Soybean genotype determines functional symbiotic outcomes with phylogenetically diverse Bradyrhizobium

Soybean genotype determines functional symbiotic outcomes with phylogenetically diverse Bradyrhizobium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Soybean genotype determines functional symbiotic outcomes with phylogenetically diverse Bradyrhizobium Dominic V.A. Agyekum, Elsie Sarkodee-Addo, Francisca Addae-Frimpomaah, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8802844/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Soybean can meet much of its nitrogen demand through biological nitrogen fixation (BNF). However, yields in sub-Saharan Africa (SSA) remain constrained by nitrogen deficiency and inconsistent responses to rhizobial inoculation. Despite widespread promotion of inoculation, the influence of host genotype on symbiotic effectiveness in African soybean cultivars remains is not well characterized. We assessed nodulation, nitrogen fixation, and growth responses of three widely cultivated Ghanaian soybean cultivars inoculated with ten phylogenetically diverse Bradyrhizobium strains under controlled, nitrogen-free conditions. Symbiotic performance was assessed using nodulation traits, acetylene reduction assay, shoot biomass, and relative symbiotic effectiveness (RSE) relative to mineral nitrogen treatment. Symbiotic outcomes were strongly dependent on the host. Two cultivars exhibited high nitrogen fixation and growth with multiple strains, whereas one showed consistently weak fixation and growth despite nodulation, indicating host-imposed post-infection constraints. Nodule weight and nitrogenase activity, but not nodule number, reliably predicted symbiotic benefits. Notably, several non-classical soybean Bradyrhizobium strains performed comparably or better to recognized soybean symbionts when paired with compatible hosts. These results demonstate that host genotype is a key determinant of soybean BNF effectiveness and highlight the need to integrate symbiotic performance traits into breeding and inoculant design for reliable BNF in low-input SSA farming systems. Biological sciences/Microbiology Biological sciences/Plant sciences Biological Nitrogen Fixation (BNF) Bradyrhizobium Soybean (Glycine max) Relative Symbiotic Effectiveness (RSE) Sub-Saharan Africa Acetylene Reduction Activity (ARA) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Soybean ( Glycine max L. Merr.) is one of the world’s most important legume crops, providing a major source of plant protein and oil for human consumption and animal feed. Beyond its nutritional and economic value, soybean plays a critical role in sustainable agriculture because of its capacity for biological nitrogen fixation (BNF) in symbiosis with soil-dwelling Bradyrhizobium soecies 1 . Through this symbiosis, soybean can meet most or all of its nitrogen requirements by converting atmospheric dinitrogen into a plant-available forms 2 , 3 . This is particularly important in low-input systems prevalent in sub-Saharan Africa (SSA), as mineral fertilizers impose a significant financial burden on smallholder farmers and contribute to environmental degradation via nutrient leaching, eutrophication, and greenhouse gas emissions 4 – 6 . Optimizing soybean- Bradyrhizobium symbiosis is therefore central to enhancing productivity while advancing environmentally sustainable intensification of cropping systems. The agronomic potential of BNF in soybean is well demonstrated in major producing regions, including Brazil, the United States, and Argentina, which together account for almost 80% of global soybean production 7 . In these systems, long-term investments in cultivar improvement, agronomic management, and rhizobial strain selection have enabled the consistent exploitation of BNF, substantially reducing nitrogen fertilizer requirements without compromising yield 8 , 9 . In particular, Brazil represents a global benchmark for successful soybean inoculation technology. Coordinated efforts in elite strain development, host-strain compatibility testing, inoculant quality control 10 , and farmer adoption have allowed Brazilian soybean production to rely almost entirely on BNF, generating large economic savings while maintaining high and stable yields 3 , 9 , 11 – 13 . These successess demonstrate that when the host genotype, microbial symbionts, and management practices are well aligned, soybean can reliably achieve high productivity through biological nitrogen inputs. In contrast, soybean productivity in SSA remains low and highly variable, despite the increasing demand for food, feed, and cash crops. Across the region, yield is constrained by poor soil fertility, inconsistent nodulation, and nitrogen fixation 14 – 16 . Field and greenhouse studies have frequently reported divergent yield responses to rhizobial inoculation, ranging from substantial gains to negligible or even negative effects, depending on location, cultivar, and management history 17 – 21 . Such inconsistencies have persisted even when commercial inoculants are deployed, undermining farmer confidence and limiting adoption of BNF-based technologies 22 – 25 . These contrasting outcomes relative to major soybean-producing regions suggest that inoculation success in SSA is governed by complex biological and environmental interactions rather than inoculant quality alone. Soybean nodulation and nitrogen fixation are primarily mediated by slow-growing Bradyrhizobium species through finely regulated molecular interactions involving Nod factor signaling, host receptor recognition, and downstream nodule organogenesis 26 – 28 . Historically, soybean symbiosis has been considered taxonomically narrow, dominated by a narrow group of classical symbionts, notably Bradyrhizobium japonicum , B. diazoefficiens , and B. elkanii , which form the basis of most commercial inoculants worldwide 29 , 30 . However, recent phylogenomic and ecological studies have fundamentally revised this perspective. The genus Bradyrhizobium encompasses a broader phylogenetic diversity, including lineages associated with tree legumes, wild herbaceous legumes, non-legume hosts, and bulk soils. Many of these non-classical lineages harbor functional symbiosis gene clusters and can effectively nodulate soybean when host compatibility permits 31 – 36 . This expanded diversity represents a largely untapped resource for improving soybean symbiosis, particularly in tropical regions where indigenous rhizobial communities differ markedly from those in temperate agroecosystems 37 , 38 . Among the determinants of symbiotic success, host genotype plays a central role in regulating rhizobial recognition, infection, nodulation, and nitrogen fixation 39 – 41 . Soybean lines exhibit considerable variation in their ability to recognize and sustain symbiosis with specific Bradyrhizobium strains governed by genetic determinants commonly known as Rj genes. Restrictive alleles such as Rj2 , Rj4 , or related loci can selectively block nodulation or reduce symbiotic effectiveness with particular strains through effector-triggered immune responses 42 – 44 . Although such genetic restriction may help maintain symbiosis with elite strains in highly managed systems, it can be detrimental in environments where compatible, competitive rhizobia are absent or poorly adapted. Consequently, high agronomic performance of a cultivar does not necessarily translate into high symbiotic efficiency, especially when breeding programs in SSA have traditionally prioritized agronomic traits such as yield stability, maturity, and stress tolerance without explicit selection for BNF-related traits 15 , 45 , 46 . In SSA, and particularly in Ghana, the genetic control of soybean- Bradyrhizobium compatibility remains poorly characterized. Inoculation studies in Ghana have reported yield responses ranging from negligible to substantial, often without clear relationships to nodulation intensity or plant nitrogen accumulation 20 , 24 , 47 , 48 . These variable outcomes are frequently attributed to soil fertility constraints, competition from indigenous rhizobia, and inoculant quality 49 , 50 . However, an underexplored but potentially critical factor is the compatibility between locally grown soybean cultivars and the phylogenetic diversity of the available Bradyrhizobium strains. Most soybean breeding programs in SSA have not systematically evaluated or selected for symbiotic performance, raising the possibility that some widely cultivated varieties possess restrictive genetic backgrounds that limit effective nodulation and nitrogen fixation under low-input conditions. Despite extensive research on soybean inoculation, a critical gap remains in understanding how African soybean cultivars differ in their symbiotic compatibility with diverse Bradyrhizobium lineages and how this variability influences nitrogen fixation and plant performance under nitrogen-free conditions. In particular, the extent to which the host genotype constrains nitrogen fixation independently of nodulation remains poorly resolved. In this study, we evaluated the symbiotic performance of three commonly cultivated Ghanaian soybean cultivars inoculated with a phylogenetically diverse panel of Bradyrhizobium strains under controlled, nitrogen-free conditions. By integrating plant growth responses, nodulation, acetylene reduction activity, and relative symbiotic effectiveness, we aimed to (i) quantify cultivar-specific responses to rhizobial diversity, (ii) identify restrictive and permissive host genotypes, and (iii) assess the implications of host genetic control on soybean inoculation strategies in low-input African systems. Materials and Methods Plant Materials and Experimental Design Three soybean cultivars widely cultivated in Ghana (Jenguma, Favour, and Suong Pungun) were used in this study. These cultivars were selected to represent their contrasting agronomic characteristics, particularly differences in maturity and field performance, and to capture potential variations in symbiotic responsiveness. Certified seeds were obtained from the Council for Scientific and Industrial Research - Savanna Agricultural Research Institute (CSRI-SARI), Ghana. The experiment followed a completely randomized factorial design, with soybean variety and Bradyrhizobium treatment as the main factors. Twelve treatments were evaluated per cultivar: ten Bradyrhizobium strains, an uninoculated nitrogen-free control (negative control), and a mineral nitrogen treatment supplemented with 5mM KNO 3 (positive control). Each treatment was replicated thrice per cultivars, resulting in 108 experimental units. All plants were grown under strictly nitrogen-free conditions except for mineral nitrogen control, allowing for an unambiguous assessment of biological nitrogen fixation. Bradyrhizobium Strains and Culture conditions Ten Bradyrhizobium strains representing both classical and non-classical soybean symbionts were obtained from the NITE Biological Resource Center (NBRC) and used in this study. These included B. diazoefficients USDA110, B. elkanii USDA61, B. japonicum NKS4, B. betae (NBRC 103048), B. lablabi (NBRC 108826), B. canariense (NBRC 103049), B. yuanmingense (NBRC 100594), B. iriomotense (NBRC 102520), B. liaoningense (NBRC 100396), and