The use of inoculants to improve grain legume yield and quality is a key strategy for sustainable agriculture, reducing reliance on synthetic nitrogen fertilizers (Rani et al., 2019). This practice Supports Development Goals (SDGs) 2, 13 and 15 by enhancing food security, climate resilience and biodiversity (Stagnari et al., 2017). Although nitrogen is essential for plant growth, excessive use of synthetic fertilizers negatively impacts ecosystems and human health (Ahmed et al., 2017). Furthermore, recent geopolitical disruptions have affected global fertilizer availability (Hailes et al., 2023). These issues underscore the importance of Biological Nitrogen Fixation through legume-rhizobia symbiosis as a sustainable alternative.
       
Cowpea, a grain legume of African origin, plays a vital economic and environmental role due to its resilience to drought, heat and poor soils (Narayana and Angamuthu, 2021; Pavithra et al., 2025), making it suitable for arid, climate-affected regions like coastal Peru (Murga-Orrillo et al., 2024). In 2023, global production reached 9.78 × 10⁶  tons, with Peru contributing 3.21 ×  10⁴  tons (FAO, 2025). Although cowpea is a promiscuous legume, forming symbioses with diverse rhizobia, studies show specificity at species, strain and varietal levels. Effective symbionts include Bradyrhizobium yuanmingense, B. liaoningense (Valdez et al., 2016), B. pachyrhizi (Leite et al., 2018), B. uaiense (Cabral-Michel et al., 2020), B. amazonense (Moreira et al., 2024), among others.
       
Bradyrhizobium-based inoculants significantly enhance symbiotic efficiency and agronomic performance in cowpea under controlled conditions (Valdez et al., 2016; Araújo et al., 2017). Inoculation effectiveness depends on chemical (pH, salinity, nutrient availability), physical (soil texture, compaction) and biological factors, including native rhizobia presence, inoculant strain efficacy and cowpea genotype. Cowpea varieties vary in their Biological Nitrogen Fixation (BNF) capacity, influenced by environmental factors such as phosphorus and water availability (Jemo et al., 2017). Genetic regulation of BNF involves dominant-recessive epistatic genes (Seido et al., 2019). Mohammed et al. (2020) reported BNF efficiencies ranging from 27% to 97% among Ghanaian cultivars.
       
In Peru, rhizobial inoculation research in cowpea is limited. Valdez et al. (2016) identified effective Bradyrhizobium strains from floodplain soils in Ucayali-two B. yuanmingense and two B. liaoningense-that increased grain yield by 29.5% compared to the control and by 6.3% over nitrogen fertilization (60 kg N ha^-1). However, no rhizobial strains are currently recommended for cowpea in Peru’s arid coastal zones. Evaluating Biological Nitrogen Fixation (BNF) potential in commercial varieties across agroecological zones is crucial, accounting for genotype-rhizobium-environment interactions (Seido et al., 2019). We hypothesize that strains adapted to arid coastal conditions will exhibit superior symbiotic performance.
       
This study presents the first evaluation of the symbiotic and agronomic potential of seven native strains from three Bradyrhizobium species, effective in symbiosis with cowpea and isolated from the arid coastal zone of Peru. The objectives were: (1) to assess the symbiotic effectiveness of seven selected Bradyrhizobium strains under non-sterile soil conditions; and (2) to evaluate the influence of inoculating these strains on the agronomic performance of two commercial cowpea varieties under the same soil conditions.

Origin of rhizobia strains and cowpea varieties
 
The Bradyrhizobium strains were previously isolated from the arid northern and central coast of Peru and selected them based on their symbiotic effectiveness, osmotolerance and salt tolerance by Valdez-Nuñez et al. (2025). The strains are currently preserved in the Collection of Beneficial Microorganisms for Agriculture and Livestock at Universidad Nacional de Barranca (ColMiBAP-UNAB). The strains were reactivated from cryovials stored at -80^oC and cultured them on YEM agar at 28^oC for 7 days. We evaluated two cowpea varieties: Vaina Verde INIA-432, a commercially demanded cultivar provided by the National Institute of Agrarian Innovation (INIA, 2013) and Vaina Verde CAR-9, an experimental line donated by Granos y Legumbres-Peru.
 
