Comparative Phenotypic and Transcriptomic Analysis Reveals Distinct Yet Partially Convergent Mechanisms of Daphnetin and 6-Methylcoumarin Against Eucalyptus-Derived Ralstonia pseudosolanacearum

Eucalyptus bacterial wilt in China, caused predominantly by Ralstonia pseudosolanacearum, severely threatens Eucalyptus plantation management in China and demands plant-derived alternatives to conventional bactericides. This study evaluated the in vitro antibacterial activities and underlying molecular mechanisms of two differentially substituted coumarin derivatives, daphnetin (DAP) and 6-methylcoumarin (MC), against a eucalyptus-derived R. pseudosolanacearum strain. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using broth microdilution assays. Quantitative phenotypic assays were conducted to evaluate the effects of sub-lethal concentrations on biofilm formation, swimming motility, and extracellular polysaccharide (EPS) accumulation. Furthermore, high-throughput RNA sequencing integrated with GO and KEGG enrichment analyses was employed to characterize the genome-wide transcriptomic responses. Antimicrobial susceptibility testing demonstrated that DAP possessed superior bactericidal efficacy, yielding lower MIC (62.5 μg/mL) and MBC (250 μg/mL) values than MC. Conversely, sub-lethal MC exhibited a more pronounced, early-stage suppression of flagellum-dependent swimming motility and biofilm maturation. Both compounds consistently attenuated EPS production. Transcriptomic analysis revealed distinct yet partially convergent regulatory networks: DAP primarily exerted metabolic strangulation by downregulating genes governing aerobic respiratory chains and peripheral carbon/nitrogen pathways, whereas MC targeted collective behavior and active pathogenesis, systematically downregulating the expression of genes associated with flagellar assembly, quorum sensing, and Type III and VI secretion systems. Notably, both pathways converged downstream to repress the master virulence regulator xpsR and the epsA-P operon. These findings provide valuable insights into the potential molecular pathways affected by DAP and MC, offering a useful reference for future exploration of botanical formulations for ecologically responsible forest disease control.