B. denitrificans (NBRC 105663). The strains were selected to capture broad phylogenetic diversity within the genus Bradyrhizobium , enabling evaluation of host cultivar responses across a wide range of symbiotic backgrounds rather than focusing on known soybean microsymbionts. Phylogenetic relationships among the selected strains were assessed using reference 16S rRNA gene sequences retrieved from NCBI database and aligned using MEGA12. Multiple sequence alignments were performed using CLUSTALW, and phylogenetic trees were constructed using the Neighbor-joining method implemented in MEGA12 51 . Branch support was evaluated using 1,000 bootstrap replicates 52 , confirming that the selected strains spanned multiple Bradyrhizobiium clades ( Supplementary Fig. S1 ) . All strains were initially streaked on yeast mannitol agar (YMA) and incubated at 28±2°C for 5–7 days. Single colonies were transferred to yeast mannitol broth (YMB) and cultured at 28°C with shaking at 150 rpm for 5 days. Prior to inoculation, the bacterial cells were harvested by centrifugation, washed twice with sterile 0.85% (w/v) saline and resuspended to a final density of approximately 10 7 CFU mL − 1 , as estimated by the optical density at 600 nm(OD 600 ). Seed Surface Sterilization and Germination Soybean seeds were surface-sterilized by immersion in 70% ethanol for 1 min, followed by immersion in 3% sodium hypochlorite for 1 min, and then rinsed five times with sterile distilled water. Sterilized seeds were pre-germinated on moist sterile tissue paper in Petri dishes and incubated in the dark at 28°C for 48 h. Uniformly germinated seedlings were transferred into a plant box (CUL-JAR300; Iwaki, Japan) filled with autoclave vermiculite as an inert growth substrate. Each seedling was inoculated with I mL of a bacterial suspension (1 ⋅ 10 7 cells/mL) of each Bradyrhizobium strain directly into the root zone. The uninoculated control received only sterile saline, whereas the nitrogen-supplemented control received 5mM KNO 3 in the nutrient solution. The plants were maintained in a controlled growth chamber under tropical conditions (12 h light/ 12 h dark) at 28°C day/25°C night temperature and relative humidity of 60–70%. The pots were watered with sterile nitrogen-free B&D nutrient solution to maintain adequate moisture. Plants were grown for 30 days after inoculation (DAI). Assessment of nodulation and plant growth The plants were carefully harvested at 30 DAI. The root systemes were gently washed under running tap water to remove the vermiculite. Shoots were separated from the roots and oven-dried at 65°C for 72h to obtain shoot dry weight (SDW), which was used as the primary indicator of plant growth response. Roots were used for nitrogenase activity assessments after which the nodule number was counted manually and nodule fresh weight was determined using an analytical balance. Measurement of Nitrogenas Activity via Acetylene Reduction Assay (ARA) Nitrogenase activity was estimated using acetylene reduction assay (ARA). Intact root systems with attached nodules were washed to remove vermiculite and blotted dry. The root systems of each plant were placed in 500 mL airtight glass jars fitted with gas-tight rubber septa. To initiate the assay, 50 mL (10% v/v) of the headspace of the jar was withdrawn using a gas-tight syringe and replaced with 50 mL of acetylene gas (C₂H₂). The jars were then incubated in the dark at 28°C for 45 minutes. Following incubation, 1 mL of headspace gas was sampled and injected into a gas chromatograph (GC) equipped with a Porapak Q column and a flame ionization detector (FID). The GC was calibrated using standard concentrations of ethylene (C₂H₄). The amount of ethylene produced was quantified based on peak area comparisons with the standard curve, and expressed as micromoles of C₂H₄ produced per plant per hour. All ARA assays were conducted within 2 h of harvesting to ensure the integrity of enzyme activity. Relative symbiotic effectiveness (RSE) Relative symbiotic effectiveness (RSE) was calculated using shoot dry weight according to the following formula: $$\:\text{R}\text{S}\text{E}\:\left(\text{%}\right)=\left(\frac{\text{S}\text{D}\text{W}\text{i}\text{n}\text{o}\text{c}\text{u}\text{l}\text{a}\text{t}\text{e}\text{d}-\text{S}\text{D}\text{W}\text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n}\:\text{f}\text{r}\text{e}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}{\text{S}\text{D}\text{W}\text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n}\:\text{f}\text{e}\text{r}\text{t}\text{i}\text{l}\text{i}\text{z}\text{e}\text{d}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}-\text{S}\text{D}\text{W}\text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n}\:\text{f}\text{r}\text{e}\text{e}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\right)100$$ Where SDW inoculated is the shoot dry weight of inoculated plants, SDW N−free control is the mean SDW of uninoculated nitrogen-free controls, and SDW N−fertilized control is the mean SDW of plants receiving 5mM KNO 3 . A negative RSE was interpreted as growth suppression relative to the uninoculated control, whereas values ≥ 75% were considered indicative of highly effective symbioses. Statistical Analysis All statistical analyses were performed using R (version 4,3.0). A two-way analysis of variance (ANOVA) was used to evaluate the effects of the soybean cultivar, Bradyrhizobium strain, and their interaction on nodulation traits, nitrogenase activity and shoot dry weight. Where significant effects were detected, Tukey’s HSD test was applied for post-hoc comparisons at α = 0.05. Spearman correlation analyses were conducted to examine the relationships among nodulation parameters, nitrogenase activity, and plant growth traits. Principal component analysis (PCA) was performed on the scaled trait data to visualize multivariate relationships and identify major axes of variation in symbiotic performance. All graphical outputs were obtained using the ggplot2 package. Results Nodulation responses vary strongly among cultivar-strain combinations All inoculated soybean plants developed nodules under nitrogen-free conditions, whereas the uninoculated controls remained non-nodulated, confirming the sterility of the experimental setup. Analysis of nodule number and nodule weight across the three soybean cultivars revealed that infection frequency did not consistently predict symbiotic tissue development. All Bradyrhizobium strains successfully elicited nodule formation; however, the quantitative and qualitative outcomes were hightly dependent on the host genotype. In Favour, classical soybean symbionts such as B. diazoefficiens , B. elkanii , and B. japonicum induced prolific nodulation, with counts exceeding 80 nodules per plant (Fig. 1A). Interestingly, while B. diaziefficiens produced the highest number of nodules in Favour, the resulting total nodule biomass was statistically comparable to that of other high-performing strains, such as B. denitrificans , which produced significantly fewer nodules (Fig. 1B). This suggests that in Favour, certain strains compensate for the lower infection frequency by producing larger, more substantial nodules. In contrast, Jenguma exhibited a suppressed nodulation profile across the entire bacterial panel. Nodule counts for Jenguma were consistently low, generally failing to exceed 50 nodules per plant regardless of the strain used (Fig. 1A). Furthermore, the nodule weight data for Jenguma showed a marked reduction compared to the other two cultivars, with total biomass often falling below 0.5 g. Even when inoculated with B. diazoefficiens , a strain that induced over 100 nodules in Favour, Jenguma produced only half that number, and the resulting nodules were significantly smaller (Fig. 1A, B). This indicates systemic restriction in both the initiation of symbiotic organs and their subsequent development within the Jenguma genetic background. Suong Pungun displayed a unique generalist nodulation pattern, maintaining relatively stable nodule numbers (~ 40–70 nodules/plant) across nearly all the tested strains (Fig. 1A). Notably, this cultivar maximized nodule biomass with non-traditional soybean rhizobia. For example, B. denitrificans induced the highest total nodule weight in Suong Pungun (~ 1.2g), despite producing nodule counts similar to those of the classical soybean symbiont B. diazoefficiens (Fig. 1A, B). These results highlight a significant host-driven effect where cultivars such as Suong Pungun prioritize nodule maturation and biomass over rew infection count, particularly when interacting with diverse rhizobial lineages. Functional nitrogen fixation and biomass accumulation across host genotypes Functional nitrogen fixation activity, measured via an acetylene reduction assay (ARA), as well as shoot dry weight (SDW) further confirmed the superiority of the Favour and Suong Pungun cultivars in forming productive symbioses compared to Jenguma. In Favour, B. denitrificans elicited the highest nitrogenase activity of approximately 18µmol/h/plant (Fig. 2A), which translated into a robust growth response, with SDW statistically comparable to that of the nitrogen-fertilized control (Fig. 2B). While B. diazoefficiens also demonstrated high functional activity in this cultivar, other strains, including B. lablabi and B. yuanmingense , exhibited significantly reduced ARA levels, resulting in SDW values that were not significantly different from those of the non-inoculated control (CTL). In Jenguma, functional nitrogen fixation was severely limited, with ARA values generally remaining below 10µmol/h/plant across all the strains (Fig. 2A). This functional deficiency was reflected in the growth data, as almost all inoculation treatments in Jenguma resulted in SDW significantly lower than that of the nitrogen-fertilized reference (Fig. 2B). Even the most effective strain for this cultivar, B. denitrificans , only achieved a fraction of the biomass potential observed in the other cultivars. Notably, the inoculation of Jenguma with B. lablabi resulted in the lowest SDW recorded across the study, falling even below that of the non-inoculated CTL, suggesting a high metabolic cost of unproductive nodulation. Suong Pungun exhibited a highly flexible functional response to rhizobia inoculation, achieving its peak ARA activity with B. canariense (~ 18µmol/h/plant) and similarly high activity with B. japonicum . These functional peaks were closely associated with biomass accumulation, as both strains produced SDW values comparable to the 5mM KNO 3 control (Fig. 2A, B). In contrast, B. lablabi , B. elkanii , and B. yuanmingense , were consistenly ineffective in Suong Pungun, yielding shoot biomass that was not significantly different from the uninoculated control (Fig. 2B), as observed in the other cultivars. Collectively, these results indicate that while cultivars such as Suong Pungun can translate effective nitrogen fixation across diverse rhizobial lineages into vegetative growth, cultivars such as Jenguma appear to be constrained by a physiological bottleneck that limits the conversion of nitrogenase activity into biomass. Accordingly, plant growth responses were more strongly linked to nitrogen fixation efficiency than to nodulation intensity, underscoring