An antifungal polypeptide from Bacillus amyloliquefaciens FS6 and its biocontrol potential against ginseng gray mold caused by Botrytis cinerea

• Ba-AFP produced by Ba. amyloliquefaciens FS6 effectively inhibits Botrytis cinerea. • Ba-AFP exhibits stable activity under a range of temperatures, UV light, and pH. • Ba-AFP exhibits strong control of gray mold ( Bo. cinerea ) on ginseng. Gray mold, caused by Botrytis cinerea , is a globally devastating plant disease that severely threatens the economic sustainability of ginseng cultivation in Northeast China. Our previous field studies demonstrated that Bacillus amyloliquefaciens FS6 (BaFS6) exhibits strong biocontrol efficacy against ginseng gray mold. However, the underlying mechanism of its antifungal activity remained unclear. In this study, we identified an antifungal polypeptide (AFP) from Ba. amyloliquefaciens FS6 through comparative proteomic analysis. Prokaryotic expression of the afp gene in Escherichia coli yielded a recombinant protein (RP BaAFP) that significantly inhibited B. cinerea hyphal growth, conidial germination, germ tube elongation and disruption of cell wall integrity. Notably, RP BaAFP exhibited exceptional stability across a broad pH range (4–9), tolerated 2–8 h of UV irradiation, and retained activity at elevated temperatures (40–80 °C), outperforming the reference protein bovine serum albumin (BSA) in stability. Fermentation broth (FB) from Ba. amyloliquefaciens FS6 strain overexpressing afp (FS6- BaAfp OE) demonstrated superior antifungal activity compared to the wild-type Ba. amyloliquefaciens FS6 FB. Both RP BaAFP and the FS6- BaAfp OE cell-free FB effectively protected detached ginseng tissues and whole plants in greenhouse trials, significantly reducing B. cinerea infection. These findings elucidate the functional role of AFP in Ba. amyloliquefaciens FS6-mediated biocontrol and highlight its potential, along with the FS6 strain, for sustainable management of ginseng gray mold.