Ergosterol is an important component of the fungal cell membrane and represents an effective target of chemical pesticides. However, the current understanding of ergosterol biosynthesis in the soybean root rot pathogen Fusarium oxysporum remains limited. In addition, the regular use of fungicides that inhibit ergosterol synthesis will seriously harm the ecological environment and human health. Bacillus subtilis is gradually replacing chemical control as a safe and effective biological agent; to investigate its effect on ergosterol synthesis of F. oxysporum, we verified the biological function of the FoERG3 gene of F. oxysporum by constructing knockout mutants. The results showed that knocking out FoERG3 blocked ergosterol biosynthesis, restricted mycelial growth, and increased the sensitivity to external stressors (NaCl, D-sorbitol, Congo Red, and H₂O₂). The increased permeability of the cell membrane promoted increased extracellular K⁺ levels and decreased mitochondrial cytochrome C contents. Treatment with suspension of B. subtilis HSY21 cells resulted in similar damage as observed when treating FoERG3-knockout F. oxysporum cells with ergosterol, which was characterised by deformity and swelling of the mycelium surface; increased membrane permeability; decreased pathogenicity to soybeans; and significantly decreased activities of cellulase, β-glucosidase, amylase, and pectin-methyl galactosylase. Notably, deleting FoERG3 resulted in a significant lag in the defense-response time of soybeans. Our results suggest that FoERG3 strongly influences the virulence of F. oxysporum and may be used as a potential antimicrobial target by B. subtilis HSY21 to inhibit ergosterol synthesis, which supports the use of B. subtilis as a biological control agent for protecting against F. oxysporum infection.

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