substantial functional variation in symbiotic effectiveness among specific cultivar-strain combinations. Variation in symbiotic effectiveness across soybean cultivars and Bradyrhizobium strains To integrate nitrogen fixation and plant growth responses, relative symbiotic effectiveness (RSE) was calculated as the shoot biomass relative to the mineral nitrogen control (Fig. 3). The results demonstated a wide range of variation in the relative symbiotic effectiveness across the ten Bradyrhizobium speices and the three soybean cultivars tested. Symbiotic performance was highly dependent on the specific strain-cultivar combinations, with RSE values ranging from approximately − 10% to 90%. Among the host plants, Suong Pungun generally exhibited the highest symbiotic potential, with RSE values above 75% when inoculated with B. diazoefficiens , B. japonicum , B. canariense , and B. denitrificans (Fig. 3). Conversely, Jenguma exhibited the lowest symbiotic potential overall, as it failed to reach an RSE of 30% with any of the tested strains and showed negligible or negative responses to B. canariense and B. lablabi (Fig. 3). Strain-specific analysis revealed that B. diazoefficiens was the most broadly effective symbiont, yielding the highest RSE for Favour (~ 90%) and Jenguma (~ 23%), while maintaining high efficacy in Suong Pungun (~ 77%) (Fig. 3). In contrast, the other strains showed more pronouced host specificity. For instance, B. canariense was highly effective for Suong Pungun but only moderately effective for Favour and ineffective for Jenguma. Furthermore, certain strains such as B. lablabi and B. yuanmingense were poorly suited for these cultivars, with B. lablabi inducing a negative RSE in both Favour and Jenguma (Fig. 3), indicating a lack of productive nitrogen fixation in those specific pairings. Overall, the results highlight a distinct hierarchy in symbiotic receptivity, with Suong Pungun being the most promising effective host and Jenguma the most restricted. General and cultivar-specific trait correlations To assess the relationships among symbiotic traits, pairwise correlation analyses were performed using pooled data and cultivar-specific subsets (Fig. 4, Supplementary Tables S1-S4 ). Analysis of the combined data across all three cultivars revealed strong positive correlations between symbiotic performance and plant growth metrics. Relative symbiotic effectiveness (RSE) exhibited a near-perfect correlation with SDW ( r = 0.95), identifying biomass accumulation as the most reliable indicator of symbiotic success across the study (Fig. 4A, Supplementary Table S1 ). Nodule weight also showed high predictive value for both RSE ( r = 0.87) and SDW ( r = 0.87). Although nitrogenase activity (ARA) was positively associated with RSE ( r = 0.63) and shoot biomass ( r = 0.69), these functional correlations were notably weaker than those observed for physical nodule parameters. Nodule number displayed the weakest overall associations, particularly with the RSE ( r = 0.57) When disaggregated by cultivar, distinct physiological strategies and integration levels were observed. In Suong Pungun, the correlation between RSE and nodule weight reached its maximum ( r = 0.90) and the ARA activity remained a robust predictor of growth ( r = 0.88) (Fig. 4D, Supplementary Table S4 ). Favour displayed a similarly strong link between RSE and SDW ( r = 0.90), although its reliance on ARA for overall effectiveness was moderate (r = 0.58) (Fig. 4B, Supplementary Table S2 ). In contrast, Jenguma exhibited a significant decoupling of functional and growth traits. Most notably, the correlation between RSE and ARA in Jenguma was negligible ( r = 0.14) (Fig. 4C, Supplementary Table S3 ), suggesting that nitrogenase activity was not the primary limiting factor for symbiotic effectiveness in this poorly performing cultivar. These cultivar-specific variations indicate that while general trends favor biomass as a proxy for RSE, the underlying efficiency of symbiosis is highly dependent on the host geneotype. Multivariate analysis of symbiotic performance traits Principal component analysis (PCA) was performed to further elucidate the multivariate relationships between plant growth and symbiotic traits across the three soybean cultivars. The first two principal components (PCs) collectively accounted for 89.6% of the total variance, with PC1 explaining 78.3% and PC2 explaining 11.3% (Fig. 5). Trait loading analysis revealed that PC1 was strongly influenced by SDW, RSE, nodule weight, and ARA activity, all of which were clustered along the positive axis of the first dimension (Fig. 5). This indicates that PC1 primarily captures the overall efficiency and productivity of the symbiosis. In contrast, nodule number contributed more significantly to PC2 (Fig. 5), suggesting that the number of nodules is a distinct factor from their individual size or functional quality. The PCA score plot demonstrated a clear separation of the cultivars based on their symbiotic performance profiles. Jenguma clustered almost exclusively in the negative quadrant of PC1 (Fig. 5), reflecting its consistently low symbiotic effectiveness and reduced biomass accumulation. Conversely, Favour and Suong Pungun displayed broader distributions along the positive PC1 axis, indicating higher overall symbiotic potential. Although Favour and Suong Pungun showed overlapping ranges along PC1, they exhibited partial separation along the PC2 axis (Fig. 5), which was likely driven by differences in nodule number and specific nitrogenase activity. This multivariate separation confirms that the poor performance of Jenguma is a distinct phenotypic state characterized by the decoupling of symbiotic traits, whereas Favour and Suong Pungun achieve high productivity through more integrated trait expressions. Discussion The present study was designed to move beyond assessments of rhizobial strain efficiency alone and to clarify how soybean host genetype governs nodulation, nitrogen fixation, and growth outcomes, especially in the context of sub-Saharan soybean cultivars. Host genotype emerged as the major determinant of symbiotic performance, influencing not only the extent of nodulation but, more critically, whether nodulation translated into effective nitrogen fixation and plant growth. Although all cultivars formed nodules with multiple Bradyrhizobium strains, their functional outcomes differed markedly. Suong Pungun exhibited broad symbiotic permissiveness, achieving a high RSE across phylogenetically diverse strains, including non-classical soybean symbionts (Fig. 3 ). In contrast, Jenguma consistently exhibited low nitrogenase activity and poor growth responses, often with negative RSE values, despite forming nodules. Favour showed an intermediate phenotype, responding positively to some strains while restricting others. These cultivar-specific patterns are consistent with earlier reports of strong host control over soybean- Bradyrhizobium compatibility under standardized conditions and indicate that symbiotic failure mostly reflects host-mediated restriction rather than intrinsic strain efficiency 41 . 53–55 . Evidence from legume genomics and transcriptomics suggests that such incompatibility is often accompanied by sustained defence signaling, impaired bacteroid differentiation, reduced expression of symbiotic transporters, and incomplete metabolic integration 39 . Liu et al. (2020) reported that domestication and modern breeding, often conducted under nitrogen-replete conditions, can inadvertently reshape symbiotic traits and reduce BNF potential when selection does not explicitely consider host-microbe interactions 56 . Our results indicate that some African soybean cultivars may harbor latent symbiotic constraints that become apparent only under nitrogen-limiting conditions. This provides a biologically plausible explanation for the persistent inconsistency in inoculation responses reported across SSA, where cultivar choice is often decoupled from symbiotic performance 21 , 25 . Large-scale field studies and meta-analyses in SSA have documented highly variable yield responses to inoculation, frequently attributing poor performance to soil fertility limiations, competition with indigenous rhizobia, or inoculant quality 18 , 20 , 21 , 49 , 57 , 58 . While these factors undoubtedly contribute, our controlled nitrogen-free experiment demonstrated that the host genotype alone can impose a ceiling on symbiotic benefits. When inoculants are poorly matched to host compatibility profiles, even biologically effecient strains may fail to deliver consistent BNF benefits gains. This perspective complements existing explanations and helps reconcile why inoculation responses remain unpredictable even when elite strains are used, particularly in systems relying on promiscuous soybean cultivars 20 , 21 , 58 . The Jenguma cultivar in this study represents a clear case of functional symbiotic restriction, where infection and nodule initiation occurred, but did not culminate in effective nitrogen fixation. Several molecular mechanisms are likely to be responsible for this phenotype. First, Jenguma may impose post-infection barriers that disrupt bacteriod differentiation, a process essential for nitrogenase expression and sustained BNF production. In several legume-rhizobium systems, host-mediated interference at this stage results in nodules that are anotomically present but metabolically ineffective, and are often associated with altered oxygen diffusion, impaired leghemoglobin synthesis, or incomplete bacterial termial differention 39 , 59 , 60 . Such nodules typically contribute little to plant nitrogen nutrition despite appearing normal during developmental stages. Second, Jenguma may activate host sanctioning mechanisms that limit carbon allocation or oxygen supply to underperforming symbionts. Host sanctions are now well established as a means by which legumes regulate efficiency and selectively reward effective nitrogen fixers while penalizing less cooperative strains 61 , 62 . In Jenguma, the consistently low nitrogenase activity across diverse Bradyrhizobium strains suggests that sanctions may be triggered broadly rather than in a strain-specific manner, potentially reflecting a low tolerance threshold for symbiotic inefficiency or altered carbon-nitrogen feedback regulation. Third, Jenguma may possess R j-like genetic constraints or heightened immune sensitivity that permits early infection but restict downstream symbiotic compatibility. Rj genes and related resistance loci are known to mediate incompatibility between soybean cultivars and specific Bradyrhizobium strains by recognizing bacterial effectors and triggering defense-like response 40 , 42 – 44 , 63 . Such responses can suppress nitrogen fixation without completely abolishing nodulation, resulting in structurally intact but functionally compromised nodules. The persistence of low relative RSE, including negative values in some treatments (Fig. 3 ), strongly supports this interpretation and positions Jenguma as a cultivar with inherent symbiotic constraints, rather than one limited by strain availability. Similar patterns have been observed in other legume systems, where nodules form but fail to mature functionally, underscoring that