1 Introduction

Soybean is a cornerstone crop for global food, feed, and oil production, and its high protein content makes it a major biological sink for nitrogen (N). Despite its capacity for biological nitrogen fixation (BNF), soybean cultivation frequently relies on mineral N inputs, which interact in complex ways with symbiotic fixation and soil N supply (La Menza et al., 2020). In many high-yield systems, BNF and soil N alone are insufficient to satisfy crop N demand, leading to negative N balances and potential soil N mining over time. At the same time, low fertilizer use efficiency and N losses through leaching and gaseous emissions raise environmental concerns and economic costs, highlighting the need to optimize fertilization strategies that enhance nitrogen use efficiency (NUE) while sustaining yield and seed quality. Understanding how different N sources and application regimes affect soybean N uptake, fixation dynamics, and allocation is therefore critical for designing sustainable production systems (Helios et al., 2025).

Soybean N nutrition is characterized by the simultaneous use of soil mineral N and symbiotically fixed N₂, with BNF often contributing 50%-85% of the crop’s total N requirement under well-managed conditions. Field syntheses across environments show a tight positive relationship between aboveground N accumulation and seed yield, but also reveal that fixed N rarely fully compensates for N removed in harvested seed, particularly at high yield levels. Detailed temporal analyses indicate that the maximum rate of N fixation typically occurs around the beginning pod stage (R3), while the proportion of N derived from the atmosphere often peaks later, between full pod and seed-filling stages, emphasizing the importance of synchronizing N supply with reproductive demand (Ciampitti et al., 2021). However, BNF is sensitive to environmental and management factors such as soil moisture, temperature, and N fertilization, which can alter nodule formation, fixation rates, and seasonal N dynamics (Wu et al., 2020). These characteristics create both opportunities and constraints for fertilization strategies aimed at improving NUE in soybean.

Soybean’s N requirements are high because of its large biomass production and the substantial N contained in seeds, where most plant N is ultimately allocated by maturity (Wysokinski et al., 2024). Studies in high-yield environments show that when N supply from BNF and soil is limiting, crop growth rate, leaf area development, radiation interception, and N accumulation during early and mid-season are constrained, leading to reduced seed set and smaller seeds (La Menza et al., 2020). Quantitative analyses have identified biomass production and carbon supply as key drivers of total BNF, with approximately 0.013 kg N fixed per kg of biomass, indicating that strategies enhancing early growth can indirectly increase N acquisition through both fixation and soil uptake. At the same time, soybean efficiently reallocates N from vegetative organs to seeds, with nitrogen harvest index values often exceeding 85%-88%, suggesting that internal remobilization and allocation efficiency are central determinants of NUE at the whole-plant level (Helios et al., 2025). Fertilization regimes must therefore account not only for total N inputs, but also for how timing and rate influence source-sink relationships, BNF activity, and N partitioning to grain.

Current research shows that N fertilization can both complement and compete with BNF, leading to context-dependent effects on yield and NUE. Meta-analyses and multi-site studies indicate that soybean yield responses to N fertilization are more likely in high-yield (>4.5 Mg/ha) environments where crop N demand exceeds the combined supply from soil and BNF (La Menza et al., 2020). However, increasing fertilizer rates typically suppress BNF in a dose-dependent manner and often reduce nitrogen use efficiency, with diminishing yield gains per unit of applied N (Helios et al., 2025). Process-based modeling and field experiments suggest that moderate, well-timed N applications—such as small starter doses before sowing, split applications, or targeted N supply during reproductive stages—can improve yield and seed N content while limiting negative impacts on BNF and soil N budgets. Despite these advances, substantial knowledge gaps remain regarding optimal fertilization strategies across diverse climates, soils, and management systems, particularly with respect to integrating mineral N, biological inputs, and other nutrients to maximize NUE and long-term sustainability (Shome et al., 2022).

