In vitro activity of neem oil [Azadirachta indica A. Juss (Meliaceae)] on Aspergillus flavus growth, sporulation, viability of spores, morphology and Aflatoxins B1 and B2 production


The effectiveness of neem (Azadiracta indica) oil on the growth, morphology, sporulation, viability of spores, aflatoxin B1 and B2 production by A. flavus on Yeast Extract-Sucrose medium was determined. Neem oil inhibited the fungal growth (i.e. mycelia dry weight, diameter of colony and growth rate) on solid media at concentrations from 0.5 to 5.0% v/v, although it significantly increased sporulation in the same conditions. Spores obtained from cultures grown without neem oil reduced germination when incubated in a neem oil supplemented medium. Colonies grown on solid media and in submerged cultures in the presence of neem oil exhibited morphological alterations, including granular cytoplasm, atypical hyphae branching pattern, abnormal and undifferentiated conidiophores. High Performance Liquid Chromatography was used to measure aflatoxins. In submerged cultures, neem oil at concentrations from 0.5 to 4.0% v/v caused approximately 95% inhibition in Aflatoxin B1 and B2. On other hand, these conditions failed to suppress fungal growth. Current research emphasized that neem oil was not fungistatic or fungicidal, but exhibited anti-aflatoxigenic activity.

Share and Cite:

