Genomic characterization of Bacillus velezensis BP5 and BP103: insights into biocontrol potential against bacterial leaf spot on pepper

Abstract

Bacillus velezensis strains BP5 and BP103, isolated from pepper rhizosphere soil in the Mekong Delta, Vietnam, displayed potent antagonistic activity against Xanthomonas euvesicatoria, which causes pepper bacterial spot. In dual-culture assays, BP5 formed the largest inhibition zone (36.7 mm), outperforming BP103 (32.1 mm) and oxolinic acid (31.3 mm). Both Gram-positive, rod-shaped strains produced extracellular protease, lipase, amylase, cellulase, and siderophores. Whole-genome sequencing revealed compact and genetically stable genomes: BP5 (4.08 Mb, 46.04% GC, 4,047 protein-coding genes, 5 prophages) and BP103 (3.91 Mb, 46.46% GC, 3,793 genes, 2 prophages). Phylogenetic reconstruction and average nucleotide identity (ANI of more than 98%) confirmed their assignment to B. velezensis, with closest relatedness to strain 160. Orthologous gene analysis across 28 B. velezensis genomes and B. subtilis DSM10 identified 2,593 single-copy genes. Compared to BP103, BP5 harbors greater numbers of multi-copy orthologs, other orthologs, and unique paralogs (388, 910, and 5 in BP5 with 336, 763, and 1 in BP103, respectively). Pangenome analysis showed that the core genome size decreased progressively with the addition of new genomes; BP5 showed more pronounced genetic divergence driven by expanded unique paralogs and shell gene families (with 456 in BP5 compared with 203 in BP103). Both strains encode 13 secondary metabolite biosynthetic gene clusters, including surfactin, fengycin, bacillaene, macrolactin, difficidin, bacilysin, bacillibactin, mersacidin, and locillomycin, plus four previously undescribed terpene/PKS. The CAZyme repertoire was richest in BP5 (133 genes), exceeding BP103 and the commercial strain FZB42 (both 129 genes), thereby enhancing adhesion and adhesion and rhizosphere colonization. These findings establish BP5 and BP103 as highly promising biocontrol agents, combining high genetic stability with diverse secondary metabolite profiles, suitable for development into sustainable microbial bio-bactericides.