Optimization of fermentation conditions and identification of active substances from Bacillus velezensis HZ33 for inhibiting Rhizoctonia solani in potato black scurf

Abstract BACKGROUND Bacillus velezensis HZ33, isolated from the potato rhizosphere, exhibits significant inhibitory activity against Rhizoctonia solani AG‐3, the pathogen causing potato black scurf. To determine the antifungal compounds responsible for this inhibitory effect, we optimized the fermentation process and conducted bioassay‐guided fractionation. RESULTS Whole‐genome sequencing revealed that this strain harbors 12 known biosynthetic gene clusters for secondary metabolites, indicating substantial biosynthetic potential. To enhance the yield of antifungal substances, single‐factor experiments and response surface methodology were used to optimize the fermentation conditions. Among the crude extracts obtained from fermentation supernatant, the hydrochloric acid‐precipitated fraction exhibited the strongest antifungal activity. This extract was fractionated by Mitsubishi Chemical Industries gel column chromatography and Sephadex gel chromatography, purified by high‐performance liquid chromatography, and characterized by high‐resolution mass spectrometry and nuclear magnetic resonance. Two cyclic lipopeptides (marihysin A and B) and 10 surfactin derivatives were identified. Notably, this is the first report of marihysin A and B in B. velezensis . Among these, marihysin B showed the highest yield (43.04 mg L −1 ) and the strongest antifungal activity against R. solani AG‐3, with an EC 50 of 9.78 μg mL −1 . Furthermore, marihysin B demonstrated preventive and curative efficacy against potato black scurf. Its primary mechanism of action involves disrupting hyphal structure and cell membrane integrity, as well as inducing the accumulation of reactive oxygen species. CONCLUSION This study presents the first evidence that marihysin B, the most active constituent of B. velezensis HZ33, has strong biocontrol potential against the potato black scurf pathogen, providing a basis for developing new biocontrol agents and fungicide lead compounds. © 2026 Society of Chemical Industry.