2 Concepts and Evaluation Indices of Soybean Nitrogen Fertilizer Use Efficiency

2.1 Definition and classification of nitrogen fertilizer use efficiency

Nitrogen use efficiency (NUE) in soybean generally describes the capacity of a cropping system to convert applied or available nitrogen into harvested yield with minimal loss to the environment. Conceptually, NUE is often decomposed into nitrogen uptake efficiency (the proportion of available N taken up by the crop) and nitrogen utilization or physiological efficiency (the yield produced per unit of N taken up), which together determine the overall productivity of applied N (Anas et al., 2020). In soybean-maize rotations, long-term manure and mineral N trials highlight that improved soil organic matter and N retention can raise system-level NUE by simultaneously enhancing N uptake and reducing leaching losses (Hua et al., 2020).

Within this broad concept, further classifications distinguish indices focused on total N supply from those explicitly tied to fertilizer N input. For example, agronomic studies separate indices that consider total N uptake (including residual soil N and biological N fixation) from indices such as N agronomic efficiency and N recovery efficiency, which relate yield gains or N uptake increases directly to the amount of fertilizer N applied (Niu et al., 2023). This distinction is important in soybean systems where symbiotic N fixation and residual N from rotations or organic amendments contribute substantially to crop N supply, and where fertilization strategies must be evaluated against both productivity and environmental objectives (Anas et al., 2020).

2.2 Commonly used evaluation indices (e.g., NUE, NRE, NPFP, etc.)

A range of quantitative indices is used to characterize nitrogen fertilizer use efficiency in soybean and soybean-based rotations. Common agronomic indices include NUE (often expressed as yield per unit of applied N), N agronomic efficiency (yield increase per unit of applied N), N recovery efficiency (proportion of applied N taken up by the crop), and partial factor productivity of N (yield per unit of N applied without subtracting the control yield) (Niu et al., 2023). In long-term soybean-maize systems, manure plus mineral fertilizer increased partial factor productivity and physiological efficiency relative to mineral fertilizer alone, reflecting more efficient conversion of applied N into grain yield and improved soil N retention (Hua et al., 2020).

Soybean-focused studies also employ N harvest index (NHI) to describe the proportion of plant N allocated to seed, and sometimes distinguish physiological versus agronomic and recovery efficiencies to separate internal N use from external N capture (Kakabouki et al., 2020). Under different tillage and N rates, indicators such as NUE, NHI, N agronomic efficiency, and N absorption and uptake effects were sensitive to fertilization level and soil management, effectively expressing how N fertilization influenced yield formation and the balance between soil-derived N and fertilizer-derived N in soybean.

2.3 Key factors influencing the determination of evaluation indices

The choice of evaluation indices depends strongly on study objectives, N sources, and system complexity. In corn-soybean rotations receiving mineral fertilizer and various manure forms, indices emphasizing yield and total N uptake (NUE, N uptake efficiency, N utilization efficiency) were most informative for comparing overall N supply among treatments, whereas agronomic and recovery efficiencies better distinguished responses specifically attributable to fertilizer N (Niu et al., 2023). In this context, physiological indices like N harvest index or N physiological efficiency were less sensitive, underscoring that different indices capture different components of N behavior and may vary in diagnostic value depending on fertilization strategy and crop sequence.

In soybean systems with multiple N inputs (biological fixation, soil mineralization, chemical and organic fertilizers), experimental design and time scale also influence index selection. Long-term experiments show that manure plus mineral N improved partial factor productivity and physiological efficiency by increasing soil organic matter, water storage, and N retention, suggesting that indices integrating crop yield with soil N storage are particularly valuable for assessing sustainable fertilization strategies (Hua et al., 2020). Conversely, short-term soybean fertilization or tillage trials may prioritize indices such as NUE, NHI, and agronomic or recovery efficiency to diagnose immediate crop responses and to disentangle the relative contribution of fertilizer versus symbiotically fixed or residual soil N to plant N uptake (Kakabouki et al., 2020).

3 Types of Fertilization Strategies and Their Theoretical Basis

3.1 Theory of nitrogen fertilizer rate control and optimization

The core principle of nitrogen rate optimization in soybean is to balance mineral N supply with biological nitrogen fixation (BNF) and soil N, so that crop demand is met without suppressing nodulation or wasting fertilizer. Under mulched drip irrigation in Xinjiang, stepwise increases in N rate showed that 180 kg N/ha maximized nodule number, nodule dry weight, N₂ fixation (%Ndfa), seed yield, and agronomic N use efficiency, whereas 240 kg N ha^-1 reduced nitrogenase activity, leghemoglobin content, and NUE, illustrating a clear optimum and the penalties of over-fertilization (Xu et al., 2025). Similarly, a Central European field study comparing 0, 30, and 60 kg N ha^-1 found the highest NUE at the moderate rate (30 kg N ha^-1), while 60 kg N ha^-1 increased total N uptake but sharply reduced NUE, again indicating diminishing returns at high N input (Helios et al., 2025).

