Synergistic Rhizobium–PSB Consortium (NRA1–JCA-5) Optimizes Groundnut Productivity and Nutrient Use Efficiency (NUE) under Reduced Fertilization

¹Department of Agronomy, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, ²Department of Agriculture, Government of West Bengal

Amrita Sengupta¹²* ; Sunil Kumar Gunri¹

Nadia, West Bengal 741252, India

Corresponding* Author’s email: amritawork88@gmail.com

Abstract

Excessive synthetic fertilizer use undermines soil health, profitability, and sustainability in legume production. Here we validated a novel Rhizobium sp. NRA1 (99.2% identity to R. phaseoli; GenBank PP355674)–PSB (Phosphate solubilizing bacteria) Priestia megaterium JCA-5 (99.6% identity to P. megaterium; GenBank PP809390) consortium enabling 25% N-P fertilizer reduction in groundnut field trials while maintaining yields and enhancing nutrient efficiency. Two-year randomized block trials (loamy Inceptisol) demonstrated T₉ (Novel Rhizobium PSB consortium + 75% N-P + 100% K) achieved yield parity with 100% NPK (3679 kg ha⁻¹ vs. 3854 kg ha⁻¹ predicted), superior agronomic efficiency (AE: 23.76 kg yield kg⁻¹ nutrient), partial factor productivity (PFP: 36.79 kg kg⁻¹), and highest profitability (B:C 3.05). Enhanced nodulation (NEI > 640%), seed quality (oil 45.6%, protein 26.1%), and soil nutrient retention confirmed synergistic N₂ fixation and P solubilization. Multivariate analyses (heatmap–dendrogram) revealed mechanistic complementarity, with T₉ optimizing carbon partitioning and resource use. This field-validated consortium establishes a scalable model for fertilizer substitution, advancing climate-smart legume intensification.

Keywords:

Rhizobium consortium

fertilizer reduction

groundnut

nutrient-use efficiency

field validation

sustainable agriculture

1. Introduction

Legumes constitute a cornerstone of sustainable agriculture through biological nitrogen fixation (BNF), yet suboptimal soil nutrient status frequently constrains symbiotic efficiency (Abd-Alla et al., 2023; Owaresat et al., 2023). Groundnut (Arachis hypogaea L.), a pivotal oilseed legume contributing 25% of global edible oil supply, exemplifies this challenge, demanding balanced N-P nutrition for optimal productivity, seed quality, and economic viability (Brar & Manhas, 2023; Kamal et al., 2024). Its nutrient-dense composition—harboring 25–30% protein, 45–50% healthy fats, and essential minerals—positions groundnut as indispensable for food security, malnutrition alleviation, and smallholder livelihoods across developing regions (Drewnowski & Conrad, 2024; Rajput et al., 2024; Tabe-Ojong et al., 2023). Nevertheless, excessive synthetic fertilizer dependence escalates production costs, accelerates soil degradation, and amplifies N₂O emissions (constituting 6% of global greenhouse gases), compelling innovative biofertilizer interventions that optimize nutrient-use efficiency while safeguarding soil health (Kurniawati et al., 2023; Kvakkestad et al., 2023; Shahzad et al., 2025).

Rhizobium-phosphate-solubilizing bacteria (PSB) consortia deliver synergistic N₂ fixation and P mobilization (Rabani et al., 2023), yet field-scale fertilizer substitution remains the definitive performance metric. Thilakarathna and Raizada (2017) synthesized 28 field trials, documenting 17% average yield increments under reduced N regimes, with cowpea (61.7%) and groundnut (19.8%) registering highest responses. Buernor et al. (2022) analyzed 62 grain legume studies, confirming 13–25% N fertilizer savings attributable to strain-specific inoculation efficacy. Empirical field validations substantiate extremes: Wei et al. (2023) achieved 33–69% N reduction in soybean through Bradyrhizobium japonicum, sustaining yields via enhanced nodulation and soil pH stabilization; Gueguen et al. (2022) demonstrated 25–50% P substitution in faba bean, attaining record grain yields (4.4 t ha⁻¹); Mbaye et al. (2025) validated 84% total chemical fertilizer displacement in Senegalese common bean with comparable productivity. Native strains exhibit superior long-term stability over commercial inoculants (Castellano-Hinojosa et al., 2022).

