Abstract: 【Objective】The aim of this study was to investigate the effect of the nitrogen input amount on diversity of denitrifying bacterial community in paddy soil by nirS gene based on field location test of rice. 【Method】 Composition and abundance of denitrifying bacterial community in paddy soil were studied with the aid of denaturing gradient gel electrophoresis, cloning sequencing and real-time PCR by assay nirS gene in the forth year of a field experiment.【Result】Shannon-Wiener index of denitrifying bacterial nirS gene community in paddy field was calculated according to the image of DGGE. In the three treatments of nitrogen fertilizer (N1: 75 kg N•hm-2, N2: 150 kg N•hm-2 and N3: 225 kg N•hm-2), the Shannon-Wiener index was higher than that in the treatment of CK (without fertilizer), and the differences reached a significant level at heading and maturate stages of rice (P＜0.05). Whereas, among treatments of N1, N2 and N3 only Shannon-Wiener index of N3 was markedly higher than that of N1 in surface soil (P＜0.05) at tillering and heading stages of rice. Redundancy discriminate analysis (RDA) showed that the denitrifying bacterial nirS gene community structure in surface soil or in root zone soil all markedly correlated to growth stages of rice (P=0.002, 0.002), while there was a significantly correalation (P=0.002) between different levels of nitrogen fertilizer treatments and denitrifying bacterial nirS gene community structure only in surface soil. Real-time PCR analysis of nirS genes copies manifested that the increase in nitrogen input amount enhanced the abundance of denitrifying bacteria. At heading or maturate stages of rice, there was a upward trend of CK＜N1＜N2＜N3 in nirS genes copies in surface soil or in root zone soil, especially in surface soil at heading stage of rice (P＜0.05). At the same time, 8 DNA sequences of nirS gene cloning from DGGE band were got and logged in GenBank (Accession Number: JX997923, JX997924, JX997926-JX997931). In addition, the yield of rice in treatments of N1, N2 or N3 was 59%, 92%, or 107% more than that of CK. NO3--N in surface soil or in root zone soil increased with the increase of nitrogen input amount.【Conclusion】The findings demonstrated that the increase in nitrogen input amount promoted the enhancement in abundance and Shannon-Wiener index of denitrifying bacterial nirS gene community in paddy field, especially in surface soil. Changes in community structure of denitrifying bacterial nirS gene among different levels of nitrogen fertilizer treatments also took on in the surface soil. So there is an obvious response of denitrifying bacterial nirS gene community to nitrogen input amount in surface soil of the paddy field.

Key words: nitrogen fertilizer levels , denitrifying bacteria , nirS gene , community structure , community abundance