertilizer showed lower flowering percentage at 70 days after pruning. However, at 90 days after pruning, there is more flowering percentage because at 90 days leaves are fully developed. Flower induction should be done on fully develop shoot, showing dark green leaves and have enough accumulated carbohydrate. (Chaunhan and Pandy, 1984). Percentages Percentages 81 a 90 71 a 73 a 80 69 a 80 65 a 70 70 60 60 45 b 50 50 40 40 30 30 20 20 0b 0c 10 10 (b) 0 (a) 0 control 0-52-34 0-40-22 0-40-22 Treatments control 0-52-34 0-40-22 0-40-22 Treatments 100 g 50 g 100 g 100 g 50 g 100 g
Figure 2 Flowering percentages at 45 days after flower induction of shoots sprayed with 052-34 and 0-40-22 fertilizers at 70 days (a) and 90 days (b) leaf stage a
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Figure 3 Shoots applied with 5.0 g/L of 0-52-34 (b), 50 g of 0-40-32 (c) and 100 g of 0-4032 (c) fertilizers, compared with control (a) Inflorescence Shoots sprayed with 5 g/L of 0-52-34, 2.5 and 5 g/L of 0-40-22 fertilizer at 70 days leaf stage after pruning showed an inflorescence length of 42.0, 49.2 and 49.2 cm (p<.01) and existing peduncles of 22.78, 24.22 and 27.67 cm (p<.01), respectively. Spraying with 2.5 and 5 g/L of 0-40-22 caused a reddish healthy inflorescence and more peduncles. The red color of an inflorescence indicated food accumulation in an inflorescence. There is more total nonstructural carbohydrate (TNC) in a reddish inflorescence than a pale one where a red color is caused by anthocyanins (Nartvaranan, 1997). However, there are other factors that can influence fruit set such as pollinator, climate, calcium and boron content. (Figure 4 and 5).
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Lengths (cm) 50
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Figure 4 Length (a) and peduncles (b) of inflorescence at 30 days after flowering induction
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Figure 5 Inflorescence applied with 5.0 g/L of 0-52-34 (a), 2.5 g/L (b) and 5 g/L of 0-40-22 (c) fertilizer compared with control (a) Conclusion 1. Sprays of 0-40-22 and 0-52-34 fertilizers at the rate 5.0 g/L can appreciably inhibit leaf flushing when compared with control. 2. Sprays of 0-40-22 fertilizer at the rate 2.5 g/L can increase flowering of 90 days leaf stage after pruning. 3. Sprays of 0-40-22 fertilizer at the rates 2.5 and 5 g/L can increase the quality of inflorescence when compared with spray of 0-52-34 fertilizer. References Nartvaranan, P. 1997. Relatinship between Inflorescence Color and Carbohydrate Content in the Inflorescences and Shoots of Mango (Mangifera indica L.) cv. ‘Nam Dok Mai. M.S. Special problem, Kasetsart University. Nakhonpathom. (In Thai) Sruamsiri, P. 2011. Mineral Nutrients in Horticultural Crop Production. Bangkok 314 p. Phaopadetkarn, N. 1992. Effect of Ethephon and Monopatassium phosphate on Flowering of ‘Nam Dok Mai’ Mango. Special problem, Kasetsart University. Nakhonpathom (In Thai) Chaunhan, P.S. and R.M. Pandy. 1984. Relative 14CO2 fixation by leaves and fruits and translocation of 14C sucrose in mango. Scientia Hortic. 22: 121-128. Martin, P.P., J. Gagnard and P. Gaulter (ed). 1984. Plant Analysis: As a Guide to the Nutrient of Temperate and Tropical Crops. Lavoisier, New York. 722 p. Suton, V. 1994. Effect of Foliar fertilizers on Nutritional Contents and Flowering of Two Mango Cultivars. M.S. Thesis, Kasetsart University, Nakhonpathom. (In Thai)
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P-26
Sand-Size Distributions of Soils on the So-Called Aeolian Sand Splay Landform in the Lower Mun-Chi Basin, Northeast Thailand Pornthiwa KANYAWONGHA1* and Anongnat SRIPRACHOTE2 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut 's Institute of Technology, Ladkrabang, Bangkok 10520, Thailand 2 Department of Plant Science and Natural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: kkpornth@kmitl.ac.th
ABSTRACT This study aimed at determining the sand-size distribution of soils on the so-called aeolian sand splay landform located on the right side of the Lam Se Bai, the Chi, Lam Sieo Yai and the Mun rivers, northeast Thailand. Four pedons were collected. After particle size analysis, the sand particles were separated into 5 sizes: the very coarse sand (VCS: 2.00-1.00 mm), the coarse sand (CS: 1.00-0.50 mm), the medium sand (MS: 0.50-0.25 mm), the fine sand (FS: 0.25-0.106 mm) and the very fine sand (VFS: 0.106-0.053 mm). The soils had similar morphology, which homogeneous coarse textures throughout the depth. Their sand contents ranged from 60% to more than 90% whilst approximately 1% to 15% of the clays were determined. The FS and MS pronounced, they occupied more than 80% of the total sand. By contrast, summation of the VFS+CS+VCS was less than 20%. The MS dominated in Pedon 2 and 3 whilst Pedon 1 and 4 the FS pronounced. The MS-dominated soils had less the fine sand to coarse sand ratio [Fi-S/CoS = (VFS+FS)/(MS+CS+VCS)] than the FS-pronounced soils (0.169-0.961 and 1.105-2.765, respectively). The rations of the other sizes of the sands to the FS or MS (VFS/FS, MS/FS, VFS/MS, FS/MS and CS/MS) of all pedons revealed similar ranges for the VFS/FS, CS/FS and CS/MS. The MS-pronounced soil had higher MS/FS and lower VFS/MS rations than those on the FS-dominated soils. On the other hand, the FS/MS rations of the FS-dominated soils had trend to be higher than those on the MS-pronounced soils. It could be concluded that the MS and FS contents and their relative rations had high possibility to be the indicators of the aeolian deposited soils. Keywords: Aeolian sand splay landform; Fine sand/Coarse sand ratio; Lower Mun-Chi Basin; Northeast Thailand; Rations of VFS/FS, MS/FS, VFS/MS, FS/MS and CS/MS
Introduction The aeolian landform was the landform originated in the desert, the place which continuously dry and rare of land cover to protect wind erosion. The major process of wind erosion is saltation which carried the fine sand and the coarse sand particles (Brady and Weil, 2008). The so-called "aeolian sand splay landform" is the one of the miscellaneous landform in the Khorat Basin, northeast Thailand. It was detected on the right side of the Lam Se Bai, the Chi, Lam Sieo and the Mun river. The origin of the particles on this landform was the alluvial deposits by the afore-mentioned rivers. Year by year, on the dry season-around December to March, the seasonal northeast monsoon-the winter monsoon blown southwest together with eroded the surface of bare soil on the alluvial landform, carried the particles and deposited on the presented places. The velocity of the seasonal northeast monsoon was somewhat similar, at approximately 30 to 50 km hr-1 (The National Research Council of Thailand, 1995), thus, the particle size that moveable by the wind should be in the same range. According to the similar geologic carrier-the northeast monsoon, soils on this landform should have some similar morphology. The objective of this study was to determine the sand-size distribution of July 1-3, 2015
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soils on the so-called aeolian sand splay landform in the Lower Mun-Chi Basin, northeast Thailand. Materials and Methods The aeolian sand splay landforms located in Yasothon, Sri Saket and Roi Ed provinces were selected. Four pedons were systematically collected sampling, 20 cm increment, downed to 5 meters depth or more, after field descriptions were recorded. Before laboratory analyses, the samples were prepared. The particle size analysis by pipette method was processed. Then sand particles were separated by dry-sieving method into 5 grades: the very coarse sand (VCS: 2.00-1.00 mm), the coarse sand (CS: 1.00-0.50 mm), the medium sand (MS: 0.50-0.25 mm), the fine sand (FS: 0.25-0.106 mm) and the very fine sand (VFS: 0.106-0.053 mm) (Gee and Bauder, 1986). Results and Discussion All pedons had similar morphology, which homogeneous coarse textures throughout the depth, brownish, reddish and yellowish colors. Seldom of nodules or concretions were observed. Their textures were sand, loamy sand and sandy loam, in most, which the sand contents ranged from 60% to more than 90% whilst approximately 1% to 15% of the clays were determined. The exception was from 450 cm of Pedon 1 down to the bottom which the finer texture pronounced (Figure 1a). To eliminate the soil forming process, clay illuviation, the clay-free silt to clay-free sand ratio (Cl-FSi/Cl-FS) which proposed by Chapman and Horn (1986) was used. Accordingly, the adjacent depth with doubling or halving different indicated the lithologic discontinuity. In this case, the material of the depth below 450 cm of pedon 1 differed from the overlying part because the Cl-FSi/Cl-FS of the 430-450 cm was 0.537 and 1.093 for the 450-470 cm depth. For the other pedons, no signs of lithologic discontinuity were detected (Figure 1c). For the sand-sized distribution, the FS and MS pronounced (Figure 1b). The MS dominated in Pedon 2 and 3 whilst Pedon 1 and 4 -the FS pronounced. To identify the difference of parent materials or geologic carriers, the fine sand to coarse sand ratio [Fi-S/Co-S = (VFS+FS)/(MS+CS+VCS)] was used (Tulaphitak, et al., 1996). The MS-dominated soils had less the Fi-S/Co-S ratio than the FS-pronounced soils (Figure 1c) (0.287-0.961, 0.169-0.908, 1.211-1.947 and 1.206-2.765 for pedon 2, 3, 1 (0-450 cm) and 4 respectively). This ratio of 450-620 cm of pedon 1 was 2.118-4.255. Because the fine sand and medium sand are the major sizes of the sand that eroded by saltation, thus the rations of the other sizes of the sands to the FS or MS (VFS/FS, MS/FS, VFS/MS, FS/MS and CS/MS) were used (Figure 1d). All pedons revealed similar ranges for the VFS/FS, CS/FS and CS/MS. The MS-pronounced soil had higher the MS/FS and lowered the VFS/MS rations than those on the FS-dominated soils (Table 1). On the other hand, the FS/MS rations of the FS-dominated soils had trend to be higher than those on the MS-pronounced soils. Conclusion The MS and FS contents and their relative rations had high possibility to be the indicators of the soils on the so-called aeolian sand splay landform in the northeast Thailand. References
Brady, N.C. and R.R. Weil. 2008. The Nature and Properties of Soil. revised 14th ed. Prentice Hall. New Jercy. 975 p. Chaoman S.L. and M.E. Hall. 1986. Parent Material Uniformity and Origin of Silty Soil in Northeast Arkansas based on Zircon-titanium Content. Soil Sci. Soc. Am. Proc., 32:262-271.
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Gee, G.W. and J.W. Bauder. 1986. Particle-size Analysis, pp. 383-409. In A. Klute (ed). Methods of Soil Analysis, Part I. Physical and Mineralogical Methods. 2nd ed. No. 9 in Agronomy. Soil Sci. Soc. Am., Inc., Wisconsin. The National Research Council of Thailand. 1995. Eolian Landform, pp 225-231. In The National Research Council of Thailand. Landforms of Thailand from Space. Darnsutha Press, Bangkok. (in Thais). Tulaphitak, T., K. Miura. K. Sakurai and K. Kyuma. 1996. Some Plateau Soil and Their Materials in Khon Kaen Area, Northeast Thailand. II. Soil Material Characterization and Classification. Japanese Journal of Tropical Agriculture, 40: 84-88. a
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0
0
Clay Silt sand
600
2
4
6
8
VFS/FS MS/FS VFS/MS FS/MS CS/MS
Figure 1 The distribution with depth of the particle size (%) (a), the sand size (%) (b), the rations of clay-free silt to clay-free sand and fine sand to coarse sand (c), and the rations of the other sizes of the sand to fine sand and medium sand.
July 1-3, 2015
d
4
700
0
VFS FS MS CS VCS
2
Pedon 1
50
d
Pedon 1
Depth (cm)
0 0
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0
50
100
a
0
0
20
40
60
80
100
b
0
1
2
3
c
0
0
0
0
50
50
50
100
100
100
150
150
150
200
200
200
250
250
250
300
300
300
350
350
350
400
400
450
450
2
4
6
d
100
300
Pedon 3
Depth (cm)
200
400
400 500 450
Silt 600
sand
Depth (cm)
0
50
100
500
a
0
20
40
60
Cl-FSi/Cl-FS Fi-S/Co-S
500
b
0
1
2
3
500
c
0
0
0
0
0
100
100
100
100
200
200
200
200
300
300
300
300
400
400
400
400
500
500
500
500
600
600
600
600
700
700
700
700
800
Clay Silt sand
VFS FS MS CS VCS
800
VFS/FS MS/FS VFS/MS FS/MS CS/MS 1
1
2
VFS/FS MS/FS VFS/MS FS/MS CS/MS
800
800
2
Cl-FSi/Cl-FS Fi-S/Co-S
Table 1 Summarized of the particle size, the sand size and their relations. (Note 1: 0-450 cm) %Clay
%Sand Cl-FSi/Cl-FS %VFS
%FS
%MS
%CS Pedon 1
%VCS Fi-S/Co-S VFS/FS
MS/FS
CS/FS VFS/MS FS/MS
Max
1
31.49
75.94
0.54
11.39
56.51
41.81
3.99
0.210
1.947
0.246
0.944
0.082
0.344
1.718
Min
1
4.14
44.57
0.24
8.03
44.29
32.04
1.40
0.005
1.211
0.153
0.582
0.027
0.216
1.059
Ave 1
14.86
65.34
0.31
10.25
50.04
36.91
0.051
1.528
0.206
0.742
0.056
0.279
1.365
Max Min Ave
11.54 1.27 7.01
95.44 76.85 84.28
0.18 0.03 0.11
15.37 3.49 9.33
34.65 16.26 25.59
75.25 46.70 59.82
0.373 0.016 0.084
0.961 0.250 0.560
0.499 0.186 0.354
4.627 1.348 2.492
0.436 0.103 0.216
0.328 0.047 0.166
0.742 0.216 0.445
Max Min Ave
8.10 2.80 6.03
90.16 83.78 86.35
0.11 0.06 0.09
6.82 3.00 5.49
41.42 11.49 30.51
73.80 48.16 58.73
0.450 0.019 0.083
0.908 0.169 0.587
0.261 0.135 0.185
6.424 1.163 2.216
1.014 0.082 0.215
0.135 0.041 0.096
0.860 0.156 0.542
Max Min Ave
11.93 1.88 8.74
86.64 78.04 80.58
0.15 0.11 0.13
14.04 7.39 9.27
56.19 40.72 51.35
44.96 32.47 37.90
2.76 Pedon 2 9.55 2.93 5.31 Pedon 3 11.65 3.24 5.19 Pedon 4 1.81 0.89 1.29
0.287 0.000 0.053
1.934 1.105 1.560
0.304 0.145 0.182
1.084 0.578 0.746
0.036 0.016 0.025
0.407 0.179 0.247
1.731 0.922 1.371
.
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d
Pedon 4
Clay
VFS FS MS CS VCS
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P-27
Parent Material Affecting Phosphorus Distributions in Acid Sulfate Soils of Thailand
1
Pornthiwa KANYAWONGHA1, Nutcharee BOONPLANG1 and Anongnat SRIPRACHOTE2 a
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut 's Institute of Technology, Ladkrabang, Bangkok 10520, Thailand 2 Department of Plant Science and Natural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: kkpornth@kmitl.ac.th
ABSTRACT To study the influence of parent materials on phosphorus distributions in acid sulfate soils of Thailand, eight pedons were collected from different physiographic positions and parent materials. They were the active tidal flat with marine deposits (Pedon 1), the former tidal flat with recent marine and brackish water deposits (Pedon 2) and the former tidal with old marine and brackish water deposits (Pedon 3 to 8). The selected pedons consisted of the potential acid sulfate soils (Pedon 1 and 2), the actual acid sulfate soils with and without jarosite (Pedon 3, 4, 5 and Pedon 6, 7, respectively) and the para acid sulfate soil (Pedon 8). They had clayey texture which the clay and the silt particles occupied more than 90% of the fine earths, in most. Because of low-developing soils, the parent materials had strongly influenced on soil properties, P contents and P forms. The distribution of the organic P more or less followed the organic carbon while the available P similar to the total P throughout the depth. The potential acid sulfate soils contained the highest total P (326 mg kg-1 to 1675 mg kg-1). The actual acid sulfate soils with jarosite had lower the total P than those on the ones without jarosite (71 mg kg-1 to 412 mg kg-1 and 119 mg kg-1 to 663 mg kg-1, respectively). The total P content of the para acid sulfate soil ranged from 169 mg kg-1 to 883 mg kg-1. For fractionation P (SL-P, Fe-P, Al-P, Ca-P), mainly the Fe-P -the highest and the SL-P -the lowest. The variations of fractional P which observed in Pedon 2, 3 and 8 might implied their origin differed. The residual P (= total P - residual P) values were higher than the fractionation P indicating that P in acid sulfate soils were mainly unavailable forms. The former brackish water deposits had the lowest total P whilst the recent marine deposits -the highest one. Keywords: Acid sulfate soils, Marine deposits, Brackish water deposits, Phosphorus forms, Thailand Introduction Phosphorus (P) is one of the essential elements. Naturally, soil had low P resulting in low P availability. For soil fertility, 3 major problems of P were recognized: soil had low P contentthe total P, soil P compound mainly in unavailable forms, and applied available P rapidly converted to unavailable form (Brady and Weil, 2008). Factors affecting P contents and P forms were parent material (Hanley and Murphy, 1970), the clay content (Longanathan and Sutton, 1987), organic matter, soil reaction and land use (Brady and Weil, 2008). Acid sulfate soil is one of the problem soils of Thailand. The major morphology is extremely acid which pH might as low as 2.5 when dried. Jarosite-the pale yellow mottles were observed within 1.5 m from surface. Besides low pH, the main problem of this soil is low P availability. Acid sulfate soils distributed in the southern part of the central plain on which their parent July 1-3, 2015
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materials were recent- and former- marine and brackish deposits. According to their different ages, the P contents might be differed. This study aimed at determining the influences of parent materials on P distribution in acid sulfate soils of Thailand. Materials and Methods The study area was the southern part of the central plain, close to the Gulf of Thailand to Nakhorn Nayok province. Four pedons were collected from the different physiographic positions and parent materials. They were the former tidal flat with recent marine and brackish water deposits (Pedon 1) and the former tidal with old marine and brackish water deposits (Pedon 2 to 4). The selected pedons consisted of the potential acid sulfate soils (Pedon 1), the actual acid sulfate soils with jarosite were detected in different depths (Pedon 2: 40-138 cm, 3: 70-140 cm) and the para acid sulfate soil (Pedon 4). The profiles were manually dug, field morphology were recorded. The samples were collected from each horizon. Laboratory analyses consisted of the particle size analysis by pipette method (Gee and Bauder, 1986), soil reaction (pH), organic carbon, organic phosphorus, available phosphorus, total phosphorus and fractionation phosphorus (soluble and loosely P: SL-P, aluminum P: Al-P; iron P: Fe-P, calcium P: Ca-P) and residual P: res-P (= total P- fraction P) using standard methods (IITA, 1979; Kuo, 1996). Results and Discussion The profile development stage differed. They had clay and silty clay textures which the clay and the silt particles occupied about 80 to 90% of the fine earths. Pedon 1 had the highest organic carbon, available P, organic P and total P whilst Pedon 2 and 3 the lowest (Table 1). The patterns with depth of organic P more or less similar to the organic carbon while the available P similar to the total P (Figure 1). Apart from the developed profile which the total P had the same trend of the clays throughout the depth, the total P represented the parent materials. According to field morphology and some basic properties (not shown), Pedon 1 had recent brackish water deposits (0-68 cm) and underlain by recent marine deposits. They had the highest the total P (326 mg kg-1 to 1675 mg kg-1). However, the pyrite-containing depth (68-100 cm and 130-170 cm), the total P contents were lower than the adjacent depths (Figure 1). The patterns of fraction P revealed different times of deposition, as Fe-P>CaP>Al-P>>SL-P (0-100, and 130-170 cm), Ca-P>Al-P>SL-P>Fe-P (100-130, and 170-190 cm), and Ca-P>Fe-P>Al-P>SL-P (190-220 cm). Pedon 2 and 3, parent materials were the former brackish water and marine deposits, their available P were the lowest (0.91-28.33 mg kg-1 and 0.85-71.88 mg kg-1, respectively) especially in the jarosite-containing depths. The total P also the lowest (89 -282 mg kg-1 and 118- 412 mg kg-1). The parent material horizon (Cg) had higher the available P and total P than the jarosite-containing parts. Pedon 2, the fractionation P differed among 0-156 cm (Fe-P>>Ca-P>Al-P>SL-P) and 156-210 cm (FeP>Al-P>Ca-P>>SL-P). For Pedon 3, Fe-P>Ca-P>Al-P>SL-P (0-140, and 180-220 cm) and Ca-P>Fe-P>Al-P>>SL-P (140-180 cm). The contents of Fe-P between 0-80 cm and 80-220 cm also indicated the parent materials. Pedon 4, the particle size distribution revealed the difference between 0-135 cm and 135-220 cm. According to the total P, they should have some differences among 0-52 cm, 52-135 cm and 135-220 cm (214.8-284.6, 433.5-832.8 and 168.5-436.6 mg kg-1, respectively). The fractionation P confirmed this finding. The patterns of fractionation P differed among depths, as Fe-P>Ca-P>Al-P>>SL-P (0-100 cm and 135-210 cm), Ca-P>Fe-P>>Al-P>>SL-P (100-135 cm) and Ca-P>>Al-P>Fe-P>>SL-P (210-220 cm). The parent materials might be the former marine deposit (135-220 cm) and overlain by the former brackish water deposit together with the alluvial deposit (0-135 cm). The residual P occupied almost half of the total P or more which indicated that major forms of soil P were unavailable. Page 282
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Conclusion Because of low-developing soils, the parent materials had strongly influenced on soil properties, P contents and P forms. The recent marine and brackish water deposits had the highest the total P whilst the former brackish water and marine deposits-the lowest one. The Ca-P pronounced in the recent marine deposits. For the former ones, the Fe-P dominated, in most. References Brady, N.C. and R.R. Weil. 2008. The Nature and Properties of Soil. revised 14th ed. Prentice Hall. New Jercy. 975 p. Gee, G.W. and J.W. Bauder. 1986. Particle-size Analysis, pp. 383-409. In A. Klute (ed). Methods of Soil Analysis, Part I. Physical and Mineralogical Methods. 2nd ed. No. 9 in Agronomy. Soil Sci. Soc. Am., Inc., Wisconsin. Hanley, P.K. and M.D. Murphy. 1970. Phosphate forms in particle size separates of Iris soils in relation to drainage and parent materials. Soil Sci. Soc. Am. Proc., 34:587-590. IITA. 1979. Selected Methods for Soil and Plant Analysis. Revised Edition. Manual Series No. 1. International Institute of Tropical Agriculture, Ibadan, Nigeria. 70 p. Kuo, S. 1996. Phosphorus, pp 869-920. In D.L. Spark, et al. (eds.). Methods of Soil Analysis Part 3. Chemical Methods. No 5 in the Soil Sci. Soc. Am. Book Series. Soil Sci. Soc. Am. Inc., Wisconsin. Longanathan, P and P.M. Sutton. 1987. Phosphorus fractions and availability in soil formed on different geological in the Niger Delta Area of Nigeria. Soil Sci. 143 (1): 16-25. Table 1. Summarized of the soil properties related to phosphorus in acid sulfate soils. Clay Silt Sand (---------%----------) Mean 61.70 Min 43.82 Max 74.69
37.77 24.47 55.94
0.53 0.17 1.42
Mean 69.86 Min 49.81 Max 74.00
28.54 25.48 48.28
1.67 0.02 4.17
Mean 51.71 Min 32.61 Max 58.47
47.05 41.02 65.94
1.23 0.21 1.86
Mean 63.37 Min 45.13 Max 75.41
36.23 24.32 54.80
0.40 0.05 3.41
pHf
pHw OC Avail. P Org-P Total-P SL-P Al-P Fe-P Ca-P Res-P (1:1) (%) (-----------------------------mg kg-1------------------------------) Pedon 1 Potential acid sulfate soil: Apg-ACg-Cg 7.8 5.86 2.25 148.8 122.7 641.3 4.30 70.00 152.7 242.1 172.2 6.0 3.41 0.28 28.87 22.92 326.50 0.25 9.81 8.55 24.78 42.49 8.5 7.45 4.54 621.4 366.4 1675 22.67 217.8 502.6 1184 292.6 Pedon 2 Actual acid sulfate soil: Apg-BAg-Bjg-BCjg-Cg 4.2 3.40 1.25 9.02 48.49 186.7 0.32 5.39 54.13 11.68 115.2 4.0 2.17 0.28 0.91 13.36 118.6 0.14 0.40 22.49 6.46 63.36 4.5 4.08 5.57 28.33 170.5 412.2 0.78 13.76 153.5 37.38 263.6 Pedon 3 Actual acid sulfate soil: Apg-Bg-Bjg-BCjg-Cg 6.0 4.15 0.77 17.44 41.03 162.3 0.23 6.31 41.56 27.62 86.59 4.5 2.70 0.17 0.85 5.74 89.03 0.10 1.33 13.19 2.37 67.93 8.5 6.16 2.44 71.88 83.08 281.6 0.62 25.67 98.46 120.5 116.9 Pedon 4 Para acid sulfate soil: Apg-ABg-Bg-BCg-Cg 7.2 4.78 1.48 28.84 75.72 373.1 0.50 15.53 81.60 61.31 217.4 5.5 2.76 0.12 2.41 22.25 168.5 0.06 0.83 37.72 8.52 11.30 8.5 7.23 3.30 80.23 195.8 832.8 3.84 65.47 143.1 170.6 650.1
Explanation of Figure 1 (next page) The explanations above each graph revealed the major x axis whilst the ones below them represented the minor x axis. For more information, see table 1 and text.
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OC
Avail P
Org P, Total P
Fractionation P
Pedon 2
Depth (cm)
Depth (cm)
Pedon 1
Depth (cm)
Pedon 3
Depth (cm)
Pedon 4
Org P
Total P
Clay
Residual P
Figure 1 Distributions with depth of soil properties related with phosphorus. Page 284
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P-28
Screening of Ametryn Resistant Bacteria from Sugarcane Cultivation Soils Pattrarat TEAMKAO*, Nipaporn ROENGANAN, and Siratee PONGPOUN Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok, Thailand *Corresponding email: ktpattra@kmitl.ac.th
ABSTRACT Ametryn is a selective systemic herbicide and widely used for the pre- and post-emergence control of weeds in plantations of sugarcane, maize, and banana. In this work, study the amount of ametryn resistance bacterial and its diversity in two types of sugarcane cultivation soil with long term of ametryn application were conducted. The result showed different bacterial number in two soil types, red- and black-soil. The occurrence of bacteria in red-soil was 2.3x105 CFU/g soil with significantly lower than that of the black-soil (9.2x105 CFU/g soil). The upper red-soil (0-15 cm in depth) had higher amount of bacteria than the lower redsoil (15-30 cm in depth). The diversity of bacteria in black-soil was lower than that of the red-soil, and all bacteria that found in both soils were gram-negative bacteria. The use of minimal medium (basal salt agar) was appropriated for ametryn resistant bacterial screening than enriched medium (nutrient agar). Keywords:Ametryn, Bacteria, Soil, Sugarcane Introduction Ametryn ((2-ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine) is triazine herbicide that is widely used in agriculture. In Thailand, ametryn is used to control broadleaf weeds and annual grasses in corn, pineapple, and sugarcane fields. Most ametryn is used in sugarcane in more than 45 countries. Thailand has more than 1.6 million hectares of sugarcane plantations and is a top five country using this chemical (Farland and Burnside, 2011). Ametryn is Class III chemical with low toxic to fish and mammals but high toxic to crustaceans and mollusks. Ametryn moves vertical and horizontal in soil. It is highly mobile in loam, sandy loams, and sand soil, but slightly mobile in clay soil. The widely application of herbicide leads to presence of them in fresh water (Farré et al., 2002). The contaminated ametryn in water came from leaching by flooding and raining from agriculture area (US EPA, 2005). Ametryn is resistance herbicide, degradation half-life in surface soil and water are 85-123 days and 368 days, respectively (US EPA, 2005). Microbial plays various roles in soil such as soil formation, nutrient cycling, and decomposition of organic matter including organic contaminants. Many indigenous microorganism in soil and water are capable to degrade organic contaminants. The persisted ametryn compound could be lost from soil by microbial degradation (Farré et al., 2002). Contaminated soil is a good source of degrading microbial for bioremediation purpose. The aim of this study was to screen the ametryn resistance bacteria from sugarcane field. Materials and Methods Soil Soil samples were collected at a depth of 0-15 cm and 15-30 cm for red-soil, and 0-15 cm for black-soil, from sugarcane fields with a long term of ametryn application in Ratchaburi July 1-3, 2015
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province, Thailand. The soil was passed through a 2 mm sieve and air dried at room temperature before chemical properties analysis of the soil. The soils for bacterial analysis were kept in ice-box immediately after being collected, then transferred to laboratory and kept at 4째C until analysis. Media preparation The culture media used in this study were carbon limited basal salt agar (BSA) medium and nutrient agar (NA) medium. BSA medium consisting of (all in g/L): 5.57, NaHPO4; 2.44, KH2PO4; 2.00, NH4Cl; 0.20, MgCl2.6H2O; 0.0004, MnCl2.4H2O; 0.001, FeCl3.6H2O; 0.001, CaCl2; 15, bactoagraose. NA medium consisting of (all in g/L): 5.0 peptone; 3.0 beef extract; 15, bactoagraose. Ametryn solution, in water, was added into the media as a carbon source with final concentration of 1.3 mg/L. The pH of the media was adjusted to 7 before bactoagarose was added and sterilized. Bacterial isolation Bacteria were collected from soil sample by serial dilution and spread plate method using BSA and NA media. Ten gram of soil sample was serially diluted in sterilized distill water to get a concentration range of 10-1 to 10-4. A volume of 0.2 mL of each dilution was transferred aseptically to agar media. The sample was spread uniformly. The plates were incubated at room temperature for 3 days. The bacterial isolates were further sub-cultured to obtain pure culture. Microscopic observation The bacterial isolates were gram stained and observed under a high power magnifying lens in light microscope. Statistic analysis Data were analyzed by Minitab program Version 16.0. The significance of treatments was set at a Pvalue of less than or equal to 0.05.
Results and Discussion
Soil properties The properties of soil samples showed in Table 1. The black-soil had higher soil fertility for plant growth with appropriate pH, higher organic matter, and higher plant nutrients (nitrogen, phosphorus, and potassium). However, sugarcane growing in both soils did not show unusual sign of growth. Table 1 Chemical property of soil samples. Soil type
pH (1:1)
OM (%)
Total N (%) 0.30
Extractable P (mg/kg) 98.34
Extractable K (mg/kg) 260
Black soil
7.44
5.86
Red soil
5.73
4.53
0.25
9.83
66
Bacterial number Regard to the study of bacteria in black- and red-soil with a long term of ametryn application, it was found that red-soil tended to have lower amount of bacteria than the black-soil by both NA medium and BSA medium. The higher numbers of soil bacteria were related to the fertility of black-soil that over the red-soil in organic matter, nitrogen, and phosphorus, that is essential for microbial growth. The depth of soil affected the number of bacteria. The surface soil (0-15 cm) had higher bacterial count than the deeper soil (15-30 cm). Higher bacterial number in surface soil related to soil fertility that usually had higher nutrient in upper than lower or deeper soil. Approximately 65% of microbial biomass found in top 2 meter of soil
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was found in a depth of 0-25 cm (Fierer et al., 2003). Bacterial number cultured in NA medium was higher than BSA medium because of nutrients in the medium. NA with enriched of bacterial nutrient could culture all of bacteria that resistant to ametryn while BSA, which is minimal medium, could culture only bacteria that could use ametryn as a carbon source. Table 2 Bacterial number in soil samples screening by BSA and NA medium. Soil type Black soil (0-15 cm) Red soil (0-15 cm) Red soil (15-20 cm)
Bacteria (CFU/g soil) BSA NA 9.2x105 a 2.0x106 a 2.3x105 b 1.1x106 a 1.8x105 b 5.9x105 b
Figure 1 Bacterial colony isolated by BSA (B-) and NA (N-) from black-soil at a depth of 015 cm (BU), red-soil at a depth of 0-15 cm (RU), and red-soil at a depth of 15-30 cm (RL). Table 3 Bacterial isolate in soil samples. Soil type Black soil (0-15 cm); BU Red soil (0-15 cm); RU Red soil (15-20 cm); RL
Bacterial strain BSA 4 5 3
NA 3 4 4
Bacterial diversity Bacterial strains were isolated on the bases of colony morphology in each soil sample (Figure 1). The number of bacterial isolates found in black- and red-soil cultured in each medium shown in Table 3. Generally, the diversity of soil bacteria isolated by BSA medium was
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higher than NA medium except in 15-30 cm deep red-soil. The ametryn resistance bacteria in the depth of 0-15 cm of red-soil were higher than in black-soil both in BSA and NA medium, although red-soil had lower amount of bacteria (Table 2). The deeper soil tended to have lower bacterial diversity (Table 3). PLFA analysis pointed that microbial diversity declined about 33% from soil surface down to 2 meter (Fierer et al., 2003). Soil depth affected the community composition of soil-bacteria, typically bacterial diversity was highest in the top 10 cm of soil profile (Eilers, 2012). All of bacterial isolates were gram-negative bacteria. Gram-negative bacteria dominated in organic compound contaminated environments, and amount of the bacteria declined with soil depth (MacNaughton et al., 1999; Männisto et al., 2001). This bacterial isolate may possibly be used in remediated contaminated soil and water however further study on potential remediation and stablity in environment is required. Conclusion Base on the screening of bacteria in two soil types, black- and red-soil, from sugarcane field in Ratchaburi province, it found that bacteria in red-soil was 2.3x105 CFU/g soil with lower than that of the black-soil that was 9.2x105 CFU/g soil. The red-soil at the depth of 0-15 cm had higher amount of bacteria than red-soil the depth of 15-30 cm. The diversity of bacteria in black-soil was lower than that of the red-soil, although bacterial amount was higher. All of bacteria isolated in both soils were gram-negative bacteria. These bacteria might have potential in ametryn bioremediation. References Eilers, K.G., S. Debenport, S. Anderson, and N. Fierer. 2012. Digging deeper to find unique microbial communities: The strong effect of depth on the structure of bacterial and archaeal communities in soil. Soil Biol. Biochem. 50: 58-65. Farré, M., J. Fernandez, M. Paez, L. Granada, L. Barba, H.M. Gutierrez, C. Pulgarin, and D. Barceló. 2002. Analysis and toxicity of methomyl and ametryn after biodegradation. Anal. Bioanal. Chem. 373: 704-709. Fierera, N., J.P. Schimela, and P.A. Holden. 2003. Variations in microbial community composition through two soil depth profiles. Soil Biol. Biochem. 35: 167–176. LeBaron, H.M., J.M. Farland, and O. Burnside. 2011. The Triazine Herbicides. Elsevier. New York. 600 p. MacNaughton, S.J., J.R. Stephen, A.D. Venosa, G.A. Davis, Y.J. Chang, and D.C. White. 1999. Microbial population changes during bioremediation of an experimental oil pill. Appl. Environ. Microbiol. 65(8): 3566-3574. Männisto, M.K., M.S. Salkinoja-Salonen, and J.A. Puhakka. 2001. In situ polychlorophenol bioremediation potential of the indigenous bacterial community of boreal groundwater. Water Res. 35(10): 2496-2504. United State Environmental Protection Agency (US EPA). 2005. Reregistration eligibility document and risk assessment for the use of ametryn on corn, pineapple and sugarcane. Washington, DC.
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P-29
Effect of Organic Fertilizers on Soil Phosphorus Sorption Capacity Sukunya YAMPRACHA1* and Parichart WATHAKIATTIKUL1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kysukuny@kmitl.ac.th
ABSTRACT Phosphorus (P) sorption capacity is an important soil property using to recommend P fertilizer for crops. Soil properties have an effect on P sorption capacity including soil organic matter. Therefore, continuous use of organic fertilizers may affect soil P sorption capacity. A pot experiment was conducted for two sweet corn crops on Kamphangsan soil series to study the effect of sources and rates of organic fertilizer on phosphorus sorption capacity. The experimental design was the 3x3 factorials in a randomized complete block design (RCBD) with three replications. Pellet compost, chicken and cattle manure were applied at three different rates i.e. 0, 156 and 312 kg P ha-1. First and second crops were planted during April-June 2013 and August-October 2013, respectively. The soil samples before planting and after second crop harvesting were measured P sorption isotherm by laboratory batch experiment. Langmuir equation was used to describe P sorption capacity and estimated maximum P sorption (Smax) of each soil. The results showed that an application of chicken and cattle manure was significantly increased maximum sorbed P, but the application of pellet compost was decreased. The highest P sorption maximum was found in the soil applied with cattle manure. Soil pH, organic matter, cation exchange capacity, iron, zinc, and manganese were positive correlation with Smax (p-value<0.01). Although, exchangeable calcium was a negative correlation with Smax (r =-0.68**). However, application rates of three organic fertilizers had no significant effect on maximum P sorption. Keywords: Phosphorus, Sorption, Organic fertilizer Introduction Phosphorus (P) is an important plant nutrient for crop growth and tends to limit crop growth in high acidic and weathering soils because of high P sorption. Study of P sorption capacity may improve P fertilizer management. P sorption data have been related to plant growth and yield to estimate P requirement. Besides, P sorption capacity is used to predict P leaching for preventing P contamination in water resources. Kamprath and Watson (1980) reported that the concentration of 0.2 mg L-1 of P in soil solution was an index of optimum P for plant growth. Ozanne and Shaw (1967) used the P sorption curve technique to determine a similar level of P in the soil solution as the P requirement for pasture growth. Phosphorus sorption data can be characterized by sorption curves determined by adding graded levels of P to the soil and shaking to allow the reaction to occur. Simple Langmuir was usually used to characterize P sorption phenomena in the soils and to estimate the P sorption maxima and bonding energy (Olsen and Watanabe, 1957). Soil properties such as pH, organic matter, soil colloid, extractable cation and soil management could affect on change of P sorption capacity in the soil. So, long-term application of organic manure that increases organic matter in the soil may have an effect on P sorption capacity. Organic fertilizer has been applied by consideration only on nitrogen (N) content. It increases other plant nutrient accumulated in the soil especially P because it is less required by the plant than N. Continuous application of organic fertilizer may increase P in the soil and organic matter which effect on P sorption July 1-3, 2015
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capacity. The objective of this study was to investigate the effect of sources and rates of organic fertilizer on the change in P sorption capacity. Materials and Method Pot experiment A pot experiment was conducted for two growing periods of sweet corn in a loamy soil as Kamphangsan soil series (Ks) that collected from a sweet corn field in Nakhonpathom province. The experimental design was the 3x3 factorials in a randomized complete block design (RCBD) with three replications. Pellet compost (PC), chicken (CK) and cattle manure (CM) were applied at three different rates i.e. 0, 156 and 312 kg P ha-1 (0, 0.8, 1.6 g P pot-1). Ten kilograms of soil were mixed with three organic fertilizers in each treatment at first cropping. Nitrogen and potassium chemical fertilizer were applied at the rate 1.6 g N pot-1 crop-1 and 1.2 g K pot-1 crop-1. First and second crops were planted during April-June 2013 and August-October 2013, respectively. The soil samples before planting and after second crop harvesting were collected for measuring P sorption isotherm by laboratory batch experiment. Some soil chemical properties were analyzed. Soil properties analysis The soil samples were air-dried, ground and sieved to 2 mm. Soil pH was determined in a soil: water ratio of 1:1 by glass electrode (Black, 1965). Soil texture was analyzed using the hydrometer method. Available P was extracted by Bray 2 (Bray and Kurtz, 1945) and determined using the method of Murphy and Riley (1962). Cation exchange capacity (CEC) was measured by leaching with NH4OAc, pH 7 (Yoshida et al., 1972). Soil organic matter was determined by the method of Walkley and Black (1934). Exchangeable potassium (K), calcium (Ca) and magnesium (Mg) were extracted by NH4OAc, pH 7 and then analyzed using atomic adsorption spectrophotometer (AA). Extractable iron (Fe), zinc (Zn), copper (Cu) and manganese (Mn) were extracted by DTPA, pH 7.3 and determined using AA. The soil samples were digested by the acid mixture of HClO4 and HNO3 and total P was determined by vanadomolybdate yellow color (Jackson, 1967). Soil properties before planting were shown in Table 1. Table 1 Soil properties before planting. pH 7.22
OM
Avai. P Total P CEC -1 -1 (g kg ) (cmol kg- (mg kg ) (mg kg ) 1 ) 33.5 12.2 173.4 470 -1
Exchangeable(mg kg-1) Ca Mg K 2,779
269.3
214.7
Extractable (mg kg-1) Fe Zn Cu Mn 16.5
2.10
13.5
24.2
Organic fertilizer analysis Organic fertilizers were sampled one day before being applied to the soils. The samples were air-dried and sieved to 2 mm. Saturated pH and electrical conductivity (EC) were measured using glass electrode and EC meter. Organic carbon was analyzed by the method of Walkley and Black (1934). Total nitrogen was measured by Kjeldahl method. The sample of organic fertilizers were digested by an acid mixture of HClO4 and HNO3 to determine total P and total K. Total P was determined by vanadomolybdate yellow color and total K was measured using AA (Jackson, 1967). C:N ratio was calculated from total N and organic carbon. Organic fertilizer properties was shown in Table 2. Table 2 Organic fertilizer properties. Fertilizer
pH
PC CK CM
7.18 7.89 8.40
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EC (dS m-1) 8.2 6.8 2.6
OC (g kg-1) 226.6 425.5 551.5
C:N ratio 7.79:1 19.7:1 30.1:1
Total N (g kg-1) 29.10 21.60 18.30
Total P (g kg-1) 3.90 9.70 4.50
Total K (g kg-1) 13.0 15.9 19.8 July 1-3, 2015
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Phosphorus sorption isotherm Phosphorus sorption isotherms were determined using the method of Nair et al. (1984). Two grams of the soil before planting and after second crop harvesting were taken into 50 ml of a centrifuge tube. Forty ml of 0, 0.081, 0.161, 0.323, 0.646 and 1.292 mmol P L-1 in 0.01M CaCl2 was added to the soil samples with three replications. The soil suspensions were shaken for 24 h and then centrifuged at 3000 R min-1. Phosphorus in the supernatant was determined using the method of Murphy and Riley (1962). The P adsorbed by soil was calculated as the amount of P added into soils minus the P remained in soil solution. The P sorption was characterized by Langmuir equation (Olsen and Watanabe, 1957). The Langmuir equation is defined as follow: ď&#x192;Š S max kC ď&#x192;š Sď&#x20AC;˝ď&#x192;Ş ď&#x192;Ť 1 ď&#x20AC;Ť kC ď&#x192;şď&#x192;ť Where S is the P sorbed by soil (mg kg-1), C is P concentration in equilibrium solution (mg L-1), Smax is the maximum P sorbed, and k is the constants related to the affinity energies of sorption. Langmuir equation was fitted using non-linear model. The soil properties were correlated to Smax and k constant. Results and Discussion Results indicated that Langmuir equation fit well with P sorption isotherm (Figure 1). The predicted maximum P sorption (Smax) of the soil before planting by Langmuir equation was 1,275 mg P kg-1, and K constant was 0.6653, indicating that havinghigh P sorption capacity and affinity energies.
