Antibacterial mechanism of hypocrellin against Ralstonia solanacearum in tobacco and its modulation of the endophytic microbiome

The photosensitive compound hypocrellin, derived from the traditional Chinese medicinal fungus Shiraia bambusicola Henn., shows potential as a novel pesticide. To evaluate its efficacy against bacterial wilt disease caused by Ralstonia solanacearum in tobacco, this study examined its antibacterial mechanisms through in vitro experiments and analyzed their impact on tobacco endophytic microbiota. An optimized hypocrellin offering a promising sustainable formulation—with a 2:3 ratio of hypocrellin A to hypocrellin B—was developed for enhanced antibacterial activity, and its interaction with bacterial DNA, intracellular components, and membrane integrity was systematically investigated. Hypocrellins exhibited strong antibacterial effects by disrupting the cell wall and membrane integrity of R. solanacearum , increasing membrane permeability, and inducing the leakage of intracellular K ⁺ , soluble proteins, and reducing sugars. Dual DNA-binding mechanisms were suggested: electrostatic adsorption to phosphate groups, followed by groove insertion, and intercalation between base pairs. In tobacco plants, hypocrellin treatment significantly altered the endophytic microbial community, increasing microbial richness and diversity, increasing the proportion of Bacillota and Pseudomonadota, and significantly reducing pathogen abundance. Predictive functional analysis based on PICRUSt2 suggested that hypocrellin may have influenced metabolic pathways associated with microbial basal metabolic activity and proliferation potential. Hypocrellins mitigate R. solanacearum infection through multi-target mechanisms: physical disruption of bacterial membranes and DNA, restoration of beneficial endophytic microbiota, and activation of metabolic pathways critical for plant defense.