Other work confirms that excessive N undermines soybean’s intrinsic capacity to rely on BNF without improving yield. In a pre-sowing rate trial (0-180 kg N ha^-1), increasing N systematically reduced root nodule number and N₂-fixation potential at flowering and maturity, and high N doses did not increase seed yield, total biomass, or seed protein yield (Wysokinski et al., 2024). Process-based modeling with SPACSYS also showed only modest yield gains (2.4%-5.2%) when 50-100 kg N ha^-1 was added, while BNF declined by 6%-33%, suggesting that relatively low starter rates (15-30 kg N ha^-1, depending on year type) best reconcile yield, seed N content, and soil N balance (Wu et al., 2020).

3.2 Strategies for fertilization timing and split application

Theoretical and empirical evidence highlight that synchronizing N supply with crop demand is central to improving NUE. In Xinjiang, applying the optimized N rate (180 kg/ha) at the beginning pod stage (R3) under mulched drip irrigation promoted carbohydrate allocation to nodules, maintained nitrogenase activity, and increased both %Ndfa and yield, indicating that targeted N during reproductive stages can support both BNF and productivity (Xu et al., 2025). A large U.S. synthesis of 207 soybean N experiments showed that, averaged across environments, split N applications yielded about 110 kg/ha more than zero-N controls, with split applications outperforming single applications, though the overall contribution of timing and rate to yield variation was small compared with environment effects (Mourtzinis et al., 2017).

Detailed timing studies further refine these concepts. In Brazil, single and split doses of 20-40 kg N ha^-1 applied at sowing and at R4 demonstrated that split application (sowing + R4) could increase yield by up to 47% relative to an unfertilized control, whereas single high doses (>40 kg/ha) risked inhibiting BNF. Combined N-S split applications (25 + 25 kg/ha of each, at sowing and R2) enhanced seed and straw yield, N and S uptake, NUE, and seed Zn and Fe content compared with basal-only or unsplit treatments, underscoring that temporally synchronized, moderate splits can improve both macronutrient efficiency and micronutrient nutrition (Khalili et al., 2024).

3.3 Formulated fertilization and precision fertilization technologies

Formulated fertilization strategies aim to match N (and other nutrients) to site-specific soil fertility, crop demand, and system design, often integrating organic and inorganic sources. Long-term soybean-maize rotations in Northeast China showed that combining manure with NPK (MNPK) improved crop yields, N recovery, and soil organic matter, while retaining a larger share of applied N in the 0-20 cm layer, indicating improved soil fertility and reduced leaching risk compared with mineral fertilizers alone (Hua et al., 2020). A 40-year soybean-maize system similarly demonstrated that manure plus mineral fertilizers increased biomass, NUE indices, and soil water and mineral N storage, allowing lower effective N losses while sustaining high yields (Liu et al., 2024).

Precision fertilization extends this concept by using spatial and temporal targeting, guided by models or field sensors. In maize-soybean relay intercropping, reduced N input combined with optimized placement (15-30 cm from maize rows) increased total system yield by 8%-12% and raised NUE and agronomic N efficiency by over 50% relative to conventional higher-rate N, while also enhancing soybean nodulation and BNF (Yong et al., 2018). Crop models such as SPACSYS support such precision strategies by simulating site- and climate-specific scenarios; for Northeast China, simulations recommended 15-30 kg N ha^-1 before sowing for yield optimization and higher but still moderate rates (45-60 kg/ha) for maximizing seed N content, thus framing N decisions within explicit trade-offs among yield, seed quality, BNF, and soil N budgets (Wu et al., 2020).