Experimental design under controlled conditions
 
The study, conducted from January to May 2024, evaluated the symbiotic efficiency of seven Bradyrhizobium strains using a completely randomized 10 × 2 factorial design. Twenty treatments with eight replicates assessed nitrogen fixation in two cowpea varieties at two growth stages, 45 days after sowing and the remaining at grain maturity.
       
The soil used in this study was collected from the Buena Vista experimental field at the Universidad Nacional de Barranca (UNAB), Peru (10^o452′47.5″S; 77^o442′21″W; 64 m.a.s.l.). The soil, with a history of sugarcane (Saccharum officinarum) cultivation and no prior rhizobia inoculation. Soil was taken from the top 20 cm, air-dried, ground, sieved (4 mm mesh) and placed (4 kg) into 5 kg-capacity pots. An aliquot was analyzed at the Soil Laboratory of Universidad Nacional Agraria La Molina for texture and chemical composition, including pH, EC (1:1), CaCO₃, OM, N, P, K, CEC, exchangeable cations (Ca²⁺, Mg²⁺, K²⁺, Na²⁺ ) and micronutrients (Cu²⁺ , Mn²⁺, Fe²z , Zn²+, B⁺) (Supplementary Table 1). All treatments received uniform phosphorus and potassium fertilization (60:30 kg ha^-1) using 1250 mg triple superphosphate and 150 mg potassium chloride per pot.


 
Quantification of rhizobial numbers in the experimental soil
 
The native rhizobial population capable of nodulating cowpea was quantified using the Most Probable Number (MPN) technique (Somasegaran and Hoben, 2012). Eight decimal dilutions (10^-1 to 10^-8) were prepared from 10 g of soil in 9 mL of 10 mM MgCl₂, homogenized with a vortex mixer (Scientific Industries, USA). Surface-disinfected seeds of cowpea variety Vaina Verde INIA-432 were germinated following Valdez et al. (2016) and sown individually in growth pouches containing nitrogen-free Jensen’s solution. Each pouch received a specific soil dilution, with four replicates per dilution. Pouches were incubated at 25± 2^oC with a 12/12 h photoperiod for 21 days. Nodulation was assessed by nodule presence and MPN values were estimated using probability tables Somasegaran and Hoben (2012).
 
Inoculum preparation and standardization
 
The strains were reactivated in 3 mL YEM broth tubes, incubated for 5 days at 150 rpm and 28±1^oC on an orbital shaker (MRC, Israel). Subsequently, each strain was transferred to 50 mL flasks containing 27 mL of YEM broth and incubated them under the same conditions for 7 days. Each culture was standardized an OD₆₀₀ ~ 1.0 using 10 mM MgCl₂. For the consortium treatment, four standardized inocula were mixed in equal ratios (1:1:1:1). Cell concentrations were quantified via the drop plate method (Ferreira et al., 2024).
 
Experimental setup
 
Seeds of cowpea varieties Vaina Verde INIA-432 and CAR-9 were selected and surface-disinfecting them with 70% ethanol (1 min), 2.5% sodium hypochlorite (5 min) and rinsing them eight times with sterile distilled water. Six seeds per pot were sowed and thinned to three plants five days post-emergence. Each seed received 1/ mL of standardized inoculum based on the treatment. To maintain uniform moisture, they calculated the water-holding capacity (WHC) and maintained it at 60% by monitoring pot weight. Irrigation used only distilled water. Control treatments (T9 and T10) received 1 mL of 10 mM MgCl₂. The experiment was conducted on metal tables in a greenhouse from Buena Vista experimental field at the Universidad Nacional de Barranca (UNAB), Peru (10^o45′55.7″S; 77^o44′27.6″W; 64/ m.a.s.l.), where temperatures ranged from 23^oC to 30^oC.
 
