Application of several plant essential oils as postharvest plant fungicides in the control of mango anthracnose
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https://www.eduzhai.net/ International Journal of A griculture and Forestry 2013, 3(1): 1-6 DOI: 10.5923/j.ijaf.20130301.01 Use of Some Plant Essential Oils as Post-harvest Botanical Fungicides in the Management of Anthracnose Disease of Mango Fruits (Mangi Feraindica L.) Caused by Colletotrichum Gloeosporioides (Penz) M. A. Abd-AllA, Wafaa M. Haggag* Department of Plant Pathology, National Research Centre, Egypt Abstract Mango suffers fro m several diseases at all stages of its life. Anthracnose, caused by the fungus Colletotrichum gleosporioides is the most important post harvest disease of mango. The aim of this s tudy was to test the poss ibility of the use of some plant essential oils i.e. Basel oil (Ocimum basilicum), Orange oil (Citrus sinensis), Le mon oil (Citrus Medica) and Mustard oil (Brassica juncea L.) to reduce postharvest losses induced by Colletotrichum gloeosporioides (Penz.) in mango fruits. In this study, the antifungal activity of essential oils under in vitro condition were assayed by tested various concentrations (0, 50,100 and 150µg/ ml)) and under in vivo condition by used different essential oil concentrations (0, 250,500 and 1000pp m) on inoculated mango fruits. Results of the present study showed that orange oil at all tested concentrations were a significant reducing the fungal linear growth if co mpared with other tested essential oils. At low concentration 50 (µg/ ml) orange oil caused 10.0% reduction in fungal growth, while at 100 (µg/ ml) caused 72.2% and at high tested concentration 150 (µg/ ml) caused a comp lete reduction in myce liu m linear growth of pathogenic fungus. Meanwhile, at low tested concentration 50 (µg/ ml), mustard oil caused a highly significantly reduction of the percentage of fungal spore germination by 70.8 % followed by basil oil by 64.7%. Results of in vivo studies showed that, at low concentration 250 ppm, mustard oil caused a highly reduction of anthracnose incidence of mango fru its by 79.9% fo llo wed by basil o il with 66.7% .On the other hand, orange and lemon oil at low concentration (250ppm) were showed a highly effect to reducing the percentage of rotting fruit tissue by 84.5 and 75.0%, respectively if co mpared with other treat ments and un-treated fruits. Keywords Plant Essential Oils, Mango Fru its, Anthracnose Disease, Colletotrichum Gloeosporioides 1. Introduction Mango (Mangifera indica L.) is one of the top five fruit crops in the world. It is adaptable to a wide range of climates, ranging fro m wet trop ical to dry subtropical. A mong the various constraints, the most important is anthracnose caused b y Col l eto t ri ch u m glo eospo rio id es Pen z. A nd Sacc. (teleo morph Glomerellac ingulata). Flower blight, fruit rot, and leaf spots are among the symptoms of this disease. Severe infection destroys the entire in florescence resulting in no setting of fru its. Young in fected fru its develop black spots, shrivel and drop off. Fru its infected at mature stage carry the fungus into storag e and cause cons iderab le loss du ring storage, transit and marketing. Disease control methods include the prophylactic use of fungicides such as benomyl, man co zeb , carb en d azim, an d t h iab en d azo l. Th e * Corresponding author: email@example.com (Wafaa M. Haggag) Published online at https://www.eduzhai.net Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved postharvest use of chemicals as fungicides is restricted in most countries. Consumer demand for agricultural commodit ies without pesticide residues is high[7,24] however, pesticides may also kill various beneficial organisms and their toxic forms may persist in soil and increase the incidence of resistance among pathogens towards synthetic chemicals[3,23]. Thus, a new preservation technologies are needed, which have to be considered as human-safe and environmentally friendly. A mong the various alternatives, natural plant products, including essential oils that are biodegradable and eco-friendly, are catching the attention of scientists worldwide. Such products fro m higher plants are b io-efficacious, economical, and environmentally safe and can be ideal candidates for use as agrochemicals. Nu merous reports showed that oils fro m some plant species are harmful to fungal