Microbial Alliance of Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 Enhances Nitrogen Fixation, Yield, and Salinity Tolerance in Black Gram Under Saline, Nutrient-Depleted Soils

Abstract

Salinity is a major abiotic stress limiting black gram (Vigna mungo) productivity, particularly in arid and semi-arid regions. Saline soils negatively impact plant growth, nodulation, nitrogen fixation, and yield. This study evaluated the efficacy of co-inoculating salt-tolerant plant growth-promoting bacteria Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 on black gram performance under saline field conditions (EC: 8.87 dS m⁻¹; pH: 8.37) with low organic carbon (0.6%) and nutrient deficiencies. In vitro assays demonstrated the biocontrol potential of SPR11, inhibiting Fusarium oxysporum and Macrophomina phaseolina by 76% and 62%, respectively. Germination assays and net house experiments under 300 mM NaCl stress showed that co-inoculation significantly improved physiological traits, including germination rate, root length (61.39%), shoot biomass (59.95%), and nitrogen fixation (52.4%) in nitrogen-free media. Field trials further revealed enhanced stress tolerance markers: chlorophyll content increased by 54.74%, proline by 50.89%, and antioxidant enzyme activities (SOD, CAT, PAL) were significantly upregulated. Electrolyte leakage was reduced by 55.77%, indicating improved membrane stability. Agronomic performance also improved, with co-inoculated plants showing increased root length (7.19%), grain yield (15.55 q ha⁻¹; 77.04% over control), total biomass (26.73 q ha⁻¹; 57.06%), and straw yield (8.18 q ha⁻¹). Pod number, seed count, and seed weight were also enhanced. Nutrient analysis showed elevated uptake of nitrogen, phosphorus, potassium, and key micronutrients (Zn, Fe) in both grain and straw. To the best of our knowledge, this is the very first field-based report demonstrating the synergistic benefits of co-inoculating Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 in black gram under saline, nutrient-poor conditions without external nitrogen inputs. The results highlight a sustainable strategy to enhance legume productivity and resilience in salt-affected soils.

1. Introduction

Global food security is increasingly challenged by rapid population growth and the degradation of arable land. Among the critical constraints to sustainable agriculture are abiotic stresses, such as salinity and alkalinity, which are often compounded by biotic pressures from soil-borne pathogens. Soil salinization alone affects over 20% of the world’s irrigated land and significantly reduces crop productivity by disrupting essential physiological processes, including water and nutrient uptake, photosynthesis, seed germination, and cell expansion [1,2]. Legumes such as black gram (Vigna mungo), widely cultivated in South and Southeast Asia, are particularly vulnerable to both salinity and pathogen stress. Salinity poses a major barrier to legume productivity due to its adverse impact on legume rhizobium symbiosis. Elevated salt concentrations inhibit rhizobial survival, root colonization, nodulation, and nitrogen fixation, key processes for legume growth and yield [3,4,5]. Black gram (Vigna mungo) is notably susceptible to soil-borne pathogens under biotic stress. Macrophomina phaseolina, causing charcoal rot and seedling blight, persists in soil for years, complicating control strategies. Likewise, Fusarium oxysporum induces vascular wilt and root decay, collectively undermining plant health and yield potential [6]. Although plants possess intrinsic antioxidant defense systems, including enzymatic (e.g., superoxide dismutase, catalase, ascorbate peroxidase) and non-enzymatic (e.g., glutathione, flavonoids) components, these are often insufficient under severe saline conditions [7]. Nitrogen (N) is a vital macronutrient required for amino acid, protein, and nucleic acid synthesis, as well as chlorophyll production. Legumes contribute significantly to nitrogen cycling through symbiotic nitrogen fixation, offering agronomic and environmental benefits such as improved soil fertility and reduced dependence on synthetic fertilizers. However, salinity disrupts the efficacy of biological nitrogen fixation, thereby limiting these advantages. Moreover, soil salinity negatively affects microbial N-cycling processes, including nitrification, denitrification, mineralization, and nitrogen fixation, by reducing the abundance and expression of key functional genes such as nifH, ureC, and napA [8].

Given these challenges, microbial-based strategies are gaining traction as sustainable alternatives to chemical inputs. Salt-tolerant plant growth-promoting rhizobacteria (STPGPR) improve plant resilience under saline conditions by colonizing the rhizosphere, enhancing nutrient availability, producing phytohormones and siderophores, and suppressing pathogens [9,10]. STPGPR inoculation has been shown to enhance plant biomass, chlorophyll content, antioxidant activity, and stress tolerance under saline stress [11]. Recent studies suggest that co-inoculation of rhizobia with non-rhizobial STPGPR enhances plant growth, nodulation, and nitrogen fixation more effectively than single-strain inoculation [12]. Among these beneficial microbes, Paenibacillus spp. have gained attention for their salinity tolerance, nitrogenase activity, and biocontrol properties [13]. Strains such as Paenibacillus polymyxa and P. yonginensis have demonstrated potential in alleviating abiotic stress through the production of cytokinins, osmoprotectants, and stress-responsive enzymes [14,15]. However, the role of non-rhizobial PGPR in combination with rhizobia under saline field conditions, particularly in black gram, remains insufficiently explored.

