Updated September 30, 2025:
A unique characteristic of soybean [Glycine max (L.) Merr.] is its ability to meet most of its nitrogen (N) demand of about 5 lbs of N per bushel through symbiosis with bacteria, a process known as biological N fixation. The N fixation, however, is highly dynamic and sensitive to environment. From a system perspective, these uncertainties have direct consequences for N budgets. While soybean provides substantial N inputs through N fixation, harvest removes large amounts of seed N, often resulting in negative N budgets. This imbalance raises questions about long-term soil fertility and sustainability, highlighting the importance of accurate quantification methods that capture both seasonal fixation dynamics and overall crop N demand. This uncertainty complicates soybean N management. The critical N dilution curve, a tool widely used in other crops, defines the minimum plant N concentration required for maximum shoot biomass. Yet, in soybean, N uptake derives from both soil and fixation, making the curve difficult to establish in field conditions. In soybean, these responses are less understood due to dynamic N fixation. Advancing soybean N management requires two main components, which we address through independent studies. The first quantifies the seasonal dynamics of N fixation and the associated N budget, while the second defines a true critical N dilution curve for soybean using non-nodulating varieties. Together, these approaches provide complementary knowledge to better diagnose N deficiency, enhance N use efficiency, and inform sustainable soybean-based management strategies and policies.
Study 1: Seasonal N fixation and N budget
Seven soybean field experiments were conducted across Kansas during the 2021 and 2022 growing seasons. The experimental design was a randomized complete block with four to five replications. Each plot was 50 ft long by 20 ft wide, with 30-inch row spacing. Seeds were inoculated with Bradyrhizobium japonicum. The specific treatments were as follows: the Check received no fertilizer; the N (nitrogen) treatment received 27 lbs N/acre as urea at planting; the S (sulfur) treatment received 30 lbs S/acre as gypsum at R3 (beginning pod); and the NS treatment received 27 lbs N/acre as ammonium sulfate at planting and 30 lbs S acre?¹ as ammonium sulfate at R3. Adjacent unfertilized corn strips served as the non-fixing reference crop.
Key Results:
Across all sites, fertilizer additions (N, S, or N+S) did not significantly increase yields compared to the unfertilized Check. Mean yields were 61.1 bu/ac (KS1), 60.0 bu/ac (KS2), 37.56 bu/ac (KS3), 64.2 bu/ac (KS4), 60.0 bu/ac (KS5), and 50.18 bu/ac (KS6), highlighting a limited response to supplemental nutrients under these conditions.
Across the six experiments, N fixation in soybean reached its peak between 34% and 41% of total plant N, with an average peak of approximately 38%, and showed no relationship with yields. The timing of peak fixation varied among locations but generally occurred during the mid to late reproductive stages, from R4 to R6. At KS1, KS4, and KS6, maximum fixation occurred around R4 (full pod), whereas at KS2, KS3, and KS5, peaks were closer to R6 (full seed), corresponding to 0.52 and 0.74 of the relative GDD. This variability highlights the adaptive nature of biological N fixation in response to differing environmental conditions, including climate, soil characteristics, and management practices, and emphasizes that single samplings can misrepresent the dynamics, underscoring the need to avoid broad generalizations
Across all observations, N budgets were consistently negative, with experiments showing an average N budget of –113 lbs N/acre (range: –250 to –3 lbs N/acre). Greater N fixation tended to reduce the negative budgets, but higher yields did not necessarily correspond to proportionally higher N fixation, often increasing the N deficit. To fully offset seed N removal, N fixation would need to contribute between 57 and 62% of total crop N, with a mean requirement of approximately 60%. Despite substantial contributions from N fixation, these levels were rarely sufficient to achieve a neutral N budget, and could have long-term implications for soil fertility, soil health and soybean-based rotations sustainability.
Study 2: Critical N dilution curve
This experiment, conducted in a split-plot design with four replications, took place in Junction City, Kansas, during the 2023 growing season. Soybeans were sown on May 17 and harvested on September 28. During the growing season, 10 inches of precipitation were accumulated, with an average maximum and minimum temperature of 88°F and 63°F, respectively. The main plots received one of four N fertilizer rates (0, 90, 180, and 270 lbs N/acre applied as urea at planting). Subplots included four early maturity soybean genotypes: two non-nodulating near-isogenic lines (Non-nod1 and Non-nod2) and two nodulating lines (Nod1 and Nod2). The non-nodulating lines were genetically similar to their nodulating counterparts but had inhibited nodulation genes. Nodulating genotypes were inoculated with Bradyrhizobium japonicum before sowing. Plots measured 65 ft² (6 m × 9 m) with 2.5 ft row spacing. The average plant density was 25 plants/m².