infection success alone is insufficient for agronomic benefit 55 , 64 . Consistent with this interpretation, nodulation traits alone were poor predictors of symbiotic performance. While nodule weight showed a strong positive association with RSE (Fig. 4 A; Supplementary Table S1 ), nodule number was only weakly correlated, particularly in restrictive host backgrounds, such as Jenguma (Fig. 4 C). Mutlivariate analysis further supported this distinction by separating infection intensity from symbiotic quality, with plant growth and nitrogenase activity clustering independent of nodule counts (Fig. 5 ). These results reinforce the view that functional metrics, rather than nodulation frequently, are more reliable indicators of BNF effectiveness. From a practical standpoint, reliance on nodule number as a screening criterion may lead to misleading conclusions in both breeding and inoculant evaluation, whereas measurements of shoot biomass, nitrogenase activity, or tissue nitrogen content provide more direct insights into symbiotic value. An important insight from this study is the strong performance of non-classical Bradyrhizobium lineages in symbiotically permissive soybean hosts. These strains, including B. denitricans and B. canariense supported high nitrogen fixation and plant growth comparable to or exceeding that achieved by canonical soybean symbionts (Fig. 2 A, B). This observation aligns with growing evidence that symbiotic competence within Bradyrhizobium is phylogenetically broad, and that effective soybean symbiosis can arise from lineages outside traditional soybean-associated clades 36 , 65 – 68 . In SSA, where indigenous Bradyrhizobium populations are both diverse and well adapted to local soil conditions 17 , 29 , these non-traditional symbionts should therefore be viewed not merely as competitors of introduced inoculant strains, but as a potentially valuable and undexploited resource. When paired with compatible or broadly permissive cultivars, locally adapted strains may confer additional advantages, including tolerance to soil acidity, low phosphorus availability, and other abiotic stresses characteristic of African agroecosystems. These findings highlight the need for more integrated approaches to inoculant development and soybean improvement. Although controlled screening under nitrogen-free conditions is essential for identifying effective strain-cultivar combinations, such evaluations must extend beyond nodulation counts to include functional assessements of nitrogen fixation and growth. Reliance on nodulation alone risks overestimating symbiotic performance and obscuring host-mediated constraints. In parallel, soybean breeding programs should explicitly incorporate symbiotic responsiveness into selection pipelines, either through direct phenotyping under low-nitrogen conditions or as genomic resources advance, through marker-assisted approaches. Selection conducted exlusively under nitrogen-replete environments risks releasing cultivars with a limited capacity to benefit from BNF, thereby undermining the effectiveness of inoculation strategies in low-input systems. Although the controlled conditions used here are essential for isolating host-strain interactions, field validation remains necessary to account for soil heterogeneity, nutrient limitations, and competition with native rhizobia. Further genetic and transcriptomic characterization of restrictive cultivars would help elucidate the molecular basis of host-mediated incompatibility, whereas genomic and functional analyses of effective non-classical soybean symbionts could inform the development of locally adapted inoculants. Integrating host genetics, rhizobial diversity, and functional phenotyping will be key to stabilizing BNF performance across SSA and improve the reliability of soybean inoculation strategies in smallholder farming systems. Collectively, these results reinforce three key conclusions. First, soybean host genotype is a major determinant of symbioic outcome and is capable of enabling or constraining nitrogen fixation irrespective of strain identity. Second, nodulation alone is an insufficient indicator of symbiotic effectiveness, as functional integration between host and symbiont ultimately governs contribution of BNF to plant growth. Third, non-classical and locally adapted Bradyrhizobium strains represents a valuable yet underexplored resource, especially when paired with permissive cultivars. Declarations Author contributions D.V.A.A: conceptualization, investigation, formal analysis, writing (original draft), and visualization. E.S.A: conceptualization, writing (review and editing). F.A.F: resources, writing (review and editing). S.O: conceptualization, resources, supervision, and writing (review and editing). Conflict of interest The authors declare that there is no potential conflict of interest that may be perceived to influence the results or discussions in this paper. Funding Declaration This study was financially supported by Japan Society for the Promotion of Science (Kakenhi No. 23KK0108 and No. 23K26804), New Energy and Industrial Technology Development Organization, (Grant No. JPNP18016), and Japan Society for the Promotion of Science, Bilateral Joint Research Project (Grant Number JPJSBP120259917). Data availability All data generated or analysed during this study are available from the corresponding author upon reasonable request. References Hartman, G. L., West, E. D. & Herman, T. K. Crops that feed the World 2. Soybean—worldwide production, use, and constraints caused by pathogens and pests. Food Secur. 3 , 5–17 (2011). Herridge, D. F., Peoples, M. B. & Boddey, R. M. Global inputs of biological nitrogen fixation in agricultural systems. Plant. Soil. 311 , 1–18 (2008). 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Supplementary Files SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx SupplementaryTable4.docx SupplementaryFig.S1.jpg Supplementary Figure S1. Phylogenetic relationships among Bradyrhizobium strains used in this study based on 16S rRNA gene sequences. The tree was constructed using the Neighbor-joining method in MEGA12 with 1,000 bootstrap replicates. The scale bar represents number of substitutions per site. Reference type strains and representative soybean symbionts are included for context. The selected strains span multiple Bradyrhizobium clades, including both classical soybean symbionts and non-classical lineages. Mesorhizobium loti strain LMG 17826t2 was used as an outgroup to root the tree. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 05 May, 2026 Reviews received at journal 03 May, 2026 Reviewers agreed at journal 25 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviews received at journal 10 Mar, 2026 Reviewers agreed at journal 05 Mar, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 25 Feb, 2026 Editor invited by journal 25 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 23 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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18:16:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16349,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable4.docx","url":"https://assets-eu.researchsquare.com/files/rs-8802844/v1/28ebe9018ea628f2892916d2.docx"},{"id":104400032,"identity":"be44bd0a-56fc-43f8-b7d6-3e4feb389955","added_by":"auto","created_at":"2026-03-11 12:08:36","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1197785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure S1. Phylogenetic relationships among Bradyrhizobium strains used in this study based on 16S rRNA gene sequences\u003c/strong\u003e. The tree was constructed using the Neighbor-joining method in MEGA12 with 1,000 bootstrap replicates. The scale bar represents number of substitutions per site. Reference type strains and representative soybean symbionts are included for context. The selected strains span multiple \u003cem\u003eBradyrhizobium\u003c/em\u003eclades, including both classical soybean symbionts and non-classical lineages. \u003cem\u003eMesorhizobium loti\u003c/em\u003e strain LMG 17826t2 was used as an outgroup to root the tree.\u003c/p\u003e","description":"","filename":"SupplementaryFig.S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8802844/v1/287064215ee24d23b74e4e8a.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Soybean genotype determines functional symbiotic outcomes with phylogenetically diverse Bradyrhizobium","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoybean (\u003cem\u003eGlycine max\u003c/em\u003e L. Merr.) is one of the world\u0026rsquo;s most important legume crops, providing a major source of plant protein and oil for human consumption and animal feed. Beyond its nutritional and economic value, soybean plays a critical role in sustainable agriculture because of its capacity for biological nitrogen fixation (BNF) in symbiosis with soil-dwelling \u003cem\u003eBradyrhizobium\u003c/em\u003e soecies \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Through this symbiosis, soybean can meet most or all of its nitrogen requirements by converting atmospheric dinitrogen into a plant-available forms \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This is particularly important in low-input systems prevalent in sub-Saharan Africa (SSA), as mineral fertilizers impose a significant financial burden on smallholder farmers and contribute to environmental degradation via nutrient leaching, eutrophication, and greenhouse gas emissions \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Optimizing soybean-\u003cem\u003eBradyrhizobium\u003c/em\u003e symbiosis is therefore central to enhancing productivity while advancing environmentally sustainable intensification of cropping systems.\u003c/p\u003e \u003cp\u003eThe agronomic potential of BNF in soybean is well demonstrated in major producing regions, including Brazil, the United States, and Argentina, which together account for almost 80% of global soybean production \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In these systems, long-term investments in cultivar improvement, agronomic management, and rhizobial strain selection have enabled the consistent exploitation of BNF, substantially reducing nitrogen fertilizer requirements without compromising yield \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In particular, Brazil represents a global benchmark for successful soybean inoculation technology. Coordinated efforts in elite strain development, host-strain compatibility testing, inoculant quality control \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and farmer adoption have allowed Brazilian soybean production to rely almost entirely on BNF, generating large economic savings while maintaining high and stable yields \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. These successess demonstrate that when the host genotype, microbial symbionts, and management practices are well aligned, soybean can reliably achieve high productivity through biological nitrogen inputs. In contrast, soybean productivity in SSA remains low and highly variable, despite the increasing demand for food, feed, and cash crops. Across the region, yield is constrained by poor soil fertility, inconsistent nodulation, and nitrogen fixation \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Field and greenhouse studies have frequently reported divergent yield responses to rhizobial inoculation, ranging from substantial gains to negligible or even negative effects, depending on location, cultivar, and management history \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Such inconsistencies have persisted even when commercial inoculants are deployed, undermining farmer confidence and limiting adoption of BNF-based technologies \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These contrasting outcomes relative to major soybean-producing regions suggest that inoculation success in SSA is governed by complex biological and environmental interactions rather than inoculant quality alone.