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International Journal of Agricultural Science and Research (IJASR) ISSN 2250-0057 Vol. 2 Issue 4 Dec 2012 53-62 © TJPRC Pvt. Ltd.,

IMPROVE POSTHARVEST QUALITY OF FRESH FRUIT CITRUS WITH PLANT EXTRACT HARBAN SINGH, GHASSAN.F AL-SAMARRAI & MUHAMMAD SYARHABIL School of Bioprocess Engineering, Universiti Malaysia Perlis (UinMAP), Perlis, Malaysia

ABSTRACT Different concentrations (500 -5000 ppm) of five ethanol extracts of Azadirachta indica L (Neem), Cerebra odollam L. (Pong-pong) ,Capsicum frutescence L (Chili), Cymbopogon nardus L (Lemon grass) and Zingiber officinale L (Ginger) were compared with fungicide (Guazatine) for their anti-fungal activity inhibition zone Penicillium digitatum in vitro on PDA media and during storage conditions in vivo. Lethality test LC50 (BST) was followed to determine the lethal dose from plant extract compared with lethal dose for synthetic chemicals (Guazatine). Crude extraction from Neem, Chili and, Pong-pong showed a complete inhibition zone at 3000ppm (100%) in the green mold in vitro. In vivo, concentrations (4000 and 5000ppm) from Neem, Chili, and Pong-pong showed a high effect on prevention development of mycelia growth Penicillium digitatum on surface fruits in storage condition at 250C±2.

Also spraying fruit has improved postharvest quality characteristics in the store by reducing the weight loss and numbers of the undesirable fruits percentage. In addition, the lethal concentration (LC50) values of the crude extracts were investigated via using the Brine-shrimp (Artemia salina Leach) lethality test (BST). At 20.5 and 30 µg/ml-1 Neem, Pongpong and hot Chili showed very high lethal toxicity on brine and effect. Lemon grass and Ginger in killed 50% were at 495 and 473 µg/ml- 1 respectively with compared controls .

KEYWORDS: Postharvest Quality, Natural Plant Extracts, Modified Atmosphere Storage, Environmental Management INTRODUCTION The problems caused by synthetic pesticides and their residues have increased the need for effective biodegradable pesticides with greater selectivity. Alternative strategies have included the search for new types of pesticides, which are often effective against a limited number of specific target species, are biodegradable into nontoxic products and suitable to be used in integrated pest management programs (Guptan and Dikshit, 2010) Applied chemical pesticides are one of the means to reduce the loss of post-harvest diseases for being effective and fast. Nevertheless, the use of these chemicals, excessively for controlling mold fungal in fruit has led to counterproductive, causing damage to the environment and humans with increased claims to reduce the use of these chemicals that accumulate in fruits and vegetables (Nichoison, 2007).

This damage increased significantly with the improper use and randomly led to grow in order to reduce the use of these chemicals that accumulate in fruits and vegetables. It is claimed that these fruits were the major health problems after they became a crop of major commodities exported to the different countries all over the world. As persistent hazardous chemicals the use of many of these pollutants by the government and international organizations such thiabendazole (TBZ) and imazalil ( Eckert, 1990). to gravity, which was previously used to control a wide range of fungi, which led to an imbalance in the balance environmental and low rate of natural enemies, which of course helps to maintain the pathogen without the influence (Eckert , 1967).

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Harban Singh, Ghassan.F. Al-Samarrai & Muhammad Syarhabil

The natural plant products derived from plants effectively meet this criterion and have enormous potential to influence modern agrochemical research. When extracted from plants, these chemicals are referred to as botanicals. The use of botanical pesticides is now emerging as one of the prime means to protect crops and their products and the environment from pesticide (Sanjay and Tikul, 1967). Botanicals degrade more rapidly than most chemical pesticides, and are, therefore, considered friendly to the environment and less likely to kill beneficial pests than synthetic pesticides with longer environmental retention. Most of the botanical pesticides generally degrade within few days and sometimes within a few hours (Siddiqui and Gulzar, 2003).