Based on this background, we hypothesize that co-inoculation with the compatible, salt-tolerant strains Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 can synergistically enhance plant growth, nodulation, nitrogen fixation, and physiological resilience under saline conditions more effectively than either strain alone or uninoculated controls. This study aims to evaluate these microbial interactions through a two-phase approach: (i) greenhouse trials to assess growth, nodulation, and nitrogen fixation under controlled salinity stress (300 mM NaCl), and (ii) field experiments to measure grain yield, physiological and biochemical responses, and nutrient uptake in saline and nutrient-depleted soils.

2. Materials and Methods

2.1. Microorganisms and Antagonistic Activity Against Plant Pathogens

The antagonistic activity of Paenibacillus sp. strain SPR11 against two major soil-borne phytopathogens, Fusarium oxysporum and Macrophomina phaseolina, was assessed using an in vitro dual-culture assay with slight modification [16]. A 5 mm mycelia disc of the target fungal pathogen was placed at the center of a potato dextrose agar (PDA) and nutrient agar (1:1) with 2% NaCl. Strain SPR11 was streaked in a straight line approximately 2.5 cm away from the fungal disc. The inoculated plates were incubated at 30 ± 2 °C for 7 days. Antagonistic activity was determined by measuring both the radial growth of the fungal colony and the width of the inhibition zone between the bacterial and fungal colonies. Control plates containing only the fungal pathogens (without SPR11) were included for comparison. The percent inhibition of fungal growth was calculated using the following formula:

$$Inhibition \left(\%\right) =\left({\displaystyle \frac{R_1-R_2}{R_1}}\right)\times 100$$

where R₁ is the radial growth of the fungal colony in the control, and R₂ is the radial growth in the presence of SPR11.

2.2. Seed Germination Assay and Net House Evaluation of Growth and Nitrogenase Activity in Black Gram

2.2.1. Preparation of Inoculum

Bradyrhizobium yuanmingense PR3 was incubated in a shaker incubator (Thermo Fisher Scientific, Waltham, MA, USA) for five days in yeast mannitol broth (~10⁹ CFU mL⁻¹), while Paenibacillus sp. SPR11 was grown for 24 h in a nutrient broth (~10⁹ CFU mL⁻¹). Both cultures were centrifuged, washed twice with sterile water, and resuspended in 0.1 M phosphate buffer (pH 7.0) for seed inoculation.

2.2.2. Seed Surface Sterilization and Inoculation

Seeds of black gram variety PU-6 (sourced from ICAR-IISS, Mau Nath Bhanjan, and Uttar Pradesh) were surface-sterilized by immersion in 70% ethanol for 3 min, followed by 2% sodium hypochlorite for 5 min, and rinsed five times with sterile Milli-Q water. The seeds were then shade-dried for one hour prior to inoculation. Four treatments, PR3, SPR11, PR3 + SPR11, and an uninoculated control, were selected for seed germination and net house evaluation, based on their efficacy demonstrated in our earlier study [17].

2.2.3. Seed Germination Assay

Black gram seeds were treated with a specific combination and placed in sterilized Petri dishes containing two layers of filter paper soaked in nitrogen-free nutrient solution. Dishes were incubated under saline (300 mM NaCl) and non-saline conditions at 30 °C with a 10 h light/14 h dark cycle. Each treatment comprised three replicates with five seeds each. Germination and growth parameters were assessed following protocols [17].

2.2.4. Measurement of Nitrogenase Activity and Growth Parameters in Black Gram

In the net house evaluation, a modified Leonard jar setup was used, with the upper chamber filled with autoclaved sand (sieved through a 2 mm mesh) and the lower chamber containing nitrogen-free nutrient solution. To simulate salinity stress, the solution was supplemented with 300 mM NaCl [17].

Nitrogenase activity was estimated using the acetylene reduction assay (ARA) as described in [18], with slight modifications. At 45 days after germination, blackgram root samples with intact nodules were carefully collected and individually placed into 300 mL airtight glass bottles. Ten percent of the air volume in each bottle was replaced with acetylene (C₂H₂) gas using a gas-tight syringe. The bottles were then sealed and incubated at room temperature (28 ± 2 °C) for 1 h. The amount of ethylene (C₂H₄) produced in each bottle was measured using a gas chromatograph (GC) equipped with a flame ionization detector (FID)(Agilent Technologies, Santa Clara, CA, USA) and a capillary column. Since each bottle contained the root system of a single plant, the total ethylene produced was considered to represent the nitrogenase activity of that individual plant. Nitrogenase activity was calculated based on the fresh weight of the entire root system (including nodules) and expressed as micromoles of ethylene (C₂H₄) produced per milligram of root fresh weight per hour (µmol C₂H₄ mg⁻¹ FW h⁻¹), using the following equation:

$$Nitrogenaseactivity={\displaystyle \frac{Amountof{\mathrm{C}}_2{\mathrm{H}}_4\left(\mu g\right)\times ContainerVolume\left(mL\right)}{Rootfreshweight\left(mg\right)\times IncubationTime\left(h\right)}}$$

(Here, C₂H₄ (µg) refers to the mass of ethylene measured by gas chromatography. To convert this value to micromoles (µmol), the molecular weight of ethylene (28.05 g/mol) was used).

