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Article

Seed Coating Synergies: Harnessing Plant Growth Regulators to Strengthen Soybean Nodulation and Stress Resilience

by
Saranyapath Pairintra
1,
Nantakorn Boonkerd
2,
Neung Teaumroong
2 and
Kamolchanok Umnajkitikorn
1,*
1
School of Crop Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
2
School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(12), 2876; https://doi.org/10.3390/agronomy15122876
Submission received: 13 November 2025 / Revised: 4 December 2025 / Accepted: 11 December 2025 / Published: 14 December 2025
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
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Abstract

Soybean (Glycine max) is a globally important crop, but its productivity is often limited by suboptimal nodulation and nitrogen fixation, particularly under stress conditions. Bradyrhizobium diazoefficiens strain USDA110 is widely applied to enhance nodulation, yet its efficiency can be further improved by phytohormone modulation. This study examined the effects of seed coatings containing plant growth regulators (PGRs)—acetylsalicylic acid (ASA), aminoethoxyvinylglycine (AVG), Indole-3-butyric acid (IBA), and 6-benzylaminopurine (BAP)—at varying concentrations (5, 50, and 500 nM), in combination with USDA110, on nodulation, nitrogenase activity, ethylene emission, physiological traits, and yield of soybean cultivar CM60. Laboratory assays identified 50 nM AVG, 5 nM IBA, and 5 nM ASA as optimal treatments, significantly enhancing nodule number and nitrogenase activity more than 32% and 28%, as, respectively, compared to untreated seeds. Greenhouse trials in pots, both under well-watered and water stress conditions, showed that USDA110 + AVG/IBA significantly improved photosynthetic rate (+21 and +18% compared to USDA110 alone) and increased plant height. Notably, USDA110 + AVG/IBA treatments sustained higher seed weight under drought, increasing it by over 25%, indicating strong synergistic effects in mitigating stress impacts. These findings highlighted that integrating USDA110 with specific PGRs represented a promising strategy to optimize nitrogen fixation and enhanced soybean productivity under both favorable and challenging conditions.

1. Introduction

Soybean (Glycine max) consumption continuously increases worldwide and is expected to reach a value of USD 226.28 billion by 2032, which is 40% more than the current market value [1]. Thai soybean, Chiang Mai 60 (CM 60), is one of the most widely cultivated cultivars of soybean in Thailand for food industries, including for tofu, soymilk, and plant-based protein food production, which significantly grow in this last decade. It has been commercially important for its high protein content and resistance to soybean rust caused by the fungus, Phakopsora pachyrhizi [2]. However, the constraints of production due to nodulation and biological nitrogen fixation efficiency in field condition are still prominent, particularly under stress conditions [3,4]. The field efficiency of rhizobium colonization is still decreased by competition with other bacterial normal flora and suboptimal conditions in the field [3,4].
Bradyrhizobium diazoefficiens strain USDA110 has been used as the commercial rhizobium for soybean production. Inoculation with B. diazoefficiens has been shown to enhance nodulation on roots and positively influence soybean yield, irrespective of the type of bacterial preparation employed [5,6,7]. It offers advantages over nitrogen fertilizers, primarily due to its cost-effectiveness and nitrogen provision efficiency [8]. A key feature of this interaction is the formation of specialized organs called nodules, facilitating the transfer of carbon from the plant and fixed nitrogen from the bacteria [9]. Additionally, USDA110 is highly effective in promoting nodule formation compared to other strains due to its specific genetic characteristics, superior nitrogen fixation capabilities, and higher biological compatibility with soybeans [10,11,12,13]. Drought severely reduces soybean productivity by suppressing nodulation and nitrogen fixation efficiency. Inoculation with USDA110 helps plants recover nitrogen fixation capacity more rapidly, maintaining yield under drought and potentially increasing grain yield by more than 56% [12,13].
Nodule formation in legumes is a highly coordinated developmental process controlled by both signaling pathways and rhizobial infection, and plant hormones act as the central regulators that integrate these signals to initiate and shape nodule development. Hormones modulate early molecular communication, infection thread progression, and organogenesis, ensuring that nodules develop only when energetically favorable for the plant. It is well known that nodule formation is crucial for symbiotic nitrogen fixation and depends on a complex interplay of plant hormones that regulate the infection process, cortical cell division, and vascular connection [14]. The major hormones involved are auxin, cytokinin, and ethylene. Cytokinin initiates nodule formation by acting as the start signal. It promotes rapid cortical cell division near the root’s vascular bundle, leading to nodule primordium formation [15]. Auxin contributes to bacterial entry and nodule development by promoting infection thread formation in root hairs and maintaining meristematic activity during nodule growth [16]. In contrast, ethylene serves as a negative regulator, limiting excessive nodulation to help the plant balance energy costs with nitrogen needs [17].
Understanding soybean hormonal control on rhizobia compatibility and the autoregulation of nodulation remains limited. Modulation of phytohormones in soybeans holds promise for increasing nodulation and nitrogen fixation efficiency of the Bradyrhizobium sp. in soybean cultivation, thereby enhancing yield and reducing production costs. 6-Benzylaminopurine (BAP), indole acetic acid (IAA), salicylic acid (SA), gibberellic acid (GA), and jasmonic acid (JA) have the potential to increase nodule number per plant, albeit at higher concentrations resulting in a significant decrease of over 70% in soybean [18]. Application of PGRs significantly influenced soybean growth and yield and could potentially increase global productivity by 10–15 million tons annually if applied effectively [19]. For example, GA3 applied at the vegetative stage enhanced plant height and internode length, while application at flower initiation increased chlorophyll content and dry weight. Similarly, cytokinin promoted plant height, pod count, and weight, and increased branching [20]. Salicylic acid applied at flower or pod initiation maximized branching, seed yield, leaf area, and root nodules [20,21]. Foliar application of these PGRs improved reproductive biomass, pod and seed number, and 100-seed weight, resulting in a 12% increase in grain yield [22].
Additionally, low concentrations of cytokinins enhanced nodule development while improving biological nitrogen fixation [23]. Ethylene inhibitors such as silver nitrate (AgNO3) and aminoethoxyvinylglycine (AVG) play a crucial role in inhibiting ethylene synthesis and activity, thereby influencing growth and nodulation in leguminous plants. These inhibitors, commonly used in horticulture for extending shelf life, target 1-aminocyclopropane-1-carboxylate synthase (ACS) and act antagonistically to ethylene in legume plants. It was found that AVG could delay nodule senescence to a certain extent under heat stress in Pisum sativum [24].
PGRs can either promote or inhibit nodule formation depending on their concentration or their ratio to other PGRs. At low to intermediate PGR concentrations, specific auxin–cytokinin ratios stimulate cell division and dedifferentiation, promoting the initiation of callus, tumor-like structures, or early nodule primordia. However, high PGR concentrations shift the developmental response toward organogenesis—high auxin inducing roots and high cytokinin inducing shoots—while excessively high levels of any single PGR can become inhibitory or toxic, reducing cell division and limiting nodule formation [25,26].
The symbiotic signaling pathway involving plant hormones and rhizobium nodulation is not fully understood. More studies are needed to identify the specific types and concentrations of plant growth regulators (PGRs) that most effectively enhance rhizobium nodulation and nitrogen fixation. To better understand how PGRs affect the symbiosis between Bradyrhizobium and soybean cultivar CM 60, this research aimed to investigated different concentrations of various PGRs to enhance nodulation and nitrogen fixation efficiency under laboratory condition and improve the physiological parameters and yield components of soybeans in greenhouse conditions under drought stress.

