Objectives The objective of this study was to investigate the comprehensive effects of nitrogen fertilization amounts on legumes crop N fixation, nitrogenase activity in green manure season and rice nutrient absorption and yield in later rice season.

Methods The long-term Chinese milk vetch-rice rotation system was adopted in this experiment, with four field treatments established: rice-winter fallow-N0 (RF-N0), rice-milk vetch-N0 (RM-N0), rice-milk vetch-N1 (RM-N1), rice-milk vetch-N2 (RM-N2). N0, N1 and N2 represented the application of 0, 50, 100 kg/hm² of N during the milk vetch green manure season, respectively, while 200 kg/hm² of N was uniformly applied during the subsequent rice season. Field observations on symbiotic nitrogen fixation, nitrogenase activity, nutrient nitrogen uptake by milk vetch, and nitrogen uptake, yield and qualities of grains by rice were conducted for 2 consecutive rotations.

Results During the green manure crop season, the average symbiotic nitrogen fixation of RM-N0 reached 78.3 kg/hm² of N and the average symbiotic nitrogen fixation rate being 91.1%. Compared with the RM-N0, the average symbiotic nitrogen fixation in the RM-N1 and RM-N2 was reduced by 6.5% and 50.8%, respectively. The average symbiotic nitrogen fixation rate of the RM-N1 and RM-N2 was reduced by 22.1% and 72.1%, respectively as compared with the RM-N0 treatment. The nitrogenase activity per unit nodule was improved with the increase of nitrogen application during green manure season. While the Chinese milk vetch nodule biomass was significantly reduced under high nitrogen addition at 100 kg/hm² of N. In the green manure season, the average nitrogen uptake in the RM-N0 was 86.0 kg/hm² of N. Compared with RM-N0 treatment, the average nitrogen uptake of the RM-N1 and RM-N2 treatments increased by 25.5% and 62.7%, respectively. Compared with the RF-N0 treatment, the yield of rice was significantly increased by 6.1%−10.8% (2021) and 6.7−10.9% (2022) in all three RM treatments. Moreover, the protein contents in the rice grains increased by 11.5% and 21.5% for the RM-N1 and RM-N2, respectively, without obvious influences on the amylose contents or edibility values as compared to the RF-N0. Additionally, the weeds growth was significantly inhibited by growing milk vetch and soil total nitrogen content was improved as well. The RM-N1 produced the highest nifH gene copies among all treatments, while the RM-N2 produced better in AOB gene copies, and the RM-N0 was more suitable for AOA gene copies.

Conclusions Compared with the winter fallow field, growing Chinese milk vetch would significantly increase the rice yield, improve grain qualities and inhibit weeds growth. Adding moderate amount of nitrogen fertilizer in the RM-N1 at 50 kg/hm² of N can improve the biomass of Chinese milk vetch nodules, nitrogenase activity and nitrogen uptake, with greater nifH gene copies than other treatments. Thus, the RM-N1 is recommended based on comprehensive evaluations for the winter milk vetch-summer rice rotation cropping system.