Objectives Nitrogen (N) application regulates nodulation, N₂ fixation, yield and quality of soybeans. We studied the effects of N application time and rate for high-yield and high-quality soybean production.

Methods A pot experiment was conducted using soybean cultivar Dongsheng 35 as the test material. The treatments include two N application periods (V2 and R1) and three nitrogen application rates 0, 5, 100 mg/(kg, soil). At R2 stage (full bloom) and R5 stage (beginning seed-setting), the shoot dry matter accumulation, nodule number, nodule dry weight and nitrogenase activity of soybean were analyzed. At R8 stage (full maturity), the grain yield and protein content of soybean were investigated.

Results Both the N application rate and time had significant effects on shoot dry matter accumulation, nodulation and nitrogenase activity of soybean. N application increased the shoot dry matter accumulation but decreased the nodule dry weight and number. Applying N at R1 stage, the recorded nodule number and dry weight in N100 treatment were 42.3% and 32.8% lower than N0 treatment, while N5 treatment achieved the highest nitrogenase activity. Applying N at V2 stage, the nitrogenase activity in N5 treatment was 15.3% and 27.1% higher than N0 treatment at the R2 and R5 stages, respectively. N5 treatment produced the highest soybean yield and protein content, and the protein content was 6.3% and 9.4% higher than N0 treatment at the N application times of R1 and V2 stages. According to the structural equation model analysis, N application rate positively regulated nitrogenase activity and affected soybean yield indirectly, but negatively regulated the nodule number and affected soybean protein content indirectly. The N application time negatively regulated soybean yield but positively regulated protein content.

Conclusions In general, earlier N application (V2 stage) is beneficial to soybean yield formation, while later N application (R1 stage) is good for nitrogen fixation and soybean protein content. The effect of nitrogen application rate on soybean yield and protein content is greater than that of application time in pot experiment. And the rate of N application should be controlled at N 5 mg/(kg, soil).