S=
1275.9(0.6653đ??ś) (1+0.6653đ??ś)
Figure 1 Relationship between P in soil solution at equilibrium (C) and P sorbed (S) in soil before planting. Experimental data, Langmuir prediction High P sorption in Ks soil may be due to high exchangeable Ca and Mg in the soil that high precipitated with P (Table 1). Furthermore, organic matter in Ks soil was high which may enhance high P sorption. Tisdale et al. (1985) suggested that P sorption has been found to increase with OM content of soils due to adsorption involved an exchange of P anions with OH groups in the organic matter. Usually, certain organic anions arising from the decomposition of organic matter will form stable complexes with Fe and aluminum (Al) thus preventing their reaction with P and increase the available P to plants. However, the soil in this study was slightly basic with less Al and Fe. The results from P sorption isotherm of soil after second crop harvesting indicated that organic fertilizer source significantly affected on Smax and k constant (Table 3). Smax increased with CK and CM application but decreased with PM application as compared to the soil before planting. The highest and lowest Smax were found in the soil applied with CM and PM, respectively. High organic carbon in CM may increase P sorption (Table 2). Whereas, an effect of organic fertilizer sources was opposite to Smax. It indicated that an application of CM decreased the affinity energies of P
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sorption resulting in high P leaching and losses to the environment (Olsen and Watanabe, 1957). Application rates had no significant different effect on Smax and k constant. Table 3 Effects of organic fertilizer source and application rates on Smax and k constant. Factor Organic fertilizer source(O) PC CK CM Application rates (A) 0 g P pot-1 0.8 g P pot-1 1.6 g P pot-1 AxO PC 0 g P pot-1 PC 0.8 g P pot-1 PC 1.6 g P pot-1 CK 0 g P pot-1 CK 0.8 g P pot-1 CK 1.6 g P pot-1 CM 0 g P pot-1 CM 0.8 g P pot-1 CM 1.6 g P pot-1 CV (%) F-test Organic fertilizer Application rate OxA
Smax (mg P kg-1)
k
1129.8a 1338.5b 1436.0c
0.9121a 0.5782b 0.4653c
1257.4 1289.7 1348.0
0.6487 0.6806 0.6261
1180.8cd 1097.7d 1110.6d 1259.9cbd 1389.4b 1366.1b 1331.5bc 1409.0b 1567.4a 5.95 ** ns *
0.7819b 0.9848a 0.9694a 0.6280c 0.6027c 0.5038cd 0.5362cd 0.4545d 0.4051d 11.79 ** ns *
ns = not significant, *significant at 0.05 level (P<0.05), **significant at 0.01 level (P<0.01) Means followed by the same letter in each column within the same factor are not significantly different at P<0.05 by Duncanâ&#x20AC;&#x2122;s multiple range test.
Smax and k constant were correlated with soil properties after second crop harvesting (data not shown). Soil pH, organic matter, cation exchange capacity, iron, zinc, and manganese were a positive correlation with Smax (p-value<0.01). Although, exchangeable calcium was a negative correlation with Smax (r =-0.68**). However, the highest correlation was found in organic matter (r =0.74**). Whereas, exchangeable Ca was a positive correlation with k constant(r =0.83**). Conclusion A different source of organic fertilizers affected on the change of P sorption capacity. Cattle and chicken manure gave maximum P sorption. High organic carbon in organic fertilizers increased the maximum of P sorption and decreased the affinity energies. Reference Black, C.A. 1965. Methods of Soil Analysis. American Society of Agronomy, Madison, Wisconsin Kamprath, E.U., and M.E. Watson. 1980. Conventional soil and tissue tests for assessing the phosphorus status of soils., pp.433-469. In F.E. Khasawneh., E.C. Sample, E.U. Kamprath, eds. The Role of Phosphorus in Agriculture. Tennessee Valley Authority, Amer. Soc. Agron., Crop Sci. Soc. And Soil Sci. Am., Wisconsin. Murphy, J., and J.P. Riley. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta. 27: 31-36. Nair, P.S., T.J. Logan, A.N. Sharpley, L.E. Sommers, M.A. Tabatabai and T.L. Yuan. 1984. Interlaboratory comparison of a standardized phosphorus adsorption procedure. J. Environ. Qual. 13: 591-595. Olsen, S. R., and F. S. Watanabe. 1957. A method to determine a phosphorus adsorption maximum of soils as measured by the Langmuir isotherm. Soil Sci. Soc. Am. J. 21: 144- 149. Ozanne P.G., and T.C. Shaw. 1967. Phosphate sorption by soils as a measure of the phosphate requirement for pasture growth. Aust. J. Agric. Res. 18: 601-612. th
Tisdale, S.L., W. L. Nelson and J.D. Beaton. 1985. Soil Fertility and Fertilizers. 4 ed. Macmillan Publ. Com. New York. Walkley, A., and C.A. Black. 1934. An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic titration method. Soil Sci. 37: 29-38.
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P-30
A Relationship between Loss on Ignition and Total Organic Carbon Determined by Wet Digestion to Assess C/N Ratio of Plant Materials and Organic Fertilizers Nukoon TAWINTEUNG1* 1
Department of Plant Production Technology, Faculty of Agriculture Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: ktnukoon@kmitl.ac.th
ABSTRACT Loss on ignition (LOI) is a simple method for determining ash and organic matter content of compost and manure. However, only few reported on the correlation between LOI and carbon concentration of plant materials and organic fertilizers in order to assess C/N ratio. One hundred fifty-four organic materials were analyzed for organic carbon by wet digestion (modified Walkley-Black and Graham methods) and LOI. Organic materials consisted of plant materials (119 samples of rice straw) and organic fertilizers (35 samples, including: 18 samples of green-manuring, 8 samples of compost and 9 samples of animal dung) Total nitrogen was analyzed by Kjeldahl method. The result showed that carbon content and LOI were 36.4-42.3% and 82.3-92.5% for plant materials and 14.4-53.2% and 32.1-96.2% for organic fertilizers, respectively. A linear relationship between LOI and carbon content by wet digestion was highly significant in both plant materials and organic fertilizers (carbon of plant materials (%) = 0.425 x LOI (%) + 2.223; R2 = 0.633, p-value < 0.01 and carbon of organic fertilizers (%) = 0.549 x LOI (%) â&#x20AC;&#x201C; 5.704; R2 = 0.900, p-value < 0.01). The C/N ratio calculated by carbon content from the linear regression and wet digestion was not significant difference by parried sample t-test (p < 0.05). Therefore, LOI can be used to estimate of carbon concentration of both plant materials and organic fertilizers for C/N ratio assessment. Keywords: Loss on ignition (LOI), Total organic carbon (TOC), Plant material, Organic fertilizers, C/N ratio Introduction Microorganisms that decompose organic material such as organic fertilizers and plant debris use carbon as a source of energy and nitrogen for building cell structure. They need more C than N. If there is too much C, the microorganisms that break down the organic material will very quickly run out of N and therefore will start to consume the N in the soil. This reduces the amount available to the plants and therefore depresses crop yield. Decomposition takes longer, however, when the initial C/N ratio is much above 30 (USDA NRCS, 1977). The C/N ratio is based on total organic carbon and total nitrogen which there are many devices that can be used to measure this ratio such as C/N analyzer and the continuous-flow isotope ratio mass spectrometer (CF-IRMS) (Brenna et al., 1997). However, for more practical applications, desired C/N ratios can be achieved by blending common used substrates and equipments, which are readily available and easy to use. The Kjeldahl method is commonly used for total nitrogen analysis of organic materials (Jones, 1991). Whereas, the loss on ignition (LOI) is a simple and relatively inexpensive method for determining organic matter, which has been widely used in soil science (Nelson and Sommers, 1996; Howard and Howard, 1990; Konen et al., 2002; Bhatti and Bauer, 2002). However, the correlation between TOC and LOI has not much been reported. Therefore, the aim of this study was to construct a correlation
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between TOC determined by wet digestion and LOI which can be run together with dry ashing during nutrients analysis to assess C/N ratio of plant materials and organic fertilizers. Materials and Methods The plant materials (rice straw 119 samples) and organic fertilizers (35 samples, including: green-manuring 18 samples, compost 8 samples and animal dung 9 samples) were analyzed for TOC, LOI and total nitrogen. TOC was analyzed by wet digestion followed Office of Science for Land Development (2004) which modified the method of Walkley-Black and Graham. A 100 mg ground sample was added with25 mL of 1 N K2Cr2O7. Add 20 mL of H2SO4 (conc.) and allow standing for overnight. Dilute the suspension with 100 mL distilled water. Titrate with 0.5 N Fe(NH4)2(SO4)2 using 0.025 N O-phenantholine ferrous sulfate as indicator. LOI was analyzed follow Mark et al. (2005). A 1.000 g ground sample was taken into porcelain crucibles and placed in an oven at 105째C for 4 h. On removal from the oven, the samples were placed in a desiccator and allowed to cool for 30 min before the initial oven-dry weight was recorded. The samples were then placed in a preheated muffle furnace set at 550째C for 2 h. On removal from the furnace, the samples were cooled in a desiccator as before, and the final weight was recorded and then LOI percent was calculated. Total nitrogen was analyzed by Kjeldahl method (Jones, 1991). Data were subjected to regression analysis to construct a correlation between LOI and TOC. Then the C/N ratios calculated by TOC from the regression and wet digestion were compared using parried sample t-test at p < 0.05. Results and Discussion Loss on ignition, total organic carbon, total nitrogen and C/N ratio LOI and TOC of plant material (straw) were higher than those of organic fertilizer whereas total nitrogen of organic fertilizer (1.23-1.85%) was higher than that of plant material (0.430.46%) as shown on Table 1. Consequently, C/N of organic fertilizer (19-26) was lower than plant material (89-96). Studies to date have generally shown that the decomposition rate of high quality (i.e. with low lignin content and/or narrow C/N ratio) of litter is stimulated by elevated N deposition, but that the decomposition of low-quality litter is retarded (Knorr et al., 2005). Table 1 Lost on ignition, total organic carbon, total nitrogen C/N ratio of plant material and organic fertilizer. Plant material (straw) Organic fertilizes TOC (%) TOC (%) LOI (%) TN (%) C/N ratio LOI (%) TN (%) C/N ratio (wet digestion) (wet digestion) Minimum 86.88 39.16 0.430 88.52 62.31 27.75 1.23 19.44 Maximum 87.71 39.61 0.463 95.79 76.92 34.45 1.85 26.27 Mean 87.29 39.39 0.446 92.16 69.61 31.1 1.54 22.86 SE 0.21 0.11 0.008 1.84 3.55 1.63 0.15 1.66 N 119 119 119 119 27 27 27 27 LOI = lost on ignition, TOC = total organic carbon, TN = total nitrogen Data
A Relationship between loss on ignition and total organic carbon Data were subjected to regression analysis to construct a correlation between LOI and TOC as shown on Figure 1. It was found the strong linear relationship in both plant material and organic fertilizer. TOC of plant material (%) = 0.421 x LOI (%) + 2.6428; R 2 = 0.6023, pvalue < 0.01 and TOC of organic fertilizers (%) = 0.4372 x LOI (%) +0.6631; R2 = 0.9076, pvalue < 0.01. Baldock and Nelson (2000) pointed that SOM derived form LOI had a high relationship to soil organic carbon (SOC) therefore, SOM of soil substrates can be calculated Page 294
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of SOC contents by using 1.72 to 2.0 as a conversion factors depending on soil type. In comparison to peats, the average SOM/SOC ratio including organic and mineral soil substrates was 2.2 (Pribyl, 2010).
45.0
[B] Organic fertilizer
y = 0.421x + 2.6428 2
R = 0.6023**
42.5 40.0 37.5 35.0 80
85 90 Loss On Ignition (%)
95
50
TOC (wet digesttion)(%)
TOC (wet digestion) (%)
[A] Plant material
y = 0.4372x + 0.6631 R2 = 0.9076**
40 30 20 10 30
40
50
60 70 80 Loss On Ignition (%)
90
100
Figure 1 A relationship between LOI and TOC [A] plant material and [B] organic fertilizer Comparisons of TOC and C/N ratio from the equation and wet digestion It was found that TOC from the regression and wet digestion were not significantly different (p < 0.05) resulted in none significant difference for C/N ratio as shown on Table 2. Table 2 Paired samples statistic of TOC and C/N ratio. Paired samples test Plant material (straw) Pair1 TOC (wet didestion) - TOC (equation) Pair2 C/N (wet didestion) - C/N (equation) Oranic fertilizer Pair1 TOC (wet didestion) - TOC (equation) Pair2 C/N (wet didestion) - C/N (equation)
df
Sig.(2-tailed)
118 118
0.987 0.631
26 26
0.995 0.665
The high correlations (p < 0.01) were found on the graph plotted against between C/N ratio (wet digestion) and C/N ratio (equation), which R2 = 0.993 and 0.9797 for plant material [A] and organic fertilizer [B], respectively as shown on Figure 2. [A] plant material
[B] organic fertilizer 50
C/N ratio from TOC (equation)
C/N ratio from TOC (equation)
150 R2 = 0.993**
125
R2 = 0.9797**
40 30
100
20
75
10
50 50
75 100 125 C/N ratio from TOC (wet digestion)
150
10
20 30 40 C/N ratio from TOC (wet digestion)
50
Figure 2 A relationship between C/N ratio (wet digestion) and C/N ratio (equation) [A] plant material and [B] organic fertilizer. Conclusion A linear relationship between LOI and TOC by wet digestion was highly significant in both plant materials and organic fertilizers (TOC of plant material (%) = 0.421 x LOI (%) + 2.6428; R2 = 0.6023, p-value < 0.01 and TOC of organic fertilizers (%) = 0.4372 x LOI (%) July 1-3, 2015
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+0.6631; R2 = 0.9076, p-value < 0.01). The C/N ratio calculated by TOC from the linear regression and wet digestion was not significant different by parried sample t-test (p < 0.05). Therefore, LOI can be used to estimate of TOC for both plant materials and organic fertilizers in order to assess C/N ratio. References Baldock, J.A., P.N. and Nelson. 2000. Soil organic matter. In: Sumner, M.E. (Ed.), Handbook of soil science. CRC Press, Boca Raton, pp. 36–42. Bhatti, J.S. and I.E. Bauer. 2002. Comparing loss-on-ignition with dry combustion as a method for determining carbon content in upland and lowland forest ecosystems. Commun. Soil Sci. Plant Anal., 33: 3419–3430. Brenna, J. Thomas, 1997. High‐precision continuous‐flow isotope ratio mass spectrometry, Mass spectrometry reviews 16.5: 227-258. Howard, P.J.A. and D.M. Howard. 1990. Use of organic carbon and loss-on-ignition to estimate soil organic matter in different soil types and horizons. Biol. Fertil. Soils, 9: 306–310. Jones, Jr., J.B. 1991. Kjeldahl Method for Nitrogen (N) Determination. Micro-Macro Publishing, Inc., Athens, GA. Knorr, M., S.D. Frey and P.S. Curtio, , 2005. Nitrogen additions and litter decomposition: a meta analysis. Ecology 86, 3252–3253. Konen, M.E., P.M. Jacobs, C.L. Burras, B.J. Talaga and J.A. Mason. 2002. Equations for predicting soil organic carbon using loss-on-ignition for North Central U.S. soils. Soil Sci., 66: 1878–1881. Mark, K. M., J. L. Francis, L. B. Selinge and F. O. Andrew. 2005. Influence of loss-onignition temperature and heating time on ash content of compost and manure. Communications in Soil Science and Plant Analysis, 36: 2561–2573. Nelson, D.W. and L.E. Sommers. 1996. Total carbon, organic carbon, and organic matter. In Methods of Soil Analysis. Part 3. Chemical Methods; Sparks, D.L., ed.; SSSA Book Series No. 5; Soil Science Society of America: Madison, Wisconsin, 961–1010. Office of Science for Land Development. 2004. Laboratory Manuals for Soil, Water, Fertilizer and Plant Samples Analysis to Certify Commodity Standards. Department of Land Development. Bangkok. Thailand. (in Thai). Pribyl, D.W., 2010. A critical review of the conventional SOC to SOM conversion factor. Geoderma 156 (3), 75–83. USDA NRCS. 1977. Conservation Agronomy Technical Notes, No. 30: Relationships of carbon to nitrogen in crop residues. Available at http://www.nm.nrcs.usda.gov/ Technical/tech-notes/agro/AG30.pdf. [verified 1.05.15]
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P-31
Relationship between Total Calcium and Pectin in Asparagus (Asparagus officinalis L.) Cell Wall Nutcharee BOONPLANG1* 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kbnutcha@kmitl.ac.th
ABSTRACT Calcium (Ca) is a pre-harvest nutrient that affected on plant quality. Usually, high calcium status in asparagus results in better quality and fewer problems with post-havest rots. The purpose of this research was to study the relationship between total calcium and pectin on cell wall in asparagus. Asparagus spears and leaves were harvested from a commercial farm and trimmed to 180 mm in length and divided into two sections for total calcium and cell wall preparation for total pectin. Total Ca concentrations were determined using ICP-OES, after ashing the samples at 550° C for 5 hr and dissolving the ashes in 10 ml 1 N HCl for 1 hr and 10 ml distilled water overnight. Total pectin in asparagus cell wall was hydrolysed using the method of Ahmed and Labavitch (1977) and uronic acids were detected using the assay method of Blumenkrantz and Asboe-Hansen (1973). The relationship of total calcium and pectin on cell wall was slightly high but not significantly different. Keywords: Asparagus (Asparagus officinalis, L.), Calcium, Pectin Introduction Asparagus (Asparagus officinalis, L.) is a fleshly monocotyledon which good source of nutrients and contains manifold elements that a kind of health-caring vegetable contributing both to nutrition and fitness. The edible quality parameters of green asparagus include its texture, namely hardness and tenderness. Texture is a crucial sensory attribute of fruit and vegetables that influenced by cell wall structure, cell wall thickness, cell wall chemical composition, pectin degradation level and turgor pressure within cells (Jackman and Stanley, 1995). Green asparagus deteriorates quickly after harvest, losing sensory and visual quality. With the rapidity of changes in physiological and gene expression associated with harvest in mind (O’Donoghue and Somerfield, 1998). The very short shelf-life of green asparagus is mainly related to its high respiratory activity which continues after harvesting that quickly leads to the maturation and the senescence of the vegetable. Calcium is an essential plant macronutrient with key structural and signaling roles. Calcium ions act as a stabilizing element of membranes, a strengthening agent in cell walls and a secondary messenger for a multitude of signals. The Ca in membrane stability is importance, typical features of senescence and increases in respiration. Marschner (1978) suggested that the low Ca content of storage organs induces a high membrane permeability and allows solute diffusion in the tissue. This is obviously of importance in fruits and storage organs which accumulate large amounts of sugars from the phloem. In plant tissues, Ca occurs as free Ca2+, as Ca2+ adsorbed to indiffusible ions such as carboxylic, phosphorylic and phenolic hydroxyl groups. It is also present in Ca oxalates, carbonates and phosphates. These compounds often occur as deposits in cell vacuoles. In seeds, Ca present predominantly as the salt of the inositol hexaphosphoric acid (phytic acid). As already indicated, Ca in the cell wall is associated with the free carboxylic groups of the pectin and saturates most of these sites (Mengel and Kirkby, 1987). Uronic acid in the form of galacturonic acid is a major July 1-3, 2015
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component of the pectic polysaccharide rhamnogalacturonan, which is present in large amounts in the cell wall of most fruits and vegetables. The purpose of this research was to study the relationship between total calcium and pectin on cell wall in asparagus. Materials and Method Asparagus was obtained from a commercial asparagus farms at Nakhonpathom province. The asparagus leaf samples collected at the fern-growth stage were 30 cm long from the upper fern and spears collected from upper 9 cm of the spears tips (RJ Hill Laboratories Limited, 2002). Total collected samples were 144 samplings. The leaf and spear samples were washed in distilled water and air dry and then divided 2 sections. The first section of leaves and spear samples was dried in an oven at 70°C to constant weight. The dried leaf and spear samples were then ground to pass through 0.42 mm. sieve (40 mesh) in stainless steel mill. Total calcium concentrations were determined using ICP-OES (PerkinElmer Optima 4300DV), after ashing the samples at 550 °C for 5 hr and dissolving the ashes in 10 ml of 1 N HCl for 1 h and 10 ml distilled water overnight. The other leaf and spear samples were analyzed for total uronic acids in cell wall (CW). For cell wall preparation, approximately 5 g of fresh tissue from each sample was ground in 100 ml of 80% cold ethanol with mortar and pestle. The tissue was then boiled with 100 ml of 80% ethanol for 30 min to inactivate the potential wall-modifying enzymes, homogenized and filtered through a glass microfiber filter (GF/C, Whatman). The insoluble residue was washed sequentially with 50 ml. of 80% ethanol, 50 ml. of 95% ethanol, 50 ml. of chloroform: methanol (1:1, v/v) and 50 ml. of acetone. The pellets were dried at room temperature, weighed and stored in desiccator. The white pellet was considered as alcohol insoluble solid cell wall material. Total pectin in asparagus cell wall was hydrolyzed using the method of Ahmed and Labavitch (1977) and total uronic acids in cell wall were detected using the assay method of Blumenkrantz and Asboe-Hansen (1973). Total Ca in both asparagus parts was correlated with total uronic acid and fitted a linear equation using Microsoft excel. Results and Discussion The relationship between total calcium concentrations in flesh asparagus spears) and total uronic acid in cell walls were presented in Figure 1. The uronic acid content in asparagus spears were positive correlated with total Ca (r=0.72**, p≥0.01). Although, the total calcium in the asparagus leaf was also a positive correlated with the total uronic acid in cell walls (r=0.45**, p≥0.01). Total Ca in spear was significantly correlated to total uronic acid more than total Ca in leaves. It indicated that Ca in asparagus tissue was significantly related to pectin in a cell wall. Leaves and spears samples were obtained from 7 commercial asparagus farms that different in soil management, the age of plants, fern and spear. Asparagus spears are rapidly growing and rapidly respiring immature vegetative shoots. Physiological changes occur quickly in harvest asparagus spears (King et al., 1990). O’Donoghue and Somerfield (1998) suggested that harvest-related changes in asparagus cell wall are initially manifested as slowing down and eventual cessation of wall accumulation, rather than extensive wall breakdown.
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Figure 1 The relationship between total Ca and pectin in spear (a) and leaf (b) asparagus cell walls. A linear regression between total Ca and total uronic acid in cell wall of asparagus spear and leave as follows: Spear Y=7.0103x+39.361 R2=0.51 Leaves Y=0.5319x+59.183 R2=0.21 Where Y = total Ca in spear or leaves (mg/g) x = total uronic acid in cell wall of spear or leaves(mg/g CW) The both linear equation indicated that total uronic acid in spear and leaves can be predicted by total Ca but less accurate in asparagus leaves. Then, analysis of total Ca in asparagus spear can be predicted pectin in the asparagus. Conclusion The relationship of total calcium and pectin on cell wall of asparagus spear was high but less correlated with leaves. A linear equation can be predicted for pectin in asparagus spear using total Ca which is more convenient to analyze. Acknowledgments The authors gratefully acknowledge the financial support of the Faculty of Agricultural Technology, KMITL. Reference Ahmed, A. and J.M. Labavitch. 1977. A simplified method for accurate determination of cell wall uronide content. Journal of Food Biochemistry 1:361-365. Blumenkrantz, N. and G., Asboe-Hansen. 1973. New method for quantitative determination of uronic acies. Analytical Biochemistry 54:484-489. Jackman, R.L. and D.W. Stanley. 1995. Perspectives in the textural evaluation of plant foods. Trends in Food Science and Technology 7(6):187-194.
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Jarvis M.C., W. Forsyth, and H.J. Duncan. 1988. A survey of the pectic content of nonlignified monocot cell walls. Plant Physiol. 88(2):309-14. King, G.A., D.C. Woollard, D.E. Irving, and W.M., Borst. 1990. Physiological changes in asparagus spear tips after harvest. Physiologia plantarum. 80:393-400. Marschner, H. 1978. Nutritional and yield physiological aspects of plant nutrition. Angew. Botanik. 52:71-87. Mengel, K. and E.A. Kirkby. 1987. Principles of Plant Nutrition. 4th ed. International Potash Institute. Switzerland. 687p. Oâ&#x20AC;&#x2122;Donoghue, E.M. and S.D. Somerfield. 1998. Cell walls of asparagus after harvest. pp. 447450. In R.L. Bieleski, W.A. Laing and C.J. Clark, (eds.) ISHS Acta Horticulturae 464: International Postharvest Science Conference Postharvest 96. Taupo, New Zealand. RJ Hill Laboratories Limited. 2002. Crop guide: Asparagus. RJ Hill Laboratories Limited, Private Bag, Hamilton, New Zealand. Available online at http://www.hilllaboratories.com/file/21650.
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P-32
Antibacterial Activity of Roselle (Hibicus sabdariffa Linn.) Flower Extract Napaporn KONGKARN1, Pussadee TANGWATCHARIN1, Montinee TEERARAK2, Kanokporn CHANGSAWAKE2 and Komkhae PILASOMBUT1* 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kpkomkha@kmitl.ac.th
ABSTRACT The objectives of this study were to investigate total content of phenolic compounds and antibacterial activity of water and ethanol roselle (Hibicus sabdariffa Linn.) flower extracts. It was found that total phenolic content was 1262.80 and 675.80 mgGAE/100g DW for water and ethanol extracts, respectively. Various concentrations (6.25, 12.5, 25 and 50 mg/ml) of both extracts were studied for antibacterial activity against 16 bacterial strains by the agar well diffusion method. At the concentration of 50 mg/ml of ethanol extract showed antibacterial activity against all tested isolates, while antibacterial activity of water extract was not observed. However, the effectiveness of extract depended on bacterial strains (inhibition zone 11-23.33 mm). Keywords: Antimicrobial activity, Roselle, Agar well diffusion Introduction Pathogens are the major source of disease and mortality to human that become a major public health concern (Khalaphallah and Soliman, 2014; Fullerton et al., 2011). A large number of plants are used to possess antimicrobial activity against many pathogens and food spoilage bacteria (Arora and Kaur, 1999; Fullerton et al., 2011). Hibicus sabdariffa L. belongs to family Malvaceae, is commonly known in English as roselle or red sorrel (Jung and Joo, 2013). Roselle is an edible plant used in various applications, including foods, local drink and medicinal herb (Al-Hashimi, 2012; Khalaphallah and Soliman, 2014; Tolulope, 2007). This plant is widely grown in central and west Africa, South East Asia and elsewhere (Jung and Joo, 2013). Roselle is rich source of vitamin C, anthocyanin and phenolic compounds (Jung and Joo, 2013; Fullerton et al., 2011) Several studies of roselle extract have shown antimicrobial properties. In addition, AlHashimi (2012), Tolulope (2007), Khalaphallah and Soliman (2014) found that roselle extract exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus mutans, Micrococcus luteus, Bacillus cereus, Klepsiella pneumonica and Pseudomonas aeruginosa. Therefore, we were interested in investigating roselles extract for inhibiting the growth of pathogenic and food spoilage bacteria. Material and Method Preparation of roselle flower extract Water extraction Twenty grams of roselle flower powder were extracted with 80 ml of distilled water at 10°C for 72 h. Then, the extract was filtered through Whatman No. 1 filter paper and made to a concentration of 200 mg/mL and stored in a refrigerator at 4°C until assay (Changsawake et al., 2014). July 1-3, 2015
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Ethanol extraction One kilogram of dried roselle was extracted with 95% ethanol at 12°C for 72 h to yield a final concentration of 100 g dry roselle per liter (g/L). The resultant extract was filtered through four layers of cheesecloth to remove any fiber debris, followed by a second filtration through Whatman No. 1 filter paper, and set as original concentration (100 g/L). This original stock extract was kept at 5°C until used (Yonsawad et al., 2012). Determination of the total phenolic content Determination of total phenolic content in the extract was done using Folin-Ciocalteu reagent as described by Ainsworth and Gillespie (2007). Properly diluted extract of 1 ml was added to 0.5 mL of the 2N Folin-Ciocalteu reagent. The reaction mixture was mixed with 4 mL of sodium carbonate solution (7.5%, w/v) and incubated in dark at room temperature for 30 min. The absorbance of the resulting blue color was measured at 765 nm. The total phenolic contents were determined from the linear equation of a standard curve prepared with gallic acid. The content of total phenolic compounds expressed as mg gallic acid equivalent (GAE)/ 100 g dry weight (DW). Determination of antibacterial activity of roselle flower extracts A test of antibacterial activity was carried out by using agar well diffusion method followed by Perez et al. (1990). Each bacterial strain was suspended in MRS broth (Merck, Germany) and Tryptic Soy Broth (TSB) (Merck, Germany). Samples of tested bacteria were prepared to have 0.5 McFarland standard turbidity and final concentration of bacteria was 105 cfu/ml. The inoculated agar was poured into the assay plate. In each of these plates, 5 wells (6 mm) were cut out using sterile cork borer. Using sterilized dropping pipettes, 50 µl of ethanol extract was carefully added into the wells and incubated at a specified temperature. Ten percent of ethanol solution were used as positive control. After incubation, inhibition zones were observed. Inhibition of bacterial growth was measured. Lists of bacterial strains, media, and their growth condition were shown in Table 2. Results and Discussion The result showed that total phenolic contents of roselle flower extract were 1262.60 mg GAE/100 g DW and 675.80 mg GAE / 100 g DW for water and ethanol extracts, respectively (Table 1). Extracts of Hibiscus sabdariffa Linn. (roselle) were evaluated for their antimicrobial activity against 16 tested strains of bacteria. At the concentration of 50 mg/ml, ethanol extract of roselle showed all antimicrobial activity against : Lcatobacillus sakei TISTR 890, Lactococcus cremoris TISTR 1344, Leuconostoc mesenteroides subsp. mesenteroides TISTR 942, Enterococcus faecalis TISTR 888, Streptococcus sp. TISTR 1030, Lb. plantarum ATCC 14947, Lactococcus lactis 19435, Enterococcus faecalis TISTR 1344L, E. coli TISTR 780, Bacillus coagulans TISTR 1447, S. aureus TISTR 118, Bacillus subtilis JCM 1465, Listeria innocua ATCC 33090T, Pseudomonas fluorescens TISTR 358, Salmonella Typhimurium TISTR 292, and Aeromonas hydrophila TISTR 1321 (inhibition zone 11-23.33 mm), where 10% ethanol solution was used as control. On the other hand, at the concentration of 25 mg/ml, ethanol extract of roselle showed considerable antimicrobial activity against the 14 following strains: Lb. sakei TISTR 890, Le. mesenteroides subsp. mesenteroides TISTR 942, E. faecalis TISTR 888, Lb.plantarum ATCC 14947, Lactococcus lactis 19435, E. faecalis TISTR 1344L, E. coli TISTR 780, Bacillus coagulans TISTR 1447, S. aureus TISTR 118, Bacillus subtilis JCM 1465, L. innocua ATCC 33090T, P. fluorescens TISTR 358, S. typhimurium TISTR 292, and A. hydrophila TISTR 1321 (inhibition zone 8-22 mm). At the concentration of 12.5mg/ml, ethanol extract of roselle showed considerable antimicrobial activity against the 5 following strains: S. aureus TISTR 118, B. subtilis JCM 1465, P. fluorescens TISTR 358, S. Page 302
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typhimurium TISTR 292, and A. hydrophila TISTR 1321 (inhibition zone 11-12.5 mm) and at the concentration of 6.25 mg/ml, ethanol extracts of roselle inhibited only A. hydrophila TISTR 1321 (inhibition zone 10 mm) (Table 2). However, no antimicrobial activities of water extract were observed at all, as shown in Table 3. The result of this study indicated that only ethanol extract of roselle inhibited the growth of tested microorganisms. This result supported by Al-Hashimi (2012), Khalaphallah and Soliman (2014) who reported that ethanol extract of roselle showed more efficiency than water extract. There were serveral reports on antimicrobial activity of roselle extract. Tolulope (2007) reported that roselle calyces extract exhibited antibacterial activity (MIC 0.3-1.30 mg/ml) against S. aureus, B. stearothermophilus, Micrococcus luteus, Serratia mascences, E. coli, B. cereus and P. fluorescence. Al-Hashimi (2012) also reported that aqueous extract and alcoholic extract showed antibacterial activity against E. coli, S. aureus, Streptococcus mutans and P. aeruginosa. In addition, the extract exhibited antioxidant activity. The phenolic content was 77.2 mg/g and 87.7 mg/g for aqueous and alcoholic extract. Conclusion The results clearly showed that ethanol extract of roselle had strong antimicrobial activity. The study suggested that this extract could be used as an antimicrobial activity in meat and meat product. Table 1 Total phenolic content of roselle flower extracts. Extract Water extract Ethanolic extract
Total Phenolic content (mg GAE / 100 g DW) 1262.60 675.80
Table 2 Inhibitory activity of ethanol extracts of roselle against food pathogenic and food spoilage bacteria. Bacterial strains
Media
MRS Lactobacillus sakei TISTR 890 MRS Lactococcus cremoris TISTR 1344 MRS Leuconostoc mesenteroides subsp. mesenteroides TISTR 942 MRS Enterococcus faecalis TISTR 888 MRS Streptococcus sp. TISTR 1030 MRS Lb. plantarum ATCC 14947 MRS Lactococcus lactis 19435 MRS Enterococcus faecalis TISTR 1344L TSB-0.6%YE Escherichia coli TISTR 780 TSB-0.6%YE Bacillus coagulans TISTR 1447 TSB-0.6%YE Staphylococcus aureus TISTR 118 TSB-0.6%YE Bacillus subtilis JCM 1465 TSB-0.6%YE Listeria innocua ATCC 33090T TSB-0.6%YE Pseudomonas fluorescens TISTR 358 TSB-0.6%YE Salmonella typhimurium TISTR 292 TSB-0.6%YE Aeromonas hydrophila TISTR 1321 ATCC = American Type Culture Collection, Rockville, Md JCM = Japanese Culture of Microorganism, Wako, Japan TISTR = Thailand Institute of Scientific and Technological Research, Thailand - means no inhibitory activity MRS = DE MAN ROGOSA and SHARPE TSB = Tryptic Soy Broth, YE = Yeast extract granulated
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37 37 30
Inhibition zone (mm.) Concentration (mg/ml) 50 25 12.5 6.25 14.33 10 11.5 13 10 -
37 30 30 30 37 37 37 37 37 37 26 37 30
18 12 11 12 22.33 20.66 19.66 22.66 21 20.66 21 21.33 23.33
ŕš?
C
22 9 8 14 13 15 15 15 15 12 15 16
12.5 11 11 12 12
10
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Table 3 Inhibitory activity of water extracts of roselle against food pathogenic and food spoilage bacteria. Bacterial strains Lactobacillus sakei TISTR 890 Lactococcus cremoris TISTR 1344 Leuconostoc mesenteroides subsp. mesenteroides TISTR 942 Enterococcus faecalis TISTR 888 Streptococcus sp. TISTR 1030 Lb. plantarum ATCC 14947 Lactococcus lactis 19435 Enterococcus faecalis TISTR 1344L Escherichia coli TISTR 780 Bacillus coagulans TISTR 1447 Staphylococcus aureus TISTR 118 Bacillus subtilis JCM 1465 Listeria innocua ATCC 33090T Pseudomonas fluorescens TISTR 358 Salmonella typhimurium TISTR 292 Aeromonas hydrophila TISTR 1321
Media
ŕš?
C
MRS MRS MRS
37 37 30
MRS MRS MRS MRS MRS TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE TSB-0.6%YE
37 30 30 30 37 37 37 37 37 37 26 37 30
Inhibition zone (mm.) Concentration (mg/ml) 50 25 12.5 6.25 -
-
-
-
ATCC = American Type Culture Collection, Rockville, Md JCM = Japanese Culture of Microorganism, Wako, Japan TISTR = Thailand Institute of Scientific and Technological Research, Thailand - means no inhibitory activity MRS = DE MAN ROGOSA and SHARPE; TSB = Tryptic Soy Broth; YE = Yeast extract granulated
References Ainsworth, E.A. and K.M. Gillespie. 2007. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nature Protocols 2 (4): 875-877. Al-Hashimi, A.G. 2012. Antioxidant and antimicrobial activity of Hibiscus sabdariffa L. extract. Journal of Food Science 6 (21): 506-511. Arora, D.S. and J. Kuar. 1999. Antimicrobial activity of spices. International Journal of Antimicrobial Agents 12 (3): 257-262. Changsawake, K., W. Krusong, C. Laosinwattana and M. Terarak. 2014. Antioxidant properties and DNA damage protective potential of RD6 glutinous rice. In 12th International Sysmposium on Biocontrol and Biotechnology.70 p. Jung, E. and N. Joo. 2013. Roselle (Hibiscus abdariffa L.) and soybean oil effects on quality characteristics of pork patties studied by response surface methodology. Meat Science 94 (3): 391-401. Khalaphallah, R. and W.S. Soliman. 2014. Effect of henna and roselle extracts on pathogenic bacteria. Asian Pacific Journal of Tropical Diseases 4 (4): 292-296. Perez C., M. Paul and P. Bazerque. 1990. Antibiotic assay by agar-well diffusion method. Acta Biologiae Medica Experimaentalis 15: 113-115. Tolulope, O.M. 2007. Cytotoxicity and antibacterial activity of methanolic extract of Hibiscus sabdariffa. Journal of Medicinal Plants Research 1 (1): 9-13. Yonsawad, N., P. Wichittrakarn, M. Teerarak and C. Laosinwattana. 2012. Antioxidant activity from crude extract of Etlingera elatior (JACK) R.M. SMITH. In 10th International Symposium on Biocontrol and Biotechnology. Harbin,P.R.China. 39-43.
