Unveiling the genetic basis of biochemical pathways of plant growth promotion in Bacillus pumilus and the first genomic insights into B. pseudomycoides as a biostimulant

<i>Bacillus</i> species are among the most promising plant growth-promoting bacteria (PGPB) due to their adaptability to various environmental niches and extensive biosynthetic capabilities. Despite the available data on the PGP-traits (PGPTs) of <i>Bacillus</i>, the genetic basis underlying their beneficial effects remains largely unexplored. In this study, a comparative genomic analysis of three <i>B. pumilus</i> and one <i>B. pseudomycoides</i> strains, isolated from the maize rhizosphere, is presented to elucidate the molecular mechanisms behind their PGP-traits. All strains exhibited multiple PGP-traits, including phosphate solubilization, phytohormone and siderophore production, growth in nitrogen-free medium, stress tolerance, and biofilm formation. Phylogenomic analysis revealed that plant-associated strains have higher genetic similarity, emphasizing niche-specific evolution. Genome analyses revealed strain- and species-specific adaptations, particularly in relation to nutrient acquisition and abiotic stress response mechanisms. <i>B. pumilus</i> strains encoded alternative sigma factors (SigB, SigM, SigW) enabling enhanced salt tolerance, whereas <i>B. pseudomycoides</i> lacked this system and relied on conventional osmoprotective strategies. The strains utilized different tryptophan-dependent (IAN, IAM or IPyA) pathways for auxin biosynthesis and differed in phosphate solubilization ability, which can be attributed to upstream and missense variants in genes affecting acid metabolism (<i>gltA, acnA, acnB, citM</i>, and <i>citS)</i> and phosphatase (<i>phoA</i>) activity. Iron uptake <i>via</i> bacillibactin-siderophores was exclusive to <i>B. pumilus</i>. The inability of the <i>B. pseudomycoides</i> strain to acquire iron was associated with structural variants (absence of <i>bsaA</i> gene) within the bacillibactin biosynthetic gene cluster. This work provides new insights into the molecular basis of PGP traits in <i>Bacillus</i> and supports the development of <i>Bacillus</i>-based bioinoculants for sustainable agriculture.