Genomic sequencing of vineyard-isolated Bacillus strains and comparison with closely related Bacillus species reveals a putative new lineage, distinct pan-genome architecture and secondary metabolism potential

Abstract

Introduction Bacillus species are ubiquitous in nature, and they display diverse traits of biotechnological importance, including the production of various enzymes and secondary metabolites. In this study, we profiled the genomes of vineyard-derived Bacillus strains shown to inhibit Botrytis cinerea mycelial growth and spore germination through the production of cyclic lipopeptides and cell wall-degrading enzymes. Methodology Long-reads-whole genome sequencing was conducted using PacBio Sequel II Single-Molecule Real-Time platform, followed by genome assembly, phylogenetic analysis and functional annotation using different bioinformatics pipelines. Results The strains possess ~3.900,000 bp genome size and %GC ranging between 45%–46.7%, as expected in Bacillus species. Phylogenetic analysis separated the vineyard-derived strains into two lineages. Strains B4003 and B4005 grouped with B. velezensis. Notably, three Bacillus strains (B4022, B4001, and B4023) clustered with the undescribed strains with placeholders “sp018613535”, an undescribed taxon in the genome taxonomy database, supported by the Average Nucleotide Identity (ANI) score above 96%. Putative biosynthetic gene clusters (BGCs) involved in secondary metabolite production were identified by AntiSMASH analysis among the isolates. Orthologous cluster analysis further identified 3203 genes conserved across all strains, constituting a stable core genome that supports essential cellular and metabolic functions. Comparative pan-genome analysis of the Bacillus velezensis and Bacillus spp. revealed clear species-level differentiation alongside a conserved functional backbone. Furthermore, functional annotation revealed strain-specific enrichment in metabolic and environmental response pathways, emphasizing the ecological specialization and adaptive diversification. Discussion Overall, these findings emphasize the dynamic interplay between genomic conservation and flexibility within the Bacillus genus and support the biological control potential of these strains. Furthermore, genomic evidence strongly indicates that B4001, B4022 and B4023, represent members of a putatively novel species.