In addition to this, nodules were carefully detached from the roots of each plant. The total number of nodules was counted manually for each individual plant. Fresh nodule weight was recorded immediately, and the nodules were then oven-dried at 65 °C for 48 h to determine their dry weight. At the time of harvest, biometric parameters were also recorded on a per-plant basis.

2.3. Field Site Description and Soil Analysis

The field experiments were conducted over two consecutive summer seasons (April 2022 and April 2023) at the ICAR–National Bureau of Agriculturally Important Microorganisms (ICAR–NBAIM), Mau, Uttar Pradesh, India. The experimental site is located at 25.8978° N latitude and 83.4882° E longitude, with an elevation of approximately 236 feet above sea level. The mean ambient temperature during the cropping period averaged approximately 35 °C. Prior to sowing, composite soil samples were collected from the experimental site to evaluate its physicochemical properties. Soil pH was determined using the method described in [19], and electrical conductivity (EC) was measured as per [20] and expressed in dS m⁻¹. Soil organic carbon was estimated using the Walkley and Black rapid dichromate oxidation method [21]. Available nitrogen was analyzed using a semi-automatic nitrogen analyzer (Pelican Equipments, Chennai, India)

India following the procedure in [22], while available phosphorus was determined using Olsen’s method [23]. Available potassium was measured through flame photometry as described in [24]. Micronutrients, including calcium (Ca), magnesium (Mg), iron (Fe), and zinc (Zn), were quantified by atomic absorption spectrophotometry according to [25].

Preliminary analyses showed minor variations in soil properties between the two seasons; however, these were within normal agronomic ranges and unlikely to affect experimental outcomes.

2.4. Experimental Setup for Field Trials

Field trials were conducted to evaluate the effect of microbial inoculation on black gram under saline soil conditions. Four treatments were tested: T1—inoculation with Bradyrhizobium yuanmingense PR3, T2—inoculation with Paenibacillus sp. SPR11, T3—co-inoculation with PR3 and SPR11 (1:1, v/v), and T4—uninoculated positive control. These treatments were selected based on earlier results showing improved germination, growth, nodulation, and nitrogen fixation under saline nitrogen-free conditions. Seeds were surface-sterilized as previously described and inoculated with the respective bacterial formulations. The experiment was arranged in a randomized block design (RBD) with five replications per treatment, comprising 20 plots in total. Each plot measured 2 m × 2 m with 20 cm spacing between rows and between plots. The field was properly plowed and leveled prior to sowing, and weed management was performed manually without using chemical herbicides.

2.5. Estimation of Physiological Parameters

2.5.1. Chlorophyll Content

Chlorophyll content was estimated non-destructively using an Apogee MC-100 chlorophyll meter (Apogee Instruments, Logan, UT, USA), which provides SPAD (Soil and Plant Analysis Development) index values. Measurements were taken from the fourth fully expanded leaf (counted from the base) of mature black gram plants at 45 days after sowing. For each treatment, SPAD readings were recorded from ten randomly selected leaves, and the average of these values was used to represent the chlorophyll content for that treatment, following the methodology [26].

2.5.2. Carotenoid Content

Carotenoids were extracted following the method described in [27]. Leaf samples (0.1 g) were ground with 80% acetone and then centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 470 nm to quantify carotenoid content. The total carotenoid content was calculated using the following formula:

$$Carotenoid Content \left(\mu g/g\right)={\displaystyle \frac{A \times V \times {10}^{-4}}{{\mathrm{A}}_1{\%}_1\mathrm{c}\mathrm{m}\times P}}$$

where A = Absorbance, V = Total extract volume (mL), A₁%₁cm = 2592 (extinction coefficient), P = Sample weight (g)

2.5.3. Electrolyte Leakage (EL)

Electrolyte leakage (EL) was measured to evaluate cell membrane integrity during the grain-filling stage following the described method [28]. Fresh leaf discs (approximately 0.5 g) were rinsed with deionized water and incubated in 10 mL of distilled water at room temperature for 24 h. The initial electrical conductivity (EC₁) of the bathing solution was measured using a digital EC meter (Labman Scientific Instruments Pvt. Ltd. Chennai, India). Subsequently, the samples were boiled for 20 min to release all electrolytes and cooled to room temperature before recording the final conductivity (EC₂). Electrolyte leakage was calculated using the following formula:

$$EL \left(\%\right) =\left({\displaystyle \frac{{EC}_1}{{EC}_2}}\right)\times 100$$

where

• EC₁ = Conductivity before incubation;
• EC₂ = Conductivity after boiling water incubation.

