ISSN 1330-7142 UDK = 632.937.14:665.5 IN VITRO ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS ON GROWTH OF PHYTOPATHOGENIC FUNGI Jasenka Ćosić, Karolina Vrandečić, Jelena Postić, Draženka Jurković, Marija Ravlić Original scientific paper Izvorni znanstveni ~lanak SUMMARY Eleven essential oils (clove, rosemary, cinnamon leaf, sage, scots pine, neroli, peppermint, aniseed, caraway, lavander, common thyme) were tested for in vitro antifungal activity on twelve plant pathogenic fungi (Fusarium graminearum, F. verticillioides, F. subglutinans, F. oxysporum, F. avenaceum, Diaporthe helianthi, Diaporthe phaseolorum var. caulivora, Phomopsis longicolla, P. viticola, Helminthosporium sativum, Colletotrichum coccodes, Thanatephorus cucumeris). The results indicated that all oils except scots pine and neroli had antifungal activity against some or all tested fungi. The best antifungal activity had common thyme, cinnamon leaf, clove and aniseed oils. When compared to control, scots pine, neroli and sage oils stimulated mycelium growth of some investigated fungi. Key-words: essential oil, inhibition, plant disease, mycelial growth INTRODUCTION MATERIAL AND METHODS More than 1300 plant species are known to be potential sources of antimicrobial components but only some of them have been studied scientifically (Wilkins and Board, 1989, Paster et al., 1990.). For instance, some previous studies evaluated the inhibitory activity of essential oils on fungi. Cardwell and Dongo (1994) tested extracts from nine plant species on mycelial growth of Apergillus flavus L.; Manohar et al. (2001) researched origano commercial oil against Candida albicans (Robin) Berkhout; Marin et al. (2004) tested cinnamon, clove, oregano, palmarosa and lemongrass oils against Fusarium graminearum Schw., Hadizadeh et al. (2009) analyzed antifungal potential of five essential oils against Alternaria alternata (Fr.) Keissl. Burgiel and Smaglowski (2008) described complete growth inhibition of Fusarium culmorum (W.G. Smith) Sacc. and B. cinerea on media with a 0.5% addition of tea tree oil. In addition, some studies reported positive effect of essential oils in reducing mycotoxin accumulation in maize grain (Marin et al. 2004, Velluti et al. 2004). The aim of this research was to test in vitro the effect of 11 commercial essential oils on mycelial growth of 12 plant pathogenic fungi. The effect of clove (Eugenia caryophyllus (Sprengel) Bullock & Harr.), rosemary (Rosmarinus officinalis L.), cinnamon leaf (Cinnamomum verum Presl.), sage (Salvia officinalis L.), scots pine (Pinus sylvestris L.), neroli (Citrus aurantium L. ssp. amara Engl.), peppermint (Mentha piperita L.), aniseed (Pimpinella anisum L.), caraway (Carum carvi L.), lavander (Lavandula angustifolia Mill. ssp. angustifolia) and common thyme (Thymus vulgaris L.) oils on growth of F. graminearum, Fusarium verticillioides (Sacc.) Nirenberg, Fusarium subglutinans (Wollenw. & Reink.) Nelson, Toussoun & Marasas, Fusarium oxysporum Schlecht. emend Snyder and Hansen, Fusarium avenaceum Fr. Sacc., Diaporthe helianthi Munt.-Cvet. et al., Diaporthe phaseolorum var. caulivora (Lehman) Wehmeyer, Phomopsis longicolla Hobbs., Phomopsis viticola (Sacc.) Sacc., Helminthosporium sativum Pamm., King and Bakke, Colletotrichum coccodes (Wallr.) Hughes and Thanatephorus cucumeris (Frank) Donk was evaluated. Fungi were isolated from wheat grains (F. graminearum, F. avenaceum, H. satiProf.DSc. Jasenka ]osi} (jasenka.cosic@pfos.hr), DSc. Karolina Vrande~i}, Jelena Po{ti}, BSc., Prof. DSc. Dra`enka Jurkovi}, Marija Ravli} - J. J. Strossmayer University of Osijek, Faculty of Agriculture in Osijek, Trg Sv. Trojstva 3, 31000 Osijek POLJOPRIVREDA 16:2010 (2) 25-28 26 Jasenka ]osi} et al.:IN VITRO ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS ON GROWTH ... vum), maize stalks (F. verticillioides, F. subglutinans, F. oxysporum), sunflower stalks (D. helianthi), soybean stalks (D. phaseolorum var. caulivora), soybean grains (P. longicolla), grape canes (P. viticola), common bean roots (T. cucumeris) and Abutilon theophrasti Med. roots (C. coccodes). The essential oils were produced in Pranarom International (Belgium) and analysed in Pranarom laboratory by GC-MC (gas chromatographymass spectrometry). Antifungal assays were carried out in vitro according to Saikia et al. (2001). A 5-mm diameter sterilized filter paper disc was placed in the center of Petri dish (9 cm diameter) with PDA and