Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens

Abstract

Background Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles. Results Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin. Conclusion The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Use of rpoB gene phylogenetic marker-based distinction of abiotic stress tolerant and plant-growth promoting Bacillus paralicheniformis isolates from their closely related Bacillus licheniformis

“…(Figueroa-Brambila et al, 2023). Although 16S rRNA gene sequencing has traditionally been considered as the gold standard for bacterial identification and phylogenetic inferences; however, the misidentification of these bacterial species may be attributed to the insufficient sequence variation between the closely related species, as they share more than 99.4 % of sequence similarity (Ashajyothi et al, 2024).…”

“…Microorganisms residing in the plant rhizosphere possess the capacity to notably augment the plant growth and ameliorate the various challenges encountered in the modern agriculture, thus presenting a promising pathway towards achieving agricultural sustainability (Hakim et al, 2021). The primary mechanisms through which these plant growth-promoting rhizobacteria (PGPR) enhance the plant growth include the associative nitrogen fixation, ethylene level reduction, production of siderophores and phytohormones, induction of resistance against pathogens, nutrient solubilization, promotion of mycorrhizal symbiosis, and alleviation of the soil pollutant toxicity (Ashajyothi et al, 2024;Khade and Sruthi, 2024). In this respect, the cultivation and production of olives and olive oil hold profound significance within the Moroccan and the broader Middle East and North Africa (MENA) regions.…”

“…Bacillus paralicheniformis, a species within the genus Bacillus, is a Gram-positive, facultatively anaerobic, motile, rod-shaped, and endospore forming bacterium (Dunlap et al, 2015). This species was previously referred to B. licheniformis and has recently been separated and described as a new species, based on the phylogenetic and phylogenomic studies (Ashajyothi et al, 2024). The genus Bacillus is recognized for its large use and effectiveness in producing various biochemicals and molecules that are crucial in the biotechnological industries, such as the enzymes (i.e., proteases, lipases, etc.)…”

“…Hence, there is an urgent necessity for distinct differentiation of B. paralicheniformis from its closely related counterpart, B. licheniformis. This differentiation is facilitated by the presence of a potent factor; mainly urease activity, which is a characteristic feature of B. paralicheniformis (Ashajyothi et al, 2024). Urease activity has been described as a phenotypic criterion enabling the discrimination between these two bacterial species (Dunlap et al, 2015) until Jeong et al, (2018) disclosed that the urease activity of B. paralicheniformis is not a reliable discriminator between these two species.…”