Bradyrhizobium japonicum and Bacillus subtilis alleviate soybean herbicide stress by altering shoot and root growthAbstract:
Inoculation of soybean plants promotes a symbiotic relationship that improves the plant's ability to tolerate abiotic stresses, including herbicide exposure. However, the mechanisms by which rhizobia influence herbicide tolerance, particularly root growth and development, remain poorly understood. Therefore, the objective of this study was to evaluate the effects of single inoculation and co-inoculation with rhizobacteria on soybean growth and root development following exposure to residual corn herbicides under greenhouse conditions. A greenhouse experiment was conducted in a randomized complete block design with six treatments and four replications to examine the impact of single inoculation with Bradyrhizobium japonicum and co-inoculation with Bacillus subtilis on soybean (Glycine max L.) growth under stress from atrazine, mesotrione, and a nicosulfuron + rimsulfuron mixture. Plant height, shoot biomass, root morphological traits, and nodule number were evaluated. Results indicated that co-inoculation tended to improve shoot and root growth and nodulation while reducing injury caused by atrazine, with increases of 6% in height, 3% in shoot biomass, 51% in root length, and 94% in nodule number. Similar responses were observed with Steadfast: 5% in height, 45% in shoot biomass, 63% in root length, and 64% in nodulation. Single inoculation with B. japonicum was especially effective under mesotrione exposure, resulting in higher shoot height (23%), shoot biomass (41%), and nodule number (75%) compared with the control treatment. Significant positive correlations were observed between shoot and root parameters in treatments with B. japonicum alone or in combination with B. subtilis. Overall, the results suggest that bacterial inoculants may improve soybean growth under residual corn herbicide exposure, indicating their potential to mitigate herbicide carryover effects in soybean production systems.
Keywords:
Root Growth; Residual Herbicides; Microbial Inoculants; Rhizobacteria; Abiotic Stress Tolerance
1. Introduction
Soybean (Glycine max L.) plays a crucial role in global food production and is commonly grown in rotation with corn, a practice that offers numerous benefits, including increased yields over monocropping (Reddy, Saravanan, 2013). In these rotation systems, herbicides are widely used for weed management, as weed interference can significantly reduce soybean establishment and yield. However, the persistence of certain herbicides in the soil may result in carryover effects that influence weed management and interfere with the establishment and early growth of soybean in rotational systems. Herbicide application has therefore become an essential agronomic strategy for weed management in maize–soybean rotations, as weed interference can significantly reduce soybean establishment and yield. Photosystem II (PSII), 4-hydroxyphenylpyruvate dioxygenase (HPPD), and acetolactate synthase (ALS) inhibiting herbicides are widely used residual herbicides in the USA, particularly in corn production, due to their broad-spectrum weed control capabilities (Dweikat et al., 2023). However, the long-term persistence of herbicides such as atrazine in soil can lead to carryover effects that harm subsequent crops (Jiang et al., 2024). The residual effects of sulfonylurea herbicides have been shown to impair crop development, leading to significant reductions in shoot and root growth (Masoumeh et al., 2014). Furthermore, residual herbicides can reduce soybean yield in corn-soybean rotation systems (Jiang et al., 2024; Stephenson et al., 2022).
Numerous studies have reported that residual herbicides can negatively affect soybean physiology and early plant development. Exposure to herbicides such as atrazine, mesotrione, and sulfonylureas may reduce root growth, plant height, chlorophyll content, and biomass accumulation, ultimately impairing crop establishment and yield (Arruda et al., 2001; Novais et al., 2019; Jiang et al., 2023; Stephenson et al., 2022).
Because root systems are central to water and nutrient acquisition, any impairment caused by herbicide residues can compromise plant establishment and productivity. In addition, herbicide persistence in soil may interact with the soil microbial community, influencing plant–microbe relationships in the rhizosphere. The plant root system, however, has the potential to significantly increase crop adaptability to diverse environments by indirectly influencing the soil microbial community, which can, in turn, enhance nutrient uptake, thereby improving the growth of crop roots (Wang et al., 2022). Recent studies have shown that the soil microbiome plays a crucial role in crop growth. Crop roots secrete a diverse range of chemicals, including organic acids, phenolic compounds, amino acids, and sugars, which recruit beneficial microorganisms and promote positive responses to both biotic and abiotic stresses (Jingjing et al., 2022; Pantigoso et al., 2022; Chauhan et al., 2023).