2.5.4. Proline Content

Proline content in black gram leaves was determined following the described method [29]. Dried leaf samples were finely ground and homogenized in 3% aqueous sulfosalicylic acid. The homogenate was centrifuged at 8000 rpm for 10 min, and the clear supernatant was collected. To 2 mL of the supernatant, 2 mL of acid Ninhydrin and 2 mL of glacial acetic acid were added. The reaction mixture was then incubated in a boiling water bath for 30 min, followed by rapid cooling on ice for 5 min. The resulting mixture was extracted with 4 mL of toluene by vigorous mixing, and the chromophore-containing toluene layer was separated. The absorbance of the toluene extract was measured at 520 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Proline concentration was quantified against a standard curve and expressed on a fresh weight basis.

2.6. Antioxidant Enzyme Activities

2.6.1. DPPH Radical Scavenging Activity

The antioxidant capacity of black gram leaf tissues was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, following the described protocol [30]. For the assay, 1 mL of leaf extract was mixed with 1.98 mL of 80% methanol and 1 mL of 0.3 mM DPPH solution. The reaction mixture was incubated in the dark at room temperature for 30 min to allow scavenging of the DPPH radicals. After incubation, the absorbance was measured at 517 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was analyzed in triplicate. The percentage of DPPH radical scavenging activity was calculated using the following formula:

$$DPPH scavenging activity \left(\%\right)=100\times \left({\displaystyle \frac{Ab sample - Ab blank}{Ab control}}\right)$$

where Ab sample = Absorbance of the test sample, Ab blank = Absorbance of the blank sample, and Ab control = Absorbance of the control containing DPPH and 80% methanol.

2.6.2. Catalase Activity (CAT)

Catalase activity was determined at room temperature by measuring the decrease in absorbance at 240 nm due to the decomposition of hydrogen peroxide (H₂O₂), following the described method [31]. The reaction mixture contained 100 mM sodium phosphate buffer (pH 7.0), 30 mM H₂O₂, and 100 µL of crude enzyme extract, and was brought to a final volume of 3 mL. One unit of catalase activity was defined as the amount of enzyme causing a decrease in absorbance of 0.001 per minute at 240 nm.

2.6.3. Superoxide Dismutase Activity (SOD)

Superoxide dismutase (SOD) activity was determined using a modified method that measures the enzyme’s ability to inhibit the photoreduction of nitro blue tetrazolium (NBT) [32]. The assay mixture (3 mL) consisted of 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 130 mM methionine, 0.75 mM NBT, 0.02 mM riboflavin, and 0.1 mL of enzyme extract. Parallel control samples without enzyme extract—both illuminated and non-illuminated were used as standards. The reaction was initiated by exposure to light, and absorbance was recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of NBT photoreduction. Enzyme activity was expressed as units per milligram of protein.

2.6.4. Estimation of Phenylalanine Ammonia Lyase (PAL)

The enzyme extract (0.1 mL) was mixed with 1 mL of borate buffer (pH 8.8) and 1 mL of L-phenylalanine, then incubated at 37 °C for 60 min. The reaction was stopped by adding 0.1 mL of HCl. The formation of trans-cinnamate was measured spectrophotometrically at 290 nm. Enzyme activity was expressed as µmol trans-cinnamate produced per gram of fresh weight (FW), following the described method [33].

2.6.5. Polyphenol Oxidase Activity Assay (PPO) Activity Assay

Polyphenol oxidase (PPO) activity was determined following the described method with minor modifications [34]. Fresh plant material (2.5 g) was ground in liquid nitrogen using a pre-chilled mortar and pestle. The homogenate was mixed with 5 mL of 0.05 M sodium phosphate buffer (pH 6.0) containing 5% polyvinylpolypyrrolidone (PVPP), then filtered through four layers of muslin cloth and centrifuged at 13,000 rpm for 5 min at 4 °C. For the assay, 1 mL of the enzyme extract (supernatant) was mixed with 2.9 mL of 0.05 M sodium phosphate buffer and 1 mL of 0.1 M catechol. A control was prepared by replacing the extract with 1 mL of sodium phosphate buffer. Absorbance was recorded at 546 nm every 20 s for 4 min. PPO activity was calculated based on the rate of absorbance increase and expressed as enzyme units per microliter per minute (U µL⁻¹ min⁻¹).

2.6.6. Estimation of Peroxidase (POD) Activity

Peroxidase (POD) activity was determined following the described method with minor modifications [35]. An aliquot of 50 µL of crude enzyme extract was mixed with 2.5 mL of a reaction solution containing: 100 mM potassium phosphate buffer (pH 7.0), 1% (v/v) guaiacol, and 3% (v/v) hydrogen peroxide (H₂O₂). The enzymatic reaction was initiated upon the addition of H₂O₂, and the increase in absorbance, corresponding to the oxidation of guaiacol, was recorded at 470 nm using a spectrophotometer. Measurements were taken at 30 s intervals over a period of 5 min. Peroxidase activity was expressed as micromoles of guaiacol oxidized per minute per milligram of protein (μmol min⁻¹ mg⁻¹ protein).

