Abstract: Understanding the impacts of long-term fertilization regimes on soil microbial ecology in paddy field can provide theoretical basis for optimal fertilization and farming. Here, an 8-year experiment was conducted to examine the effects of N application levels (N0, N100, N200) under the condition of no returning milk vetch in winter on the abundance and structure of soil microbial community during the flowering of milk vetch in a double cropping system in Hunan Province. Soil microbes were quantified using Illumina MiSeq high-throughput sequencing platform and fluorescence quantitative PCR technique. The results showed that soil pH, nitrate content and soil C/N ratio significantly decreased with increasing N application level under the condition of no returning milk vetch in winter. Moreover, N fertilization significantly increased the contents of total N, soil organic carbon and ammonium N in paddy soil. The microbial quantity in paddy field was 1.66×10⁸-1.23×10¹⁰ copies·g^-1 dry soil. N fertilization increased the 16S rDNA gene abundance, and the copy number of 16S rDNA gene in N0, N100 and N200 fertilization treatments was 1.0, 72.4 and 32.6 times of that in CK, respectively. The diversity index (Shannon index and Simpson index) and species richness index (Ace and Chao1) of the three N fertilization treatments were higher than those of CK, among which N200 had the highest diversity index and N100 hand the highest species richness. Three major taxonomic groups of 16S rDNA in different samples were Proteobacteria, Chloroflexi and Nitrospirae, which accounted for 33%-44.4%, 17%-22.3% and 9%-10% of the total OTU, respectively. The relative abundance of Proteobacteria and Nitrospirae in N0, N100 and N200 soil samples was lower than that of CK, while the relative abundance of Chloroflexia in N0, N100 and N200 was 1.2, 1.3 and 1.2 times of CK,respectively. Correlation analysis showed that the copy number of bacterial 16S rDNA gene was significantly correlated with soil organic carbon, total N and ammonium contents. There was a close correlation between soil bacterial groups and soil chemical properties. In conclusion, planting Chinese milk vetch in winter without returning to field and N fertilizer application significantly increased the yield of rice and the abundance of soil microbes in the flowering period of Chinese milk vetch, changed soil microbial community structure, and improved soil chemical properties, which is of guiding significance for planting green manure scientifically and reasonably in double cropping rice area of Hunan Province.

Key words: maize, soil, layered fertilization, slow-release urea, enzyme activity, nitrogen