Although microbial isolation and agronomic evaluations proliferate independently, integrated investigations—from novel strain isolation and phylogenetic authentication (GenBank PP355674, PP809390) through multi-year field fertilizer substitution—represent exceptional rarities. Commercial inoculants manifest erratic field efficacy owing to edaphic maladaptation (Hossain et al., 2023; Ibáñez et al., 2023; Symanczik et al., 2023), compelling development of site-attuned, ecologically robust consortia (Baraza et al., 2024; Guardiola-Márquez et al., 2023; Khan et al., 2023; Souza et al., 2025). Indigenous microbes consistently surpass exotic counterparts across soil gradients (Fadiji et al., 2024; Zhou et al., 2024), yet holistic validation integrating phylogenetics, multivariate mechanisms, and economic metrics remains virtually absent.

The present investigation redresses this deficiency via systematic isolation, molecular characterization, and two-year randomized block field trials on loamy Inceptisol, validating novel Rhizobium sp. NRA1 (99.2% R. phaseoli)– PSB Priestia megaterium JCA-5 (99.6% P. megaterium) consortium efficacy. Pairing these bioinoculants with a 25% reduction in chemical N and P fertilizers elicited substantial gains in pod yield, kernel quality, and economic viability, while attenuating reliance on synthetic inputs. In contrast to antecedent studies restricted to commercial inoculants or greenhouse assays, this research delineates a scientifically rigorous and economically pragmatic paradigm for augmenting productivity and nutrient-use efficiency via indigenous microbial strains.

The investigation also carries global import, proffering a replicable, low-input paradigm to elevate legume productivity across heterogeneous agroecologies. By substantiating that native, genetically authenticated Rhizobium–Priestia bioinoculants sustain yields under curtailed fertilizer application, it propels the paradigm shift toward climate-adaptive, resource-efficient agriculture—in consonance with global imperatives for carbon-neutral crop production, soil vitality restoration, and diminished synthetic fertilizer dependency.

2. Methods

2.1 Strain Isolation, Screening, and Characterization

Native Rhizobium and phosphate-solubilizing bacterial (PSB) strains were isolated from groundnut nodules and rhizosphere soil sampled across varied sub tropical agro-ecosystems, encompassing acidic lateritic soils (pH 4.8–5.6, organic C < 0.4%, available P < 10 kg ha⁻¹), calcareous red soils (pH 6.5–7.2, high Fe/Al-P fixation), saline coastal alluvium (pH 7.8–8.4, EC 2–4 dS m⁻¹), and loamy Inceptisols (pH 6.95, N-P₂O₅-K₂O: 205-15-105 kg ha⁻¹; Supplementary Table A1). These soil gradients reflect semi-arid and humid tropical and subtropical constraints prevalent in 70% of global groundnut cultivation areas.

Pure cultures were maintained on yeast extract mannitol agar (YEMA) for Rhizobium and Pikovskaya's agar for PSB at 28°C. All isolates were screened for their biochemical properties at laboratory and under controlled pot culture conditions for nodulation efficiency, phosphorus solubilization, and plant growth promotion. Based on superior performances (Sengupta et al., 2024),(Sengupta et al., 2018) two strains were selected for field testing: Rhizobium sp. NRA1 (GenBank PP355674; 99.2% identity to Rhizobium phaseoli ATCC 14482, NR_044112.1) and Priestia megaterium JCA-5 (PP809390; 99.6% identity to P. megaterium NBRC 15308, NR_112636.1). Liquid inoculants were prepared at 10⁸ CFU mL⁻¹ (verified by plate dilution) and used within 48 h.

2.2 Experimental Design and Crop Management

Ten integrated nutrient management treatments were evaluated in a randomized block design with four replicates (4 × 5 m plots, 30 × 10 cm spacing; 33,333 plants ha⁻¹) over two consecutive summer seasons on a moderately fertile loamy Inceptisol (Supplementary Table A1) under subtropical humid conditions (annual rainfall 1530 mm, mean temperature 25.8°C).