2.7. Collection and Evaluation of Yield Parameters

Five plants from each plot were selected randomly from the middle row to record all agronomic data such as each. Growth parameters measured included root length (cm), shoot length (cm), fresh weight of roots and shoots (g), and both fresh and dry weights of nodules (mg). At the time of harvest, detailed biometric and yield-related data were recorded to assess the overall performance of black gram under different inoculation treatments. Yield attributes were evaluated by recording the number of pods per plant (PD), number of seeds per plant (SN), seed weight per plant (SW, g), grain yield (q ha⁻¹), and straw yield (SY, q ha⁻¹). Grain yield was computed on a per-hectare basis by extrapolating from the net plot area, while total biomass yield was derived by summing grain and straw yields.

2.8. Analysis of Plant Nutrient Uptake and Grain Nitrogen Content in Black Gram

To evaluate the macronutrient and micronutrient content in black gram, including nitrogen (N), phosphorus (P), potassium (K), copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) were collected from each plot during harvesting. A total of 0.5 g of oven-dried and finely ground plant tissue (grain and straw separately) was subjected to acid digestion. The digestion was performed using a di-acid mixture of nitric acid (HNO₃) and hydrochloric acid (HCl) in a 1:3 ratio to ensure complete mineralization of organic matter. Nitrogen content was quantified using the Kjeldahl method with a KEL PLUS Nitrogen Analyzer (Pelican Instruments, Chennai, India). Phosphorus was determined by the Olsen method, which is suitable for neutral to alkaline soils. Potassium concentration was measured using a flame photometer, and micronutrient concentrations (Zn, Cu, Mn, Fe) were analyzed through atomic absorption spectrophotometry (AAS) as per standard protocols [36]. Total nutrient uptake (kg/ha) was calculated by multiplying the concentration of each nutrient (in % or ppm) with the respective dry biomass of grain and straw, followed by conversion to a per-hectare basis.

$$Crude Protein \left(\%\right)=Total Nitrogen \left(\%\right)\times 6.25$$

2.9. Statistical Analysis

All experimental data were expressed as mean values of five replicates. Differences among treatment means were assessed using one-way analysis of variance (ANOVA) at a 95% confidence level. Where significant differences (p < 0.05) were detected, Duncan’s Multiple Range Test (DMRT) was applied for post-hoc comparisons using SPSS software (version 16.0; IBM Corp., Armonk, NY, USA). Since the field experiments were conducted over two consecutive years (2022 and 2023), initial statistical analyses were performed to evaluate year-to-year variability. No significant differences were found in key physiological, growth, or yield parameters between the two years. Consequently, to avoid data redundancy and maintain clarity, results from the 2023 growing season are presented throughout the manuscript, as they are representative of the consistent trends observed across both years.

3. Results and Discussion

3.1. Antagonistic Activity of Paenibacillus sp. SPR11 Against Plant Pathogens

Paenibacillus sp. SPR11 exhibited strong antagonistic activity against two major soil-borne pathogens: Fusarium oxysporum and Macrophomina phaseolina. In vitro dual culture assays revealed that SPR11 inhibited the radial growth of F. oxysporum by 76% and M. phaseolina by 62% (Figure 1), indicating significant biocontrol potential. Importantly, the biocontrol function of SPR11 is particularly valuable under saline conditions, where ionic and osmotic stress often compromise plant immune responses.

In such environments, microbial allies capable of both stress mitigation and pathogen suppression offer a crucial advantage for sustainable crop production. These results are consistent with earlier findings that several Paenibacillus strains, including UV79, suppressed phytopathogenic fungi such as Fusarium oxysporum and Macrophomina phaseolina through lytic enzyme and antibiotic production [16]. Similarly, many Paenibacillus spp. have demonstrated fungal biocontrol efficacy against plant pathogens [37].

Isolates	Inhibition in Dual Plate Assay (%)
	F. oxysporum	M. phaseolina
Paenibacillus sp. SPR11	76%	62%

3.2. Effect of Co-Inoculation on Germination, Nodulation, and Nitrogen Fixation in Blackgram Under Salt Stress (Net House Study)

Co-inoculation of black gram (Vigna mungo) with Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 significantly enhanced germination, early growth, and symbiotic traits under both non-saline and 300 mM NaCl-induced saline conditions. In germination assays, co-inoculated seeds achieved 100% germination in both conditions, outperforming single inoculations and the uninoculated control. Under salt stress, SPR11+PR3 and SPR11 alone exhibited 1.77-fold higher germination rates than the control (100% vs. 56.6%), while PR3 alone showed a 1.24-fold increase.