2. Materials and Methods

2.1. Plant Materials

Soybean seeds (cv. CM 60) were obtained from the Chiang Mai Agricultural Research and Development Center. The experiment was conducted at the laboratory of the Suranaree University of Technology, Thailand, during 2022 and 2023, with two crop repetitions per year performed over this period. Commercial strains of the Bradyrhizobium diazoefficiens strain, USDA110, were grown for 3 to 5 d in yeast extract-mannitol (YM) broth at a 30 °C. Seeds were sterilized by immersion in 70% ethanol for 30 s, repeated twice, followed by a two-time rinse with sterilized water for 2 min each. Then, seeds were disinfected with 3% NaOCl for 5 min and washed with sterilized water. Following this process, seeds were coated with USDA110 along with PGRs.

2.2. Bradyrhizobium and PGRs Inoculant

Bradyrhizobium diazoefficiens commercial strain USDA110 was cultivated in YM broth at 30 °C under constant orbital shaking (200 rpm) for 3–5 days. Subsequently, 10 mL of bacterial culture was centrifuged at 5000 rpm (≈2907 g) for 10 min. After centrifugation, bacterial strain, equivalent to 106 and 108 cells [27,28], was mixed with 0.6% xanthan gum and PGRs. Preliminary results (see Supplementary Figures S1–S6) showed that the 106 concentrations had a clearer effect on nitrogenase activity, number of nodules, and dry weight of shoot and root, as well as a significant difference in treatment compared to the 108 concentrations under environmentally controlled laboratory conditions. Therefore, the 106 concentration was chosen for this experiment.
A 0.6% xanthan gum solution (0.06 g per 10 mL distilled water) was prepared following the method of Dissanayake et al. [29]. Next, 2.16 mL of this solution (for 60 seeds per treatment) was pipetted into a sterilized glass bottle [30,31]. This was performed immediately prior to coating the seeds with acetylsalicylic acid (ASA), aminoethoxyvinylglycine (AVG), and Indole-3-butyric acid (IBA) at 5, 50, and 500 nM. The components were mixed together thoroughly to ensure a uniform solution.
This mixture was then used to thoroughly coat 60 seeds per treatment (see Supplementary Figure S1). The coated seeds were subsequently germinated in sterilized modified Leonard jars containing NFb medium inside a growth chamber under environmentally controlled laboratory conditions, and in 12″ plant pots with sandy loam soil under greenhouse conditions (see Supplementary Figures S1 and S2).

2.3. Methods

2.3.1. Screening of Plant Growth Regulators’ Concentrations Affecting Nitrogenase Activity and Nodule Numbers in Soybean Roots

This experimental design followed a Completely Randomized Design (CRD) with three replications per treatment. B. diazoefficiens commercial USDA110, equivalent to 106 cells, was mixed with 0.6% xanthan gum, along with plant growth regulators (PGRs) at concentrations of 5, 50, and 500 nM of acetylsalicylic acid (ASA), aminoethoxyvinylglycine (AVG), Indole-3-butyric acid (IBA), and 6-Benzylaminopurine (BAP), and coated onto sterilized soybean seeds to facilitate the screening of suitable PGRs for studying the effect of plant hormones on rhizobium nodulation and nitrogen fixation under laboratory condition. The germination process occurred within sterilized modified Leonard jars (see in Figure S2) and NFb medium, situated within a growth chamber (Biosyn Series 6000 Tissue Growth Chamber, Contherm Scientific Ltd., Lower Hutt, New Zealand). Environmental parameters were meticulously controlled, maintaining a temperature of 28 ± 2 °C, and a photoperiod of 12 h of light and darkness, with a light intensity of 300 µE m−2 S−1 and humidity set at 50%. The NFb medium, in accordance with Baldani et al. [32], comprised the following constituents per liter: malic acid (5.0 g), K2HPO4 (0.5 g), MgSO4·7H2O (0.2 g), NaCl (0.1 g), CaCl2·2H2O (0.02 g), and FeEDTA solution (16.4 g L−1). The final volume was adjusted to 1 L with distilled water, and the pH was subsequently adjusted to 6.5.
At 30 days after inoculation (DAI), soybean seedlings were observed for nodule numbers, and nitrogenase activity and ethylene emission were measured using the Acetylene Reduction Assay (ARA) with a gas chromatography (GC) machine (SRI Instruments, Torrance, CA, USA) (Supplementary Figure S7). Additionally, measurements of nodule number per plant, shoot length, and root length were conducted. The most effective PGRs were then selected and tested under greenhouse conditions.

