3,5-Di-tert-butylphenol from Bacillus siamensis controls kiwifruit leaf spot by disrupting membrane integrity and interfering with energy metabolism of Fusarium graminearum.

Fusarium graminearum, a notorious multi-host pathogen threatening global agriculture, has recently expanded its host range to become a dominant and aggressive causal agent of kiwifruit leaf spot, causing severe yield losses and threatening industrial sustainability. To address this challenge, a biocontrol bacterium exhibiting broad-spectrum antimicrobial activity, Bacillus siamensis strain BsiaSC07, was isolated from the kiwifruit phyllosphere. Notably, the potent broad-spectrum antifungal compound 3,5-di-tert-butylphenol (3,5-DTBP) was identified from the fermentation metabolites of this strain using gas chromatography-mass spectrometry (GC/MS). To the best of our knowledge, this represents the first report of 3,5-DTBP production by Bacillus spp. 3,5-DTBP demonstrated significant inhibitory activity against six kiwifruit leaf spot pathogens, with particularly high efficacy against F. graminearum. It effectively suppressed mycelial growth, sporulation, and spore germination, and significantly alleviated disease severity in detached leaf assays. Furthermore, transcriptomic analysis and validation experiments elucidated the hierarchical mechanism of action: 3,5-DTBP exerts its antifungal effect primarily by destroying cell membrane integrity. This primary structural damage may act as an initiating trigger that precipitates a secondary irreversible collapse in energy metabolism, ultimately driving systemic cell failure. These findings demonstrate the biocontrol potential of B. siamensis BsiaSC07 and suggest that 3,5-DTBP is a promising candidate fungicide for the management of kiwifruit leaf spot.