Long-term application of cattle manure alters functional N cycling genes and improves maize yield and nitrogen use efficiency

• Cattle manure significantly boosts maize yield by improving NUE. • Cattle manure shifted nitrogen-related microbial communities. • Genes associated with nitrogen cycling related with NUE and yield. • Double-dose manure amendment did not further increase yield but induce more narG genes and smaller MAGs. Using livestock manure as organic fertilizer on farmlands has resulted in increased crop yields in many areas; however, the impact of long-term manure application on N cycling microorganisms and their effect on nitrogen utilization efficiency (NUE) associated with increased maize yield remains unclear. In this study, a long-term field trial was performed to compare four fertilization regimes: no fertilizer (CK), conventional synthetic fertilizer (CF), CF with 15 Mg ha −1 (FM1) and 30 Mg ha −1 (FM2) of dry-weight cattle manure, applied annually. After 10 and 12 years, FM1 and FM2 significantly boosted maize yield, especially after cycles of soybean–maize rotation, and improved NUE by 19–33 % and 17–53 %, respectively, compared with CF, with a positive correlation with increases in yield. Principal coordinate analysis showed that different fertilizer regimes were partitioned into four groups of N-related microbial communities. Proteobacteria, Actinobacteria, and Acidobacteria were the main phyla involved in N cycling. Among the genes involved in N cycling, gdhA , narB , nasD , norB , napA , nirB , nifA and hao demonstrated significant correlations with both plant N uptake and NUE. The insignificant differences between FM1 and FM2 in the relative abundance of most N genes in 2021 and 2023 helped to explain the similar crop outcomes between these two treatments. Compared with FM1, FM2 had a higher relative abundance of narG and a smaller size of constructed metagenome-assembled genomes (MAGs).