The objective of this study is to evaluate the use of botanical pesticides of five plants extracts Neem, Pong-pong, Chili, Lemon grass and Ginger as natural products and examine their effectiveness as anti-fungal, eco-friendly, low-cost, and easy to prepare.

MATERIALS AND METHODS Preparation of Plants for Extraction Leaves from Neem and Pong-pong were collected from trees growing at the river side park near the Kangar town.

The other plants, leaves and stems of lemon grass, chili fruits, and ginger tubers were purchased from the wet market at Kangar Table.1. These samples were brought to laboratory and washed under running water in order to get rid of dirt, insects and plankton. They were dried overnight in the laboratory- electric oven at 40oC. One hundred grams of the materials were pulverized by an electric mixer, and preserved in labeled glass bottles that were sealed to be used in the right time.

Table 1: “Plant Species” Parts and Concentration Used in Experiment Vitro and Store Common name

Plant species

Family name

Neem Chili Pong-Pong Lemon grass

Azadirachta indica L. Capsicum frutescence L. Cerbera odollam L. Cymbopogon nardus L.

Meliaceae Solanaceae Apocynaceae Poaceae

Ginger

Zingiber officinale L

Zingiberaceae

Nature of the extract Leaf powder Fruit powder Leaf powder Leaf powder Tuber powder

Conc .(ppm)

500 to 5000

Preparation of Plant Extracts The extraction technique that was used was a modification of Ruch’s (2001) method .Up to 50g each of the oven dried and pulverized powered material from plants were treated with 500 ml of 95% alcohol with constant stirring for 30 minutes. After stirring, the solutions were filtered through 2 layers of cheese-cloth gauze and Whitman’s (No.2) filter paper before the filtrates were subjected to evaporation using a rotary evaporator at 60 oC degree for 60 minutes to remove the ethanol.

The dark spongy materials were dried in an oven at 37 oC for 2 days. The dried powder was stored in small and sterilized 5ml screw-capped glass bottles that were refrigerated (4oC) in order to be used further.

Preparation of Plant Extract-Dilutions Powder from each plant (0.5, 1. 2, 3,4 and gm) was dissolved on 2ml organic solvent DMSO (solvent dim ethyl sulfoxide ,99.5%) with concentrations mg/mL .It was diluted further by mixing with water to get a concentration of 500, 1000, 2000, 3000, 4000 and 5000ppm.

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Pathogen Using taxonomic and morphological references, the identified pathogen was Penicillium digitatum. Highly aggressive, single-spore isolates of P. digitatum, originally isolated from citrus fruits were grown on potato dextrose agar (PDA) at 25oC for 7 days. Spores were harvested by flooding the media surface with sterile distilled water and gently agitating the plate to dislodge spores (Mconie, 1964; Serrano et al., 2005). Concentration spores were determined by using a hemacytometer and adjusted to 1x106 spores mL-1

IN VITRO SCREEN The Effect of Plant Extracts on Mycelium Growth of Penicillium Digitatum PDA media were incorporated into forty-five in glass flasks (50 ml) and autoclaved for 20 minutes at 121OC. After autoclaving, the flasks were cooled down to about 45oC. Five ml of each plant extract, (500 , 1000 , 2000, and 3000 ppm) was added in flasks by using pipette that were gently agitated by hand for 2 minutes in order to mix the extracts properly. Media cultures were amended into 9cm in Petri-dishes. Chloramphenicol (250 mg-1 per Petri dish) was added to the medium to prevent bacterial growth (Falah et al., 2007). The experiment was performed under aseptic lamina conditions and replicated thrice. One ml, of P. digitatum, spore suspensions (conc.1 × 106 spores/ml-1) was added by pipette on to the centre of the amended PDA extracts. Inoculated plates were incubated at 25 oC for 10 days. The Petri-dish inoculated without the extract concentration (water) as negative control. Colony diameter was determined by measuring the average radial growth. The inhibition zone was measured using the formula of (Francisco and Lippa, 2010) as follows: Mycelial growth (control/water) –- Mycelial growth (treatment) % Mycelial inhibition =---------------------------------------------------------------------X 100 Mycelial growth (control/water) In storage Conditions Healthy freshly citrus fruit was washed with tap water and then was air dried and sterilized by immersion in 70% ethanol for 1 minute before spraying. Fruits randomly divided into 5 equal groups (5 fruits); all groups were wounded in depth of 5.0 mm with a 1.25 mm diameter needle. At the equator all treatments were inoculated by spray in suspension of P.digitatum (1x106 spores’ mL-1). Six treatments were carried out after one hour as follows.