\u003c/p\u003e \u003cp\u003eSoybean nodulation and nitrogen fixation are primarily mediated by slow-growing \u003cem\u003eBradyrhizobium\u003c/em\u003e species through finely regulated molecular interactions involving Nod factor signaling, host receptor recognition, and downstream nodule organogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Historically, soybean symbiosis has been considered taxonomically narrow, dominated by a narrow group of classical symbionts, notably \u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e, \u003cem\u003eB. diazoefficiens\u003c/em\u003e, and \u003cem\u003eB. elkanii\u003c/em\u003e, which form the basis of most commercial inoculants worldwide \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, recent phylogenomic and ecological studies have fundamentally revised this perspective. The genus \u003cem\u003eBradyrhizobium\u003c/em\u003e encompasses a broader phylogenetic diversity, including lineages associated with tree legumes, wild herbaceous legumes, non-legume hosts, and bulk soils. Many of these non-classical lineages harbor functional symbiosis gene clusters and can effectively nodulate soybean when host compatibility permits \u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This expanded diversity represents a largely untapped resource for improving soybean symbiosis, particularly in tropical regions where indigenous rhizobial communities differ markedly from those in temperate agroecosystems \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the determinants of symbiotic success, host genotype plays a central role in regulating rhizobial recognition, infection, nodulation, and nitrogen fixation \u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Soybean lines exhibit considerable variation in their ability to recognize and sustain symbiosis with specific \u003cem\u003eBradyrhizobium\u003c/em\u003e strains governed by genetic determinants commonly known as \u003cem\u003eRj\u003c/em\u003e genes. Restrictive alleles such as \u003cem\u003eRj2\u003c/em\u003e, \u003cem\u003eRj4\u003c/em\u003e, or related loci can selectively block nodulation or reduce symbiotic effectiveness with particular strains through effector-triggered immune responses \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Although such genetic restriction may help maintain symbiosis with elite strains in highly managed systems, it can be detrimental in environments where compatible, competitive rhizobia are absent or poorly adapted. Consequently, high agronomic performance of a cultivar does not necessarily translate into high symbiotic efficiency, especially when breeding programs in SSA have traditionally prioritized agronomic traits such as yield stability, maturity, and stress tolerance without explicit selection for BNF-related traits \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn SSA, and particularly in Ghana, the genetic control of soybean-\u003cem\u003eBradyrhizobium\u003c/em\u003e compatibility remains poorly characterized. Inoculation studies in Ghana have reported yield responses ranging from negligible to substantial, often without clear relationships to nodulation intensity or plant nitrogen accumulation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These variable outcomes are frequently attributed to soil fertility constraints, competition from indigenous rhizobia, and inoculant quality \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, an underexplored but potentially critical factor is the compatibility between locally grown soybean cultivars and the phylogenetic diversity of the available \u003cem\u003eBradyrhizobium\u003c/em\u003e strains. Most soybean breeding programs in SSA have not systematically evaluated or selected for symbiotic performance, raising the possibility that some widely cultivated varieties possess restrictive genetic backgrounds that limit effective nodulation and nitrogen fixation under low-input conditions.\u003c/p\u003e \u003cp\u003eDespite extensive research on soybean inoculation, a critical gap remains in understanding how African soybean cultivars differ in their symbiotic compatibility with diverse \u003cem\u003eBradyrhizobium\u003c/em\u003e lineages and how this variability influences nitrogen fixation and plant performance under nitrogen-free conditions. In particular, the extent to which the host genotype constrains nitrogen fixation independently of nodulation remains poorly resolved. In this study, we evaluated the symbiotic performance of three commonly cultivated Ghanaian soybean cultivars inoculated with a phylogenetically diverse panel of \u003cem\u003eBradyrhizobium\u003c/em\u003e strains under controlled, nitrogen-free conditions. By integrating plant growth responses, nodulation, acetylene reduction activity, and relative symbiotic effectiveness, we aimed to (i) quantify cultivar-specific responses to rhizobial diversity, (ii) identify restrictive and permissive host genotypes, and (iii) assess the implications of host genetic control on soybean inoculation strategies in low-input African systems.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant Materials and Experimental Design\u003c/h2\u003e\n \u003cp\u003eThree soybean cultivars widely cultivated in Ghana (Jenguma, Favour, and Suong Pungun) were used in this study. These cultivars were selected to represent their contrasting agronomic characteristics, particularly differences in maturity and field performance, and to capture potential variations in symbiotic responsiveness. Certified seeds were obtained from the Council for Scientific and Industrial Research - Savanna Agricultural Research Institute (CSRI-SARI), Ghana. The experiment followed a completely randomized factorial design, with soybean variety and Bradyrhizobium treatment as the main factors. Twelve treatments were evaluated per cultivar: ten \u003cem\u003eBradyrhizobium\u003c/em\u003e strains, an uninoculated nitrogen-free control (negative control), and a mineral nitrogen treatment supplemented with 5mM KNO\u003csub\u003e3\u003c/sub\u003e (positive control). Each treatment was replicated thrice per cultivars, resulting in 108 experimental units. All plants were grown under strictly nitrogen-free conditions except for mineral nitrogen control, allowing for an unambiguous assessment of biological nitrogen fixation.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eBradyrhizobium Strains and Culture conditions\u003c/h3\u003e\n\u003cp\u003eTen \u003cem\u003eBradyrhizobium\u003c/em\u003e strains representing both classical and non-classical soybean symbionts were obtained from the NITE Biological Resource Center (NBRC) and used in this study. These included \u003cem\u003eB. diazoefficients\u003c/em\u003e USDA110, \u003cem\u003eB. elkanii\u003c/em\u003e USDA61, \u003cem\u003eB. japonicum\u003c/em\u003e NKS4, \u003cem\u003eB. betae\u003c/em\u003e (NBRC 103048), \u003cem\u003eB. lablabi\u003c/em\u003e (NBRC 108826), \u003cem\u003eB. canariense\u003c/em\u003e (NBRC 103049), \u003cem\u003eB. yuanmingense\u003c/em\u003e (NBRC 100594), \u003cem\u003eB. iriomotense\u003c/em\u003e (NBRC 102520), \u003cem\u003eB. liaoningense\u003c/em\u003e (NBRC 100396), and \u003cem\u003eB. denitrificans\u003c/em\u003e (NBRC 105663). The strains were selected to capture broad phylogenetic diversity within the genus \u003cem\u003eBradyrhizobium\u003c/em\u003e, enabling evaluation of host cultivar responses across a wide range of symbiotic backgrounds rather than focusing on known soybean microsymbionts. Phylogenetic relationships among the selected strains were assessed using reference 16S rRNA gene sequences retrieved from NCBI database and aligned using MEGA12. Multiple sequence alignments were performed using CLUSTALW, and phylogenetic trees were constructed using the Neighbor-joining method implemented in MEGA12 \u003csup\u003e51\u003c/sup\u003e. Branch support was evaluated using 1,000 bootstrap replicates \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, confirming that the selected strains spanned multiple \u003cem\u003eBradyrhizobiium\u003c/em\u003e clades (\u003cstrong\u003eSupplementary Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/strong\u003e. All strains were initially streaked on yeast mannitol agar (YMA) and incubated at 28\u0026plusmn;2\u0026deg;C for 5\u0026ndash;7 days. Single colonies were transferred to yeast mannitol broth (YMB) and cultured at 28\u0026deg;C with shaking at 150 rpm for 5 days. Prior to inoculation, the bacterial cells were harvested by centrifugation, washed twice with sterile 0.85% (w/v) saline and resuspended to a final density of approximately 10\u003csup\u003e7\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, as estimated by the optical density at 600 nm(OD\u003csub\u003e600\u003c/sub\u003e).\u003c/p\u003e\n\u003ch3\u003eSeed Surface Sterilization and Germination\u003c/h3\u003e\n\u003cp\u003eSoybean seeds were surface-sterilized by immersion in 70% ethanol for 1 min, followed by immersion in 3% sodium hypochlorite for 1 min, and then rinsed five times with sterile distilled water. Sterilized seeds were pre-germinated on moist sterile tissue paper in Petri dishes and incubated in the dark at 28\u0026deg;C for 48 h. Uniformly germinated seedlings were transferred into a plant box (CUL-JAR300; Iwaki, Japan) filled with autoclave vermiculite as an inert growth substrate. Each seedling was inoculated with I mL of a bacterial suspension (1 \u0026sdot; 10\u003csup\u003e7\u003c/sup\u003e cells/mL) of each \u003cem\u003eBradyrhizobium\u003c/em\u003e strain directly into the root zone. The uninoculated control received only sterile saline, whereas the nitrogen-supplemented control received 5mM KNO\u003csub\u003e3\u003c/sub\u003e in the nutrient solution. The plants were maintained in a controlled growth chamber under tropical conditions (12 h light/ 12 h dark) at 28\u0026deg;C day/25\u0026deg;C night temperature and relative humidity of 60\u0026ndash;70%. The pots were watered with sterile nitrogen-free B\u0026amp;D nutrient solution to maintain adequate moisture. Plants were grown for 30 days after inoculation (DAI).\u003c/p\u003e\n\u003ch3\u003eAssessment of nodulation and plant growth\u003c/h3\u003e\n\u003cp\u003eThe plants were carefully harvested at 30 DAI. The root systemes were gently washed under running tap water to remove the vermiculite. Shoots were separated from the roots and oven-dried at 65\u0026deg;C for 72h to obtain shoot dry weight (SDW), which was used as the primary indicator of plant growth response. Roots were used for nitrogenase activity assessments after which the nodule number was counted manually and nodule fresh weight was determined using an analytical balance.