4 Mechanisms by Which Fertilization Strategies Affect Soybean Nitrogen Fertilizer Use Efficiency

4.1 Processes of soil nitrogen transformation and supply

Fertilization strategies first act by altering soil N pools and transformation rates, thereby changing the balance between fertilizer-derived, soil-derived, and biologically fixed N available to soybean. Long-term fertilization in maize-soybean rotations has shown that mineral N strongly increases soil ammonium and total N availability, with soil N dynamics explained more by fertilization than by climatic variability, underscoring the dominant role of fertilizer in driving soil N supply patterns. At the same time, microbial carbon use efficiency and microbial community structure were tightly linked to nitrate and ammonium availability, indicating that fertilization indirectly modifies N mineralization, nitrification, and immobilization through shifts in soil microbiology (Luo et al., 2023).

In cereal-legume systems, fertilizer N applied to the preceding maize crop contributes relatively little to N nutrition of the following soybean, with 15N tracing showing that most soybean N uptake comes from native soil N rather than residual fertilizer or maize stover, even under no-tillage. These findings emphasize that fertilization strategies need to consider not only immediate fertilizer N pools but also their limited short-term transfer through residues and the central role of intrinsic soil N mineralization in supplying soybean (Bruno et al., 2025). Thus, N rate, placement, and interactions with residue and microbial processes jointly determine the temporal and quantitative pattern of soil N supply available for soybean uptake and symbiotic fixation.

4.2 Regulation of soybean root uptake and nodule nitrogen fixation

Fertilization regimes directly regulate the relative contributions of root N uptake and nodule N₂ fixation by modifying soil N concentration around roots and nodules. Experiments with dual-root systems demonstrated that increasing fertilizer N concentration on the non-nodulated side systematically inhibited nitrogenase activity and reduced the percentage of N derived from the atmosphere, with short-term supply acting mainly through specific nitrogenase activity and long-term supply through reduced nodule growth. Nevertheless, the absolute amount of N fixed by nodules was often maintained, suggesting that moderate external N can enhance total plant N without fully suppressing fixation if concentrations are carefully controlled (Lyu et al., 2020).

Field studies further show that moderate N doses combined with inoculation can enhance nodulation quality, N uptake, and yield, whereas higher N rates increasingly shift the plant toward direct root N absorption and reduce the efficiency of symbiotic fixation. Under mulched drip irrigation, applying 180 kg N ha^-1 at the beginning pod stage increased nodule number, biomass, and ureide export to shoots, and maximized N agronomic efficiency, while higher rates (>180 kg/ha) depressed nitrogenase activity, leghemoglobin, and ureide translocation, leading to reduced %Ndfa and nodule senescence. Thus, fertilization strategies modulate a physiological trade-off between low-energy root uptake and energy-expensive fixation, with moderate, well-timed N supporting both processes and excessive N favoring uptake at the expense of nodulation and N₂ fixation (Xu et al., 2025).

4.3 Nitrogen Allocation and Utilization within the Soybean Plant

Once absorbed or fixed, N must be efficiently allocated among organs and over time to support growth and seed filling; fertilization strategies influence this internal partitioning and thereby nitrogen use efficiency. Across diverse environments, 50%-60% of soybean N demand is typically met by symbiotic fixation, and there is a strong linear relationship between aboveground N accumulation and seed yield, but fixed N often fails to fully replace N removed in harvested seed, especially at high yields. High-yield systems exhibit greater late-season N uptake and higher nitrogen harvest index, indicating that fertilization strategies that sustain N supply through reproductive stages can enhance remobilization from vegetative tissues and allocation of N to seeds per unit of total N uptake (Gaspar et al., 2017) (Figure 1).

Figure 1 Trellising methods influence cowpea yield and yield components through canopy, structre, light interception, resaurce fliciency

Detailed partitioning studies using dual-root systems and ¹⁵N tracers showed that 81.5%-87.1% of both fertilizer-derived and nodule-fixed N is allocated to shoot growth, with only 12.9%-18.5% retained in roots and nodules, and that N required for root and nodule growth is largely supplied by locally absorbed or fixed N rather than direct fertilizer N transfer to nodules (Zhang et al., 2020). After reaching shoots, both fertilizer N and fixed N are redistributed, with a significant fraction retranslocated back to nodulated roots and nodules during reproductive development, demonstrating a dynamic circulation that fertilization can modify by changing the relative importance of each N source. Consequently, fertilization strategies that avoid excessive suppression of fixation while ensuring adequate N during seed filling can optimize both total N accumulation and its partitioning to grain, thereby improving overall nitrogen fertilizer use efficiency.