2.3.2. Plant Growth Regulators’ Effects on Physiological Parameters, Yield, and Yield Components of Soybean in Greenhouse Condition

Five to three seeds were initially sown per pot then thinned to a single healthy plant after 10 days of growth, which was maintained until harvest in 12 inches plant pots with 9 kg of sandy clay loam soil per pot under greenhouse condition (see Supplementary Figure S2, Table S1). Under well-watered conditions, irrigation was applied twice a week throughout the growing period to maintain optimal soil moisture, typically 50–60% of the available water-holding capacity (AWHC). Under water stress conditions, irrigation was applied only until the anthesis stage, followed by a 7-day drought stress period. Soil moisture content (SMC) was monitored daily using a soil moisture meter (Theta Probe with HH2 Soil Moisture Meter, Delta-T Devices Ltd., Cambridge, UK). Measurements for well-watered plants were taken at 30 DAI (the plants were in pre-flowering stage: approximately 10–14 days before flowering), while measurements for water-stressed plants were taken after the 7-day drought induction period, when SMC had dropped to a significantly low level (≤20% of AWHC), calculated from field capacity (FC) and permanent wilting point (PWP) (see Supplementary Table S1 for the chemical composition and physical properties of the soil used in the greenhouse experiments).
Physiological parameters including net photosynthesis rate (A), stomatal conductance (gs), transpiration rate (E), and water use efficiency (WUE), calculated as the ratio of A to E, were measured using an LI-6800 Portable Photosynthesis System (LI-COR Biosciences, Lincoln, NE, USA). For all measurements, the third or fourth fully expanded leaves from the top of each plant were used. The leaf chamber conditions were maintained at a reference CO2 concentration of 400 ppm, a Photosynthetically Active Radiation (PAR) of 1200 µmol m−2 s−1 provided by the instrument’s internal light source and a constant leaf temperature of 28 ± 1°C. Six replicates were performed per treatment. Plant height was also measured.
Dry weight per plant, nodule number per plant, pod number per plant, 100-seed weight, and seed weight per plant were measured at the harvest stage, or when the plants reached full maturity (when at least 95% of the pods had turned a tan or brown color).

2.4. Statistical Analyses

The SPSS 25 statistical package was used for statistical data analyses. The experiments were based on a Completely Random Design. The data was analyzed using a two-way ANOVA, and mean differences were compared statistically using the Tukey HSD method. The differences between the means were tested at 0.05 significance level (p < 0.05).

3. Results

3.1. Effects of Seed Coating and Plant Growth Regulators on Nitrogenase Activity and Nodule Numbers in Soybean Roots

The nodule numbers at 30 DAI with USDA110 and treated with plant growth regulators (PGRs) at concentrations of 5, 50, and 500 nM of AVG, BAP, IBA, and ASA are depicted in Figure 1 and Supplementary Figures S3–S6. Seedlings treated with PGRs, including AVG, IBA, and ASA, exhibited a significant increase in nodule number per plant compared to the control group. Seedlings treated with 50 nM of AVG showed the highest nodule number per plant at 28.67, surpassing those treated with 50 nM of AVG (Figure 1a). The nodule number of seedlings treated with USDA110 after 30 DAI was higher than those treated with 5 nM BAP (Figure 1b). Seedlings treated with 5 nM of IBA and ASA in combination with USDA110 exhibited increased nodule numbers (27.67 and 26.00, respectively), surpassing those treated with other concentration levels (Figure 1c,d).
The nitrogenase activity at 30 DAI with USDA110 and various plant growth regulators concentration are presented in Figure 2 and Supplementary Figures S3–S6. Seedlings treated with PGRs showed a significant increase in nitrogenase activity compared to the control and untreated PGR groups. Seedlings treated with 50 nM of AVG exhibited a nitrogenase activity of 2262.72 nmol h per plant, surpassing those treated with other concentration levels (Figure 2a). Seedlings treated with USDA110 and 500 nM of BAP at 30 DAI showed an increased nitrogenase activity of 2079.23 nmol h per plant (Figure 2b). Additionally, seedlings treated with 5 nM of IBA and ASA enhanced nitrogenase activity, reaching 1616.96 nmol h per plant and 538.84 nmol h per plant, respectively, exceeding other concentration levels (Figure 2c,d). Based on the aforementioned findings presented in Figure 1 and Figure 2, it can be inferred that the application of 50 nM of AVG, 5 nM of IBA, and 5 nM of ASA significantly impacts the nitrogen fixation and nodulation processes in soybean cultivar CM60. AVG significantly suppressed ethylene emission in soybean roots compared to control and USDA110 treatments over seven days (0 to 7 DAI), as detailed in Table S2. Ethylene emission levels varied significantly across all four treatments at every time point (p = 0.039 to p = 0.000). The AVG-alone treatment yielded the lowest mean emission rate by 7 DAI, confirming its high effectiveness in inhibiting the hormone’s production (Table S2).

