Bacillus velezensis RF2 Rescued from Citrus Phyllosphere: Dual Mechanisms and Broad-Spectrum Activity for Controlling Citrus Bacterial Canker

Citrus bacterial canker (CBC), caused by <i>Xanthomonas citri</i> subsp. <i>citri</i> (<i>Xcc</i>), threatens citrus production worldwide. Long-term dependence on copper-based bactericides not only poses environmental risks but also accelerates the emergence of copper-resistant <i>Xcc</i> strains. To develop safe and efficient alternative control strategies, 72 bacterial strains were isolated from the phyllosphere of citrus plants naturally infected by CBC and identified by 16S rRNA sequencing. Using an <i>Xcc</i>-GFP-based screening method, we systematically screened a highly effective strain, which was identified as <i>Bacillus velezensis</i> RF2 (Bv-RF2). Both inhibition zone assays and bioactivity tests of the crude methanolic extract of Bv-RF2 demonstrated stable antibacterial activity under UV irradiation, protease treatment, high temperature, and across a wide pH range. Whole-genome sequencing and antiSMASH analysis revealed multiple predicted NRPS/PKS-type biosynthetic gene clusters (BGCs). Together with metabolomic profiling, these data provide hypotheses for candidate metabolites that may contribute to antagonism. Bv-RF2 was associated with the induction of <i>PR</i> gene expression in immune-related pathways implicated in CBC responses. In sweet orange leaves, Bv-RF2 infiltration was associated with transient induction of defense-related (PR) genes, consistent with an ISR-like, priming-related response. In addition, Bv-RF2 inhibited the growth of fungal pathogens associated with citrus anthracnose and brown spot in vitro, indicating broad inhibitory potential under the tested conditions. Collectively, Bv-RF2 represents a promising candidate for developing environmentally friendly strategies against CBC and other citrus diseases.