1.

Spraying with 2000ppm

2.

Spraying with 3000ppm

3.

Spraying with 4000ppm

4.

Spraying with 5000ppm

5.

DMSO (99.5%)

6.

Fungicides (Guazatine).

Treated fruits were packed in plastic boxes and incubated at 25ºC with >85% RH for 21 days. Evaluation of the treatment was done every week. Efficacy of treatment application was determined according to (Ibtesam et al.,2011). There were three replicate trials of 5 fruits. Representative samples of five fruits per replicate were taken each weekly. Severity of decay was visually evaluated during a spray to crude plant extract, at 28 °C/85% RH. The degree of infection on fruit

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Harban Singh, Ghassan.F. Al-Samarrai & Muhammad Syarhabil

was rated using a scale of 1 to 5, where [1–clean, no infection; 2–trace infection; 3–slight infection; 4–moderate infection; and 5–severe infection].

Studies Character in Vivo 1.

Weight Loss%: This character was determined by weighing 5 fruits in each replicate after three week .Percentage of weight loss was calculated by determination of progressive reduction in fruit weight during storage period relative to the original fresh weight at the beginning of storage. .

2.

Undesirable Fruits Percentage: Calculated by dividing the number of decayed fruits by the total number of fruits as following equation:

Undesirable fruits% = (The number of undesirable fruits/Total number of fruit) x 100

CYTOXICITY SCREENING (LC50) Test Brine shrimp lethality is widely used in the bioassay to investigate the cytoxicity of plant extracts as a method of simple screening (Chandra et al., 2008). The crudes from plants extracts were used to test (LC50). The eggs of the brine shrimp were collected from a fish shop (Kangar, Perlis). 5mg of Artemia salina (Leach) eggs were added to flasks containing Ocean/Sea water (50ml). A flask was kept under an inflorescent bulb for 48 hours for the eggs to hatch into shrimp larvae following the method (Agrios, 2005) .The concentrations for test were prepared by dissolving them in DMSO to attain concentrations (5, 10, 20, 40, 130, 260, 390, and 520 µg ml-1). DMSO and fungicide (Guazatine) were used as negative and positive control. Solutions were transferred to all flasks and dosage was tested in triplicate for all flasks (9 per test fraction). After 24 hours the number of dead larvae was counted. The data were collected and analyzed by using Excel 2007.

STATISTICAL ANALYSIS The experimental data was subjected to analysis of variance (ANOVA). Significant differences between a value s were determined using Duncan’s Multiple Range test (P= 0 .05) following ANOVA. Statistical analyses were performed using SPSS (SPSS Inc., Chic ago, and US A).

RESULTS Effect of Plant Extracts on Development Mycelium Growth of Penicillium Digitatum in Vitro The study showed different significances between treatments and controls at (P≤0.05). Generally the results from Table.2 showed completed inhibition growth of P. digitatum reached to (100, 93%) of crude plant extracts concentration at 3000ppm from Chili, Pong-pong. In addition, crude extract from Neem, Lemon grass, and Ginger were highly effective at same concentration which amounted to 88.3, 88.3, and 81.5% respectively. Negative control was performed with serial dilutions of DMSO it was observed that there was any effect on fungal growth (0%).

Table 2: Percentage of Inhibition Colony Diameter (Cm2) P. digitatum Caused by Plant Extracts (ppm) Compared with Controls ( [ Guazatine (P.C) and water ( N.C) ] in Petri Dishes PDA Inoculated and Incubated at 25oc For 10 Days .

