Antifungal activities and chemical characterization of Neem leaf extracts on the growth of some selected fungal species in vitro culture

The efficacy of different extracts of neem leaf on seed borne fungi Aspergillus, Rhizopus and chemical characterization of the neem leaf extracts were studied in vitro on the culture medium. The growth of both the fungal species was inhibited significantly (p<0.01) and controlled with both alcoholic and water extract of all ages and of the concentrations used. The alcoholic extracts of neem leaf was most effective in comparison to aqueous extract for retarding the growth of Rhizopus and Aspergillus. The crude aqueous and alcoholic leaf extracts of neem was more effective in inhibitions of growth of the fungi Aspergillus in comparison to inhibitory effects on Rhizopus growth in the artificial culture medium. Leaf extracts of neem which are cheap and environmentally safe are promising for protecting crop species against the fungal infestation and leading towards improvement of the crop in terms of yield and productivity. @ JASEM A large number of chemicals have been developed for the control of plant diseases. But due to overgrowing awareness of the hazardous side effects of these chemicals, more and more emphasis is being given to the use of biocontrol agents. Now major challenge is felt in the field of plant pathology to introduce some ecofriendly and safe alternative control strategies for agriculture, which led researchers to turn their attention to plants and microorganisms as sources of biocontrol agents. As the sources of biocontrol agent, neem has already emerged at the top of the list of plants with the highest potential. The following species of neem trees of Meliaceae family have been the subject of botanical biocontrol research: Azadirachta indica A. Juss., A. excelsa Jack, A. siamens Valeton, Melia azadirachta L, M. toosendon Sicb. and Zucc. and M. volkensii Gurke. The Meliaceae specially A. indica (Indian neem tree), contains at least 35 biologically active principals of which Nimbin and azadirachtin (T.D. Pennigton el al., 1981) are the most active insecticidal ingradients and are present predominantly in the seeds, leaves and other parts of the neem tree (Mulla et at., 1999). In this study the effect of the leaf extracts of various ages of neem leaves along with different extractants. On in vitro culture medium of Aspergillus and Rhizopus were studied and subsequent chemical characterization of the need leaf extracts were mediated for its antifungal activity. MATERIAL AND METHODS Extraction of leaf extracts: Juvenile and mature leaves were collected separately from Azadirachta indica plants growing in University campus, Burdwan University. For antifungal and secondary metaibolite studies fresh leaves of 2-4 days old and 79 days old were collected during emergence time (February-March.). Collected fresh leaves of A. indica (Neem) were washed thoroughly in tap water and sterile distilled water, air-dried at 27°C, weighted (l00g) and ground in a sterile mortar. The paste was added to 100ml of sterile distilled water in 250 ml beaker, stirred vigorously and allow to stand for 1 hour and then filtered through four folds of sterile cheese cloth to obtain water extract. Percentage inhibition of fungi growth by the leaf extracts was calculated using the formula: % FG = Dc-Dr/Dc x100 Where: %FG = % inhibition of fungi growth Dc = diameter of control Dr = diameter of test In vitro tests: Species of Aspergillus and Rhizopus were collected from the Mycology and Plant Pathology Laboratory, Department of Botany, The University of Burdwan and are maintained in pure live on potato dextrose agar (PDA) slants at 4 C. For evaluation of in vitro antifungal activity of the biocide (plant extract of Azadirachta indica) the phytoextracts were added to Potato Dextrose Agar (PDA) medium in different concentrations (0.1%; 0.5% and 1%) in separate sterilized petriplates. Each plate was inoculated with a mycelial disc (5mm diameter) taken from 7-day-old culture raised on PDA. The inoculated plates were incubated at 30 ± 1°C and the diameter of colony of the pathogen was measured in each case for successive 7 days (Dutta, 2001). The results are shown in table 1. Table: l: Effect of crude aqueous and alcoholic leaf extracts of Neem (A. indica) of different ages on the growth of Rhizopus sp. and Aspergillus sp. in artificial culture medium. Antifungal activities and chemical characterization of Neem leaf extracts on the growth of some selected fungal species in.......... * Corresponding author: *Mondall , N. K. 50 Measurement of percentage inhibition of Growth (mm) of fungi in artificial medium after hours of incubation Experimental material Nature of leaf extract Ages of leaf (days) Conc. of extract (%) 24 48 72 96 120 144 196 Alcoholic Extract Young 2-4 days 0.1 0.5 1.0 SD(1%) 15.79 31.58 100.0 1.30 12.0 24.0 100.0 1.071 36.84 47.36 81.58 1.08 32.69 36.54 78.85 1.21 46.91 49.38 82.72 1.28 45.56 48.89 78.89 1.29 4.0 42.22 68.89 1.31 Mature 7-9 Days 0.1 0.5 1.0 LSD(1%) 21.05 42.10 100.0 1.19 24.00 36.0 100.0 0.95 44.74 5.00 84.21 1.05 34.92 38.46 84.62 1.89 49.38 56.79 87.65 0.88 47.78 57.78 84.44 0.68 42.22 51.11 73.33