5 Impacts of Different Fertilization Strategies on Soybean Growth and Yield

5.1 Relationship between nitrogen fertilizer application rate and soybean growth and development

Nitrogen rate strongly shapes soybean vegetative growth, photosynthetic capacity, and nodulation, with clear evidence of both positive and negative effects depending on dose. Under Central European conditions, increasing mineral N from 0 to 60 kg/ha improved seed yield but the highest nitrogen use efficiency occurred at the moderate rate of 30 kg/ha, highlighting diminishing returns and the risk of inefficiency at higher inputs (Helios et al., 2025). In contrast, studies with very high N rates up to 180 kg/ha found that elevated pre-sowing N systematically reduced nodule number at flowering and maturity and suppressed N₂-fixation potential, without increasing seed yield or biomass, indicating that excessive N can impair symbiosis while failing to enhance growth (Wysokinski et al., 2024).

In high-input irrigated systems, a similar trade-off appears: under mulched drip irrigation in Xinjiang, applying 180 kg N ha^-1 at R3 significantly increased nodule number, nodule biomass, and yield compared with 0 or 120 kg N ha^-1, but further increasing N to 240 kg/ha inhibited nitrogenase activity and leghemoglobin synthesis and reduced symbiotic N fixation. At very high N supply (up to 300 kg N ha^-1), growth and photosynthetic traits such as chlorophyll content, photosynthetic rate, stomatal conductance, and leaf area index improved markedly at an intermediate optimal rate (225 kg N ha^-1), with grain yield strongly associated with these enhanced physiological parameters, but declines or plateaus at the highest rate emphasized the environmental and agronomic costs of over-fertilization (Kubar et al., 2021).

5.2 Effects of Fertilization Methods on Yield Components

Fertilization methods and timing significantly modify yield components such as pod number, seeds per pod, seed weight, and canopy distribution of yield. A global synthesis of 207 U.S. environments showed that both single and split N applications increased mean seed yield by 60 and 110 kg/ha, respectively, relative to zero-N controls, with split applications, often combining soil and foliar methods, giving the largest gains and indicating benefits for pod and seed set across diverse conditions. In high-yield irrigated systems, a Full-N treatment that alleviated N limitation increased yield by 984 kg/ha via higher seed number (+253 seeds/m²) and greater individual seed weight (+16 mg/seed), particularly enhancing pod and seed numbers in middle-upper canopy sections and reducing seed abortion (Bonfanti et al., 2025).

At more moderate N rates, combined nutrient strategies can further improve yield structure. Split application of 25 + 25 kg N ha^-1 with an equivalent split of sulfur (S) significantly increased seed and straw yield, N and S uptake, and seed Zn and Fe accumulation compared with unsplit or lower-rate treatments, demonstrating positive effects on both pod and seed formation and nutrient density (Khalili et al., 2024). Similarly, combining low mineral N doses (30 kg N ha^-1) with effective Bradyrhizobium inoculation increased the number of pods, seeds per plant, and seed weight per plant compared with higher N alone, illustrating that integrating biological inputs with modest fertilization can optimize yield components (Szpunar-Krok et al., 2023).

5.3 Synergistic relationship between nitrogen fertilizer use efficiency and yield

The relationship between yield and nitrogen fertilizer use efficiency (NUE) is synergistic when rates, timing, and sources are aligned with crop demand and symbiotic fixation. A comprehensive review across 637 data sets showed a strong linear relationship between N accumulation and seed yield, with yield more likely to respond to N fertilization in high-yield (>4.5 Mg/ha) environments; however, increasing N rate followed a negative exponential relationship with N₂ fixation, underscoring that maximizing yield requires balancing fertilizer N with BNF to avoid depressing fixation and mining soil N (Xu et al., 2025). Field studies with ¹⁵N-labeled fertilizer demonstrated that applying N at reproductive stages (R3) increased total plant N uptake and yields, particularly in tropical conditions, while fertilizer recovery efficiency declined at higher rates, indicating that moderate, well-timed N can raise both yield and fertilizer recovery but excessive N reduces efficiency (Pierozan et al., 2020).

