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Endophytic fungi live inside the plant without causing any negative effect on the host; rather they protect the host plant from biotic and abiotic stresses. The ability of endophytic fungi of grasses to provide protection from insect herbivore drew the attention of researchers to exploit the endophytic fungi for better plant health management. Endophytic fungi have been isolated from almost every plant tried for isolation of endophytic fungi. Bio-activity of endophytic fungi isolated from plants has been reported against wide range of pests and pathogens.
In this study, endophytic fungi were isolated from leaf and stem of Indian medicinal plants Withania somnifera, Jatropha curcas and Tylophora indica. They were grouped into 20 morphotypes. Further identification was done by rDNA sequencing of ITS region. Based on molecular and morphological characters these fungi were identified as Dothideomycetes sp. isolate EF1 and Alternaria tenuissima isolate EF2 isolated from leaf of T. indica while Thielavia subthermophila isolate EF3, Alternaria sp. isolate EF4, Nigrospora oryzae isolate EF5, Colletotrichum truncatum isolate EF6 and Chaetomium sp. isolate EF7 isolated from stem of T. indica. From W. somnifera, Dothideomycete sp. isolate EF19, A. tenuissima isolate EF20, and Chaetomium globosum isolate EF18 were isolated from leaf and isolate of C. globosum EF17 was isolated from the stem. N. oryzae isolate EF8, C. truncatum isolates EF9 and EF 10, Fusarium proliferatum isolate EF11, Chaetomium sp. isolate EF12, C. truncatum isolates EF13 and EF 14, Guignardia camelliae isolate EF15 and Alternaria destruens isolate EF16 were obtained from leaves of J. curcas.
These endophytic fungi were subjected to dual culture bioassay against plant pathogenic fungi Fusarium oxysporum, Sclerotinia sclerotiorum and Rhizoctonia solani. Isolates EF1, EF2, EF3, EF4, EF6, EF7, EF9, EF10, EF12, EF13, EF14, EF17, EF18 and EF20 exhibited good activity against S. sclerotiorum and F. oxysporum, while EF15 and EF19 were active against S. sclerotiorum only. None of the isolated exhibited activity against R. solani.
Based on dual culture assay of endophytic fungi, EF18 from W. somnifera, EF9, EF10, EF12, EF13, EF15 from J. curcas and EF1 and EF3 from T. indica were subjected to metabolites extraction and further bioassays. Ethyl acetate extract of C. globosum EF18 showed 75.68% growth inhibition (GI) at concentration of 500 μg/ ml. Methanol extract of C. truncatum EF13 was found most active with 83.33% GI at concentration of 500 μg/ml while methanol TERI University-Ph.D. Thesis, 2012 extract of C. truncatum EF10 showed 70% GI at similar concentration. Methanol extract of Dothediomycetes sp. EF1 showed 66.5% GI at 500 μg/ml concentration.
Hexane extracts of C. truncatum isolates EF9, EF10 and EF13 yielded triglyceride oil. Fatty acid profile of the oil from EF10 was similar to the oil produced by the seeds of the host plant i.e. J. curcas. These species of endophytic fungi have not been reported to produce oil so far.
Due to promising antifungal activity of ethyl acetate extract of C. globosum isolate EF18, bioassay guided fractionation of metabolites was performed to identify active principle responsible for antifungal activity. Each fraction was analyzed by High Performance Liquid Chromatography (HPLC). Active peaks were identified based on correlation of the peak area and bioactivity of the fractions. The fractions of C. globosum EF 18 were found effective against S. sclerotiorum showing >80% GI. Fraction no. VIII obtained from VLC column of ethyl acetate extract was most active with IC50 value of 35.4 μg/ml. The active fraction was further purified by Preparative HPLC and characterized by Mass and NMR spectroscopy. The active compound was characterized as compound similar to antibiotic Sch 210971 (m/z 445 and λmax 290) and found to be the principle component responsible for antifungal activity against S. sclerotiorum. This is the first report of antifungal activity of this compound isolated from endophytic fungus C. globosum.
Present work provides evidence that endophytic fungus C. globosum isolate EF18 per se and its culture filtrate has potential to be used for management of Sclerotinia-stem and root rot of chickpea. Other endophytic fungi found active in dual culture bioassay as well as in bioassay of their extract can also be exploited for development of bio-rational pesticide after studying their non-target effects. Oil producing endophytic fungus C. truncatum isolate EF10 can be exploited for oil production after optimization of its cultural conditions.
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