\u003c/p\u003e\n\u003ch3\u003eMeasurement of Nitrogenas Activity via Acetylene Reduction Assay (ARA)\u003c/h3\u003e\n\u003cp\u003eNitrogenase activity was estimated using acetylene reduction assay (ARA). Intact root systems with attached nodules were washed to remove vermiculite and blotted dry. The root systems of each plant were placed in 500 mL airtight glass jars fitted with gas-tight rubber septa. To initiate the assay, 50 mL (10% v/v) of the headspace of the jar was withdrawn using a gas-tight syringe and replaced with 50 mL of acetylene gas (C₂H₂). The jars were then incubated in the dark at 28\u0026deg;C for 45 minutes. Following incubation, 1 mL of headspace gas was sampled and injected into a gas chromatograph (GC) equipped with a Porapak Q column and a flame ionization detector (FID). The GC was calibrated using standard concentrations of ethylene (C₂H₄). The amount of ethylene produced was quantified based on peak area comparisons with the standard curve, and expressed as micromoles of C₂H₄ produced per plant per hour. All ARA assays were conducted within 2 h of harvesting to ensure the integrity of enzyme activity.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eRelative symbiotic effectiveness (RSE)\u003c/h2\u003e\n \u003cp\u003eRelative symbiotic effectiveness (RSE) was calculated using shoot dry weight according to the following formula:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$$\\:\\text{R}\\text{S}\\text{E}\\:\\left(\\text{%}\\right)=\\left(\\frac{\\text{S}\\text{D}\\text{W}\\text{i}\\text{n}\\text{o}\\text{c}\\text{u}\\text{l}\\text{a}\\text{t}\\text{e}\\text{d}-\\text{S}\\text{D}\\text{W}\\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n}\\:\\text{f}\\text{r}\\text{e}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}{\\text{S}\\text{D}\\text{W}\\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n}\\:\\text{f}\\text{e}\\text{r}\\text{t}\\text{i}\\text{l}\\text{i}\\text{z}\\text{e}\\text{d}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}-\\text{S}\\text{D}\\text{W}\\text{n}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n}\\:\\text{f}\\text{r}\\text{e}\\text{e}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\right)100$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eWhere SDW\u003csub\u003einoculated\u003c/sub\u003e is the shoot dry weight of inoculated plants, SDW\u003csub\u003eN\u0026minus;free control\u003c/sub\u003e is the mean SDW of uninoculated nitrogen-free controls, and SDW\u003csub\u003eN\u0026minus;fertilized control\u003c/sub\u003e is the mean SDW of plants receiving 5mM KNO\u003csub\u003e3\u003c/sub\u003e. A negative RSE was interpreted as growth suppression relative to the uninoculated control, whereas values\u0026thinsp;\u0026ge;\u0026thinsp;75% were considered indicative of highly effective symbioses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using R (version 4,3.0). A two-way analysis of variance (ANOVA) was used to evaluate the effects of the soybean cultivar, \u003cem\u003eBradyrhizobium\u003c/em\u003e strain, and their interaction on nodulation traits, nitrogenase activity and shoot dry weight. Where significant effects were detected, Tukey\u0026rsquo;s HSD test was applied for post-hoc comparisons at \u0026alpha;\u0026thinsp;=\u0026thinsp;0.05. Spearman correlation analyses were conducted to examine the relationships among nodulation parameters, nitrogenase activity, and plant growth traits. Principal component analysis (PCA) was performed on the scaled trait data to visualize multivariate relationships and identify major axes of variation in symbiotic performance. All graphical outputs were obtained using the ggplot2 package.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eNodulation responses vary strongly among cultivar-strain combinations\u003c/h2\u003e\n \u003cp\u003eAll inoculated soybean plants developed nodules under nitrogen-free conditions, whereas the uninoculated controls remained non-nodulated, confirming the sterility of the experimental setup. Analysis of nodule number and nodule weight across the three soybean cultivars revealed that infection frequency did not consistently predict symbiotic tissue development. All \u003cem\u003eBradyrhizobium\u003c/em\u003e strains successfully elicited nodule formation; however, the quantitative and qualitative outcomes were hightly dependent on the host genotype. In Favour, classical soybean symbionts such as \u003cem\u003eB. diazoefficiens\u003c/em\u003e, \u003cem\u003eB. elkanii\u003c/em\u003e, and \u003cem\u003eB. japonicum\u003c/em\u003e induced prolific nodulation, with counts exceeding 80 nodules per plant (Fig. 1A). Interestingly, while \u003cem\u003eB. diaziefficiens\u003c/em\u003e produced the highest number of nodules in Favour, the resulting total nodule biomass was statistically comparable to that of other high-performing strains, such as \u003cem\u003eB. denitrificans\u003c/em\u003e, which produced significantly fewer nodules (Fig. 1B). This suggests that in Favour, certain strains compensate for the lower infection frequency by producing larger, more substantial nodules.\u003c/p\u003e\n \u003cp\u003eIn contrast, Jenguma exhibited a suppressed nodulation profile across the entire bacterial panel. Nodule counts for Jenguma were consistently low, generally failing to exceed 50 nodules per plant regardless of the strain used (Fig. 1A). Furthermore, the nodule weight data for Jenguma showed a marked reduction compared to the other two cultivars, with total biomass often falling below 0.5 g. Even when inoculated with \u003cem\u003eB. diazoefficiens\u003c/em\u003e, a strain that induced over 100 nodules in Favour, Jenguma produced only half that number, and the resulting nodules were significantly smaller (Fig. 1A, B). This indicates systemic restriction in both the initiation of symbiotic organs and their subsequent development within the Jenguma genetic background.\u003c/p\u003e\n \u003cp\u003eSuong Pungun displayed a unique generalist nodulation pattern, maintaining relatively stable nodule numbers (~ 40–70 nodules/plant) across nearly all the tested strains (Fig. 1A). Notably, this cultivar maximized nodule biomass with non-traditional soybean rhizobia. For example, \u003cem\u003eB. denitrificans\u003c/em\u003e induced the highest total nodule weight in Suong Pungun (~ 1.2g), despite producing nodule counts similar to those of the classical soybean symbiont \u003cem\u003eB. diazoefficiens\u003c/em\u003e (Fig. 1A, B). These results highlight a significant host-driven effect where cultivars such as Suong Pungun prioritize nodule maturation and biomass over rew infection count, particularly when interacting with diverse rhizobial lineages.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eFunctional nitrogen fixation and biomass accumulation across host genotypes\u003c/h2\u003e\n \u003cp\u003eFunctional nitrogen fixation activity, measured via an acetylene reduction assay (ARA), as well as shoot dry weight (SDW) further confirmed the superiority of the Favour and Suong Pungun cultivars in forming productive symbioses compared to Jenguma. In Favour, \u003cem\u003eB. denitrificans\u003c/em\u003e elicited the highest nitrogenase activity of approximately 18µmol/h/plant (Fig.\u0026nbsp;2A), which translated into a robust growth response, with SDW statistically comparable to that of the nitrogen-fertilized control (Fig.\u0026nbsp;2B). While \u003cem\u003eB. diazoefficiens\u003c/em\u003e also demonstrated high functional activity in this cultivar, other strains, including \u003cem\u003eB. lablabi\u003c/em\u003e and \u003cem\u003eB. yuanmingense\u003c/em\u003e, exhibited significantly reduced ARA levels, resulting in SDW values that were not significantly different from those of the non-inoculated control (CTL).\u003c/p\u003e\n \u003cp\u003eIn Jenguma, functional nitrogen fixation was severely limited, with ARA values generally remaining below 10µmol/h/plant across all the strains (Fig. 2A). This functional deficiency was reflected in the growth data, as almost all inoculation treatments in Jenguma resulted in SDW significantly lower than that of the nitrogen-fertilized reference (Fig. 2B). Even the most effective strain for this cultivar, \u003cem\u003eB. denitrificans\u003c/em\u003e, only achieved a fraction of the biomass potential observed in the other cultivars. Notably, the inoculation of Jenguma with \u003cem\u003eB. lablabi\u003c/em\u003e resulted in the lowest SDW recorded across the study, falling even below that of the non-inoculated CTL, suggesting a high metabolic cost of unproductive nodulation.\u003c/p\u003e\n \u003cp\u003eSuong Pungun exhibited a highly flexible functional response to rhizobia inoculation, achieving its peak ARA activity with \u003cem\u003eB. canariense\u003c/em\u003e (~ 18µmol/h/plant) and similarly high activity with \u003cem\u003eB. japonicum\u003c/em\u003e. These functional peaks were closely associated with biomass accumulation, as both strains produced SDW values comparable to the 5mM KNO\u003csub\u003e3\u003c/sub\u003e control (Fig. 2A, B). In contrast, \u003cem\u003eB. lablabi\u003c/em\u003e, B. \u003cem\u003eelkanii\u003c/em\u003e, and \u003cem\u003eB. yuanmingense\u003c/em\u003e, were consistenly ineffective in Suong Pungun, yielding shoot biomass that was not significantly different from the uninoculated control (Fig. 2B), as observed in the other cultivars. Collectively, these results indicate that while cultivars such as Suong Pungun can translate effective nitrogen fixation across diverse rhizobial lineages into vegetative growth, cultivars such as Jenguma appear to be constrained by a physiological bottleneck that limits the conversion of nitrogenase activity into biomass. Accordingly, plant growth responses were more strongly linked to nitrogen fixation efficiency than to nodulation intensity, underscoring substantial functional variation in symbiotic effectiveness among specific cultivar-strain combinations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eVariation in symbiotic effectiveness across soybean cultivars and Bradyrhizobium strains\u003c/h2\u003e\n \u003cp\u003eTo integrate nitrogen fixation and plant growth responses, relative symbiotic effectiveness (RSE) was calculated as the shoot biomass relative to the mineral nitrogen control (Fig. 3). The results demonstated a wide range of variation in the relative symbiotic effectiveness across the ten \u003cem\u003eBradyrhizobium\u003c/em\u003e speices and the three soybean cultivars tested. Symbiotic performance was highly dependent on the specific strain-cultivar combinations, with RSE values ranging from approximately − 10% to 90%. Among the host plants, Suong Pungun generally exhibited the highest symbiotic potential, with RSE values above 75% when inoculated with \u003cem\u003eB. diazoefficiens\u003c/em\u003e, \u003cem\u003eB. japonicum\u003c/em\u003e, \u003cem\u003eB. canariense\u003c/em\u003e, and \u003cem\u003eB. denitrificans\u003c/em\u003e (Fig. 3). Conversely, Jenguma exhibited the lowest symbiotic potential overall, as it failed to reach an RSE of 30% with any of the tested strains and showed negligible or negative responses to \u003cem\u003eB. canariense\u003c/em\u003e and \u003cem\u003eB. lablabi\u003c/em\u003e (Fig. 3).