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P-34
In vitro Antagonistism of Talaromyces flavus and Neosartorya pseudoficheri Against Anthracnose Disease on Coffee Mayamor SOYTONG1 and Supattra POEAIM1* 1
Department of Biology, Faculty of Science, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: poeaim@hotmail.com
ABSTRACT Six isolates of Talaromyces flavus (EU05, EU07, EU16, EU18, EU25 and EU26) and three isolates of Neosartorya pseudoficheri (EU27, EU35 and EU36) were proved to be antifungal activity against Colletotrichum coffeanum causing coffee anthracnose var. Arabica. T. flavus EU05 expressed the highest significantly difference inhibition of pathogen spore at 92.49 %, and followed by T. flavus EU26 (84.95 %). T. flavus EU18, EU25, EU16, EU07 and N. pseudoficheri EU27 and EU35 showed high antagonistic activity which inhibited spore production of 81.89, 80.28, 75.80, 74.39, 71.59 and 67.64 %, respectively. N. pseudoficheri EU36 was recorded as the lowest antifungal activity. This research finding is the first report of antagonism of T. flavus and N. pseudoficheri against anthracnose disease in coffee. Keywords: Biological control, Colletotrichum coffeanum, Neosartorya pseudoficheri and Talaromyces flavus Introduction Talaromyces sp. and Neosartorya sp. are saprophytic fungi belong to Trichocomaceae. They are found to disperse in food, indoor environment, dust and forest. T. flavus is a telomorph of Penicillium dangeardii; sometimes reported as Penicillium vermiculatum and Neosartorya is a telomorph of Aspergillus fumigatus. They play an important role in agriculture and as biocontrol agent against fungal pathogens including Sclerotium rolfsii and Verticillium dahlia and their bioactive compound inhibited pathogenic fungi causing human disease (Shiozawa et al., 1995; Suzuki et al., 2000). Presently, there are several hundred million people drink coffee although toxic chemical pesticide still remain in coffee beans and pathogen become resistant to chemicals (Soytong et al., 2001). A problem which damages coffee plants is anthracnose caused by Colletotrichum coffeanum which causing low quality of coffee bean (Vilavong and Soytong, 2013). Biological control of plant disease is increasingly interesting to replace the chemicals in agriculture. So, the aim of present study was to evaluate in vitro antifungal activity of T. flavas and N. pseudoficheri against C. coffeanum causing anthracnose disease on coffee var. Arabica. Materials and Methods Antagonists: Neosartorya pseudoficheri and Talaromyces flavus Neosartorya spp. and Talaromyces spp. were isolated from soil in Doi Suthep and Doi Inthanon mountains (Chiang Mai, Thailand) by soil plate technique. Each species was identified based on morphology and DNA sequencing (Soytong and Poeaim, 2014) which EU05, EU07, EU16, EU18, EU25 and EU26 were identified as Talaromyces flavus and EU27, EU35 and EU36 were identified as Neosartorya pseudoficheri. These were selected by random for screening the fungal activity of each isolate. Pure cultures were maintained on potato dextrose agar (PDA) medium in petri dish and incubated at 25ď&#x201A;°C.
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Pathogen: Colletotrichum coffeanum C. coffeanum was isolated from anthracnose of coffee var. Arabica planted in Paksong highland, Laos PDR by isolation techniques of Vilavong and Soytong (2013). Pure culture was maintained on PDA medium in petri dishes and incubated at 25C. Antagonistic activity in vitro Nine isolates of promising antagonists, T. flavus (EU05, EU07, EU16, EU18, EU25, and EU26) and N. pseudoficheri (EU27, EU35 and EU36) were screened for antagonistic effect against anthracnose of coffee var. Arabica which caused by C. coffeanum. To test the abilities of antagonistic fungi to inhibit the growth of C. coffeanum, bi-culture test was performed as the following method of Soytong (1992). The experiment was designed in Completely Randomized Design (CRD) with three replications. The antagonistic fungi (T. flavus and N. pseudoficheri) and coffee anthracnose pathogen (C. coffeanum) were separately cultured on PDA petri dishes. The edge of radial growth of pathogenic fungus and each antagonist was separately cut with 5 mm diameter sterilized cork borer and one agar plug of antagonist was transferred to PDA plate at one side and agar plug of pathogen was transferred to opposite site. The antagonist and pathogen were separately cultured on PDA plates served as the controls. The tested plates were incubated at room temperature (25C) until pathogen in control growing in full plate. Data were collected and calculated as spore number of pathogen and inhibition. The spore number of pathogen in bi-culture, and control plates were counted by using haemacytometer. Spore inhibition of pathogen (%) was calculated as the following formula: % inhibition = (A-B) / A ×100, where A = spore number of pathogen in control and B = spore number of pathogen in bi-culture plate. All data were statistically computed for analysis of variance (ANOVA). Treatment means were compared by using Duncan’s New Multiple Range Test (DMRT) at P=0.05 and P=0.01. Results and Discussion Bi-culture antagonistic test of T. flavus and N. pseudoficheri and C. coffeanum were investigated. Result showed that the number of spores which produced by C. coffeanum, T. flavus isolate EU05, EU07, EU16, EU18, EU25, EU26, N. pseudoficheri isolate EU27, EU35 and EU36 showed significantly inhibited number of spores which were 4.63×105, 15.80×105, 8.00×105, 0.94×105, 1.15×105, 0.86×105, 5.37×105, 19.20×105 and 21.87×105 spores, respectively, when compared to the control plate (Table 1). The research showed T. flavus EU05 gave the highest significantly difference inhibition of pathogen spore at 92.49 % and followed by T. flavus EU26 which spore inhibition of 84.95 %. Moreover, T. flavus EU18 and EU25 gave high antagonistic activity and were not significantly different in spore inhibition of 81.89 and 80.28 %, respectively. T. flavus EU07 and EU16 showed high antagonistic activity and were not significant difference which spore inhibition of 74.39 and 75.80 %, respectively. The tested antagonistic activity, T. flavus EU05 expressed the highest antifungal activity against coffee anthracnose that would become the first priority of promising antagonist to investigate for biocontrol of coffee anthracnose. The other isolates showed high antagonistic activity were T. flavus EU07, EU16, EU18, EU25, EU26 and N. pseudoficheri EU27 and EU35 that would be interesting antagonists in further research. N. pseudoficheri EU36 was recorded as the lowest antifungal activity against coffee anthracnose. This study is similar to the works of Stosz et al. (1996) and Madi et al. (1997) who reported that T. flavus can give a good control of plant pathogens: Sclerotium rolfsii and Verticillium dahlia both in laboratory and field and it is known that the enzymes released from T. flavus can inhibit the growth of Botrytis fabae (faba bean chocolate spot) (Haggag et al., 2006). Dethoup et al. (2007) reported that T. flavus has ability to control Colletotrichum capsici and C. gloeosporioides (chilli anthracnose). These research findings of T. flavus and N. pseudoficheri would be further testing with other plant pathogens and develop as Page 306
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biological fungicide (Soytong et al., 2001). Sibounnavong et al. (2012) found that Emericella rugulosa which related species to tested antagonists could express antifungal activity against Fusarium oxysporum f. sp. lycopersici (tomato wilt). However, Jun et al. (1999) found that T. flavus could not inhibit the growth of Colletotrichum gossypii that would be noticed. In addition, there is an interesting report that crude extract of N. pseudoficheri has ability to inhibit Phytophthora palmivora and C. capsici at 100 ppm (Boonsang et al., 2014). Kanomedhahul et al. (2011) found that bioactive meroterpenoids and alkaloids are produced from Eurotium chevalieri and Moosophon et al. (2009) found prenylxanthones and a bicycle (3.3.1) nona-2, 6-diene derived from Emericella rugulosa which related species to tested antagonists that the compounds expressed antimicrobial activity against human pathogens. It is recommended for future studies to investigate their metabolites for antifungal activity against pathogens. Table 1 Number of pathogen spores on antagonistic bi-culture tests.
Antagonists
Collectotrichum coffeanum Number of spore Inhibition of spore production % SE (× 105)
T. flavus EU05 4.63 + 0.33 92.49e T. flavus EU07 15.80 + 0.65 74.39bc T. flavus EU16 8.00 + 1.31 75.80bc T. flavus EU18 0.94 + 0.26 81.89cd T. flavus EU25 1.15 + 0.17 80.28cd T. flavus EU26 0.86 + 0.62 84.95de N. pseudoficheri EU27 5.37 + 0.12 71.59b N. pseudoficheri EU35 19.20 + 0.47 67.64b N. pseudoficheri EU36 21.87 + 0.61 48.61a The average of three replications and means followed by a common letter were not significantly different by DMRT at P = 0.05.
Conclusion T. flavus EU05 expressed the highest antifungal activity against C. coffeanum causing coffee anthracnose. Other isolates of T. flavus (EU07, EU16, EU18, EU25 and EU26) and N. pseudoficheri (EU27 and EU35) showed high antifungal activity and N. pseudoficheri EU36 was recorded as the lowest antifungal activity. So, T. flavus EU05 would become the first priority of promising antagonist to be investigated for biocontrol of coffee anthracnose and future studies should investigate its metabolites. Acknowledgements We would like to thank Assoc. Prof. Dr. Kasem Soytong for his comment and suggestion in our experiment and Asst. Prof. Donlachart Tantivanit for his suggestion about statistics. This paper is a part of the author’s Master of Science Thesis. References Boonsang, N., T. Dethoup, N. Singburaudom, N.G.M. Gomes and A. Kijjoa. 2014. In vitro antifungal activity screening of crude extracts of soil fungi against plant pathogenic fungi. Journal of Biopesticides 7(2): 156-166. Dethoup, T., L. Manoch, N. Visarathanonth, C. Chamswarng and A. Kijjoa. 2007. Morphology and distribution of Talaromyces flavus from soil and potential use as a July 1-3, 2015
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biological control agent against plant pathogenic fungi. Thai Journal of Agricultural Science 40(1): 37-50. Haggag, M.W., A.L. Kansoh and M.A. Aly. 2006. Proteases from Talaromyces flavus and Trichoderma harzianum: purification, characterization and antifungal activity against brown spot disease on Faba bean. Plant Pathology Bulletin 15: 231-239. Jun, W.S., L.G. Qin, J. DaoHong and W. DaoBen. 1999. Study on the antagonism of Talaromyces flavus against pathogenic fungi infecting cotton. Journal of Huazhong Agricultural University 18(1): 16-19. Kanokmedhakul, K., S. Kanokmedhakul, R. Suwannatrai, K. Soytong, S. Prabpai and P. Kongsaeree. 2011. Bioactive meroterpenoids and alkaloids from the fungus Eurotium chevalieri. Tetrahedron 67: 5461-5468. Madi, L., J. Katan and Y. Henis. 1997. Biological control of Sclerotium rolfsii and Verticillium dahliae by Talaromyces flavus is mediated by different mechanisms. Phytopathology 87:1054-1060. Moosophon, P., S. Kanokmedhakul, K. Kanokmedhakul and K. Soytong. 2009. Prenylxanthones and a bicycle (3.3.1) nona-2, 6-diene derive from the fungus Emericella rugulosa. Journal of Natural Product 72: 1442-1446. Shiozawa, H., M. Takahashi, T. Takatsu, T. Kinoshita, K. Tanzana, T. Hosoya, K. Furuya, S. Takahashi, K. Furihataand and H. Seto. 1995. Trachyspic acid, a new metabolite produced by Talaromyces trachyspermus, that inhibits tumor cell heparanase: taxonomy of the producing strain, fermentation, isolation, structural elucidation, and biological activity. The Journal of Antibiotics 48(5): 357-562. Sibounnavong, P., S. Kanokmedhakul and K. Soytong. 2012. The role of Emericella rugulosa as a bio-control agent for controlling Fusarium wilt of tomato. African Journal of Agricultural Research 7(34): 4782-4789. Soytong, K. 1992. Biological control of rice blast disease by seed coating with antagonistic fungi. Songklanakarin. Journal of Science and Technology 14: 59-65. Soytong, K., S. Kanokmedhakul, V. Kukongviriyapan and M. Isobe. 2001. Application of Chaetomium species (Ketomium) as a new broad spectrum biological fungicide for plant disease control: A review article. Fugal Diversity 7: 1-15. Soytong, M. and S. Poeaim. 2014. Isolation and identification of Trichocomaceae from soil by morphology and DNA sequencing. Proceeding: The 3rd ICIST, Pakse, Laos, PDR 27-28 November 2014. 51-62. Stosz, S.K., D.R. Fravel and D.P. Roberts. 1996. In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium Dahlia. Applied and Environmental Microbiology 62(9): 3183-3186. Suzuki, S., T. Hosoe, K. Nozawa, K. Kawai, T. Yaguchi and S. Udagawa. 2000. Antifungal substances against pathogenic fungi, Talaroconvolutins, from Talaromyces convolutus. Journal of Natural Product 63: 768-772. Vilavong, S. and K. Soytong. 2013. Plant pathogenic fungi and pathogenicity test on Arabica Coffee plantation at Paksong in Lao PDR and preliminary biocontrol test in vitro. Proceeding: ICIST 2013, KMITL, November 28-29, 2013. 444-452.
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P-35
Identification of Blast Resistance Gene in Yang Mawng Variety of Thai Indigenous Rice Siriporn PRAMRIT1,2*and Nonglak PARINTHAWONG3 1
Department of Agricultural Biotechnology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Center of Excellence on Agricultural Biotechnology: (AG-BIO/PERDO-CHE), Bangkok 10900, Thailand 3 Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: siripornpramrit@gmail.com
ABSTRACT Rice blast, caused by Pyricularia oryzae, is a major disease of rice almost worldwide. KDML105 is a good quality cultivar, however, this cultivar is susceptible to rice blast disease. Therefore, finding of new rice blast resistance gene is a major approach for rice breeding program. A Thai indigenous rice variety,Yang mawng, is highly resistant to the infection of P. oryzae. In this study, an F2 population was developed from a cross between KDML105 and Yang mawng. Two hundred and twenty eight F2 plants were inoculated with conidia suspension of P. oryzae and the disease was scored 7 days later. The segregation of resistance and susceptible phenotype showed a goodness of fit to the ratio 15:1. Two hundred and thirty simple sequence repeat (SSR) markers were screened for polymorphism. The result showed that 111 markers showed polymorphism between the parents KDML105 and Yang mawng. Bulk segregant analysis (BSA) was conducted using 111 SSR markers and four markers included RM495, RM431, RM443 and RM543 were obtained. This suggested that, the gene that control blast disease resistance in Yang mawng variety might locate on chromosome 1 of rice genome. The four markers will be used in further analysis of an individual F2 population in order identify the SSR markers linked to blast resistance gene. Keywords: Rice blast, Blast resistance gene, Pyricularia oryzae Introduction Blast disease caused by a fungal pathogen, Pyricularia oryzae, is one of the important rice diseases because of its devastating effects on yield under favorable conditions. Effective and durable use of blast resistance lines has been limited and lost within a few years of initial cultivation due to the emergence of more virulent forms of the rice blast fungus (Zhou et al., 2007). However, development of blast resistant varieties of rice is yet the most effective and economical method of controlling this disease. Thai indigenous rice variety provides potentially valuable resources for the improvement of cultivated rice. In addition, Thai indigenous rice variety is a donor of several other resistance genes. To develop an effective resistance, it is essential to determine spectrum of resistance mediated by resistance (R) genes. To date, more than 70 R genes and some quantitative trait loci (QTL) have been identified and mapped on rice chromosomes (Liu et al., 2010; Koide et al., 2009; Ballini et al., 2008). The use of molecular markers in many aspects of rice studies has been increasing considerably. Microsatellites or simple sequence repeats (SSRs) are widely used in rice genetic studies. SSRs are highly polymorphic genetic markers and because of their widespread distribution in the genome. The objective of this study was to analyze SSR
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markers associated with rice blast resistance genes in an F2 population derived from Yang mawng resistant variety and KDML105, a susceptible rice cultivar. Materials and Methods Plant material and phenotypic evaluation The F1 plant, which obtained from the cross between KDML105 and Yang mawng varieties were self-fertilized. A total of 228 F2 plants from a cross were harvested and used for developing the mapping population. Seeds were pre-germinated by soaking in water at 30 ºC for 2 days and were, sown in plastic trays (30×60×4 cm) filled with soil. The parental cultivars KDML105 and Yang mawng were also included as controls. Seedlings were grown in a greenhouse for 3 weeks before inoculation. Nineteen isolates of P. oryzae, were cultured on Rice Flour Agar (RFA) for 10 days. Conidia were induced by adding sterilized water on to mycelium and scraping with L-shape glass rod. Plates were incubated on room temperature for another 2 days. The conidia were harvested and the conidial suspension was adjusted to 105 conidia/mL using hemacytometer and mixed together. Seedlings in each tray were sprayed with a 100 ml conidia suspension and incubated at 25 ºC under high humidity for 18 hours, and sprayed three or four times a day with distilled water to maintain high humidity for 3 days. Disease incidence was observed 7 days after inoculation using the visual rating scale 0 to 6 rating system as described by Roumen et al. (1997). The plants showing scores 02 were considered resistant, 3-4 were considered moderate resistant and 5-6 as susceptible. KDML105 and Yang mawng were used as a susceptible and resistant control, respectively. In the inheritance study, the frequencies of the classes obtained were tested for significance by Chi-square. DNA extraction and PCR conditions Total genomic DNA was extracted from frozen leaves of the seedlings using CTAB method described by Doyle and Doyle (1990). Two hundred and thirty simple sequence repeat (SSR) markers distributed evenly across the twelve chromosome of rice were screened for polymorphisms between KDML105 and Yang mawng. Those markers that showed polymorphism between parents were used for bulk segregant analysis (BSA). DNA pools of susceptible (B1) and resistant (B2) bulks were generated by mixing equimolar amounts of DNA from either 5 resistant or 5 susceptible F2 individuals, respectively. The BSA was carried out to identify molecular markers putatively associated with the resistant and susceptible phenotype using polymerase chain reaction (PCR). The PCR mix consisted of 10 μL of reaction mixture containing 20 ng of total DNA, 5 μmol each of primers, 2.5 mM dNTPs, 1.5 mM MgCl2 10X Taq polymerase buffer and 1 unit Taq polymerase. The PCR was conducted with thermocycler (Biometra, Germany), with the following temperature profiles, the initial denaturation was at 95 ºC for 5 min, followed by 30 cycles of denaturing at 95 ºC for 30 seconds, annealing at 55 ºC for 30 seconds, extension at 72ºC for 30 seconds, and 5 min at 72ºC for final extension. The PCR products were separated on 6% polyacrylamide gels and visualized using the silver staining method described by Benbouza et al. (2006). The target bands were scored and analyzed. Results and Discussion Phenotypic evaluation in F2 populations Nineteen isolates of P. oryzae were collected from northern, eastern, northeast and southern region in Thailand. The conidia suspensions were pooled together and used to evaluate susceptible and resistance frequencies in F2 population. The resistant trait obtained will indicate broad-spectrum resistance to phenotype P. oryzae., as reported previously by Chen et al. (2005) and Zhou et al. (2004). Among the 228 tested F2 seedlings, 223 seedlings were characterised as resistant, while only 5 seedlings were found to be susceptible to fungal Page 310
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pathogen isolates. The numbers of resistant and susceptible lines at the ratio of 3:1 (2 = 63.24; P = 0.00) were unreliable. While, the segregation of resistance and susceptible phenotype showed a goodness of fit to the ratio 15:1 (2 = 6.13; P = 0.011) (Table 1). Chisquare data on the segregation analysis suggested that the resistant phenotype of the Yang mawng variety on blast disease was controlled by more than one gene. Two hundred and thirty SSR markers were screened for polymorphisms between parents KDML105 and Yang mawng varieties, and 111 markers were obtained as they showed polymorphism between the two parents. BSA was conducted using 111 SSR markers and four markers included RM495, RM431, RM443 and RM543 on chromosome 1 were found polymorphism between susceptible and resistant parents (Figure 1) and corresponding bulks indicating their possible association with blast resistance in the mapping population. Similarly, Lin et al. (2007) reported that RM543 linked to the resistance gene Pi37 and Chen et al. (2005) reported that RM543 linked to the resistance gene Pi37(t). The F2 mapping population will be genotyped with these four markers to study their possible association with blast resistance. Table 1 Segregation in the F2 population obtained from a cross between KDML105 and Yang mawng varieties at 7 days after inoculation with 19 isolates of P. oryzae. Total no. of seedlings
Expected ratio
Expected No. R
S
R
S
2
p
F2 (228)
3:1
214
14
223
5
63.24
0.00
F2 (228)
15:1
214
214
223
5
6.13
0.011
P2 B1 B2
A
P1 P2 B1 B2
A
B
A
B
P1
P2
P1
B1 B2 A
RM543
B
A
B
B
A
A
RM443
RM431 P1
Observed No.
B
A
H
P2
B1 B2 A
RM495
Figure 1 DNA patterns of markers analyzed by bulked segregant analysis (BSA). P1; KDML105, P2; Yang mawng, B1; resistance bulk and B2; susceptible bulk. A = susceptible parent allele; B = resistant parent allele and H = heterozygous.
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Conclusion In summary, an F2 population was developed from a cross between KDML105 and Yang mawng. Two hundred and twenty eight F2 plants were inoculated with the mix of conidia suspension of P. oryzae and the disease was scored 7 days later. The segregation of resistance and susceptible phenotype showed a goodness of fit to the ratio 15:1 (2 = 6.13; P = 0.011). Chi-square data on the segregation analysis suggested that the resistant phenotype of the Yang mawng variety on blast disease was controlled by more than one gene. Two hundred and thirty simple sequence repeat (SSR) markers were screened for polymorphism. The result showed that 111 markers showed polymorphism between the parents KDML105 and Yang mawng. Bulk segregant analysis (BSA) was conducted using 111 SSR markers and four markers included RM495, RM431, RM443 and RM543 which located on chromosome 1 were obtained. Acknowledgments This research is partially supported by the Center of Excellence on agricultural Biotechnology Science and Technology Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of Education (AG-BIO/PERDOCHE), Thailand Advanced Institute of Science and Technology. Thanks also to King Mongkut’s Institute of Technology Ladkrabang for financial support for presenting this research at the symposium. References Ballini, E., Morel J.B., Droc G., Price A., Courtois B., Notteghem J.L. and Tharreau D. 2008. A genome-wide meta-analysis of rice blast resistance genes and quantitative trait loci provides new insights into partial and complete resistance. Molecular Plant-Microbe Interact 21(7): 859–868. Benbouza, H., Jacquemin J.M., Baudoin J.P. and Mergeai G. 2006. Optimization of a reliable,fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnology, Agronomy, Society and Environment 10(2): 77-81. Chen, F., Wang L., Que Z., Pan R. and Pan Q. 2005. Genetic and physical mapping of Pi37(t),a new gene conferring resistance to rice blast in the famous cultivar St. No. 1. Theoretical and Applied Genetics 111: 1563-1570. Doyle, J.J. and Doyle J.L. (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15. Koide, Y., Kobayashi N., Xu D. and Fukuta Y. 2009. Blast resistance genes and their selectionmarkers in rice (Oryza sativa L.), JIRCAS Working Report 63: 95-122. Lin, F., Chen S., Que Z., Wang L., Liu X. and Pan Q. 2007. The blast resistance gene Pi37 encodes a nucleotide binding site–leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1. Genetics 177: 1871-1880. Liu, J., Wang X., Mitchell T., Hu Y., Liu X., Dai L. and Wang G. 2010. Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction. Molecular Plant Pathology 11: 419–427. Zhou, E., Jia Y., Correll J. and Lee F.N. 2007. Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence. Fungal Genetics and Biology 44: 1024-1034. Zhou, J.H., Wang J.L., Xu J.C., Lei L.C. and Ling Z.Z. 2004. Identification and mapping of a rice blast resistance gene Pi-g(t) in the cultivar Guangchangzhan. Plant Pathology 53: 191-196.
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P-36
Acaricidal Activity of Paracress (Acmella oleracea (L.) R.K. Jansen) Extract on the Mushroom Mite (Dolichocybe indica Mahunka) Jarongsak PUMNUAN1*, Jakkrapong AREEWONG2, Pornprapa KONGTRAGOUL2 and Ammorn INSUNG1 1
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Program in Horticulture, Department of Agricultural Technology, King Mongut’s Institute of Technology Ladkrabang, Prince of Chumphon Campus, Chumphon 86160, Thailand *Corresponding email: kpjarong@kmitl.ac.th
ABSTRACT Acaricidal activities of ethanol, acetone and hexane extracts ofleaf, stem, and root from paracress (Acmella oleracea (L.) R.K. Jansen) against adults of mushroom mite (Dolichocybe indica Mahunka) were investigated by residual contact method. The bioassay was conducted withvarious concentrations of the extracts (10.6, 21.2, 31.8, 42.4, 53.0 and 63.6 µg/cm2) in glass tubes sized 0.4 cm in diameter and 3 cm in length, withboth ends sealed with filter paper. Standard ethanol, acetone and hexane were used as the controls. Percentages of mortality were examined at 12 h after application. The results showed that the acetone and hexane extracts from leaf, stem, and root of paracress were highly effective in controlling mushroom mite. The applications at 53.0 µg/cm2from all extracts showed more than 80% mortalities. The LC50was obtained at 14.90-35.38 µg/cm2. The acetone root extract showed the highest toxicity against the mite with the LC50at 14.90 µg/ cm2, while mortality of the mite treated with acetone extracts of leaf and stem showed the LC50at 20.12 and 22.73 µg/ cm2, respectively. Keywords: Residual contact method, Crude extract, Local plant, Spilanthes acmella Introduction The mushroom mite (Dolichocybe indica Mahunka) is generally considered as the major pest causing extensive yield losses in mushroom production, particularly in tree ear (Auricularia auricular Judae), split gill (Schizophylum commune Fr), yanagi matsutake (Agrocybe cylindracea (Dc.Ex.Fr.) Maire.) and golden needle (Flammulina velutipes Karst.) (Grudloyma et al., 2010). Unfortunately, the management of mite infestation has generally been limited to the application of chemical substances, particularly the phosphine fumigation (Kulpiyawat et al., 2008). In particular, phosphine has been frequently reported for resistance of insects, while also causing damages on produces. Normally, a series of particular pesticide applications will increase the risk of the pesticide resistance in insects (Daglish, 2004). Consequently, plant essential oils and crude extracts have recently been considered a potential alternative acaricide and have become a rich source of pesticide studies. For example, extracts from clove and cinnamon were also found highly toxic against a mushroom mite (Luciaphorus perniciosus Rack) (Pumnuan et al., 2008). In addition, acaricidal activities of some wild plants in Thailand (Justicia adhatoda L. and Litsea cubeba (Lour.) Pers.) were also observed presenting high potency in controlling L. perniciosus and Formicomotes heteromorphus Magowski (Insung et al., 2008). Pumnuan et al. (2010) reported the toxicity of L. cubeba essential oil against L. perniciosus by contact and fumigation methods. Paracress (Acmella oleracea (L.) R.K. Jansen) is member of herbaceous plants in the Compositae family. This is non-ingested weed that commonly appears in cattle pastures, but people used it as medicinal herb (Ponpornpisit et al., 2011). The paracress is renamed from the two plants, Spilanthes acmella (L.) Murray and Spilanthes oleracea Jacquin. The plant has shown anti-inflammatory,
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antibacterial, antifungal, diuretic, sialagogic and larvicidal properties (Phrutivorapongkul et al., 2008). Regard to the extracts or its constituents, this plant showed toxic activity against larvae of diamondback moth (Sharma et al., 2012), rice weevil (Broussalis et al., 1999), mosquito (Pandey et al., 2007) and corn earworm (Ramsewak et al., 1999). This study was to investigate the acaricidal activities of ethanol, acetone and hexane extracts from various parts of paracress (Acmella oleracea (L.) R.K. Jansen) including leaf, stem and root in controlling an economic mushroom mite (Dolichocybe indica Mahunka) under laboratory condition by residual contact method. Materials and Methods Stock culture of mushroom mite Colonies of mushroom mite (D. indica) were obtained from a mushroom farm in Phetchaburi province, Thailand. The mites were reared on sorghum grain infested with mycelia of Schizophylum commune Fr. under the temperature of 252C in laboratory conditions. Crude extracted preparation Fresh parts of paracress (A. oleracea) including leaf, stem and root samples (1 kg) were extracted with 2.5 L of hexane using soaking method allowed to stand at room temperature about 2 days, then filtered through filter paper. After filtering, the hexane was removed at 50C using a rotary evaporator, to obtain the crude hexane extract. The remaining materials were then extracted with 2.5 L of acetone and ethanol, respectively. By the same method, acetone and ethanol crude extracts were also obtained. The final yield of crude extract was re-suspended in 10% Tween-20 in water to a volume of 10 ml, to make a 10% (w/v) stock solution and kept at 10C for subsequent use. Bioassay The contact treatment method applied in this study was modified from Pumnuan et al. (2010). A glass tube, 0.4 cm in diameter and 3 cm long with fine nylon mesh on both ends, was used to confine the mite samples. In general, 20 µl of the extract at various concentrations (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2% extract solutions equaling to 10.6, 21.2, 31.8, 42.4, 53.0 and 63.6 µg/cm2, respectively) and 1.2% Tween-20 in water was used as the control. The solution was distributed evenly around the inner wall of the test tube and allowed to air dry, before 15-20 non-physogastric mites were introduced into each glass tube. Observations were made 12 h after treatment and the number of dead mites was recorded. The experiment was CRD with 3 replications (3 glass tube per replication). Statistical analysis The experiment was a completely randomized design with five replicates. The actual death rates were calculated using Abbot’s formula (Abbott, 1987). The data obtained were statistically analyzed by applying analysis of variance (ANOVA) and Duncan’s multiple range tests (DMRT) in SAS. Then, the statistical outputs were presented. The median lethal concentration and 90% lethal concentration (LC50 and LC90, respectively) were calculated by the probit analysis in SPSS. Results and Discussion The preliminary results showed that acetone and hexane extracts of paracress leaf, stem, and root generally presented high contact toxicity against adults of mushroom mite (D. indica Mahunka). Basically, 53.0 µg/ cm2 of all extracts showed more than 80% mortality at 12 h after treatment (Figure 1). Furthermore, the results showed that acetone extracts from all parts at 63.6 µg/cm2 showed 100% mortalities. In particular, the acetone root extract showed the highest toxicity with LC50 and LC90 at 14.90 and 32.23µg/cm2, respectively. Besides, the acetone extracts from the leaf and stem resulted in LC50 at 20.12 and 22.73 µg/cm2 and the LC90 at 42.10 and 46.32 µg/cm2, respectively. In hexane solvent, hexane leaf extract was more effective than the extracts from stem and root with the LC50 at 29.52, 31.25 and 35.38 µg/cm2, respectively (Table 1).
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Figure1 Mortality percentages (Means±SD) of the adults mushroom mite (D. indica Mahunka)at 12 h after treatments with parts of paracress (A. oleracea (L.) R.K. Jansen) at 53.0 µg/cm2 by residual contact method. Table 1 Mortality percentages (Means±SD) of the adults mushroom mite (D. indica Mahunka) at 12 h after treatments with paracress (A. oleracea (L.) R.K. Jansen) at the various concentrations by residual contact method. Parts of plant 0.0
10.6
% Mortality* Concentration (µg/cm2) 21.2 31.8 42.4
LC50 LC90 53.0
Slope ± SE
63.6
Hexane extract Leave 0.0±2.5 16.2±4.6c 22.6±8.0d 58.4±7.3c 84.7±5.3ab 93.8±6.7a 96.8±3.1ab 29.52 48.97 0.066±0.004 Stem 0.0±2.5 28.7±2.6bc 39.3±5.2b 47.1±7.2cd 63.5±10.0c 84.0±13.9a 92.8±5.1ab 31.25 60.33 0.044±0.003 Root 0.0±2.5 18.0±8.1c 25.5±8.9cd 36.6±11.5d 68.3±17.2bc 81.1±7.7a 89.7±9.0b 35.38 61.67 0.049±0.003 Acetone extract Leave 0.0±2.5 41.3±9.1ab 58.1±12.7b 78.3±4.2a 86.0±3.1ab 94.6±9.4a 100.0±0.0a 20.21 42.10 0.059±0.004 Stem 0.0±2.5 31.1±1.9bc 62.3±6.6ab 70.0±8.0ab 81.2±8.9abc 90.2±3.9a 100.0±0.0a 22.73 46.32 0.054±0.004 Root 0.0±2.5 55.7±15.7a 75.8±5.5a 82.9±6.9a 95.7±7.5a 100.0±0.0a 100.0±0.0a 14.90 32.23 0.074±0.005 * Means±SD in column followed by the same common letter were not significantly different (P<0.05) according to DMRT
Polarity differences of solvents used in plant extraction normally showed different effects (Raveen et al., 2014). For examples, hexane extract of Anethum graveolens and Polygonum odoratum was more effective than the acetone and ethanol extracts in controlling Spodoptera litura by leaf dipping method. Moreover, Bakavathiappan et al. (2012) studied antifeedant activity of different solvent extracts from Calatropis procera leaves against adults of S. litura and found that chloroform extract showed the highest anti-feedant activity followed by extracts from hexane, ethanol, acetone, ethyl acetate and methanol solvents, respectively. Besides, Ohimain et al. (2014) found that methanolic extract of Jatropha curcas leaf showed the highest results against Anopheles gambiae in final screening test with LC50at 15.0 ppm followed by stem and root extracts at 47.5 and 62.5 ppm, respectively. In addition, extracts from different parts of plant also showed different effects. Achio et al. (2012) reported that extracts from neem root showed higher insecticidal acitivity against Macrotermes sp., Phaseouslus sp., Periplaneta sp. and Anopheles sp. by direct spray method, when compared to the extracts from stem, leaf and seed, with LC50at 10-30, 30-40, 30-45 and 40-55%, respectively. In the present study, extracts from particular parts of paracress also showed particular results. Different parts of plants normally contain different amounts of particular components. For example, Tiwari et al. (2011) reported that several secondary volatile metabolites like sesquiterpenes, alkamides and oxygenated compounds have been isolated and identified from parts of paracress. In particular, the most abundant alkamide found was spilanthol root. In conclusion, acetone extracts of paracress flower, leaf and root are highly effective in controlling adults of mushroom mite. In addition, field application is also recommended as well as effects on the extracts on mushroom growth should also be concerned.
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References Abbott, W.S. 1987. A method of computing the effectiveness of an insecticide. 1925. Journal of the American Mosquito Control Association 3(2): 302-3. Achio, S., E. Ameko, F. Kutsanedzie and S. Alhassan, 2012. Insecticidal effects of various neem preparations against some insects of agricultural and public health concern. International Journal of Biosciences 1(2): 11-19. Bakavathiappan, Ga., S. Baskaran, M. Pavaraj and S. Jeyaparvathi 2012. Effect of Calotropis procera leaf extract on Spodoptera litura (Fab.). Journal of Biopesticides 5 (Supplementary): 135-138. Broussalis A.M., G.E. Ferraro, V.S.I. Martino, R. Pinzon, J.D. Coussio and J.C. Alvarez. 1999. Argentine plants as potential source of insecticidal compounds. Journal of Ethnopharmacology 67(2): 219-223. Daglish, G.J. 2004. Effect of exposure period on degree of dominance of phosphine resistance in adults of Rhyzopertha dominica (Coleoptera: Bostrichidae) and Sitophilus oryzae (Coleoptera: Curculionidae). Pest Management Science 60(8): 822-826. Grudloyma, P., T. Kulpiyawat, A. Payapanon, M. Kongchuensin, and P. Konvipasruang, 2010. Studies on Biology and Control of Dolichocybid Mite, Dolichocybe indica Mahunka on Mushrooms by Aplication of Some Acaricides. DOA Research Database. 2243-2255. [Online]. Available: http://www.doa.go.th/research/index.php (in Thai). Insung, A., J. Pumnuan, and A. Chandrapatya 2008. Acaricidal activities of wild plant extracts against Luciaphorus perniciosus Rack (Acari: Pygmephoridae) and Formicomotes heteromophus Magowski (Acari: Dolichocybidae). Systematic and Applied Acarology 13(3-4): 188-194. Kulpiyawat, T., A. Payapanon, M. Kongchuensin, P. Grudloyma, and P. Konvipasruang. 2008. Control of dolichocybid mite, Dolichocybe indica Mahunka on mushroom by application of phosphine fumigant. Entomology and Zoology Gazette 26(1): 24-32 (in Thai with English). Ohimain, E.I., T.C.N. Angaye and S.E. Bassey. 2014. Comparative larvicidal activities of the leaves, bark, stem and root of Jatropha curcas (Euphorbiaceae) against malaria vector Anopheles gambiae. Sky Journal of Biochemistry Research 3(3): 029-032. Pandey, V., V. Agrawal, K. Raghavendra, and A.P. Dash. 2007. Strong insecticidal activity of three species of Spilanthes (Akarkara) against Malaria (Anopheles stephensi Liston, Anopheles culicifacies, species C) and filaria vector (Culex quinquefasciatus Say). Parasitology Research 102(1): 171-174. Phrutivorapongkul, A., Chaiwon, A., Vejabhikul, S., Netisingha, W. and Chansakaow, S. 2008. An anesthetic alkamide and fixed oil from Acmella oleracea. Thai Journal of Health Research 22(2): 97-99. Ponpornpisit, A., N. Pirarat, W. Suthikrai, and A. Binwihok 2011. Toxicity Test of Kameng (Eclipta prostrate Linn.) and Kradhuawean (Spilanthes acmella (Linn.) Murr.) to Early Life Stage of Zebrafish (Danio rerio). The Thai Journal of Veterinary Medicine 41(4): 523-527. Pumnuan, J., A. Chandrapatya, and A. Insung. 2010. Acaricidal activities of plant essential oils from three plants on the mushroom mites, Luciaphorus perniciosus Rack (Acari: Pygmophoridae). Pakistan Journal of Zoolog. 42(3): 247-252. Pumnuan, J., A. Insung, and A. Chandrapatya. 2008. Acaricidal effects of herb extracts on the mushroom mites, Luciaphorus perniciosus Rack and Formicomotes heteromorphus Magowski. Systematic and Applied Acarology 13(1): 33â&#x20AC;&#x201C;38. Ramsewak, R.S., A.J. Erickson and M.G. Nair. 1999. Bioactive N-Isobutylamides from the flower buds of Spilanthes acmella. Phytochemistry 51(6): 729-732. Raveen, R., K.T. Kamakshi, M. Deepa, S. Arivoli and T. Samuel. 2014. Larvicidal activity of Nerium oleander L (Apocynaceae) flower extracts against Culex quinquefasciatus Say (Diptera: Culicidae). International Journal of Mosquito Research 1(1): 38-42. Tiwari, K.L., S.K. Jadhav and V. Joshi. 2011. An updated review on medicinal herb genus Spilanthes. Chinese Journal of Integrative Medicine 9(11): 1170-1178.
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P-37
A Degree-Day Simulation Model for the Population Dynamics of the Brown Planthopper, Nilaparvata lugens Stal. (Homoptera: Delphacidae) Wirote KHLIBSUWAN1, Yupa HANBOONSONG1 and Krirk PANNANGPETCH 1 1
Department of Plant Science and Agriculture Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: wirote@kku.ac.th
ABSTRACT The population dynamics of the brown planthopper (BPH), Nilaparvata lugens Stal was simulated based on insect life cycle and weather data. The computer model followed the state variable approach by using thermal requirements of each insect stage as the driving variables. Subsequently, stages and numbers of insect were predicted. Simulated nymphs and adults of BPH were compared with survey data from the field. The model has reasonable predictive value. In time forecasting of the BPH population may help farmers in their decision making process. Simulation using future weather patterns and the influence of global warming on population dynamics of BPH can be predicted. Keywords: Brown planthopper (BPH), Simulation model, Thermal requirements Introduction The brown plant hopper (BPH), Nilaparvata lugens is one of the important rice pests, which and is distributed in all the rice-growing tracts in Thailand. The long winged adults migrate to the rice fields when the rice is in its growing stage. The nymphs and adults damage rice directly through feeding on stem and severe infestations cause plants in the drought stages to gradually yellow from the tip, brown, dry out and a wilt, known as hopperburn (Minister of Agriculture and Livestock, Solomon island, 2012). Simulation of insect systems is based on the assumption that the state of the system at any particular time can be expressed quantitatively and that changes in the system can be described in mathematical terms (De Wit and Goudriaan, 1974). In the present study, a mechanistic model for simulating BPH population dynamics was developed by incorporating thermal requirements. Materials and Methods Collection and rearing of brown planthopper, N. lugens: The experiment was carried out in the laboratory of the Entomology Branch, Faculty of Agriculture, Khon Kaen University, Thailand. The collected insects were brought to the laboratory and reared to build up a large population. For a continuous supply of 15-20 day old rice plants, TN1 seeds were sown in seed beds at an interval of 15 days. Gravid females were released on 15-20 day old rice plants of TN1 variety grown in a 1.5x15 in test tube (diameter x length) (2 hills/tube) which was moistened with wet cotton to one third of its height, so that the plant did not dry. The tubed rice plants were separately kept in incubator at temperature 20, 25, and 30 째C. The eggs were observed everyday and records were taken of the number of eggs, survival and development from eggs to nymphs and the number that reached the adult or gravid stage until the insect died. The results were employed to calculate the thermal constant (Sharov, 1997). The development of this model employed a linear chain of differential equations to represent each of the BPH life cycle stages. (De Wit and Goudriaan 1974; Vansickle 1977). (Equation 1).