pathogens. Wilson et al.,, tested 49 essential oils fro m various plants and found that the oils fro m palmarosa (Cymbopogon martini), red thyme (Thymus zygis),cinnamon leaf (Cinnamomum zeylanicum), and clove buds (Eugenia caryophyllata) were effective in the control o f Botrytis 2 M . A. Abd-AllA et al.: Use of Some Plant Essential Oils as Post-harvest Botanical Fungicides in the M anagement of Anthracnose Disease of M ango Fruits (Mangi Feraindica L.) Caused by Colletotrichum Gloeosporioides (Penz) cinerea. Oils fro m Eucalyptus globules and Ocimum canum at 2000 pp m were effective in reducing mycelial gro wth and sclerotial production of Sclerotium rolfsii. This study aimed to evaluate the effectiveness of a number of plant essential oils as alternative method against growth and spore germination of Colletotrichum gloeosporioides the causal agent of anthracnose disease of mango fru its under vitro conditions , and study their effect on the d isease incidence under vivo conditions. Fi gure 1. Ant hracnose disease symptoms on mango fruit s 2. Materials and Methods 2.1. Fruits Mango (Mangifera indica L.) fruits Zabdia cv. were harvested at the mature stage, and sorted based on size and the absence of physical injuries or disease infection. Befo re treatments, fruit were surfaced disinfected with 2% sodium hypochlorite for 3 min, then rinsed with tap water, and air-dried. 2.2. Pathogen Culture Colletotrichum gloeosporioides was cultured fo r 1–2 weeks on potato dextrose agar (PDA) at 25 ◦ C. The isolate used was obtained from infected mango fru it in Egypt. Spores were harvested by adding 3–4 ml of sterile, de-ionized water (d iH2O) to the Petri dish. The spores were then rubbed with a sterile glass rod to free them fro m the PDA med iu m, and the spore suspension was passed through two layers of cheese cloth. The suspension was diluted with water to obtain the spore concentrations (106spores ml−1) according to determination with a Haemocytometer slid. 2.3. Source of Tested Plant Essential Oils Pure-grade (not containing synthetic chemicals and/or non-natural co mponents) essential oils of Basel oil (Ocimum basilicum), Orange o il ( Citrus sinensis), Lemon o il(Cit rus Medica) and Mustard oil (Brassica juncea L.), were obtained from Cairo Co mpany for Oils and Aromatic Extractions CID, Egypt. These essential oils were stored in dark bottles at 4ºC for further studies. 2.3.1. In Vitro Screening of Plant Essential Oils Against C. Gloeosporioides Mycelium Linear Growth and Spore Germination The antifungal tests were carried out in vitro according to the method described by Pitarokili et al. using Petri dishes 9 cm in d iameter containing potato dextrose agar (PDA ). The essential oils were d ispersed individually as an emu lsion in sterilized water using Tween 20 (0.05%) and added to PDA immediately before it was filled into the Petri dishes at a temperature of 45-50°C. The concentrations tested were0, 50,100 and 150µg/ ml. The controls included the same quantity of Tween 20 mixed with PDA. The tested fungus was inoculated immediately after preparation of the Petri dishes by placing in the centre of each plate a 5 mm diameter disk of the test fungus, cut with a sterile cork borer fro m the periphery of actively growing cultures on PDA plates. The Petri dishes were incubated in the dark at a temperature of 25°C. Mean growth rates were calculated fro m five rep licates of fungus every 24 h until fungal growth in the control filled the Petri d ishes completely, the percentage mycelial inhib ition was calculated by the following formu la: % mycelial inhib ition = dc _ dt X100 dc: where dc is mean colony diameter of control sets and dt is mean co lony diameter of t reatment sets conidial germination inhib ition test was performed by the cavity slide technique and the results were exp ressed in percentage. 