Uncovering the Genomic and Phenotypic Landscape of Nitrogen-Fixing Rhizobium miluonense WD29 in Free-Living Conditions

Abstract The endophytic bacterial strain Rhizobium miluonense WD29, isolated from the giant Mexican landrace maize Jala, demonstrates remarkable nitrogen fixation capabilities in free-living conditions. Through comprehensive genomic and phenotypic analyses, we characterized its unique attributes and potential applications in sustainable agriculture. Whole genome sequencing revealed a 6.8 Mb genome with 59.7% GC content, comprising 6,908 protein-coding genes, 3 rRNA genes, 46 tRNA sequences, and 146 insertion elements. Comparative genomic analysis showed that WD29 contains unique gene clusters associated with nitrogen fixation, biofilm formation, and plant growth promotion. The strain exhibits notable plant growth-promoting characteristics, including phosphate solubilization (26.1 ± 1.9 µg/mL), IAA production (19.7 ± 2.5 µg/mL), and multiple metallophore production capabilities (21.1-62.4% chelation for various metals). Significantly, R. miluonense WD29 demonstrates efficient nitrogen fixation in free-living conditions, with rates up to 21.7 ± 2.3 nmol h−1 of reduced acetylene at optimal galactose concentrations (1 g/L), coupled with substantial exopoly-saccharide production (0.8 ± 0.076 g/mL). The strain’s genome harbors at least 9 genes involved in exopolysaccharide biosynthesis, likely contributing to its biofilm formation capability and enhanced nitrogen fixation efficiency. Additionally, WD29 shows remarkable environmental adaptability, possessing genes for heavy metal resistance and various stress responses. These findings highlight R. miluonense WD29’s potential as a valuable biofertilizer for sustainable agriculture, particularly for non-leguminous crops like maize, and demonstrate the importance of studying nitrogen-fixing bacteria isolated from traditional agricultural systems. Competing Interest Statement The authors have declared no competing interest.