Groundnut cv. TG-51 (high-yielding, rainfed-adapted) received recommended dose fertilizer (RDF: 20-60-40 kg N-P₂O₅-K₂O ha⁻¹ via urea, single superphosphate, muriate of potash Treatment structure and description of integrated nutrient management combinations evaluated under field conditions is as follows.

(Details of all ten nutrient management treatments combining Rhizobium NRA1, Priestia megaterium JCA-5, and graded fertilizer levels.)

Treatment	Description
T1	Control
T2	Rhizobium inoculation (NRA1)
T3	PSB inoculation (JCA5)
T4	Rhizobium + PSB (Consortium of NRA1 and JCA5)
T5	50% Recommended N & P + 100% K
T6	75% Recommended N & P + 100% K
T7	100% Recommended NPK
T8	T4 + 50% NP + 100% K
T9	T4 + 75% NP + 100% K
T10	T4 + 100% NPK

Seeds were surface-sterilized (0.1% HgCl₂, 1 min), rinsed (5× sterile water), and coated with inoculants (5 mL kg⁻¹ seed). Coated seeds were shade-dried (30 min) before sowing. Supplementary soil drenches (100 mL plot⁻¹, 10⁸ CFU mL⁻¹) were applied at 15–20 days after sowing (DAS) and at flowering stage. Nitrogen was split-applied twice (50% basal, 50% at 30 DAS); P and K were basal. Uniform management included critical-stage irrigation, pre-emergence herbicide application (pendimethalin 1 kg a.i. ha⁻¹), manual weeding with earthing up after gypsum application, and integrated pest control as per institutional protocols.

2.3 Soil, Nutrient, and Economic Analyses

Composite soil samples (0–15 cm depth, 10 cores plot⁻¹) were collected pre-sowing and post-harvest, air-dried, sieved (2 mm), and analyzed for available nutrients: N by alkaline KMnO₄ distillation, P₂O₅ by Olsen's method (0.5 M NaHCO₃, colorimetric), K₂O by 1 N NH₄OAc extraction (flame photometry), and organic C by Walkley–Black wet digestion. Nutrient-use efficiencies were computed as agronomic efficiency (AE = (Yₜ – Y₀)/Fₜ) and partial factor productivity (PFP = Yₜ/Fₜ), where Yₜ is treatment yield, Y₀ is control yield, and Fₜ is total nutrient applied (kg ha⁻¹).

Nodule Efficiency Index (NEI)

To quantify the relative enhancement of symbiotic performance under varying nutrient regimes, the Nodule Efficiency Index was calculated using the absolute control (T₁) as a baseline. The index represents the percent increase in nodule biomass relative to the untreated control and was computed as follows

NEI (%) =	Nodule Dry Weight _Treatment	X 100
	Nodule Dry Weight _Control

This metric allows for a standardized comparison of how the microbial consortium (NRA1–JCA-5) optimizes nodule development even under curtailed synthetic fertilizer applications.

Economic returns were computed using existing market prices (groundnut pods: ₹35 kg⁻¹; haulm: ₹2 kg⁻¹) as follows: gross returns = (pod yield × ₹35) + (haulm yield × ₹2); net returns = gross returns − total cultivation costs; benefit:cost (B:C) ratio = gross returns / cultivation costs.

2.4 Molecular Characterization and Statistical Analysis

Genomic DNA was extracted via CTAB protocol from 48-h cultures. The 16S rRNA gene (~ 1.5 kb) was amplified using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), purified, and sequenced bi-directionally (Sanger). Sequences were assembled (BioEdit v7.2), aligned (ClustalW), and identified via NCBI BLASTn. Phylogenetic trees were constructed by neighbor-joining (MEGA X; Kimura-2-parameter model; 1,000 bootstraps, nodes > 70% retained).

Pooled two-year data (n = 4 replicates treatment⁻¹) underwent ANOVA for randomized block design (SPSS v.27). Variance homogeneity was verified (Levene's test, P > 0.05) prior to pooling. Treatment means were separated by Duncan's Multiple Range Test (DMRT) at P ≤ 0.05. Figures report means ± standard error of the mean (SEm).

