Objectives Asymbiotic nitrogen fixation is one of the important sources of nitrogen input in farmland ecosystems. Investigating the responses of asymbiotic nitrogen fixation rate, abundance of nitrogen-fixing microorganisms and diversity of community structure to nitrogen fertilizer application rates will provide fertilization reference for sustainable agricultural production.

Methods The experiment was conducted in black soil area of Northeast China, the test field was under conservation tillage and straw return since 2012. Six N application rate treatments were setup, 0 (N0), 120 (N120), 150 (N150), 180 (N180), 210 (N210), and 240 (N240) kg/hm². At harvest of maize, 0−5 cm and 5−20 cm soil samples were collected, conventional chemical methods were used to determine soil pH and available N, P and K contents, and acetylene reduction method, quantitative PCR, and high-throughput sequencing technology were used to analyze the soil asymbiotic nitrogen fixation rate, diazotroph community abundance and structure diversity.

Results Under the six N rate treatments, the asymbiotic nitrogen fixation rate ranged from C₂H₄ 0.26−0.63 nmol/(g·d) in both 0−5 cm and 5−20 cm soil layers, and N150 treatment exhibited the maximum asymbiotic nitrogen fixation rate, C₂H₄ 0.53 nmol/(g·d) in 0−5 cm layer and C₂H₄ 0.63 nmol/(g·d) in 5−10 cm layer. The nifH gene abundance ranged from 5.34×10⁶ to 15.64×10⁶ copies/g, and N150 treatment was recorded the maximum nifH gene abundance as well, they were 15.64×10⁶ and 8.74×10⁶ copies/g in the two aforementioned layers. Principal component analysis (PCoA) revealed notable differences among fertilization rates regarding the composition of soil diazotroph community. The dominant bacterial genera at species level were unclassified_o_Burkholderiales, unclassified_k_norank_d_Bacteria, Skermanella, and Methylocystis in all the six treatments. Spearman correlation analysis between environmental factors and indicators for soil diazotroph community showed that asymbiotic nitrogen fixation was positively correlated with available K and negatively correlated with pH. nifH gene copy number was positively correlated with AK and negatively correlated with fertilizer application rates. Redundancy analysis indicated that nifH community structure had a significant correlation with soil pH, NH₄⁺-N, NO₃⁻-N, soil organic C, total N, available P, and available K.

Conclusions Application of N 150 kg/hm² is beneficial for increasing the asymbiotic nitrogen fixation and nitrogen-fixing microbial abundance, enriching the community structure diversity, so is thought an optimal fertilizer rate in black soil farmland under long-term no-tillage and full-crop return.