Abstract
A Gram-positive, rod-shaped, spore-forming, aerobic bacterium, with biocontrol potential designated as RMG6T, 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 RMG6T is closely associated to Bacillus siamensis KCTC 13,613 (99.93% similarity). Additionally, multilocus sequence analysis (MLSA) suggested that RMG6T represents a novel Bacillus species. The morphological characteristics of RMG6T colonies further distinguished it from closely related Bacillus species. RMG6T remained unidentified through Vitek-2 analysis. Fatty acid methyl ester (FAME) profiling revealed the predominant cellular fatty acids in RMG6T were anteiso-C15:0 (28.13%), iso-C15:0 (20.72%), C18:0 (10.72%), iso-C17:0 (7.00%), iso-C16:0 (5.88%), and anteiso-C17:0 (5.87%). MALDI-TOF MS analysis validated that RMG6T belongs to Bacillus genus. RMG6T 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 RMG6T for cross-species bioaugmentation. Antibiotic susceptibility profiling revealed that RMG6T 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 RMG6T, leading to its proposed designation as Bacillus ayatagriensis sp. nov., with RMG6T (= MCM-B-1537). We propose Bacillus ayatagriensis RMG6T as a novel species with significant biocontrol potential and plant growth-promoting capabilities, advocating its expanded use in agriculture and industry.
Keywords:
Bacillus; Biocontrol; Comparative genomics; Fatty acid methyl ester profiling; Genome sequencing; Multilocus sequence analysis.