Metagenomic study on microbial function in increasing total nitrogen content in soil under crop rotation systems

Leguminous crops, which are vital bioresources, increase total nitrogen (TN) levels in soil, effectively improving microbial and plant nutrition and achieving environment-sustainable agricultural production. However, the specific processes of the nitrogen cycle and the role of soil microorganisms in increasing TN levels after soybean planting remain unclear. Herein, a comparative analysis of microbial nitrogen-cycling genes in soil was conducted based on a 12-year crop rotation involving wheat–maize (WM), wheat–cotton (WC), and wheat–soybean (WS) rotations. After 12 years, the WS treatment increased soil TN content by 9.1 % and 19.4 % compared with the WC and WM treatments, respectively, highlighting its unique nitrogen-enriching effects. Microbial community analysis indicated that the WS treatment significantly increased Shannon and β-diversity indices, both of which were positively correlated with TN content. Network analysis revealed three characteristic modules: module 1 (dominated by Proteobacteria/Actinobacteria) exhibited no significant correlation with TN; module 2 (dominated by Acidobacteria/Cyanobacteria), negative correlation; and module 3 (dominated by Thaumarchaeota), positive correlation. The analysis of nitrogen-cycling functional genes revealed that the nifH (nitrogen fixation) and nxrB (nitrite oxidation) genes were significantly enriched in WS soils, whereas the hao (hydroxylamine oxidation) gene abundance increased in WM soils. Further tracing indicated that WM promoted the accumulation of the hao gene by facilitating the proliferation of the Gemmatimonadetes phylum, whereas WS enhanced the expression of the nxrB gene by enriching Actinobacteria and key genera (e.g., Modestobacter, Pediococcus) and increased the abundance of the nifH gene by promoting Bradyrhizobium proliferation. This study confirmed that the WS system enhanced soil TN through a triple mechanism of constructing high-diversity microbial communities, optimizing functional microbial structures, and the promotion of functional gene expression to promote N accumulation in the soil, thereby providing a theoretical basis for improving soil nitrogen reservoirs via coordinated crop configuration and microbial regulation.