LAUSR

Abstract Excessive nitrogen (N) fertilization (ENF) and low utilization efficiency of fertilizer-derived N in high-input, high-yield cropping systems are serious ecological and economic problems in China. To examine the effects of long-term ENF on soil diazotrophs in relation to maize growth, we performed a 5-year field trial whereby 0–300 kg N ha−1 were added to black soil under a maize monocropping system in Northeast China. At the end of the 5-year field trial, the abundance and community structure of diazotrophs in the bulk soil and rhizosphere were investigated at the maize jointing stage by real-time quantitative polymerase chain reaction (qRT-PCR) and high-throughput sequencing. The results showed that: 1) ENF (N300) limited maize growth in the early stages and did not increase grain yield compared with moderate N supplementation (N180); 2) ENF significantly reduced nifH copy number, species richness, and Shannon index values of soil diazotrophs by 53.7%–79.7%, 37.2%–47.6%, and 20.0%–31.6%, respectively, reduced the relative abundance of Burkholderia, and increased the relative abundance of Sphingobium; 3) ENF decreased soil pH and increased electrical conductivity, which were the main factors affecting soil diazotrophic community structure as determined by Mantel test; and 4) diazotrophs were 1.3–3.0 times more abundant in the rhizosphere than in bulk soil, but no differences in α-diversity and community composition of diazotrophs were observed between rhizosphere and bulk soil. Thus, long-term ENF has negative impacts on the soil diazotrophic community, inhibiting maize growth in the early stage. Split N fertilization or slow/controlled-released N fertilizer should be used to avoid the negative effects of ENF on the soil diazotrophic community structure and early maize growth.