Morpho-biochemical and molecular characterization of Bacillus subtilis and Priestia megaterium isolates from eastern Indian farmlands.

BACKGROUND

The study aimed to investigate the microbial diversity of soils from distinct environments, an urban park (SJ1) and an agricultural research field (SJ2) using serial dilution and spread plate techniques on nutrient agar, to identify region-specific bacterial strains with potential agricultural or biotechnological applications.

METHODS AND RESULTS

Bacterial isolates were purified through selective sub-culturing, characterized via Gram staining and biochemical tests (MR-VP, catalase, oxidase), and preserved at 4 °C. Genomic DNA was extracted, PCR-amplified targeting the 16S rRNA gene, and sequenced for BLAST-based identification. Phylogenetic analysis (MEGA 12, neighbor-joining, 1000 bootstraps) and evolutionary rate estimation (maximum likelihood, gamma-distributed site variation) were performed to assess genetic relationships and divergence. Morphological and biochemical analyses revealed Gram-positive rods in both isolates, exhibiting catalase positive, oxidase negative, methyl red negetive, and Voges-Proskauer positive activity. Genomic DNA was extracted, and the 16S rRNA gene was amplified (~ 1500 bp), sequenced, and analyzed via BLASTN. SJ1 showed 99.84-99.92% identity with Bacillus spp., while SJ2 matched Priestia megaterium (99.92%). Phylogenetic analysis placed SJ1 within Bacillus subtilis strain SBIS1 (GenBank: PV523294.1), and SJ2 within P. megaterium strain PIIS1 (GenBank: PV523528.1), indicating distinct evolutionary lineages. Maximum likelihood estimation revealed differing evolutionary rates, i.e., SJ1 exhibited high rate heterogeneity, while SJ2 showed uniform rates. Mean relative evolutionary rates across five discrete gamma categories for SJ1 ranged from 0.08 to 2.75, while for SJ2 they ranged from 0.90 to 1.10. SJ1 showed 25.23% (A), 20.09% (T), 23.43% (C), and 31.26% (G); while SJ2 had 24.87% (A), 21.26% (T), 24.20% (C), and 29.68% (G). Maximum log-likelihood values were -3315.740 for SJ1 (1268 aligned sites) and -1034.491 for SJ2 (750 aligned sites), confirming robust phylogenetic inference.

CONCLUSION

The study successfully identified distinct bacterial strains with unique evolutionary rates, genetic compositions, and robust divergence between the isolates, supporting their suitability for further agricultural or biotechnological exploration.