Evaluation of Antifungal Effect of Thyme and Peppermint Essential Oils and Their Major Monoterpenes in Controlling Verticillium Fungus on Pistachios

Document Type : scientific research article


1 Corresponding Author, Plant Protection Research Department, Agricultural and Natural Resources Research Center of Semnan Province (Shahrood), AREEO, Shahrood, Iran

2 Food Technology Research Department, Agricultural Engineering Research Department, Agricultural and Natural Resources Research and Education Center of Semnan Province (Shahrood), AREEO, Shahrood, Iran

3 Agricultural Engineering Research Department, Agricultural and Natural Resources Research and Education Center of Semnan Province (Shahrood), AREEO, Shahrood, Iran

4 . Plant Protection Research Department, Agricultural and Natural Resources Research and Education Center of Semnan Province (Shahrood), AREEO, Shahrood, Iran


Background and purpose: Wilt disease caused by the pathogenic fungus Verticillium dahliae is one of the dangerous factors threatening the cultivation of pistachio trees in the country, which has spread in different parts of the country in the last two decades, including Semnan province. Lack of suitable fungicides to control V. dahliae and continuous use of several common fungicides in the country has contributed to the spread of the fungus. Therefore, in the current situation, we need to use new compounds with high efficiency to control this pathogenic fungus. Plant essential oils and their monoterpene compounds are considered as one of the important options due to the comparative advantage of the production of medicinal plants in the country.
Materials and Methods: In this study, V. dahliae native isolates obtained from gardens of Semnan province were used. Isolation and identification process was performed using conventional methods and isolates were identified using general ITS primer. Then, the essential oils of thyme (Zataria multiflora) and Peppermint (Mentha piperita) were used to evaluate the antifungal effect. After preparing the herbal plants, the essential oil extraction process was performed using Clevenger method. The antifungal effect was investigated by mixing with culture medium at concentrations of 20, 40, 60, 80 and 100 μg/ml. Also, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MBC) were determined for the two essential oils. Then, using thymol and carvacrol monoterpenes, antifungal effects were tested by mixing with culture medium at three concentrations of 15, 150 and 1500 μg/ml against V. dahliae.
Results: Isolation and identification tests confirmed the presence of V. dahliae in the sampled areas. The use of ITS primer confirmed the fungal species V. dahliae. The results of antifungal effect showed that both essential oils were effective in preventing the growth of fungi, but Zataria multiflora essential oil was evaluated as stronger, so that in the third week, growth was observed only at a concentration of 20 μg/ml, but in the case of peppermint essential oil, fungus growth was observed in all concentrations. The results showed that the minimum inhibitory (MIC) and minimum fungicidal (MFC( concentrations were 20 and 30 μg/ml for thyme and 100 and 150 μg/ml for peppermint essential oils, respectively. In addition, the results showed that thymol was more potent than carvacrol in preventing the growth of V. dahliae.
Conclusion: The results of this study showed that plant essential oils and monoterpene compounds of essential oils had a very high ability to control Verticillium fungus, so that compared to common fungicides in the country, their inhibitory concentration was 10 to 15 times lower