loaded with 5 µl of essential oil. Four discs (5 mm diameter) of mycelial plugs were cross placed into each Petri dish. Petri dishes were kept in thermostat at 22ºC and for 12/12 h light/dark regime. The inhibition zones around the paper disc were measured after eight days as the distance from the margine of the colony to the filter paper. The experiment was performed in four replications. Research results were analyzed statistically by ANOVA and LSD using Statistica for Windows version 9. RESULTS AND DISCUSSION The inhibitory effect of twelve essential oils against some economically important plant pathogenic fungi are shown in Table 1. Our research showed that all investigated oils except scots pine and neroli had antifungal activity against some or all researched fungi. The highest degree of antifungal activity showed common thyme oil followed by cinnamon leaf oil (Photo 1), clove oil and aniseed oil. Table 1. Inhibition zone (mm) eight days after inoculation Tablica 1. Zona inhibicije (mm) osam dana nakon inokulacije DPC PV DH PL HS CC TC FO FA FG FS FV Clove/ klin~i}evac 23.06 27.13 26.38 20.75 21.63 23.25 0 16.70 8.50 0 7.81 10.50 Rosemary/ ru`marin 0.56 7.50 10.13 0 12.25 9.75 0 0 0 0 1.19 0 Cinnamon leaf / list cimeta 18.56 32.31 28.06 22.82 18.38 22.25 0 16.80 8.80 0 7.00 8.50 Sage/ kadulja 1.75 8.88 2.94 0 4.44 11.90 0 0 0 0 0.88 4.44 Scots pine/ bor 0.63 10.50 4.06 0 3.94 8.31 0 0 0 0 1.13 0 Neroli/ gorka naran~a 1.06 7.31 4.94 0.56 17.25 8.19 0 0 0 0 0.50 0 Peppermint/ metvica 1.63 18.75 11.31 0 9.50 12.75 0 9.80 0 3.25 4.00 8.06 8.63 Aniseed/ anis 6.06 17.69 16.75 11.69 19.38 21.44 2.63 17.20 25.00 0 8.88 Caraway/ kim 1.00 17.75 17.69 0.38 17.50 14.38 0 4.70 0 0 6.31 3.69 Lavander/ lavanda 1.63 14.50 10.31 1.31 3.56 10.94 0 0 0 0 0.81 1.81 Common thyme/ timijan 35.00 35.00 35.00 34.38 29.31 31.50 0 24.50 16.00 24.38 26.00 24.38 Control/ kontrola 0.31 8.25 10.88 0 0 1.50 0 0 0 0 8.06 0 LSD 0.05 0.01 2.50 3.29 3.51 4.61 3.48 4.57 2.02 2.65 1.09 1.43 2.22 1.43 1.73 2.28 1.90 2.60 1.10 1.40 3.44 4.52 3.13 4.12 1.10 1.45 DPC - D. p. var. caulivora; PV – P. viticola; DH – D. helianthi; PL – P. longicolla; HS – H. sativum; CC – C. coccodes; TC – T. cucumeris; FO – F. oxysporum; FA – F. avenaceum; FG – F. graminearum; FS – F. subglutinans; FV - F. verticillioides They had a statistically significant negative impact on mycelium growth of all investigated fungi except T. cucumeris and F. graminearum. Common thyme essential oil showed complete inhibition on mycelial growth of D.p. var. caulivora, D. helianthi, P. viticola, 98.23% inhibition on mycelial growth of P. longicolla and 90% inhibition on mycelial growth of C. coccodes. Those results are in accordance with the strong toxic properties of thyme oil and its active compounds, such as thymol and carvacrol, against a large number of microorganisms described by Soliman and Badea (2002). The major compounds found in the thyme oil used in our research were thymol (38.61%) and POLJOPRIVREDA 16:2010 (2) 25-28 p-cymene (25.02%). In our research cinnamon leaf oil showed 53.02, 80.17 and 92.31% inhibition on mycelial growth of D.p. var. caulivora, P. viticola and D. helianthi, respectively. The major compound of our cinnamon leaf oil was eugenol (75.43%) having, according to Wang et al. (2010), strong inhibitory effect against Botrytis cinerea (De Bary) Whetzel and Sclerotinia sclerotiorum (Lib.) De Bary. Edris and Ferrag (2003) indicated synergistic effect of different compounds present in natural essential oils. Bartyńska and Budzikur-Ramza (2001) described high toxicity of eucalyptus, lavender and rosemary oils against Fusarium spp. On the contrary, in our research lavander (linalol 37.04%) and rosemary (1-8 cineole 44.40%) oils did not have inhibitory effect on Jasenka ]osi} et al.:IN VITRO ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS ON GROWTH ... 