Comparisons(CONCENTRATION ppm) 500 1000 2000 3000 Treatments Inhibition zone (cm) Neem a 3.9 a2.9 a 2.33 a1.33 Pong-Pong b 2.76 b1.99 b 1.46 b 0.63 Chili a 3.9 a 2.96 a 2.1 c 0.00

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Comparisons(CONCENTRATION ppm) 500 1000 2000 3000 Treatments

Inhibition zone (cm)

Lemon grass a 3.9 a 2.96 a 2.33 a1.33 Ginger c 4.16 a 2.67 a 2.23 a1.38 Guazatine b 2.2 b 1.9 b1.86 d2 Water e 8.56 c 9 c 8.56 e 8.4 *Positive control [fungicide (Guazatine)], * Negative control (DOSM) Effect of Plant Extracts on Fruit Decay Data presented in Table. 3 Figure.1 showed the treatments from Neem, Pong-pong, and Chili at concentrations (4000 and 5000ppm) which prevented the full growth of green mold on surface fruits stored at 25±2°C for 21 days. In addition, number of undesirable fruits were (0%) in concentrations (4000 and 5000ppm) result to curb the development of fungal growth. Spraying extracts from Lemon grass and Ginger did not show a considerable statistical significance in applying on fruits compared with controls. Percentage of damaged fruits was between (66.6 to 46%) for all concentrations from extracts of Lemon grass and Ginger.

Table 3: Effect Spray Plant Extracts at Different Concentrations (Ppm) on Undesirable Fruits (%) Result Development Mycelium Growth Penicillium digitatum on Orange Surface fruits Under Condition Store at 25oc±2 Compared with Controls Plants Comparisons 2000 3000 4000 5000 Guazatine DOMS Undesirable fruits (%) Neem 20 a 0a 0a 0a 6.7 a 93.3 a Pong-Pong 6.7 b 0a 0a 0a 6.7 a 80 a Chili 13.4 c 0a 0a 0a 6.7 a 80 a Lemon grass 66.6 d 60 b 60 b 46 b 0b 86.7 a Ginger 66.6 d 60 b 60 b 46 b 6.7 a 100 a [Clean, no infection= 0, Trace infection= 6.7, Slight infection= 13.4- 20, Moderate infection= 46, Severe infection= 60100).]

Effect of Plant Extract Spray on Fruit Weight loss Result showed that fruit weigh loss percentage increased by extending in storage period. These results could be due to the loss in moisture content. The results indicated that all treatments significantly decreased fruit weight loss percentage during the period of storage for 8 weeks, compare with controls. Experiments tested plant extracts spraying with Neem, Pong-pong and hot Chili showed significantly reduced fruit weight loss percentage by 21.3, 18.98 and 19.8% .Lemon grass and Ginger also recorded low fruit weigh loss percentage reached to 38.8 and39.6 % respectively (Fig.2).

Figure 1: Effect of Spraying Plant Extracts at Different Concentrations (Ppm) on Weight Loss (%) After 21 Days During Storage Condition(25±2OC) Compared with Controls[ Fungicides (Guazatine) and DOMS]

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Harban Singh, Ghassan.F. Al-Samarrai & Muhammad Syarhabil

Effect of Plant Extract Spray on Undesirable Fruits Percentage Results indicated that all treatments significantly reduced the undesirable fruits percentage during storage for 8 weeks. The best treatments were spraying with plant extract from Neem, Pong-pong and hot Chili at conc: 4000 and 5000ppm significantly reduced undesirable fruits percentage by 53.58, 52.50, 55.6, 53.16 and 52.90, 55.5% respectively compare with controls, .Also showed the treatments spraying with Lemon grass and Ginger at 5000ppm some effect through reduce the undesirable fruits by 42.9 and 43.6%, Sequentially (Fig.2).

Plant

extract

Figure 2: Effect of Spraying Plant Extracts at Different Concentrations (ppm) on Mean Number Orange Undesirable Fruits (%) after 21 Days During Storage Condition (25±2OC) Compared with Controls[ Fungicides (Guazatine) and DOMS].

Brine Shrimp lethality Bioassay The cytotoxic activities of all the extracts of Azadirachta indica L., Cerbera odollam L., Capsicum frutescence L., Zingiber officinale L., and Cymbopogon nardus L. were studied by brine shrimp lethality bioassay .The LC50 values of parts of plant extracts were 20 (32-6), 5(15-0), 30(37-9), 473 (1818 - 212) 495 (858 – 306)µg mL-1, respectively compared with values of controls (Guazatine and DOMS) that reached to (326 and >1000) Table.4.

