Journal of Agricultural Technology Antifungal activity of a known medicinal plant Mimusops elengi L. against grain moulds S. Satish, M.P. Raghavendra, D.C. Mohana and K.A Raveesha *. Agricultural Microbiology Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore - 570006, India Satish, S., Raghavendra, M.P., Mohana, D.C. and Raveesha, K.A. (2008). Antifungal activity of a known medicinal plant Mimusops elengi L. against grain moulds. Journal of Agricultural Technology 4(1): 151-165. The aqueous and different solvent extracts viz., petroleum ether, benzene, chloroform, methanol and ethanol extracts and isolated constituents of Mimusops elengi L. (Sapotaceae) was screened in vitro for antifungal activity, by poisoned food technique against wide array of seed borne phytopathogenic fungi. The test organisms included Alternaria alternata, two species of Drechslera, eight species of Fusarium, ten species of Aspergillus and three species of Penicillium, which are frequently associated with sorghum [Sorghum bicolor (L.) Moench], maize (Zea mays L.) and paddy (Oryza sativa L.) seeds. Aqueous, methanol and ethanol extract recorded highly significant antifungal activity against all the tested fungi. Methanol extract was subsequently fractioned and monitored by antifungal activity guided assay leading to the isolation of an active fraction and confirmed as alkaloids by further phytochemical analysis. The results indicated that the antifungal activity of alkaloid fraction is highly significant compared to Dithane M-45 and other fungicides. Mimusops elengi L. has significant medicinal value, hence the results of the present investigation indicate that, it could be exploited in the management of seed borne pathogenic fungi and prevented biodeterioration of grains and mytoxin elaboration during storage. Key words: Mimusops elengi, antifungal activity, poisoned food technique, sorghum Introduction Plants have been formed the basis of natural pesticides, that make excellent leads for new pesticide development (Newman et al., 2000). The potential of higher plants as a source of new drugs is still largely unexplored. Hence, last decade witnessed an increase in the investigation on plants as a source of new biomolecules for human disease management (Grierson and Afolayan, 1999). Traditionally plants have been well exploited by man for the treatment of human diseases, Ayurveda is a good example, but not much *Correspondence to: Raveesha, K.A.; e-mail: [email protected] 151 Journal of Agricultural Technology information is available on the exploitation of plant wealth for the management of plant diseases, especially against phytopathogenic fungi. Fungi cause severe damage to stored food commodities. Among different species of fungi Aspergillus sp., Fusarium sp. and Penicillium sp. are associated with heavy loss of grains, fruits, vegetables and other plant products during picking, transit and storage rendering them unfit for human consumption by producing mycotoxins and affecting their nutritive value (Miller, 1995; Janardhana et al., 1999; Galvano et al., 2001). Many seed borne fungi, which cause severe damage to stored food commodities, were generally managed by synthetic chemicals, which were considered both efficient and effective. The continuous use of these synthetic fungicides started unraveling nonbiodegradability and known to have residual toxicity to cause pollution (Pimentel and Levitan, 1986). Pesticide pollution of soil and water bodies is well documented (Nostro et al., 2000). Hence in recent time application of plant metabolites for plant disease management has become important viable component of Integrated Pest Management, as plant metabolites are eco-friendly. Mimusops elengi is a wild plant distributed in tropical and subtropical region belonging to the family sapotaceae. Earlier report reveals that the fruits are used in chronic dysentery, constipations; flowers are used as snuff to relive headache, lotion for wounds and ulcers. Barks are used to increase fertility in women and known to have antiulcer activity (Shah et al., 2003). They are rich source of tannin, saponin, alkaloids, glucoside, ursolic acid (Anonymous, 1969). A pentacylcic triterpene 3β, 6β, 19α, 23-tetrahydroxy-urs-12-ene reported from bark recorded moderate inhibiting activity against β– glucuronidase enzyme associated with gastric ulcers (Jahan et al., 2001). Seeds of Mimusops is known to contain several saponins such as mimusin Mi- saponin A and 16α-hydroxy Mi-saponin A (Sahu et al., 1997), taxifolin, α- spinasterol glucoside, Mi-glycoside 1, mimusopside A and B (Sahu, 1996). Seed kernel also reported to have saponins (Lavaud et al., 1996). A scientific and systematic phytochemical investigation of leaves with regard to the various biological activities in general and antifungal activity against phytopathogenic fungi in particular of this plant is lacking, hence the present study. Considering these, higher plants are routinely screened in our laboratory; during this routine screening Mimusops elengi recorded highly significant antifungal activity. Thus detailed investigations were conducted to test the efficacy of M. elengi against important wide array of seed borne phytopathogenic fungi. 