Nitrogen (N) fertilizers can promote soybean growth, nodulation, and nitrogen fixation to a certain extent. However, excessive nitrogen application inhibits the nitrogen fixation capacity of soybean nodules. In this study, three experimental materials were used to investigate the direct and indirect effects of localized exogenous nitrogen (Ammonium Nitrate, NH₄NO₃) on nodule nitrogen fixation in soybean. Three nitrogen supply methods were applied: bilateral nodulation dual-root soybeans, unilateral nodulation dual-root soybeans, and upper- and lower-layered soybeans. The root nitrogen accumulation of direct contact with exogenous nitrogen reached 72.61 mg/plant, 30.59 mg/plant, and 88.48 mg/plant, respectively, and its nitrogen accumulation ability was higher. Exogenous nitrogen inhibited nodule growth and nitrogen accumulation. Nodule development and nitrogenase activity were regulated both directly and indirectly by exogenous nitrogen, with a more pronounced inhibitory effect observed in the roots directly exposed to nitrogen. Experiment I demonstrated that the number and dry weight of nodules on the nitrogen supply side decreased by 35.04% and 40.00%, respectively, while the difference was not significant on the non-nitrogen supply side. Furthermore, the nodule system exhibited a substantial buffering effect on exogenous nitrogen. In Experiment I, no significant differences were observed in the number, dry weight, or nitrogenase activity of nodules on the non-nitrogen-supplying side. The number and dry weight of nodules in Experiment II decreased by 61.55% and 35.91%, respectively. The specific nitrogenase activity (SNA) and acetylene reduction assay (ARA) also decreased by 32.28% and 67.20%, respectively, showing significant differences. In Experiment III, the number and dry weight of nodules in the upper layers decreased by 23.70% and 15.12%, respectively. Furthermore, significant differences in nitrogenase activity were detected, indicating that the nodules exposed to exogenous nitrogen spontaneously initiated the nitrogen regulation mechanism. This partially offsets the inhibitory effect on the nitrogen fixation function of nodules on the indirectly exposed side. This study revealed that exogenous nitrogen supply significantly affected the growth efficiency and nodule nitrogen fixation function of soybean plants by regulating nitrogen absorption and resource allocation. The use of deep unilateral fertilization can ensure the nitrogen fixation capacity of nodules and nitrogen accumulation in soybean plants and provide theoretical support for improving nitrogen use efficiency and realizing scientific fertilization.

Biological nitrogen fixation is an important support for sustainable agricultural development. The symbiotic nitrogen fixation system between legumes and rhizobia is the core of biological nitrogen fixation in nature. As a crucial signaling molecule, NO plays a complex regulatory role in concentration and stage dependence in the nodulation and nitrogen fixation of legumes, and its direct and indirect regulatory effects are not yet clear. In this study, a unilateral nodulating dual-root soybean was used as the experimental material. Different concentrations of NO donor sodium nitroprusside (SNP) were supplied to the nodulating side and the non-nodulating side, and measurements were taken of nodule growth, nitrogen-fixation-related indicators, and NO content in various plant organs. The results showed that exogenous NO inhibited the growth of soybean nodules in a contact-, concentration-, and time-dependent manner. The direct supply of NO on the nodulating side showed a more significant inhibitory effect. After 10 days of treatment, the number and dry weight of the nodules in T5 decreased by 53.9% and 33.3%, respectively, compared with the control treatment T1, while the nitrogenase activity (ARA) in the nodules was significantly lower than that in the control treatment T1 by 58.4%. There were concentration differences in how NO regulates nitrogen fixation capacity. A high concentration or long-term supply of NO significantly reduced nitrogen fixation efficiency, which may be due to the excessive accumulation of NO in the nodules. We found that the four SNP application methods significantly increased the NO content in all parts of the plant. The NO content in the plants increased with higher SNP concentrations and longer treatment durations. The effect of direct NO supply on plant NO content was notably stronger than that of indirect NO supply. After 7 days of treatment, the NO content of the T4 and T5 nodules increased by 44.2% and 62.8%, respectively, compared with T1, while T2 and T3 only increased by 23.5% and 45.3%, respectively. After 10 days of treatment, the same significant results were also obtained. This study clarified the role of NO in regulating soybean nodulation and nitrogen fixation. It provided a theoretical basis for improving soybean symbiotic nitrogen fixation efficiency and guiding agricultural fertilizer-saving production.