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Equation 1:
dNnet dt
Rne Mne Dne
(ta * na) * MF te * ne de * ne
t
dNne ( Rne Mne Dne) * dt
net
t
t
t0 t0 ( Rne Mne Dne) * t ne0
dNnnt dt
nnt
Rnn Mnn Dnn te * ne tn * nn dn * nn
t
t dNnn t ( Rnn Mnn Dnn) * dt t0 t0
te * ne tn * nn dn * nn
dNnat dt
nat
Rna Mna Dna tn * nn ta * na da * na t t dNnat ( Rna Mna Dna) * dt t0 t0 ( Rna Mna Dna) * t na 0 + mra
The first term on the right hand side of equation 1 is the new individuals that are only recruited from the previous stage "Rne, Rnn, and Rna", the second and the last term are the maturing "Mne, Mnn, and Mna" and the mortality "Dne, Dnn, and Dna" out of class, respectively. The "MF" is reproductive rate of the adult. New individuals are introduced through immigration at the rate "mra". Some mortality is indicated from each sub-stage at rate of ''-de''. Insect samples were collected weekly at the sites of part 4-CHI basin, Thailand, from 2010 to 2012. The observed sample data time series were then compared to the computer model output for validation. Model validation: The data on sampling of BPH recorded at the sites of part 4-CHI basin, Thailand, from 2010 to 2012 were used for validating the model. The mean number of nymphs and adults sampled from in-season and out-season rice fields was estimated. The model was initiated with the immigration number of long-wing adults as observed in the weekly average sampled for the first month of the crop. A simulation run was performed for 3 generations of the BPH using the weather data of each site. Results and Discussion Thermal requirements: After initial oviposition, development proceeded on TN1 variety (BPH susceptible) and all life stages are shown in Table 1. Eggs were found to have a thermal constant of 98.4 degreedays and a threshold of development of 12.3°C. There were five nymphal instars and the thermal constant were 146.4 degree-days. The lower development threshold was estimated as 9.4°C. The thermal requirement of adults was assumed to be 111.6 degree-days over a
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development threshold of 10.0°C. The maximum effective temperature for eggs and nymphs was 35°C, while that for the adult stages was 38°C. These were the upper threshold temperatures beyond which no development was expected to occur. The sex ratio was 1:1.4 and the mean number of eggs laid per day per female during the oviposition period was 74.8. Table 1 Effect of temperature on the development of BPH on rice variety TN1. Growth stages Egg Nymph 1st Nymph 2nd Nymph 3rd Nymph 4th Nymph 5th Male Female Egg-Adult Sex Ratio (Male:Fe male)
20°C Average±SD (Day) 12.54±0.86 5.16±1.52 4.33±3.12 5.43±2.84 4.07±2.12 5.02±2.51 10.16±1.01 13.02±1.12 46.55±4.52 1:1.3
Range (Day) 10-14 3-6 3-8 3-8 3-7 4-7 8-11 10-14 44-48
25°C Average ±SD (Day) 6.75±1.49 5.08±0.29 3.00±0.43 2.58±0.67 2.50±0.52 3.51±1.62 13.12±1.21 16.11±2.13 44.12±1.10 1:1.51
Range (Day) 5-8 5-6 3-4 2-4 2-3 5-6 11-15 13-18 42-49
30°C Average±SD (Day) 7.21±0.53 3.75±0.28 3.39±0.52 5.56±0.68 4.02± 0.78 7.12±3.12 14.11±1.60 18.12±2.11 43.13±0.12
Range (Day) 7-9 3-5 2-5 4-7 2-5 5-10 12-15 16-19 42-46
1:1.58
1/
The value was obtained under constant temperature of 25oC in the laboratory.
Simulation of population dynamics: The population dynamics of the BPH was simulated. They are active in the field from August to November and hence the simulation was done from 1 August to 31 December (214th to 366th Julian days) using weather data at each site. The model was initialized with 0.02 immigration adults because the incidence of this pest starts with adults, which immigrate from other rice fields or weeds. The number of eggs and nymphs at the start of the simulation was assumed to be zero. The peak population of nymphs and adults was predicted to occur around 65 and 75 Julian days, respectively (Figure 1a-d).
Figure 1 Population growth of nymphs and adults of BPH as predicted by the model from 4 areas of CHI basin; a= Nampong, b= Khon Kaen, c= Chiang Yuen, d= Kosum Phisai. Model validation: The population growth simulated by the model for this period was compared with the observed catches (Figure 2). The overall performance of the model was acceptable as it simulated the trend in population growth (r2=0.707). Degree-day models for insect
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development are largely based on empirical functions of temperature, which are liable to change spatially and temporally (Hilbert 1995). The present model is a mechanistic one, with the underlying physiological processes and follows a state variable–rate variable approach. The mortality factors such as parasitism and predation were not incorporated into the model.
Figure 2 The scatter plot of BPH data: modelled and observed showing a correlation and a linear regression line. Models facilitate understanding of the factors affecting outbreak and damage caused by pests. They help to understand the pest as an element of the crop ecosystem and to assess the pest status from a holistic point of view (Graf et al., 1992). Population dynamics models linked to crop growth models can simulate pest population peaks and are useful for timing spray schedules based on economic thresholds (Benigno et al., 1988). Conclusion This work was incorporate the thermal degree day to life cycles of the BPH. This methodology deserves further exploration as a practical tool to study the influence of global warming on population dynamics of BPH. Acknowledgements This project was funded from Thailand Research Fund (TRF) and Thailand Global Warming (T-GLOB). We wish to acknowledge the support of the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University, for their assistance. References Benigno E.A., Shepard B.M., Rubia E.G., Arida G.S., Penning devries F.W.T. and J.P. Bandong. 1988. Simulation for rice leaf folder population dynamics in lowland rice. IRRI, Manila. De Wit C.T. and J. Goudriaan. 1974. Simulation of ecological processes. Simulation monographs. Centre for Agricultural Publishing and Documentation (Pudoc), Wageningen. Graf B., Lamb R., K. L. Heong and L. Fabellar. 1992. A simulation model for the population dynamics of rice leaf-folders (Lepidoptera: Pyralidae) and their interactions with rice. J. Appl. Ecol. 29: 558–570. Hilbert D.W. 1995. Growth based approach to modeling the development rate of arthropods. Environ Entomol. 24: 771–778. Minister of Agriculture and Livestock, Solomon island. 2012. Extension Fact Sheet 64 Nilaparvata lugens. [Leaflet]. Helen Tsatsia, MAL & Graham e Jackson: Authors. Sharov A. 1997. Quantitarive population ecology. http://home.comcast.net/~sharov/PopEcol /popecol.html. Accessed 15 Feb 2013. Vansickle J. 1977. Attrition in distributed delay models. IEEE Transactions on Systems, Man, & CYhernetcs. 7: 635-638. Page 320
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P-38
Preliminary Study on Used Microalga (Nostoc commune) as a Protein Supplement in Dry-Wet Mixtures Feed for Juveniles Snakehead (Channa striata) Suneerat RUANGSOMBOON* Program in Fisheries Science, Department of Animal Production Technology and Fisheries King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520 Thailand *Corresponding email: krsuneer@kmitl.ac.th
ABSTRACT Snakehead (Channa striata) is a valuable food fish cultured in Thailand. Farms generally produced their own dry-wet mixtures feed on-site each day for snakehead. Farm-made feed is combinations of dry pelleted feed (i.e. commercial feed) and a wet meal containing a variety of feed ingredients. The dry-wet mixtures feed containing microalga, N. commune as a protein supplement for juvenile snakehead was studied. Commercial fish feed was used as a reference diet. Diets containing 0 (control), 10, 20, and 30% of N. commune were used to feed C. striata for 14 weeks. Experimental fish was hand-fed at 5% total body weight twice a day. The optimum inclusion level of N. commune was 10 % with significant highest growth rate, including feed conversion ratio (2.79±0.02), feed conversion efficiency (35.89±0.25), protein efficiency ratio (0.82±0.01), and specific growth rate (2.46±0.04 %/day). An increased N. commune levels negatively affected the growth response of fish while it did not influence the survival rate of fish. The carotenoid content significantly increased with increasing N. commune level. Flesh of C. striata fed with 30% of N. commune contained highest total carotenoid (2.51±0.33 mg/g FW) and protein (73.89±0.06 % DW). C. striata fed with control diet produced significant higher lipid (9.24±0.18 %DW). Additional studies with isoproteic and energetic diets are further recommended to confirm the effect of this culturemicroalga on snakehead. Keywords: Channa striata, Fish feed, Nostoc commune, Snakehead Introduction Snakehead (Channa striata) is cultured commercially in Thailand. The production of this species in 2010 was 26,700 ton (Fisheries Statistic, 2015). This fish has high market demand, rapid growth rates, coupled with high price. The researchers try to find the method to enhance its immune and production (Samantaray and Mohanty, 1997; Verma et al., 2012; Talpur et al., 2014) as well as the need of high quality diets for this. Nowadays, aquaculture techniques are relatively well developed. The preparation of low cost feeds, including microalgae, is becoming of increasing attraction. Microalgae and their products support fish growth and it might be practical to feed microalgae to fish with little or no processing. Moreover, it has high conversion efficiencies (Benemann, 1992). The most popular product of snakehead is dried fish, which the consumers prefer red flesh color. However, fish do not possess the ability to synthesize carotenoids which cause red color of their flesh. The carotenoid pigmentation of fish results from the pigments present in the diet and accumulated in the tissues (Benemann, 1992). Since fish are unable to synthesize carotenoids by themselves, synthetic astaxanthin is commonly used in the diets of farmed fish to produce coloration to gain market acceptance, however, it cause a high cost in aquaculture. Cyanobacteria, Nostoc, is one of microalgae, it is ubiquitous naturally, successfully masscultured (Ruangsomboon et al., 2005) and serves as one of the biomaterials with high July 1-3, 2015
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pigment content (Patricia et al., 1996), for use as a source of pigments for fish. Mass-cultured microalgal biomass has been tested as a food source for a number of aquaculture animals because of its low cost and convenience (Ruangsomboon et al., 2005). In this study cyanobacterium N. commune was fed to snakehead fish to assess the effect of this cyanobacterial on this fish. Materials and Methods Cyanobacterium, N. commune (Desikachary, 1959) was grown in BG-11 medium under the continuous illumination. The medium was autoclaved before use. Upon harvesting, the N. commune cells were washed three times with tap water, then dried at 60oC and ground. Commercial fish feed was used as a reference diet. The experimental diets were made by inclusion of N. commune powder at 0-30% in the reference diet. Snakehead (C. striata) fry were acclimated to laboratory conditions for 1 week and maintained by commercial diet prior to the experiment. Twelve (0.7x0.8x1 m) aquaria were each stocked with 30 fry and provided with aeration. Three replicate groups of fish were fed their diets twice daily at the rate of 5% of fish weight for 14 weeks. Throughout the feeding trial, temperature, pH and dissolved oxygen were measured. Temperature, pH and dissolved oxygen ranges were 27.5–28.0 °C, 7.2-7.8, and 5.0-6.5 mg/l, respectively. The fish were weighed every 2 weeks, the growth parameters were calculated. Feed and flesh samples were analyzed for chemical composition (AOAC). Significant differences were determined using ANOVA with 95% confidence. Results and Discussion Snakehead fish growth and survival: The initial weight of C. striata was 1.60±0.03 g. At the end of the experiment, the average weight of the fish increased to 23.17±0.59 g. The snakehead fed the diet containing 10% N. commune was significantly higher than other treatments. However, 20 and 30% N. commune had a negative effect on final weight of snakehead (Table 1). The mean specific growth rate of snakehead fed the diets containing 10% N. commune (2.46±0.04%) was significantly higher than other treatments. The feed conversion ratio (FCR) for the control diet (2.99±0.01) was significantly higher than the diets containing 10% N. commune. Table 1 Growth of C. striata (14 weeks) and its indicators of test diets containing N. commune.
Mean final weight (g) Mean final length (cm) Mean specific growth rate (%) FCR FCE PER Survival (%)
Diets containing N. commune (%) 0 10 20 30 21.08±0.36b 23.17±0.59c 19.08±0.53a 19.17±0.49a 13.28±0.50a 15.01±0.42b 12.87±0.50a 12.84±0.53a 2.31±0.02a 2.46±0.04b 2.26±0.04a 2.24±0.03a b a b 2.99±0.01 2.79±0.02 3.13±0.09 3.07±0.05b 33.39±0.84a 35.89±0.25b 31.94±0.51a 32.59±0.56a 0.72±0.02a 0.82±0.01b 0.72±0.01a 0.72±0.01a a a a 74.44±1.11 76.67±1.92 76.67±5.09 73.33±1.92a
The same superscript letter in each row denotes no significant difference (p>0.05).
Food conversion efficiency (FCE) (35.89±0.25) and protein efficiency ratio (PER) (0.82±0.01) of snakehead fed the diets containing 10% N. commune were significantly higher than other treatments. The experimental diets containing 10-30% N. commune did not significantly affect snakehead survival rate (between 73.33–76.67 %) (Table 1). Proximate composition of N. commune, diet and snakehead flesh: The total protein content of N. commune powder was 58.7±1.2% of the dry wt. The diets which prepared using supplements of N. commune powder were chemical composition analyzed. The protein in Page 322
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control diets (47.46±0.02%) was significantly higher than other treatments. Protein content in diet containing 10% N. commune was not significantly different from 20 and 30% N. commune (Table 2). Diet containing N. commune showed significantly higher lipid and fiber than the control. The highest N. commune gave the highest lipid, fiber and calcium in diet. Protein, calcium and ash content of snakehead given diet containing N. commune was significantly higher than control, while lipid was significantly lower. There was no significant difference of phosphorus in all treatments (Table 3). Table 2 Proximate composition (% dry wt.) of diet containing N. commune. N. commune 0 10 20 30
protein
lipid
Fiber
P
Ca
Ash
47.46±0.02a 43.95±0.16b 44.29±0.15b 44.73±0.03b
1.75±0.02a 3.35±0.00b 3.30±0.12b 3.48±0.02b
2.66±0.03a 3.01±0.05b 3.67±0.01c 4.52±0.03d
1.08±0.01a 1.44±0.01b 1.13±0.00c 1.09±0.00a
2.45±0.02a 2.45±0.02a 2.47±0.01a 2.55±0.03b
12.24±0.02a 12.10±0.03a 12.17±0.01a 12.12±0.04a
The same superscript letter in each column denotes no significant difference (p>0.05).
Table 3 Proximate composition (% dry wt) of snakehead fed with diet containing N. commune. N. commune 0 10 20 30
protein 69.16±1.44a 71.99±0.93b 72.75±0.21b 73.89±0.06b
lipid 9.24±0.18d 5.34±0.02a 6.94±0.05b 8.00±0.07c
P 0.87±0.00a 1.02±0.00a 0.92±0.01a 0.91±0.00a
Ca 0.39±0.00a 0.62±0.00b 0.63±0.00bc 0.69±0.01c
Ash 4.35±0.01a 5.27±0.00b 5.01±0.01b 5.15±0.00b
The same superscript letter in each column denotes no significant difference (p>0.05).
Pigmentation of N. commune powder and snakehead flesh: The pigments; chlorophyll, phycocyanin, phycoerythrin, and carotenoid in N. commune were 261.47±8.66, 0.04±0.00, 0.33±0.01 and 0.16±0.01 mg/g on a dry basis, respectively. Depending on the N. commune containing in diets, the carotenoid in flesh of snakehead was increase with increasing N. commune biomass. The average carotenoid in fresh flesh of snakehead given 30% N. commune was significantly highest (2.51±0.33 mg/kg), followed by the group given 20% (1.30±0.15 mg/kg), 10% (0.89±0.23 mg/kg), and 0% (0.81±0.13mg/kg) of N. commune. Cyanobacteria have been found to be a good source of protein for fish (Nandeesha et al., 2001). In addition, cyanobacteria have been reported to have no cell wall, which results in improved digestion and absorption. N. commune is a cyanobacteria owing high protein content (46.9-51.2%) (Ruangsomboon et al., 2005). There is no positive effect observed on survival of snakehead in this study. However, the survival improvement in kuruma prawn, Marsupenaeus japonicus (Chien and Jeng, 1992), and black tiger prawn, Penaeus monodon, by dietary carotenoid; astaxanthin supplementation was reported (Chien and Shiau, 2005). N. commune appeared to be effective as a feed for snakehead. The higher protein in snakehead given diet containing N. commune reflected its high quality as a food. The snakehead fed with diets containing N. commune have better carotenoid than those fed with control (commercial diet without N. commune) because the pigment in diets containing N. commune have higher carotenoid than control diet. Conclusion The incorporation of 10% N. commune in snakehead diet have positive effects on growth, FCR, FCE, PER, survival, and protein. The total carotenoid levels were significantly related to the level of N. commune in the diet. The results of the present study clearly demonstrate that cyanobacterium N. commune could be exploited as a pigment and protein source for incorporation in snakehead diets. July 1-3, 2015
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Acknowledgments The author is grateful to the Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang for financial support. References Association of official analytical chemists (AOAC). 2000. Official Methods of Analysis. Washington, DC. Benemann, J.R.1992. Microalgae aquaculture feeds. Journal of Applied Phycology.4:233245. Chien, Y.H. and W.C. Shiau. 2005. The effects of dietary supplementation of algae and synthetic astaxanthin on body astaxanthin, survival, growth, and low dissolved oxygen stress resistance of kuruma prawn, Marsupenaeus japonicus Bate. Journal of experimental Marine Biology and Ecolog. 318:201-211. Chien, Y.H. and S.C. Jeng. 1992. Pigmentation of kuruma prawn, Penaeus japonicus Bate, by various pigment sources and levels and feeding regimes. Aquaculture. 102:333-346. Desikachary, T.V. 1959. Cyanophyta. Indian Council of Agricultural Research.Fisheries Statistic. 2015. Available online: http://www.fisheries.go.th/it-stat/. Nandeesha, M.C., B. Gangadhara, J.K. Manissery and L.V. Venkataraman. 2001. Growth performance of two Indian major carps, catla (Catla catla) and rohu (Labeo rohit2001.a) fed diets containing different levels of Spirulina platensis. Bioresource Technology. 80: 117-120. Patricia, A., I.S. Ross and J. D. Mills. 1996. Regulation of pigment content and enzyme activity in the cyanobacterium Nostoc sp. Mac growth in continuous light, a light-dark photoperiod, or darkness. Biochimica et Biophysica Acta. 1277:141-149. Ruangsomboon, S., J. Buppha, S. Choochote and P. Thaweekijakarn. 2005. Nutritional values of Nostoc commune cultured under various media. King Mongkutâ&#x20AC;&#x2122;s Agricultural Journal 23:38-47. Samantaray, K. and S.S. Mohanty. 1997. Interactions of dietary levels of protein and energy on fingerling snakehead, Channa striata. Aquaculture 156:241-249. Talpur, A.D., M.B. Munir, A. Mary and R. Hashim. 2014. Dietary probiotics and prebiotics improved food acceptability, growth performance, haematology and immunological parameters and disease resistance against Aeromonas hydrophila in snakehead (Channa striata) fingerling. Aquaculture 426-427:14-20. Verma, V.K., K.V. Rani, N. Sehgal and O. Prakash. 2012. Immunostimulatory response induced by supplementation of Ficus behghalensis root powder, in the artificial feed the Indian freshwater murrel, Channa punctatus. Fish & Shellfish Immunology 33:590596.
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P-40
Protein and some other Constituents of Sucker Catfish’s Fish Meal Kalkullanutch PATRARASRIPONG1 and Kanok LERTPANICH2* 1
Soil and Environment management Program, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand 2 Department of Agricultural Development and Resource Management, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand *Corresponding email: klkanok@kmitl.ac.th
ABSTRACT The Sucker catfish (Hypostomus plecostomus) is an invasive species in Thailand. Moreover, the Pavesburirom canal, Ladkrabang district, Bangkok especially found the massive numbers of this catfish (Lertpanich and Aranyavali, 2008). The rising population of sucker catfish has been effected to the local fish species. The proper management urgently need for this invasive catfish. However, the production of fish meal from this catfish is regarded. Then, the Sucker catfish’s fish meals are investigated by proximate analysis (AOAC, 1995). The amount of protein, moisture, fat, ash, fiber, calcium and phosphorus were quantified and 3 replications are considered. The results showed the percentage of protein and some other constituents 58.22 % of crude protein, 3.62 % of moisture, 12.34 % of crude fat, 23.20 % of ash, 0.44 % of crude fiber, 8.03 % of calcium and 3.99 % of phosphorus. The Sucker catfish’s fish meal indicated that the protein and some other constituents quality are quite similar to the fish meal standard. However, the higher amount of fat may reduce the quality and delectable. Keywords: Sucker catfish, Fish meal, Proximate analysis, Invasive species management Introduction Alien species were defined as any organisms from the other part of the world that transport beyond their habitats and established in the new area. If they can reproduce the abundance numbers and capable to reduce the population of native species and/or destroy the natural habitat, which will call invasive alien species. In Thailand, there were numerous invasive alien species such as Water Hyacinth (Eichhornia crassipes), Golden apple snail (Pomacea canaliculata), Yellow crazy ant (Anopolepis gracilipes), and especially Sucker catfish (Hypostomas spp). The Sucker catfish invasion can be found all over Thailand, such as Nong Yay Pa swamp, Tak province can found 5,000 kilogram per catching (Tak Inland Fisheries Research and Development Center, 2008). Chaichana et al. (2011) and Lertpanich and Aranyavalai (2013) found that sucker catfish highly increase the population in the low quality water. Moreover, they can occupied the habitat and reduce the native fish population to 30 to 1 percentage. The sucker catfish in Pravesburirom canal is Hypostomus plecostomus and had average 408.37 ± 52.01 gram weights, 34.21 ± 2.15 centimeter lengths and 6.46 ± 0.40 centimeter width (Lertpanich and Aranyavalai, 2013). The Suckers catfish are urgently need the management. The highly number of sucker catfish consumption may the solution of the problem. The fish meal production is one of many ways to decrease the sucker catfish population. However, the quality of the fish meal from this fish is needed to know, and then this research is setting up for that consideration. Materials and Methods The fish meal processing was based on Thai fish meal producer association (1996) method. The samples of Sucker catfish were get rid of entrails and cleaned with water. The samples July 1-3, 2015
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were steam cooking, drying in 60 - 70 degree Celsius and grinding. The fish meal samples were analyzed the nutrients by the proximate analysis method (AOAC, 1995). The amount of protein, moisture, fat, ash, fiber, calcium and phosphorus were quantified and 3 replications are considered. The fish meal sample also physical evaluated by 3 animal nutrition experts form Department of Animal production and fisheries, Faculty of Agricultural Technology, KMITL Results and Discussion The fish meal analysis results showed that the average of protein and some other constituents are 58.22 % of crude protein, 3.62 % of moisture, 12.34 % of crude fat, 23.20 % of ash, 0.44 % of crude fiber, 8.03 % of calcium and 3.99 % of phosphorus. (Table 1) Table 1 Percent composition of Sucker catfish’s fish meal. Sample 1
Sample 2
Sample 3
Average
Crude protein
59.58
58.46
56.63
58.22
Moisture
3.68
3.54
3.63
3.62
Crude fat
12.25
12.28
12.20
12.34
Ash
23.15
22.98
23.47
23.20
Crude fiber
0.41
0.47
0.44
0.44
Calcium
8.07
7.61
7.98
8.03
Phosphorus
4.06
3.97
3.94
3.99
The physical evaluation focuses on color, odor, and texture properties. The results showed that the Sucker catfish’s fish meal had similar color when compare with common fish meal product. However the odor had specific smelling and quite rancidity. The sample also found in sticky agglomerate texture and found a lot of bone composition (Figure 1). Regarding to the proximate analysis, the amount of crude fat and ash had high percentage and affected to fish meal odor and texture. The improvement of fish meal quality can do by eliminate the head part and skin, which contained more bone and fat.
Figure 1 Sucker catfish’s fish meal texture. The comparison of fish meal composition (Table 2) showed that Sucker catfish’s fish meal had crude protein and crude fiber lower than other marine fish, but had more crude fat, calcium and phosphorus. However, the Sucker catfish’s fish meal composition is closed to Thai average fish meal (Wongsang and Akkagraisri, 2000) but had slightly more crude fat. Page 326
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Regarding to the Thai fish meal standard (Ministry of commerce, 1985), the Sucker catfish’s fish meal composition is reach the grade 2 level. However, the crude protein is only one parameter that obstructs to get to grade 1 (Table 2). The process of production of fish meal can change the proportion of protein. In this case, the head of Sucker catfish contain more bone and fat, which increase the composition of ash and crude fat. Then the fish preparation by exclude the head part is necessary for protein proportion increasing. Table 2 The comparison of fish meal composition. Sucker catfish
Trash fish
Threadfin Anchovy Bream fish fish
Average Thai fish meal
Thai fish meal Standard Grade1
Thai fish meal Standard Grade2
Crude protein
58.22
61.97
60.34
65.00
59.39
≥ 60.00
≥ 55.00
Moisture
3.62
6.06
3.18
-
8.62
≤ 10.00
≤ 10.00
Crude fat
12.34
12.07
9.82
10.20
7.82
-
-
Ash
23.20
18.78
24.95
20.00
25.88
≤ 26.00
≤ 28.00
Crude fiber
0.44
1.11
1.35
1.00
0.78
≥ 2.00
≥ 2.00
Calcium
8.03
-
-
5.00
-
-
-
Phosphoru s
3.99
-
-
3.00
-
-
-
Source: Hanbanchong et al. (2001), Wongsang and Akkagraisri (2000) and Ministry of Commerce, (1985) Conclusion The Sucker catfish’s fish meal had average of protein and some other constituents are 58.22 % of crude protein, 3.62 % of moisture, 12.34 % of crude fat, 23.20 % of ash, 0.44 % of crude fiber, 8.03 % of calcium and 3.99 % of phosphorus. The composition of Sucker catfish’s fish meal reaches the Thai fish meal standard grade 2. However, the high amount of crude fat lead to rancid odor. The Sucker catfish’s fish meal composition indicated that can use in the animal production business. Then this invasive species can manage the population by give an impetus to fish meal production. However, the quality of fish meal can improve by get rid of the head part and skin Acknowledgements We would like to thanks the fish meal experts, Assist. Prof. Dr. Uscharee Reangdet, Dr. Norrathus Prachum and Ms. Janya Kongrit for Physical evaluation and Dr.Varanya Aranyavalai for manuscrpt preparation. References AOAC. 1995. Official Methods of Analysis. Association of Official Analytical Chemists, Thirteenth edition, Washington, D.C Chaichana, R., S. Pouangcharean and R. Yoonphand. 2011. Habitat, abundance and diet of invasive suckermouth armored catfish (Loricaiidae ptergoplichthys) in the Nond Yai Canal, East Thailand. Opical Zoology 24 (1): 49-62. (In Thai) July 1-3, 2015
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Hanbanchong, A., D. Singermsiri and K. Atsawayenjai. 2001. Study of Fish meal quality. Department of Animal husbandry, Faculty of Agriculture. Kasetsart University. (In Thai) Lertpanich, K. and V. Aranyavalai. 2013. Ecological Study of Sucker catfish in Pravesburirom canal, Ladkrabang District, Bangkok. The First Higher Education Research Promotion Congress (HERP CONGRESS I) (In Thai) Thai fish meal producer association. 1996. Thai Fish Meal Industry. Thai fish meal producer association. Bangkok. Wongsang, S. and S. Akkagraisri. 2000. Quality of Thai Fish meal. Animal Business 17(74): 1634. (In Thai) Ministry of commerce. 1985. Ministry announce. 102 section 198. 27 December 1985. (In Thai)
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P-44
Genetic Diversity of Seagrasses across the Eastcoast of Thailand Based on Sequence-Related Amplified Polymorphism (SRAP) Technique Pattama SRINAMNGOEN1* and Kanok-on DUANGPAKDEE1 1
Faculty of Science and Art, Burapha University, Chanthaburi Campus, Chanthaburi 22170, Thailand *Corresponding email: pattama@buu.ac.th
ABSTRACT Seagrasses have a long evolution and adaptation to become a marine flowering plant, and also have high impact on marine ecosystem. The identification of seagrasses was classified using the functional group, not a taxonomic group. Because, the morphology and phenotypic can be changed by the environment. Genetic diversity can be measured by genetic variability. So, SRAP technique was investigated to detect the variability of seagrass genome. Twentysix samples of six seagrass species from the eastcoast of Thailand including Chonburi, Rayong, Chanthaburi and Trad provinces were collected. The result showed that 12 out of 25 primers pairs produced clear polymorphic patterns and showed highly polymorphism due to locations and environment. According to the genetic relationship and cluster analysis, the phylogenetic tree was constructed and the similarity coefficient was 0.93. The phylogenetic tree was divided into 2 major groups (or 4 minor groups). The first major group, elliptic short leaf blade, was Halophila ovalis from Trad province. The second major group, having acuminate long leaf blade, included 5 species, namely Cymodocea serrulata, Halodule uninervis, H. pinifolia and Enhalus acoroides. Although the large variability of seagrasses within species and also between species was detected, the genetic diversity showed a correspondence with the morphological identification as well. Keywords: Seagrass, SRAP, Phylogenetic tree, Genetic variability Introduction Seagrasses, a functional group of marine flowering plant, have an important role in coastal habitat, nurseries for crustacean, by providing food for green sea turtles and dugong and they also have been proved as a carbon sink for carbon accumulation (Greiner et al., 2013). The seagrasses are divided into 5 families: Cymodoceaceae, Hydrocharitaceae, Posidoniaceae, Ruppiaceae and Zosteraceae, with 12 genera and 60 species. In Thailand, 12 species of them have been found. Some specie in the Indo-Pacific Ocean is listed as a critical endangered one such as Halodue pinifolia (Short et al., 2011). Damage of the seagrasses occurs from both natural causes, such as storm and climate change, and human activity, such as coastal fishery. So far, there ishypothezied that the taxonomic and genetic diversity are uncertainly identified among the seagrass species. There are several techniques to access genetic diversity between plant genome such as random-amplified polymorphic DNA (RAPD; Williams et al., 1990), amplified fragment length polymorphism (AFLP; Vos et al., 1995), and sequence-related amplified polymorphism (SRAP; Li and Quiros, 2001). The advantage of the SRAP technique was PCR-based technique and used to amplify coding regions of the DNA with primers targeting open reading frame (ORF) of the genome, which are useful in map construction, breeding program, taxonomy and also evolution. The aim of this study was to explore genetic diversity of the seagrass species that were collected from the Eastcoast of the Gulf of Thailand based on SRAP approaches and also analyzed the environmental effect on the differential gene expression. July 1-3, 2015
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Materials and Methods Sample collection and DNA extraction The population of samplings in this study consists of 6 species throughout the natural distribution in eastcoast of Thailand. These samplings are distributed in different regions from Chonburi, Rayong, Chanthaburi and Trat province along the Gulf of Thailand. The regions were determined by province border. For each location, the sampling was randomly collected at least 1 km apart in order to prevent a collection of the same single genotype, as shown in Table 1. Young leaves were harvested, cleaned of epiphytes, stored on ice and transferred to the laboratory. The genomic DNA was extracted using the CTAB method that described by Doyle and Doyle (1990). The DNA quality determined using 0.8% agarose gel with ethidium bromide straining and DNA concentration was measured using spectrophotometer. SRAP analysis The set of 25 primer pairs were used to estimates genetic diversity of these seagrass population. The SRAP loci were amplified in 10 µl reaction volume containing 1X PCR buffer, 0.25 µM of each SRAP primer, 0.1 mM dNTP, 2 mM MgCl2, 1 U Taq Polymerase, 25 ng of diluted DNA template and water up to 10 µl. The termal cycling profile consisted of first five cycles were run at 94 ˚C, 1 min, 30 ˚C, 45 sec, and 72 ˚C, 1 min, for denaturing, annealing and extension, respectively. Then the annealing temperature was increased to 45˚C for another 35 cycles. The PCR products were separated using 5% naturing polyacrylamide gel, detected by ethidium bromide straining and visualized by Gel Documentation Genetic Diversity Analysis The polymorphic fragments were scored as binary data (1, band present; 0, band absent). The phylogenetic tree was analyzed using Euclidean matrix distance similarity and coephenetic correlation (r) was calculated using PAST ver.2.17c (Hammer et al., 2001). Table 1 Population of seagrass samplings in this study. Collection sites Aoh Sattahep, Chonburi province Ban Chom Ta-wan Resort, Rayong province Rock Garden Community, Rayong province
Latitude
Longitude
No. of sampling
Species
12°39'42.0"N
100°53'48.8"E
1
Halodule pinifolia
12°39'10.3"N
101°39'06.0"E
2 Halodule pinifolia
101°39'39.7"E
12°36'06.2"N
101°53'25.9"E
12°34'34.8"N
101°54'23.1"E
11°51'05.9"N
102°31'44.4"E
4
11°49'57.5"N
102°31'19.3"E
5
Aoh Khung Kraben, Chanthaburi province
Kra-dad Island; Trad province 11°50'07.8"N
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5
12°39'58.4"N
102°31'47.6"E
1
Halodule pinifolia
1
Enhalus acoroides
1
Enhalus acoroides
1
Halodule pinifolia Cymococea serrulata
1
Halodule uninervis
3
Halophila ovalis
1
Thalassia hemprichii
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Results and Discussion The genetic diversity of the seagrass has an important consequence for marine ecosystem. However, there is few information about the genetic diversity experiments to generate genetic patterns between species and environment. Here, we investigated the SRAP marker to access the seagrass genome to study the genetic diversity based on transcribed genes. The 25 SRAP markers were used to screen these six seagrass population to identify polymorphic band. Only 12 SRAP markers could generate polymorphism patterns, which was an average of 23 bands per marker. The SRAP bands were ranging in length from 100-2000 bp and showed 100% of polymorphism. For diversity analysis, the phylogenetic tree was generated with Euclidean matrix similarity of distance. The coephenetic correlation showed 0.93, which referred to excellent clustering. The genetic distance was 4.5-11.6. The phylogenetic tree was divided into 2 major groups or 4 minor groups (Fig. 1). First minor group, elliptic short leaf blade, was Halophila ovalis from Thad province. The second minor group, acuminate long leaf blade included 3 species; Cymodocea serrulata, Halodule uninervis and Thalassis hemprichii from Trad province. The third minor group, acuminate long leaf blade, was Halodue pinifolia from Chonburi, Chanthaburi and Rayong province. The fourth minor group, acuminate long leaf blade, was Enhalus acoroides from Chanthabuti province. SRAP has been proved as a powerful molecular marker system used in transcriptome map (Que et al., 2012), gene tagging, marker-assisted selection and evolution (Li et al., 2014). So, the polymorphism or genetic variability of these seagrasses occurred due to differential species and also environmental effect. Because the genetic variability was detected among individual sampling resulting from the genetic change due to surviving adaptation in each environment. Limitations of SRAP markers have not yet been described (Robarts and Wolfe, 2014). Therefore, SRAP marker is a valuable technique for amplification between coding and non-coding regions, and also actual genes, and cDNA fingerprint could be generated (Yu et al., 2008). It would be another good option to get the novel genes and genome analysis of transcribed genes at the transcriptional level.
Figure 1 Phylogenetic tree of genetic distance among individuals based on Euclidean matrix similarity value within 6 species of seagrass population.
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Conclusion The study of genetic diversity and variability of the seagrasses is a fundamental understanding of the characteristic of seagrass habitat, which significantly changes due to environmental condition. SRAP is a reliable technique to identify the seagrasses species and environmental relationship. Our findings have an important effect on seagrass identification, conservation and restoration. Acknowledgments The financial support of the research was provided by Faculty of Science and Arts, Burapha University, Chanthaburi Campus. And we would like to thank Marine and Coastal Resources Research and Development Center, the Eastern Gulf of Thailand for their help on samples collection. References Greiner J.T., K. J. McGlathey, J. Gunnell and B.A. McKee. 2013. Seagrass restoration enhances “Blue Carbon” sequestration in coastal water. PLoS ONE 8(8): e72469. doi:10.1371/journal.pone.0072469 Hammer Ø., D.A.T. Harper and P.D. Ryan. 2001. Past: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica 4(1), 4:9 p. Li, G., and C. F. Quiros. 2001. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics 103: 455-461. Li X. Y., J. Li, Z. J. Zhao, F. Yang, Q. W. Fu, H. S. Liu, D. D. Wang, Y. C. Yang and R. Y. Wang. 2014. Sequence-Related Amplified Polymorphism (SRAP) for studying genetic diversity and population structure of plant and other living organisms: Protocol. The Journal of Animal and Plant Sciences 24(5): 1478-1786. Que Y., L. Xu, J. Lin, J. Luo, J. Xu, J. Zheng and R. Chen. 2012. cDNA-SRAP and its application in differential gene expression analysis: a case study in Erianthus arundinaceum. Journal of Biomedicine and Biotechnology ID: 390107. doi:10.1155/2012/390107 Robarts D.W.H. and A.D. Wolfe. 2014. Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology. Application in Plant Science 2(7): 1400017. Short F.T, B. Polidoro, S. R. Livingstone, K. E. Carpenter, S. Bandeira, J.S. Bujang, H.P. Calumpong, T.J.B. Carruthers, R.G. Coles, W.C. Dennison, P.L.A. Erftemeijer, M.D. Fortes, A.S. Freeman, T.G. Jagtap, A.H. M. Kamal, G.A. Kendrick, W.J. Kenworthy, Y.A. La Nafie, I. M. Nasution, R.J. Orth , A. Prathep, J.C. Sanciangco, B.V. Tussenbroek, S.G. Vergara, M. Waycottt and J. C. Zieman. 2011. Extinction risk assessment of the world’s seagrass species. Biological Conservation 144: 1961-1971 Vos P., R. Hogers, M. Bleeker, M. Reilans, T. Lee, M. Hornes and M. Zabeau. 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407-4414. Williams J., A. Kubelik, K. Livak, J. Rafalski and S. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18: 6531-653. Yu M., B. Ma, X. Luo, L. Zheng, X. Xu and Z. Yang. 2008. Molecular diversity of Auricularia polytricha revealed by inter-simple sequence repeat and sequence-related amplified polymorphism markers. Current Microbiology 56: 240-245.
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P-46
Aquatic Polyculture Farm in Bangsaothong District, Samutprakarn Province, Thailand Nipon JITTAMNAN1,2* and Panneepa SIVAPIRUNTHEP2 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Agricultural Education, Faculty of Industrial Education, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kjnipon@gmail.com
ABSTRACT Direct observation, interview, and discussion with a selected aquatic polyculture farmer was conducted to collect data regarding which kind of fish and shrimp to be cultured, how to culture and manage aquatic polyculture farm, how much of yield, the cost and profit of the investment, and etc. Data were analyzed using descriptive statistics and it was then described to be the knowledge for polyculture farming as a sustainable aquaculture. Aquatic polyculture farm is the most popular aquaculture farming in Bangsaothong district, Samutprakarn province, Thailand because of the occurrence of natural food sources such as phytoplankton, zooplankton, aquatic insect, and benthos for feeding the fish and shrimp. Thus, the aquatic polyculture farming is the method to use all natured food. In addition, the investment cost of aquatic polyculture farming is low and the return is high. The results of this study revealed that 3 types of herbivorous fish namely, Tilapia (Oreochromis niloticas), Rohu (Labeo rohita), Bighead carp (Aristichthys nobilis (Richardson)) and the Vannamei shrimp (Litopenaeus vannamei) were cultured in this aquatic polyculture farm. Vannamei shrimp was cultured to marketable size and sold every 2 months while fishes were harvested after 8-10 months of the culturing. The advantages of this aquatic polyculture were the maximum utilization of natural food in the pond by fish and shrimp and no accumulation of waste and disease thanks to the utilization of all organic residues by Vannamei shrimp. Keywords: Herbivorous fish, Vannamei shrimp, Aquatic polyculture, Natural food Introduction Aquatic polyculture farm is the most popular aquaculture farming in Bangsaothong district, Samutprakarn province, Thailand because of the occurrence of natural food sources such as phytoplankton, zooplankton, aquatic insect, and benthos for feeding the fish and shrimp. The aquatic polyculture farming is the method to use all natured food by aquatic animals which cultured in the same pond. The investment cost of aquatic polyculture farming is low and the return is high. According to the maximize profit of the investment, the aquatic polyculture farming is the one of doing sustainable aquaculture farming. In order to educated farmers, the knowledge for aquatic polyculture farming should be available. Thus, in this study would reveal information about kind of fish and shrimp to be cultured, how to culture and manage aquatic polyculture farm, how much of yield, the cost and profit of the investment, and so on, to help the farmers who interested in doing aquatic polyculture farming use for decision making in doing aquatic farming.