3. Results

3.1. Strain Identity and Edaphic Context

Phylogenetic analysis of 16S rRNA sequences confirmed the isolates as Rhizobium sp. NRA1 (GenBank PP355674; 99.2% identity to R. phaseoli) and Priestia megaterium JCA-5 (PP809390; 99.6% identity), establishing them as native, edaphically adapted strains (Plate 1). The experimental site featured a loamy Inceptisol characterized by suboptimal fertility, representing a typical phosphorus-limited agroecosystem.

3.2 Fertilizer Substitution Efficacy (25% N-P Reduction) and Yield Equivalence

Co-inoculation with Rhizobium–Priestia consortium fully compensated 25% synthetic N-P fertilizer reduction (T₉: 75% N-P + 100% K + Co-I), achieving pod yield equivalence to 100% NPK alone (3679 ± 40 vs 3513 ± 48 kg ha⁻¹; P > 0.05; LSD(0.05) = 164 kg ha⁻¹) across two Summer seasons [Plate 2; Table 1]. T₉ demonstrated superior agronomic efficiency (AE: 23.76 ± 0.3 kg yield kg⁻¹ nutrient) versus 100% NPK alone (19.25 ± 0.5 kg kg⁻¹; +23%) and maximal economic returns (B:C ratio 3.0 ± 0.05 versus 2.8 ± 0.05; +7%).

Table 1

Concise summary of key agronomic and economic parameters of groundnut.
Treatment	Description	Pod Yield (kg ha⁻¹)	AE (kg yield kg⁻¹ nutrient)	B:C Ratio
T₁	Absolute Control	2456 ± 89ᵉ	N/A	1.0 ± 0.02ᵉ
T₇	100% NPK only	3513 ± 48ᵇ	19.25 ± 0.5ᶜ	2.8 ± 0.05ᵇ
T₈	50% NPK + Co-I	3384 ± 42ᵇ	25.00 ± 0.4ᵃ	2.9 ± 0.05ᵃᵇ
T₉	75% NPK + Co-I	3679 ± 40ᵃ	23.76 ± 0.3ᵃ	3.0 ± 0.05ᵃ
T₁₀	100% NPK + Co-I	3675 ± 43ᵃ	20.75 ± 0.3ᵇᶜ	3.0 ± 0.05ᵃ

3.3 Economic Viability, Resource Optimization, and Fertilizer Responsiveness

Integrated co-inoculation strategies significantly enhanced profitability and nutrient-use efficiency (P < 0.05) [Plate 2]. T₉ and T₁₀ co-inoculated treatments attained highest B:C ratios (3.0 ± 0.05), with T₈ exhibiting peak agronomic efficiency (AE: 25.00 ± 0.4 kg kg⁻¹ versus T₇ 19.25 kg kg⁻¹; P < 0.05). Cubic regression modeling (R² = 0.918) confirmed microbial consortium fundamentally altered fertilizer responsiveness [Plate 2], expressed as:

y = − 16.61x ³ + 254.9x² − 688.1x + 1484

This model demonstrates T₉ optimizes production at reduced synthetic inputs (8.63 nutrient units), enabling 25% N-P fertilizer substitution while maintaining maximal economic returns and establishing field-scale thresholds for microbial intervention.

3.4 Symbiotic-Yield Mechanistic Linkage

Nodulation performance exhibited strong linear correlation with pod yield (r = 0.95; n = 48; P < 0.001) across treatments [Plate 3]. T₉ maximized nodule efficiency index (646.7% relative to T₁ control) and nodule biomass (42.81 mg plant⁻¹), confirming biological N₂ fixation as the primary mechanism compensating synthetic N reduction (detailed parameters: Supplementary Table S2).

3.5 Seed Quality Under Optimized Management

Co-inoculated reduced-input regime maintained superior seed quality without yield dilution [Plate 3]. T₉ achieved oil content (45.6 ± 1.2%) and protein (26.1 ± 0.8%) comparable to full NPK (P > 0.05) while reducing input costs by 25% (comprehensive attributes: Supplementary Table S3).

