Shikimic acid pathway drives the phase-dependent antibacterial activity of Bacillus amyloliquefaciens against Xanthomonas citri subsp. citri

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease affecting citrus species worldwide. Although Bacillus amyloliquefaciens ZJLMBA1908 has demonstrated significant potential as a biocontrol agent, the metabolic basis underlying its phase-dependent efficacy remains poorly understood. This study aims to elucidate the key metabolic pathways driving these fluctuations to optimize its agricultural application. The inhibitory effects of cell-free supernatants (CFS) from B.amyloliquefaciens ZJLMBA1908 at three growth phases—exponential (EP), stationary (SP), and decline (DP)—were evaluated using agar-well diffusion (mm) and the Minimum Inhibitory Concentration (MIC)-like growth inhibition assays. Untargeted LC-MS/MS metabolomics (MSI Level 2) were employed to identify phase-specific metabolites, followed by functional validation using the metabolic perturbagen curcumin and gene expression analysis by quantitative real-time PCR. The SP-CFS exhibited the highest antibacterial activity against Xcc, with an inhibition zone of 24.94 ± 0.03 mm and MIC of 3.13% (1/32) dilution. Metabolomic analysis identified 103 differential metabolites primarily enriched in the shikimic acid (SA) and phenylalanine metabolic pathways. Curcumin treatment (a putative shikimate dehydrogenase inhibitor) significantly downregulated SA-pathway genes (e.g., aroE, aroK) and reduced the inhibition zone diameter by 4.25 mm. Notably, phenylalanine-derived metabolites were significantly depleted following pathway perturbation. These findings indicate that the SA and phenylalanine pathways are key associated drivers of the phase-dependent antibacterial activity in strain B. amyloliquefaciens ZJLMBA1908. This study provides a mechanistic basis for the timing of Bacillus-based biopesticide production.