Transformative impact of three decades of nitrogen-based fertilization on diazotrophic communities and co-occurrence patterns in soils of Northeast China

Biological nitrogen (N) fixation (BNF) is a crucial biogeochemical process on Earth, in which diazotrophs play a significant role. The impact of long-term nitrogen (N) input from fertilization on diazotroph responses remains unclear. In this study, we investigated diazotrophic abundance, diversity, and potential N₂-fixation activity in a 29-year maize (Zea mays L.) grown brown soil experiment with CK (unfertilized control), N4 (270 kg N ha^-1 a^-1 urea), and M2N2 (135 kg N ha^-1 a^-1 urea, 135 kg N ha^-1 a^-1 pig manure). We analyzed the nifH gene abundance and diversity using quantitative polymerase chain reaction (qPCR) and high-throughput sequencing, and determined the nitrogenase activity with the acetylene reduction method. Our results showed that N fertilization inhibited the soil's potential N₂-fixation activity compared to CK. Specifically, N4 had no impact on diazotrophic abundance and alpha diversity, while M2N2 increased both. Prolonged fertilization inhibited the phylum Cyanobacteria, Nitrospirae, and an unclassified phylum, while improving the phylum Proteobacteria. Additionally, N4 enhanced the phylum Deinococcus-Thermus. Monte Carlo test and canonical correlation analysis revealed that factors such as available N and pH played crucial roles in community construction. Network analysis indicated that fertilization enhanced diazotroph relationships, with N4 having the most complex network. The genera actively connected, or "common species," differed in moderate abundance, contributing to diazotrophic community stability. Long-term fertilization reduced soil N₂-fixation activity, prompting diazotrophs to form intricate co-occurrence networks in response to environmental shifts induced by nitrogen accumulation.IMPORTANCEFertilization has the potential to impact nitrogen (N) cycling within soil ecosystems. However, the response of diazotrophs to environmental changes induced by long-term N input remains largely unknown. Our research has uncovered that while inorganic N fertilizer did not significantly alter diazotrophic abundance and diversity, a combination of organic manure and mineral N fertilizer resulted in elevated levels of both. Moreover, the community structure of diazotrophs was found to be significantly influenced by nearly 30 years of N-based fertilizer application, with the key driving factors being the availability of N and soil pH. Network analysis revealed various co-occurrence patterns, indicating that fertilization led to increased interactions among diazotrophs, suggesting a more competitive dynamic as a result of prolonged N fertilization. We anticipate that these novel and significant findings will contribute to the development of more sustainable practices for N fertilizer utilization and enhance our understanding of soil diazotrophic diversity.