\u003c/p\u003e\n \u003cp\u003eStrain-specific analysis revealed that \u003cem\u003eB. diazoefficiens\u003c/em\u003e was the most broadly effective symbiont, yielding the highest RSE for Favour (~ 90%) and Jenguma (~ 23%), while maintaining high efficacy in Suong Pungun (~ 77%) (Fig. 3). In contrast, the other strains showed more pronouced host specificity. For instance, \u003cem\u003eB. canariense\u003c/em\u003e was highly effective for Suong Pungun but only moderately effective for Favour and ineffective for Jenguma. Furthermore, certain strains such as \u003cem\u003eB. lablabi\u003c/em\u003e and \u003cem\u003eB. yuanmingense\u003c/em\u003e were poorly suited for these cultivars, with \u003cem\u003eB. lablabi\u003c/em\u003e inducing a negative RSE in both Favour and Jenguma (Fig. 3), indicating a lack of productive nitrogen fixation in those specific pairings. Overall, the results highlight a distinct hierarchy in symbiotic receptivity, with Suong Pungun being the most promising effective host and Jenguma the most restricted.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eGeneral and cultivar-specific trait correlations\u003c/h2\u003e\n \u003cp\u003eTo assess the relationships among symbiotic traits, pairwise correlation analyses were performed using pooled data and cultivar-specific subsets (Fig. 4, \u003cstrong\u003eSupplementary Tables S1-S4\u003c/strong\u003e). Analysis of the combined data across all three cultivars revealed strong positive correlations between symbiotic performance and plant growth metrics. Relative symbiotic effectiveness (RSE) exhibited a near-perfect correlation with SDW (\u003cem\u003er\u003c/em\u003e = 0.95), identifying biomass accumulation as the most reliable indicator of symbiotic success across the study (Fig. 4A, \u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e). Nodule weight also showed high predictive value for both RSE (\u003cem\u003er\u003c/em\u003e = 0.87) and SDW (\u003cem\u003er\u003c/em\u003e = 0.87). Although nitrogenase activity (ARA) was positively associated with RSE (\u003cem\u003er\u003c/em\u003e = 0.63) and shoot biomass (\u003cem\u003er\u003c/em\u003e = 0.69), these functional correlations were notably weaker than those observed for physical nodule parameters. Nodule number displayed the weakest overall associations, particularly with the RSE (\u003cem\u003er\u003c/em\u003e = 0.57)\u003c/p\u003e\n \u003cp\u003eWhen disaggregated by cultivar, distinct physiological strategies and integration levels were observed. In Suong Pungun, the correlation between RSE and nodule weight reached its maximum (\u003cem\u003er\u003c/em\u003e = 0.90) and the ARA activity remained a robust predictor of growth (\u003cem\u003er\u003c/em\u003e = 0.88) (Fig. 4D, \u003cstrong\u003eSupplementary Table S4\u003c/strong\u003e). Favour displayed a similarly strong link between RSE and SDW (\u003cem\u003er\u003c/em\u003e = 0.90), although its reliance on ARA for overall effectiveness was moderate (r = 0.58) (Fig. 4B, \u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e). In contrast, Jenguma exhibited a significant decoupling of functional and growth traits. Most notably, the correlation between RSE and ARA in Jenguma was negligible (\u003cem\u003er\u003c/em\u003e = 0.14) (Fig. 4C, \u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e), suggesting that nitrogenase activity was not the primary limiting factor for symbiotic effectiveness in this poorly performing cultivar. These cultivar-specific variations indicate that while general trends favor biomass as a proxy for RSE, the underlying efficiency of symbiosis is highly dependent on the host geneotype.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eMultivariate analysis of symbiotic performance traits\u003c/h2\u003e\n \u003cp\u003ePrincipal component analysis (PCA) was performed to further elucidate the multivariate relationships between plant growth and symbiotic traits across the three soybean cultivars. The first two principal components (PCs) collectively accounted for 89.6% of the total variance, with PC1 explaining 78.3% and PC2 explaining 11.3% (Fig. 5). Trait loading analysis revealed that PC1 was strongly influenced by SDW, RSE, nodule weight, and ARA activity, all of which were clustered along the positive axis of the first dimension (Fig. 5). This indicates that PC1 primarily captures the overall efficiency and productivity of the symbiosis. In contrast, nodule number contributed more significantly to PC2 (Fig. 5), suggesting that the number of nodules is a distinct factor from their individual size or functional quality.\u003c/p\u003e\n \u003cp\u003eThe PCA score plot demonstrated a clear separation of the cultivars based on their symbiotic performance profiles. Jenguma clustered almost exclusively in the negative quadrant of PC1 (Fig. 5), reflecting its consistently low symbiotic effectiveness and reduced biomass accumulation. Conversely, Favour and Suong Pungun displayed broader distributions along the positive PC1 axis, indicating higher overall symbiotic potential. Although Favour and Suong Pungun showed overlapping ranges along PC1, they exhibited partial separation along the PC2 axis (Fig. 5), which was likely driven by differences in nodule number and specific nitrogenase activity. This multivariate separation confirms that the poor performance of Jenguma is a distinct phenotypic state characterized by the decoupling of symbiotic traits, whereas Favour and Suong Pungun achieve high productivity through more integrated trait expressions.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study was designed to move beyond assessments of rhizobial strain efficiency alone and to clarify how soybean host genetype governs nodulation, nitrogen fixation, and growth outcomes, especially in the context of sub-Saharan soybean cultivars. Host genotype emerged as the major determinant of symbiotic performance, influencing not only the extent of nodulation but, more critically, whether nodulation translated into effective nitrogen fixation and plant growth. Although all cultivars formed nodules with multiple \u003cem\u003eBradyrhizobium\u003c/em\u003e strains, their functional outcomes differed markedly. Suong Pungun exhibited broad symbiotic permissiveness, achieving a high RSE across phylogenetically diverse strains, including non-classical soybean symbionts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, Jenguma consistently exhibited low nitrogenase activity and poor growth responses, often with negative RSE values, despite forming nodules. Favour showed an intermediate phenotype, responding positively to some strains while restricting others. These cultivar-specific patterns are consistent with earlier reports of strong host control over soybean-\u003cem\u003eBradyrhizobium\u003c/em\u003e compatibility under standardized conditions and indicate that symbiotic failure mostly reflects host-mediated restriction rather than intrinsic strain efficiency \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. \u003csup\u003e53\u0026ndash;55\u003c/sup\u003e. Evidence from legume genomics and transcriptomics suggests that such incompatibility is often accompanied by sustained defence signaling, impaired bacteroid differentiation, reduced expression of symbiotic transporters, and incomplete metabolic integration \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Liu et al. (2020) reported that domestication and modern breeding, often conducted under nitrogen-replete conditions, can inadvertently reshape symbiotic traits and reduce BNF potential when selection does not explicitely consider host-microbe interactions \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Our results indicate that some African soybean cultivars may harbor latent symbiotic constraints that become apparent only under nitrogen-limiting conditions. This provides a biologically plausible explanation for the persistent inconsistency in inoculation responses reported across SSA, where cultivar choice is often decoupled from symbiotic performance \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLarge-scale field studies and meta-analyses in SSA have documented highly variable yield responses to inoculation, frequently attributing poor performance to soil fertility limiations, competition with indigenous rhizobia, or inoculant quality \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. While these factors undoubtedly contribute, our controlled nitrogen-free experiment demonstrated that the host genotype alone can impose a ceiling on symbiotic benefits. When inoculants are poorly matched to host compatibility profiles, even biologically effecient strains may fail to deliver consistent BNF benefits gains. This perspective complements existing explanations and helps reconcile why inoculation responses remain unpredictable even when elite strains are used, particularly in systems relying on promiscuous soybean cultivars \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The Jenguma cultivar in this study represents a clear case of functional symbiotic restriction, where infection and nodule initiation occurred, but did not culminate in effective nitrogen fixation. Several molecular mechanisms are likely to be responsible for this phenotype. First, Jenguma may impose post-infection barriers that disrupt bacteriod differentiation, a process essential for nitrogenase expression and sustained BNF production. In several legume-rhizobium systems, host-mediated interference at this stage results in nodules that are anotomically present but metabolically ineffective, and are often associated with altered oxygen diffusion, impaired leghemoglobin synthesis, or incomplete bacterial termial differention \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Such nodules typically contribute little to plant nitrogen nutrition despite appearing normal during developmental stages.