Abstract Aim: To investigate antifungal activity of the extract and major metabolite of the endophytic fungus Acrophialophora jodhpurensis (belonging to Chaetomiaceae) against crown and root rot caused by Rhizoctonia solani (teleomorph: Thanatephorus cucumeris), as an important pathogen of tomato. Methods and Results: The endophytic fungus A. jodhpurensis, has high inhibitory effect against R. solani AG4-HG II in vitro and in vivo. The media conditions were optimized for production of the endophyte’s metabolites. The highest amounts of secondary metabolites were pro- duced at pH 7, 30◦C temperature, and in the presence of 0.5% glucose, 0.033% sodium nitrate, and 1 gl−1 asparagine as the best carbon, nitrogen, and amino acid sources, respectively. The mycelia were extracted by methanol and the obtained extract was submitted to various chromatography techniques. Phytochemical analysis via thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy showed that ergosterol peroxide was the major component in the extract of this endophyte. Antifungal activities of the methanolic extract and ergosterol peroxide in the culture media were studied against R. solani. Minimum inhibitory concentrations of the extract and ergosterol peroxide against the pathogen were 600 and 150 μg ml−1 , respectively. Ergosterol peroxide revealed destructive effects on the pathogen struc- tures in microscopic analyses and induced sclerotia production. Histochemical analyses revealed that it induced apoptosis in the mycelia of R. solani via superoxide production and cell death. Application of ergosterol peroxide in the leaf disc assay reduced the disease severity in tomato leaves. Conclusions: Antifungal metabolites produced by A. jodhpurensis, such as ergosterol peroxide, are capable of controlling destructive Rhizoctonia diseases on tomato. Impact Statement Ergosterol peroxide could be used as a bioactive compound and replaced with hazardous chemical fungicides for protecting tomato against Rhizoctonia solani.

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