Denitrification is one of the major processes causing nitrogen loss from arable soils. This study aimed to investigate the responses of nirS-type denitrifier communities to different chronic fertilization regimes across the black soil region of Northeast China. Soil samples were collected from sites located in the north (NB), middle (MB), and south (SB) of the black soil region of Northeast China, each with four chronic fertilization regimes: no fertilizer (NoF), chemical fertilizer (CF), manure (M), and chemical fertilizer plus manure (CFM). Methods of quantitative polymerase chain reaction (qPCR) and Illumina MiSeq sequencing were applied to assess the abundance and composition of denitrifier communities by targeting the nirS gene. The results showed that the M and CFM regimes significantly increased the abundances of nirS-type denitrifiers compared with NoF at the three locations. The majority of nirS sequences were grouped as unclassified denitrifiers, and the different fertilizers induced little variation in the relative abundance of known nirS-type denitrifier taxa. Over 90% of the sequences were shared among the four fertilization regimes at each location, but none of the abundant operational taxonomic units (OTUs) were shared among the three locations. Principal coordinate analysis (PCoA) revealed that the communities of nirS-type denitrifier were separated into three groups that corresponded with their locations. Although similar fertilization regimes did not induce consistent changes in the nirS-type denitrifier communities, soil pH and NO3--N content simultaneously and significantly influenced the structure of nirS-type denitrifier communities at the three locations. Our results highlight that geographical separation rather than chronic fertilization was the dominant factor determining the nirS-type denitrifier community structures, and similar chronic fertilization regimes did not induce consistent shifts of nirS-type denitrifier communities in the black soils.

Denitrification is one of the major processes causing nitrogen loss from arable soils. This study aimed to investigate the responses of nirS-type denitrifier communities to different chronic fertilization regimes across the black soil region of Northeast China. Soil samples were collected from sites located in the north (NB), middle (MB), and south (SB) of the black soil region of Northeast China, each with four chronic fertilization regimes: no fertilizer (NoF), chemical fertilizer (CF), manure (M), and chemical fertilizer plus manure (CFM). Methods of quantitative polymerase chain reaction (qPCR) and Illumina MiSeq sequencing were applied to assess the abundance and composition of denitrifier communities by targeting the nirS gene. The results showed that the M and CFM regimes significantly increased the abundances of nirS-type denitrifiers compared with NoF at the three locations. The majority of nirS sequences were grouped as unclassified denitrifiers, and the different fertilizers induced little variation in the relative abundance of known nirS-type denitrifier taxa. Over 90% of the sequences were shared among the four fertilization regimes at each location, but none of the abundant operational taxonomic units (OTUs) were shared among the three locations. Principal coordinate analysis (PCoA) revealed that the communities of nirS-type denitrifier were separated into three groups that corresponded with their locations. Although similar fertilization regimes did not induce consistent changes in the nirS-type denitrifier communities, soil pH and NO3--N content simultaneously and significantly influenced the structure of nirS-type denitrifier communities at the three locations. Our results highlight that geographical separation rather than chronic fertilization was the dominant factor determining the nirS-type denitrifier community structures, and similar chronic fertilization regimes did not induce consistent shifts of nirS-type denitrifier communities in the black soils.