How soil organic carbon structures diazotrophic communities: Insights from apple orchards across China

Biological nitrogen fixation (BNF) mediated by diazotrophs is critical for soil nitrogen cycling. However, the effect of variations in soil organic carbon (SOC) on the regulation of diazotroph diversity and community assembly in orchard ecosystems remains unclear, limiting our ability to optimize nutrient management for sustainable orchard production. Here, we investigated the effects of SOC on diazotrophic communities across 299 apple orchards in 11 major apple-producing provinces of China. Soil samples collected from the 0-40 cm depth were analyzed for key physicochemical properties including pH, organic carbon, and available nitrogen/phosphorus/potassium, and high-throughput sequencing of the nifH gene was used to characterize diazotrophic diversity, community composition, assembly processes, and co-occurrence network patterns. The results showed that SOC was the principal factor influencing diazotrophic diversity. Diversity increased significantly with SOC in low-SOC orchards (R = 0.22), but reached a plateau under high-SOC conditions. Across all regions, generalist species-including generalist-abundant (R = 0.85, R = 0.73) and generalist-rare (R = 0.90, R = 0.83) taxa had a greater influence on overall diazotrophic diversity, whereas only specialist species exhibited significantly higher diversity in high-SOC orchards than in low-SOC orchards. Furthermore, SOC significantly altered diazotrophic community structure, particularly by enriching Bradyrhizobium and Microvirga in high-SOC orchards. Stochastic processes dominated community assembly (50.87-86.08 %), however, deterministic processes increased for generalist-abundant (22.23 %) and specialist taxa (36.56 % and 31 %) in high-SOC regions. Co-occurrence networks in high-SOC orchards showed reduced connectivity (30.82) and modularity (0.042), indicating weaker microbial interactions, whereas generalist species contributed to network stability in low-SOC orchards. Overall, organic carbon management is critical for enhancing biological nitrogen fixation and reducing reliance on synthetic fertilizers. Increasing SOC through organic amendments benefits low-SOC orchards, while high-SOC orchards should prioritize carbon maintenance to preserve microbial interactions, -providing targeted guidance for sustainable orchard nutrient management.