Diazotrophs play a critical role in converting air-inactive nitrogen to bio-available nitrogen. Assessing the influences of different fertilization regimes on diazotrophs is essential for a better understanding of their maintenance of soil fertility and agricultural sustainability. In this study, we targeted the nifH gene to investigate the effects of different long-term fertilization on the diazotrophic community in a vertisol, using real-time quantitative polymerase chain reaction (PCR) and MiSeq sequencing. Five fertilization regimes were tested: no fertilizer (CK), chemical nitrogen, phosphorus, and potassium fertilizer (NPK), organic fertilizer (O), chemical NPK plus organic fertilizer with an equivalent application rate of nitrogen (NPKO), and chemical NPK plus organic fertilizer with a high application rate of nitrogen (HNPKO). Our results showed that fertilization significantly affected the diazotrophic activity, abundance and composition. NPK tended to reduce the activity, abundance, operational taxonomic units (OTU)-richness and alpha-diversity of the diazotrophs, while O had the opposite effect. The effects of inorganic and organic fertilization on the diazotrophs depended on the N application rate, showing that the diazotrophic activity, abundance, and alpha-diversity in NPKO were higher than that of HNPKO. For the diazotrophic community structure, CK, O, and NPKO were grouped and separated from NPK and HNPKO. The diazotrophic community structure strongly correlated with the soil pH, electrical conductivity (EC), total carbon content (TC), and total nitrogen content (TN), among which pH was the major factor shaping the diazotrophic community structure. Different network patterns were observed between the long-term organic and non-organic fertilizers, suggesting that the organic amendment resulted in a more complicated diazotrophic community than the non-organic amendments. Rhizobium was the most important hub connecting members in the community. These results indicated that organic amendments are beneficial to diazotrophic activity, abundance, OTU richness, alpha-diversity, and the diazotrophic communities' potential interactions, which may enhance biological nitrogen fixation in vertisols.