1.AL-Saghir, M.G. and Porter, D.M. 2012. Taxonomic revision of the genus Pistacia L. (Anacardiaceae). Am. J. Plant Sci.
3: 1. 12-32.
2.Taghizadeh-Alisaraei, A., Alizadeh Assar, H., Ghobadian, B. and Motevali, A. 2017. Potential of biofuel production from pistachio waste in Iran. Renew. Sustain. Energy Rev. 72: 510-522.
3.Eskalen, A., Küsek, M., Danıstı, L. and Karada, S. 2001. Fungal diseases in pistachio trees in East-Mediterranean and Southeast Anatolian regions. In ‘11 GREMPA Seminar on pistachios and almonds. Zaragoza, CIHEAM-IAMZ’. (Ed. BE Ak) pp. 261-264.
4.Jafary, H., khanmohammadi, S. and Mehri, N. 2014. Detection of pathotypes of Verticillium dahliae, the causal agent of olive Verticillium wilt in olive orchards of Tarom using Nested-PCR technique.J. Appl. Res. Plant. Prot. 2: 2. 47-58.
(In Persian)
5.Nasimi, Z., Bani Hashmi, Z. and Mostofi zade, R. 2013. In vivo interaction of Verticillium dahliae, the causal agent of pistachio Verticillium with Acremonium kiliense. Iran. J. Plant Path. 49: 4. 127-129. (In Persian)
6.Mohammadi, A.H., Haghdel, M., Moghaddam, M.M. and Banihashemi, Z. 2006. Current status of Verticillium wilt disease of pistachio trees in Iran, IV International Symposium on Pistachios and Almonds.
7.Fotoohiyan, Z., Rezaee, S., Shahidi Bonjar, G.H., Mohammadi, A.H. and Moradi, M. 2015. Induction of systemic resistance by Trichoderma harzianum isolates in pistachio plants infected with Verticillium dahliae. J. Nuts. 6: 2. 95-111. (In Persian)
8.Bajpai, V.V., Shukla, S. and Kang, S.C. 2008. Chemical composition and antifungal activity of essential oil and various extract of Silene armeria L. Bioresource Tech. 99: 18. 8903-8908.
9.Aali, E., Mahmoudi, R., Kazeminia, M., Hazrati, R. and Azarpey, F. 2017. Essential oils as natural medicinal substances: review article. Tehran Univ. Med. J. 75: 7. 480-489. (In Persian)
10.Kamatou, G.P. and Viljoen, A.M.2010. A review of the applicationand pharmacological properties ofα-bisabolol and α-bisabolol-rich oils. J. Am. Oil Chem. Soc. 87: 1. 1-7.
11.Mansoori, S., Dakhili, M. andZargar, M. 2014. The comparison of concentration in-vitro effects of Zataria multiflora Boiss and Mentha pulegium essential oil plants composition with one of them Nystatin on Candida albicans isolates of vaginal candidiasis and comparing their with standard. Appl. Biology .4: 2. 1-14. (In Persian)
12.Pina-Vaz, C., Rodrigues, A.G., Pinto, E., Costa-de-Oliveira, S., Tavares, C., Salgueiro, L.R., Cavaleiro, C., Goncalves, M.J. and Martinez-de-Oliveira, J. 2004. Antifungal activity of Thymus oils and their major compounds. J. Eur. Acad. Dermatol. 18: 1. 73-78.
13.Arslan, M. and Dervis, S. 2010. Antifungal activity of essential oil against three vegetative compatibility groups of Verticillium dahliae. World J. Microbiol. Biotechnol. 26: 1813-1821.
14.Amini, J., Farhang, V., Javadi, T. and Nazemi, J. 2016. Antifungal effect of plant essential oils on controlling Phytophthora species. Plant Path. J.32: 16-24.
15.Hillocks, R.J. 1992. Cotton Diseases. CAB International, Wallingford, UK. 415p.
16.Damjanovi´c-Vratnica, B., Dakov, T., Sukovic, D. and Damjanovic, J. 2011. Antimicrobial effect of essential oil isolated from Eucalyptus globulus Labill. from Montenegro. Czech J. Food Sci. 29: 3. 277-284.
17.Sharifi-Rad, J., Miria, A., Hoseini-Alfatemic, S.M., Sharifi-Radd, M., Setzere, W.N. and Hadjiakhoondif, A. 2014. Chemical composition and biological activity of Pulicaria vulgaris essential oil from Iran. Nat. Prod. Commun. 9: 11. 1633-1636.
18.Shariari, F., Tanhaeian, A., Akhlaghi, M. and Nazifi, N. 2018. Comparison of antimicrobial activity of essential oils and plant extracts with recombinant peptide in controlling of some pathogens of cultivated white button mushrooms.
J. Hort. Sci. 32: 4. 615-628. (In Persian)
