Objectives This paper studied the effect of nitrogen levels on the community and quantity of soil bacteria during winter fallow period of double cropping rice region, in order to present the theoretical basis for sustainable rice production in this region.

Methods Based on a 8-years’ continuous localized experiment in double cropping rice area, Hunan Province, soil samples in 5–20 cm layer were collected in treatments of nitrogen fertilizer levels of CK (N0), 100 kg/hm² (N1) and 200 kg/hm² (N2) during winter fallow period. The abundances and community of soil bacteria were determined using high throughput sequencing and fluorescence quantitative PCR methods.

Results Compared with CK, both N1 and N2 significantly increased the yield of double cropping rice, increased soil total N and total C contents, but decreased soil pH, NO₃⁻-N concentration and C/N ratio (P < 0.05). Nitrogen application significantly increased the total bacterial abundance in soils. The copy number of bacteria16s rDNA gene in N1 and N2 was 48.25 and 40.31 times of that in CK, respectively. The richness of soil bacteria was positively correlated with soil total N and C/N ratio, but negatively correlated with soil total C. Nitrogen application changed the diversity and community structure of soil bacteria in paddy field during winter fallow period. Compared with CK, N1 increased the soil bacterial diversity, N2 significantly increased bacterial abundance, but decreased soil bacterial diversity significantly. In addition, the highest relative abundance in all the three treatments was Proteobacteria, up to 40.16% – 51.16%. The relative abundance of Proteobacteria in N2 were significantly higher than those in CK and N1 treatments, while the relative abundance of Proteobacteria in N1 treatment was significantly lower than that in CK. In all the three treatments, Anaerolineaceae_uncultured and Nitrospira had higher relative abundance at genus, reaching 8.6%–14.56% and 8.16%–11.46%, respectively. Spearman correlation analysis showed that the 11 dominant phylum groups were significantly correlated with soil chemical properties. The diversity and community structure of bacteria in paddy soils are dependent on soil chemical properties.

Conclusions  Nitrogen application causes decreased soil pH and soil C/N ratio in the tillage layer. Low nitrogen application level will increase the diversity of soil bacteria, while high nitrogen application level will decrease the diversity of bacteria, although increase the total number of bacteria.