Root length in co-inoculated seedlings was 13.96-fold greater than in the control, with SPR11 and PR3 alone producing 10.58-fold and 3.31-fold increases, respectively. In net house conditions using nitrogen-free Leonard jars with 300 mM NaCl, co-inoculation enhanced root length by 72.7% over the control and 25.8% over SPR11 alone. Shoot length also increased to 41.37 cm—59.9% higher than the control and 4.8% above SPR11 alone.

Nodulation was absent in the control and PR3-only treatments, while co-inoculated plants developed 61.56 nodules per plant—78.9% more than SPR11 alone. Nitrogenase activity in co-inoculated plants reached 8.32 µmol mg⁻¹ FW h⁻¹, representing a 110% increase over PR3 (3.96), with no detectable activity in SPR11-alone or control treatments (Figure 2 and

Table 1. Biometric observations and measurements of black gram seedlings after 45 days under Nethouse conditions.

Seed Germination Test					Net House Evaluation (300 mM NaCl)
Treatment	Seed Germination (%) Non-Saline Saline		Seed Root Length (cm) Non-Saline Saline		RL (cm)	RFW (mg)	SL (cm)	SFW (mg)	NC (mg)	NFW (mg)	NDW (mg)	NF (μmol/mg FW h−1)
PR3	100%	70%	2.16 c	0.86 c	17.13 c	105.40 c	22.37 c	248.30 c	13.00 b	106.37 b	9.54 b	3.96 b
SPR11	100%	100%	3.92 b	2.75 b	31.25 b	312.23 b	39.47 a	417.57 b	0.00 c	0.000 c	0.00 c	0.00 c
PR3 + SPR11	100%	100%	5.95 a	3.63 a	39.30 a	386.24 a	41.37 a	553.20 a	61.56 a	723.23 a	66.87 a	8.32 a
Control	86%	56.56%	1.88 cd	0.26 cd	15.17 cd	89.52 d	19.57 d	221.54 cd	0.00 c	0.00 c	0.00 c	0.00 c

Note: The table presents the effects of different treatments (PR3, SPR11, PR3 + SPR11, and Control) on seed germination (%) under non-saline and saline (300 mM NaCl) conditions, as well as on key growth parameters evaluated in a net house. Measured parameters include root length (RL), root fresh weight (RFW), shoot length (SL), shoot fresh weight (SFW), nodule count (NC), nodule fresh weight (NFW), nodule dry weight (NDW, mg), and nitrogenase activity (NF). Data are means of three replicates (n = 3). Different letters within each column indicate statistically significant differences according to Duncan’s multiple range test at p < 0.05.

).

These results are consistent with previous reports that salt stress (100–150 mM NaCl) impairs black gram germination and nitrogen fixation [38,39]. In contrast, our co-inoculation strategy mitigated these negative effects, significantly enhancing plant performance. These findings support an earlier study where the same bacterial combination showed synergistic effects under saline, nitrogen-free conditions [17].

The observed improvements are likely due to Paenibacillus sp. SPR11 plant growth-promoting mechanisms, including IAA production, ACC deaminase activity, EPS secretion, and the solubilization of phosphorus, potassium, and zinc [17]. Supporting evidence showed similar benefits of co-inoculation in alfalfa, where Sinorhizobium meliloti GL1 combined with Enterobacter ludwigii doubled growth and nitrogen fixation under saline conditions, further underscoring the potential of microbial consortia in salinity management [40].

3.3. Soil Analysis

Prior to the field trials, the physicochemical properties of the experimental soil were assessed to determine baseline fertility and salinity status. The soil was classified as saline clay loam, with a moderately alkaline pH (8.37) and an electrical conductivity (EC) of 8.87 dS m⁻¹, indicating strong salinity. The analysis revealed low organic carbon content (0.60–0.80%) and deficient levels of available macronutrients, including phosphorus (2.87–3.29 kg ha⁻¹), potassium (48.56–51.88 kg ha⁻¹), and nitrogen (3.23–4.67 kg ha⁻¹). Micronutrient concentrations were also generally low, with iron ranging from 0.020 to 0.23 kg ha⁻¹, calcium between 4.04 and 6.06 kg ha⁻¹, magnesium at 20.20–20.21 kg ha⁻¹, and zinc at 0.073–0.56 kg ha⁻¹.

These results align with previous observations of constrained nutrient availability and moderate salinity under similar field conditions, including low organic carbon (0.16–0.36%), low available nitrogen (186 kg ha⁻¹), low phosphorus (23.5 kg ha⁻¹), moderate potassium, and nutrient deficiencies in the Jaunpur district of eastern Uttar Pradesh [41].

3.4. Effect of Co-Inoculation on Biochemical Parameters in Blackgram Under Saline Stress

Co-inoculation of black gram with Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 significantly enhanced biochemical responses under saline and low-nutrient field conditions. Proline accumulation increased by 50.89% over the control, exceeding the increases observed with SPR11 (22.19%) and PR3 (47.81%) alone (Figure 3A). Chlorophyll (SPAD) and carotenoid contents increased by 54.74% and 41.17%, respectively, compared to the uninoculated control (Figure 3B,D). Electrolyte leakage was reduced by 55.77%, reaching 23%, which was also lower than in single inoculations (Figure 3C).