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Materials and Methods Direct observation, interview, and discussion with a selected aquatic polyculture farmer was conducted to collect data regarding which kind of fish and shrimp to be cultured, how to culture and manage aquatic polyculture farm, how much of yield, the cost and profit of the investment, and etc. A selected aquatic polyculture farmer was the owner of an aquatic polyculture farm in Bangsaothong District, Samutprakarn Province, Thailand with the pond size of 10 Rai. Data of one production cycle of 10 months were collected. Results and Disscussion The results of this study revealed that 3 types of herbivorous fish namely, Tilapia (Oreochromis niloticus), Rohu (Labeo rohita), Bighead carp (Aristichthys nobilis (Richardson)) and the Vannamei shrimp (Litopenaeus vannamei) were cultured in this aquatic polyculture farm as shown in Figure 1. The amount of fish and shrimp and their cost were shown in Table 1. Apart from natural food sources such as phytoplankton, zooplankton, aquatic insect, and benthos in the pond for fish and shrimp, farmer fed fish with fish feed and rice bran. Shrimp had only natural food for growing and at the same time farmer supplemented calcium carbonate for making shrimp shell. The farmer helped filling natural food by adding organic fertilizer and urea into the pond. Antibiotic was used to protect against diseases. Nylon strings were hanged and covered over the pond to protect fish and shrimp from the birds. Hydro test kits were used to check pH and alkaline in the water. Water was added to the pond every two months. Vannamei shrimp was cultured to marketable size and sold every 2 months while fishes were harvested after 8-10 months of the culturing. The production of fish and shrimp were shown in table 2. The cost, income, and profit of one production cycle were shown in table 3. Table 1 Type, number, and cost of fish and shrimp cultured in the farm. Type
Size
Number
1. shrimp larva for 1st culture 2. shrimp larva for 2nd culture 3. shrimp larva for 3rd culture 3. shrimp larva for 4th culture Total shrimp larva 4. Tilapia 5. Rohu 6. Bighead carp Total of 1-6
Post larva 10
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200,000
Cost per unit (Baht) 0.04
Total cost (Baht) 8,000
Post larva 10
200,000
0.04
8,000
Post larva 10
200,000
0.04
8,000
Post larva 10
120,000
0.04
4,800
-
720,000
0.04
28,800
2-3 cm. 3 inch 3 inch -
3,500 800 200 -
0.40 0.20 0.20 -
1,400 160 40 30,400
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Figure 1 Fish and shrimp from aquatic polyculture farm. Table 2 Amount and income of fish and shrimp. Type of fish and shrimp 1. shrimp 2. Tilapia 3. Rohu and Bighead carp
Number of harvesting 27 2 1
Amount (kg) 712.80 13,632.00 1,613.00
Price/kg (Baht) 180.50 37.28 25
Total income (baht) 129,785.00 499,079.00 40,325.00
Total
669,189.00
Table 3 Cost, income, and profit of aquatic polyculture farming for one production cycle (crop). Cost of 1. fish and shrimp 2. feed 3. fertilizers 4. chemical 5. labor 6. others Total
Cost (Baht) 30,400.00
Income (Baht) 669,189.00
Profit -
347,310.00 13,260.00 3,090.00 45,000.00 Not available 439,060.00
669,189.00
230,192.00
According to the different living and finding food behavior of Tilapia (Oreochromis niloticas), Rohu (Labeo rohita), Bighead carp (Aristichthys nobilis (Richardson)), and the Vannamei shrimp (Litopenaeus vannamei), they could use all natured food in the different level of water in the pond. The Rohu and Bighead carp fish and Vannamei shrimp find food from any level of the pond. The Vannamei shrimp prefers to find natural food at the bottom of the pond or land surface and eat all left over fish feed and waste from fish (Department of Fishery, 2014). Tilapia fish is herbivorous fish. It lives and finds food in the middle of the July 1-3, 2015
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pond (Choochote, 1993). Water predators such as small crab and shrimp are the disease carrier but fish eat them all so there is no chance of the disease outbreak in Vannamei shrimp (Administrator, 2014). Regarding the maximum utilization of natural food in the pond by fish and shrimp and no accumulation of waste and disease, the production from aquatic polyculture farming is more economics. Conclusion To run aquatic polyculture farming, farmer got good profit. The fish and shrimp that could be cultured in the same pond should live and find natural food in different level of water. They could use all natural food, the waste, and the organic residues. The advantages of this aquatic polyculture were the maximum utilization of natural food in the pond by fish and shrimp and no accumulation of waste and disease thanks to the utilization of all organic residues by Vannamei shrimp. Acknowledgments The data in this paper came from Mrs. Pornpana and Mr. Suriya Aiemsumang. Without their support the study would not be performed. The authors thank to them for their cooperation. References Administrator. 2014. Herbivorous fish and Vannamei shrimp culturing. Online: www.fisheries.go.th/fpo-samutpra/index.php? November 10, 2014 Department of Fishery. 2014. The knowledge of Vannamei shrimp culturing. Online:www.fisheries.go.th/train-gr/coastal/002/GuidlineFGAP.pdf. November 12, 2014 Choochote, S. 1993. Fresh Water Fish Culturing. Bangkok: O. S. Printing House.
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P-47
Microbiological Safety of Ready-to-Eat Semi-Dried Nham, an Innovation of Thai Fermented Meat Product Thanapa CHETAWAN1, Komkhae PILASOMBUT1* and Supaluk SORAPUKDEE1 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: komkhae@yahoo.com
ABSTRACT Semi-dry Nham is an innovation of Thai fermented meat product. The objective of this study was to determine microbiological contamination in semi-dry Nham. Semi-dry Nham was prepared by a fermentation process at 30 ºC for 3 days and subsequently dried in an oven to reach core temperature at 71 ºC. The experiment was composed of control and 2 treatments: adding 5% and 10% glycerol to Nham. The numbers of Staphylococcus aureus, yeast/mold, and lactic acid bacteria were determined as well as pH and total acidity. The results showed that after 3 days of fermentation, the number of S. aureus and yeast/mold were not observed. The numbers of lactic acid bacteria in control and treatments increased progressively after 3 days of fermentation. On the other hand, lactic acid bacteria S. aureus and yeast/mold were not found in any groups after the Nham was dried. The pH value of the 10% glycerol treatment was higher than those of the other two groups, whereas the total acidity of the 10% glycerol treatment was lower than those of the other groups. Hence, this study indicated that ready-to-eat semi-dry Nham prepared by our process would be safe from pathogenic bacteria. Keyword: Semi-dry Nham, Fermented meat product Introduction Nham is a popular traditional fermented meat product in Thailand. It is prepared by natural fermentation at surrounding temperature of 30°C for 3 days (Luxananil et al., 2009). Under air-limited condition, the fermentation is complete naturally by lactic acid bacteria. Therefore, the final product is acidic (pH of Nham approximately 4.5) with a sour flavor (Pringsulaka et al., 2011). According to Nham standard TIS 1219-2547 (2004) issued by the Thai Industrial Standard Institute, Ministry of industry, pH of ready-to-eat Nham is lower than 4.6 and S. aureus is not found in 0.1 g of Nham. This pH is useful to inhibit the growth of pathogenic bacteria such as S. aureus (Chokesajjawadee et al., 2009). S. aureus is one of the leading causes of foodborne disease. Ready-to-eat product contamination may occur directly from infected raw materials or may result from poor hygiene during production processes (Normanno et al., 2007). Moreover, S. aureus growth was observed during the early phase of Nham fermentation, when the pH in this phase is within the tolerable growth range of S. aureus (Bergdoll and Lee Wong. 2006). Therefore, the objective of this study was to control the pathogenic bacteria in Nham with cook in an oven to reach core temperature of 71 °C. Materials and Methods Semi-dried Nham preparation The ingredients of semi-dried Nham were pork ham, cooked rice, salt, sugar, nitrite, and minced garlic. Ham portion from culled sow pig were used. It was minced using a meat grinder after that fat and connective tissues were removed. The minced pork ham was mixed with the other ingredients. The mixture divided into 3 groups: i) control (no glycerol added), ii) treatment with 5% glycerol added, and iii) treatment with 10% glycerol added. Each July 1-3, 2015
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mixture was then formed into a strip (3 x 10 x 0.5 cm2) and fermented at 30ºC for 3 days in vacuum packed. Subsequently, strip samples were dried in an oven until the internal temperature reached of 71ºC. Determination of microbiological and chemical analysis in semi-dried Nham The numbers of S. aureus, yeast/mold, and lactic acid bacteria in the mixtures were counted at the following times: before each mixture was fermented, after it was fermented for 3 days, and after it was dried. All microbe counts were carried out according to AOAC (2006) and expressed as log10 colony forming unit per gram of Nham (log10 cfu/g). The pH of each mixture of Nham was detected according to the modified AOAC (1984) and the mixture’s total acidity was determined according to Friedrich (2001). Statistical analysis All experimental design in this study were carried out by completely randomized design. Mean values was compared by the Duncan's multiple range test. Analysis was performed using the SPSS package (SPSS 16.0 for windows, SPSS Inc., Chicago, IL, USA). Results and Discussion Determination of microbiological quality during fermentation and semi-dried Nham The result showed that, the numbers of lactic acid bacteria after fermentation (3 days) were not significantly different among sample (p>0.05), as shown in Table 1. Table 1 The number of microbial detected in semi-dried Nham during processes (Mean±SD).
Microorganisms
Time (day)
Lactic acid bacteria
Number of microbial (log CFU/g) Control
Glycerol5%
Glycerol 10%
0 3 After drying
5.11 ± 0.13b 7.49 ± 0.48a <10*
4.19 ± 0.37a 7.40 ± 0.02a <10*
4.28 ± 0.19a 7.43 ± 0.05a <10*
S. aureus
0 3 After drying
3.42 ± 0.06a <10* <10*
2.92 ± 0.18b <10* <10*
1.34 ± 1.89c <10* <10*
Yeast/mold
0 3 After drying
3.32 ± 0.01ab <10* <10*
3.40 ± 0.08b <10* <10*
3.23 ± 0.02a <10* <10*
abc
Values with different letters within the same row differ significantly (p<0.05) Note * Means number of bacteria less than 10 cfu/g
It was interesting that S. aureus or yeast/mold were not observed (the limit of detection less than 10 cfu/g) at 3 days of fermentation as well as after drying. The number of lactic acid bacteria increased significantly during fermentation (p<0.05), which effectively inhibited pathogenic bacteria. Lactic acid bacteria are commonly used as starter culture producing fermented meat product (Luxananil et al., 2009). Moreover, appropriate fermentation of the product to pH less than 4.6, and additional refrigeration storage was shown to reduce the number of S. aureus in Nham (Chokesajjawadee et al., 2009).
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Determination of chemical quality of Nham during fermentation The pH during fermentation of the 3 groups of Nham is shown in Table 2. At the beginning, the pH was not significantly different between 3 groups (p>0.05). On 1 and 2 day of fermentation, the pH of 10% glycerol added sample was significantly higher than those of the other groups. After 3 days of fermentation, pH of the control group was the lowest. Glycerol can keep water in food (Chen et al., 2000), so the high pH of 10% glycerol was obtained. The pH of Nham was gradually decreased during fermentation because organic acid was produced from carbohydrate by lactic acid bacteria, contributing the inhibition of undesirable microorganisms (Visessanguan et al., 2004). Table 2 The pH value of Nham during fermentation (Mean±SD)ใ Time of fermentation Control Glycerol 5% Glycerol 10% (day) 0 5.98 ± 0.13a,z 5.87 ± 0.16a,y 5.95 ± 0.10a,y 1 4.73 ± 0.12a,y 4.77 ± 0.14 a,x 5.19 ± 0.18b,x 2 4.60 ± 0.03a,xy 4.77 ± 0.13ab,x 4.92 ± 0.12b,w 3 4.50 ± 0.05a,w 4.74 ± 0.15b,x 4.95 ± 0.07c,w abc Values with different letters within the same row differ significantly (p<0.05) xyz Values with different letters within the same column differ significantly (p<0.05) Total acidity varied with pH. After 3 days of fermentation, the total acidity of the control group was significantly higher than those of the other 2 groups with added glycerol, as shown in Table 3. Table 3 Total acidity during fermentation (Mean±SD). Time of fermentation control Glycerol 5% Glycerol 10% (day) 1.17 ± 0.13a,x 1.09 ± 0.03a,x 1.14 ± 0.06a,x 0 1.43 ± 0.04c,y 1.38 ± 0.01b,y 1.33 ± 0.02a,y 1 1.66 ± 0.05 b,z 1.54 ± 0.02a,z 1.46 ± 0.02a,z 2 1.89 ± 0.05b,z 1.60 ± 0.06a,z 1.54 ± 0.07a,z 3 abc Values with different letters within the same row differ significantly (p<0.05) xyz Values with different letters within the same column differ significantly (p<0.05) Conclusion According to the result, the process to produce ready-to-eat semi-dried Nham by drying in an oven to reach the core temperature of 71ºC is safe from S. aureus and yeast/mold. References AOAC. 2006. Chapter 17 AOAC Official Method 966.23c-24. p. 5-6. In Horwitz, W. and Latimer, G.W. Official methods of analysis of AOAC International. Maryland. Bergdoll, M.S., and A.C. Lee Wong. 2006. Staphylococcal intoxications. In: Rieman, H.P.,Cliver,D.O. (Eds.), Foodborne Infections and Intoxications. Academic Press,Elsevier Inc., San Diego, California, pp. 523–562. Chen, W.S., D. C. Liu, M. T. Chen and H. W. Ockerman. 2000. Improving texture and storage stability of Chinese-sytle pork jerky by the addition of humectants. Asian-Aus. J. Anim. Sci. 13: 1455-1460. July 1-3, 2015
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Chokesajjawatee, N., S. Pornaem, Y.G. Zo, S. Kamdee, P. Luxananil, S. Wanasen and R. Valyasevi. 2009. Incidence of Staphylococcus aureus and associated risk factors in Nham, a Thai fermented pork product. Food Microbiol. 26:547â&#x20AC;&#x201C;551. Friedrich, J.E. 2001.Tritratable Activity of Acid Tastants. Current Protocols in Food Analytical Chemistry. John Wiley&Sons, Inc, Cargill In corporated Minneapolis, Minesota. Luxananil P., R. Promchai, S. Wanasen, S. Kamdee, P. Thepkasikul,V. Plengvidhya,W. Visessan guan and R. Valyasevi. 2009. Monitoring Lactobacillus plantarum BCC 9546 starter culture during fermentation of Nham, a traditional Thai pork sausage. Int. J. Food Microbiol. 129 (2): 312-315. Normanno G, G. La Salandra, A. Dambrosio, N.C. Quaglia, M. Corrente, A. Parisi, G. Santagada, A. Firinu, E. Crisetti and G.V. Celano. 2007. Occurrence, characterization and antimicrobial resistance of enterotoxigenic Staphylococcus aureus isolated from meat and dairy products. Int. J. Food Microbiol. 115:290â&#x20AC;&#x201C;296. Pringsulaka, O., N. Patarasinpaiboon, N. Suwannasai, W. Atthakor and A. Rangsiruji. 2011. Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage. Int. J. Food Microbiol. 28(2): 518-525. Visessanguan, W., S. Benjakul, S. Riebroy and P. Thepkasikul. 2004. Change in composition and functional properties of proteins and their contributions to Nham Characteristics. Meat Sci. 66: 579-588.
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P-48
Comparison of Carcass Traits in Duroc and Two Commercial Crossbred Pigs Numfon TAJASRI1*, Chanporn CHAOSAP2, Ronachai SITTHIGRIPONG1 and Rutcharin LIMSUPAVANICH1 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Agricultural Education, Faculty of Industrial Education, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: numfont.j@gmail.com
ABSTRACT Carcass traits of Duroc (DR), DR x [Large White x Landrace] 1 (DLL1) and DR x [Large White x Landrace] 2 (DLL2) crossbreds were studied. Thirty pigs were randomly distributed into 3 treatments, with 5 barrows and 5 gilts in each. All pigs were reared under the same housing and feeding condition for 6 months prior to slaughter at 110±5 kg. live weight. After slaughter, carcass traits were evaluated. Loin eye area (LEA) was measured and marbling score was determined on Longissimus muscle between the 10th and 11th ribs from the left side of each carcass. Results showed that there were no significant differences (P>0.05) in live weight, hot carcass weight, carcass length, LEA, backfat thickness, FOM grading and lean percentage among breeds. DR had the highest (P<0.05) marbling score and leg percentage, whereas DLL2 had the lowest (P<0.05) trimmed fat. Gilts had higher (P<0.05) dressing percentage, belly and trimmed fat than barrows. However, barrows had higher (P<0.05) marbling score than gilts. Comparing to DLL1 and DLL2, DR showed excellent marbling but no significant difference in lean percentage. Keywords: Carcass Traits, Duroc, Crossbred Pigs, Lean Percentage, Marbling Introduction The quality of pork carcass can be evaluated from the meatier carcass, backfat depth and carcass length. Several factors can affect carcass quality such as breed, feed, age and production system. However, breed is one of the most important factors affecting carcass quality. There are three main purebred pigs including Large White (LW), Landrace (LR) and Duroc (DR). LR and LW are highly prolific and have a good mothering ability while Duroc has fast growth, good feed efficiency, a meatier carcass and good meat quality (Sorapukdee,. 2012; Choi et al., 2014). To gain the effect of a genetic phenomenon called heterosis, which usually gives crossbred pigs an improvement over the average of its parent purebreds in a certain trait. Therefore, most commercial swine producers use crossbred pigs rather than purebreds. There are several planned crossbreeding systems that producers commonly use. Pure breeds for crossbreeding systems are selected for their ability to add some traits to the final crossbred market pig. Fertility, milking ability and litter size are inherited through the maternal line, while meat productivity and meat quality are inherited through the paternal line (Kim et al., 2006; Lee et al., 2011). A white breed such as LW or LR is usually included in the crossbreeding system for its maternal traits. A colored breed such as DR or Hampshire is normally included for its growth rate, feed efficiency, or carcass traits. A planned crossbreeding system may include as few as two or as many as four breeds. Johnson et al. (2002) stated that average daily gain (ADG) is highest in DR compared to Hampshire, LR, and Yorkshire (0.88±0.13 kg, 0.83±0.13 kg, 0.85±0.15 kg, and 0.87±0.14 kg, respectively). July 1-3, 2015
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Oh (2005) reported that the eye muscle ares (EMA) heritability of LR, Yorkshire and DRwas estimated to be 0.33, 0.18, and 0.37, respectively. Three way crossbred pigs (LR Ă&#x2014; Yorkshire Ă&#x2014; DR) are mainly utilized for production of commercial pork and have greater production efficiency than purebred or two-way crossbreds (Nelson and Robison, 1976). The DR breed is used as a terminal sire for commercial pig production not only for its growth and slaughtering performance but also for its fattening performance (Suzuki et al., 2003). The breeding system of combining breeds is necessary to gain a better performance of the offsprings. Breed selection is an important factor for obtaining desirable characteristics of carcasses. Therefore, the aim of this study was to investigate the impact of DR purebred and crossbreds on carcass traits. Materials and Methods Carcass traits of DR, DR x [LW x LR] (DLL1) and DR x [LW x LR] (DLL2) crossbreds, whose parent sows were from different sources, were studied. Thirty pigs were randomly distributed into 3 treatments, with 5 barrows and 5 gilts in each. All pigs were reared for 6 months prior to slaughter under the same housing and feeding condition in contract farming in Lopburi province, Thailand. At 110Âą5 kg live weight, they were transported to a commercial slaughter house in Lopburi province and, after 2 h resting, were electrically stunned before exsanguination. After slaughter, hot carcass weight and backfat depth of the left side of the empty pig carcasses were measured at 45 minutes after death. The backfat depth was measured 65 mm. from the top line at the level of the last rib using the UltraFom300 (SFK technology A/S, Denmark). Carcasses were then chilled immediately and held at 2-4oC for 24 h. before deboning and splitting into primal cuts. Primal cuts including ham, loin, Boston Shoulder, Picnic Shoulder, and belly were weighed. Percentages of each primal cut based on carcass weight were calculated. Marbling of the longissimus muscles in all samples were evaluated at the 10th rib by an industry expert. Marbling scores were 1 (devoid to practical devoid of marbling, <1% intramuscular lipid) to 6 (moderately abundant, >6% intramuscular lipid) (NPPC, 1991). Statistical analysis was done using SPSS statistics 19.0 (SPSS Inc., 2010). Data were analysed by the analysis of variance (ANOVA) to test for the effects of breed, sex and the interaction between them. A Duncan's multiple range test procedure was used to determine significant differences among means at a 5% level of significance. Results and Discussion The carcass traits of DR purebred and crossbred pigs are shown in Table 1. Carcass traits among breeds including live weight, carcass weight, dressing percentage, carcass length, LEA and backfat thickness were not significantly different (p>0.05). But DR and DLL2 seemed to provide greater LEA than DLL1 (p=0.083). DR had higher (P<0.001) leg percentage than those of crossbreds. The dressing percentage of gilts were higher (P<0.05) than those of barrows. In contrast to this study, Peinado et al. (2008) showed no difference in dressing percentages between gilts and barrows. There was no interaction (p>0.05) between breeding and gender on carcass traits. DLL2 had higher (P<0.01) Boston Shoulder percentage but lower trimmed fat than DR and DLL1. No difference among breeds was detected (p>0.05) in other lean proportion. However, crossbred pigs tended to provide greater loin percentages (p=0.054). According to the research of Franco et al. (2014), crossbreeding improved growth, carcass yield and percentage of lean meat. Gilts had higher (p<0.05) belly percentage and lower (p<0.05) trimmed fat than barrows. DR had higher (p<0.05) marbling scores than crossbreds. But barrows had higher (p<0.05) marbling scores than gilts. This was in accordance with Choi et al. (2014) who reported that DR breed had higher intramuscular fat content than crossbred pigs. Page 342
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Table 1 Carcass traitsψ of Duroc and two types of commercial crossbred pigs breeding effect (B)
Trait
P- valueγ
Gender effect (S)
Duroc 112.2+2.26
DLL1¥ 110.7+5.78
DLL2¥ 112.2+4.19
Barrow 113.07+3.02
Gilt 110.34+4.90
B 0.655
S 0.086
BxS 0.449
87.14+2.18
85.45+4.91
87.38+3.12
87.28+2.93
86.04+4.12
0.457
0.370
0.787
77.66+0.70
77.19+1.34
77.90+1.24
77.18+0.99
77.98+1.15
0.302
0.042
0.139
85.70+3.10
84.10+1.80
84.10+3.67
83.87+3.05
85.40+2.75
0.335
0.137
0.091
Loin eye area (cm )
58.49+3.91
54.29+4.77
59.39+4.85
56.60+4.46
58.78+5.21
0.083
0.389
0.835
Back fat thickness (mm) Marbling score
19.26+5.17
21.93+4.06
19.82+1.67
21.33+3.80
19.35+3.98
0.292
0.179
0.598
3.45+1.43a
1.70+0.83b
2.30+1.34b
2.94+1.54
1.93+1.00
0.011
0.035
0.291
Lean percentage (%)
43.49+1.67
43.87+1.47
44.95+1.54
43.58+1.48
44.62+1.66
0.113
0.077
0.947
Ham (%)
15.89+0.79
15.30+0.65
15.76+1.12
15.37+0.97
15.92+0.71
0.291
0.093
0.913
Picnic Shoulder (%)
9.21+0.47
9.24+0.60
9.73+0.49
9.34+0.46
9.45+0.66
0.064
0.572
0.408
Boston Shoulder (%)
5.59+0.34b
5.89+0.32ab
6.20+0.47a
5.90+0.42
5.89+0.49
0.007
0.915
0.479
Loin (%)
7.64+0.51
8.20+0.46
8.07+0.62
7.80+0.37
8.14+0.69
0.054
0.078
0.306
Tender loin (%)
1.25+0.05
1.23+0.11
1.28+0.08
1.24+0.09
1.27+0.08
0.427
0.340
0.887
Belly (%)
11.69+0.69
11.65+0.67
11.27+0.49
11.77+0.56
11.30+0.62
0.220
0.036
0.551
Rib (%)
3.98+0.3
4.01+0.26
4.20+0.31
4.02+0.25
4.11+0.33
0.163
0.367
0.086
Bone (%)
7.89+0.49
7.61+0.26
7.89+0.50
7.71+0.35
7.88+0.51
0.228
0.267
0.207
Leg (%)
9.73+0.66a
8.64+0.38b
8.91+0.37b
8.97+0.66
9.22+0.67
0.000
0.161
0.513
Skin (%)
6.48+0.30
6.7+0.31
6.76+0.36
6.67+0.45
6.62+0.17
0.097
0.647
0.019
Trimmed meat (%)
3.92+0.38
4.04+0.32
3.94+0.27
3.95+0.31
3.98+0.34
0.679
0.828
0.346
Trimmed Fat (%)
10.64+1.65a
10.87+0.97a
9.61+0.79b
10.88+1.34
9.87+1.02
0.028
0.013
0.157
Slaughtered weight (kg.) Carcass weight (kg.) Dressing percentage (%) Carcass length (cm) 2
ψ
γ
Values were presented as mean+standard deviation. P<0.05 was considered as significantly different. DLL1= Duroc x (Large White x Landrace) 1; DLL2 = Duroc x (Large White x Landrace)
¥
Conclusion As expected, the Duroc purebred and barrows showed higher marbling scores, a generally accepted indicator for meat palatability. Although there was no difference in lean percentages among breeds, DLL1 and DLL2 tended (p=0.054) to provide greater loin percentages. In addition, DLL2 resulted (p<0.05) in greater percentages of Boston Shoulder and less trimmed fat than those in the purebred. Therefore, it had a trend to make more profit in carcass yield. At the same time, its similar marbling score to that in Duroc may indicate to its palatability. Acknowledgements The researchers thank the Faculty of Agricultural Technology and Faculty of Industrial Education, King Mongkut’s Institute of Technology for their technical and research facility supports. References Choi, J.S., H.J. Lee, S.K. Jin, Y.I. Choi and J.J. Lee. 2014. Comparison of carcasscharacteristics and meat quality between Duroc and crossbred Pigs. J. Food Sci. 34(2): 238-244.
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Franco, D., J.A. Vaquezand. and J.M. Lorenzo. 2014. Growth performance, carcass and meat quality of celta pigs crossbred with Duroc and Landrace genotypes. Meat Sci. 96: 195202. Kim, I. S., S. K. Jin, Y. M. Song, K. H. Park, S. M. Kang, J. H. Ha, I. J. Kim, Y. S. Park, and J. H. Kim. 2006. Quality characteristics of pork by sex on crossbred pigs. Korean J. Intl. Agri. 18:34-39. Lee, Y. H., Kwon, S. G., Park, D. H., Kwon, E. J., Cho, E. S., Bang, W. Y, Park, H. C. Park, B. Y., Choi, J. S., and Kim, C.W. 2011. Development of high meat quality using microsatellite markers in Berkshire pigs. J. Anim. Sci. Tech. 52:89-97. NPPC. 1991. Procedures to evaluate market hogs (3rd ed.). Des Moines: National Pork Producers Council, USA. Nelson, R. E. and O. W. Robison. 1976. Comparisons of specific two- and three-way crosses of swine. J. Anim. Sci. 42, 1150-1157. Oh, H. S. 2005. Estimation of genetic trend for economic traits of the integrated swine group led by kaya GGP in Gyeongsangnamdo, Korea, Gyeongsang Univ., Doctorate Thesis. Peinado, J., P. Medel, A. Fuentetaga. and G.G. Mateos. 2008. Influence of sex and castration of female on growth performance and carcass and meat quality of heavy pigs destined for the dry-cured industry. J. Anim. Sci. 86: 1410-1417 Sorapukdee, S. 2012. Quality of pork meats from different breeds and production systems: Their post-mortem changes and effect of processing. Ph.D. dissertation, Prince of Songkla University. SPSS Inc. Advanced Statistics. Version 19.0. SPSS Inc., Chicago, IL. Suzuki, K., T. Shibata, H. Kadowaki. and T. Toyoshima. 2003. Meat quality comparison of Berkshire, Duroc and crossbred pigs sired by Berkshire and Duroc. Meat Sci.64, 35-4
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P-49
Optimization of Culture Conditions of Bacteria Isolated from Buffalo Rumen for Cellulase and Xylanase Production Kanya JIRAJAROENRAT1*, Jutatip CHALERMWANICHWONG2, Tipaporn NGAMSANGA2, and Kanokrat SRIKIJKASEMWAT1 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand 2 Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kjkanya@kmitl.ac.th
ABSTRACT Cellulase and xylanase producing bacteria were isolated from swamp buffalo rumen. The bacterial isolate ID2R1P1 showing a clear zone on both CMC agar and xylose agar was selected. Optimum concentration of substrates, pH, temperature and incubation time for cultivation of the isolate to maximize the production of cellulase and xylanase enzymes were determined. The results indicated that optimal cellulase production was carried out by incubation of the isolate in a broth containing 1% CMC using yeast extract as a nitrogen source, at pH 7.0 and 39°C for 2 days. Maximum cellulase yield was 2.034±0.023 U/ml. Optimal condition for production of the xylanase enzyme was an incubation of the isolate in a broth adding yeast extract and 2% xylan at pH 3.0 and 37°C for 2 days. Maximum xylanase yield was 183.272 ± 0.015 U/ml. The optimal condition of the enzyme activities was incubation at pH 7.0 and temperature 50oC. Keywords: Cellulase, Xylanase, Buffalo, Ruminal bacteria Introduction Rumen ecosystem contained a highly diverse population of obligate anaerobic microorganisms including fungi, protozoa, bacteria and archaea. The ruminants utilized the nutrients released by the ruminal microorganisms after the digestion of animal feed fibers consisted of cellulose, hemicellulose and other plant materials (Hobson and Stewart 1997). The microorganism population is controlled under the balance of the ecosystem in anaerobic condition. For the growth of rumen bacteria, optimum pH in the range 6.0-6.9 and temperature 39ºC were required (Kamra, 2005). Among a variety of ruminal microorganisms, cellulolytic bacteria are the main nutrition providers in the rumen such as Fibrobacter succinogenes, Ruminococcus albus, R. flavefaciens, Clostridium lochheadii and Butyrivibrio fibrisolvens (Kamra, 2005). Moreover, hemicellulose-degrading bacteria in rumen were also identified such as Butyrivibrio fibrisolvens, Prevotella ruminicola, Eubacterium xylanophilum, E. uniformis (Kamra 2005). Cellulase and xylanase enzymes are commonly used in various industries, for example: animal feed, textile, bleaching and cleaning powder, food, pulp and paper production (Subramaniyan and Prema, 2002; Sukumaran et al, 2005). However current available commercially enzymes are not suitable for all applications. It is therefore important to identify new enzymes, in order to increase the diversity of cellulase and xylanase enzymes for various industries. Booppata et al. (2011) isolated a total of 21 bacterial isolates from the buffalo ruminal fluid, 19 isolates could degrade cellulose whereas 4 isolates could degrade xylan. Among these isolates, 4 of them produced both enzymes. However, cultivation of the bacterial isolates from the animal rumen in the laboratory is limited due to the specificity of July 1-3, 2015
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rumen environment. Therefore, this work observed the optimal culture conditions of bacterial isolate ID2R1P1 which produced both cellulase and xylanase enzymes in order to obtain the maximum amount of enzyme production for further industrial applications. Materials and Methods Isolation and screening for cellulase and xylanase producing bacteria Rumen fluid samples were collected from slaughtered buffaloes at an abattoir in Bangkok. The rumen fluid samples were suspended and serially diluted in peptone media solution up to 10-4. Two hundred microlitres of each dilution were spread on Rumen Fluid Glucose Cellobiose Agar (RFGCA) modified from Kobayashi et al (2000) and incubated at room temperature under anaerobic conditions for 48 hours. The bacterial colonies were transferred in four replicates onto CMC agar and xylan agar and incubated at room temperature under anaerobic conditions for 72 hours. The plates were stained with Lugol's iodine solution (2 % (w/v) KI and 1% (w/v) I2) for 5 minutes at room temperature (modified from Kasana et al., 2008) and scored for a clear halo surrounding the colony (Lu et al 2005). One of the isolate, named ID2R1P1 showed the clear zone on both CMC and xylan agars, was used in this study (Booppata, 2011). Determination of optimal condition of bacterial cultivation Bacteria were cultured on Lubia-Bertani (LB) agar by incubation at 37째C for two days. A single colony was sub-cultured into LB broth and incubated at 37째C for one day at the shaking speed of 230 rpm. The bacterial culture was added in liquid CMC broth or xylan broth containing 1%, 2%, and 3% to produce cellulase and xylanase respectively. Addition of nitrogen sources including 0.1% yeast extract, 0.1% urea or 0.1% tryptone was performed. The temperature of incubation was varied from 33, 35, 37, 39 and 41 째C in individual experimental flasks. Influence of the cultured pH was studied by variation of the pH in the culture media from 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0. The bacterial culture was incubated for 1-5 days at shaking speed of 230 rpm. Enzyme activity assay The bacterial culture media was centrifuged and the supernatant was collected as a crude enzyme for analysis of reducing sugars by DNS method (Miller, 1959). Quantity of the reducing sugars was calculated by the measurement of absorbance at 540 nm and calibrated with the glucose or xylose standard curves. The unit of enzyme activity means the amount of enzymes that degrade the substrate into a reducing sugar which is released one micromole for 1 minute under defined conditions. Results and Discussion Optimal conditions for cultivation of the bacterial isolate ID2R1P1 When cultivation of bacterial isolate ID2R1P1 in CMC broth or a xylan broth for 1-5 days, both cellulase and xylanase enzymes were produced at the highest quantity after incubation for two days (Table 1). The cellulase activity showed at the highest value when incubated the culture at temperatures 39oC, while the maximal production of xylanase enzyme was found when incubation at 37oC. The maximal production of cellulase enzyme was found when using CMC broth with a pH of 7.0. While the production of xylanase enzyme was in a maximum magnitude when cultivation in the xylan broth with a pH of 3.0. The cellulase was produced at a highest quantity when using recipes with CMC concentration of 1%, while the highest production of xylanase was found when using 2% xylan as a car bon source. The bacterial isolate ID2R1P1 produced both cellulase and xylanase in the media containing yeast extract, tryptone and urea as a nitrogen source, respectively. When incubation under appropriate conditions, the results showed that the bacteria isolate ID2R1P1 produced the Page 346
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cellulase enzyme and the xylanase enzyme at the maximal magnitude of 2.034 ± 0.023 and 183.272 ± 0.015 units per milliliter, respectively. Table 1 Optimal conditions for cellulase and xylanase enzyme production by bacterial isolate ID2R1P1 from buffalo ruminal fluid. Optimal condition Incubation time pH Temperature Carbon source Nitrogen Source Maximum yield
Cellulase production
Xylanase production
2 days 7.0 39oC 1% CMC 0.1% yeast extract 2.034±0.023 U/ml
2 days 3.0 37oC 2% xylan 0.1% yeast extract 183.272 ± 0.015 U/ml
Optimal conditions for enzyme activities Cellulase and xylanase activities of the bacterial isolate ID2R1P1 were measured based on the DNS method (Table 2). The optimal cellulase activity was observed when incubation of the crude enzyme at pH 7.0, temperature 50oC for 10 minutes. The enzyme digested both CMC and filter paper. This indicated that the cellulase enzyme contain both endoglucanase and exoglucanase activities. The optimal xylanase activity was found when incubation of the crude enzyme with xylan substrate at pH 7.0 and at temperature 50oC for 5 minutes. Table 2 Optimal conditions of cellulase and xylanase enzyme activities of bacterial isolate ID2R1P1 from buffalo ruminal fluid. Optimal condition
Cellulase activity
Xylanase activity
Incubation time pH Temperature Substrate
10 min. 7.0 50oC CMC, filter paper
5 min. 7.0 50oC xylan
Conclusion This study revealed that the bacterial culture isolate ID2R1P1 from the rumen of buffalo can be adapted to grow in the controlled laboratory environment and produced cellulase and xylanase enzymes. However maximization of the enzyme production by alteration of carbon sources and variation of the oxygen concentration in the cultivation system seems to be necessary to study in order to employ these enzymes to industrial applications. Acknowledgement This research was partially supported by the Center for Agricultural Biotechnology, Postgraduate Education and Research Development Office (AG-BIO/PERDO-CHE), Commission on Higher Education, Ministry of Education, Thailand. References Booppata M., A. Jindaprasert, K. Jirajaroenrat and K. Srikijkasemwat. 2011. Selection of Cellulase and Xylanase Producing Bacteria from Buffalo Rumen. In Proceedings of The 4th International Conference on Fermentation Technology for Value Added Agricultural Products (FerVAAP2011). Fer4P34. 29-31th August 2011, Khon Kaen, Thailand.
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Hobson P.N. and C.S. Stewart. 1997. The Rumen microbial ecosystem. Springer. 719 p. Kamra, D.N. 2005. Rumen microbial ecosystem. Current Science 89:124–135. Kasana, R.C., R. Salwan, H. Dhar, S. Dutt and A. Gulati. 2008. A rapid and easy method for the detection of microbial cellulases on agar plates using gram’s iodine. Current Microbiology 57:503–507. Kobayashi, Y, J. F. Robert and M.T. Ronald. 2000. ‘Development of a competitive polymerase chain reaction assay for the ruminal bacterium Butyrivibrio fibrisolvens OB156 and its use for tracking an OB156-derived recombinant,’ FEMS Microbiology, Vol. 188, pp. 185–190. Lu, W.J., W. Hong-Tao, Y. Shi-Jian, W. Zhi-Chao and N. Yong-Feng. 2005. Isolation and characterization of mesophilic cellulose-degrading bacteria from flower stalks-vegetable waste co-composting system. The Journal of General and Applied Microbiology 51(6):353-360. Miller, L.G. 1959. Use of dinitrosalicylicacid reagent for determination of reducing sugar. Analytical Chemistry 31(3):246-248. Subramaniyan, S. and P. Prema. 2002. Biotechnology of microbial xylanases: enzymology, molecular biology and application. Critical Reviews in Biotechnology 22(1):33-64. Sukumaran, R.K., R.R. Singhania and A. Pandey. 2005. Microbial cellulases - Production, applications and challenges. Journal of Scientific and Industrial Research 64(11):832844.
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P-50
Consumption of Energy, Water and Consumables in Pon-Yang-Kham Beef Cutting Process Chalermsak SAKDAPECHSIRI1, Thierry TRAN2, Matana OSOTHONGS3 and Kanya JIRAJAROENRAT1* 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 QUALISUD, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), 34398 Montpellier Cedex 5, France 3 Pon-Yang-Kham Livestock Breeding Cooperative NSC., Ltd., Pathum Thani 12130, Thailand *Corresponding email: kjkanya@kmitl.ac.th
ABSTRCT Pon-Yang-Kham or Thai-French beef is well known as a premium beef product especially as steak in Thailand modern trade. To generate the environmental friendly production process, we investigated the consumption of energy, water and consumables during the production from farming to packaging of the product. In the cutting process, energy consumption included diesel for truck and electricity. Consumables include clothes, cleaning equipment, cleaning chemicals, cutting equipment and packaging equipment. The data were collected during year 2010-2014. The results showed that half carcass about 2,000 ton/year or 163 ± 10 ton/month entered the cutting process. After cutting, boneless beef was about 480 ton/year. During the cutting process, consumption of electricity was 0.423 kWh/kg of beef. Consumption of water was 9.56 litre/kg of beef. Diesel used by truck for delivery of beef to supermarket was 0.019 litre/kg of beef. In packaging, plastic bags including low density polyethylene (LDPE), high density polyethylene (HDPE) were used about 10 ton/year. Moreover, booth shoe made of polyvinyl chloride (PVC), foam box made of polystyrene were used. The emission factor of all equipment will be further analyzed. Finally, life cycle assessment (LCA) of Pon-Yang-Kham beef production will be analyzed and also the carbon footprint of the product will be reported. Keywords: Beef, Emission factor, Life cycle assessment (LCA), Carbon footprint Introduction Thailand economy bases on agricultural production from cultivation of crops and livestock. Thailand Greenhouse Gas Management Oraganization (TGO) has found that agriculture's greenhouse gas emissions compared to the other manufacturing sectors was approximately 24.1% of the greenhouse gas emissions of the entire country. The trend of greenhouse gas emissions was rising. Low GHG emission livestock production and meat production is to be aware of (TGO, 2005). Livestock sector was highly related to the greenhouse gas (GHG) emissions. By use of the space and farmland, the GHG was equivalent to 2.5 GtCO2-eq. Feed production including fuel combustion in manufacturing processes and the use of chemical fertilizers in the cultivation of forage led to the GHG emission of 0.4 GtCO2-eq. Animal farming including the use of fuel on farms and methane emissions from the digestive system of animals, the emission was around 1.9 Gt CO2-eq. Moreover, the management of manure decomposition released the GHG as high as 2.2 Gt CO2-eq (Steinfeld et al., 2006). Especially in beef production in the United States, the emissions of greenhouse gases were at 22.0 kgCO2-eq to 1 kg of beef (Johnson et al., 2003; Subak, 1999). Production of organic July 1-3, 2015
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beef in Sweden released greenhouse gas more than the 22.3 kgCO2eq (Koneswaran and Nierenberg, 2008). Production of Kobe beef with high intramuscular fat by longer fattening and a long way to transport had high impact on greenhouse gas emissions as high as 36.4 kgCO2-eq per 1 kg Kobe beef (Ogino et al., 2007). The environmental impact of meat production of cattle throughout the life of the product by Life Cycle Assessment (LCA) to develop sustainable production systems and social responsibility was studied. The production of Pon-Yang-Kham (PYK) Livestock Breeding Cooperative was selected as a prototype. The cooperative consist about 5,000 members of which are in the provinces: Sakon Nakhon, Nakhon Phanom and Nong Khai (Cooperative Phon Yang Kham, 2009). The PYK management system has been well established from cattle farming to beef cutting and packaging. The consumption of electricity, water, fuel and consumable during the cutting and packing process was reported in order to evaluate the LCA of product in the future. Materials and Methods
Data collection Historical data included the use of water, electricity, fuel and consumables, regular transport by pick-up car and by refrigerated truck were recorded at, Pon-Yang-Kham Livestock Breeding Cooperative NSC., Ltd., Wang Thong Branch, Moo 8 Phaholyothin Road, Khukhot, Pathum Thani Province, Thailand. Consumables include clothes, cleaning equipment, cleaning chemicals, cutting equipment and packaging equipment. The data were collected during year 2009 to 2014. Results and Discussion Input and output quantity of beef in the cutting process The average amount of half-carcass of PYK beef delivered by refrigerated trucks from the PYK slaughterhouse at Sakon Nakhon province to PYK Livestock Breeding Cooperative NSC., Ltd., Pathum Thani Province was as high as 163 ± 10 tons/month or 1,961.93 ± 2.63 tons/year. After cutting, boneless beef was about 480 ton/year. Half carcass was cut into 21 main parts, in which T-bone and Stew were the most types of 14.78% and 13.44% respectively. Consumption of electricity, water and diesel The average consumption of electricity in the cutting and packaging process of PYK was 191,199 ± 12,312 kWh annually or 0.423 kWh/ 1 kg of beef. Water consumption was 4,357 ± 619 M3 annually or 9.56 litre/ 1 kg of beef. Fuel was used for transportation of meat to customers at 10,324 ± 2,362 liters annually or 0.019 litre/ 1 kg of beef. However, in year 2011, the consumption of electricity and water was lower than other years due to the flood crisis in the Central of Thailand effecting the production at Wang Thong Branch (Figure 1a-1c). Consumption of plastic materials Different types of plastic materials were used in packaging of boneless beef before delivery to customers by refrigerated pick-up car. Polyethylene plastic bags made by low-density polyethylene (LDPE) and high-density polyethylene (HDPE) were purchased as high as 7.79 tons and 2.76 tons annually respectively. Per 1 kg of PYK beef, LDPE plastic bag was used at the highest amount, 17.69 g. Moreover, foam trays and foam box, stretch film were also used in a large quantity (Figure 2).