2.3.2. In Vivo Applicab ility of the Plant Essential Oils of Anthracnose Incidence of Mango Fruits The Mango Fruits cv. Zabdeia were treated with d ifferent concentrations of plant essential oils by the standard techniques followed by Chandra and Sharma & Yadav  in order to find out the efficacy of the oils against anthracnose rot disease caused by C. gloeosporioides. Mature healthy mango fruits of med iu m size were used for the e xperiment. The fresh mango fruits of control as we ll as of treat ment sets were washed in running water and were surface sterilized with 0.1% sodiu m hypochlorite solution and were then washed with distilled water. Fruit inoculation with C. gloeosporioides spores obtained from a 7 day old culture, spores was suspended in sterile d istilled water and 0.03% Tween- 80. Mango fruits were wounded on two sides to a depth of 1.5 mm by puncturing them with a pin. Each wound site was then inoculated with 40µl of spore suspension (106 spores/ml) of C. gloeosporioides , and kept at ambient temperature for drying. Air-dried fru its were dipped for 2-3 min in different concentrations of tested essential oils (250,500 and 1000pp m) individually and again kept at ambient for drying. After applicat ion of treatments mango fru its were packed in cardboard boxes and stored (13±1°C, 80± 5% RH) for 28 days. The effect of various plant essential oils on disease incidence and disease severity (percentage of fruit rotting tissue) was evaluated weekly for 28 days during cold storage. Six replicates were kept for treatment and control sets. International Journal of A griculture and Forestry 2013, 3(1): 1-6 3 3. Statistical Analysis Tukey test for mult iple co mparisons among means was utilized (Neleret al.,1985). 4. Results 4.1. In Vitro Screening of Plant Essential Oils Ag ainst C. Gloeosporioides Mycelium Linear Growth Results in Table (1) showed that, orange oil at all tested concentrations was a significant reducing the fungal linear growth if co mpared with other tested essential oils . At low concentration 50 (µg/ ml) orange oil caused 10.0% reduction in fungal growth, wh ile at 100 (µg/ ml) caused 72.2% and at high tested concentration 150 (µg/ ml) caused a complete reduction in myceliu m linear gro wth of pathogenic fungus. On the other hand, basil oil gave a moderate effect to reducing the fungal linear gro wth at all tested concentrations by 5.5, 20.8 and 38.5%, respectively. Lemon and mustard oil showed a lower effect at all tested concentrations if compared with the other oils and control treat ments. significantly reduction of the percentage of fungal spore germination by 70.8 % followed by basil o il by 64.7% and orange oil by 61.2%. Meanwhile, at h igh tested concentration 150 (µg/ ml) , orange oil caused 63.0% reduction of fungal spore germination , followed by basil o il by 39.9% and mustard oil by 22.4% . Lemon oil showed a lower effect to reducing the fungal spore germination than other tested oils at all tested concentrations. Table 2. Effect of various concentrations of some plant essential oils against the percentage of fungal spore germination of C. gloeosporioides in v it ro Essential oils Orange oil ( Citrus sinensis) Con c. (µg/ml ) Fungal spore ge rmination % Re duction % 50 31.7 d 63.0 100 30.0 d 65.0 150 33.3 d 61.2 Lemon oil (Citrus Medica) 50 100.0 a 00.0 Table 1. Effect of various concentrations of some plant essential oils against the mycelium linear growth of C. gloeosporioidesin vitro Essential oils Orange oil ( Citrus sinensis) Con c. (µg/ml ) 50 Fungal linear growth (mm) 81.0 a Re duction % 10.0 100 25.0 d 150 00.0 e 72.2 100.0 Lemon oil (Citrus Medica) 50 90.0 a 00.0 100 71.8 b 20.2 150 53.0 c 41.1 Mustard oil (Brassica juncea 50 90.0 a 00.0 L.) 100 90.0 a 00.0 150 77.1 b 14.3 Basil oil (Ocimum 50 85.0 a 5.5 basilicum) 100 71.2 b 20.8 150 55.3 c 38.5 Con trol 0.0 90.0 a --- 0.0 90.0 a --- 0.0 90.0 a --- Figures with the same letter are not significantly different (P=0.05). 4.2. In Vitro Screening of Plant Essential Oils Ag ainst C. Gloeosporioides Spore Germination Results in Table (2) showed that, at low tested concentration 50 (µg/ ml) ,mustard oil caused a highly 100 90.5 a 6.28 150 51.6 c 39.9 Mustard oil (Brassica junceaL.) 50 66.6 b 22.4 100 31.9 d 62.8 150 25.0 d 70.8 Basil oil (Ocimumbasilicum) 50 51.6 c 39.9 Con trol 100 33.0 d 61.5 150 30.3 d 64.7 0.0 85.9 a ---- 0.0 85.9 a ---- 0.0 85.9 a ---- Figures with the same letter are not significantly different (P=0.05). 4.3. In Vi vo Applicability of the Plant Essential Oils of Anthracnose Inci dence of Mang o Frui ts Results in Table (3) and Fig.2. showed that , at low concentration 250 pp m , mustard oil caused a highly reduction of anthracnose incidence of mango fruits by 79.9% followed by basil oil with 66.7% reduction of disease incidence, while orange oil and le mon oil showed a moderate effect . At high concentration 1000 pp m, basil oil caused 94.4% reduction of disease incidence fo llowed by orange and mustard oils by 91.3 and 90.0 %, respectively. On the other hand, orange and lemon oil at low concentration (250pp m) were showed a highly effect to reducing the percentage of rotting fruit tissue by 84.5 and 75.0%, respectively if compared with other treatments and un-treated fruits. Mustard and basil oils showed moderate effect at all tested concentrations. 