2',4′-dimethylacetophenone from Streptomyces roseofulvus St29 suppresses tobacco bacterial wilt by targeting virulence regulation and membrane integrity of Ralstonia solanacearum

Abstract

Tobacco bacterial wilt, caused by Ralstonia solanacearum , is a destructive soil-borne disease that severely threatens global tobacco production. In this study, fifteen actinobacteria-like strains were isolated from the rhizosphere soil of healthy tobacco plants. Among them, ten isolates exhibited antagonistic activity against R. solanacearum , with strain St29 displaying the most potent and broad-spectrum inhibition. Based on morphological observations, physiological-biochemical tests, and molecular identification, strain St29 was classified as Streptomyces roseofulvus . Greenhouse pot experiments revealed that treatment with St29 significantly mitigated disease severity, achieving a biocontrol efficacy of 64.86%. Twelve metabolite compounds were identified by gas chromatography-mass spectrometer (GC-MS) analysis, predominantly consisting of terpenoids, benzenoids, and ketones. Among them, 2′,4′-dimethylacetophenone showed the strongest antibacterial activity against R. solanacearum , with an IC 50 value of 253.40 µg·mL⁻¹ . Mechanistic studies indicated that 2′,4′-dimethylacetophenone disrupted bacterial cell morphology and membrane integrity, leading to cytoplasmic leakage and significant inhibition of biofilm formation and exopolysaccharide production. Furthermore, 2′,4′-dimethylacetophenone significantly downregulated key genes associated with virulence regulation, type III secretion system (T3SS), motility, chemotaxis, and EPS biosynthesis. High-precision molecular docking further confirmed that 2′,4′-dimethylacetophenone stably bound to critical protein targets involved in membrane biosynthesis and virulence regulation-specifically enoyl-ACP reductase (FabI), PhcS, and VsrB— via hydrophobic encapsulation and π–π stacking interactions. Overall, this study highlights 2′,4′-dimethylacetophenone produced by S. roseofulvus St29 disrupts a critical virulence mechanism and membrane integrity in R. solanacearum , providing a promising alternative for controlling the tobacco bacterial wilt.