In this study, 0 - 10.5 m soil samples were taken below experimental plots where N-fertilizer was applied for nearly 20 years under wheat-maize double cropping in the North China Plain (NCP). N-fertilizer application rates of 0 (N0) and 600 kg N ha-1yr-1 (N600) were applied to different plots. Multidisciplinary approaches were employed to profile the vertical distribution of denitrifer genes and soil microbial communities responding to long-term N fertilization.

Soil microbiomes played essential roles in nitrogen cycling of agricultural ecosystems. However, little was known of their vertical responses to long-term N fertilization and their contribution to denitrification process. In this study, 0 - 10.5 m soil samples were taken below experimental plots where N-fertilizer was applied for nearly 20 years under wheat-maize double cropping in the North China Plain (NCP). N-fertilizer application rates of 0 (N0) and 600 kg N ha-1yr-1 (N600) were applied to different plots. Multidisciplinary approaches were employed to profile the vertical distribution of denitrifer genes and soil microbial communities responding to long-term N fertilization. The decreasing abundance of denitrifier genes illustrates the low denitrification potential in the lower part of the vadose zone and denitrification in deep soils appeared to be substrate limited. The higher relative abundance of nosZ gene in N fertilized soil indicated N2 was the main product during denitrification process. High-throughput sequencing of the 16S rRNA gene revealed that overuse of N fertilizer caused a decrease of bacterial diversity. In addition, soil microbiomes also showed significant soil-depth dependence. The changes of soil properties significantly related with the shift in microbial structure, while soil pH and soil organic matter were the most marked factors impacting the microbial community in N0 and N600 treatments. It was also found that 2.5 m underground is an important watershed, whether for the vertical distribution of denitrification genes or for the profile of microbial community from surface soil to deep vadose soil, which may be caused by plant root exudates. In general, this study clearly elucidates the loss of N from agricultural soil subject to long-term overuse of N fertilizers, as increasing interest on the accumulation of nitrate and nitrous oxide which had potential threat on ground water pollution and climate change, respectively.