Aims This study aimed to obtain an antagonistic endophyte against Sclerotium rolfsii from peanut seeds, evaluate the biocontrol efficacy towards peanut stem rot and explore its antifungal mechanism against S. rolfsii. Methods and Results Thirty‐seven endophytic bacteria were isolated from peanut seeds, six of which exhibited stronger antagonistic activities against S. rolfsii (inhibition rate, IR of hyphae growth ≥70%). Strain LHSB1, the strongest antagonistic strain, was identified as Bacillus velezensis. LHSB1 showed 93·8% of radial growth inhibition of S. rolfsii hyphae and exhibited obvious antagonistic activity against another six pathogenic fungi of peanut. Pot experiments showed two different LHSB1 treatments both significantly reduced the disease incidence and severity of stem rot (P < 0·05) compared to the controls, and the biocontrol efficacy reached 62·6–70·8%, significantly higher than that of Carbendazim control (P < 0·05). Further analyses revealed LHSB1 culture filtrate significantly inhibited sclerotia formation and germination, caused the abnormalities and membrane integrity damage of S. rolfsii hyphae, which might be the possible mode of action of LHSB1 against S. rolfsii. Three antifungal lipopeptides bacillomycin A, surfactin A and fengycin A, were detected in LHSB1 culture extracts by UPLC‐ESI‐MS, which could be responsible for the biocontrol activity of LHSB1 against S. rolfsii. Conclusion Our results suggested that the seed‐borne endophytic B. velezensis LHSB1 would be a tremendous potential agent for the biocontrol of peanut stem rot caused by S. rolfsii. Significance and Impact of the Study This comprehensive study provides a candidate endophytic biocontrol strain and reveals its antifungal mechanism against S. rolfsi. To the best of our knowledge, this is the first time that seed‐borne endophytic B. velezensis was used as the biocontrol agent to control peanut stem rot.

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