Abstract: A five-year field experiment was conducted at Dingxi in the Gansu Province of China from 2013 to 2018 to determine the effects of continuous application of different nitrogen rates on bacterial community structure and denitrification mechanisms. The experiment had six nitrogen application rates, namely: No nitrogen application (control, N₀), 75 kg N·ha^-1 (N₇₅), 150 kg N·ha^-1 (N₁₅₀), 225 kg N·ha^-1 (N₂₂₅), 300 kg N·ha^-1 (N₃₀₀) and 375 kg N·ha^-1 (N₃₇₅). It was found that nitrate ($NO_{3}^{-}$-N) content and nitrite reductase activity significantly increased with increasing nitrogen application rates. Treatment N375 increased $NO_{3}^{-}$-N content by 86.87%-856.12% when compared with the other treatments. Results from high-throughput sequencing showed that N375 significantly reduced the Alpha diversity of the bacterial community and changed the bacterial community composition of the rhizosphere. The control increased the relative abundance of Proteobacteria and thick-walled bacteria by 1.66%-26.53% and 22.59%-85.52%, respectively when compared with the nitrogen treatments. The relative abundance of nirK and nirS increased with increasing nitrogen application rates. Treatment N375 increased the relative abundance of nirK and nirS by 116.26% and 28.24%, respectively when compared with the control. Pearson correlation analysis showed that the relative abundance of nirK was significantly and positively correlated with $NO_{3}^{-}$-N while soil pH was negatively correlated with the abundance of nirK and nirS. The accumulation of $NO_{3}^{-}$-N and the reduction of soil pH due to the high nitrogen application rate changed the bacterial community structure and nirK and nirS abundance.

Key words: $NO_{3}^{-}$-N, bacterial community structure, denitrification, nirK, nirS