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(a)
(b)
Annual consumption of electricity (kWh)
Annual consumption of water (M3)
210,000 200,000 190,000 180,000 170,000 160,000 150,000
6,000 5,000 4,000 3,000 2,000 1,000 2009
2010
2011
2012
2013
2009
2010
2011
2012
2013
(c) Annual consumption of diesel (Litre) 20,000 15,000 10,000 5,000 2009
2010
2011
2012
2013
Figure 1 Annual consumption of electrictity (a), water (b) and diesel (c) in the cutting process and packaging of Livestock Breeding Cooperative NSC., Ltd., Wang Thong Branch, Pathum Thani, Thailand during year 2009-2013. Consumption of plastic (g/1 kg of beef) 20
17.69
15 10 5
6.26 2.85
1.85
0.38
0.56
PP
EPS
0 PVC
LDPE
HDPE
PE Foam
Figure 2 Quantity of plastic materials used in Pon-Yang-Kham beef packaging (gram/1 kilogram of beef). PVC = polyvinylchloride; LDPE = low density polyethylene; HDPE = high density polyethylene; PE = polyethylene; PP = polypropylene; EPS = expanded polystyrene. Conclusion Consumption of electricity, water, fuel, and consumables of Pon-Yang-Kham beef production was related to amount of beef production each year. Complete analysis of life Cycle Assessment from farming, slaughtering, cutting, packaging, distribution, and waste management will reveal greenhouse gas emission.
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Acknowledgement This project was sponsored by Higher Education Research Promotion, The Commission on Higher Education, Ministry of Education. References Johnson, D.E., H.W. Phetteplace, A.F. Seidl, U.A.Schneider and B.A. McCarl. 2003. Management variations for U.S. beef production systems: Effects on greenhouse gas emissions and profitability. In: Proceedings of the 3rd International Methane and Nitrous Oxide Mitigation Conference, 17–21 November 2003, Beijing, China. Beijing: China Coal Information Institute, 953–961. Koneswaran, G. and D. Nierenberg. 2008. Global farm animal production and global warming: impacting and mitigating climate change. Environ Health Perspect 116:578–582. Ogino, A., H.Orito, K. Shimada and H. Hirooka. 2007. Evaluating environmental impacts of the Japanese beef cow-calf system by the life cycle assessment method. Animal Sci J. 78:424–432. Pon-Yang-Kham Livestock Breeding Cooperative. 2009. History. [Online] http://www.coopthai.com (cited on 10/05/2015). Steinfeld, H., P. Gerber, T. Wassenaar, V. Castel, M. Rosales and C. de Haan. 2006. Livestock’s long shadow – Environmental issues and options. Rome, Italy, Food and Agriculture Organization of the United Nations,. Subak, S. 1999. Global environmental costs of beef production. Ecol Econ. 30:79–91. TGO. 2005. Carbon label and carbon footprint of organization. Thailand greenhouse gas management organization (public organization). [Online] http://www.tgo.or.th. (cited on 10/05/2015).
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P-52
Changes in Physicochemical, Microbiological and Sensorial Quality of Jerky Processed From Spent Laying Hen Meat during Storage Chanpen UESAKULRUNGRUENG, Supaluk SORAPUKDEE* and Komkhae PILASOMBUT Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: kssupalu@kmitl.ac.th
ABSTRACT The objective of this study was to evaluate the quality deterioration of jerky produced from spent laying hen meat during 90 days of storage. Reformed jerky product was packed in vacuum packaging, stored at room temperature and then collected at day 0, 30, 60 and 90 for determination of shear force, color, water activity, lipid oxidation, microbiological content and sensory evaluation. The result showed that texture of products stored at 60 and 90 days had higher shear force value than those stored at 0 and 30 days (P<0.05). Regarding color of jerky, lightness value increased, while redness and yellowness values decreased during prolong storage (P<0.05). There were no significant differences in lipid oxidation as indicated by TBARS and water activity during 90 days (P>0.05). The number of total plate count, yeast/mold and S. aureus of product along 90 days was less than limit of detection. It was indicated that jerky product was stable to micro-organisms that can cause product spoilage and poisoning. Finally, the hedonic score of jerky stored at 90 days was similar to those stored at day 0 (P>0.05), which refer to the acceptability of product. In conclusion, jerky product was stable to microbiological and chemical changes during 90 days of storage, but some discoloration and tough texture was observed. Keywords: Humectants, Semi-dried meat, Meat product Introduction Spent laying hen is bird aged last about 60 weeks or chicken give egg production less than 60% of herd. Their prices are likely to low as compared to brolier chicken. However, the meat looks quite tough, does not meet the needs of consumers. It can be use to produce jerky, because the production process is not complicated. It is popular consumer products and can be stored at room temperature for a long period. Jerky is one of the most popular in United States. Ready to eat meat products, with sales increasing from 631.6 million US dollars in 1994 to 2.7 billion in 2004, mainly sold at convenience stores, shopping malls and gas stations (Konieczny et al., 2007; Han-Sul Yang et al., 2009, USDA-FSIS, 2012). It's manufacturing is simple process. The finished product can be stored at room temperature for a long period. However, The moisture and the water activity (aw) decreased make jerky beef has a dark color, brittle and dry. Several descriptions and investigations of jerky by Guilbert et al. (1981) found that the addition of 5 or 10% glycerol to a reduction in the aw of the product. Later, Kim and Sung (1989) showed that the addition of glycerol was effective in reducing aw and moisture content. The influence of moisture as a ubiquitous plasticizer has been extensively reviewed (Levine and Slade, 1992). Also, glycerol has been an effective plasticizer in certain protein systems (Kalichevsky et al., 1992). Due to the problem of dark color, brittle and dry of jerky made from spent laying hen meat, jerky formulating with 15% (w/w) glycerol were prepared.
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The objective of this study was to investigate physicochemical, microbiological and sensorial quality of jerky processed from spent laying hen meat during storage. Materials and Methods Preparation of dried jerky samples Spent laying hen meat (breast, boneless leg meat and fillet) were used formulation of curing ingredients (w/w) included 3.70% (w/w) sugar, 0.74% (w/w) sodium chloride, 0.19% (w/w) monosodium glutamate, 1.85% (w/w) soybean sauce, 0.5% (w/w) BBQ, 0.5% (w/w) paprika, 0.1% (w/w) potassium sorbate and 15% (w/w) glycerol. The jerky was processed by the following procedure: (i) grinding meat; (ii) mixing with ingredients; (iii) forming jerky with jerky gun; (iv) subsequent drying in an oven to reach core temperature of 71 ºC and until the water activity of product below than 0.70; (v) packing final products in aerobic packaging with oxygen absorber and store at room temperature. Physicochemical, microbiological and sensorial qualities were measured at 0, 30, 60 and 90 days of during storage. Determination of physicochemical, microbiological and sensorial quality analysis in jerky processed from spent laying hen meat Drying yield was determined by calculating the weight differences of jerky before and after drying. Water activity was determined with a water activity meter (LabMASTER aw, Novasina, Switzerland). Shear force value was measured on jerky sample (1 x 3 x 0.5 cm) using a Warner-Bratzler shear (Instron, model 1011. USA). The surface color of the jerky samples was measured by the CIE L*, a*, b* system using a Colorimeter MiniScan EZ 4000L (Hunter Lab Inc, Reston, VA, USA) standardized with a white plate and black plate. Thiobarbituric acid (TBA) was determined according to the method of Buege and Aust (1987). The numbers of plate count, yeast/mold and S. aureus of product were measured according to AOAC (2005), AOAC (2006) and BAM (1987), respectively. Microbial counts were expressed as log CFU/g. The sensory panel used a seven point hedonic score test in this experiment. Ten panelists were recruited and trained .The panelists evaluated of the sample using a 7-point hedonic scale, where one (1) was “dislike extremely” and (7) was “like extremely.”The sensory characteristics in the present study can be divided into the following parameters: color, appearance, texture, sweet, flavor and overall. Statistical analysis All experiments in this study were carried out by completely randomized design. Mean values was compared by the Duncan's multiple range test. Analysis was performed using the SPSS package (SPSS 11.5 for windows, SPSS Inc., Chicago, IL, USA). Results and Discussion The result of table 1 showed that drying yield of products, glycerol can keep water in food (Chen et al., 2000), so drying yield up to 47.72%. While pork jerky without glycerol as reported by Yang et al. (2009) showed low drying (38.25%). jerky product not added glycerol drying yield as 38.25%. Shear force values of products stored at 60 and 90 days was higher shear force value than those stored at 0 and 30 days (P<0.05). It might be caused by protein oxidation via cross-linked of myosin heavy chain. There were no significant differences in water activity during 90 days of storage (P>0.05). Color of the jerky samples was discolored at 30, 60 and 90 days, where lightness value increased, while redness and yellowness values decreased during prolong storage (P<0.05). There were no significant differences in lipid oxidation as indicated by TBARS during 90 days (P>0.05). This event might be due to low fat products with the addition of spices that help to control the lipid oxidation of jerky processed from spent laying hen meat. An et al. (2010) found that the percentage of protein, carbohydrates, fat, water and ash were 40.27%, 37.45%, 8.90%,
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7.95%, and 5.43% respectively, suggested that jerky products retired high energy and low fat. The number of total plate count, yeast/mold and S. aureus of product along 90 days was less than limit of detection. It was indicated that jerky product was stable to micro-organisms that can cause product spoilage and poisoning. Previously trails pointed out that without the addition of potassium sorbate, products are experiencing a short shelf life due to the growth of yeast and mold. The addition of 0.1% potassium sorbate could extend the shelf life of the product. Similarly, research of Kim et al. (2014) with the addition of potassium sorbate 0.1% in jerky provided shelf stable products. Finally, the hedonic score of jerky stored at 90 days was similar to those stored at 0 day (P>0.05), which refer to the acceptability of the product (Table 2). Table 1 Changing in physicochemical and microbiological in jerky spent laying hen meat during storage at room temperature. Parameter
Day 0
Drying yield (%) Water activity (aw) Shear force (N) Lightness(L*) Redness (a*) Yellowness(b*) TBARS (mg MDA/kg meat) Total plate count Yeast/mold S. aureus
0.580 ± 0.00a 30.49 ± 1.15b 22.73 ± 0.79c 11.79 ± 0.74a 14.81 ± 0.64a 10.20 ± 0.85a ND* ND ND
Storage time (days) Day 30 Day 60 47.72 ± 0.65 0.579± 0.00a 0.586 ± 0.01a b 30.29± 1.84 33.54 ± 1.24a 24.42± 0.45b 25.17 ± 0.79ab 6.46± 0.48b 6.04 ± 0.30b b 9.23± 0.40 9.55 ± 0.54b 10.92± 0.28a 9.90 ± 1.01a ND ND ND ND ND ND
Day 90 0.582± 0.01a 32.95± 1.64a 25.77± 0.30a 5.49± 0.14b 8.98± 0.28b 10.96± 1.71a ND ND ND
abc
Mean values with different superscripts within a same row are significantly different (P < 0.05). ND* = less than limit of detection
Table 2 Changes in sensory attributes in jerky spent laying hen meat during storage at room temperature. Parameter Colour Appearance Texture Sweet Flavour Overall abc
Storage time (days) Day 0 5.10 ± 1.28 a 5.50 ± 0.85 a 4.40 ± 1.07 a 4.00 ± 1.41 a 4.10 ± 1.19 a 4.90 ± 0.99 a
Day 90 4.40 ± 1.35 a 4.30 ± 1.06 b 4.70 ± 0.97 a 3.90 ± 1.07 a 3.60 ± 1.36 a 4.10 ± 0.99 a
Mean values with different superscripts within a same row are significantly different (P < 0.05).
Conclusion The results showed that the 15% glycerol formulated jerky product made from spent laying hen meat was stable to microbiological and chemical changes during 90 days of storage, but some discoloration and tough texture was observed.
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References AOAC. 2005. Chapter 17 AOAC Official method 940.36B. p. 2. In W. Horwitz and G.W. Latimer. Official methods of analysis of AOAC International. Maryland: AOAC International. AOAC.2006. Chapter 17 AOAC Official method 966.23c-24.P. 5-6 In W. Horwitz and G.W. Latimer. Official methods of analysis of AOAC International. Maryland: AOAC international. BAM. 2001. Bacteriological analytical manual online, Chapter 12 on Staphylococcus aureus U.S. Food and Drug Administration. January 2001. [Online] Available: http://www.fda.gov/Food/FoodScienceResearch/Laboratorymethods/BacteiologicalA nalyticalManualBAM. [27-10-2014]. Barrett, A. H., J. Briggs, M. Richardson and T Reed. 1998. Texture and storage stability of processed beef sticks as affected by glycerol and moisture levels. J. Food Sci. 63(3):84-87. Chen, W.S., D. C. Liu, M. T. Chen and H. W. Ockerman. 2000. Improving texture and storage stability of Chinese-style pork jerky by the addition of humectants. J. Anim. Sci. 13(2):1455-1460. Guilbert S., O. Clement, and J. C. Cheftel. 1981. Relative efficiency of various aw-lowing agents in aqueous solutions and in intermediate moisture foods. Lebensm. Wiss. uTechnol.14: 245-251. Han-Sul Yang, Young-Hwa Hwang, Seon-Tea Joo and Gu-Boo Park. 2009. "The physicochemical and microbiological characteristics of pork jerky in comparison to beef jerky". Meat Sci. 82 : 289â&#x20AC;&#x201C;294. Kim S. M. and S.K. Sung. 1989. Effects of level of glycerol addition on physicochemical characteristics of intermediate moisture meat. Korean J Anim Sci. 31: 342â&#x20AC;&#x201C;352. Kim, J. H., H. H. Chun, H. J. Song, K. B. Song. 2014. The physicochemical and microbiological characteristics of pork jerky. Meat Sci. 82: 289-294. Konieczny P., J.Stangierski and J. Kijowski. 2007. Physical and chemical characteristics and acceptability of home style beef jerky. Meat Sci. 76: 253-257. USDA-FSIS. 2012. FSIS Compliance Guideline for Meat and Poultry Jerky Produced by Small and Very Small Establishments. [Online]. Available: www.fsis.usda.gov/PDF/ Compliance_Guideline_Jerky_2012.pdf. [26-05-2014].
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P-53
Screening of Lactic Acid Bacteria from Traditional Fermented Meat Products Jiraroj NITHISANTAWAKHUPT1, Pussadee TANGWATCHARIN1* and Kan SUKSUPATH1 1
Department of Animal Production Technology and Fisheries, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: putang3009@hotmail.com
ABSTRACT The aim of this study was to screen lactic acid bacteria (LAB) from 22 traditional fermented meat products which were 6 Thai fermented sausages and 16 Nhams, produced from different factories. They were collected from 12 local markets in Bangkok, Thailand. LAB was isolated on MRS agar by pour and streak plates. They were confirmed as LAB by catalase test, gram stain and cell shape. The results showed that 167 isolates of LAB were identified as 135 (80.84%) isolates of gram-positive cocci and 32 (19.16%) isolates of gram-positive rods. This study found that LAB isolate numbers were ranged in 6.00 - 8.50 isolates per sample. Thus, traditional fermented meat products are the major source of LAB. Keywords: Screening, Lactic acid bacteria, Traditional fermented meat products Introduction Fermented foods, which were invaded or overgrown by edible microorganisms whose enzymes, particularly amylases, proteases and lipases hydrolyze the polysaccharides, proteins and lipids to non-toxic products with flavors, aromas and textures pleasant and attractive to the human consumer (Steinkraus, 1997). Meat fermentation is a low energy, biological acidulation, preservation method, which results in unique and distinctive meat properties, such as flavor and palatability, color, microbiological safety, tenderness, and a host of other desirable attributes of this specialize meat item (Ocherman and Basu, 2007). Traditional fermented meat products in Thailand such as Nham and fermented sausage were the products of a biotechnological process. Nham was the traditional salt-fermented ground meat, a sausage-like product commonly made of pork (fermented pork) with garlic, pepper, salt and cooked rice (Klayruang et al., 2008). Fermented sausages were defined as a mixture of ground lean meat and minced fat, curing salts, sugar and spices, embedded into a casing and subjected to fermentation and drying (Ammor and Mayo, 2007). Meat is the major source of protein for humans and microbial species. Meat spoilage and various toxins produced by the bacteria occur, therefore the processing and preservation of meat are employed extensively both at home and abroad. In each country, the specific and varied characteristics of meat products were developed. Lactic acid bacteria (LAB), the most commonly used microorganisms in food preservation techniques such as fermentation. Lactic acid produced by LAB is a useful compound for food preservation because it maintains the acidic conditions of the fermented products, and it is lethal to food spoilage and food poisoning bacteria (Kobayashi et al., 2004). LAB was identified as Gram positive bacteria which could produce organic acids such as lactic acid, acetic acid and propionic acid during the fermentation of carbohydrate. In addition, hydrogen peroxide and diacetyl could also be produced which caused the smell and taste of fermented foods. LAB are usually present in high hygienic quality raw meat at low number (102â&#x20AC;&#x201C;103 colony forming units, cfu/g), but they rapidly dominate the fermentation due to the anaerobic July 1-3, 2015
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environment and the presence of NaCl, nitrate and nitrite, that favor their growth. The pHreducing of the sausages and the lactic acid producing from carbohydrates, were the main metabolic activity (Hammes et al., 1990). The objective of this study was to screen isolates of LAB from traditional fermented meat products from local markets in Bangkok, Thailand. Materials and Methods Collection of sample A total of 22 traditional fermented meat products were purchased from 12 local markets in Bangkok, Thailand. They were 6 Thai fermented sausages and 16 Nhams produced from different factories. Isolation and screening of LAB from fermented meat products The modified method of Murray et al. (1994) and Axelsson (2004) was applied in this study. Samples of 25 grams were added to 225 ml of normal saline dilution (0.85% NaCl) and mixed in stomacher (Stomacher Bag Mixer 400 model VW, France). Appropriate decimal dilutions were diluted the mixture 1 ml and poured and streak on a sterile petri dish on de Man Rogosa and Sharpe agar (MRS agar, Merck, Darmstadt, Germany), added of 0.4% bromocresol purple and then incubated at 35 °C for 24 h. Three of single colonies (yellowround colonies, different sizes, shapes and color) were selected and taken from each plate. Then, each colony was further determined for catalase test by placing a drop of 3% hydrogen peroxide solution on the cells. Immediate formation of bubbles indicated the presence of catalase in the cells. All colonies were gram-stained and tested for catalase reaction. Colonies exhibited gram-positive with catalase negative were screened as LAB isolates. These isolates were cultured in MRS broth and stored at -20°C. Results and Discussion Screening of lactic acid bacteria Screening of LAB from traditional fermented meat products in local markets were cultured on MRS agar and incubated at 35°C for 24 h. Total expected LAB colonies on MRS agar were 212 colonies. However, 175 and 167 isolates were identified as LAB using catalase negative and gram positive, respectively. Furthermore, the total of LAB isolates were 135 (80.84%) bacilli (rod shape) and 32 (19.16%) cocci shape (Table 1). Table 1 Screening number of LAB from fermented meat products in Bangkok’s local markets Expected LAB colony no.* LAB isolate no. Fermented meat MRS agar Catalase test Gram stain (gram products Bacilli Cocci (-) positive) Nham (pork)
125
109
97
76
19
Nham (beef)
20
15
13
8
-
Nham (chicken tendon) Fermented sausages Total * no. = number
10
6
6
6
-
57
55
51
45
13
212
175
167
135
32
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Isolation of LAB LAB isolates from traditional fermented meat products from local market in Bangkok, Thailand were cultured on MRS agar. The total of 167 LAB isolates were presumptive lactic acid bacteria due to their gram-positive-staining, absence of catalase, non-spore forming and non-motile characteristics. The numbers of LAB isolates from 13 pork, 2 beef and 1 chicken tendon Nhams were 97, 13 and 6 isolates, respectively. In addition, the 51 isolates were isolated from 6 Thai fermented sausages (Table 2). According to Oki et al. (2011), they observed diversity of species of lactic acid bacteria, bacilli and cocci, from ethnic preserved meat products of the Western Himalayas. They found 182 LAB isolates from 16 samples of ethnic preserved meat products. Furthermore, we found that LAB isolate numbers were ranged in 6.00 - 8.50 isolates per sample in this study. Table 2
Isolate number of LAB from traditional fermented meat products in Bangkok’s local markets.
Traditional fermented meat products Nham (pork) Nham (beef) Nham (chicken tendon) Thai fermented sausage Total * no. = number
Sample no.*
LAB isolate no.
13 2 1
97 13 6
6 22
51 167
LAB isolates no. per sample 7.46 7.00 6.00 8.50
Conclusion The 167 isolates of 212 positive colonies from MRS agar were identified as LAB by catalase test and gram stain. There were 32 gram-positive cocci and 135 gram-positive rods. The screening number similarly ranged in 6.00 - 8.50 (fermented meat products). These LAB isolations will be further study for the probiotic properties and application in fermented meat. Acknowledgment This research work was financial supported by the Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Thailand. References Ammor, M. and B. Mayo. 2007. Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: An update. Meat Science 76 (1): 138-146. Axelsson, L. 2004. Lactic acid bacteria: classification and physiology. pp. 1-66. In S. Salminen, A. Von Wright and A. Ouweland (eds). Advance in Lactic Acid Bacteria: Microbiological and Functional Aspects. New York, U.S.A., Marcel Dekker. Hammes, W.P., A. Bantleon and S. Min. 1990. Lactic acid bacteria in meat fermentation. FEMS Microbiology 87(1):165–173. Hwanhlem, N., S. Buradaleng, S. Wattanachant, S. Benjakul, A. Tani and S. Maneerat. 2011. Isolation and screening of lactic acid bacteria from Thai traditional fermented fish (Plasom) and production of Plasom from selected strains. Food Control. 22: 401-407. Kobayashi, T., M. Kajiwara, M. Wahyuni, N. Hamada-Sato, C. Imada and E. Watanabe. 2004. Effect of culture conditions on lactic acid production of Tetragenococcus species. Journal of Applied Microbiology 96: 1215–1221. July 1-3, 2015
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Klayraung, S., H. Viernstein, J. Sirithunyalug and S. Okonogi. 2008. Probiotic properties of lactobacilli isolated from Thai traditional food. Scientia Pharmaceutica 76:485â&#x20AC;&#x201C;503. Murray, J.M., M. Tavassoli, R. Al-Harithy, K.S. Sheldrick, A.R. Lehmann, A.M. Carr and F.Z. Watts. 1994. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Molecular and Cellular Biology 14: 4878-4888. Nanasombat, S., Phunpruch S., and T. Jaichalad. 2012. Screening and identification of lactic acid bacteria from raw seafoods and Thai fermented seafood products for their potential use as starter cultures. Songklanakarin Journal of Science and Technology 34 (3): 255-262. Ockerman, H.W. and L. Basu. 2007. Production and consumption of fermented meat products. pp. 9-15. In Handbook of fermented meat and poultry. Blackwell publishing Asia, Australia. Oki K., A.K. Rai , S. Sato, K. Watanabe and J.P. Tamang. 2011. Lactic acid bacteria isolated from ethnic preserved meat products of the Western Himalayas. Food Microbiology 28 (1): 1308-1315. Ratanaburee, A. 2010. Production of fermented beverage from Phom-nang seaweed (Gracilaria fisheri) using ď §-Aminobutyric acid (GABA) producing lactic acid bacterium. Degree of Master of Science in Microbiology, Prince of Songkla University. Steinkraus, K.H. 1997. Classification of fermented foods: worldwide review of household fermentation techniques. Food Control 8 (5): 311-317.
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P-54
Screening and Identification of Lactic Acid Bacteria with Antimicrobial Activity to Mastitis Pathogen in Dairy cows; Staphylococcus aureus Khakhanang RATANANIKOM1, *, NantiyaSUWANPANYA1, Yupaporn KHONNALAO1 and Sarayut SRIROD1 1
Faculty of Agro-Industrial Technology, Rajamangala University of Technology Isan, Kalasin Campus, Thailand *Corresponding email: khakhanang_r@yahoo.com
ABSTRACT This study aimed to screen and identify of Lactic acid bacteria which are capable of inhibiting the growth of Staphylococcus aureus, a common dairy cows Mastitis pathogen. A total 250 bacterial strains were isolated from 60 raw milk samples in Kalasin province. The differences in antimicrobial activity against S. aureus among these isolates were found when performing the agar-disc diffusion technique. Five isolates, showing the dominant inhibitory efficiency to the indicator strain were chosen for biochemical test. The results exhibited that all five isolates were gram positive, catalases-negative microorganisms and identified as Lactobacillus spp. by using 16s rDNA sequencing. The inhibitory efficiency was described as percentage of inhibition ratio. Their ratios of inhibitory activity were in the range of 25.3231.37ď&#x20AC;Ľ, compared with commercial antimicrobial drug. Bacterial growth, pH change and stability in high temperature condition as well as protease treatment were also studied. It clearly showed that productions of antimicrobial activity by these five isolates in MRS medium followed a growth-associated pattern. The pH change from as about 5.6 to as about 4.2 during 24 hours led the inhibitory activity to the highest and then remained gradually stable. High temperature and protease treatment of lactic acid culture indicated that heating and protease had no effect on their antimicrobial activity. On the other hand, pH adjustment in lactic acid culture to 7.0 exhibited no anti-S. aureus activity, implying that the acidic condition, produced by lactic acid bacteria is one of the mechanism for inhibiting the growth of dairy cow-Mastitis pathogen. Keywords: Antimicrobial activity, Cow-Mastitis pathogens, Lactic acid bacteria Introduction Mastitis is one of the endemic diseases that commonly bring problems to dairy farms. It is usually coursed by bacteria infection to udder tissue, leading to the inflammatory responds. One of the common bacteria, causing dairy-cows Mastitis is Staphylococcus aureus (Bradley, 2002). Milk from dairy cows suffering from Mastitis is undoubtedly in a low quality because of high amount of white blood cells, released from mammary gland against bacteria invasion. This disease is considered to be the most expensive disease worldwide in dairy industries. In the US, Mastitis has made economic loss about 2,000 billion US dollars in the last 15 years (Gruet et al., 2001). Similar situations have been reported in Thailand (Ajariyakhajorn et al., 2003). To treat the Mastitis in dairy cows, using antibiotic drug is the most popular method. Although, using antibiotics does not consume a lot of time and labors, it can create hidden health problems to consumers. Lactic acid bacteria are Generally Recognized As Safe (GRAS) Organisms. They have been used for various food fermentations such as dairy, fish, meat and vegetable for centuries (Chagnaud et al., 2001). They do not only contribute to improve flavor, texture and prolong food-shelf life, but they also own ability to produce antimicrobial substances such as organic July 1-3, 2015
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acids, hydrogen peroxide, ethanol, diacetyl and bacteriocin which play important roles in balance microfloral and lowering the indecent of bacterial infections (Sung-Mee et al., 2009 and El-Shefei et al., 2000). The objectives of this study were to screen and identify of Lactic acid bacteria which are capable of inhibiting the growth of Staphylococcus aureus. The knowledge gained from this study would beneficially provide the alternative means for prevention or treatment of Mastitis in dairy cows. Materials and Methods Isolation of Lactic Acid Bacteria All raw milk samples were diluted with MRS and 0.1 mL aliquot was spread-plated onto MRS agar, containing 1 CaCO3. Colonies forming a clear zone in the MRS agar were selected and stocked in 30 glycerol at -20C. Antibacterial activity of Lactic acid bacteria Antibacterial activity was tested against S. aureus TISTR 746 by agar-disc diffusion technique. Briefly, cell-free culture of bacteria suspension was prepared by centrifugation and filtration through 0.45 m pore-size filter. The aliquot of 10 l of cell-free culture was dropped on sterile blank discs with 6 mm diameter, which were individually placed on NA agar plates, covered with 100 µL of the indicator strain. The plates were incubated at 37ºC for 24 hours. Cell-free cultures showing a clear zone were chosen for the study of the nature of inhibiting agents. The determination of antimicrobial property was done in triplicate and evaluated by measuring diameter of inhibition zone of each cell-free culture against the indicator strains in millimeter (mm). The inhibitory efficiency was reported as the percentage of Inhibitory Ratio (IR), shown in equation (1). The “Da”, “Db” and “Dc” were the diameter of inhibition zone of cell-free culture, oxytetracycline (positive control, 50g/disc) and paper disc against indicator strain, respectively
Study of the nature of inhibiting agents Sensitivity of cell-free culture to pH change Selected cell-free cultures were normalized to pH 7.0 by 1 M NaOH and subsequently tested for the remaining inhibitory activity. Sensitivity of cell-free culture to proteolytic enzyme Selected cell-free cultures were incubated with proteinase K at the final concentration of 1 mg/ml for 12 hour. The proteinase K-treated cultures were heated to kill proteolytic enzyme and tested for the remaining inhibitory activity. Sensitivity of cell-free culture to high temperature Selected cell-free cultures were incubated at 50 and 100 C for 10 min. After cooling down, the high temperature-treated cultures were tested for the remaining inhibitory activity. Identification of lactic acid bacteria Bacteria isolated with dominant antibacterial activity were subject to identification by using the sequence of 16s rDNA. Results and Discussion Sixty raw milk samples, collected from Kalasin province area were screened for lactic acid bacteria. It was found that the total bacteria count from samples ranges from 107-108 CFU/ml. After screening by media selection, there were 250 bacteria isolates, showing a clear zone on
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MRS agar with 1 CaCO3. All 250 isolates were selected for antimicrobial activity test against S. aureus. The amount of isolates found from raw cow milk samples was different from that found from raw milk of goat (59 isolates) and camel (352 isolates) (Saidi et al., 2011 and Jans et al., 2012). It might be as a result of differences in environment condition such as climate, season and temperature. Apart from the environment factors, type and age of sample also had the impact on amount and diversity of lactic acid bacteria (Kwantrairat et al., 2009). After screening for the inhibition activity of cell-free cultures, it was found that each cell-free culture possessed different degree of anti-S. aureus property. Five cell-free cultures, showing dominant inhibitory efficiency to indicator strain, were chosen for further tests. Five isolates with dominant inhibitory efficiency, including A27, A29, A39, B29 and C35, were gram positive and catalase-negative bacteria. Selected cell-free cultures were tested for their anti-S. aureus property under different condition in order to identify type of anti-S. aureus agent, produced from each strain. The anti-S. aureus efficiency was presented in the percentage of Inhibitory Ratio (IR), which compared to the anti-S. aureus activity of each cell-free culture to oxytetracycline; the commercial antibiotic drug (50 g/disc). It was found that no any cell-free culture exhibited the anti-S. aureus activity better than commercial drug. Their IR ranged from 25.3231.37. The IR of cell-free cultures was in about the same range among normal condition, proteinase K-treated condition and high temperature-treated condition. On the other hand, the IR of cell-free cultures dramatically dropped to 0 when adjusting pH of these cell-free cultures to 7.0. This information indicated that protease and heating had no effect on the antimicrobial activity, but pH naturalization destroyed their ability of anti-microorganisms (Table 1). Following the antimicrobial activity of these 5 isolates further indicated that the production of anti-S. aureus property followed the growth-associated patterns. It was found that pH fell from as about 5.6 to as about 4.2 within 24 hour, which led the IR to the highest (data not shown). This data implied that the antimicrobial property of these selected 5 isolates might come from the acidic condition, produced by lactic acid bacteria. Similar result had been reported from lactic acid bacteria, isolated from gastrointestinal tract of Nile tilapia (Oreochromis niloticus) (Kwantrairat et al., 2009). Though, some isolated lactic acid bacteria had been reported to produce another antimicrobial agents, apart from acidic condition, such as bacitracin (Saidi et al., 2011; Petsuriyawong and Khunnajark, 2010; Khunnajark et al., 2008; Todorov et al., 2006). Sequence of 16s rDNA of these 5 isolated indicated that 4 out of 5 isolates were Lactobacillus rhamnosus and another was Lactobacillus paracasei subsp. tolerans (data not shown). Table 1 Percentage of inhibitory ratios against S. aureus.
Strain A27 A29 A39 B29 C35 H2O Oxytetracycline
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normal 26.10 25.32 28.37 31.37 28.90 0.00 100.00
pH adjustment to 7.0 0.00 0.00 0.00 0.00 0.00 0.00 -
Condition Proteinase K treatment 25.03 26.30 26.27 31.71 30.31 0.00 -
High temperature treatment (50C) (100C) 22.10 23.30 26.84 27.77 27.77 24.90 29.17 28.70 28.50 29.77 0.00 0.00 -
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Conclusion The lactic acid bacteria, isolated from raw milk samples exhibited different anti-S. aureus activity. The IR against S. aureus was in the range of 25.32-31.37. Their anti-S. aureus abilities come from the acidic condition, produced by lactic acid bacteria. Acknowledgments This research was financially supported by Higher Education Research Promotion (HERP), Office of the Higher Education Commission. References Ajariyakhajorn K., Samngamnim S., Boonserm T., Inchaisri C., Thirapatsakun T. and R.J. Farnsworth. 2003. Mastitis in small dairy holders of Nakhonpathom provinces, Thailand, pp.122-123. The 11th International Symposium of the World Association of Veterinary Laboratory Diagnosticians and OIE Seminar on Biotechnology, November 9-13, 2003. Bradley A.J. 2002. Bovine mastitis: an evolving disease. Vet. J. 164: 116-128. Chagnaud P., K. Machinis, L.A. Coutte, A. Marecat and A. Mervenier. 2001. Rapid PVRbased procedure to identify lactic acid bacteria: application to six common Lactobacillus species. J. Microbiol. Methods 44: 139-148. El-Shafei H.A., H. A .Ei-Sabour, N. Ibrahim and Y. Mostafa. 2000. Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food. Microbiol. Res. 154: 321-331. Gruet P., P. Maincent, X. Berthelot and V. Kaltsatos. 2001. Bovine mastitis and intermammary drug delivery: review and perspectives. Adv. Drug Delivery Rev. 50: 245-259. Jans C., Bugnard J., Njage P.M.K., Lacroix and L. Meile. 2012. Lactic acid bacteria diversity of African raw and fermented camel milk products reveals a highly competitive, potentially health-threatening predominant. LWT-Food Sci. Technol. 47:371-379. Khunajakr N., A. Wongwicharn, D. Moonmanagmee and S. Tantipaiboonvut. 2008. Screening and identification of lactic acid bacteria producing antimicrobial compounds from pig gastrointestinal tracts. KMITL Sci. Tech. 8: 8-16. Kwantrairat S., W. Yossiri and S. Siri. 2009. Isolation of lactic acid bacteria producing organic acids and possessing Anti- Aeromonas hydrophila activity from gastrointestinal tract of Nile tolapia (Oreochromis niloticus). KKU Res. J. 14: 811-821. Petsuriyawong B. and N. Khunajak. 2010. Screening of lactic acid bacteria isolated from piglet faces for antimicrobial activity. KKU. Res. J. 15: 446-48. Saidi N., M. Hadadji and B. Guessas. 2011. Screening of bacteriocin-producing lactic acid bacteria isolated from west Algerian goat’s milk. Global Journal of Biotechnology Biochemistry, 6: 154-161. Sung-Mee L. and D. S. Im. 2009. Screening and characterization of probiotic lactic acid bacteria isolasted from Korean fermented foods. J. Microbiol. Biotechnol. 19: 178186. Todorov S.D. and L.M.T. Dicks. 2006. Screening for bacteriocin-producing lactic acid bacteria from boza, a traditional cereal beverage from Bulgaria comparison of the bacteriocins. Process Biochem. 41: 11-19.
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P-55
Comparison of Stomoxys calcitrans (Diptera: Muscidae) Attacked and Behavioral Responses on Genetic Blood Differences of Holstein-Friesian Dairy Cattle in Thailand Ubon TANGKAWANIT1*, Varangrat SENASING1, Wirote KHLIBSUWAN1, and Tassanee JAMJANYA1 1
Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand *Corresponding email: ubonta@kku.ac.th
ABSTRACT Fourteen dairy cows in each genetic blood levels of 75-87.5%, >87.5-93.8% and >93.8-100% Holstein-Friesian were examined the number of stable fly attacked and the behavioural responses during November 2009 to October 2010. Stable flies attacked were studied by counting flies from the front leg within 2 min. Then, the following four behaviours of tail flick, skin twitch, leg stamp and head throw were observed in each genetic blood level within 3 min. The results showed statistically significant for the number of fly attacked. The genetic blood level of >93.8-100% Holstein-Friesian dairy cow was less attacked than the others. For the behavioural responses, 75-87.5% Holstein-Friesian showed the highest frequencies of behavioural responses. The correlation between fly number and fly repelling behaviours is high, except head throw (r2=0.12). The number of stable fly attacked dairy cattle was highest on July 2010 and lowest on March 2010. The information of population dynamic of stable fly and behavioural responses of different blood level of Holstein-Friesian cross breed to stable fly are useful for the farmer in order to selecting suitable percentage of genetic blood level of crossbred Holstein-Friesian dairy cattle and awareness the disease and the fly attack in stable fly season. Keywords: Stable flies, Holstein-Friesian, Behaviour, Dairy cow Introduction Stable flies are haematophagous insects which belong to family Muscidae. In Thailand, six species were recorded. However, Stomoxys calcitrans is the most abundant (Muenworn et al., 2010). Both male and female of stable fly is blood feeder on animals such as poultry and livestock. For cattle, the economic losses in term of weight gain and milk production were reported (Campbell et al., 2001). An average of milk production loss was 0.7% per stable fly per cow (Bruce and Decker 1958). Additionally, behavioural responses of cattle to biting fly such as bunching leg can increase heat stress which indirectly reduced in cattle weight gain (Wieman et al., 1992). Stable fly abundance can monitor by counting on animal. Lysyk (1995) suggested the number of stable flies on front legs is about 45% of stable flies on a whole body. Furthermore, the frequency of fly repellent behaviors such as head throw, leg stamp, tail flick, and skin twitch are used for determining stable fly abundance (Mullen et al., 2006; Gerry et al., 2007). Mullen et al. (2006) estimated ten or more tail flicks per min is equivalent to a count of five stable flies per leg. In Thailand, Holstein-Friesian (HF) cross-breeds are widely bred in dairy farms in order to develop and produce cross-breed dairy cows suitable for the Thai environment, and produce greater dairy products such as raw milk. At present, the HF bloods at 75% up to 100% are distributed in the dairy farms in Thailand. Kaewkamchan et al. (2002) reported high July 1-3, 2015
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percentage of HFcross-breeds provided higher milk yield than the lower percentage. While, milk components such as protein and fat are decrease. Thailand's climate is tropical which is high both of temperature and humidity. This climate results in insect abundant and disease. The dairy cow which tolerate with this condition is the most suitable for dairy farm in Thailand. The possible mechanisms which cause fly attacked are the alteration of fly attraction and repellency to cow. Present study was assessed the possibility by counting the number of stable fly attacked and the frequency of fly-repelling behavior among genetics level of crossbred HF dairy cows. Materials and methods Crossbred HFdairy cows were divided into three genetic levels; 75-87.5%, >87.5-93.8%, and >93.8-100%. Forty- two adults female (14 cows of each levels) with four to eight year old were studied from six farms in Ban-Kho district, Khonkaen province. Each farm has the similar environment and management (such as food, medicine providing). Individual cows were monitored the number of stable fly attacked and behavioral responses once a month during November 2009 to October 2010 from 2.00 to 4.00 pm before milking. The numbers of stable flies on individual dairy cows were counted within 2 min below the elbow on the outside of one front leg and the inside of the other front leg (Mullens et al., 2006). The space between observer and cow is 2-3 m in order to avoid the cow’s stress. After stable fly were counted, monitoring of head throw, leg stamp, tail flick, and skin twitch for fly repelling were determined by two counters within 3 min. The monitoring of fly repelling behaviour was followed the procedures of Mullen et al. (2006) and Garry et al. (2007). Tail flicks were counted when the tail’s tip pass over cow’s body and was a musclepowered motion. Leg stamps were observed if the foot of any leg was raise from the ground. Head throws were recorded when the cow toss the head backward and the nose cross the cow’s chest. One skin twitch was counted when skin continuously move less than 2 s, twice skin twitch was counted, if the movement is continuous between 2-4 s. Data in the months that have high population of stable fly (June to September) were analyzed by ANOVA using PROC GLM (SAS Institute, 2001) to determine significance. Results from significant ANOVAs were followed by Duncan’s Multiple Rang Test (P=0.05). Correlations between fly number and behaviours were analyzed for July and August which were most fly abundant by regression using PROC reg (SAS Institute, 2001). Results and Discussion The population of stable fly is highest in July (6.29 flies per 2 min) and lowest in March (0.21 flies per 2 min) (Figure 1). Mean of stable flies in the months that have high stable fly population are shown in Table 1. Mean of stable flies attacked in the genetic blood level of >93.8-100% dairy cow (2.63 flies per leg per 2 min), was significantly lower than means of the genetic blood levels of 75-87.5%, and > 87.5-93.8% (ANOVA, P < 0.05) (Table 1).