3.2. Effects of Seed-Coated Plant Growth Regulators on Physiological Parameters, Yield, and Yield Components of Soybean Under Greenhouse Condition

The effects of coating soybean seeds with ASA, AVG, BAP, and IBA at concentrations of 5, 50, and 500 nM were evaluated in combination with USDA110. Seeds treated with USDA110 and PGRs (including ASA at 5 nM, AVG at 50 nM, BAP at 500 nM, and IBA at 50 nM) significantly increased nitrogenase activity, nodule numbers, and the dry weight of roots and shoots at 30 days after inoculation (DAI) compared to the USDA110-only control group (Figure 1 and Figure 2 and Supplementary Figures S3–S6). However, the increase in nodule number and dry weight for IBA and BAP-treated seeds was relatively low compared to other treatments (Figure 1 and Supplementary Figures S5 and S6). As a result, only 5 nM ASA, 50 nM AVG, and 5 nM IBA were selected to determine the potential of PGRs and USDA110 on the physiological and yield components in soybean under a greenhouse experiment, both in normal and water-stressed conditions (Figure 1 and Figure 2 and Supplementary Figures S3–S6).

3.2.1. Physiological Parameters

Soybean plants subjected to water stress when SMC had dropped to less than 20% of AWHC during the pre-flowering stage, showed a decrease in all physiological parameters. These differences were also statistically significant across treatments. Soybean plants treated with AVG/IBA, along with the USDA110 at 30 DAI, resulted in a significantly higher transpiration rate (E), photosynthetic rate, stomatal conductance (gs), plant height, nitrogenase activity, and number of nodules per plant compared to other treatments and the control under both well watering and water-stressed conditions (Figure 3, Figure 4 and Figure 5), with the treatment combining AVG and USDA110 showing a particularly strong effect. In contrast, soybean plants treated with USDA110 or PGR alone were negatively affected, showing a decline below the levels of other treatments where USDA110 was combined with a PGR.

3.2.2. Yield and Yield Components

The effects of USDA110 and plant growth regulator (PGR) seed coatings on yield and yield components of soybean cultivar CM60 at 30 DAI under greenhouse conditions are presented in Table 1. Water stress significantly reduced all measured parameters across treatments compared with well-watered plants. Although USDA110 alone promoted nodule formation under stress, the number of nodules was lower than under non-stressed conditions (Figure 5 and Table 1). Combination treatments (USDA110 + PGRs) similarly enhanced nodulation and yield components, but values remained reduced relative to well-watered controls. Seeds treated with PGRs alone failed to induce nodulation or improve yield components compared with the untreated control. Overall, USDA110 inoculation substantially improved yield performance, particularly nodule number, under both normal and water-stressed conditions. Under well-watered conditions, the integration of USDA110 with PGRs produced the strongest responses. Specifically, USDA110 + 50 nM IBA and USDA110 + 50 nM AVG treatments achieved the highest values for nearly all parameters, including nodule number (12.78 and 13.33, respectively) and seed weight per plant (8.41 g and 8.24 g, respectively). Notably, under water stress, USDA110 + 50 nM AVG produced the highest seed weight per plant (7.55 g), surpassing several well-watered treatments, while USDA110 + 5 nM IBA also maintained a high seed weight (5.73 g) (Table 1). The most significant finding was the synergistic effect of combining USDA110 with PGRs, specifically AVG and IBA, which resulted in the highest yield even under severe drought conditions. This synergistic effect suggests that PGRs might enhance the efficacy of the Bradyrhizobium strain under water deficit conditions.