27 mycelium growth of the investigated Fusarium species. Only thyme oil showed statistically significant inhibition on mycelial growth of all researched Fusarium species. On the other hand, only aniseed oil (anethole 83.50%) had statistically significant negative impact on mycelium growth of T. cucumeris and peppermint (menthol 41.06%) and common thyme oils on mycelium growth of F. graminearum. When compared to control, scots pine, neroli and sage oils stimulated mycelium growth of D. helianthi, scots pine, sage, peppermint and lavander oils stimulated mycelium growth of H. sativum (Photo 2) while rosemary oil stimulated mycelium growth of F. subglutinans. Photo 1. Influence of common thyme and cinnamon leaf essential oils on mycelium growth of P. viticola Slika 1. Utjecaj eteri~nog ulja timijana i cimeta na porast micelija P. viticola Photo 2. Influence of lavander and scots pine essential oils on mycelium growth of H. sativum Slika 2. Utjecaj eteri~nog ulja lavande i bora na porast micelija H. sativum ACKNOWLEDGEMENTS 7. The authors would like to express their gratitude to the Croatian Ministry of Science, Education and Sports for financial support awarded to the scientific project No.079-0730718-0578. 8. References 1. 2. 3. 4. 5. 6. Bartyńska M. and Budzikur-Ramza R. (2001): The action of some essential oils on fungi. Bull. Polish Acad. Sci. Biol. Sci. 49(4): 327-331. Burgieł Z.J., Smagłowski M. (2008): Fungistatyczne właściwości olejku z drzewa herbacianego [Fungistatic properties of tea tree oil]. Zesz. Probl. Post. Nauk Roln. 529: 13-18. Cardwell, K.F., Dongo, L. (1994): Effect of extracts from nine plant species found in Africa on the mycelial growth of Aspergillus flavus Link. Proc. of the 6th IWCSPP, Canberra, Australia. 978-980. Edris, A.E., Ferrag, E.S. (2003): Antifungal activity of peppermint and sweet basil essential oils and their major aroma constituents on some plantpathogenic fungi from the vapor phase. Food 47(2): 117-121. Hadizadeh, I., Pivastegan, B., Hamzehzarghani, H. (2009): Antifungal activity of essential oils from some medicinal plants of Iran against Alternaria alternata. Am. J. Applied Sci., 6: 857-861. Manohar, V., Ingram, C., Gray, J., Talpur, N.A., Echard, B.W., Bagchi, D., Preuss, H.G. (2001): Antifungal activities of origanum oil against Candida albicans. Mol. Cell. Biochem. 228: 111-117. 9. 10. 11. 12. 13. Marin, S., Velluti, A., Ramos, A.J., Sanchis, V. (2004): Effect of essential oils on zearalenone and deoxynivalenol production by Fusarium graminearum in non-sterilized maize grain. Food Microbiology 21: 313-318. Paster N., B.J. Juven, E. Shaaya, M. Menasherov, R. Nitzan, H. Weisslowicz, U. Ravid (1990): Inhibition effect of oregano and thyme essential oils applied as fumigants against fungi attacking stored grain. Journal of Food Protection 58: 81-85. Saikia D., S.P.S. Khanuja, A.P. Kahol, A.P. Gurta, S. Kumar (2001): Comparative antifungal activity of essential oils and constituents from three distinct genotypes of Cymbopogon spp. Current Science 80: 1264–1266. Soliman K.M., Badea R.I. (2002): Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food. Chem. Toxic. 40: 1669-1675. Velluti, A., Sanchis, V., Ramos, A.J., Turon, C., Marin, S. (2004): Impact of essential oils on growth rate, zearalenone and deoxynivalenol production by Fusarium graminearum under different temperature and water activity conditions in maize grain. Jour. of Applied Microbiology 96: 716-724. Wang, C., Zhang, J., Chen, H., Fan, Y., Shi, Z. (2010): Antifungal activity of eugenol against Botrytis cinerea. Tropical Plant Pathology 35(3): 137-143. Wilkins K.M., R.G. Board (1989): Natural antimicrobial systms. In G.W. Gould (Eds.), Machanisms of Action of Food Preservation Procedures. Elsevier, London POLJOPRIVREDA 16:2010 (2) 25-28 28 Jasenka ]osi} et al.:IN VITRO ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS ON GROWTH ... ANTIFUNGALNO DJELOVANJE ETERI^NIH ULJA NA PORAST FITOPATOGENIH GLJIVA IN VITRO SA@ETAK U radu je testiran utjecaj jedanaest vrsta eteričnih ulja (klinčićevac, ružmarin, list cimeta, kadulja, bor, gorka naranča, metvica, anis, kim, lavanda, timijan) na porast micelija dvanaest fitopatogenih gljiva (Fusarium graminearum, F. verticillioides, F. subglutinans, F. oxysporum, F. avenaceum, Diaporthe helianthi, Diaporthe phaseolorum var. caulivora, Phomopsis longicolla, P. viticola, Helminthosporium sativum, Colletotrichum coccodes, Thanatephorus cucumeris). Sva eterična ulja koja smo koristili, izuzev ulja bora i gorke naranče, pokazala su određeno inhibitorno djelovanje prema nekim ili svim istraživanim gljivama. Najbolje antifungalno djelovanje imala su ulja timijana, lista cimeta, klinčićevca i anisa. U usporedbi s kontrolom, ulja bora, gorke naranče i kadulje pozitivno su utjecala na porast micelija nekih gljiva. Ključne riječi: eterična ulja, inhibicija, biljna bolest, porast micelija (Received on 12 October 2010; accepted on 8 November 2010 - Primljeno 12. listopada 2010.; prihva}eno 08. studenoga 2010.) POLJOPRIVREDA 16:2010 (2) 25-28