Metagenomic insights into Bacillus-Bradyrhizobium co-inoculation enhancing soybean nodulation

Co-inoculation with plant growth promoting rhizobacteria (PGPR) and rhizobia can enhance nodule number and yield in leguminous crops. To the best our knowledge, this is the first study combined microbial diversity analysis and metagenomics to systematically investigate, from a soil microbial ecology perspective, the mechanism by which Bacillus subtilis 8-32 and Bradyrhizobium liaoningense CCBAU 05525 co-inoculation increase soybean nodules. Co-inoculation significantly boosted shoot and root biomass and increased nodule number by 108.6% through three synergistic pathways. First, it improved soil physicochemical properties and enzyme activities, increasing nitrogen and phosphorus availability and enhancing urease and cellulase activities. Second, it restructured the rhizosphere bacterial community, enriching three representative PGPR genera (Bacillus, Sphingomonas, and Stenotrophomonas), and recruiting three potentially functional genera (Massilia, Ensifer, and Noviherbaspirillum). Third, it significantly enhanced carbon and nitrogen cycling, as evidenced by higher abundances of genes related to carbon metabolism and fixation, providing ample carbon and energy for rhizobial colonization and nodule development. Furthermore, key nitrogen metabolism genes, including nifH, vnfH, and those for ammonia assimilation, nitrification, and nitrogen transport, were significantly upregulated, helping maintain rhizosphere nitrogen homeostasis. Therefore, these findings offer theoretical insights into the synergistic mechanisms of B. subtilis 8-32 and B. liaoningense CCBAU 05525 in promoting soybean nodulation and support the development of efficient microbial inoculants for sustainable agriculture.