Bacillus ayatagriensis sp. nov., a novel plant growth-promoting rhizobacteria strain isolated from mulberry rhizosphere.

A Gram-positive, rod-shaped, spore-forming, aerobic bacterium, with biocontrol potential designated as RMG6, was isolated from the rhizosphere of the mulberry plant located in the mulberry garden of Raiganj University, West Bengal, India. The strain demonstrated significant antimicrobial activity against Enterococcus faecalis MTCC 439 (1.92 cm inhibition zone) and Escherichia coli MTCC 43 (1.62 cm inhibition zone) in agar-well diffusion assays. Scanning electron microscopy (SEM) confirmed bacterial cell lysis and cytoplasmic leakage. Phylogenetic analysis of the 16S rDNA sequence (1545 bp) revealed that RMG6 is closely associated to Bacillus siamensis KCTC 13,613 (99.93% similarity). Additionally, multilocus sequence analysis (MLSA) suggested that RMG6 represents a novel Bacillus species. The morphological characteristics of RMG6 colonies further distinguished it from closely related Bacillus species. RMG6 remained unidentified through Vitek-2 analysis. Fatty acid methyl ester (FAME) profiling revealed the predominant cellular fatty acids in RMG6 were anteiso-C (28.13%), iso-C (20.72%), C (10.72%), iso-C (7.00%), iso-C (5.88%), and anteiso-C (5.87%). MALDI-TOF MS analysis validated that RMG6 belongs to Bacillus genus. RMG6 exhibited optimal growth at 35 °C, pH 6, and up to 14% NaCl tolerance. The strain produced indole-3-acetic acid (IAA) through the indole-3-pyruvic acid pathway, with a maximum yield of 73.4 µg/mL at 0.2% L-tryptophan supplementation. The Vigna radiata seed germination assay validated its plant growth-promoting potential, with significantly enhanced radicle growth compared to control treatments, highlighting the potential of RMG6 for cross-species bioaugmentation. Antibiotic susceptibility profiling revealed that RMG6 was resistance to beta-lactam antibiotics, aligning with the genomic identification of resistance genes. Whole-genome sequencing (3,818,111 bp; G + C content 46.62%) identified 3723 coding sequences, including biosynthetic gene clusters (BGCs) associated with antimicrobial compound biosynthesis. Moreover, the highest recorded values for average nucleotide identity (ANI) and Genome-to-Genome Distance Calculator 3.0 analysis were 94.44 and 56.80%, respectively, with Bacillus siamensis KCTC 13,613. Comparative genomic and phylogenomic analyses confirmed the novelty of RMG6, leading to its proposed designation as Bacillus ayatagriensis sp. nov., with RMG6 (= MCM-B-1537). We propose Bacillus ayatagriensis RMG6 as a novel species with significant biocontrol potential and plant growth-promoting capabilities, advocating its expanded use in agriculture and industry.