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Endophytic fungi inhabit a unique biological niche and are categorized as highly diverse, polyphyletic group of primarily ascomycetous fungi, capable of colonizing tissues of plants asymptomatically. Endophytic fungi have been known to impart abiotic and biotic stress tolerance to host plants. They have been proved beneficial to host plant by enhancing plant nutrient uptake, restriction of phytopathogenic attack and reducing disease severity. Several metabolites depicting antifungal activity against plant pathogens have been isolated and characterized from endophytic fungi.
In the present study, Indian medicinal plants based on their previously reported antiphytopathogenic properties were selected as host for isolation of endophytic fungi. Two categories of host plants were selected i.e. essential oil producing plant species such as Ocimum sanctum and Mentha piperita and saponins containing plant species such as Chlorophytum borivilianum and Asparagus racemosus. Both saponins and essential oils have been documented antifungal activities in literature. In our pursuit of finding bioactive endophytic fungi and characterization of their metabolites, we strategically collected host plants from different agroclimatic zones endophytic fungi isolation, namely Hyderabad, Mukteshwar and Delhi at two different sampling times in two consequent years (2010 and 2011).
A total of 264 endophytic fungal isolates were obtained and broadly classified into 28 species and 18 genera based on ITS rDNA analysis. Majority of endophytic fungi reported in the present study belonged to phylum Ascomycota and one belonged to Zygomycota (Rhizopus oryzae). Each medicinal host plant behaved as a distinct ecosystem. Acremonium sp. was exclusively recovered from M.piperita while Colletotrichum capsici occurred only from A.racemosus. Noted plant pathogens such as Chaetomium coarctatum, Rhizoctonia bataticola and Setosphaeria rostrata are reported for the first time in the present study as endophytes.
All the endophytic fungal isolates recovered depicted considerable antiphytopathogenic activity against broad spectrum plant pathogens investigated. Quite noteworthy antiphytopathogenic activity against Rhizoctonia solani were exhibited by endophytic fungi isolated from O. sanctum collected from Mukteshwar in 2010 (73.3%) and Hyderabad in 2011 (50%). Based on the confrontation bioassay 37 fungal endophytes were further mass cultured on rice medium to evaluate antifungal activity of their extract concentrate.
Extract concentrate of Acremonium sp. (MPM-2.1) displayed strongest antifungal activity against Sclerotinia sclerotiorum and Rhizoctonia solani with IC50 value of 0.056 and 0.292 mg/ml respectively. While ethyl acetate extract of Penicillium sp. (ARDS-2.3) exhibited highest antifungal activity towards Botrytis cinerea having 0.055 mg/ml IC50 value whereas extract of Alternaria alternata (OSDSL-5.6) depicted best inhibition against Fusarium oxysporum with IC50 value of 0.076 mg/ml. “Strain specific” differences in antifungal activities have been well documented in the present study.
GC-MS chromatography of hexane extracts of selected endophytic fungal isolates revealed presence of volatile compounds, fatty acids, and aliphatic constituents reported to have antifungal activity in literature such as palmitic acid, oleic acid, linolenic acid, hexadecanoic acid and 1-heptacosanol. Also, sulfamethazine and sulfadimidine metabolites are identified by LC-MS/MS dereplication from endophytic S. rostrata bioactive fraction. This study is the first report of production of an antifungal compound 2H-pyran-2-one 5, 6 dihydro-6-pentyl from an endophytic fungus Macrophomina phaseolina.
Metabolites originated from fungal endophytes in the present study hold promise to be further developed as greener and safer biocontrol agent in crop disease management. It can be concluded that fungal endophytes harbored inside leaf and stem tissues of indigenous Indian medicinal plants collected from different geographical locations in different sampling times hold great promise not only as biocontrol agents against broad spectrum and economically significant phytopathogens, but also as sustainable resource of novel antifungal secondary metabolites.
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