Phenotypic and Genomic Characterization of Bacillus sp. DLB3, a Black Pepper Rhizosphere Isolate with Antagonistic, Abiotic Stress-Tolerant, and Antibiotic-Susceptible Traits

Rhizosphere bacteria combining biocontrol activity with resilience to environmental stress are valuable candidates for sustainable agriculture. This study characterized strain DLB3, isolated from black pepper rhizosphere soil, through dual-culture and disk-diffusion antagonism assays, enzymatic and siderophore screening, physiological tolerance tests, antibiotic susceptibility testing, whole-genome sequencing, and genome mining. Phylogenomic analysis and average nucleotide identity (>97%) and digital DNA–DNA hybridization (>79%) values identified the isolate as Bacillus velezensis. The strain inhibited Fusarium falciforme, Fusarium oxysporum, and Colletotrichum gloeosporioides (47.6–71.2% radial growth inhibition) and produced an inhibition zone (8.4 mm) against Ralstonia solanacearum, alongside extracellular protease, amylase, CMCase, and catecholate-type siderophore production. DLB3 tolerated a broad range of temperatures (20–50 °C, with optimal growth at 25–40 °C, peaking at 35 °C), pH (5–9), and salinity (up to 10% NaCl), and exhibited minimum inhibitory concentrations (MICs) of 1800, 500, 450, and 250 mg L−1 for Pb, Zn, Cu, and Cr, respectively, while remaining susceptible to all nine antibiotics tested. The 3,913,361 bp draft genome was assembled into 44 contigs (N50 = 990,239 bp; L50 = 2), with 99.81% completeness and 0.12% contamination according to CheckM. It harbored 18 biosynthetic gene clusters, carbohydrate-active enzymes, peptidases, and genes associated with heavy metal resistance, broadly consistent with the observed phenotypes, as well as a homolog of the cfr methyltransferase gene (97.4% identity) that was not phenotypically expressed. These findings provide a comprehensive genomic and phenotypic profile of strain DLB3 and its biocontrol-related potential.