da Costa, C. , Geraldo, M. , Arrotéia, C. and Kemmelmeier, C. (2010) In vitro activity of neem oil [Azadirachta indica A. Juss (Meliaceae)] on Aspergillus flavus growth, sporulation, viability of spores, morphology and Aflatoxins B1 and B2 production. Advances in Bioscience and Biotechnology, 1, 292-299. doi: 10.4236/abb.2010.14038.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Pitt, J.I. and Hocking, A.D. (1977) Fungi and Food Spoilage. 2nd Edition, Blackie Academic & Professional, London, United Kingdom.
[2] Hedayati, M.T, Pasqualotto, A.C, Warn, P.A, Bowyer, P. and Denning, D.W. (2007) Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology, 153, 1677-1692.
[3] Holmes, R.A., Boston, R.S. and Payne, G.A. (2008) Diverse inhibitors of aflatoxin biosynthesis. Applied Microbiology and Biotechnology, 78(4), 559-572.
[4] Martinez, S.S. (2002) O Nim-Azadirachta indica. Natureza, Usos Múltiplos, Produ??o. Martinez, S.S. Ed., IAPAR, Londrina, PR, Brazil.
[5] Locke, J.C. (1995) Fungi. In Schmutterer, H. Ed., The Neem Tree. VHC. Weinheim, Germany, 118-126.
[6] Bhatnagar, D. and McCormick, S.P. (1988) The inhibitory effect of neem (Azadirachta indica) leaf Extracts on aflatoxin synthesis in Aspergillus parasiticus. Journal of American Oil Chemical Society, 65(7), 1166-1168.
[7] Zeringue, H.J. and Bhatnagar, D. (1994) Effects of neem leaf volatiles on submerged cultures of aflatoxigenic Aspergillus parasiticus. Applied and Environmental Microbiology, 60(10), 3543-3547.
[8] Zeringue, H.J. and Bhatnagar, D. (1990) Inhibition of aflatoxin production in Aspergillus flavus infected cotton bolls after treatment with neem (Azadirachta indica) leaf extracts. Journal of American Oil Chemical Society, 67(4), 215-216.
[9] Allameh, A., Razzaghi-Abyaneh, M., Shams-Gahahfarokhi, M., Rezzar, M.B. and Jaimand, K. (2001) Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase, isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus. Mycopathologia, 154(2), 79-84.
[10] Razzaghi-Abyaneh, M., Allameh, A., Tiraihi, T., Shams- Ghahfarokhi, M. and Ghorbanian, M. (2005) Morphological alterations in toxigenic Aspergillus parasiticus exposed to neem (Azadirachta indica) leaf and seed aqueous extracts. Mycopathologia, 159(4), 565-570.
[11] Mossini, S.A.G., de Oliveira, K.P. and Kemmelmeier, C. (2004) Inhibition of patulin production by Penicillium expansum cultured with neem (Azadirachta indica) leaf extracts. Journal of Basic Microbiology, 44(2), 106-113.
[12] Arrotéia, C.C., Kemmelmeier, C. and Machisnki, Jr., M. (2007) Effect of aqueous and oily extracts of Neem [Azadirachta indica A. Juss (Meliaceae)] on patulin production in apples contaminated with Penicillium expansum. Ciência Rural, 37, 1518-1523.
[13] Mossini, S.A.G. and Kemmelmeier, C. (2008) Inhibition of citrinin production in Penicillium citrinum cultures by neem [Azadirachta indica A. Juss (Meliaceae)]. International Journal of Molecular Science, 9(9), 1676-1684.
[14] Costa, C.L. and Kemmelmeier, C. (2008) Effect of aqueous and oily extracts from Neem [Azadirachta indica A. Juss (Meliaceae)] on the production of mycotoxins by the polyketide pathway (penicillic acid and sterigmatocystin). Current topics in Biotechnology, 4, 35- 40.
[15] Fandohan, P., Gbenou, J.D., Gnonlonfin, B., Hell, K., Marasas, W.F.O. and Wingfield, M.J. (2004) Effect of essential oils on the growth of Fusarium verticillioides and fumonisin contamination in corn. Journal of Agricultural and Food Chemistry, 52(22), 6824-6829.
[16] Mossini, S.A.G., Arrotéia, C.C. and Kemmelmeier, C. (2009) Effect of Neem Leaf extract and Neem oil on Penicillium growth, sporulation, morphology and ochratoxin a production. Toxins, 1, 2-13.
[17] Govindachari, T.R., Suresh, G., Gopalakrishnan, G., Banumathy, B. and Masilamani, S. (1998) Identification of antifungal compounds from the seed oil of Azadirachta indica. Phytoparasitica, 26(2), 109-116.
[18] Smith, D. and Onions, A.H.S. (1983) The preservation and maintenance of living fungi. Commonwealth Mycological Institute, Page Bros Ltd., Great Britain.
[19] Betina, V. (1984) Prodution, Isolation, Separation and Purification. In: Betina, V. Ed., Mycotoxins. Elsevier Science Publishers BV, Amsterdam, Netherlands, 183-215.
[20] Davis, N.D., Diener, U.L. and Eldridge, D.W. (1966) Production of aflatoxin B1 and G1 by a Aspergillus flavus in a semi-synthetic medium. Applied Microbiology, 14(3), 378-380.
[21] Farag, R.S., Daw, S.Y. and Abo-Raya, S.H. (1989) Influence of some spice essential oils on Aspergillus parasiticus growth and production of aflatoxins in a synthetic medium. Journal of Food Science, 54, 74-76.
[22] Passone, M.A., Resnik, S.L. and Etcheverry, M.G. (2005) In vitro effect of phenolic antioxidants on germination, growth and aflatoxin B1 accumulation by peanut Aspergillus section Flavi. Journal of Applied Microbiology, 99(3), 682-691.
[23] Sutton L.M. and Starzyk, M.J. (1972) Procedure and analysis of a useful method in determining mycelial dry weights from agar plates. Applied Microbiology, 24(6), 1011-1012.
[24] Bluma, R.V, Etcheverry, M.G. (2008) Application of essential oils in maize grain: Impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation. Food Microbiology, 25(2), 324-334.
[25] López-Malo, A., Alzamora, S.M. and Argaiz, A. (1995) Effect of natural vanilin on germination time and radial growth of moulds in fruit-based agar systems. Food Microbiology, 12, 213-219.
[26] Vargas-Isla, R. and Ishikawa, N.K. (2008) Optimal conditions of in vitro mycelial growth of Lentinus strigosus, an edible mushroom isolated in the Brazilian Amazon. Mycoscience, 49(3), 215-219.
[27] Gusmán-De-Pe?a, D. and Ruiz-Herrera, J. (1997) Relationship between Aflatoxin Biosynthesis and Sporulation in Aspergillus parasiticus. Fungal Genetics and Biology, 21(2), 198-205.
[28] Bischoff, T.W. and Garraway, M.O. (1985) Ammonium production by races and mating types of Bipolaris maydis and its relationship to mycelium dry weight, sporulation and pH of the culture medium. Ohio Journal of Science, 85, 155-158.
[29] Marques, R.P., Monteiro, A.C. and Pereira, G.T. (2004) Crescimento, esporula??o e viabilidade de fungos entomopatogênicos em meios contendo diferentes concentra??es do óleo de Nim (Azadirachta indica). Ciência Rural, 34, 1675-1680.
[30] Deacon, J.W. (2006) Fungal Biology, 4th edition. Blackwell Publishing, Oxford, UK.
[31] Bankole, S.A. (1997) Effect of essential oils from two Nigerian medicinal plants (Azadirachta indica and Morinda lucida) on growth and aflatoxin B1 production in maize grain by a toxigenic Aspergillus flavus. Letters in Applied Microbiology, 24(3), 190-192.
[32] Ghorbanian, M., Razzaghi-Abyaneh, M., Allameh, A., Shams-Ghafarokhi, M. and Qorbani, M. (2008) Study on the effect of neem (Azadirachta indica A. juss) leaf extract on the growth of Aspergillus parasiticus and production of aflatoxin by it at different incubation times. Mycoses, 51(1), 35-39.
[33] Sithisarn, P., Supabphol, R. and Gritsanapan, W. (2005) Antioxidant activity of Siamese neem tree (VP1209). Journal of Ethnopharmacology, 99(1), 109-112.
[34] Gowda, N.K.S., Malathi, V. and Suganthi, R.U. (2004) Effect of some chemical and herbal compounds on growth of Aspergillus parasiticus and aflatoxin production. Animal Feed Science and Technology, 116, 281-291.
[35] Suberu, H. (2004) Preliminary studies of inhibitions in Aspergillus flavus with extracts of two lichens and Bentex-T fungicide. African Journal of Biotechnology, 3(9), 468-472.
[36] Wilson, D.M., Gueldner, R.C., McKinney, J.K., Lievsay, R.H., Evans, B.D. and Hill, R.A. (1981) Effect of β-ionone on Aspergillus flavus and Aspergillus parasiticus growth, sporulation, morphology and aflatoxin production. Journal of the American Oil Chemists’ Society, 58, A959-A961.
[37] Kale, S.P., Cary J.W., Bhatnagar, D. and Bennet J.W. (1996) Characterization of experimentally induced, nonaflatoxigenic variant strains of Aspergillus parasiticus. Applied and Environmental Microbiology, 62(9), 3075- 3092.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.