Figure 1 Relationship between mean of stable flies attacked Holstein-Friesian dairy cows for the genetic level of 75-87.5%, >87.5-93.8%, and > 93.8-100% during November 2009 to October 2010. Page 366
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Table 1 Comparison for the average of stable fly number on front leg and frequencies of stable fly defensive behaviours; head throw, leg stamp, tail flick and skin twitch of Holstein-Friesian (HF) dairy cow in the percentage of >93.8-100%, >87.5-93.8%, and 75-87.5%. Percentage range of HF dairy cow >93.8-100% >87.5-93.8% 75-87.5%
Stable flies Head throw Leg stamp Tail flick Skin twitch per leg (mean±SEM) (mean±SEM) (mean±SEM) (mean±SEM) (mean±SEM)a 2.63±0.48a 0.80± 0.09a 1.82± 0.19a 2.63± 0.37b 1.93 ±0.35b 4.25±0.17b 1.07 ±0.08b 2.30± 0.14b 3.69± 0.19b 2.75 ±0.18b 5.66 ±0.65a 0.68±0.03ab 1.84± 0.25a 5.73± 0.41a 5.02 ±0.36a a Within each column, values followed by the same letter indicate no significantly different (P>0.05) . The frequencies of four behaviours; head throw, leg stamp, tail flick, and skin twitch, are shown in Table 1. The linear regression (r2) between the number of stable fly and head throw, leg stamp, tail flick and skin twitch during June to September were 0.126, 0.871, 0.863 and 0.863, respectively (P = 0.05), and August were 0.0576, 0.0634, 0.5848 and 0.4989, respectively. The correlations between stable fly and each behaviours are high, except correlations between stable fly and head throw (r2 = 0.126). Visual cues (such as size, contrast, colour, and pattern), and airborne chemical cues of endothermic hosts are important factors for host-seeking haematophagous insects finding host (Mooring et al., 2007). In our study, HF dairy cows in each genetic blood level are not different in size, and in pattern of skin color. However, the lowest of host attacked for the genetic level of >93.8-100% may involve with the less attractant of chemical cues than the other levels. The number of stable fly on front leg of dairy cow is significantly highest in July which is the rainy season in Thailand, whereas there is significantly lowest on March in dry season (Figure 1). This corresponds to the report of stable fly abundance in Nakhonpathom Province, Thailand (Masmeatathip et al., 2006). In addition, Taylor et al. (2007) suggested that reduction of the precipitation levels explained the observed midsummer drop of the stable fly populations in Eastern Nebraska. Therefore, rainy season, (especially in July), should be watched for stable fly outbreak in order to protect the cow from biting. The percentages of HF blood strain seem to be a factor in frequency of certain fly defensive behavior. It was clearest in the percentage of >87.5-93.8% and >93.8-100% for tail flick and skin twitch behavior which related to the number of stable flies attacked. The frequency of insect repelling behavior is correspondence to the number of stable fly which is similar to the study of Mullen et al. (2006) except head throw. The less significant of r2 value may result in the experimental methods. Stable flies were counted only in the front leg which may not the representative for stable fly attacked in the whole body. Additionally, the observation of fly repelling behaviour was delayed until fly counting was done. Making accurate counts is difficult, if fly repelling behaviours were counted at the same time. Gerry et al. (2007) suggested cows may flick a tail because of the irritation. Most of stable fly feed on lower body and leg, so that tail flick is not very effective for fly defence. Correlation of fly number and bite rate depend on the location. Dougherty et al. (1993) found that stable fly on head of beef cattle were positively correlated with bite rate where as stable flies on the hind legs were negatively correlated. Conclusion The measurements of this study recorded only fly number and four repelling behaviours that established on the host. However, the fate of the mechanism in host preference was unknown. Dairy cow which is annoy when stable flies even small amount and they express high
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frequency of the behaviors may be affected their health. A cow may express those behaviors for repelling horse fly and horn fly. However, there were only house flies, stable flies and a few mosquitoes attacked on a cow during the study. Acknowledgements This work was financially supported by Khon Kaen University. We thank Assoc.Prof. Virote Pattarajinda and Assoc.Prof. Monchai Duangjinda, for their valuable advice of the research. We extend special thanks to the owner of six cooperate private farms; Sanfun farm, Khem Thong farm, Peanthong farm, Surapong farm, Suwat farm and Muangchai farm. References Bruce W.N. and G.C. Decker. 1958. The relationship of stable fly abundance to milk production in dairy cattle. J. Econ. Entomol. 52: 269â&#x20AC;&#x201C;274 Campbell J.B., S.R.Skoda, D.R.Berkebile, D.J.Boxler, G.D. Thomas, D.C. Adams, and R. Davis. 2001. Effects of stable flies (Diptera: Muscidae) on weight gains of grazing yearling cattle. J. Econ. Entomol. 94: 780-783 Dougherty C.T., F.W. Knapp, P.B. Burrus, D.C. Willis, J.G. Burg, P.L. Cornelius, and N.W. Bradley. 1993. Stable flies (Stomoxys calcitrans L.) and the behaviour of grazing beef cattle. Appl Anim Behav Sci. 35: 215 Gerry A.C., N.G. Peterson, and B.A. Mullens. 2007. Predicting and controlling stable flies on California dairies. ANR Publication 8258. http://anrcatalog.ucdavis.edu. Accessed 23 Aug 2010. Kaewkamchan S., P. Vijchulata, P. Chairatanayuth, S. Sintuvanich, V. Santisopasri, and S. Surapat. 2002. Influence of feeding management and seasons on yield and composition of milk produced from Friesian crossbred cows raised under hot and humid environment in central Thailand. Kasetsart J. (Nat Sci) 36: 392-398. Lysyk T.J. 1995. Temperature and population density effects on feeding activity of Stomoxys calcitrans (Diptera: Muscidae) on cattle. J. Med. Entomol. 32: 508â&#x20AC;&#x201C;514 Masmeatathip R., J. Gilles, C. Ketavan, and G. Duvallet. 2006. First survey of seasonal abundance and daily activity of Stomoxys spp. (Diptera: Muscidae) in Kamphaengsaen campus, Nakornpathom province, Thailand. Parasite 13: 245-250 Muenworn V., G. Duvallet, K. Thainchum, S. Tuntakom, S. Tanasilchayakul, A. Prabaripai, P. Akratanakul, S. Sukonthabhirom, and T. Chareonviriyaphap. 2010. Geographic Distribution of Stomoxyine Flies (Diptera: Muscidae) and Diurnal Activity of Stomoxys calcitrans in Thailand. J. Med. Entomol. 47: 791-797 Mullens B.A., K.S. Lii, Y. Mao, J.A. Meyer, N.G. Peterson and C.E. Szijj. 2006. Behavioural responses of dairy cattle to the stable fly Stomoxys calcitrans, in an open field environment. Med. Vet. Entomol. 20:122-137 Mooring M.S., D.T. Blumstein, and D.D. Reisig. 2007. Insect-repelling behavior in bovids: role of mass, tail length, and group size. Biol. J. Linn Soc. 91:383-392 SAS Institute. 2001. PROC GLM, version 5.1.2600 ed. SAS Institute, Cary, NC Taylor D.B, D.R. Berkebile, and P.J. Scholl. 2007. Stable fly population dynamics in eastern Nebraska in relation to climatic variables. J. Med. Entomol. 44:765-71 Wieman G.A., J.B. Campbell, J.A. Deshazer, and I.L. Berry. 1992. Effects of stable flies (Diptera: Muscidae) and heat stress on weight gain and feed efficiency of feeder cattle. J. Econ. Entomol. 85:1835-42
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Opinion Toward Agricultural Further Study of Student in Nongchok District, Bangkok Metropolis Tippawan LIMUNGGURA 1* and Panya MANKEB1 Department of Agricultural Development and Resource Management, Faculty of Agricultural Technolog, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand * Corresponding email: kltippaw@kmitl.ac.th
ABSTRACT The purposes of this study were to explore the opinion toward agricultural further study of student and factors relating to the student opinion in Nongchok district, Bangkok Metropolis. Data were collected by questionnaires from 358 sixth grade secondary school students in 3 school; namely, Nongchok Pittayanusorn Matthayom, Bordindaecha (Sing Singhasaenee)4 and Matthayom Wat Nongchok. Data were analyzed by descriptive statistics, t-test and F-test. The findings revealed that the opinion toward agricultural further study of student had medium level (Grand mean 3.28) and the important factors relating to their opinions were school (p≤ .01), religion and family occupation (p≤ .05). Keywords: Opinion toward agricultural further study, Student, Nongchok district, Bangkok Metropolis Introduction The agricultural sector is fundamentally important to the economy of Thailand and has been featured in all national social and economic development plans. However, the population in agriculture is declining steadily. In 1990, the population in the agricultural sector were 63.4 percent but it was 41.1 percent in the year 2011. In addition, the new generation young farmer, age 15-24 years in the last two decades is likely to reduce almost three times from 35.3 percent in the year 1987 to only 12.1 percent in the year 2011. (National Statistical Office, 2013). Including, the admission exam announcement of Thailand showed that children pay attention to further study in the agricultural sector decreasing. A lot of them have no willing choose to study in agriculture. Such a situation should be taken to concern the population in agricultural sector in the future, therefore, this research was manipulated to provide the fundamental data used in the planning of the agricultural sector of Thailand in the future. Nongchok district, the largest area of Bangkok, located on the northeastern side, plenty of agricultural area and near King Mongkut’s Institute of Technology Ladkrabang was selected for this research. The purposes of this study were to explore the opinion toward agricultural further study of student and factors relating to the student opinion in Nongchok district, Bangkok Metropolis. Materials and Methods Data were collected by questionnaires from 358 sixth grade secondary school students in 3 school; namely, Nongchok Pittayanusorn Matthayom, Bordindaecha (Sing Singhasaenee) 4 and Matthayom Wat Nongchok. Sample size was computed by Yamane formula (Yamane, 1973) (Table 1). Validity and reliability were examined by alpha coefficient of 0.82 (Nunnaly, 1998). Data were analyzed by descriptive statistics, t-test and F-test.
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Table 1 Population and sample of school in Nongchok district. No. 1 2 3
School Nongchok Pittayanusorn Matthayom Bordindaecha (Sing Singhasaenee)4 Matthayom Wat Nongchok Total
Population 181 190 150 521
Sample 124 127 107 358
Results and Discussion Overview of the students The research found that most students (72.35 %) were female with the average age of 17.54 years, about half (51.40 %) were Buddhists and the inferior (47.49%) were Islam. Grade point average (GPA) of student was 3.09. The most family occupation was worker (47.76%), followed by trader 18.99 % and farmers 11.74%, respectively. The results revealed that about half (57.26%) have not been trained in agriculture, however, about half (58.10 %) have ever practiced the tasks related to agriculture and about two-thirds of students (68.40 %) have ever studied in agricultural subject. The results showed that a minimum number of students (2.23 %) were interested in farmer career in the future (Table 2). Table 2 Overview of the students. Characteristics
Number
(n=358) Percent
Sex Male Female Age(year) 16 17 18 19 mean 17.54, max 19.00, min 16.00, SD. 0.55 Religion Buddhist Islam Christ GPA < 2.50 2.50-2.74 2.75-2.99 3.00-3.24 > 3.25 mean 3.09, max 3.99, min 2.00, SD.0.43 Family occupation Farmer Government officer State enterprise Private company Trader Worker
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99 259
27.65 72.35
4 160 189 5
1.11 44.70 52.80 1.40
184 170 4
51.40 47.49 1.11
37 45 63 77 136
10.30 12.60 17.60 21.51 37.99
42 36 14 27 68 171
11.74 10.06 3.91 7.54 18.99 47.76
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Agricultural training Train No train Agricultural practice Practice No practice Agricultural subject learning Learn No learning Future career Farmer Government officer Company Private sector Others
205 153
57.26 42.74
150 208
41.90 58,10
113 245
31.60 68.40
8 81 16 31 222
2.23 22.63 4.47 8.66 62.01
The opinion toward agricultural further study of student The findings revealed that the opinion toward agricultural further study of student had medium level (Grand mean 3.28) and the important factors relating to their opinions were school (pâ&#x2030;¤ .01), religion and family occupation (pâ&#x2030;¤ .05) (Table 3). Table 3 Factors relating to the student opinion Factors Sex
Male Female Factors
School Nongchok Pittayanusorn Matthayom Bordindaecha (Sing Singhasaenee)4 Matthayom Wat Nongchok Religion Buddhist Islam Christ Family occupation Farmer Government officer State enterprise Private company Trader Worker Other
Mean 3.30 3.27 Mean 3.33 3.14 3.40 3.24 3.34 2.72 3.47 3.19 3.17 3.10 3.29 3.29 3.17
Student opinion S.D. t/F Sig 0.50 0.487 .626 0.46 Student opinion S.D. t/F Sig 0.04 10.272 .000** 0.04 0.04 0.47 4.173 .016* 0.47 0.70 0.46 2.203 .042* 0.41 0.51 0.62 0.43 0.47 0.46
Conclusion Most of the students were female, mean age 17.54 years, about half were Buddhist. Secondary were Islam GPA of 3.09, family occupations were workers, about half of the students have not been trained in agriculture, but about half have performed tasks related to agriculture, the two-thirds of students have ever studied in agriculture subject. There were a few students who were interested in a career as a farmer in the future. The opinion toward
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agricultural further study of student had medium level (Grand mean 3.28) and the important factors relating to their opinions were school (p≤ .01), religion and family occupation (p≤ .05). Acknowledgments The researcher would like to thank Nongchok Pittayanusorn Matthayom, Bordindaecha (Sing Singhasaenee) 4 and Matthayom Wat Nongchok school for gathering data. We also thank the Department of Agricultural Development and Resource Management, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang for financial support. References National Statistical Office.2013. Considering the Level of Workers in Thailand. [Online]. Available: www.agricdas.org. [19/5/2015]. Nunnaly, J. 1978. Psychometric Theory. Mc Graw-Hill: New York. Yamane, T. 1973. Statistics: An Introductory Analysis. Harper : Tokyo.
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Using Augmented Reality (AR) Technology for the Promotion of Agricultural Products: A Case Study of Bang Krathum Processed Banana Product Nutthakorn SONGKRAM1* 1
Department of Agricultural Development and Resource Management, Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand *Corresponding email: Nutthakorns@gmail.com
ABSTRACT The purpose of this research was to study the application of augmented reality (AR) technology to design poster and packaging labels of Bang Krathum processed banana products. The methods used in this research were as follow: 1) To interview the five experts for their opinion about the use of augmented reality technology for the promotion of agricultural products. 2) To develop poster and packaging labels of Bang Krathum processed banana products. 3) To evaluate poster and packaging labels by five experts and improve them based on their recommendations. 4) To Survey the poster and packaging labels sampling with a group of 50 people and assess their satisfaction. The data were analyzed by using frequency, percentage, mean and standard deviation. The results of the study revealed that the samples were satisfied at the highest level (mean 4.64, SD 0.49). In addition, they also thought that using AR technology: 1) could make the products trustworthy 2) enhanced agricultural products standard in Thailand 3) easy to use. 4) provided information of products clearly 5) built brand identity 6) stimulated the customers to purchase. Keywords: Augmented reality (AR), Promotion of agricultural products, Poster and label Design Introduction Augmented reality, also known as AR, is an information technology used world-wide with potential of showing the combination of reality and virtual in a multimedia form of picture, three dimension, movies, video and audio, including the hyperlink which created new and interesting way to be used in the marketing. Moreover, the augmented reality technology can be applied in in many fields such as engineering, architecture, medical and especially in education field where it keep the student interested and self-helped because it is easy to understand. Azuma (1997) defined AR as systems that have the following three characteristics: 1) Combines real and virtual 2) Interactive in real time 3) Registered in 3D. The main purpose of the AR is to merge the reality with the virtual through the devices that have AR software installed. Such devices can scan the AR-Code as marker / image including location of the target and display additional information on screen. In general, AR usages have two ways: 1) Mobile augmented reality using a smart phone or tablet with camera and iOS, Android and other OS. 2) Desktop augmented reality using a computer with a webcam and Windows and other OS. The research in 2013 found that AR application users were about 60 million people and will expand to 200 million more in 2018 (Jupiter Research, 2013). Using AR technology is an important tool in the future that enables entrepreneurs build applications to serve customers, building brand image without much investment. Itâ&#x20AC;&#x2122;s only provider and service recipient must have the equipment or tools that are available for the virtual world (Tansiri, 2010). However, AR technology is new to Thailand and only used by large organizations for public relations and July 1-3, 2015
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promotions. The small businesses do not use this technology eventhough it is not complicated and there are a lot more AR software or apps available to be applied in business promotion to easily reach the consummers. With this reason, the researcher is interested in applying the AR technology to promote the sales of agricultural product in a case study of Bang Krathum processed banana product. The product was a dried bananas and buttered bananas from Phitsanulok province which marketed by poster and packaging labels under the brand “Unstoppable Banana” to make it interesting. The product was surveyed for customer satisfaction using AR technology. The results will lead the way for AR to be used in promoting agricultural product. Materials and Methods One poster and four labels of Bang Krathum processed banana products The components of the AR Technology used in poster and labels are: 1) Marker or image (ARCode) that printed on the poster or label of the products. 2) Mobile Devices with camera to capture the marker or image, “Layar” app to analyze the AR-Code and deliver the information, internet access to pull the products information and screen to display the information. When the poster and labels are scanned by smart phone or tablet that has a “Layar” app installed, it will show AR information on screen (Table 1). Table 1 Type, descriptions and AR information of poster and labels. Type Poster Labels
Descriptions Poster size A3 to educate the consumers about the banana product and where to buy it Dried banana label (8.7x 8 cm.) shows the product name and logo, product highlights, manufacturers and address Dried banana nutrition label (7.6 x 7.2 cm.) shows nutritional components, manufacturing date, expiry date and weight. Butter banana label (9.9 x 8.3 cm) shows the product name and logo, product highlights, manufacturers and address Dried banana nutrition label (7.4 x 7.3 cm.) shows nutritional components, manufacturing date, expiry date and weight.
AR Information Video to introduce the product and where to buy it Picture and video to introduce the product and where to buy it Hyperlink to the website for nutrition Picture and video to introduce the product and where to buy it Hyperlink to the website for nutrition
The instrument used for data collection. - Interview for the experts to study the application of AR technology for the promotion of agricultural products. A structured interview includes an explanation about the interview, related definitions and questions about the application of AR technology for the promotion of agricultural products. - Interview for the experts to evaluate the poster and labels. It has explanation about the interview and questions about quality of the poster and labels. - Questionnaire to measure the sample customer satisfaction level in the form of 5-level Likert scale from 1 (lowest) to 5 (highest) and open-ended questions. The research methodology: - Collect and study information about the products from the actual producers. Collect and study information about the poster and label, including about the AR technology.
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- Interview 5 experts in the field of poster and label design to apply the AR technology to design the poster and packaging labels of Bang Krathum processed banana products. - Decide the layout; collect the descriptions and graphic materials for the design. - Used Adobe Photoshop and Illustrator software to design the poster and labels. Used Layar creator (www.layar.com) to create the AR-Code and deliver the product information. - Print the poster and labels on the packaging of Bang Krathum processed banana. The 5 experts review and give comments. Analyze the comments, compare the pros and cons, the modify the poster and labels where at least three experts agree on the same comments. - Use the poster and labels on the Bang Krathum processed banana products and sell the products at the agriculture fair from Feb 9th to 13th 2015 at the faculty of agricultural technology, KMITL. Perform survey with at least 50 samples with accidental sampling method. The surveyors will explain about the study and let the samples check out the product’s poster and labels while noticing their behavior. The surveyor will then give the samples the questionnaire form to collect data and comments. - Analyze the survey results to find the statistic, means and standard deviation with Criteria as follows. Average score: 4.51–5.00 = Highest satisfaction, 3.51–4.50 = High satisfaction, 2.51–3.50 = Average satisfaction, 1.51–2.50 = Low satisfaction, 0.00–1.50 = Lowest satisfaction Results and Discussion The data collected from documentation and the 5 experts found that the use of AR technology to promote Bang Krathum processed banana products should be made in the form of mobile augmented reality which lead to the design of the poster and labels that include logo, picture, nutritional information, manufacture address and include hyperlink to the product website that show picture and video, the manufacturing processes and benefit of the products usage. The results of the 5 experts evaluation found that the poster and labels have enough and clear information about the products and recommended to use a larger character font for easier reading and include the information about the AR usage. The results of the survey from 50 samples showed consumer satisfaction rating in the highest level with average means score of 4.64 with 0.49 standard deviation. The results also showed that the AR technology made the products trustworthy and enhances the agricultural products standard. It helps in delivering the product’s information easier and cleared, at the same time, creates a unique brand for the products. (Table 2) Table 2 Assessment of the customer’s satisfaction (N = 50) Messurement Quality of character font and size 4.60 Quality of the graphics 4.58 Trust worthyness of the brand 4.82 Uniqueness of the brand 4.60 Easy to understand information from the AR 4.68 Easy to use AR 4.72 AR helps delivering clear information 4.60 Interesting of the products 4.50 Price value of the products 4.52 Enhances agricultural products standard 4.78 Total Means 4.64
S.D 0.49 0.50 0.39 0.53 0.47 0.45 0.53 0.58 0.58 0.42 0.49
Level of satisfaction Highest Highest Highest Highest Highest Highest Highest High Highest Highest Highest
The research results showed that the AR stimulates can enhance the customer’s interest of agricultural products and give a better perception of the products. Consistent with Azuma (1997) who said augmented reality enhances a user's perception of and interaction with the
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real world. The virtual objects display information that the user cannot directly detect with his own senses. The information conveyed by the virtual objects helps a user perform realworld tasks. The AR technology for the promotion of agricultural products can be used by mobile augmented reality and desktop augmented reality. The first type is to promote further public relation or advertisement that does not fit in the poster or small label. The customers only need to install the AR app on their mobile devices. The second type is to promote in an exhibition, campaign or brand image building activity including the dissemination of knowledge. However, considering the conditions for the promotion of Bang Krathum processed banana products. Using AR technology for mobile augmented reality is more convenient and better value. It focuses on providing more information of the product at the point of sale, which are widely available. Rather than organizing promotional events only one point. It also saves cost and personnel. Consistent with Liu et al. (2013) that said, compared to the desktop computer, the mobile devices have small memory, limited computing capability, easy to carry features, while the servers have powerful computing and storage capacity. Using client/server system architecture can make full use of advantages of both hardware environments, to allocate tasks reasonably, and it can effectively reduce the system overhead. The problems found that sometime it difficult to use AR when customers have low quality camera on the devices or the amount of light is too dark to capture the marker or image. Consistent with Jullapho and Utakrit (2014) who applied AR technique to present information of real estate house plans and found the same problem with the quality of the camera and light conditions. In addition, another important problem is the low speed access to the internet that makes the display AR information is slow or cannot show. Even though the problems may be on the customer’s side, it may cause negative perception or satisfaction. Therefore, the design of the AR and the information contents should consider this limitation on the internet speed and size of download information. Conclusion The AR technology is a new method that can promote and bring interest to Thai agricultural products. It can be a marketing strategy tool for entrepreneurs or small farmers to make the products trustworthy build products brand identity and enhances Thai agricultural products standard. References Encyclopedia Britannica. 2010. Augmented reality. Available online at: http://www.britannica.com/EBchecked/topic/1196641/augmented-reality Azuma, Ronald T. 1997. A survey of augmented reality. Presence: Teleoperators and Virtual Environments 6(4):355–385, August. Jullapho S. and Utakrit N. 2014. “Applied AR Technique to Present Information of Real Estate House Plans”. The Tenth National Conference on Computing and Information Technology (NCCIT2014): 619-624. Jupiter Research. 2013. Mobile augmented reality users to approach 200 Million Globally by 2018”, 2013. Available online at: http://www.juniperresearch.com/viewpress release.php?pr=410 Liu M, X. Li., X. Lei .and S. Wu 2013. Research of mobile augmented reality technology applied in agriculture”. International Conference on Advanced Computer Science and Electronics Information (ICACSEI 2013): 311-314. Tansiri, P. 2010. Augmented reality. Executive Journal 30(2): 169-75.
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P-58
Factors Affecting Consumer Purchasing Behavior of Toxic-Free Vegetables in Muang District, Samutprakarn Province Panya MANKEB1*, Pratompong MATONG1, Suneeporn SUWANMANEEPONG1 and Prapaporn CHULILUNG2 1
Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520,Thailand 2 Department of Skill Development, Ministry of Labour, Bangkok 10400, Thailand *Corresponding email: kmpanya@kmitl.ac.th
ABSTRACT Interest in toxic-free vegetables has grown substantially as the consumers react to the media about food safety. The purposes of this study were to investigate consumer purchasing behavior and factors affecting consumer purchasing behavior of toxic-free vegetables in Muang district, Samutprakarn province. Questionnaires were used to collect data from 405 respondents. Data analysis was conducted by descriptive statistics such as frequency, percentage, arithmetic mean and standard deviation. Hypothesis tests were applied using ttest, F-test and multiple regression analysis. The results showed that the majority of respondents were female, 45-54 years old, college-educated, married, employed in private business, and personal income greater than 40,000 THBper month. The rationale to buy toxic-free vegetables was health concern. Respondents perceived most information from television media. The average purchase volume of toxic-free vegetables was 0.86 kg. per week and the amount of money spent per each purchase was 107.22 THB. The significant factors (p<.05) affectingpurchasing behavior of toxic-free vegetables were; age, marital status, monthly income, product and marketing promotion. Keyword: Consumers purchasing behavior, Toxic-free vegetables Introduction The critical effect on human health and the environment become important concerns for many people. Chemical residues in fruits and vegetables have caused widespread anxiety among consumers who concern about the quality of food they consume (Chouichom and Yamao, 2010). Meanwhile, Thai consumers' concern with food safety has been increasing during the last decades, especially in urban areas. This is due to the fact that food safety scandals still remain a prominent issue in domestic markets, for instance a scandal related to chemical residues on some fresh produce e.g., Chinese Kale and chili (Wongprawmas et al., 2014). In order to meet consumers’ demand and to increase the level of food safety assurance provided by the market, the Thai government tried to strengthen the regulation in the domestic market and to introduce a voluntary standard for enhanced food safety assurance procedures and the related food safety label in the market (Lippe et al., 2010). In 2004, Thailand declared the year 2004 as “Food-Safety Year” to enhance the awareness of producers and consumers on safe food (Rattanasuteerakul and Thapa, 2012). The marketing mix is one of factors influences the purchasing of consumers. According to Kotler and Amstrong (2010), the marketing mix is a set of marketing tools used by companies to achieve their marketing objectives. The marketing mix is incorporated into a modern marketing system, called the four Ps of marketing: product, price, place and promotion.In previous research, it was stated that marketing mix factors such as product, price, place and promotion have significant impact to purchasing decisions (Azzadina et al., July 1-3, 2015
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2012). Therefore, the purposes of this study were to investigate consumer purchasing behavior and factors affecting consumer purchasing behavior of toxic-free vegetables in Muang district, Samutprakarn province.The results of the study will be proposed to policy makers with useful information on food safety and guiding future management and marketing strategies for the Thai fresh produce industry. Materials and Methods The study was conducted in Muang district, Samutprakarn province, Thailand. Questionnaires were used to collect data from 405 respondents who experienced with purchasing toxic-free vegetables in three department stores which located in Muang district, Samutprakarn province, Thailand.The respondents were asked their level of importance of the marketing mix factors influents on their decision-making to purchase toxic-free vegetables.The sample size was computed by Cochran (1997). Testing of the reliability was conducted by internal consistency using Cronbanchâ&#x20AC;&#x2122;s alpha coefficient with the values of 0.77 (Cronbach, 1951). Data were analysed by descriptive statistics such as frequency, percentage, arithmetic mean and standard deviation. Hypothesis tests were applied by using ttest, F-test and multiple regression analysis (MRA). Results and Discussions Demographic characteristic of respondents The majority of respondents were female (60%), had an average age of 45-54 years old(27.7%). Most of them completed bachelor degrees (61.0%), married (71.6%), employed in private business (29.6%) and had personal income greater than 40,000 Thai Baht(THB) permonth (22.2%).This may be explained by the fact that the middle-class consumers tend to consume toxic-free vegetables. Purchasing behavior of toxic-free vegetables The main rationale for buying toxic-free vegetables of respondents was a health concern (42.2%). More than half of them received toxic-free vegetables information from television media (51.9%). The average purchase volume of toxic-free vegetables was 0.86 kilograms per week and the amount of money spent per each purchase was 107.22 THB. Most of them usually preferred buying toxic-free vegetables to ordinary vegetable (67.9%) and had a trust in band that certified toxic-free vegetables at the high-level (42.0%). Importance of marketing mix on consumer purchasing behavior of toxic-free vegetables The result indicated that the overall of marketing mix factors affecting purchasing behavior toxic-free vegetables were at high level (xË&#x2030; =4.06). The marketing mix product was the highest, followed by price, place and the promotion with the average scores at 4.26, 4.19, 4.16 and 3.61, respectively (Table 1). Factors affecting consumer purchasing behavior of toxic-free vegetables Demographic characteristic factors The results showed that three factors affected statistically significant consumer purchasing behavior including: age, marital status and monthly income (Table 2). The results of this study were consistent with the research of Anathawat (2004) discovered that age and marital status were also associated statistically significant with purchasing behavior of vegetable and fruit of respondents and similar to the study of Niti-apaitham (2004) reported that income significantly associated with consumption behavior of healthy food of the respondents. The marketing mix factors The results of multiple regression analysis (MRA) revealed that the marketing mix factors affecting consumer purchasing behavior of toxic-free vegetables were statistically significant
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including: product, price and promotion. The description of the impact of marketing mix factors towards purchasing decision in term of frequency, quantity and cost of purchasing was shown in the Table 3, Table 4 and Table 5, respectively. Above all, the result is consistent with the study of Salgado-Beltrán et.al. (2012) found that product and distribution channel had a significant influence on organic food purchase. This finding also similar to the study of Sookchan (2001) discovered that distribution channels and marketing promotion were affected significantly decision purchasing of toxic-free vegetable. Moreover, the finding supports the concept that price factor is the most important factors in order to make purchasing decision (Azzadina et.al., 2012). Table 1 Repondents’ level of importance of each marketing mix factors on consumer purchasing behavior of toxic-free vegetables xˉ 4.26 4.19 4.16 3.61 4.06
Items 1. Product 2. Price 3. Place 4. Promotion Total
S.D. 0.74 0.71 0.81 0.81 0.77
Level high high high high high
Rank 1 2 3 4
Table 2 Demographic characteristic factors affecting consumer purchasing behavior. Purchasing behavior
Items t or F 1.39 5.76 0.44 2.32 1.49 2.70
Sex Age Education Marital Status Career Monthly income
Frequency p-value 0.17 0.00** 0.82 0.09 0.19 0.00**
t or F 1.29 2.69 1.73 1.19 0.73 2.78
Quantity p-value 0.89 0.02* 0.13 0.02* 0.602 0.00**
t or F 0.03 2.38 1.45 5.89 1.07 1.88
Cost p-value 0.98 0.04* 0.21 0.00** 0.37 0.06
Note: *significance at level 0.05, **significance at 0.01
Table 3
Marketing mix factors affecting consumer purchasing behavior towards the frequency of purchasing. Attribute
Constant Product Price Place Promotion
B 2.645 -.278 .062 -.072 .059
multiple R = 0.158 multiple R2 = 0.025 SEest = 0.916
Std. Error .464 .121 .089 .088 .089 F Sig F Durbin Watson
Beta -.146 .040 -.050 .035
t 6.986 -2.953 .693 -.819 .658
= = =
2.548 0.039 1.991
Sig. .000** .022* .488 .413 .511
Note: *significance at level .05, **significance at level .01
Table 4 Marketing mix factors affecting consumer purchasing behavior towards the quantity of purchasing. Attribute Constant Product Price Place Promotion multiple R = 0.237 multiple R2= 0.056 SEest = 0.538
B .792 -.145 -.021 -.022 .240
Std. Error .273 .071 .052 .052 .052 F Sig F Durbin Watson
Beta -.127 -.022 -.025 .239
t 2.904 -2.047 -.399 -.415 4.579
Sig. .004** .041* .690 .678 .000**
= 5.965 = 0.000 = 1.770
Note: *significance at level .05, **significance at level .01
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Table 5
Marketing mix factors affecting consumer purchasing behavior towards the cost of purchasing. Attribute
Constant Product Price Place Promotion multiple R = 0.248 multiple R2= 0.062 SEest = 58.856
B 102.165 -13.071 -12.268 3.268 27.008
Std. Error 29.822 7.744 5.714 5.683 5.721 F Sig F Durbin Watson
Beta -.105 -.118 .034 .246
t 3.426 -1.688 -2.113 .575 4.721
Sig. .001** .092 .035* .566 .000**
= 6.573 = 0.000 = 1.744
Note: *significance at level .05, **significance at level .01
Conclusion The finding of the study indicated that the main rationale for buying toxic-free vegetables of respondents was a health concern. The significant factors (p<.05) affecting purchasing behavior of toxic-free vegetables were: age, marital status, monthly income, product, price, and promotion. By understanding this relationship, the policy makers as well as entrepreneurs in Thai fresh produce industry can be predicted from: the customers’ behavior patterns, customers’ preferences, and formulate effective marketing strategy to enhance the awareness of producers and consumers on safe food. Acknowledgments The authors would like to gratefully acknowledge the Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand for financial support. References Ananthawat, N. 2004. Fruit and vegetable purchasing behavior of the personnel in Kasetsart University. Master Thesis, Kasetsart University. Azzadinaa, I., A.N., Hudab, CorinthiasPamatang Morgana Sianipar. Understanding Relationship between Personality Types, Marketing-mix Factors, and Purchasing Decisions. Procedia Social and Behavioral Sciences 65:352–357. Chouichom, S. and M. Yamao. 2010. Consumers’ general perception and acceptance of organic food products in the Urban Thai Marketplace. Proceedings 16th AAS and 1st ISAT, 25-27 August 2010, Bangkok, Thailand. pp.243-246. Cochran, W. G. 1997. Sampling Techniques. Third Edition, New York: John Wiley & Sons. Cronbach, L. J. 1951. Coefficient alpha and the internal structure of tests. Psychmertrika, 16(3):297-334. Kotler, P. and G.M. Armstrong. 2010. Principle of marketing. 13nd (Global) Edition. Pearson Education, Inc., Boston. Lippe, S.R., S. Isvilanonda, H. Seebens and M. Qaim. 2010. Food Demand Elasticities among Urban Households in Thailand. Thammasat Economics Journal 28(2):1-29. Niti-apaitham, K. 2004. Factors associate with consumption behavior of healthy foodsof the students. Master Thesis, Kasetsart University. Rattanasuteerakul, K. and G.B. Thapa. 2012. Status and financial performance of organic vegetable farming in northeast Thailand. Land Use Policy 29:456-463. Salgado-Beltrán, L., J. E., Espejel-Blanco and L. F. Beltrán-Morales. 2013. Marketing Mix Influencing Organic Foods Purchase of Mexican Consumers. China-USA Business Review 2(6):618-626. Sookchan, B. 2001. Perception and demand on toxic-free vegetable of people. Master Thesis, Maejo University. Wongprawmas, R., M. Canavari1 and C. Waisarayutt. 2014. Are Thai consumers willing to pay for food safety labels? choice experiment on fresh produce. The paper presentation at the EAAE 2014 Congress ‘Agri-Food and Rural Innovations for Healthier Societies’ August 26 to 29, 2014, Ljubljana, Slovenia.
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P-59
Financial Cost and Benefit Analysis of Commercial Orchid Farming in Krathumban District, Samutsakorn Province, Thailand Panya MANKEB1*, Chalida LERTKRASEMPON1, Tippawan LIMUNGGURA1 and Prapaporn CHULILUNG2 1
Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok 10520, Thailand 2 Department of Skill Development, Ministry of Labour, Bangkok, 10400, Thailand *Corresponding email: kmpanya@kmitl.ac.th
ABSTRACT The main objective of this study was to assess the financial return of orchid farming in Kratumban district, Samutsakorn province, Thailand. The results showed that the total cost of orchid investment in 10 rai (1.6 ha.) for 4 years was 1,897,096.84 Thai Baht (THB), while total benefits generated by the investment was 2,339,291.67 THB and net profit was 442,194.83 THB. The financial analysis showed that the net present value (NPV) was 3,925,028.51 THB. The benefit-cost ratio (BCR) was estimated to be 1.65. The internal rate of return (IRR) on investment was found high at 52.56 percent. These results of financial analysis indicated that the orchid farming investment was financially worth and profitable because 3 basic of financial measures were acceptable; NPV of the investment was positive, BCR was greater than 1 and the IRR was greater than the opportunity cost of investment (7 percent). Keyword: Orchid, financial cost and benefit analysis
Introduction Thailand is the worldâ&#x20AC;&#x2122;s major producer of orchids for international trade. The orchid is an important flower for Thailand economiy. The total orchid export value in Thailand is over 2,727 million Thai Baht (THB) (orchid cut-flowers and orchid plants).The export of orchid cut-flowers still predominates than orchid plants; the value was 2,305 million THB and 422 million THB, respectively (Orchid net, 2012). The major importing countries for orchid cutflowers from Thailand have been Japan followed by U.S.A., Chinese and Italy, whereas Japan, Singapore, Philippines, U.S.A. and South Korea were the major orchid plants importing countries (OAE. 2014) The estimated total area for orchid cultivation in 2010 was about 3,645 hectares (OAE, 2014). Most of the orchid growing areas are located in the Central Plain, mainly in Nakompathom, Samutsakorn, Bangkok, Ratchaburi and Nonthaburi where climatic conditions, water, transportation and marketing system are the most favorable. Many farmers have resorted to orchid growing as their main occupation, being a better earner than other crops. Krathumban district, Samutsakorn province is the main orchid cultivation area of the province. In 2010, there were 412 orchid growers, which the cultivation areas covered 2,824 rais (Krathumban District Agricultural Extension Office, 2014). Therefore, this research aimed to study the financial cost and benefit analysis of commercial orchid farming in Krathumban district, Samutsakorn province, Thailand, as well as the problems associated with orchid production. The results of the study will benefit both the farmers who are making investment decision on orchid production and the related government agencies which provide advices on the promotion and problem solving related to orchid farmers.