4. Discussions

4.1. Seed Coating–Delivered Plant Growth Regulator–Mediated Regulation of Nodulation and Nitrogenase Activity in Soybean

The effects of PGR coatings at varying concentrations combined with the B. diazoefficiens strain USDA110 were investigated after 30 DAI. It was found that seeds treated with USDA110, along with ASA, AVG, BAP, and IBA, at concentrations of 5 nM, 50 nM, 500 nM, and 50 nM, respectively, led to a statistically significant increase in nitrogenase enzyme activity, nodule number, and the dry weight of shoots and roots when compared to control groups with only USDA110 or PGR alone (Figure 1 and Figure 2 and Supplementary Figures S3–S6). The treatment combination of USDA110 and 50 nM AVG performed the best among the other treatments (Figure 1 and Figure 2). For these reasons, these treatments were the most suitable for further studies under greenhouse conditions because they positively affected the number of nodules and the nitrogen fixation process in soybean root nodules. The differences in optimal concentrations for AVG, ASA, and IBA are directly related to the sensitivity of their respective hormone signaling pathways. AVG is an ethylene inhibitor that blocks ACC synthase, reducing ethylene production. Application of AVG in the range of 50 nM to 10 mM ensures sufficient enzyme inhibition in target tissues to delaying nodule senescence, compound uptake, and ethylene signaling levels [33,34]. ASA acts as a precursor to salicylic acid (SA), a key signaling molecule in plant defense. Due to the high sensitivity of the SA pathway, only 5 nM of ASA is needed to induce or modulate the necessary defense responses [35]. IBA is an auxin involved in plant growth and development, particularly cell elongation and differentiation. Low concentrations of IBA ranging from 50 to 100 μM provide enough IBA to be converted to active IAA, maintaining optimal auxin levels for processes like root and nodule development without the negative effects observed when higher concentrations are used [36]. This finding was consistent with the experiments of Lindström & Mousavi [37] and Zhang et al. [38], who studied nodule formation and nitrogen fixation efficiency in soybean nodules. Their research showed that using USDA110 resulted in better and more efficient nodule formation and nitrogen fixation compared to other strains in both greenhouse and field conditions [37,38].
Meanwhile, many studies have shown that using plant growth regulators at appropriate concentrations also enhanced the efficiency of nodule formation and development, leading to increased nitrogen fixation in the nodules. For example, ASA is a synthetic derivative of salicylic acid (SA) and plays a crucial role in the early stages of the Rhizobium-legume symbiosis [39,40]. Hegazi & El-Shraiy [39] found that foliar application of 1–10 mM SA increased plant height and nodule numbers. However, van Spronsen et al. [41] reported that 100 μM SA inhibited early nodulation in soybean plants and repressed the indeterminate nodulation of Rhizobium leguminosarum cv. viciae. Senaratna et al. [42] also found that pre-soaking bean and tomato seeds with 0.1–0.5 mM salicylic acid (SA) or ASA enhanced tolerance to heat, chilling, and drought stress. Similar stress tolerance was also achieved by applying SA or ASA as a soil drench to seedlings. In contrast, the ASA concentration used in this experiment is quite low. Our preliminary results indicated that ASA concentrations greater than 500 nM did not result in a marked differentiation between the various treatments (Figure 1 and Figure 2 and Supplementary Figure S3).
Similarly, the IBA and BAP concentrations used in this experiment (Figure 1 and Figure 2 and Supplementary Figures S3–S6) were also lower than those reported in other studies. Radhakrishnan & Ranjithakumari [43] inoculated BAP (4.4 μM to 22.2 μM) and IBA (4.9 μM to 24.5 μM) onto B5 medium solution to half-seed explants of soybean cv. CO3 for organogenesis. The highest cell proliferation and shoot induction were obtained from explants cultivated in the presence of 13.3 μM BAP, and roots were then induced from shoots on B5 media containing IBA at 14.7 μM. Rathod et al. [44] also studied the improvement of the regeneration protocol for local soybean cv. MAUS-71 and MAUS-162 using embryonic axis and cotyledonary node explants. They found that shoot initiation and multiplication were optimized using MS medium supplemented with 3 mg/L BAP, while optimal root formation for both explants and cultivars was achieved using MS medium fortified with 0.3 mg/L IBA [44]. Moreover, the application of BAP on soybeans at the early reproductive stages in concentrations varying from 0.5 to 1.5 mM in greenhouse and field conditions increased the total number of pods. Low concentrations of BAP (0.5 mM) significantly reduced flower abortion and delayed pod abscission, while 1 mM of BAP significantly increased seed yield and 100-seed weight in field experiments [45].
van Spronsen et al. [46] investigated the role of cytoplasmic bridges, rhizobial Nod factors (NFs), and ethylene on the early determinate nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus. They discovered that AVG at 0.1 mg/L stimulated root nodulation in light-grown L. japonicus, supporting the idea that cytoplasmic bridges play a functional role in its nodulation process. Liu et al. [47] also examined the role of exogenous polyamines and ethylene in regulating amino acid levels in rice by applying 1 mmol L−1 spermidine or 50 μmol L−1 AVG to panicles at the early grain-filling stage. The results showed that the spermidine (Spd) or AVG applications significantly increased Spd contents and decreased ethylene levels in rice grain [48].
Seed coating technique is also an important factor in modern agricultural seed treatment because it allows for the effective delivery of various beneficial PGRs and B. diazoefficiens directly to the seed surface, thus enhancing plant performance [49,50]. This ensures that as soon as the root hairs emerge after germination, the rhizobia and PGRs are immediately available to initiate the infection and subsequent nodule formation.
Dissanayake et al. [29] prepared a coating by mixing powdered biochar with three different xanthan gum concentrations (0.25%, 0.5%, and 1%). Seeds were dipped in this mixture and air-dried. The results showed that seed germination was significantly higher for both coated and non-coated seeds, but initial bacterial viability dropped significantly after three days [29]. Similarly, in the Sheteiwy et al. [51] experiment, soybean seeds were soaked in B. diazoefficiens (formerly B. japonicum) USDA110 with 16% Arabic gum and allowed to air-dry before transplanting. They found that B. japonicum significantly improved the growth and yield of soybean plants, even when a two-week drought stress was induced at the early pod stage [51].
Interestingly, Jarecki [52] developed a two-layer coating (chitosan and sodium alginate) to protect seeds for early sowing. The layering, which included substances like jojoba oil or polyethylene glycol 400 (PEG 400) applied via a spraying device, resulted in a thick film and increased seed weight. For rhizobium inoculation, B. japonicum inoculant (Commercial HiStick Soy), which contains a natural polymer, was also dry-mixed with the seeds using a device. Jarecki [52] found that coating seeds with these polymers only fully improved soybean performance when combined with B. japonicum inoculation.
Our research used a thin-film coating, similar to the approaches of Dissanayake et al. [29], Sheteiwy et al. [51], and Jarecki [52]. The coating polymer, 0.6% xanthan gum, was mixed with PGR and USDA110 for our formulation (see in Materials and Methods). Xanthan gum is commonly used as a food additive. In this experiment, 0.6% xanthan gum was identified as the highest concentration that could be fully solubilized while providing suitable viscosity for thin-film coating, retaining moisture, and potentially improving germination [53,54,55]. Xanthan gum significantly enhances the water-holding capacity of seed coatings, thereby increasing moisture availability. This improvement accelerates germination and promotes better seedling growth under drought conditions [53]. In addition, xanthan gum serves as an effective carrier and protective matrix for beneficial bacteria, improving their survival rate. Low concentrations of xanthan gum (0.1–1.0%) form a physical barrier that protects microorganisms and helps maintain viable cell numbers under unfavorable environmental conditions [56]. In contrast, some of the cited studies used different coating polymers like Arabic gum, chitosan, and sodium alginate and different methods like soaking [4,5,29], dipping [51], or a coating device [52], which often resulted in a thicker layer that did not quickly dissolve in water.
Therefore, film coating is mostly recommended for soybean seed coating due to the characteristic seed morphology and physiology. This technique prevents hazardous “dust-off” during handling and sowing and allows for the precise delivery of low-dose active ingredients like PGRs and beneficial microbes [49,57]. This precise delivery, in turn, results in increased growth-promoting bacteria in the rhizosphere, increasing nutrient uptake and stress tolerance. It is the most economical and fastest process; its cost is relatively lower than encrusting and pelleting, without significantly increasing the overall cost [49]. Thicker coatings (encrusting and pelleting) can create a barrier to water uptake and gas exchange, potentially delaying or reducing germination [49,58].