In this context, inoculating plants with rhizobacteria has been explored as a strategy to increase herbicide degradation in the rhizosphere, enhance biological nitrogen fixation, and produce compounds such as antimicrobials, exopolysaccharides, and lipo-chito-oligosaccharides, which promote plant growth (Moretti et al., 2020). Rhizobacteria capable of degrading PSII herbicides, such as simazine and atrazine, have been isolated from soybean roots. Bazhanov et al. (2017) isolated 45 bacterial strains from soybean nodules grown in simazine-treated soils, 25 of which were identified as Bradyrhizobium japonicum. Seven B. japonicum strains degraded more than 40% of simazine in SMS broth. Some plant growth-promoting rhizobacteria (PGPR) can improve soybean root development by increasing its length, diameter, volume, and biomass (Moretti et al., 2020; Frasca et al., 2024).
Studies have shown that co-inoculation strategies may enhance root growth and plant performance under stress conditions. For example, the addition of Bacillus subtilis BV02 to a glyphosate application solution helped attenuate herbicide-induced stress and promoted root system development in soybean (Braz et al., 2022). Similarly, soybean plants inoculated with Bradyrhizobium japonicum have shown increases in root growth parameters, such as root length, volume, and surface area, compared with uninoculated plants (Zuffo et al., 2015; Moretti de Souza et al., 2020).
Despite the growing evidence of beneficial plant–microbe interactions, the role of microbial inoculation in mitigating the effects of residual corn herbicides on soybean growth remains insufficiently understood. In particular, limited information is available on how inoculation with rhizobacteria influences soybean root growth under herbicide stress. Therefore, the objective of this study was to evaluate the effects of single inoculation and co-inoculation with rhizobacteria on soybean growth and root development following exposure to residual corn herbicides under greenhouse conditions.
2. Materials and Methods
2.1 Planting and Inoculation
The experiment was conducted in the greenhouse of the Department of Plant and Soil Sciences at Mississippi State University, Starkville, MS (33°27’18" N, 88°47’17" W), using a randomized complete block design (RCBD) with four replications. To ensure the reliability of the results, the entire experiment was repeated over time, yielding two experimental runs treated as temporal replications. Six herbicide-inoculant treatments were evaluated: atrazine (2.24 kg a.i./ha) combined with Bradyrhizobium japonicum and Bacillus subtilis; mesotrione (0.105 kg a.i./ha) with B. japonicum; nicosulfuron + rimsulfuron (0.0258 + 0.0126 kg a.i./ha) with B. japonicum and atrazine + S-metolachlor + mesotrione (1.46 + 1.46 + 0.188 kg a.i./ha) with B. japonicum. In addition, two untreated controls were included: B. japonicum alone (31 mg−1 seed) and B. japonicum combined with B. subtilis (31 mg g−1 seed + 35.4 mg L−1 soil). Treatments, combinations, and herbicide rates are described in Table 1.
B. subtilis and B. japonicum were selected for their growth-promoting characteristics, including phosphorus solubilization, biological nitrogen fixation, and indole acetic acid production (Santos et al., 2022). Pots were filled with field soil collected for greenhouse experiments and used as the growth medium for soybean plants (AG24X7 cultivar). The soil was not chemically characterized prior to the experiment, as the study focused on the effects of herbicide treatments and microbial inoculation under controlled greenhouse conditions. The pots were 11.4 liters in size to minimize restrictions on root growth and allow the harvest of the entire root system with minimal damage. Each pot served as an experimental unit, with 24 pots per experimental run. Four soybean seeds were planted per pot, and the seedlings were thinned to one after germination.
Seeds were inoculated with B. japonicum, and the soil was inoculated with B. subtilis. Herbicides were applied the same day as seeding using a spray chamber (Generation III track sprayer, DeVries Manufacturing, Inc., Hollandale, MS, USA) equipped with an FC-GA80-02 (Teejet) nozzle spaced 50 cm apart, calibrated to deliver 187 L ha−1 at 275 kPa. Irrigation was applied daily to maintain adequate soil moisture and prevent water stress.
