Quantifying soybean biological nitrogen fixation using long-term isotope enrichment

Abstract Background and aims Biological nitrogen fixation (BNF) supplies much of soybean ( Glycine max (L.) Merr.) nitrogen (N) demand, but reported fixation rates vary widely, leading to uncertainty in the soybean N cycle. We quantified whole-plant soybean BNF and N allocation using long-term 15 N labeling to assess whether BNF can offset grain N removal in a high-yielding system, and to quantify root contributions to fixed N. Methods Field mesocosms in Iowa, USA received three legacy fertilizer N rates as 2 atom% 15 N-enriched urea for four prior seasons of maize ( Zea mays L.) production. In 2024, N-fixing and non-N-fixing soybeans were grown to physiological maturity. Grain, aboveground biomass, and roots were analyzed to quantify BNF and to estimate whole-plant N budget. Results Soybean derived 81.8% ± 1% (SE) of total plant N from BNF. Legacy N rate had no effect on whole-plant N accumulation, BNF, or N partitioning among tissues (p > 0.05). Roots contained ~2% of total fixed N, with most BNF-derived N allocated to grain. High grain N removal was largely offset by BNF (174.7 ± 8.9 kg N ha −1 ), resulting in a near-neutral partial N balance (−5.7 ± 2 kg N ha⁻ 1 ). Conclusion From this one-season mesocosm study, our finding of negligible fixed N in soybean roots indicates that neglecting root BNF does not necessarily bias conclusions about soybean N cycle. However, the combination of long-term isotope labeling and non-nodulating isolines yielded much less variable and higher BNF rates than previous studies, suggesting the possibility of underestimation for soybean BNF in historical studies.