Transformative Impact of Three Decades of Nitrogen-Based Fertilization on Diazotrophic Communities and Co-occurrence Patterns in Soils of North-East 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.