2.2 Collection of root samples
In both experiment runs, roots were sampled 28 days after sowing (DAS) and triple rinsed using tap water to separate the growing media, wrapped in moist paper towels to retain moisture, packed in re-sealable zipper storage plastic bags, and stored at 4 °C. Plant height was measured from the soil surface to the apical meristem using a ruler. Chlorophyll content was determined using a portable chlorophyll meter (SPAD-502, Konica Minolta, Japan). Visual plant injury was evaluated using a 0–100 scale based on chlorosis, necrosis, and growth reduction, where 0 indicates no visible injury, and 100 indicates plant death. Afterward, the roots were placed on a transparent acrylic tray (20 cm x 30 cm) in a thin layer of water (6–8mm height) and scanned using a root scanner (WinRHIZO Dual Scan Optical Root Scanner, Regent Instruments Inc., Quebec City, QC, Canada). Images were processed using the WinRHIZO Pro image analysis system (Regent Instruments) to estimate total root length, surface area, average root diameter, and root volume. Before taking the images, the primary root length was measured manually using a ruler. The WinRHIZO software was used to assess root morphological traits, including total root length, root surface area, average root diameter, and root volume based on root diameter distribution and length. After imaging, roots and shoots were collected and dried at 60 °C for 72 h to determine dry mass.
2.2.1 Data analysis
All analyses were performed using JMP 12.1.0 (SAS Institute Inc., Cary, NC) software for Windows. The data were analyzed using a one-way analysis of variance (ANOVA) with a significance level of P ≤ 0.05, and the means were separated using the Least Significant Difference (LSD) test. Principal component analysis (PCA) and multivariate correlations were performed using JMP software (SAS Institute Inc., Cary, NC). Data from both experimental runs were combined and analyzed to assess the effects of herbicide treatments and inoculant combinations. Percentage changes reported in the Results section were calculated using the raw data prior to statistical analysis relative to the control treatment. Tables present the treatment means used for statistical comparisons.
3. Results and Discussion
3.1 Soybean growth
No significant differences were observed (p > 0.05) for plant height, root dry weight, and shoot dry weight (Table 2, Figure 1). However, numerical variation among treatments was observed, suggesting possible trends associated with inoculation across herbicide treatments. An increase in plant height was observed in plants treated with mesotrione + B. japonicum (M-I1) (23%), followed by atrazine + B. japonicum and B. subtilis (A-I2) and Steadfast (nicosulfuron + rimsulfuron) + B. japonicum and B. subtilis (S-I2) by 6 and 5%, respectively. Treatments with Lexar (atrazine + mesotrione + S-metolachlor) + B. japonicum (L-I1) resulted in a slight numerical reduction (2%) in plant height. Treatment S-I2 increased shoot dry weight by 44.96%, followed by treatments M-I1 (41%) and A-I2 (3%), although these differences were not statistically significant. Treatment L-I1 reduced shoot weight by 3%.
Plant growth traits of soybeans under herbicide stress as affected by inoculation with B. japonicum and in combination with B. subtilis
Plant growth parameters [plant height (A), shoot dry weight (B), root dry weight (C), and chlorophyll content (D)] of soybean inoculated with B. japonicum or B. japonicum + B. subtilis under different herbicide treatments (Atrazine, Steadfast, Lexar, and Mesotrione). Plants were grown for 28 days under greenhouse conditions. Columns represent the mean of eight plants (N = 8). Error bars indicate standard error. Means followed by the same letter are not significantly different according to the LSD test at P ≤ 0.05.
The results suggest that under atrazine and Steadfast exposure, co-inoculation with B. japonicum + B. subtilis was associated with numerically higher root growth traits, while B. japonicum alone showed numerical increases in shoot growth under mesotrione exposure. Additionally, under mesotrione, atrazine, and Steadfast conditions, both single inoculation with B. japonicum and co-inoculation with B. japonicum and B. subtilis were associated with higher shoot growth values compared with the control, although the differences were not statistically significant.
