| Summary: | Fusarium wilt, caused by <i>Fusarium oxysporum</i>, is one of the most notorious diseases of cash crops. The use of microbial fungicides is an effective measure for controlling Fusarium wilt, and the genus <i>Bacillus</i> is an important resource for the development of microbial fungicides. Fusaric acid (FA) produced by <i>F. oxysporum</i> can inhibit the growth of <i>Bacillus</i>, thus affecting the control efficacy of microbial fungicides. Therefore, screening FA-tolerant biocontrol <i>Bacillus</i> may help to improve the biocontrol effect on Fusarium wilt. In this study, a method for screening biocontrol agents against Fusarium wilt was established based on tolerance to FA and antagonism against <i>F. oxysporum</i>. Three promising biocontrol bacteria, named B31, F68, and 30833, were obtained to successfully control tomato, watermelon, and cucumber Fusarium wilt. Strains B31, F68, and 30833 were identified as <i>B. velezensis</i> by phylogenetic analysis of the 16S rDNA, <i>gyrB</i>, <i>rpoB,</i> and <i>rpoC</i> gene sequences. Coculture assays revealed that strains B31, F68, and 30833 showed increased tolerance to <i>F. oxysporum</i> and its metabolites compared with <i>B. velezensis</i> strain FZB42. Further experiments confirmed that 10 µg/mL FA completely inhibited the growth of strain FZB42, while strains B31, F68, and 30833 maintained normal growth at 20 µg/mL FA and partial growth at 40 µg/mL FA. Compared with strain FZB42, strains B31, F68, and 30833 exhibited significantly greater tolerance to FA.
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