4 M . A. Abd-AllA et al.: Use of Some Plant Essential Oils as Post-harvest Botanical Fungicides in the M anagement of Anthracnose Disease of M ango Fruits (Mangi Feraindica L.) Caused by Colletotrichum Gloeosporioides (Penz) Table 3. Effect of fruits coating with different concentrations with plant essential oils on anthracnose disease incidence and percentage of fruit rotting tissue of mango fruits after 28 days of cold storage Essential oils Con c. ppm %Anthracnose % Fruit in ci dence rotting tissue Orange oil ( Citrus sinensis) 250 85.7 a 15.5 c 500 1000 Lemon oil (Citrus Medica) 250 25.3 c 8.7 e 75.6 b 10.0 d 00.0 e 25.0 c 500 1000 Mustard oil (Brassica 250 junceaL.) 500 1000 Basil oil (Ocimum 250 basilicum) 500 1000 Con trol 00 60.5 b 44.3 c 20.1 d 15.0 d 10.0 d 33.3 c 13.5 d 5.6 e 100.0 a 20.0 c 10.0 d 52.5 c 30.0 c 12.5 c 55.1 b 35.0 c 30.0 c 75.0 a Figures with the same letter are not significantly different (P=0.05). Figure 2. Effect of mango fruits coating with various concentrations of plant essent ial oils on anthracnose disease incidence after 15 days of storage 5. Discussion The essential oils are reported to have some fungicidal properties against certain postharvest diseases of tropical fruits and vegetables[30,16,13] and are also safer for the environment than synthetics chemicals .The ability of four plant essential oils to inhibit the fungal growth and their effect on spore germination of C. gloeosporioides was evaluated. The most active oils fo r reducing the growth of tested fungi with significant value under v itro condition was orange oil followed by basil oil, Lemon and mustard oil showed a lower effect at all tested concentrations if compared with the other oils and control treatments. On the other hand, at low tested concentration 50 (µg/ ml) ,mustard oil caused a highly significantly reduction of the percentage of fungal spore germination by 70.8 % followed by basil o il by 64.7% and orange oil by 61.2% .The literature is also silent on the mode of act ion of the essential o ils when used as postharvest fungi toxicants. A substance may inhibit the growth of fungi either temporarily (fungistatic) or permanently (fungicidal). In agreement with the findings, Thangavelu et al. found that the extracts of Solanumtorvum, Jatrophagl and ulifera and Emblica officinalis were highly inhibitory to mycelial gro wth of Colletotrichu mmusae in banana and the inhibitory effect was directly related to the quantity of extract added to the med iu m. In another study by Palhano et al. also confirmed that the inhib itory effects of citral on spore germination of C. gloeosporioides were higher with an increase in the concentration of essential oil. In vivo applicability o f the plant essential oils of anthracnose incidence of mango fruits was indicated that, At high o il concentration 1000pp m, coating mango fruits by basil o il caused 94.4% reduction of disease incidence fo llowed by orange and mustard oils by 91.3 and 90.0 %, respectively. On the other hand, mango fru its were coating with orange and lemon oil at low concentration (250pp m) were showed a highly effect to reducing the percentage of rotting fruit tissue by 84.5 and 75.0% , respectively if co mpared with other treatments and un-treated fruits. Maqbool et al., 2010, reported that, Cinnamon oil with fungitoxic o r fungistatic activity could be considered as a suitable alternative to synthetic fungicides for managing anthracnose in bananas. In vitro inhibit ion was d irectly related to the cinnamon o il concentrations. Dubey et al. described that essential oil fro m Eupatorium cannabinum had an antifungal act ivity against Botryodiplodia theobromae and Colletotrichum gloeosporioides causing stem end rot and anthracnose diseases in mango, respectively. In addition, they found that this essential oil had an inhibitory effect on pectinase and cellulase, t wo important enzy mes produced by phytopathog enic fungi in disease development. Farrag et al.  described that the antimicrobial activity of essential o ils could be related to the presence of an aromatic