Our results align with previous studies, which have demonstrated that shoot height in several legume species increases significantly after inoculation with Rhizobium (Jiang et al., 2023; Ju et al., 2020). Jiang et al. (2023) report that B. japonicum regulates bacterial composition in atrazine-treated soils, while Bacillus influences the rhizosphere community and promotes plant growth. Studies on soybean plants have shown that seed inoculation and subsequent root colonization by rhizobia may increase herbicide degradation in the rhizosphere (Bazhanov et al., 2017; Zhu et al., 2019).
Costa et al. (2019) found that B. subtilis plays a crucial role in stimulating soybean development, with inoculation potentially improving plant nutritional status during the first 15 days of growth. This finding corroborates the present study, as the effects of inoculation were evaluated in the early development of soybeans. These same authors attributed the increases in seedling and root length in other legumes, such as common beans, to increased phytohormone production. Although phytohormone production was not measured in the present study, similar physiological responses may contribute to the trends observed. Jiang et al. (2024) documented a 16.67% reduction in plant height in uninoculated soybean exposed to 7.5 mg kg−1 of atrazine, likely due to the absence of rhizobia, which limited root elongation, lipid-soluble content in nodules, and the proportion of extra-long-chain fatty acids essential for growth. For uninoculated treatments, atrazine significantly inhibited soybean seedling growth and plant height (Jiang et al., 2023; Akor, Ubogu, 2021), further reinforcing the potential role of microbial inoculation in mitigating herbicide stress.
Among the treatments, only the L-I1 treatment significantly increased chlorophyll content (14%). In contrast, the A-I2, M-I1, and S-I2 treatments resulted in chlorophyll reductions of 6, 8, and 3%, respectively (Figure 1, Table 2). The combination of B. japonicum and B. subtilis appears to be negatively affected by mesotrione, atrazine, and nicosulfuron + rimsulfuron with respect to chlorophyll accumulation. This highlights the potential deleterious effects of these herbicides on photosynthetic pigments, even when applied together with rhizobia. Our results are in line with those of Costa et al. (2019), who reported that chlorophyll levels in soybean are significantly reduced when subjected to stress during the first 30 days of growth and are restored to normal levels after 45 days, when the plant reaches the reproductive stage. In contrast, Braz et al. (2022) found that B. subtilis BV02, in association with glyphosate, showed potential to mitigate the adverse effects of herbicides on variables related to relative chlorophyll indices. They also reported no changes in fluorescence parameters, suggesting that glyphosate did not impair PSII proteins or components. Similarly, Zuffo et al. (2015) observed an increase in chlorophyll content in treatments inoculated with B. japonicum, attributing this effect to increased nodulation and greater nitrogen accumulation in leaf tissue.
Plants grown under L-I1 showed the highest injury (20%), followed by A-I2 with 15% (Table 2). Significant injury was observed only when Lexar was applied, although all plants survived until the end of the experiment. This suggests that seed inoculation with B. japonicum and B. japonicum + B. subtilis may partially attenuate herbicide injury, although symptoms were still observed. No visible injury was observed in soybean under the M-I1 and S-I2 treatments (Table 2).
The high level of injury observed with the Lexar application suggests that the B. japonicum inoculant dose may not have been sufficient to mitigate the combined effects of the three active ingredients present in this herbicide mixture. These findings align with those of Bazhanov et al. (2017), who reported slight growth inhibition at low inoculum levels of A. ureafaciens. Similarly, Mehdizadeh and Abadan (2018) found that sulfonylurea herbicides caused residual damage and reduced yield in crops such as rapeseed, alfalfa, flax, sorghum, and pea. Supporting our findings, these results highlight the potential role of microbial inoculation in mitigating herbicide stress.