152 Journal of Agricultural Technology Materials and Methods Collection of plant materials Fresh leaves of Mimusops elengi free from diseases were collected from Mysore (India), washed thoroughly 2-3 times with running tap water and once with sterile water, shade-dried, powdered and used for extraction. A voucher specimen of the plant is deposited in the herbarium of Department of Studies in Botany, University of Mysore, Mysore. Karnataka, India. Preparation of extracts Aqueous extract Samples (50 g) of shade dried, powder of leaves of M. elengi was macerated with 100 ml of sterile distilled water in a Waring blender (Waring International, new Hartford, CT, USA) for 10 min. The macerate was first filtered through double layer muslin cloth and then centrifuged at 4000 g for 30 min. The supernatant was filtered through Whatman No. 1 filter paper and heat sterilized at 120 ºC for 30 min (Satish et al., 1999). The extract was preserved aseptically in a brown bottle at 5 ºC until further use. Solvent extracts Fifty gram of shade dried, powder of M. elengi was filled in the thimble and extracted successively with petroleum ether, benzene, chloroform, methanol and ethanol using a Soxhlet extractor for 48 h. (Mohana and Raveesha, 2006). All the extracts were concentrated using rotary flash evaporator and preserved at 5 ºC in airtight bottles until further use. All the extracts were subjected to antifungal activity assay. Phytochemical analysis Phytochemical analysis of the evaporated methanol extract was conducted following the procedure of Anonymous (1985) and Harborne (1998). Methanol extract was fractioned in to different fractions as acidic (fraction 1), phenolic (fraction 2), alkaloid (fraction 3) and neutral (fraction 4) following the procedure of Roberts et al. (1981). All the fractions were again subjected to antifungal activity. 153 Journal of Agricultural Technology Isolation of important phytopathogenic fungi Seed samples of sorghum, paddy rice and maize were collected directly from farmer fields, regulated markets, warehouse and retail shops to isolate the important pathogenic fungi associated with these seeds. The collected seed samples were subjected to standard blotter method (ISTA, 1996) and incubated in alternative cycles of dark and light. On the seventh day of incubation, samples were screened for seed mycoflora with the help of stereobinocular microscope and compound microscope. Associated fungi were identified based on growth characteristic, mycelial morphology, spore morphology and other important characters using standard manuals. The fungi, which were frequently associated in higher percentage in sorghum were selected which served as tested fungi. Anti-fungal activity assay Different concentrations (10, 20, 30, 40 and 50%) of aqueous extract, all the solvent extracts and isolated constituents (Fraction I to IV) of Mimusops elengi were subjected to antifungal activity assay by poisoned food technique. In Czepak Dox Agar (CDA) medium, the extracts were added to the medium to achieve the desired concentrations in the medium, autoclaved and poured into Petridishes (20 ml each) and allowed to cool. After complete solidification of the medium, 5 mm disc of 7-day-old culture of the tested fungi were transferred. Four replicates were maintained for each concentration. The CDA media devoid of the extract served as control. The plates were incubated at 26±1ºC for seven days. The fungitoxicity of the extract in terms of percentage inhibition of mycelial growth was calculated by using the formula: %inhibition = dc – dt x 100/dc Where dc=Average increase in mycelial growth in control, dt=Average increase in mycelial growth in treatment (Singh and Tripathi, 1999). Synthetic fungicides, viz., Blitox, Captan, Dithane M-45 and Thiram were also tested at their recommended dosage (2000 ppm) for antifungal activity by poisoned food technique for comparative studies (Zehavi et al., 1986). 154 Journal of Agricultural Technology Results Antifungal activity Aqueous extract It is interesting to note that all the fungi recorded 50% of mycelial growth inhibition at 50% concentration of the aqueous extract, except A. tamari. Among twenty-four fungi tested Drechslera sp. recorded high susceptibility with both D. halodes and D. tetramera which showed complete inhibition at 50% concentration. F. graminearum showed maximum susceptibility among Fusarium species, which showed 100% susceptibility. A flavus and A. versicolor recorded more than 90% of inhibition compared to other Aspergillus species tested and P. chrysogenum showed maximum susceptibility among Penicillium species (Table 1). Solvent extracts Among different solvent extracts tested, methanol and ethanol extracts recorded highly significant antifungal activities. Whereas, activity was not observed in petroleum ether, benzene and chloroform extracts (Table 2). Even for the methanol extract, all tested fungi showed over 50% of susceptibility among which, D. halodes, D. tetramera, F. equiseti, F. graminearum, F. moniliformae, F. proliferatum, F. solani, A. flavus, A. fumigtus, A. versicolor, P. chrysogenum and P. griseofulvum which recorded over 80% of mycelial growth inhibition at 2 mg/ml concentration. A. niger was the least susceptible and A. flavus was the most susceptible among the 24 tested fungi. Antifungal activities were varied among different tested pathogenic fungi against ethanol extract, which recorded significant activities next to methanol extract. Phytochemical analysis Phytochemical analysis of methanol extract revealed the presence of carbohydrates and glycosides, proteins and amino acids, alkaloids, phenolic compounds and tannin. Phytosterols, saponins, oils, gum and mucilage were found absent in methanol extract (Table 3). 155 Journal of Agricultural Technology 156 Journal of Agricultural Technology 157 Journal of Agricultural Technology 158 Journal of Agricultural Technology Comparative efficacy of different fractions with fungicides Fraction III (Alkaloids) recorded highly significant antifungal activity, where as activity was not observed in fraction I, II and IV indicating the nature of active principle. The susceptibility of tested fungi to alkaloid fractions varied, D. halodes recorded high susceptibility and F. oxysporum was least susceptible. Comparative efficacy of alkaloid fraction with fungicides is presented in Table 4. Among four tested fungicides, Thiram recorded significant antifungal activity and Dithane M-45 with least antifungal activities. The activity of alkaloid fraction is highly significant compared to Dithane M-45 and other tested fungicides at recommended dosage of 2000 ppm. Table 3. Preliminary phytochemical analysis of methanol extract of Mimusops elengi. Tests Methanol extract 1. Carbohydrates/Glycosides + + 2. Proteins/Aminoacids + + 3. Alkaloids + + 4. Phytosterols - - 5. Phenolic compounds + + 6. Saponin -- 7. Tannin ++ 8. Oils -- 9. Gums and mucilage -- ++ Present, -- Absent. 159 Journal of Agricultural Technology 160