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

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), threatens citrus production worldwide. Long-term dependence on copper-based bactericides not only poses environmental risks but also accelerates the emergence of copper-resistant Xcc 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 Xcc-GFP-based screening method, we systematically screened a highly effective strain, which was identified as Bacillus velezensis 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 PR 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.