3.2 Soybean root morphological traits
Root growth indices are crucial for evaluating a plant's capacity to absorb water and nutrients. No significant differences were observed for most root morphological parameters (P > 0.05), although numerical variation among treatments was present (Table 3, Figure 2). Root length increased by 63% and 51% in treatments S-I2 and A-I2, respectively. However, a significant reduction in root length was observed in treatment C-I2, which had the smallest roots throughout the experiment. Additionally, treatments L-I1 and M-I1 showed root lengths that were 4% and 5% shorter, respectively, compared to the control (Table 3, Figure 2). These results indicate numerical variation among treatments, with co-inoculation treatments (A-I2 and S-I2) showing higher root length values, whereas treatments with mesotrione (M-I1) and Lexar (L-I1) showed slightly lower root length compared with the control. Root dry weight increased by 24% and 18% in treatments S-I2 and M-I1, respectively, while treatments A-I2 and L-I1 showed reductions of 4% and 6% compared to the control. Only slight increases in root volume were recorded in L-I1 (3%) and S-I2 (1%), whereas A-I2 and M-I1 resulted in reductions of 1% and 4%, respectively. In terms of root diameter, a slight increase (2%) was observed in A-I2, whereas L-I1, M-I1, and S-I2 exhibited decreases of 3%, 4%, and 3%, respectively. Regarding root surface area, only the S-I2 treatment showed a slight increase (4%), though it was not statistically significant. Conversely, L-I1, A-I2, and M-I1 treatments showed reductions of 7, 2, and 1%, respectively, compared to the control (Table 3, Figure 2).
Root morphological traits of soybeans under herbicide stress as affected by inoculation with B. japonicum and in combination with B. subtilis
Root morphological traits [root length (A), average root diameter (B), root surface area (C), root volume (D), and nodule number (E)] of soybean inoculated with B. japonicum or B. japonicum + B. subtilis under different herbicide treatments (Atrazine, Steadfast, Lexar, and Mesotrione). Plants were grown for 28 days under greenhouse conditions. Columns represent the mean of eight plants (N = 8) with standard error bars. Means followed by the same letter are not significantly different according to the LSD test at P ≤ 0.05.
A few studies report the relationship between corn residual herbicides and bacterial inoculants under abiotic stress conditions, indicating the ability of rhizobia to promote root growth, as verified by Jabborova et al. (2018), in which the combination of B. japonicum and P. putida promoted an increase in the length of soybean roots by 182%, in addition to a greater number of nodules, shoots, roots, total plant and nodule dry weight when compared to plants from non-inoculated seeds or with single inoculation. The inoculation promoted root growth and enhanced the plants’ tolerance to salinity stress (Jabborova et al., 2018). An increase in soybean growth through co-inoculating seedlings with B. japonicum USDA 110 and P. putida NUU 8 has also been reported by Jabborova and Davranov (2015). Moretti de Souza et al. (2020) reported that all co-inoculated plants exhibited superior root surface area and root dry weight, with increases of 17.8 and 13.5%, respectively. Additionally, they observed increases of up to 1.6% in root diameter, 28.5% in root length, and 19.7% in root volume in plants co-inoculated with SI + SMMs + Azospirillum brasilense. Similarly, Egamberdieva et al. (2017) found that B. japonicum USDA110, when combined with either B. subtilis or Serratia proteamaculans, helped alleviate abiotic stress in soybeans.
Although subtle differences were observed in the evaluated parameters, the numerical trends observed in the present study are consistent with findings reported in previous research. Santos et al. (2022) documented a 28.5% increase in root length in plants co-inoculated with B. japonicum and B. diazoefficiens, as well as Rhizobium tropici and B. diazoefficiens, and Azospirillum brasilense. Studies have also shown the benefits of endophytic bacteria, such as B. subtilis, compared to rhizospheric bacteria, in relation to the increase in the relationships and interactions between microorganisms and the plant, such as the increase in the number of nodules, nodule occupancy, and grain yield when soybean seeds were co-inoculated with B. subtilis metabolites or cells (Aung et al., 2013).
