Isolation and Genetic Enhancement of Nitrogen-Fixing Rhizobacteria for Promoting Growth in Maize

This study aimed to isolate and characterize nitrogen-fixing bacteria from the maize rhizosphere and evaluate their plant growth-promoting potential to reduce reliance on synthetic fertilizers and enhance soil fertility. Nitrogen-free selective media were used for bacterial isolation, followed by detection of the nifH gene and nitrogenase activity. Phylogenetic identification was conducted via 16S rRNA sequencing. Growth-promoting traits, stress tolerance, and pot-based plant inoculation effects were assessed. Genetic modification of strain GN8811 was performed to improve nitrogen fixation and growth promotion. Seven isolates possessed the nifH gene and nitrogenase activity, including Azotobacter chroococcum GN2001, A. vinelandii GN1202, Azospirillum brasilense GN1004, Kosakonia sacchari GN2003, Klebsiella michiganensis GN8799 and GN8801, and K. quasivariicola GN8811. Furthermore, GN8801 and GN2001 exhibited phosphate solubilization and iron chelation, while GN1004 and GN8811 showed strong IAA production and potassium solubilization. Additionally, GN2003 and GN8811 tolerated high salinity and variable pH. Maize inoculated with GN8811 showed biomass and root enhancement comparable to nitrogen-fertilized controls. The genetically modified GN8811 strain (ΔnifL::nifA) exhibited further improvement in ni-trogen fixation and plant growth, maintaining performance even under high nitrogen conditions. Diverse nitrogen-fixing bacteria were identified from the maize rhizo-sphere, possessing multiple growth-promoting functions and stress tolerance. K. quasivariicola GN8811 demonstrated the best performance, and its genetic enhancement further improved nitrogen fixation efficiency. These findings highlight the potential of combining microbial screening with genetic engineering to develop efficient bioinocu-lants for sustainable maize cultivation. Biological nitrogen fixation by plant-associated bacteria offers a promising route to reduce synthetic nitrogen fertilizer inputs in cere-al-based agroecosystems, yet its reliability is often constrained by environmental stress and nitrogen repression. In this study, we combined systematic isolation of native maize rhizosphere diazotrophs with targeted regulatory engineering of the NifL–NifA system to generate a high-performance nitrogen-fixing strain capable of promoting maize growth even under nitrogen-replete conditions. Our results demonstrate that precise genetic rewiring of indigenous plant-associated bacteria can substantially en-hance nitrogen fixation efficiency and plant growth promotion, highlighting a viable strategy for developing next-generation biofertilizers to support sustainable maize production.