These findings are supported by previous reports showing that 100 mM salinity significantly reduces chlorophyll and carotenoids while increasing protective metabolites such as proline [42]. Similarly, salinity disrupts membrane integrity by displacing calcium from the plasma membrane, leading to higher electrolyte leakage; however, inoculation with Bacillus flexus helps maintain chlorophyll content and membrane stability under salt stress [43].

Our results demonstrate that co-inoculation with PR3 and SPR11 effectively mitigated salt-induced damage by promoting osmotic adjustment, preserving photosynthetic pigments, and protecting cellular integrity, outperforming single inoculants and uninoculated controls. These observations further support the role of compatible microbial consortia in enhancing salinity tolerance in black gram.

3.5. Effect of Co-Inoculation on Antioxidant Activities in Black Gram Under Field Conditions

Co-inoculated black gram plants exhibited the highest DPPH radical scavenging activity, showing a 1.50-fold increase over the control and 14.5% and 42.9% higher than SPR11 and the control, respectively (Figure 4A). This suggests enhanced non-enzymatic antioxidant potential, likely due to increased phenolic and flavonoid content. Peroxidase (POD) activity was significantly reduced (0.47-fold of the control), with values 30.5% and 52.7% lower than SPR11 and the control, respectively (Figure 4B), indicating reduced oxidative stress.

Catalase (CAT) activity, essential for hydrogen peroxide detoxification, was 3.46-fold higher in co-inoculated plants than in the control, surpassing levels in SPR11 (2.24-fold) and PR3 (2.07-fold) (Figure 4C), reflecting improved redox homeostasis. Phenylalanine ammonia-lyase (PAL) activity, associated with phenolic biosynthesis and systemic resistance, increased by 1.38-fold over the control (Figure 4D). Superoxide dismutase (SOD) activity also peaked in co-inoculated plants (2.21-fold increase), with 10% and 28.5% higher levels than SPR11 and PR3, respectively (Figure 4E), indicating efficient reactive oxygen species (ROS) detoxification.

Conversely, polyphenol oxidase (PPO) activity, which is often linked to oxidative damage, was lowest in co-inoculated plants (0.45-fold of the control), showing reductions of 32.8% and 53.5% compared to SPR11 and the control, respectively (Figure 4F). This suggests improved regulation of oxidative metabolism.

These findings are supported by earlier studies showing that Bacillus firmus SW5 enhanced antioxidant enzyme activity under salt stress in soybean, contributing to ROS scavenging [44]. Similarly, inoculation with HSNJ4 increased SOD, POD, and CAT activities in chickpea [45], while Planococcus sp. (ST3) improved FRAP, DPPH, and SOS antioxidant parameters under salinity conditions [46], consistent with our results.

3.6. Collection and Analysis of Yield Parameters

Co-inoculation with Bradyrhizobium yuanmingense PR3 and Paenibacillus sp. SPR11 significantly enhanced black gram growth, nodulation, and yield under salt-affected field conditions. Root length increased to 27.71 cm, exceeding PR3, SPR11, and control treatments by 10.9%, 1.4%, and 7.2%, respectively. Root fresh weight increased by 57.9% over the control. Shoot length and fresh weight reached 42.54 cm and 469.66 mg, marking improvements of 45.9% and 44.1%, respectively.

Co-inoculated plants formed 31.71 nodules per plant, and nodule fresh and dry weights rose by over 80% relative to the uninoculated control (Figure 5 and

Table 2. Biometric observations and measurements of black gram plants at the time of harvest under field conditions.

Treatment	RL (cm)	RFW (mg)	SL (cm)	SFW (mg)	NC (mg)	NFW (mg)	NDW (mg)	PD (Plant−1)	SN (Plant−1)	SW (g Plant−1)	Grain yield (q ha1)	SY (q ha1)	Total biomass (q ha1)
PR3	24.68 bc	124.93 c	26.40 c	293.44 c	8.00 bc	24.66 b	7.18 b	17.67 b	82.23 c	6.37 bc	4.82 bc	8.29 bc	13.11 c
SPR11	27.33 a	223.70 b	38.34 b	437.70 b	9.32.00 b	21.300 bc	8.37 b	18.12 b	126.87 b	7.26 b	5.68 b	9.72 b	18.40 b
PR3 + SPR11	27.71 a	284.00 a	42.54 a	469.66 a	31.71 a	158.70 a	31.34 a	32.54 a	242.67 a	11.38 a	8.18 a	15.55 a	26.73 a
Control	25.72 b	119.60 cd	23.00 d	262.71 cd	5.62.00 c	15.23 c	4.97 c	12.31 c	61.57 d	5.86 bc	3.91 c	3.57 c	11.48 d

Note: Table showing the effects of different treatments (PR3, SPR11, PR3+SPR11, and control) on growth, nodulation, and yield-related parameters of black gram under field conditions. Measured parameters include root length (RL), root fresh weight (RFW), shoot length (SL), shoot fresh weight (SFW), nodule count (NC), nodule fresh weight (NFW), nodule dry weight (NDW), number of pods per plant (PD), number of seeds per plant (SN), seed weight per plant (SW), grain yield, straw yield (SY), and total biomass. Data are presented as means (n = 5) and different letters within columns denote statistically significant differences according to Duncan’s multiple range test at p < 0.05.