Key Results:
Nodulating genotypes had minimal yield variation (= 7.4 bu/ac) across N fertilization rates from 0 to 268 lbs/ac, whereas non-nodulating genotypes were highly responsive to N, exhibiting an average yield difference of ~23.8 bu/ac at the same rates. Overall, plant N dilution was 46% higher in non-nodulating genotypes (than in nodulating genotypes, while the estimated %N at 1 Mg/ha was similar for both groups (5.0% vs 4.9%).
Nodulating genotypes showed a slower rate of decline in shoot N with increasing biomass compared to non-nodulating genotypes. At the same biomass, nodulating genotypes had 38% greater specific leaf nitrogen (SLN) (1.42 vs. 1.03 g/m2) and 17% greater specific leaf area (SLA) compared to their non-nodulating counterparts. They also accumulated N in pods at a higher rate (4 vs. 2.5 g N /100 g) and exhibited a 15% reduction in specific leaf weight (SLW) (0.284 vs. 0.333 g/dm2).
Previous studies and our observations suggest that nodulating soybeans can maintain higher N content at similar biomass than non-nodulating varieties in part by temporarily storing N in leaves, stems, and pods as vegetative storage proteins (VSP) to support later reproductive demand. This storage mechanism may buffer N feedback on soil uptake and N fixation, while also supporting photosynthetic activity during seed filling. From a carbon-allocation perspective, nodulating genotypes showed reduced leaf weight but higher N per unit biomass in other shoot organs, consistent with literature documenting trade-offs between N fixation, shoot growth, harvest index, and seed composition. These mechanisms highlight potential metabolic and physiological adaptations that allow nodulating soybeans to meet N demand without compromising overall yield. While further studies are needed to explore root:shoot ratio changes, metabolic costs of N fixation, and genotype × environment interactions, these results support the use of non-nodulating isolines to establish critical N dilution curves and improve N management in soybean production.
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Overall Summary
This report investigates soybean nitrogen (N) management through two complementary studies. The first quantifies seasonal dynamics of biological N fixation across six Kansas field trails, revealing that N fixation peaks in mid-to-late reproductive stages (R4 to R6) but is variable across environments. Despite substantial N fixation, most experiments exhibited negative N budgets, indicating that fixation alone rarely offsets seed N removal. The second study establishes a true critical N dilution curve using non-nodulating soybean genotypes, isolating soil N uptake. Non-nodulating lines showed greater N dilution with biomass, while nodulating genotypes maintained higher shoot N through temporary storage in leaves, stems, and pods. Together, these findings improve understanding of N dynamics in soybean and provide tools to better diagnose N deficiency, and inform sustainable management practices.
Key Results:
Study 1: Seasonal N Fixation and N Budget
Experiments located in Kansas (Topeka, Kiro, and Junction City) showed that adding starter fertilizer to soybeans (N, S, or both) did not increase yields compared to unfertilized plots. Average yields were similar across treatments within each location. Average yields were 61.1 bu/ac (KS1), 60.0 bu/ac (KS2), 37.5 bu/ac (KS3), 64.2 bu/ac (KS4), 60.0 bu/ac (KS5), and 50.18 bu/ac (KS6).
Soybeans obtained about one-third of their N through biological N fixation (34–41%, averaging ~38%). The moment in which N fixation reached its maximum contribution relative to the total N uptake (%) occurred usually around pod set (R4) in some sites and near full-seed formation (R6) in others. These differences show how soybeans adjust their N strategy to local conditions, but they also highlight the drawbacks of quantifying N fixation using single measurements in the season, in which we might misinterpret the contribution of N fixation. Multiple samples are needed to more accurately inform the contribution of N fixation, or more accurate models need to be developed to predict N fixation across varying environments and management practices, which is still far from consolidated in soybean research.
Despite contributing to about a third or more, when looking at the N budget in these experiments (N fixation - seed N removal), it averaged –113 lbs N/acre (ranging from –250 to –3). Thus, soybeans were removing more N from our soils rather than leaving “N credits” for the following crop. Higher N fixation resulted in an N budget closer to neutral, but higher yields generally worsened the deficit because more N was removed from the field in seeds without being offset by amounts of fixed N. According to our analysis, to fully balance seed N removal, N fixation would need to supply about 60% of the plant N demand, above what we observed. Over time, this negative N budget suggests soybean-based systems risk depleting soil fertility and soil health.
Study 2: Critical N Dilution Curve
The study comparing nodulating vs. non-nodulating soybean isolines further emphasized how essential it is to boost N fixation in soybeans. The application of N fertilizer had little effect on nodulating soybeans; yields varied less than 8 bu/ac across N rates from 0 to 268 lbs/ac. In contrast, non-nodulating soybeans relied heavily on N fertilizer, gaining around 24 bu/acre when supplied with N.
Nodulating plants also managed N more efficiently. They maintained higher leaf N, developed more leaf area, and allocated more N to pods. This allowed them to sustain photosynthesis and grain fill without N fertilizer dependence.