Soil phosphorus drives variation in diazotrophic communities in a subtropical nitrogen-rich forest

Although nitrogen (N)-fixing bacteria (diazotrophs) play a vital role in the biosphere's N cycle. However, large uncertainty exists regarding how atmospheric N deposition regulates biological N fixation (BNF) in soils and which diazotrophic taxa are responsible for this process. We targeted the nifH gene to investigate the effects of long-term N addition on the abundance and composition of diazotrophic communities in subtropical N-rich forests, using real-time quantitative polymerase chain reaction (q-PCR) and MiSeq sequencing. We conducted a field trial for seven years and included three N treatment levels (+0, +50, +150 kg N ha−1 yr−1; CK, LN, HN). With increasing N addition, the average total P concentration in the whole soil profile and the topsoil P availability decreased significantly, further reducing nitrogenase activity and nifH abundance significantly, while increasing the diversity of the diazotrophic community. After seven years of N addition, the relative abundance of uncultured Cyanobacteria significantly decreased, while those of Rhizobiales and Desulfobulbaceae significantly increased. The diazotrophic communities transitioned from free-living N fixers (uncultured Cyanobacteria-dominated) to symbiotic N fixers (Rhizobiales-dominated; HN treatment). The relative abundance of heterotrophic diazotrophic bacteria and the occasional diazotrophic species increased, resulting in diazotrophic bacteria still maintaining high competitiveness in the N-rich and/or P-limited soils. Proteobacteria (e.g., Rhizobiales) had the highest potential to participate in microbial mechanisms triggered by P scarcity. Variation partitioning analyses showed that diazotrophic community composition and diversity were mainly correlated with the soil P availability, with soil AP having the highest individual effect. Overall, this study will broaden our understanding of N fixation mechanisms and the diazotrophic community variation in N-rich forests, while also highlighting soil P as a key factor driving changes in diazotrophic communities.