Several studies on co-inoculation have demonstrated increases in nodule number, nodule weight, shoot and root biomass, total nitrogen content, and grain yield compared to single inoculation or uninoculated controls (Araújo et al., 2009; Aung et al., 2013). Other research has also shown that co-inoculation of B. subtilis IMA Bs/170005 in soybeans increases total root system length, plant height, shoot and root biomass, and soybean yield up to 40 DAS. In their study, Moretti de Souza et al. (2020) observed increased root activity induced by co-inoculation, which they attributed to stimulation of several physiological processes, including root hair elongation and cell multiplication in the host plant's root meristems. They also state the importance of greater soil exploration by the plant for tolerance to abiotic and biotic stresses. This corroborates the present study, as the presence of herbicides in the soil can invariably generate stress and interfere with critical metabolic processes in plants. This was evident with the co-inoculation with B. japonicum + B. subtilis, which resulted in higher shoot dry weight values in the presence of atrazine and Steadfast, and higher root dry weight under Steadfast exposure. Braz et al. (2022) also reported that Bacillus amyloliquefaciens enhanced root system development and improved root system morphology. Their study on the effects of PGPR on soybean root length identified microbial combinations that influence cell division and differentiation, leading to a more robust root structure.
Jabborova et al. (2018) reported that single inoculation with B. japonicum led to increased root dry weight, shoot dry weight, root length, root surface area, and projected root area compared to the control. Additionally, they found that inoculation with Sinorhizobium meliloti strain 2011 enhanced root dry weight, shoot dry weight, nodule number, and nodule dry weight in Medicago truncatula. The results are in line with our study, in which a single inoculation with B. japonicum was associated with numerically higher root dry weight and nodule number under mesotrione exposure, and a slightly higher nodule number under Lexar exposure. Jabborova et al. (2018) also report an increase in plant height, root and shoot biomass, root surface area, and root projection area of soybean when seeds were inoculated with Rhizobium.
The effects of the corn residual herbicides studied on the growth of root nodules are shown in Figure 2 (E). A-I2 treatments showed a significant increase in the number of nodules (94%). This was followed by M-I1 (75%), S-I2 (64%), and L-I1 (5%). Herbicides, such as atrazine, applied alone, have been found to significantly inhibit soybean nodule number (Jiang et al., 2024). However, our results suggest an improved symbiotic relationship between rhizobia and soybean roots under herbicide stress, resulting in more nodules than in the control.
The results agree with El-Shaarawi et al. (2011), who reported the highest number of nodules and nodule mass in soybeans inoculated with B. japonicum under 10 kg N/ha. These findings are also consistent with previously reported results that plants inoculated with B. japonicum E109 alone developed 16 + 4 nodules per plant. In comparison, those inoculated with a mixture of B. japonicum and T. harzianum showed 12 + 3 nodules per plant (Iturralde et al., 2020). Iturralde et al. (2020) further report that strains of Bacillus spp. used for co-inoculation with B. japonicum improved nodulation in plants inoculated with B. japonicum alone. Increases in soybean nodulation, crop yield, and biomass production were also observed after co-inoculation with Bradyrhizobium spp. and Azospirillum brasilense or Streptomyces griseoflavus (Aung et al., 2013; Machaculeha Chibeba et al., 2015). In a related study, Egamberdieva et al. (2017) reported that co-inoculated soybeans produced 75% more nodules under salt stress than plants inoculated with B. japonicum USDA 110 alone.
Nodule formation has been attributed to the involvement of microbial phytohormones. Auxin plays a crucial role in nodule formation in host plants. For instance, the IAA-producing strain Pseudomonas putida TSAU1 enhances soybean root development and nodulation under stress conditions (Egamberdieva et al., 2017). Although phytohormone production was not evaluated in the present study, microbial production of compounds such as IAA has been reported to promote root growth and nodulation in soybean. Moreover, soybean plants inoculated with rhizobia maintained growth responses under mesotrione, atrazine, and Steadfast exposure, consistent with observations by Jiang et al. (2024).
Contrary to our findings, Arruda et al. (2001) report reductions in the nodulation and dinitrogen fixation of soybeans treated with clomazone after inoculation with Bradyrhizobium. They attributed the effects to decreased plant growth, not to decreased Bradyrhizobium growth. However, they did not rule out the possibility of a direct effect of clomazone on the development or survival of Rhizobium. Other herbicides, such as sulfentrazone and imazaquin, have strongly reduced the growth or survival of Rhizobium strains (Arruda et al., 2001).
