Opportunistic keystone diazotrophs from co-occurrence networks drive biological nitrogen fixation in peanut/cotton intercropping systems

Legume-based intercropping enhances asymbiotic biological nitrogen fixation (BNF); however, the underlying mechanisms remain unclear, including the roles of soil keystone diazotroph taxa with varying niche breadths. A field experiment was conducted to evaluate soil BNF variations between rhizosphere and bulk soils in peanut/cotton intercropping systems and monocultures. BNF activities were measured by nitrogen fixation rates, nitrogenase activity, and nifH gene abundance. Phylogenetic null models, co-occurrence networks, and niche breadth analysis were applied to investigate the roles of diazotrophic keystone taxa and their ecological niches. Rhizosphere soils exhibited 7.8–125.5% higher BNF potentials than bulk soils, whereas intercropping systems showed 11.6–323.0% increases over monocultures for nitrogen fixation rate, nitrogenase activity, and nifH gene abundance (all P<0.05). Diazotrophic community composition and diversity differed significantly, with Proteobacteria (excluding Alphaproteobacteria) enriched in intercropping and rhizosphere soils, while Cyanobacteria and Firmicutes were less abundant. Deterministic processes, particularly heterogeneous selection, dominated community assembly in the rhizosphere (91.9%) and intercropping soils (86.3%). The co-occurrence networks consistently revealed more complex and interconnected communities in intercropping and rhizosphere soils that were dominated by opportunistic diazotrophs (78.8–85.9%), followed by specialists (10.2–18.5%) and generalists (1.38–3.80%). Keystone taxa, including opportunists such as Azoarcus, Azohydromonas, and Steroidobacter, and generalists like Pseudomonas and Azotobacter, correlated positively with microbial biomass carbon and nitrate nitrogen, contributing to enhanced BNF. Peanut/cotton intercropping enhances BNF by selectively enriching the keystone diazotrophic taxa with varying ecological roles, particularly opportunists and generalists. Such targeted intercropping strategies can optimize BNF, improve soil fertility, and promote sustainable agricultural production.