Where N and S were co-managed, split applications of N25+25 and S25+25 kg/ha enhanced seed yield, N and S uptake, and N use efficiency, while also increasing soil and seed Zn and Fe levels, revealing that improved NUE can coincide with higher productivity and better micronutrient profiles (Khalili et al., 2024). In mulched drip-irrigated soybean, an optimized rate of 180 kg N ha^-1 at R3 elevated agronomic N use efficiency alongside yield by promoting nodule biomass, carbohydrate supply to nodules, and sustained nitrogenase activity, whereas higher rates reduced both %Ndfa and NUE, illustrating that within a given system there is a narrow window where fertilization simultaneously maximizes yield and nitrogen efficiency.

6 Interactions between Environmental Factors and Fertilization Strategies

6.1 Effects of soil type and fertility level

Soil type and inherent fertility strongly condition soybean responses to nitrogen fertilization and thus shape nitrogen use efficiency (NUE). In low-fertility sandy or degraded pasture soils, combined inoculation and moderate N inputs (around 50 kg N ha^-1) substantially increased grain yield compared with inoculation alone, indicating that poor soils can require supplemental N to overcome limitations to biological nitrogen fixation (BNF) and early crop growth. In contrast, on post-soybean fields with better fertility, high inoculant doses alone were often sufficient, and additional N did not improve yield, demonstrating that soil fertility history and organic matter reserves determine whether fertilizer N is beneficial or redundant.

Long-term soybean-maize rotations on brown soils showed that integrating manure with mineral NPK (MNPK) improved soil organic matter, Olsen P, and N recovery compared with mineral fertilizers alone, sustaining higher yields and retaining a larger share of applied N in the 0-20 cm layer. These results emphasize that in higher-fertility, organically enriched soils, strategies focusing on organic-mineral combinations can raise system-level NUE by enhancing soil N supply capacity and reducing leaching, whereas in inherently poor soils, optimized mineral N plus inoculation is more critical for achieving acceptable productivity (Hua et al., 2020).

6.2 Regulation of Nitrogen Efficiency by Climatic Factors (Temperature, Moisture)

Climate, especially temperature and moisture, modulates how fertilization strategies translate into nodulation, BNF, and NUE. In Central Europe, a three-year field study showed large year-to-year variation in nodulation and yield; favorable early-season moisture supported higher nodule numbers and biomass, while drought strongly reduced nodulation and aboveground biomass despite similar fertilization and inoculation regimes. Moderate N rates (30 kg N ha^-1) combined with effective inoculants maximized NUE, but their benefits were attenuated in seasons with more favorable weather, underscoring the overriding influence of climate on the efficiency of any given N strategy (Helios et al., 2025).

In tropical, unfavorable edaphoclimatic environments characterized by high temperature, low fertility, and sandy soils, water deficits at reproductive stages (e.g., R5) limited grain yield and constrained BNF efficiency, making soybean more responsive to combined N fertilization and intensive inoculation. Under such stress conditions, higher N rates (up to 50-100 kg/ha depending on site) together with multiple inoculant doses significantly increased yields relative to inoculation alone, whereas in seasons or sites with less water and heat stress, similar fertilization often had limited or no yield effect, highlighting the need to adjust N inputs to expected moisture and thermal regimes (Lumactud et al., 2022).

6.3 Synergistic Effects of Agronomic Measures (Crop Rotation, Planting Density) and Fertilization

Agronomic practices interact with fertilization to influence soil N dynamics, BNF, and NUE at the system scale. In a 40-year soybean-maize rotation, manure plus mineral fertilizers not only increased crop yields but also improved N recovery and residual soil N retention, showing that rotation systems with regular organic inputs can use fertilizer N more efficiently and buffer interannual variability in N supply (Hua et al., 2020). A broader synthesis of soybean-based rotations found that including soybean in rotation enhanced soil N content, reduced soil C:N ratio, mitigated acidification, and stimulated microbial and enzyme activities, allowing lower fertilizer N inputs while maintaining or increasing productivity and reducing greenhouse gas emissions (Figure 2) (Yang et al., 2024).