Most root and shoot traits showed a positive correlation (Figure 3). In all treatments, a positive correlation was observed among Group 2 traits, which represent plant growth parameters (root length, plant height, shoot dry biomass, and root dry weight), suggesting that increases in one variable are followed by increases in the others. However, a negative correlation was observed between Group 2 traits and Group 3 variables (plant injury and root diameter). This suggests a reduction in plant height, shoot biomass, root length, and root dry weight, with an increase in plant injury and root diameter. Group 4 shows a positive correlation between nodule number and root surface area, suggesting an increase in nodule number with an increase in surface area.
Principal component analysis (PCA) showing relationships among root and shoot traits of soybean plants inoculated with B. japonicum or B. japonicum + B. subtilis under different herbicide treatments (Atrazine, Steadfast, Lexar, and Mesotrione). Component 1 and Component 2 explained 31.7% and 17.7% of the total variance, respectively. Vectors pointing in similar directions indicate positive correlations among variables, whereas vectors pointing in opposite directions indicate negative correlations.
The observed positive correlations may reflect the potential contribution of microbial inoculation to plant tolerance to herbicide exposure, as previously suggested in studies evaluating plant–microbe interactions under chemical stress. The results align with those of Zuffo et al. (2018), who observed positive relationships between the use of B. japonicum and B. japonicum + B. subtilis in the presence of residual corn herbicides, suggesting that microbial inoculation may help maintain plant growth under herbicide exposure. Zuffo et al. (2014) also found that increased nodulation was associated with higher nitrogen content and, consequently, higher chlorophyll content. Jabborova et al. (2018) also found that nodule number significantly positively correlated with root area and root volume in soybean co-inoculated with B. japonicum and P. putida grown under high P nutrient solutions.
In our study, a positive correlation was observed between root length, volume, and diameter. The results are comparable to those of Moretti de Souza et al. (2020). Jabborova et al. (2018) found that root length is linked to root surface area, root volume, and root average diameter. It is further stated that increased root length enhances root surface area, thereby improving nutrient and water uptake from the soil (Moretti de Souza et al., 2020). A positive relationship was also observed between root dry matter and chlorophyll content. This finding is consistent with Costa et al. (2019), who also observed that higher chlorophyll levels in soybeans resulted in taller plants and established a positive correlation with root dry matter.
The number of nodules showed a positive relationship with most root and shoot traits (plant height, shoot dry biomass, root dry weight, root length, root volume). This corroborates the findings of Egamberdieva et al. (2017), who identified positive correlations between nodule numbers, shoot traits, and root architecture in soybean plants inoculated with B. japonicum USDA110 and grown under various nutrient combinations. Specifically, they reported significant correlations between nodule number and root surface area (r = 0.88) and root volume (r = 0.78) under high-nitrogen, low-phosphorus (HNLP) conditions.
Although the present study did not examine the mechanisms underlying the observed responses, several hypotheses could explain the improved plant performance under herbicide exposure. These include physiological compensation through increased absorptive root area, functional redundancy within the root system, spatial escape from localized herbicide concentrations in the soil, or shifts in shoot–root allocation patterns. Future research combining physiological, spatial, and microbial analyses will be necessary to distinguish among these mechanisms.
4. Conclusions
The results of this study show that soybean growth and root morphological responses to herbicide exposure were similar across treatments, although some growth and nodulation parameters differed numerically. Treatments including co-inoculation with B. japonicum and B. subtilis were associated with higher values for certain root traits and nodule numbers under some herbicide conditions, while single inoculation with B. japonicum was associated with numerically higher root dry weight and nodulation in some treatments. Although most variables did not display statistically significant differences, the observed trends suggest that microbial inoculation may influence soybean growth responses under herbicide exposure. Therefore, our results indicate that rhizobial inoculation may help maintain soybean growth and nodulation under herbicide stress, although further research is needed to elucidate the underlying physiological and biochemical mechanisms.
Funding
Acknowledgements
The authors thank Luis Avila, Ashwathy Rai, and Shankar Shanmugam for their technical advice and guidance (Mississippi State University).
Data Availability Statement
The data presented in this study are available on reasonable request from the corresponding author.
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