4.2. Long-Term Effects of Seed Coating–Delivered Plant Growth Regulators on Soybean Physiology and Yield

Soybean plants at the pre-flowering stage exposed to seven days of water stress exhibited significant reductions in physiological traits, yield, and yield components across all treatments. At 30 DAI, however, seeds coated with USDA110 in combination with either AVG or IBA maintained higher transpiration rates, net photosynthesis, stomatal conductance, nodule number, and plant height than other treatments and the control, under both well-watered and water-stressed conditions. In contrast, treatments with USDA110 or PGRs alone were less effective, resulting in lower values than those receiving the combined USDA110 + PGR treatments (Figure 3 and Figure 5 and Table 1).
Water stress during both vegetative and reproductive stages markedly reduced yield and yield components compared with well-watered controls. Nonetheless, the combination of USDA110 with either AVG or IBA significantly improved dry weight per plant, nodule number, pod number, 100-seed weight, and total seed yield relative to other treatments under both normal and stress conditions (Figure 4 and Figure 5 and Table 1). These results clearly demonstrate the synergistic effects of integrating USDA110 inoculation with specific PGRs in mitigating the adverse impacts of drought stress.
Ethylene is a key stress-induced hormone that negatively affects plant growth by inhibiting photosynthesis, reducing root nodule initiation and function, restricting stomatal opening, and decreasing nitrogenase activity [59]. Its production increases dramatically in response to specific developmental stages and environmental stresses, including mechanical wounding, drought, flooding, and pathogen attack, thereby triggering defense-related signaling that limits bacterial growth, spread, and colonization [60,61,62]. Conversely, ethylene inhibitors such as AVG, silver ions (Ag+), and 1-methylcyclopropene (1-MCP) are chemical compounds used to reduce the physiological effects of ethylene [63].
AVG effectively suppressed ethylene production level in soybean roots, resulting in significantly lower and more consistent emission rates in AVG and USDA110 + AVG treatments than control and USDA110 alone (Supplementary Table S2). This reduction is attributed to the inhibition of ACC synthase (1-aminocyclopropane-1-carboxylic acid synthase), the enzyme responsible for catalyzing the conversion of S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC)—a critical, rate-limiting step in the ethylene biosynthesis pathway [60,63,64,65]. By suppressing ethylene accumulation, AVG alleviates its inhibitory effects, thereby promoting nodulation and enhancing root water uptake [59,60,66]. Najeeb et al. [67] reported that AVG pretreatment effectively inhibited ethylene accumulation, enhancing leaf growth, nitrogen uptake, and photosynthesis under both waterlogged and non-waterlogged conditions in cotton. Similarly, Liu et al. [68] demonstrated that AVG inhibited ethylene production, promoted hypocotyl growth, increased ACS expression and activity, and elevated Zn and Fe concentrations.
AVG reduces the rise in ethylene emission that typically occurs during water stress (Supplementary Table S2), which would otherwise negatively affect soybean yield and its components. By limiting ethylene accumulation, AVG helps sustain vegetative growth, root development, and pod formation under water-deficit conditions [59]. Reduced ethylene levels also support the maintenance of nodule activity, ensuring continued nitrogen supply and facilitating seed filling when soil moisture is limited. This is consistent with Khalid et al. [69], who reported that the application of AVG with bacterial strain effectively inhibits ethylene synthesis, improving the mechanism of root nodule formation and overall plant growth because AVG reduces stress levels caused by ethylene accumulation. If ethylene accumulates in high amounts, it can disrupt crucial processes like root nodule formation, root development, root elongation, and root branching [59,60].
In the case of IBA, it plays a role in nodule organogenesis and ENOD gene expression, influencing root development, including size, elongation, and formation [8,66,70]. Auxin transport is disrupted during drought, leading to reduced nodule growth. However, applying IBA can partially restore the hormonal balance needed for effective symbiosis. IBA stimulates cortical cell division, initiating nodule formation in legumes and soybeans [66,70]. It could promote root development and improve the plant’s ability to absorb water and nutrients, leading to better overall soybean growth, an increased number of pods, larger seeds, and higher biomass and total yield [66,70]. Moreover, the application of USDA110 also has the ability for its stable root nodule formation and strong response to soybean root exudates, which helps the bacteria attach well to the roots [68,71]. Under drought, biological nitrogen fixation declines due to reduced water potential and limited oxygen diffusion within the nodule [72]. The combined application of AVG and IBA improves the root and nodule environment for USDA110, allowing higher nitrogenase activity and maintaining nitrogen supply for protein synthesis and seed filling (Figure 1, Figure 2 and Figure 5 and Table 1). By reducing stress and enhancing water and nitrogen uptake, the AVG/IBA + USDA110 treatment preserves physiological performance under drought, supporting better stomatal conductance and photosynthesis (Figure 5). This sustained carbon and nitrogen availability promotes better seed development and higher final seed weight.
This study advances current research by showing that extremely low doses of PGRs work synergistically with B. diazoefficiens USDA110 to improve soybean nodulation and productivity. While previous work relied on much higher concentrations, we identify optimal levels (50 nM AVG, 5 nM IBA, and 5 nM ASA) that significantly increase nodule formation and nitrogenase activity without causing adverse effects. USDA110 combined with AVG or IBA treatment improved physiological parameters and increased seed weight by at least 25%. This combined approach not only boosts N2 fixation but also strengthens plant tolerance to drought. As a result, it offers a practical strategy for improving yield stability in drought-prone regions while reducing dependence on synthetic nitrogen fertilizers. Future work should focus on mechanism studies and large-scale field trials to validate and optimize these findings.

