2.6 Statistical analysis
In this study, each group (nitrogen rates and control treatments) consisted of four physicochemical and biological replicates, and results are shown as the mean value plus/minus standard deviation and the statistical significance is accepted at p < 0.05. Using SPSS Statistics 17.0 (IBM Company, Armonk, NY, USA), one-way analyses of variance (ANOVA) and two-way ANOVA comparisons were used to identify the effect of nitrogen rates and cultivation time on soil chemical properties and microbial communities. Using the QIIME software 1.7.0 (https://qiime.org), the diversity indices including observed OTUs, Chao1, ACE, Shannon, Simpson and coverage were obtained and used to evaluate the biodiversity based upon the richness of the species and to estimate the abundance-based richness within the community (Magurran, 2013). Based upon CANOCO version 5.0 (Microcomputer Power, Ithaca, USA), principal coordinates analysis (PCoA) was employed to analyze the abundance of bacterial class to visualize differences in soil bacterial communities, and redundancy analyses (RDA) were conducted to investigate the relationship between environmental factors and bacterial community structure.

3. Results