).

Yield attributes improved significantly with co-inoculation. Treated plants produced an average of 32.54 pods and 242.67 seeds per plant, with a seed weight of 11.38 g—representing a 74.6% increase over the control. Grain yield rose to 15.55 q/ha, marking a 77% improvement, while straw and total biomass yields reached 8.18 q/ha and 26.73 q/ha, respectively. Additionally, total nitrogen and seed protein content increased by 23.9% compared to the control, indicating enhanced nitrogen fixation and assimilation under saline conditions (Table 2).

These findings align with previous studies demonstrating the benefits of microbial co-inoculation under stress conditions. Co-inoculation of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 enhanced growth, nodulation, nitrogen fixation, and seed yield in soybean [47]. Similarly, co-inoculation of Paenibacillus polymyxa with Bacillus megaterium improved nodulation in common beans, outperforming single rhizobial treatments [48]. In chickpea, co-inoculation with Rhizobium and Azotobacter enhanced shoot and root biomass, nodulation, nitrogen content, and seed protein [49].

3.7. Analysis of Plant Nutrient Uptake and Grain Nitrogen Content in Black Gram

Grain nitrogen content in co-inoculated black gram increased by 52.45% compared to the PR3 treatment, 46% over SPR11, and 47.32% relative to the control. Grain protein content reached 28%, reflecting respective increases of 26.05%, 16%, and 18.5% compared to PR3, SPR11, and the control, respectively (Figure 6A). Total nitrogen uptake rose to 33.56 kg/ha—an increase of 48.27% over the control—and surpassed the PR3 and SPR11 treatments by 42.31% and 33.76%, respectively. Co-inoculation also significantly enhanced phosphorus and potassium uptake, reaching 3.57 kg/ha and 7.61 kg/ha, which were 50.49% and 63.38% higher than the control, respectively (Figure 6B). Overall, co-inoculation consistently outperformed both individual inoculants (PR3 and SPR11) across all macronutrient parameters.

Micronutrient uptake showed a similar trend. Co-inoculated plants exhibited significantly higher concentrations of copper (41.98 ppm), zinc (57.74 ppm), manganese (54.92 ppm), and iron (254.46 ppm) compared to both single inoculations and the uninoculated control (Figure 6C). These findings align with prior research demonstrating that microbial consortia enhance nutrient uptake and stress resilience. Improved yield and nutrient accumulation in legumes co-inoculated with rhizobia and PGPR strains such as Pseudomonas and Bacillus have been reported [50]. Increased macro- and micronutrient levels under both normal and drought conditions were observed with Bradyrhizobium japonicum USDA110 and Pseudomonas putida NNU8 co-inoculation [51]. Similarly, enhanced nodulation, nitrogen content, and grain yield were found in legumes co-inoculated with Rhizobium and Azotobacter chroococcum [52]. A tetrapartite symbiosis involving rhizobia, mycorrhiza, and Stenotrophomonas improved nutrient acquisition in chickpea under saline and nutrient-deficient conditions [53].

These findings are consistent with our earlier study [17], in which the same microbial combination demonstrated strong synergistic effects on seedling growth, nodulation, and nitrogen uptake under controlled saline conditions. The current field-level validation confirms and extends those results, demonstrating the practical potential of SPR11 and PR3 co-inoculation for managing salinity stress in black gram cultivation.

4. Conclusions

This study highlights the practical use of co-inoculation with compatible, salt-tolerant microbes as an effective means of alleviating salinity stress on the legume-rhizobia symbiosis in black gram. Our work demonstrated significant nodulation and nitrogen fixation ability, nutrient uptake and use, physiological tolerance through improved chlorophyll content, proline accumulation, and antioxidant enzyme activities (SOD, CAT), reduced electrolyte leakage, and enhanced DPPH radical scavenging and grain yield for black gram inoculated with Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 under managed, and uncontrolled, saline field conditions without the use of synthetic nitrogen inputs. The enhanced nodulation and nitrogenase activity observed with co-inoculation serve as a promising eco-friendly alternative to chemical nitrogen fertilizers, supporting plant nutrition and yield without external nitrogen inputs. The productive performance of the Paenibacillus sp. SPR11 and Bradyrhizobium yuanmingense PR3 consortium illustrate the possibilities of functional microbial partnerships for stress tolerance and sustainable crop yield in soils degraded by salinity. With the urgency to develop sustainable ways forward for low-input and salinity-affected agroecosystems, this co-inoculation-based approach provides a cost-effective and scalable solution to improve soil health and legume production and establishes a strong and environmentally friendly alternative to chemical fertilizers.