\u003c/p\u003e \u003cp\u003eSecond, Jenguma may activate host sanctioning mechanisms that limit carbon allocation or oxygen supply to underperforming symbionts. Host sanctions are now well established as a means by which legumes regulate efficiency and selectively reward effective nitrogen fixers while penalizing less cooperative strains \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In Jenguma, the consistently low nitrogenase activity across diverse \u003cem\u003eBradyrhizobium\u003c/em\u003e strains suggests that sanctions may be triggered broadly rather than in a strain-specific manner, potentially reflecting a low tolerance threshold for symbiotic inefficiency or altered carbon-nitrogen feedback regulation. Third, Jenguma may possess \u003cem\u003eR\u003c/em\u003ej-like genetic constraints or heightened immune sensitivity that permits early infection but restict downstream symbiotic compatibility. \u003cem\u003eRj\u003c/em\u003e genes and related resistance loci are known to mediate incompatibility between soybean cultivars and specific \u003cem\u003eBradyrhizobium\u003c/em\u003e strains by recognizing bacterial effectors and triggering defense-like response \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Such responses can suppress nitrogen fixation without completely abolishing nodulation, resulting in structurally intact but functionally compromised nodules. The persistence of low relative RSE, including negative values in some treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e), strongly supports this interpretation and positions Jenguma as a cultivar with inherent symbiotic constraints, rather than one limited by strain availability. Similar patterns have been observed in other legume systems, where nodules form but fail to mature functionally, underscoring that infection success alone is insufficient for agronomic benefit \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConsistent with this interpretation, nodulation traits alone were poor predictors of symbiotic performance. While nodule weight showed a strong positive association with RSE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; \u003cb\u003eSupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), nodule number was only weakly correlated, particularly in restrictive host backgrounds, such as Jenguma (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Mutlivariate analysis further supported this distinction by separating infection intensity from symbiotic quality, with plant growth and nitrogenase activity clustering independent of nodule counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results reinforce the view that functional metrics, rather than nodulation frequently, are more reliable indicators of BNF effectiveness. From a practical standpoint, reliance on nodule number as a screening criterion may lead to misleading conclusions in both breeding and inoculant evaluation, whereas measurements of shoot biomass, nitrogenase activity, or tissue nitrogen content provide more direct insights into symbiotic value.\u003c/p\u003e \u003cp\u003eAn important insight from this study is the strong performance of non-classical \u003cem\u003eBradyrhizobium\u003c/em\u003e lineages in symbiotically permissive soybean hosts. These strains, including \u003cem\u003eB. denitricans\u003c/em\u003e and \u003cem\u003eB. canariense\u003c/em\u003e supported high nitrogen fixation and plant growth comparable to or exceeding that achieved by canonical soybean symbionts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). This observation aligns with growing evidence that symbiotic competence within \u003cem\u003eBradyrhizobium\u003c/em\u003e is phylogenetically broad, and that effective soybean symbiosis can arise from lineages outside traditional soybean-associated clades \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan additionalcitationids=\"CR66 CR67\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. In SSA, where indigenous \u003cem\u003eBradyrhizobium\u003c/em\u003e populations are both diverse and well adapted to local soil conditions \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, these non-traditional symbionts should therefore be viewed not merely as competitors of introduced inoculant strains, but as a potentially valuable and undexploited resource. When paired with compatible or broadly permissive cultivars, locally adapted strains may confer additional advantages, including tolerance to soil acidity, low phosphorus availability, and other abiotic stresses characteristic of African agroecosystems.\u003c/p\u003e \u003cp\u003eThese findings highlight the need for more integrated approaches to inoculant development and soybean improvement. Although controlled screening under nitrogen-free conditions is essential for identifying effective strain-cultivar combinations, such evaluations must extend beyond nodulation counts to include functional assessements of nitrogen fixation and growth. Reliance on nodulation alone risks overestimating symbiotic performance and obscuring host-mediated constraints. In parallel, soybean breeding programs should explicitly incorporate symbiotic responsiveness into selection pipelines, either through direct phenotyping under low-nitrogen conditions or as genomic resources advance, through marker-assisted approaches. Selection conducted exlusively under nitrogen-replete environments risks releasing cultivars with a limited capacity to benefit from BNF, thereby undermining the effectiveness of inoculation strategies in low-input systems.\u003c/p\u003e \u003cp\u003eAlthough the controlled conditions used here are essential for isolating host-strain interactions, field validation remains necessary to account for soil heterogeneity, nutrient limitations, and competition with native rhizobia. Further genetic and transcriptomic characterization of restrictive cultivars would help elucidate the molecular basis of host-mediated incompatibility, whereas genomic and functional analyses of effective non-classical soybean symbionts could inform the development of locally adapted inoculants. Integrating host genetics, rhizobial diversity, and functional phenotyping will be key to stabilizing BNF performance across SSA and improve the reliability of soybean inoculation strategies in smallholder farming systems.\u003c/p\u003e \u003cp\u003eCollectively, these results reinforce three key conclusions. First, soybean host genotype is a major determinant of symbioic outcome and is capable of enabling or constraining nitrogen fixation irrespective of strain identity. Second, nodulation alone is an insufficient indicator of symbiotic effectiveness, as functional integration between host and symbiont ultimately governs contribution of BNF to plant growth. Third, non-classical and locally adapted \u003cem\u003eBradyrhizobium\u003c/em\u003e strains represents a valuable yet underexplored resource, especially when paired with permissive cultivars.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.V.A.A: conceptualization, investigation, formal analysis, writing (original draft), and visualization. E.S.A: conceptualization, writing (review and editing). F.A.F: resources, writing (review and editing). S.O: conceptualization, resources, supervision, and writing (review and editing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no potential conflict of interest that may be perceived to influence the results or discussions in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Japan Society for the Promotion of Science (Kakenhi No. 23KK0108 and No. 23K26804), New Energy and Industrial Technology Development Organization, (Grant No. JPNP18016), and Japan Society for the Promotion of Science, Bilateral Joint Research Project (Grant Number JPJSBP120259917).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHartman, G. 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Strains of Bradyrhizobium barranii sp. nov. associated with legumes native to Canada are symbionts of soybeans and belong to different subspecies (subsp. barranii subsp. nov. and subsp. apii subsp. nov.) and symbiovars (sv. glycinearum and sv. septentrionale). \u003cem\u003eInt J. Syst. Evol. Microbiol\u003c/em\u003e \u003cb\u003e72\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, X., Cloutier, S., Tambong, J. T. \u0026amp; Bromfield, E. S. P. Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen fixing bacterium from root nodules of soybeans in Canada. \u003cem\u003eInt. J. Syst. Evol. Microbiol.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 3202\u0026ndash;3207 (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biological Nitrogen Fixation (BNF), Bradyrhizobium, Soybean (Glycine max), Relative Symbiotic Effectiveness (RSE), Sub-Saharan Africa, Acetylene Reduction Activity (ARA)","lastPublishedDoi":"10.21203/rs.3.rs-8802844/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8802844/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoybean can meet much of its nitrogen demand through biological nitrogen fixation (BNF). However, yields in sub-Saharan Africa (SSA) remain constrained by nitrogen deficiency and inconsistent responses to rhizobial inoculation. Despite widespread promotion of inoculation, the influence of host genotype on symbiotic effectiveness in African soybean cultivars remains is not well characterized.\u003cstrong\u003e \u003c/strong\u003eWe assessed nodulation, nitrogen fixation, and growth responses of three widely cultivated Ghanaian soybean cultivars inoculated with ten phylogenetically diverse \u003cem\u003eBradyrhizobium\u003c/em\u003estrains under controlled, nitrogen-free conditions. Symbiotic performance was assessed using nodulation traits, acetylene reduction assay, shoot biomass, and relative symbiotic effectiveness (RSE) relative to mineral nitrogen treatment.\u003cstrong\u003e\u003cbr\u003e\n \u003c/strong\u003eSymbiotic outcomes were strongly dependent on the host. Two cultivars exhibited high nitrogen fixation and growth with multiple strains, whereas one showed consistently weak fixation and growth despite nodulation, indicating host-imposed post-infection constraints. Nodule weight and nitrogenase activity, but not nodule number, reliably predicted symbiotic benefits. Notably, several non-classical soybean \u003cem\u003eBradyrhizobium\u003c/em\u003e strains performed comparably or better to recognized soybean symbionts when paired with compatible hosts. These results demonstate that host genotype is a key determinant of soybean BNF effectiveness and highlight the need to integrate symbiotic performance traits into breeding and inoculant design for reliable BNF in low-input SSA farming systems.\u003c/p\u003e","manuscriptTitle":"Soybean genotype determines functional symbiotic outcomes with phylogenetically diverse Bradyrhizobium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 18:16:36","doi":"10.21203/rs.3.rs-8802844/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-05T14:07:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T10:10:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284434404224617919411381038076199144902","date":"2026-04-25T18:07:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"90851158191359166192395477115690165459","date":"2026-04-25T01:40:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-10T07:22:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"126873510944026901832573529579004732593","date":"2026-03-05T11:11:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72512840457823976015285058823099309448","date":"2026-02-25T19:42:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T19:24:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T19:18:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-25T19:08:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-24T04:53:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-24T04:05:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2427710e-9fb9-4d36-84b2-8bf77d2aab23","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-05T14:07:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T10:10:47+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63644138,"name":"Biological sciences/Microbiology"},{"id":63644139,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-19T12:54:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 18:16:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8802844","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8802844","identity":"rs-8802844","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}