A maize-associated Rhizobium miluonense fixes nitrogen in free-living conditions through an exopolysaccharide-dependent biofilm mechanism

Rhizobium miluonense WD29, isolated from the rhizosphere of Jala landrace maize (Mexico), demonstrates FLNF, an unusual trait for this typically symbiotic genus. Whole-genome sequencing revealed a 6.8 Mb genome (59.7% GC) with 6,908 protein-coding genes, including a complete repABC plasmid replication system and type IV secretion genes. The strain exhibits plant growth-promoting traits, including phosphate solubilization (26.1±1.9 µg ml ⁻¹ ), indolic compound production (19.7±2.5 µg ml ⁻¹ ) and metallophore production. Acetylene reduction assays demonstrated nitrogen fixation rates up to 21.7±2.3 nmol C ₂ H ₄ h ⁻¹ , which correlated strongly ( r =0.973) with exopolysaccharide production (0.8±0.08 g l ⁻¹ ), suggesting that biofilm formation creates microaerobic conditions protecting nitrogenase. Genomic analysis identified 12 exo -cluster genes and 98 total polysaccharide biosynthesis genes that support this phenotype. Additionally, R. miluonense WD29 exhibits remarkable environmental adaptability, harbouring genes for heavy-metal resistance and diverse stress-response pathways. These findings highlight the potential of R. miluonense WD29 as a valuable biofertilizer for sustainable agriculture, particularly for non-leguminous crops such as maize, and underscore the importance of studying nitrogen-fixing bacteria isolated from traditional agricultural systems.