Objective This study aimed to explore the changes in nirS-type denitrifier community and network structures in response to long-term chemical fertilization for the goal of providing scientific basis for rational fertilization strategies and soil quality improvement.

Methods The experiment was conducted in the Scientific Observation and Experiment Station of Arable Land Conservation and Agriculture Environment (Heilongjiang), Ministry of Agriculture and Rural Affairs. Bulk soil samples were collected from eight fertilization treatments, including non-fertilized control (NoF), N (nitrogen), P (phosphorus), K (potassium), NK, NP, PK and NPK. The nirS gene copy numbers, communities and network structures were analyzed with qPCR (quantitative real-time PCR), Illumina MiSeq sequencing and molecular ecological network. In addition, the soil properties were determined to analyze the links with the changes of nirS-type denitrifier communities.

Results 1) All the N contained treatments, whether nitrogen alone or combined with phosphorus or potassium, significantly increased the nirS gene copy numbers and reduced the community diversity of nirS-type denitrifiers, while P and K treatments had little influence compared with control. 2) Proteobacteria was the only phylum detected in this study with the relative abundance of 16.96%–27.34%, and all the N contained treatments promoted the growth of Bradyrhizobium affiliated to this phylum. 3) The PCoA analysis showed that the whole nirS-type denitrifier communities could be clustered into two groups including N fertilization and non-N fertilization, which indicated that long-term application of N fertilizer significantly changed the community structures of nirS-type denitrifier. Mantel test analysis revealed that soil pH was the most important factor in shifting nirS-type denitrifier communities. 4) Networks of N contained treatments and non-N contained ones were separately constructed based on sequencing data, and large differences were observed between the network structures of the two groups. Long-term N fertilization simplified the network structure of nirS-type denitrifiers, and decreased the network stability with more influence from environmental disturbances.

Conclusions Long-term N fertilization, rather than P and K fertilizers, has larger influences on community and network structures of nirS-type denitrifier. The results may provide a new insight for understanding the changes of nirS-type denitrifier communities in response to different fertilization managements in a black soil of northeast China.