Morpho-biochemical and molecular identification of Bacillus licheniformis and Bacillus cereus isolates from sorghum (Sorghum bicolor L.) rhizosphere

This study isolated, characterized, and identified rhizosphere-associated soil bacteria from sorghum-cultivated soils in Bhubaneswar, India, using serial dilution and spread plate techniques on Nutrient Agar. Morphological and biochemical analyses (Gram staining, catalase, oxidase, MR, and indole tests) identified 13 bacterial isolates, predominantly Gram-positive rods (Bacillus, Paenibacillus) and cocci (Staphylococcus, Micrococcus), with one Gram-negative isolate. Molecular characterization involved 16S rRNA gene amplification (~ 1500 bp), sanger sequencing, and phylogenetic analysis. BLASTN confirmed AG3 as Bacillus licheniformis (99.37% identity) (GenBank: PV590072), and AG11 as Bacillus cereus (100% identity) (GenBank: PV590099). The phylogenetic tress analysis of bacterial isolates AG3 and AG11 revealed distinct evolutionary rate variations. For AG3, site-specific rates ranged from 0.07 to 3.64 substitutions/site (nucleotide frequencies: A = 24.50%, T/U = 19.99%, C = 24.70%, G = 30.81%), while AG11 exhibited more uniform rates ranged from 0.90 to 1.10 substitutions/site (nucleotide frequencies: A = 25.79%, T/U = 20.86%, C = 22.71%, G = 30.64%). Maximum likelihood trees (log-likelihood: - 4077.853 for AG3, - 1928.562 for AG11) with 1,531 (AG3) and 1,402 (AG11) aligned positions resolved their phylogenetic relationships. This study provides valuable insights into the diversity and evolutionary dynamics of rhizosphere-associated Bacillus spp. in sorghum-cultivated soils, contributing to a better understanding of their ecological significance and potential application in sustainable agriculture and plant growth promotion.