Combined application of organic manure and chemical fertilizers stabilizes soil N-cycling microflora

• The abundance of N-cycling genes differently responded to NPK application. • Chemical NPK application greatly altered the N-cycling microbial community structure. • Soil acidification was the main driver for the variation in the N-cycling microbial community. • Manure addition was beneficial for stabilizing the N-cycling microbial community. Straw and manure are widely applied to agricultural systems, and greatly shape soil N-cycling microflora. However, we still lack a comprehensive understanding of how these organic materials structure soil N-cycling microbial communities. In this study, metagenomic analysis was performed to investigate the compositional variation in N-cycling microbial communities in a 30-year long-term experiment under five fertilization regimes: no fertilization (Control), chemical fertilization only (NPK), and NPK with wheat straw (NPK + HS), pig manure (NPK + PM), and cow manure (NPK + CM). Long-term NPK application differentially changed N-cycling gene abundance and greatly altered N-cycling microbial community structure. NPK + HS resulted in a similar pattern to NPK in terms of gene abundance and community structure. However, NPK + PM and NPK + CM significantly increased most genes and resulted in a community similar to that of the Control. Further analysis revealed that serious soil acidification caused by long-term NPK fertilization was a major factor for the variation in N-cycling microbial communities. The addition of alkaline manure, rather than wheat straw, stabilized the N-cycling microbial community structure presumably by alleviating soil acidification. These results revealed the strong impact of soil acidification on microbial N-cycling communities and illustrated the possibility of resolving nitrogen-related environmental problems by manipulating pH in acidified agricultural soils.