5. Conclusions

The combination treatment between USDA110 and specific PGR concentrations, including 50 nM of AVG, 5 nM of IBA, and 5 nM of ASA, significantly increased the highest value of nodule numbers and nitrogenase activity. These results demonstrated that the specific concentration of these PGRs could improve biological processes related to nitrogen fixation. To better understand the plants’ physiological and yield responses to PGRs and Bradyrhizobium, these treatments are suitable for determining the potential of PGRs and USDA110 under greenhouse conditions. Soybean seeds treated with the combination of USDA110 and specific PGRs, especially AVG and IBA, resulted in the highest values for key physiological parameters and yield components, such as plant height, photosynthetic rate, and seed weight in all conditions. Moreover, the application of USDA110 with either AVG or IBA treatment yielded the highest seed weight, even under drought conditions. This synergistic effect proved crucial for mitigating the negative impact of water stress. These findings indicated that coating soybean seeds with a combination of the B. diazoefficiens strain USDA110 and specific PGRs, particularly 50 nM of AVG and 5 nM of IBA, is a highly effective strategy for improving nitrogen fixation and soybean productivity, even when water is scarce. This combined approach boosts N2 fixation and improves drought tolerance, providing a practical way to stabilize yields in drought-prone areas while reducing the need for synthetic nitrogen fertilizers. Future research should examine the molecular mechanisms behind this strong combined effect and confirm the results through large-scale field trials.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15122876/s1, Figure S1: Soybean seed coating with 0.6% xanthan gum; Figure S2: Experimental setup in laboratory and greenhouse; Figure S3: Effect of ASA and B. diazoefficiens USDA110 on soybean plants under laboratory condition; Figure S4: Effect of AVG and B. diazoefficiens USDA110 on soybean plants under laboratory condition; Figure S5: Effect of BAP and B. diazoefficiens USDA110 on soybean plants under laboratory condition; Figure S6: Effect of IBA and B. diazoefficiens USDA110 on soybean plants under laboratory condition; Figure S7: The standard curve for acetyl reductase activity assay (ARA); Table S1. Chemical composition and physical properties of the soil used for the greenhouse experiments; Table S2: The impact of AVG and USDA110 on ethylene emission levels in soybean roots (cv. CM60) after inoculation for 7 days under environmentally controlled laboratory conditions.

Author Contributions

Conceptualization, K.U. and N.T.; methodology, N.B. and K.U.; data curation, S.P.; writing—original draft preparation, S.P.; writing—review and editing, K.U., N.T. and N.B.; visualization, S.P.; supervision, K.U.; funding acquisition, K.U. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (OPS MHESI), Thailand Science Research and Innovation (TSRI) (Grant number. RGNS 63–115); (i) Suranaree University of Technology (SUT), (ii) Thailand Science Research and Innovation (TSRI), and (iii) National Science, Research and Innovation Fund (NSRF) (NRIIS number 204215).

Data Availability Statement

The datasets presented in this article are not readily available because the data are part of an ongoing study. Requests to access the datasets should be directed to the corresponding author via email at k.umnajkitikorn@sut.ac.th.

