Das Sandip, Mondal Rittick, Mandal Pankaj, Kurt Halil, Chakraborty Joydeep, Islam Md Majharul, Sarkar Biraj, Shaw Shubhajit, Manna Sanjeet, Arabaci Nihan, Jangid Kamlesh, Sadat Abdul, Gangopadhyay Debnirmalya, Kati Ahmet, Mandal Amit Kumar
Department of Sericulture, Raiganj University, North Dinajpur, West Bengal, 733134, India.
Department of Botany, School of Sciences, Netaji Subhas Open University, Kolkata, West Bengal, 700064, India.
Sci Rep. 2025 Jul 23;15(1):26693. doi: 10.1038/s41598-025-05508-w.
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.
从印度西孟加拉邦赖根杰大学桑园的桑树根际分离出一株革兰氏阳性、杆状、产芽孢、需氧细菌，命名为RMG6，具有生物防治潜力。在琼脂孔扩散试验中，该菌株对粪肠球菌MTCC 439（抑菌圈直径1.92厘米）和大肠杆菌MTCC 43（抑菌圈直径1.62厘米）表现出显著的抗菌活性。扫描电子显微镜（SEM）证实了细菌细胞裂解和细胞质泄漏。对16S rDNA序列（1545 bp）的系统发育分析表明，RMG6与暹罗芽孢杆菌KCTC 13613密切相关（相似度99.93%）。此外，多位点序列分析（MLSA）表明RMG6代表一个新的芽孢杆菌物种。RMG6菌落的形态特征进一步将其与密切相关的芽孢杆菌物种区分开来。通过Vitek-2分析未能鉴定出RMG6。脂肪酸甲酯（FAME）分析表明，RMG6中主要的细胞脂肪酸为anteiso-C（28.13%）、iso-C（20.72%）、C（10.72%）、iso-C（7.00%）、iso-C（5.88%）和anteiso-C（5.87%）。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析证实RMG6属于芽孢杆菌属。RMG6在35℃、pH 6时生长最佳，耐NaCl浓度高达14%。该菌株通过吲哚-3-丙酮酸途径产生吲哚-3-乙酸（IAA），在添加0.2% L-色氨酸时，最大产量为73.4μg/mL。绿豆种子发芽试验验证了其促进植物生长的潜力，与对照处理相比，胚根生长显著增强，突出了RMG6在跨物种生物强化中的潜力。抗生素敏感性分析表明，RMG6对β-内酰胺类抗生素耐药，这与耐药基因的基因组鉴定结果一致。全基因组测序（3818111 bp；G+C含量46.62%）鉴定出3723个编码序列，包括与抗菌化合物生物合成相关的生物合成基因簇（BGCs）。此外，与暹罗芽孢杆菌KCTC 13613相比，平均核苷酸同一性（ANI）和基因组到基因组距离计算器3.0分析的最高记录值分别为94.44%和56.80%。比较基因组和系统发育基因组分析证实了RMG6的新颖性，因此将其命名为阿亚塔芽孢杆菌（Bacillus ayatagriensis）新种，模式菌株为RMG6（=MCM-B-1537）。我们提出阿亚塔芽孢杆菌RMG6是一个具有显著生物防治潜力和促进植物生长能力的新物种，提倡在农业和工业中扩大其应用。