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Materials and Methods The study was conducted at Krathumban district, Samutsakorn province, Thailand. The structured interview was used to collect data from three commercial orchid farmers who cultivated orchid more than 10 rai (1.6 ha.) mainly for exporting selected purposively from the study area. Data were analyzed using descriptive statistics and financial analysis to determine profitability of orchids. Three techniques i.e. Net Present Value (NPV), Benefit Cost Ratio (BCR) and Internal Rate of Return (IRR) were used as discussed by Gupta and George (1974); Vaidya et al. (1991) and Bakhsh et al. (2006). It can be computed as follows: The NPV is the present value of the net return obtained from orchid production throughout the period of 4 years. If the NPV > 0 which means the orchid production is a worthwhile investment. It can be calculated as follows:
where: Bt = benefits in each year Ct = costs in each year n = number of years r = interest rate (7%) The BCR is the ratio of the present value of return relative to the present value of investment on orchid production. If BCR > 1, then the total revenue is greater than total cost, if BCR = 1, then the total revenue is equal to the total cost and if BCR < 1, then the revenue is less than the total cost (Bakhsh et al., 2006). It can be calculated as follows:
where: Bt = benefits in each year Ct = costs in each year
n = number of years r = interest rate (7%) The IRR is a rate of return of the project expressed in percentage. In other words, it is a rate of interest in the discounted calculation process which makes the net present value of the project equal to 0. When the value of r is set equal to IRR, the value of r can be calculated as follows:
Apart from the basic analyses using the afore-mentioned criteria, this research has conducted a sensitivity analysis so as to determine in the case that if the return and investment vary, what impact those factors will be on the decision making and how. The study has also analyzed the switching value test so as to find out that at what increased percentage of the investment cost or what decreased percentage of the return that the project will not be a worthwhile investment. Results and Discussion Investment cost and return in cut-flower orchid production Cut-flower orchid production of farmers in Krathumban district, Samutsakorn Province covered an area of 10rais (1.6 ha.). During the cultivation years 2011/2012, the investment Page 382
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cost was 1,897,096.84THB. The return from cut-flower orchid trading during the cultivation years (1.6 ha.) was 2,339,291.67 THB. The net benefit was 442,194.83THB. Financial project analysis on cut-flower orchid investment The size of the orchid farm used in the analysis was 10 rais (1.6 ha). The investment period starting from the planting process to the clearing of the cultivation farm for the next planting process was 4 years. The present value benefit (PVB) was 9,987,923.10THB. The present value cost (PVC) was 6,062,894.59THB. Therefore, the NPV was 3,925,028.51 baht. The BCR was 1.65 and the IRR was 0.53. Since the NPV was positive, the BCR > 1, IRR was larger than the loan interest rate (7%), the cut-flower orchid investment was a worthwhile undertaking (Table 1). Above all the results are consistent with the study of U-bolyam (2003); Kosonmethakul (2004) and Teekhaphun (2004) found that orchid cutting flower was appropriate and worthwhile investment. Table 1 Investment cost, benefit and net present benefit in cut-flower orchid investment by farmers on a 10-rai farm. Unit: baht Year 0 1 2 3 4 Total
Benefit 0.00 1,293,500.00 2,437,400.00 2,909,866.65 5,603,400.05 12,244,166.70
Investment cost 1,411,167.00 2,032,938.74 1,121,972.25 1,121,972.25 1,121,972.25 6,810,022.49
PVB 0.00 1,208,878.50 2,128,919.56 2,375,317.97 4,274,807.07 9,987,923.10
PVC 1,411,167.00 1,899,942.75 979,974.02 915,863.57 855,947.26 6,062,894.59
NPV -1,411,167.00 -691,064.24 1,148,945.54 1,459,454.40 3,418,859.81 3,925,028.51 9,987,923.10 6,062,894.59 3,925,028.51 1.65 5.30
PVB PVC NPV at7% interest loan rate BCR at 7% interest loan rate IRR(%)
Sensitivity Analysis The sensitivity analysis revealed that the 10% increase in investment cost was financially feasible and the farmers would get maximum benefit. The NPV was 3,318,739.05 THB. The BCR was 1.50. The IRR was 42.9 % (Table 2). Table 2 Sensitivity analysis on cut-flower orchid investment by farmers. Items Before the change 10% increase in investment cost 10% decrease in return 10% increase in investment cost and decrease in return
NPV (THB) 3,925,028.51 3,318,739.05 2,926,236.20 2,319,946.74
BCR 1.65 1.50 1.48 1.35
IRR (%) 52.56 42.88 41.90 32.81
Switching Value Test (SVT) The switching value test revealed that the investment cost could be increased by 64.74% of the total investment cost and the return could be decreased by 39.30% of the total return. That would bring the NPV to 0, the BCR = 1 and the IRR = the reduced calculation rate or loan interest rate (7%). This could be concluded that the risk on cut-flower orchid investment was low and more related to return than investment cost.
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Problems and difficulties in cut-flower orchid production The finding revealed that the main problems and difficulties associated with cut-flower orchid production by farmers were high initial investment cost, plant-related diseases and insects, coconut coir, expensive fertilizers and chemicals, discontinued productions and inconsistent prices. During the summer, the production is low while the price is high, whereas during the rainy season, the production rises and the price drops. This fact creates price imbalances and the gaps are large. In addition, water pollution was also a problem due to the fact that the farms were located in industrial areas. Conclusion The finding of the study indicated that the total cost of orchid investment in 10 rai (1.6 ha.) for 4 years was 1,897,096.84 THB, while total benefits generated by the investment was 2,339,291.67 THB and net profit was 442,194.83 THB. The financial analysis showed that the NPV was 3,925,028.51 THB. The BCR was estimated to be 1.65. The IRR on investment was found high at 52.56%. These results of financial analysis indicated that the orchid farming investment was financially worth and profitable. Moreover, a sensitivity analysis of investment was done to assess a financial risk by switch value test (SVT) found that orchid farming investment has low risk level while risk of benefit was greater than risk of cost. Acknowledgments The authors would like to gratefully acknowledge the Faculty of Agricultural Technology, King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang, Bangkok, Thailand for financial support and MAJ GEN Dr. Thikamporn Chulilung for valuable comments and suggestion. References Bakhsh, K, I. Hassan and M.S. Akhter. 2006. Profitability and cost in growing mango orchids. J.Agri. Soc.Sci. 2(1):46-50. Gupta, G.S. and P.S. George. 1974. Profitability of Nagpur Sangtra cultivation. Indian J. Agri. Econ. 29:150-60. Krathumban District Agricultural Extension Office. 2014. Agricultural statistics in Krathumban district, Samutsakorn province in 2009 [Online]. Available: http://krathumbaen.samutsakhon.doae.go.th/home/homepage.html [2012 Mar 5]. Kosonmethakul, N. 2004. A financial cost and benefit analysis of orchid farming investment in Sam Phran district, Nakhon Pathom province. Master of Science Thesis. Kasetsart University. Office of Agricultural Economics (OAE). 2012. The situation and trends of important agricultural year 2012. Office of Agricultural Economics. [Online]. Available: http://www.oae.go.th/bapp /main.php? filename=outlook 2011 [2012 Mar 5]. OAE. 2014. A comparison of orchid production between Good Agricultural Practice (GAP) and convention method. Bangkok: OAE. Orchid net. 2012. Value, quantity and price of orchid for export in 2008-2010. [Online]. Available: http://www.orchidnet.doae.go.th. [2014 June 15]. Teekhaphun, R. 2004. The financial analysis of orchid farming investment in Changwat Ratchaburi. Master of Science Thesis. Kasetsart University. Vaidya, M.k, S.C. Tewari and K.K. Raina. 1991. An economic analysis of apple plantation in shimla district of Himachal Pardesh, J.Agric. Devel. Policy 2:9-31. U-bolyam, S. 2003. A financial analysis of orchid farming investment in Changwat Phra Nakhon Si Ayutthaya. Master of Science Thesis Kasetsart University.
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P-60
Enhance Environment of a Historic Site, Ancient Benchama Maharat Building, For Future Public Use Warong NAIVINIT 1*, Parkpoom SUEBNUKARN1, Rukkeit SANPRASERT1 and Taweesak WIYACHAI 1 1
Faculty of Agriculture, Ubon Ratchathani University, Thailand *Corresponding email: wnaivinit@yahoo.com
ABSTRACT For a century, the Benchama Maharat School has undertaken to educate the youth of Ubon Ratchathani and other nearby provinces. In 1935, Benchama Maharat School student roll reached its limit. To accommodate the growing number of students, the school was moved to a new site. A new school building was constructed on the west side of Tung Sri Meuang field. It is a two-story Manila architectural style building and completely built of hard wood and teak on a cement base. Its design is influenced by the Dutch and Thai architecture. Because of the uniqueness of the ancient Benchama Maharat building, the entire block was registered by the Fine Arts Department as a national historic site. Not only is the history and identity of the ancient building marvelous, but also the location of this site has high potential for public use as a large green space to improve metropolitan quality of life. However, without strategic plan, this site has been being misused. The objective of this design project was to enhance the environment of this site to be a new provincial landmark. The concept is to integrate the contemporary landscape design principles into colonial garden design. Our design solution would at best be an effective public notification to stimulate discussion among prospective users. This master plan will be a design guideline to help people visualize and imagine the future look and feel of this public space. Subsequently, collective agreements among people of Ubon Ratchathani to develop this site would be generated through the use of this design in participatory process. Keywords: Benchama Maharat, Collective agreements, Green space, Historic site, Metropolis environment Introduction For a century, the Benchama Maharat School has undertaken to educate the youth of the province of Ubon Ratchathani and other nearby provinces. The Benchama Maharat School is an important institution that has a long history through the changes of the Kingdom. This school was founded in the reign of King RAMA V in 1897 and was first located inside the monastic boundary of Wat Supatwanaram and named Ubonwitaya School. Due to the increasing number of students, the school was moved to a new area twice i.e. in 1915 and 1970 and renamed to Benchama Maharat School. In 1935, a new school building, which was the second building of the school, was constructed on the west side of Tung Sri Meuang field. The construction cost of this new academic building was around 40,000 baht, which was considered a huge investment at that time. It is a two-story Manila architectural style1 building that has its serviceable space of 2,376 square
1
Manila-style building is composed of the front gambrel roof. This building style is believed that it is influenced by Dutch architecture and built during the reign of the King RAMA V. The Manila-style building is often made of front colonnade hallways. The prominent features of the Manila-style building are
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meters. It is built completely of hard wood and teak on a cement base. There are three facades in the front of the building with gambrel roof representing the Dutch colonial style. The building was designed by Phra Saroj Ratthanawiman, an architect of Ministry of Education. The total area of the new school was 6.4 ha, which was a relatively very large school. (Benchama Maharat Alumni, 2008). Measured against the key characteristics of all colonial architecture to always blend the colonial powers with the colonial countries, this new Benchama Maharat building is unexceptional. Its design is believed to be influenced by the Dutch and Thai architecture even though Thailand has never been colonized. But the style of Thai landscape architecture in the early 1900s was unclear. Thus, the landscape design and planning of the Benchama Maharat School had no concept. Because the former Benchama Maharat building was unique and remains in good condition, the entire block including the building and its surroundings was registered by the Fine Arts Department as a national historic site on September 30, 2002. On December 27, 2002, the Committee and the provincial Governor approved the strategic plan to develop this significant site to be the provincial center for ethnic and cultural knowledge. (Association of the Ubon City, 2011). Consequently, the Committee requested the Fine Arts Department to restore the building. Based on architectural conservation principles, the restoration of this building was officially launched in 2007 and completed in 2010. Multiple agencies have been working on the development of the provincial center for ethnic and cultural knowledge e.g. Ubon Ratchathani University, Rajabhat University (Ubon Ratchathani), Fine Arts Department 11, Benchama Maharat Alumni, Ubon Ratchathani Municipality, the Office of Public Works and Planning. As a result of collective agreement, the management of this historic site was handed over from the Fine Arts Department to Ubon Ratchathani University. After the restoration was complete, the Benchama Maharat building became more and more beautiful. Due to the political events on May 19, 2010, the Ubon Ratchathani City Hall was burned and demolished, and the view of the building became more open to show its architectural magnificence to the public. Later in 2011, this building won the 2011 prize as the Outstanding Conservation Architecture in the Architect â&#x20AC;&#x2122;54 Fair organized by the Association of Siamese Architects. This building has sealed its place as the pride of Ubon Ratchathani and has joined the list of national treasures. Furthermore, the location of the building is in the heart of the city providing convenient travel and the access to nearby public park, Tung Sri Meuang. This site is ideal for creating a tourist attraction, a knowledge center and large green space for metropolitan. Unfortunately, the current environment of the building downgrades the elegance of the entire historic site, for instance the large parking lot in front and lack of greenery surrounding. It causes a negative impact to attracting people to visit the building. In the light of these criticized points, the objective of this research and design was to produce a master plan of landscape architecture for this ancient site. Materials and methods First, we had visited the site to carry out the field measurement of the building and its perimeter on April 9, 2011. A draft of the layout plan was created in the Computer Aided Design (CAD) program. To double-check the accuracy of the draft, we conducted a Geographic Positioning System (GPS) operation on May 30, 2011. Subsequently, the CAD and Geographic Information System (GIS) programs were used to revise the base plan. Then, research team performed the site inventory and site analysis. 1. Gables without elegant stencil fin roof 2. Arched windows with ventilation canopy 3. Graceful round-shaped pillar for the porch or veranda
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The Benchama Maharat ancient site project involves different stakeholders since it is now a national treasure for which people have the right to make development proposals. Due to the limited time to complete this project, we decided to organize a focus group on August 1, 2011. The purpose was to brainstorm and get a picture of how the future of the site would be. We prepared a semi-structured guideline and distributed it to our team so that we all understood what information we would like to acquire. The participants were selected to cover diverse professions and experiences, and they all are considered influential clients. From this point, once the problems have been clarified through the research, and site analyzed and users canvassed, a detailed program can be made out (Lynch et al., 1994). Bottom line of this design project, we used conventional landscape design process in more participatory way to ensure that the design solution was generated from multi-actors involving in the use of this proposed site. Many formal and informal meetings were organized to publicize this project and collect information to improve the design. Results and discussion Based on our analysis, the site has high potential to be developed for the community as a new public park and for the province as a new landmark. For this design project, the general philosophical concept is formalized based on the unique colonial architectural style and the interpretation of userâ&#x20AC;&#x2122;s demands that the design should provide memorable experiences to visitors. We brainstormed and decided to integrate the contemporary landscape design principles into colonial garden design. This design concept, the colonial contemporary landscape design, was used as our framework. The design also encourages scenic dynamics throughout the year by applying multi-seasonal plant species. To comply with the colonial garden, a formal style was dominated the front of the building. To support dynamic function, two large dynamic displayed representing the Thai lotus painting in the front featuring ornamental plant materials or placing festival sculptures such as lent candles. A replica of colonial building as a meeting place was built on the North. Handicap access was available on the north wing on back of the building by passing through the new wooden terrace. However, due to the limited time and financial support, an important point that has been inadequately taken into account for this project was to involve uninformed or voiceless people into the design process. This may lead to conflicts among people who do not have a chance to be part of the development. Furthermore, this master plan is, of course, incomplete, if one would like to use it for actual construction. In such a case, more drawings and written documents are definitely required, for instance, construction drawings and written specifications of the meeting place including its elevations and sections. Some critical areas of the project e.g. site entrance, arrival court, fountains, and decks, must be studied and designed on a detailed scale (Booth, 1983). Design of utilities i.e. irrigation, electricity, and drainage including grading plan could be needed. Conclusion The Dutch colonial building style of the Benchama Maharat ancient building is unique and beautiful. Moreover, the building is in perfect condition after its restoration. Without question, it should be conserved for the next generation. The surrounding area is, on the other hand, in very poor condition. The rules and authority to manage the site are not clear. As a result, site planning and design is essential to protect this building from misuse and to encourage people to visit this ancient site. The design of contemporary colonial landscape should enhance the usersâ&#x20AC;&#x2122; experience while walking through the landscape of this national treasure site, and to serve the metropolitan as a public green space for outdoor activities. As stated by Lynch, we as the designer have the responsibility to clarify the given objectives and July 1-3, 2015
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to raise hidden ones for debate (Lynch et al., 1994). Our design solution of the Benchama Maharat ancient site would at best be an effective public notification to stimulate discussion among prospective users. We hope that this project will be a design guideline to help people visualize and imagine the future look and feel of this significant provincial area. Our ultimate wish is to see that the site is properly developed as a result of collective agreements among people of Ubon Ratchathani. Acknowledgements We wish to express our profound gratitude and honest appreciation to all participants e.g. Benchama Maharat Ancient Building Development Committee, teachers and alumni of the Benchama Maharat School for their excellent cooperation throughout this design process. Acknowledgement is also given to the financial support for this research project granted by Ubon Ratchathani University. References Association of the Ubon city. 2011. Home Day 2011. Association of the Ubon city. Ubon Ratchathani. (in Thai). Benchama Maharat Alumni. 2008. To the beauty of weeping fig: 111 anniversary of Benchama Maharat School. Benchama Maharat Alumni. Ubon Ratchathani. (in Thai) Booth, N.K. 1983. Basic elements of landscape architectural design. Illinois: Waveland Press Inc. Favretti, R., and De Wolf Jr, G.P. 1972. Colonial gardens. Barre Publishers. Halper, J. Contemporary Landscape Architecture [Online]. 2009. Available from http://www.articlesbase.com/gardening-articles/contemporary-landscape-architecture904074.html [6 September 2011]. Lynch, K., and Hack, G. 1994. Site planning. 3rd. Maple-Vail, Inc. Motloch, J.L. 2001. Introduction to landscape design. 2. Austin, Texas: John Wiley & Sons Inc. Schmidt, R. 2009. Contemporary landscape architecture - Parks and open space, Duties and responsibilities. Digital landscape architecture: A GIS-based approach for modeling the spatial and temporal development. 21 May, 2009. Valletta. The Consumer Horticulture Center. 2003. The horticulture fact sheet series: Creating a colonial garden [Online]. Available from College of Agricultural Sciences, Penn State University www.hortweb.cas.psu.edu. [16 August 2011]. Turner, S.L. 1988. Historic landscapes. In: Tishler, W.H. (ed.) American landscape architecture: Designers and places, p. 142-144. Washington, D.C.: Wiley. Wikipedia contributors. American colonial architecture [Online]. 2011. Available from Wikipedia, The Free Encyclopedia: http://en.wikipedia.org/w/index.php?title= American_colonial architecture&oldid=464311818 [7 December].
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P-61
Biodiesel Synthesis from Used Frying Oil with the Modified Catalyst from Waste Cockle Shells Sasiwimol WOOTTHIKANOKKHAN*, Buppha THAMNIYOM and Supamas CHUANOI Department of Chemistry, Rajamangala University of Technology Krungthep Sathorn, Bangkok, Thailand * Corresponding email: sasiwimol.w@rmutk.ac.th ABSTRACT
Biodiesel is an alternative energy resource obtained from plant. Recently, development of low cost manufacturing process is remarkably crucial in order to solve the production cost. In this research, biodiesel synthesis from used frying palm oil with waste Anadara granosa shells as the modified catalyst was investigated. Preparation of catalyst started from calcinations of waste A. granosa shells at 1,000ď&#x201A;°C for 2 h. following by impregnation with ammonium carbonate solution and heating again at 600ď&#x201A;°C for 2 h. The catalyst was then, tested for its trans-esterification catalytic activity by varying the volume of catalyst ranging from 0.5 to 2% w/w, molar ratio of methanol to oil at 9:1 and 12:1, and reaction time between 1 and 5 h. The results showed that the catalyst derived from waste A. granosa shells and impregnated with ammonium carbonate solution, exhibited high activity in biodiesel synthesis, where the optimum condition was found with corresponding to the molar ratio of methanol to oil at 9:1 for 1 h. upon 1% w/w of the catalyst yielding the highest methyl ester of more than 90% w/w. A. granosa shell is, then, a valuable source for the production of heterogeneous base catalyst that can be successfully utilized for the synthesis of methyl ester biodiesel. Keywords: Biodiesel, Methyl ester, Anadara granosa shells, Heterogeneous catalyst Introduction Biodiesel is an alternative energy from biomass source. This alternative energy is also environment-friendly as it is low exhaust emission, and biodegradable and nontoxic. The most popular biodiesel synthesis approach is transesterification, which uses acidic or basic catalysts for catalyzing the reaction. In general, the basic catalyst exhibits higher catalytic activity. Presently, many catalyst types have been developed for using in biodiesel production either homogeneous or heterogeneous catalysts. Compared with homogeneous catalyst, heterogeneous catalyst receives a lot of concerns. Major advantages are easily separating of catalyst from reaction and eliminating a washing step, where this step consumes large amounts of water (Boey et al., 2009). Among all catalysts used, CaO is the most popular one (Isahak, et al., 2010). Since it has high basic and non-corrosive properties, and is highly active in transesterification reaction. This catalyst is cheap because it could be prepared from any natural resources containing CaCO3, Ca(NO3)2, and Ca(OH)2. Several researches have been reported the preparation of CaO catalysts from waste materials, especially various shells for catalytic activity in biodiesel production. Essentially, improving of catalyst sensitivity is needed in order to avoid excess catalyst amount in the reaction. Huaping (2006) employed ammonium carbonate to enhance catalyst activity. The results showed that CaO impregnated with ammonium carbonate can react more efficiently in biodiesel production than the one without impregnation .
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The aim of this research was to determine utilization and efficiency of waste shells as a catalyst in biodiesel synthesis. Cockle (Anadara granosa) shell is of interest because the shellfish are widely consumed in Thailand leading in a large number of waste materials. Moreover, their shells contain high calcium carbonate contents, at more than 90 wt.%, which can be used to prepare CaO catalyst for transesterification reaction. In the study, the cockle shells have been doped with ammonium carbonate, and then tested for its catalytic activity in methylester production from used frying oil. The use of waste materials for production of biodiesel is not only reducing the cost, but also solving the problem of a large volume of wastes. Materials and Methods Raw materials and chemicals In this study, the used frying oil (FFA content < 1 wt.%) was employed as raw materials for biodiesel synthesis. The oil, which viscous and dark brown color, was sieved through cheesecloth, and stored in dried and clean container at room temperature. The cockle shells were obtained from a local market. All chemicals were of analytical grade. Catalyst preparation For CaO preparation, the cockle shells were cleaned and air dried. The shells were then, milled and sieved through a 45-63 µm to achieve consistency of particle sizes with appropriate surface area for the reaction. The mashed shell was then, calcined at 1000 C for 2 h. prior to perform XRD analysis. Impregnation of CaO was carried out by mixing 1.5 g of calcined cockle shell CaO with 1.25 M ammonium carbonate until saturated, and allowing to stand for 1 h before drying at 100C. The CaO was then, calcined again at 600 C for 2 h. Transesterification reaction The transesterification was performed using different catalyst concentration, molar ratio of methanol to oil and reaction time. All experiments were carried out in a reflux system. The reaction was started by mixing a desired amount of catalyst and methanol in a three-necked flask. The reaction was heated while stirring (700 rpm). When the temperature reached ± 70 2 C, 30 g of used frying oil was added and allowed the reaction continuing until reaching the reaction time. The mixture was kept in a separatory funnel overnight to allow complete separation between catalyst, methyl ester, and glycerol layers. Quantification of the methyl ester was determined by EN 14103 standard method. Results and discussion Characterization of catalyst Figure 1 shows the results of X-ray diffraction (XRD) patterns from cockle shell samples after calcination confirming that the transformation of CaCO3 in cockle shells into CaO was completed. The obtained CaO was further preceded by doping with ammonium carbonate following with calcinating again at 600C for 2 h. After calcination, the ammonium carbonate impregnated on CaO was decomposed into oxide, which subsequently enhanced the basic property of the catalyst. Effects of catalyst amount Figure 2 demonstrates methyl ester contents obtained from various conditions. It was found that when 0.5 wt.% catalyst was added in the reaction, this yielded less than 80 % methyl ester although the reaction time was extended into 5 h. At the reaction time of 1 h, the methyl ester apparently increased from 67.7 wt.% to 93.0 wt.% when the catalyst amount was changed from 0.5 wt.% to 1.0 wt.%. One explanation is that at 0.5 wt.% catalyst, the amount of catalyst was not enough for the reaction, thus when the catalyst amount at 1.0 wt.% was used, this led into the increase of surface area for the reaction, thereby increasing of the product. Page 390
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O
Cockle shell calcined at 1000 C
CaO
Figure 1 XRD patterns of CaO catalyst calcined at 1000 ď&#x201A;°C for 2 h. However, at 2 wt.% of catalyst, the yield decreased because the excess catalyst, which was in solid phase obstructed the interaction between substrates, consequently lowering the reaction. Hence, the optimum catalyst amount was at 1 wt.%. 100
Methyl ester content (wt%)
95 90 85 80 75 70 65 0.5 wt. % 1 wt. % 2 wt. %
60 55 50 1
2
3
4
5
Time (hr)
Figure 2 Effects of catalyst amount on methyl ester content using molar ratio of methanol to oil at 9:1, temperature at 70°C, and reaction time of 1-5 h. Effects of methanol to oil molar ratio The appropriate methanol mole is crucial in driving the direction of transesterification reaction forward leading to more methyl ester yield. As can be seen in Figure 3, at the reaction time of 1 h, the methyl ester content obtained from the reaction using molar ratio of methanol to oil at 12:1 was closely to that of the methanol to oil molar ratio at 9:1. Since statistical analysis (%RSD) of methyl ester yields obtained from both molar ratios indicated no significant difference, the methanol to oil molar ratio at 9:1 was chosen for the next experiment as it used less methanol. This would lower the cost of production and reduce time in recycling of methanol for the next production.
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Effects of reaction time The experiments were conducted at 1, 3, and 5 h. to evaluate the effects of reaction time on methyl ester yield. Basically, heterogeneous catalyst will react less efficiently compared with homogeneous catalyst, but this is not the case for this catalyst. From Figure 3, it is clearly that the reaction could occur efficiently within 1 h. and more than 90.0 wt.% methyl ester was obtained. Importantly, the methyl ester yield from this study is higher than the yield obtained from transesterification reaction of palm olein when the egg shells as a catalyst was used with the same molar ratio of methanol to oil, 9:1 (Viriya-empikul, N. et al., 2012). However, at the reaction time of more than 1 h, the methyl ester production was at a constant rate. Thus, the optimal reaction time was at 1 h.
Methyl ester content (wt.%)
100
95
90
85
80
75
9:1 12:1
70 0
1
2
3
4
5
6
Time (hr)
Figure 3 Effects of molar ratio of methanol to oil on the methyl ester content when used 1% of catalyst, temperature at 70째C, and reaction time of 1-5 h. Conclusion The results from this study indicated that cockle shells, a waste material, can be potentially used to prepare a high reactive catalyst for biodiesel synthesis. Increasing of catalyst sensitivity by doping with ammonium carbonate, results in less catalyst amount use in the reaction. It was also found that the optimal condition for the highest yield is reaction time at 1 h., molar ratio of methanol to oil at 9:1, and temperature at 70째C giving 93.0 wt% methyl ester. References Boey, P.L., G.P. Maniam and S.A. Hamid. 2009. Biodiesel production via transesterification of palm olein using waste mud crab (Scylla serrata) shell as a heterogeneous catalyst. Bioresource Technology 100(2009):6362-6368. Huaping, Z., W. Zongbin, C. Yuanxiong, Z. Ping, D. Shijie and L Xiaohua. 2006. Preparation of biodiesel catalyzed by solid super base of calcium oxide and its refining process. Chinese Journal of Catalysis 27(5): 391-396. Viriya-empikul, N., P. Krasae, W. Nualpaeng, B. Yoosuk and K. Faungnawakij. 2012. Biodiesel production over Ca-based solid catalysts derived from industrial wastes. Fuels 92(2012): 239-244. Wan Isahak, W.N.R, M. Ismail, J. Mohd Jahim, J. Salimon and M.A. Yarmo. 2010. Transesterification of palm oil using nano-calcium oxide as a solid base catalyst. World Applied Sciences Journal 9 (Special Issue of Nanotechnology) 17-22.
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List of Participants
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. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.
Abdul Salam BABJI Allah Wadhayo GANDAHI Alwani HAMAD Ammorn INSUNG Anchanok ONDEE Apisit SANLA Chalermsak SAKDAPECHSIRI Chamroon LAOSINWATTANA Chandra Prakash KHARE Chanpen UESAKULRUNGRUENG Chayaporn WONGSIRIDATCHAI Chitphan KAEWME Chokchai KITTIWONGWATTANA Chulalak TALUBNAK Duangkamol PANROSITP THUNMATIWAT Dusit AUE-UMNEOY Eakkarin SUKKAEW Fontip NUUDOM Fung Lewis Fu Uh Giang NGUYEN THIEN TROUNG Gray WILLIAMS Hariyadi JAMIN PRIYO MINARJO Hideo ISHII Hien Huu NGUYEN Hiroyuki KONUMA Isao TSUTSUI Janine CROSER Jarongsak PUMNUAN Jegan REVATHI Jindarha PREAMPRAMOTE Jirarot NITHISANTAWANKUPT Julian WISEMAN Kampanat PHESATCHA Kanchanapa KAMCHAN Kanda LOKAEWMANEE Kanet JAIKENGKAJ Kanjana SAETIEW Kannikar KAEWSONGSANG Kanok LERTPANICH Kanokporn CHANGSAWAKE Kanthee SIRIVEJABANDHU Kanya JIRAJAROENRAT Kasilingam PRAKASH Kawewat THONGSURIVONG Khakkhanang RATANANIKOM Khanitta SOMTRAKOON Kitti BOONLERTNIRUN
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48. Komkhae PILASOMBUT 49. Krisana RUNGROJWANICH 50. Lampan KHURNPOON 51. Laxmana Swamy SINGAM 52. Malatee PRADUBYART 53. Manat CHAIJAN 54. Mayamor SOYTONG 55. Metha WANAPAT 56. Monthon GANMANEE 57. Montinee TEERARAK 58. Napaporn KONGKARN 59. Nattawan BUSSABA 60. Natthakiti PHURUEN 61. Nipaporn YONSAWAD 62. Nipon JITTAMNAN 63. Nittaya PHAKAMAS 64. Nonglak PARINTHAWONG 65. Noppadol PANCHAN 66. Nukoon TAWINTEUNG 67. Numfon TAJASRI 68. Nutcharee SONGSAKUL 69. Nutcharee BOONPLANG 70. Nutthakorn SONGKRAM 71. OkumuĹ&#x; VEYSI 72. Omid SADEGHIPOUR 73. Panya MANKEB 74. Pariyaporn NETSAWANG 75. Pattama SRINAMNGOEN 76. Pattharin WICHITTRAKARN 77. Pattrarat TEAMKAO 78. Paveena TAWEEKIJAKARN 79. Peerachai KULLACHAI 80. Phatchara EAMKIJAKARN 81. Phawinee KAMSAN 82. Pongsakorn NITMEE 83. Pornpairin RUNGCHAROENTHONG 84. Pornprapa KONGTRAGOUL 85. Pornthiwa KANYAWONGHA 86. Prommart KOOHAKAN 87. Rajeev BHAT 88. Rangsan SOISOM 89. Ranyikar PORAHA 90. Ronachai SITTHIGRIPONG 91. Rungrat VAREEKET 92. Rungtawan YOMLA 93. Rutcharin LIMSUPAVANICH 94. Sakchai CHOOCHOTE
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Thailand Thailand Thailand India Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Turkey Iran Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Malaysia Thailand Thailand Thailand Thailand Thailand Thailand Thailand
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95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134.
Sakchai WUTHIMANOP Sarayut PHONPHO Sasiwimol WOOTTHIKANOKKHAN Siriporn PRAMRIT Somsak MANEEPONG Srisakul VORACHANTRA Suchada BOONLERTNIRUN Suchatvee SUWANSAWAT Sudathip CHANTORN Sudteerak SAIPLUEMCHIT Sukunya YAMPRACHA Suneeporn SUWANMANEEPONG Suneerat RUANGSOMBOON Suphachai AMKHA Surasak SAJJABUT Suriyasit SOMNUEK Sutichai SAMART Syed BILAL HUSSAIN Tanimnun JAENAKSORN Teerawat SARUTAYOPHAT Thamrong MEKHORA Thanapa CHETAWAN Thierry TRAN Thiwakorn AMPAPON Tippawan LIMUNGGURA Titi THONGKAMNGAM Tupthai NORSUWAN Ubon TANGKAWANIT Vanapron SAE-ANG Wachiraporn PEWLONG Wallop LOMLIM Wanida DUANGKONGSAN Wannasiri WANNARAT Waraporn CHOUYCHAI Warin PIMPA Warong NAIVINIT Wirote KLIBSUWAN Wisara THUTO Withan TACHAKOMOL Worawan PANPIPAT
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Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Pakistan Thailand Thailand Thailand Thailand France Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand Thailand
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Authors Index
AMKHA, S. ............................................................. 97, 265 AMPAPON, T. ............................................................... 133 AREEWONG, J. ............................................................ 313 BABJI, A.S. ..................................................................... 46 BANJONG, K. ............................................................... 137 BAOLEE, A. .................................................................. 273 BASKAR, M. ................................................................. 105 BHAT, R. ......................................................................... 42 BOONLERTNIRUN, K. ........................................ 209, 213 BOONLERTNIRUN, S. ......................................... 209, 213 BOONPLANG, N. ................................................. 281, 297 BUSSABA, N. ................................................................. 61 CHALERMWANICHWONG, J. ................................... 345 CHANGSAWAKE, K. ............................................. 73, 301 CHAOSAP, C. ............................................................... 341 CHETAWAN, T. ........................................................... 337 CHOOKAEW, S. ........................................... 225, 229, 233 CHOUYCHAI, W. ......................................................... 189 CHUANOI, S. ................................................................ 389 CHULILUNG, P. ................................................... 377, 381 CHUTIPAIJIT, S............................................................ 197 CROSER, J....................................................................... 45 DAVIE, S. ...................................................................... 109 DIXIT, A.. ...................................................................... 161 DONRODPRI, J. ............................................................ 189 DUANGDEE, S. ............................................................ 121 DUANGPAKDEE, K. .................................................... 329 EAMSIRI, J. ................................................... 225, 229, 233 GANDAHI, A. ................................................................. 93 GANDAHI, R. ................................................................. 93 GANMANEE, M. .................................................. 113, 117 HAMAD, A. ................................................................... 145 HANBOONSONG, Y. ................................................... 317 HUU NGUYEN, H. ....................................................... 101 INSUNG, A. ................................................................... 313 ISHII, H............................................................................ 54 JAENAKSORN, T. ......... 153, 157, 165, 169, 173, 177, 181 JAIKENGKAJ, K. .......................................................... 177 JAMIN PRIYO MINARJO, H. ........................................ 89 JAMJANYA, T. ............................................................. 365 JAMPATHONG, N. ....................................................... 193 JINTRAWET, A. ............................................................. 57 JIRAJAROENRAT, K. .......................................... 345, 349 JITTAMNAN, N. ........................................................... 333 JOMPUK, C. .................................................................. 209 KAEWSONGSANG, K. ................................................ 273 KAMSAN, P. ................................................................. 245 KANYAWONGHA, P. .......................................... 277, 281 KHARE, C.P. .......................................................... 161,185 KHLIBSUWAN, W. .............................................. 317, 365 KHONNALAO, Y. ........................................................ 361 KHURNPOON, L. ......................................................... 253 KITPRECHAWANICH, W. ............................................ 81 KOCHARIN, P. ............................................................. 113 KONGKARN, N. ........................................................... 301 KONGTRAGOUL, P. ............................ 149, 153, 181, 313 KOOHAKAN, P. ........................................................... 177 KOTASTHANE, A.S. .................................................... 185 KOTESTHANE, A......................................................... 161 LAOSINWATTANA, C. ........... 65, 73, 237, 241, 245, 249 LERTKRASEMPON, C. ................................................ 381 LERTPANICH, K. ......................................................... 325 LIMSUPAVANICH, R. ................................................. 341 LIMUNGGURA, T. ............................................... 369, 381 LOKAEWMANEE, K.................................................... 121 MAKLAI, A. .................................................................. 193 MALA, T. ................................................................ 97, 265 MANEEPONG, S. ................................................. 101, 261 MANKEB, P. ................................................. 369, 377, 381 MARONH, C. .................................................................. 57 MATONG, P. ................................................................. 377 MEEPOKA, C. ....................................................... 113, 117 MERRIFIELD, D. .......................................................... 109 MISHRA, A. .................................................................... 69 NAIVINIT, W. ............................................................... 385
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NETSAWANG, P. ......................................................... 249 NGAMSANGA, T. ........................................................ 345 NGUYEN THIEN TRUONG, G. .................................. 125 NIAMNUY, C. .............................................................. 141 NITHISANTAWAKHUPT, J. ....................................... 357 NITMEE, P. ................................................................... 265 NOKKOUL, R. .............................................................. 201 NORSUWAN, T. ............................................................. 57 NUR‘ ALIAH, D. ............................................................ 46 NURSYAMSI, D. ............................................................ 89 NURUL NADIA, M. ....................................................... 46 OSOTHONGS, M.......................................................... 349 PANCHAN, N. .............................................................. 141 PANHWAR, K. ............................................................... 93 PANKAEW, Y. ............................................................... 81 PANNANGPETCH, K. ................................................. 317 PARINTHAWONG, N. ................................. 165, 169, 309 PATRARASRIPONG, K. .............................................. 325 PERMHIRUN, T. .......................................................... 193 PEWLONG, W. ............................................. 225, 229, 233 PHAKAMAS, N. ................................................... 193, 197 PHESATCHA, K. .................................................. 129, 133 PHONCHAROEN, P. ...................................................... 61 PHURUEN, N................................................................ 237 PILASOMBUT, K. ...................................73, 301, 337, 353 PIMPA, C. ............................................................. 217, 221 PIMPA, W. ............................................................ 217, 221 POEAIM, A.. ................................................................. 205 POEAIM, S. ................................................................... 305 PONGJAROENKIT, S. ................................................. 205 PONGPOUN, S. ............................................................ 285 PONGTHONGKAM, P. ................................................ 205 PORAHA, R. ................................................................. 205 PRADIPTA, A.I. .............................................................. 89 PRADUBYAT, M. ........................................................ 169 PRAKASH, K.. ................................................................ 85 PRAKHONGSIL, P. .............................................. 225, 229 PRAMRIT, S. ................................................................ 309 PREMPRAMOTE, J. ..................................................... 117 PUMNUAN, J................................................................ 313 PUSPAWININGTYAS, G. and E. ................................. 145 RATANANIKOM, K. ................................................... 361 REVATHI, J. ................................................................. 105 ROENGANAN, N. ........................................................ 285 RUANGSOMBOON, St ................................................ 321 RUEN-NGAM, D. ......................................................... 201 RUNGCHAROENTHONG, P. ................................ 97, 265 SADEGHIPOUR, O. ..................................................... 257 SAE-ANG, V. ................................................................ 201 SAHILAH, A.M. ............................................................. 46 SAIPLUEMCHIT, S. ....................................................... 65 SAJJABUT, S. ............................................... 225, 229, 233 SAKDAPECHSIRI, C. .................................................. 349 SAMAKRAMAN, S. ..................................................... 117 SAMART, S. ................................................................. 197 SANPRASERT, R. ........................................................ 385 SARUTAYOPHAT, T. .................................................... 61 SENASING, V. .............................................................. 365 SERTSUNGNONE, K. .................................................. 193 SETHPAKDEE, R. ........................................................ 273 SHARMA, D. ................................................................ 161 SINGH, J.. ..................................................................... 161 SIRIVEJABANDHU, K. ............................................... 253 SITTHIGRIPONG, R. ................................................... 341 SIVAPIRUNTHEP, P. ................................................... 333 SOMNUEK, S. ...................................................... 153, 181 SOMTRATOON, K. ...................................................... 189 SONGKRAM, N............................................................ 373 SORAPUKDEE, S. ................................................ 337, 353 SOYTONG, M. .............................................................. 305 SRIKIJKASEMWAT, K. ............................................... 345 SRINAMNGOEN, P. ..................................................... 329 SRIPRACHOTE, A.. ............................................. 277, 281 SRIROD, S. ................................................................... 361 SUEBNUKARN, P. ....................................................... 385
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Authors Index
SUKKAEW, E. ................................................................ 97 SUKSUPATH, K. .......................................................... 357 SUNDHAR SINGH, S.D.. ............................................... 85 SUPANSOMPORN, S. .................................................... 81 SURANINPONG, P. ...................................................... 101 SUVANNASARA, R. .................................................... 213 SUWANMANEEPONG, S. ........................................... 377 SUWANPANYA, N....................................................... 361 SWAMY, Singam L. ........................................................ 69 TAJASRI, N. .................................................................. 341 TALUBNAK, C. ............................................................ 165 TANGKAWANIT, U. .................................................... 365 TANGWATCHARIN, P. ....................................... 301, 357 TAWAI, C...................................................................... 201 TAWINTEUNG, N. ............................................... 269, 293 TEAMKAO, P................................................................ 285 TEERARAK, M. ................. 73, 77, 237, 241, 245, 249, 301 THAMNIYOM, B. ......................................................... 389 THONGKAMNGAM, T. ....................................... 157, 173
July 1-3, 2015
THUTO, W. ................................................................... 137 TIWARI, P.K.. ............................................................... 185 TRAN, T. ................................................................. 55, 349 UESAKULRUNGRUENG, C. ...................................... 353 VIJAYAKUMAR, R.M.. ................................................. 85 VIRIYAEKKUL, P. ....................................................... 149 WANAPAT, M. ........................................41, 125, 129, 133 WANNARAT, W. ........................................................... 81 WATHAKIATTIKUL, P. .............................................. 289 WICHITTRAKARN, P.................................. 241, 245, 249 WILLIAMS, G. A.. ........................................................ 113 WISEMAN, J................................................................... 44 WIYACHAI, T. ............................................................. 385 WONGWEAN, P.a .......................................................... 81 WOOTTHIKANOKKHAN, S. ...................................... 389 YAMPRACHA, S. ......................................................... 289 YOMLA, R. ................................................................... 109 YONSAWAD, N. ............................................................ 77
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Sponsors
ISAT2015 Sponsors
Faculty of Agricultural Technology King Mongkutâ&#x20AC;&#x2122;s Institute of Technology Ladkrabang Chalongkrung Road, Ladkrabang, Bangkok 10520, THAILAND
www.agri.kmitl.ac.th