Acknowledgments

During the preparation of this manuscript, Adobe Photoshop (2024) was utilized for standard image editing and enhancement including cropping, adjusting brightness and contrast, adding text annotations, and sharpening to ensure the figures were clear and properly formatted. EndNote version X9.3.3 (Build 13966) was used to ensure accurate citation and referencing throughout the manuscript. QuillBot (free online version, 2024) was used for writing assistance, such as grammar and formatting. The authors have taken full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 15 02876 g001aAgronomy 15 02876 g001b
Figure 1. Nodule number of soybean cv. CM60 with AVG (a), BAP (b), IBA (c), and ASA (d) at 30 days after inoculation. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
Figure 1. Nodule number of soybean cv. CM60 with AVG (a), BAP (b), IBA (c), and ASA (d) at 30 days after inoculation. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
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Figure 2. Nitrogenase activity of soybean cv. CM60 with AVG (a), BAP (b), IBA (c), and ASA (d) at 30 days after inoculation. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
Figure 2. Nitrogenase activity of soybean cv. CM60 with AVG (a), BAP (b), IBA (c), and ASA (d) at 30 days after inoculation. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
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Figure 3. Response of 30-day-old soybean plants to different PGRs (a) and to the treatment of USDA110 inoculation combined with PGR (b) under normal conditions in greenhouse.
Figure 3. Response of 30-day-old soybean plants to different PGRs (a) and to the treatment of USDA110 inoculation combined with PGR (b) under normal conditions in greenhouse.
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Figure 4. Response of 30-day-old soybean plants to different PGRs (a) and to the treatment of USDA110 inoculation combined with PGRs (b) under water stress in greenhouse.
Figure 4. Response of 30-day-old soybean plants to different PGRs (a) and to the treatment of USDA110 inoculation combined with PGRs (b) under water stress in greenhouse.
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Figure 5. Physiological responses of soybean cv. CM60 at anthesis stage (after 30 DAI) under greenhouse condition. (a) Net photosynthesis rate (A), (b) stomatal conductance (gs), (c) transpiration rate (E), and (d) plant height. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
Figure 5. Physiological responses of soybean cv. CM60 at anthesis stage (after 30 DAI) under greenhouse condition. (a) Net photosynthesis rate (A), (b) stomatal conductance (gs), (c) transpiration rate (E), and (d) plant height. Different lowercase letters in the columns indicate significant differences (p < 0.05). Data are presented as mean ± standard error (SE). (n = 6 replications).
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Table 1. Yield and yield components of soybean cv. CM60 after inoculation for 30 days under well watering and water stress conditions in greenhouse.
Table 1. Yield and yield components of soybean cv. CM60 after inoculation for 30 days under well watering and water stress conditions in greenhouse.
ConditionsTreatmentsDry Weight per Plant (g)Nodule Number per PlantPod Number per Plant100 Seed Weight (g)Seed Weight per Plant (g)
Well-wateringControl1.18 ± 0.17 c0.00 ± 0.00 c10.11 ± 1.02 abc2.53 ± 0.19 bcd6.12 ± 0.39 c
5 nM IBA1.26 ± 0.16 c0.00 ± 0.00 c7.67 ± 0.37 c1.77 ± 0.17 d6.34 ± 0.39 c
50 nM AVG1.53 ± 0.12 c0.00 ± 0.00 c9.11 ± 0.56 bc2.03 ± 0.17 cd6.51 ± 0.11 bc
5 nM ASA1.41 ± 0.17 c0.00 ± 0.00 c8.56 ± 1.00 bc2.05 ± 0.13 cd6.56 ± 0.21 bc
USDA1102.54 ± 0.20 b10.33 ± 0.50 b13.56 ± 0.56 a3.34 ± 0.18 a8.06 ± 0.17 a
USDA110 + 5 nM IBA3.57 ± 0.29 a12.78 ± 0.86 a11.67 ± 0.50 ab2.70 ± 0.13 abc8.41 ± 0.10 a
USDA110 + 50 nM AVG2.54 ± 0.10 b13.33 ± 0.53 a12.00 ± 1.11 ab3.24 ± 0.25 ab8.24 ± 0.19 a
USDA110 + 5 nM ASA2.50 ± 0.15 b9.44 ± 0.41 b11.44 ± 1.06 ab2.57 ± 0.14 abc7.43 ± 0.13 ab
Sig0.000 **0.000 **0.000 **0.000 **0.000 **
%CV26.0922.2023.4320.839.88
Water stressControl0.75 ± 0.04 d0.00 ± 0.00 c5.56 ± 0.29 bc1.08 ± 0.64 b4.15 ± 0.13 cd
5 nM IBA0.87 ± 0.10 cd0.00 ± 0.00 c4.67 ± 0.33 c1.05 ± 0.06 b3.45 ± 0.13 d
50 nM AVG1.36 ± 0.20 bc0.00 ± 0.00 c6.11 ± 0.39 abc1.46 ± 0.12 ab4.49 ± 0.10 c
5 nM ASA0.85 ± 0.08 cd0.00 ± 0.00 c5.63 ± 0.38 bc1.33 ± 0.12 ab3.90 ± 0.25 cd
USDA1100.89 ± 0.10 cd5.78 ± 0.22 b5.89 ± 0.66 bc1.58 ± 0.21 ab5.60 ± 0.12 b
USDA110 + 5 nM IBA2.05 ± 0.15 a7.33 ± 0.29 a7.67 ± 0.37 a1.88 ± 0.13 a5.73 ± 0.18 b
USDA110 + 50 nM AVG1.91 ± 0.12 ab7.11 ± 0.31 a6.44 ± 0.34 ab1.66 ± 0.11 a7.55 ± 0.20 a
USDA110 + 5 nM ASA2.04 ± 0.18 a6.00 ± 0.24 b4.89 ± 0.31 bc1.35 ± 0.11 ab5.48 ± 0.19 b
Sig0.000 **0.000 **0.000 **0.000 **0.000 **
%CV29.2016.9920.3925.7210.00
Different lowercase letters in the columns indicate significant differences (p ≤ 0.05). Data are presented as mean ± standard error (SE) (n = 9 replications); CV, coefficient of variation; **, statistically significant differences at p < 0.01.
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Pairintra, S.; Boonkerd, N.; Teaumroong, N.; Umnajkitikorn, K. Seed Coating Synergies: Harnessing Plant Growth Regulators to Strengthen Soybean Nodulation and Stress Resilience. Agronomy 2025, 15, 2876. https://doi.org/10.3390/agronomy15122876

AMA Style

Pairintra S, Boonkerd N, Teaumroong N, Umnajkitikorn K. Seed Coating Synergies: Harnessing Plant Growth Regulators to Strengthen Soybean Nodulation and Stress Resilience. Agronomy. 2025; 15(12):2876. https://doi.org/10.3390/agronomy15122876

Chicago/Turabian Style

Pairintra, Saranyapath, Nantakorn Boonkerd, Neung Teaumroong, and Kamolchanok Umnajkitikorn. 2025. "Seed Coating Synergies: Harnessing Plant Growth Regulators to Strengthen Soybean Nodulation and Stress Resilience" Agronomy 15, no. 12: 2876. https://doi.org/10.3390/agronomy15122876

APA Style

Pairintra, S., Boonkerd, N., Teaumroong, N., & Umnajkitikorn, K. (2025). Seed Coating Synergies: Harnessing Plant Growth Regulators to Strengthen Soybean Nodulation and Stress Resilience. Agronomy, 15(12), 2876. https://doi.org/10.3390/agronomy15122876

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