3.6 Soil Fertility Maintenance

Post-harvest soil nutrients in T₉ (N: 209.6 ± 2.5; P₂O₅: 16.2 ± 0.2; K₂O: 119.6 ± 1.5 kg ha⁻¹) matched T₇, confirming fertility sustainability under reduced inputs (Plate 3; Supplementary Table S5).

3.7 Multivariate Treatment Profiling

Heatmap-dendrogram ordination (28 variables; Euclidean distance, Ward's linkage) clustered T₉ with highest AE/PFP/B:C, distinct from fertilizer-only controls (Plate 3). 16S rRNA phylogeny authenticated strains (NRA1: 99.2% R. phaseoli; JCA-5: 99.6% P. megaterium; Fig. 6; Supplementary Table S4).

4. Discussion

The co-inoculation of Rhizobium sp. NRA1 and Priestia megaterium JCA-5 enabled a 25% substitution of synthetic N-P fertilizer while achieving yield parity with the recommended dose of fertilizers (RDF). Treatment T₉ (75% NP + Consortium) recorded a pod yield of 3679 ± 40 kg ha⁻¹, effectively matching the performance of the full chemical control (T₇). This study establishes that the biological "yield ceiling" can be shifted toward lower chemical input intensities through targeted microbial intervention, optimized here at a calculated threshold of 8.63 nutrient units based on cubic modeling (R² = 0.918).

4.1 Fertilizer Substitution and Yield Stability Mechanisms

The ability of co-inoculation to compensate for a 25% reduction in synthetic inputs is primarily attributed to enhanced symbiotic efficiency and rhizosphere priming. We observed a highly significant linear correlation (r = 0.95) between nodule dry weight and pod yield [Plate 3], suggesting that the biological nitrogen credit provided by NRA1 was the dominant driver of productivity in reduced-input plots. The Nodule Efficiency Index (NEI) of T₉ (646.7% relative to control) confirms that the consortium maximized the metabolic output of individual nodules.

Furthermore, the yield stability across two seasons (CV = 0.8% for T₉) suggests that the microbial synergy between Rhizobium and the phosphate-solubilizing P. megaterium provides a buffer against edaphic stressors. This response exceeds typical gains from single PGPR applications (Sharma et al., 2013), likely due to the dual-action mechanism of atmospheric N₂ fixation and mobilized P-acquisition, which aligns with boundary line theory for nutrient-limited Inceptisols.

Rhizosphere Priming Mechanism

Beyond direct nutrient acquisition, the observed yield stability in T₉ is likely driven by microbial-mediated rhizosphere priming. The co-inoculation of Rhizobium sp. NRA1 and P. megaterium JCA-5 appears to stimulate a "synergistic priming effect. In this process, the bacterial consortium likely secretes low-molecular-weight organic acids and signaling molecules that trigger the release of locked-up soil carbon and phosphorus.

This microbial activity enhances root exudation, which in turn supports a more diverse and active native microbial community. By altering the biochemical niche of the rhizosphere, the consortium facilitates a more rapid turnover of organic matter, thereby sustaining nutrient availability during critical pod-filling stages despite the 25% reduction in mineral fertilizer inputs. This mechanism explains the distinct clustering of T₉ in the multivariate ordination, confirming that the integrated strategy fundamentally re-engineers the rhizosphere for higher efficiency rather than merely supplementing nutrients.

4.2 Economic and Agronomic Efficiency Superiority: The T₈ vs. T₉ Trade-off

A critical finding of this study is the divergence between the Efficiency Peak and the Production Peak. While T₉ (75% NP) achieved the highest absolute yield and a maximal B:C ratio of 3.05, the highest Agronomic Efficiency (AE) was recorded in T₈ (50% NP) at 25.00 kg yield kg⁻¹ nutrient.

This establishes a clear economic frontier:

For Profit Maximization (T ₉ ): A 25% fertilizer reduction maintains top-tier yields and maximizes net returns.