Figure 2 Soil fertility level, climate conditions, and agronomic practices profoundly interact with fertilization, strategies to condition soybean nodulation, nitrogen fixation, and nitrogen

Spatial and temporal crop arrangements also modify how added N is shared and used. In maize-soybean relay intercropping, reduced N rates in maize combined with optimized band placement increased total system yield by 8-12% and raised NUE and agronomic N efficiency by more than 50% relative to conventional higher N, partly because interspecific competition for soil N promoted greater BNF in soybean (Du et al., 2020). Similarly, intercropping and adjusted N rates increased soybean nodule dry weight, nitrogenase activity, and N₂ fixation compared with sole cropping, indicating that appropriate planting patterns and rotation designs can synergize with moderate fertilization to enhance biological N inputs, improve N recovery, and maintain soil fertility (Cheng et al., 2023).

7 Case Study: Optimizing Fertilization Strategies to Enhance Soybean Nitrogen Fertilizer Use Efficiency

7.1 Case background and experimental design

Under mulched drip irrigation in arid Xinjiang, high soybean yields have commonly been pursued with very high N inputs (240-310 kg N ha^-1), but this practice suppresses symbiotic N₂ fixation and threatens long-term sustainability (Xu et al., 2025). To identify an optimal strategy, a two-year field experiment compared four N rates (0, 120, 180, 240 kg N ha^-1) applied at the beginning pod stage (R3), focusing on yield, nitrogen agronomic efficiency, and key traits of nodules and N metabolism (Xu et al., 2025).

Treatments were arranged under otherwise uniform management, and measurements included nodule number and dry weight, nodule sucrose and starch, nitrogenase and urate oxidase activities, leghemoglobin content, stem and nodule ureide contents, and the percentage of N derived from the atmosphere (%Ndfa) during reproductive stages (Xu et al., 2025). In a contrasting long-term soybean-maize rotation on brown soil, micro-plots with ¹⁵N-labeled urea under mineral N (N, NPK) and manure plus NPK (MNPK) regimes were used to quantify crop yield, N recovery, and residual soil N, providing a systems-level reference for fertilizer-N fate and efficiency (Hua et al., 2020).

7.2 Comparison of Results from Different Fertilization Treatments

In the Xinjiang case, 180 kg N ha^-1 clearly emerged as the optimum: it significantly increased nodule number and dry weight, nodule sucrose and starch contents, stem ureide content, %Ndfa, seed yield, and agronomic N use efficiency relative to 0 and 120 kg N ha^-1 (Xu et al., 2025). By contrast, 240 kg N ha^-1 reduced nitrogenase activity, leghemoglobin, and urate oxidase, disrupted the glutamine synthetase/glutamate synthase pathway, and caused ureide accumulation in nodules, leading to lower %Ndfa and no proportional yield benefit, indicating over-fertilization (Xu et al., 2025).

Correlation and path analyses showed that nitrogenase activity, leghemoglobin, urate oxidase, and stem ureide content were strongly and positively associated with %Ndfa, while high nodule ureide content was negatively associated, confirming these traits as sensitive indicators of symbiotic performance under different N rates (Xu et al., 2025). In the 41-year rotation, manure plus NPK markedly increased soybean yield, crop N uptake, and ¹⁵N recovery (up to 32.5% in the first soybean season) compared with mineral fertilizer alone, and left higher residual ¹⁵N in the 0-20 cm layer, reflecting improved N use efficiency and soil fertility (Hua et al., 2020).

7.3 Analysis of Results and Practical Implications

Together, these cases show that moderate, well-timed N can enhance both yield and nitrogen fertilizer use efficiency by supporting, rather than replacing, symbiotic fixation. Under mulched drip irrigation, 180 kg N ha^-1 at R3 promoted carbohydrate supply to nodules, sustained high nitrogenase and leghemoglobin, and increased %Ndfa, allowing fertilizer N and fixed N to contribute synergistically to plant N status and yield. Exceeding this rate shifted metabolism toward ureide accumulation in nodules and reduced fixation, illustrating the physiological basis for declining efficiency at high N inputs (Xu et al., 2025) (Figure 3).

