The application of biocontrol biopesticides based on plant growth-promoting rhizobacteria (PGPR), particularly members of the genusBacillus,is considered a promising perspective to make agricultural practices sustainable and ecologically safe. Recent advances in genome sequencing by third-generation sequencing technologies, e.g., Pacific Biosciences' Single Molecule Real-Time (PacBio SMRT) platform, have allowed researchers to gain deeper insights into the molecular and genetic mechanisms of PGPR activities, and to compare whole genome sequences and global patterns of epigenetic modifications. In the current work, this approach was used to sequence and compare fourBacillusstrains that exhibited various PGPR activities including the strain UCMB5140, which is used in the commercial biopesticide Phytosubtil. Whole genome comparison and phylogenomic inference assigned the strain UCMB5140 to the speciesBacillus velezensis. Strong biocontrol activities of this strain were confirmed in several bioassays. Several factors that affect the evolution of active PGPRB. velezensisstrains were identified: (1) horizontal acquisition of novel non-ribosomal peptide synthetases (NRPS) and adhesion genes; (2) rearrangements of functional modules of NRPS genes leading to strain specific combinations of their encoded products; (3) gain and loss of methyltransferases that can cause global alterations in DNA methylation patterns, which eventually may affect gene expression and regulate transcription. Notably, we identified a horizontally transferred NRPS operon encoding an uncharacterized polypeptide antibiotic inB. velezensisUCMB5140. Other horizontally acquired genes comprised a possible adhesin and a methyltransferase, which may explain the strain-specific methylation pattern of the chromosomal DNA of UCMB5140.