Endophytic Microbiome Diversity in Citrus Leaves and the Biocontrol Potential Bacteria against Xanthomonas citri subsp. citri

Citrus is a pivotal economic crop in southern China, while citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a devastating quarantine disease that severely threatens the sustainability of the citrus industry. Endophytic bacteria represent a promising sustainable alternative for disease management, yet systematic exploration of their potential against Xcc in citrus leaves remains limited. To address this, we characterized the leaf endophytic microbiome from major citrus-growing regions in southern China via high-throughput sequencing, revealing significant differences in the endophytic bacterial community structure between symptomatic and asymptomatic citrus leaves, with a significantly elevated relative abundance of Proteobacteria and prominent enrichment of the genera Xanthomonas, Enterobacter, and Pseudomonas in symptomatic tissues. Furthermore, symptomatic leaves harbored significantly higher endophytic bacterial diversity than asymptomatic leaves from the same production region, and the Xanthomonas abundance in the samples was highly consistent with the actual field disease severity. From 519 bacterial isolates, four strains-A1 (Bacillus altitudinis), A3 (Bacillus velezensis), A6 (Pseudomonas parafulva), and A7 (Delftia tsuruhatensis) demonstrated strong in vitro and detached-leaf antagonism against Xcc. Strain-specific fermentation parameters were optimized, and all four strains were successfully formulated into wettable powders. In a field trial, these formulations achieved control efficacies ranging from 25.8% to 53.5%, with strain A1 showing the highest and most sustained activity. Strains A3, A6, and A7 also exhibited broad-spectrum antibacterial activity against several other phytopathogenic bacteria. This study not only elucidates shifts in the endophytic microbiome associated with citrus canker but also provides novel, efficacious biocontrol resources and a practical development pipeline for sustainable disease management.