BACKGROUNDBacterial leaf blight caused by the bacterium Xanthomonas oryzae pv. oryzae has a substantial effect on the yield of rice crops. The secretory (Sec) pathway, essential for efflux transport in bacteria, remains insufficiently studied in X. oryzae pv. oryzae, especially regarding its roles in drug resistance and physiology.RESULTSThis study involved transcriptome analysis on two X. oryzae pv. oryzae strains: a secG deletion strain (∆secG) and its complemented strain (C: ∆secG). In comparison to the parental strain PXO99A, ∆secG exhibited slower growth, with reductions in swimming (20.67%) and swarming (12.59%), while maintaining 76.7% of its biofilm formation capacity and 63.6% of exopolysaccharide production. The minimum inhibitory concentration (MIC50) values for an n‐butanol extract of Bacillus velezensis HN‐2 (HN‐2E) and bacitracin against ∆secG were 0.426 μg/mL (5.3% lower than that of PXO99A) and 10.905 μg/mL, respectively. Notably, ∆secG exhibited increased susceptibility to hydrogen peroxide (H2O2), being inhibited at 0.25 mm compared to 0.3 mm for PXO99A and C: ∆secG. In the presence of 0.2 mM H2O2, the susceptibility of ∆secG to HN‐2E increased by 31.22% (MIC50 = 0.159 μg/mL), while PXO99A and C: ∆secG exhibited MIC50 values of 0.280 and 0.291 μg/mL, respectively.CONCLUSIONOur findings demonstrate that Bacillus‐induced H2O2 production enhances the sensitivity of X. oryzae pv. oryzae to biocontrol agents, providing valuable insights for the prevention of bacterial leaf blight. These results highlight the significance of the Sec pathway in the behavior and resistance of X. oryzae pv. oryzae, as well as potential areas for further research on plant diseases. © 2024 Society of Chemical Industry.