nucleus and OH group that can affect hydrogen bonds of enzymes in microorganis ms. Feng and Zheng , 2007, studied the effects of cassia oil on decay development in artificially inoculated and wounde tomatoes fruits. The results indicate that when wounded cherry tomatoes were treated with cassia oil, all concentrations (except 100 pp m) significantly inhibited A. alternateon tomatoes stored at 20 _C for 5 days (p < 0.05). The percentages of decayed cherry tomatoes treated by 500 International Journal of A griculture and Forestry 2013, 3(1): 1-6 5 ppm cassia oil was reduced by 34.2% co mpared to the 600-611 control. Maqbool et al., 2010,reported that, different  Cakir,A. S. Kordali, H. Kilic and E. Kaya, 2005. Antifungal concentrations of cinnamon oil not only delayed the onset of properties of essential oil and crude extracts of Hypericum anthracnose disease in coating panama fruits but also linarioides Bosse, Biochemical Systematics and Ecology 33: maintained the freshness during first two weeks of storage pp. 245–256. and later on showed min imal sympto ms. The highest  Chandra, H. 1984.Evaluation of some higher plants for their fungicidal effect was observed in those bananas treated with volatile activity against blue mould rot of orange. PhD. Thesis, 0.4% cinnamon oil (d isease incidence of 8.0%) and disease Gorakhpur University. Gorakhpur, India. severity (DS) score of 1.2 indicat ing fru it surface infection close to 1.0. Shelef described that within several  Cox, S.D., M ann, C.M .,M arkham, J.L., Bell, H.C., Gustafson, J.E., Warmingt on, J.R., Wyllie, S.G., 2000. The mode of components available in essential oils, the antimicrobial antimicrobial action of essential oil of M elaleuca alternifola activity of phenolic co mpounds were h igher than alcoholic (tea tree oil). Journal of Applied M icrobiology 88, 170–175. components, this is in agreement with the results of present  Cronin, M .J., D.S. Yohalem, R.F. Harris and J.H. Andrews, study. It is known that the cell wall of pathogens is the main 1996. Putative mechanism and dynamics of inhibition of target of phenolic co mpounds and these compounds may apple scab pathogen Venturiain equalis by compost extracts. disrupt the permeability barrier of cell memb rane and inhibit Soil Biol. Biochem., 28: 1241–1249 respiration. Hydrophobic nature of essential oils and their components enables these compounds to penetrate lipid of  Cutler, H.G. and S.J. Cutler, 1999.Biological active natural products: Agrochemicals, CRC Press, Boca Raton, USA, p. fungal cell memb rane and mitochondria as a result d isturbing 299. their structure and these compounds accumulate in the cell memb rane of pathogen causing energy deletion. In addition, in so me studies, it is reported that the essential o ils  Dubey R.K., Kumar J.R. and N.K. Dubey, 2007. Evaluation of Eupatorium cannabinum Linn. oil in enhancement of shelf life of mango fruits from fungal rotting. World J. M icrobiol. may affect the metabolic pathways of microorganisms. Biotechnol. 23: 467–473. Nychas found that phenolic compounds in low concentration disrupt proteins and in high concentrations  Duru, M .E. A. Cakir, S. Kordali, H. Zengin, M . Harnandar and S. Izumi, 2003. Antifungal activities of the leaves of three damaged the enzy mes outbreak in production of energy. Pistacia species grown in Turkey, Fitoterapia,74: pp. 170–176. 6. Conclusions The results of present study showed that the possibility of the use of some p lant essential oils i.e. Basel oil (Ocimum basilicum), Orange oil (Citrus sinensis), Lemon oil (Citrus Medica)and Mustard oil (Brassica juncea L.)to reduce postharvest losses induced by Colletotrichum gloeosporioid es (Penz.) in mango fruits. So essential oils can be usedas a potential source of sustainable eco-friendly botanical fungicides, after successful comp letion of wide range trials. ACKNOWLEDGMENTS This manuscript funded fro m the project “New applied approaches to promote productivity and Quality of so me fruit crops (Mango)” Nat ional Research Centre, 2007 to 2010.  Farrag R.S, DawZ.YandS.H Abo-Raya, 1989. Influence of some spice essential oils on Aspergillus parasiticus growth and production of aflatoxins in a synthetic medium. J. Food Sci., 54: 74-76.  FengW. and X.Zheng, 2007. Essential oils to control Alternaria alternatain vitro and in vivo. Food Control 18 (2007) 1126–1130.  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