For Resource-Limited Scenarios (T ₈ ): A 50% substitution is scalable, offering the highest efficiency per unit of input, which is vital where fertilizer costs comprise up to 40% of production expenses (FAO, 2022).

These results prove that the consortium doesn't just "add" yield but "optimizes" the response curve, allowing farmers to choose between maximum output or maximum efficiency [Plate 2].

4.3 Soil Fertility Sustainability and Long-term Implications

Multivariate ordination of 28 variables [Plate 3] clustered T₉ distinctly from fertilizer-only controls, highlighting a fundamental shift in the soil-plant-microbe relationship. Unlike continuous chemical fertilization, which often leads to "fertility mining" or nutrient depletion (Lal, 2020), the post-harvest soil analysis showed that T₉ maintained nutrient equivalence with T₇ (P > 0.05). The preservation of Olsen-P (16.2 kg ha⁻¹) and Available N (209.6 kg ha⁻¹) confirms that Priestia phosphatase activity and Rhizobium residue mineralization effectively replenished the soil nutrient pool despite lower mineral inputs.

4.4 Strain Specificity and Edaphic Adaptation

The superiority of the NRA1–JCA-5 consortium over traditional reference strains is attributed to their native origin and adaptation to the loamy Inceptisols of the study site (Initial N: 205 kg ha⁻¹; P: 15 kg ha⁻¹). Molecular authentication (99.2% identity for NRA1 and 99.6% for JCA-5) confirms their taxonomic placement within the Rhizobium phaseoli and Priestia megaterium clades [Supplementary Table A2]. The ability of NRA1 to enhance nodulation under phosphorus limitation—complemented by JCA-5’s IAA and phosphatase production—creates a synergistic effect that single-strain applications often fail to achieve in field conditions (Vejan et al., 2016).

4.5 Implications for Sustainable Groundnut Production

This field-scale validation provides a proof-of-concept for the systematic reduction of synthetic N-P fertilizers by 25%. By saving approximately 20–30 kg N ha⁻¹ without sacrificing profitability, this strategy directly addresses the environmental concerns of nitrate leaching and phosphate runoff. While these results are robust for the studied Inceptisol, future research using ¹⁵N isotope dilution is recommended to quantify the exact percentage of plant nitrogen derived from atmosphere, and to validate these microbial intervention thresholds across diverse agro-climatic zones.

Conclusion

The field-scale validation of the indigenous Rhizobium sp. NRA1–Priestia megaterium JCA-5 consortium confirms that high-productivity groundnut farming is achievable with significantly reduced chemical dependency. By establishing a precise threshold for microbial intervention, this research provides a definitive roadmap for substituting 25% of synthetic N-P inputs with high-efficiency bio-inoculants while ensuring crop yield and seed quality remain intact. This integrated strategy not only preserves soil fertility in phosphorus-limited environments but also secures a high-return pathway for resource-constrained farmers, shifting the focus from purely chemical-based systems to biologically augmented agriculture. The performance of these native strains underscores the strategic necessity of utilizing edaphically adapted microbial solutions to enhance resource-use efficiency and environmental sustainability. Consequently, the transition of this technology into mainstream agricultural practice now requires prioritizing the development of stable liquid bioformulations and conducting expanded regional trials to standardize adoption across diverse legume-based cropping systems.

Author Contribution

Amrita Sengupta worked as a research worker for the experiment and drafted the manuscript and submitted the manuscript for publication. Sunil Kumar Gunri acted as chairman (guide) to Amrita Sengupta during the course of the research experiments.

Funding

declaration: The study didn’t receive any specific funding.

Electronic Supplementary Material

Below is the link to the electronic supplementary material

Supplementary Material 1

Data Availability

All field experiment data supporting the findings of this study are available within the paper and its Supplementary Information.Nucleotide partial sequence data were deposited into the Gene Expression NCBI database under accession number PP355674 and PP809390 that are available at the following URL: https://www.ncbi.nlm.nih.gov/nuccore/PP355674 and https://www.ncbi.nlm.nih.gov/nuccore/PP809390.1/

Acknowledgement

The contributions of colleagues and mentors, whose insightful feedback and suggestions enriched this research, are gratefully acknowledged.

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