19.Riccioni, L. and Orzali, L. 2011. Activity of tea tree (Melaleuca alternifolia, Cheel) and thyme (Thymus vulgaris, Linnaeus.) essential oils against some pathogenic seed borne fungi. J. Essent. Oil Res. 23: 6. 43-47.
20.Ghezelbash, N., Abdolahi, M. and Shahriari, D. 2012. Evaluation of antifungal activity of Shirazi thyme and Chavil extracts on Fusarium oxysporum f.sp. lycopersici, the causal agent of tomato wilt under laboratory and greenhouse conditions. Plant Protec.36: 4. 53-65. (In Persian)
21.Mehran, M., Hosseini, H. Hatami, A., Taghizadeh, M. and Safaei, A. 2015. Investigation of components of seven species of thyme essential oils and comparison of their antioxidant properties. J. Med. Plants. 58: 2. 134-140. (In Persian)
22.Abbaszadeh, S., Sharifzadeh, A., Shokri, H., Khosravi, A.R. and Abbaszadeh, A. 2014. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J. Mycol. Med. 24: 2. 51-56.
23.Abdolmaleki, M., Bahraminejad, S., Salari, M., Abbasi, S., Panjeke, N.2011. Antifungal activity of peppermint (Mentha piperita L.) on phytopathogenic fungi. J. Med. Plants. 10: 38. 26-34.
24.WHO. http://apps.who.int/ medicinedocs/ en/d/Js2200e/28. html2012.
25.Jukic, M., Politeo, O., Maksimovic, M., Milos, M. and Milos, M. 2007. In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone. Phytother. Res.21: 3. 259-61.
26.Didry, N., Dubreuil, L. and Pinkas, M. 1994. Activity of thymol, carvacrol, cinnamaldehyde and eugenole on oral bacterial. Pharm. Acta. Helv. 69: 1. 25-8.
27.Akbarinia, A., Sharifi Ashoorabadi, E. and Mirza, M. 2011. Study on drug yield and essential oil content and composition of Thymus daenensis Celak. under cultivated condition. Iranian J. Med. Arom. Plants. 26: 2. 205-212.
(In Persian)
28.Gonçalves, G.M.S, Srebernich, S.M., Bragagnolo, N., Madalozzo, E.S., Merhi V.L. and Pires, D.C. 2013. Study of the composition of Thymus vulgaris essential oil, developing of topic formulations and evaluation of antimicrobial efficacy. J. Med. Plant Res. 7: 2. 1736-1745.
29.Lahoogi, A., Mirabolfathi, M. and Karami Osboo, R. 2010. Effect of Zataria multiflora and Satureja hortensis essential oils, Thymol and carvacrol on growth of Fusarium graminearum isolates and deoxynivalenol production. J. Plant Pathology. 46: 1. 37-50. (In Persian)
30.Najb-Zadeh, T., Yadegari, M., Nagdi Badi, H. and Salehnia, E. 2011. Antifungal efficacy of Myrtus communis essential oils on oral candidiasis in immunosupressed Rats. J. Med. Plants. 10: 38. 102-116. (In Persian)
31.Sharifian, M., Bolhari, B., Nosrat, A. and Aligholi, M. 2009. The effect of carvacerol on Enterococcus faecalis as an intracanal medicament-Invitro study. J. Dent. Med. 22: 1. 35-40.
32.Kumar, A., Shukla, R., Singh, P., Prasad, C.S. and Dubey, N.K. 2008. Assessment of Thymus vulgaris L. essential oil as a safe botanical preservation against post-harvest fungal infestation of food commodities. Innov. Food Sci. Emerg. Technol. 9: 4. 575-580.
33.Kale, S., Cary, J.W., Bhatnagar, D. and Bennett, J.W. 1996. Characterization of experimentally induced, non aflatoxigenic variant strain of Aspergillus parasiticus. Appl. Environ. Microbiol. 62: 9. 3399-404.
34.Rasooli, I. and Owlia, P. 2005. Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production. Phytochemistry. 66: 24. 28-36.
35.Zambonelli, A.D., Aurelio, A.Z., Bianchi, A. and Albasini, A. 1996. Effect of essential oils on phytopathogenic fungi. J. Phytopathol. 144: 9. 491-4.
36.Ultee, A., Kets E.P.W. and Smid, E.J. 1999. Mechanism of action of carvacrol on the food borne pathogen Bacillus cereus. Appl. Environ. Microbiol.65: 10. 46-10.
37.Meepagala, K.M., Sturtz, G. and Wedge, D.E. 2002. Antifungal constituents of the essential oil fraction of Artemisia drancunculus L. var. dracunculus. J. Agric. Food Chem. 50: 24. 6989-6992.