Figure 3 Moderate N fertilization optimizes, soybean yield and nitrogen fertilizer use efficiency by maximizing nodule function and balancing fertilizer-based-and symbiotic N supply

At rotation scale, manure plus NPK improved N recovery, soil organic matter, and Olsen P, increased yields, and retained more fertilizer-derived N in the upper soil profile, thereby reducing unaccounted N losses compared with mineral fertilizer alone (Hua et al., 2020). Practically, these findings support fertilization strategies that: (i) avoid excessive N, (ii) synchronize N with peak reproductive demand, and (iii) integrate organic amendments where possible, to simultaneously sustain high soybean yield, protect symbiotic N₂ fixation, and enhance nitrogen fertilizer use efficiency in diverse production systems (Xu et al., 2025).

8 Conclusions and Outlook

Across diverse environments, optimized fertilization strategies consistently enhance soybean yield and nitrogen use efficiency when they respect the crop’s reliance on biological nitrogen fixation and soil N supply. Long-term rotation experiments show that integrating organic manure with mineral fertilizers (MNPK/NPKM) improves soil organic matter, nutrient availability, NUE, and yield stability, outperforming mineral NPK alone in soybean-based systems. At the field scale, combining effective Bradyrhizobium inoculation with moderate N rates (typically 9-35 kg N ha^-1) improves nodulation, N uptake, and seed yield, whereas higher N rates tend to depress nodulation and reduce agronomic NUE.

Recent studies also highlight that well-timed N inputs and formulation choices can fine-tune the balance between soil N uptake and symbiotic fixation. Under mulched drip irrigation in Xinjiang, an intermediate rate of 180 kg N ha^-1 at R3 maximized nodule function, %Ndfa, yield, and N agronomic efficiency, while higher rates (240-310 kg N ha^-1) inhibited nitrogenase activity and increased nodule ureide accumulation. Integrated organic-mineral strategies and long-term manure amendments improve rhizosphere microbial communities, enzyme activities, soil quality indices, and water and N retention, thereby raising soybean yields and system-level NUE in rotation systems.

Despite substantial progress, existing studies exhibit important limitations in spatial coverage, experimental design, and metrics. Many field trials focus on a narrow range of soils or climates (e.g., specific brown soils in Northeast China or temperate Central Europe), limiting extrapolation to other regions with contrasting textures, fertility levels, or edaphoclimatic stress. Moreover, several N-rate studies emphasize short-term yield responses without fully quantifying belowground N dynamics, long-term soil N balance, or environmental losses, restricting the ability to judge true nitrogen use efficiency and sustainability.

Methodological constraints also hinder a comprehensive understanding of symbiotic and fertilizer N interactions. Numerous experiments rely on simple partial factor productivity or agronomic efficiency indices and lack direct measurements of N fixation (e.g., ¹⁵N natural abundance or tracer techniques), N leaching, or gaseous losses 34. Long-term organic amendment trials, while rich in soil and yield data, often provide limited information on nodulation, %Ndfa, and detailed plant N allocation, making it difficult to disentangle how manure-fertilizer combinations influence the partitioning between fixed, fertilizer-derived, and soil-derived N in soybean.

Future work should better integrate process-level measurements with system-level performance to refine fertilization recommendations. Priority areas include multi-site, multi-year trials that explicitly combine gradients of N rate, timing, and inoculation with direct measures of N fixation, soil N retention, and losses, across representative soil types and climates. Long-term studies that pair manure or straw amendments with optimized mineral N in soybean-cereal rotations are particularly valuable for clarifying how fertilization strategies drive soil quality, carbon sequestration, and NUE trajectories over decades.

On the application side, there is strong potential for precision and formulated fertilization that couples starter N, robust inoculation, and site-specific organic inputs. Emerging evidence suggests that moderate starter N with bradyrhizobia, or co-inoculation strategies, can support higher yields while conserving nodulation, although economic and environmental trade-offs remain to be fully quantified. Integrating improved inoculant formulations and application methods with manure-based or green-manure systems could deliver high yields, enhanced NUE, and better soil health, offering practical pathways for sustainable soybean intensification in both high-input and resource-limited farming systems.

Acknowledgments

Thanks to the reviewers for providing detailed comments and guidance on the manuscript of this study. The reviewers’ keen insights into the issues and attention to detail have greatly benefited the authors.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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