Table 4: Brine Shrimp Test Toxicity of Plant Extracts Under Study Plants Azadirachta indica L (Neem) Capsicum frutescence L( Pong-pong) Capsicum frutescence L. (hot Chilly) Zingiber officinale L.( Lemon grass) Cymbopogon nardus L.(Ginger) controls

Part used Leafs Leafs Fruits Leafs Tubers Fungicide (Guazatine)

Traditional use Anti-fungi Anti-fungi Anti-fungi Anti-fungi Anti-fungi

LC50 (Mg/L ) 20 5 30 473 495

Anti-fungi

326

DMSO

--------------

>1000

DISCUSSIONS Many plant and plant products have been reported to have antimicrobial activities against plant pathogenic fungi [15].The objective of the current research was to study the effect of plant extracts as alternative synthetic fungicides in controlling mycelium growth of Penicillium digitatum that is pathogens for the post- harvest diseases of citrus as reported by ( Gupta and Pikshit ,2010) These diseases could cause a loss of 10%-30% decrease in crop yield and marketing quality ( Nicholson ,2007 ; Sokovicet et al ,2009) . The use of biocontrol agents in plant disease control with plant extracts like lemon, citronella, clove, mint, thyme, and oregano oils has been employed by (Samson, 1984) as alternative control

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measures to replace the conventional synthetic pesticides. The plant extracts reported to be effective on the fungi Penicillium digitatum including garlic, Withania somnifera and Acacia seyal and mustard horseradish used for the same purpose as natural alternative (Adaskaveg et al; 2000; Serrano et al., 2005).

The activities of the plant extracts may be due to the action of their bioactive compounds against fungi growth through preventing growth spores such as Penicillium italicum and P. digitatum which can replace synthetic chemicals in future to reduce their increase in the environment. These results are in agreement with (Agrios, 2005). They reported that the fungicidal of oil obtained from thymus against several post harvest pathogens may reveal the marked fungicidal activity of carvacrol in thyme. Moreover, (Bashar and Baharat, 1992) reported that lime fruit peel essential oil components inhibit linear growth on spore germination of P. italicum, P. digitatum and Geotrichum canium (Kadota et al; 1990) In study by

(Ogawa et al., 1995) evaluation of effect of lime, thyme, comphore oils for their inhibitory effect in vitro,

different concentrations of each essential oil at 1.5 and 10% (v/v) were tested on the growth of P. digitatum. The best concentration at 10% showed the highest inhibition growth of P. digitatum for all tested oils and significantly reduced the disease severity of fruits compared with controls. Previous reports have shown that spearmint essential oil possesses antifungal properties (Chao et al., 2000) . These oils have wide range of bioactivity. Antifungal activities of essential oils from Thymus and Mentha species have been reported in other studies as well (Sokovicet et al, 2009; Bourdy et al, 2000) .

This study is a general agreement with the results of earlier investigations (Sanjay and Tiku, 1967; Mossini et al., 2009).

Some chemical compounds have been isolated from seed (Chao et al, 2000) and bark (Meyer et al, 1982) of S. mahogany, in spite, this result should encourage other researchers to further studies such as finding from natural product as alternatives to agriculture pesticides to be safer and less risky. The earlier reports of antimicrobial activities (Siddiqui and Gulzar, 2003) support the findings of present studies. Inhibition effects of crude extracts indicate that it can be selected for more applications in management of environment because there is a correlation between effectiveness of the extracts and activity against the brine shrimp nauplii using extracts.

CONCLUSIONS Pong-pong, Neem, and chili showed positive effect on the inhibition of postharvest fungi Penicillium digitatum.

These extracts or botanicals have a bright future in modern plant protection to replace conventional synthetic pesticides subject to their further toxicological studies.

ACKNOWLEDGEMENTS The researchers wish to thank the School of Bioprocess Engineering, University Perlis Malaysia for providing facilities in completing this research.

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