Experimental evaluation
 
At 45-50 days after sowing, when 50% of treatments reached flowering, were conducted the first evaluation. Four plants were harvested per treatment to assess plant height (H, cm), shoot dry weight (SDW, mg) and root dry weight (RDW, mg), with biomass dried at 70^oC for 72 hours (Valdez et al., 2016). Nodulation was assessed using a 0-4 scale based on nodule number, size and position and nodules on the root crown (NNC) were counted following Yates et al. (2016). Nodule dry weight (NDW, mg) was recorded and shoot nitrogen content (SNC, %) was determined by the Kjeldahl method (Rocha et al., 2021). Shoot nitrogen accumulation (SNA, mg N plant^-1) was calculated as SDW × SNC% (Florentino et al., 2010). Symbiotic efficiency (EFI) and effectiveness (EFV) were determined relative to controls (Valdez et al., 2016) and categorized according to Purcino et al. (2000): Highly effective (EFV > 80%), effective (50-79%), slightly effective (35-49%), or ineffective (<34%). At maturity, remaining plants were harvested to assess agronomic traits: number of pods per plant (NPP), number of grains per pod (NGP), 100-seed weight (W100), grain weight per plant (GWP) and grain nutrient content (N-G, P-G, K-G%).
 
Statistical analysis
 
The statistical analyses were conducted using InfoStat software (Di Rienzo et al., 2020). The assumptions of normality and homoscedasticity was verified for all variables. Nodulation data (PSN, NNC and nodulation score) were transformed using the formula (x + 0.5)^0.5. One-way ANOVA was applied at a significance level of P<0.05, followed by Scott-Knott or Kruskal-Wallis tests. Pearson’s correlation coefficients were used to assess relationships between vegetative and grain yield parameters.

Inoculation with Bradyrhizobium strains significantly enhanced cowpea varieties by increasing H, RDW, symbiotic efficiency (EFI, EFV), GWP and %P-G. Inoculated treatments consistently outperformed the uninoculated controls (Fig 1 and 2, Table 1 and Table 4). Native Bradyrhizobium strains effectively fix atmospheric nitrogen in symbiosis with cowpea (Kyei-Boahen et al., 2017; Nguyen et al., 2020). Understanding both the genetic affiliation of rhizobial strains and the genotype of the host cultivar is crucial for selecting rhizobia that optimize grain yield, fertilizer use efficiency and tolerance to edaphoclimatic stress (Mendoza-Suárez et al., 2020).






       
The H did not show significant differences among inoculated treatments within each cowpea variety. However, B. yuanmingense strains 19.3 and 20.1 increased H by 32.1% in INIA-432 and 40.6% in CAR-9, compared to the uninoculated control. H showed a strong correlation with grain yield in INIA-432 (r = 0.72**). Bradyrhizobium sp. 12.3 achieved the highest increase in shoot dry weight (SDW), with a 151.1% rise in INIA-432, while B. yuanmingense 8.2 increased SDW by 97.8% in CAR-9. Similarly, B. diversitatis 25.1 increased root dry weight (RDW) by 127.1% in INIA-432 and B. yuanmingense 8.2 increased RDW by 79.3% in CAR-9 relative to the controls. SDW reflects rhizobial symbiotic effectiveness and depends on the nitrogen source, the cultivar and their interaction (Fageria et al., 2014).
       
In our study, shoot nitrogen content (SNC) and shoot nitrogen accumulation (SNA) were consistently lower in CAR-9 than in INIA-432. Nitrogen-fertilized treatments showed higher SNC and SNA than most inoculated treatments in both varieties. However, in CAR-9, inoculation with Bradyrhizobium sp. 12.3, B. yuanmingense 8.1.1, 20.1, 19.3; B. diversitatis 25.1 and the consortium produced SNC values statistically comparable to the fertilized control.  Genotype-dependent differences in symbiotic performance are highlighted (Jemo et al., 2017), considering that the SNA serves as a robust indicator of symbiotic effectiveness, which depends on both biotic and abiotic factors, including strain-host compatibility (Florentino et al., 2010).
       
B. diversitatis 25.1 increased SNA by 163.8% in INIA-432 and B. yuanmingense 8.1.1 increased it by 114.4% in CAR-9. The poor SNA in control treatments suggests low native rhizobial effectiveness, likely due to their low soil density (<10² cells g^-1). Low soil nitrogen (0.05%) did not hinder symbiosis, allowing inoculated strains to perform efficiently and contribute significantly to biological nitrogen fixation and nitrogen accumulation (Nguyen et al., 2020).
       
We modified the formulas from Purcino et al. (2000) to calculate EFI and EFV, using SNA instead of SDW, as a more accurate measure of symbiotic performance. Future comparative studies should validate this approach. In our study, EFI values in INIA-432 were significantly higher than those in CAR-9, likely due to genotypic differences between varieties. Our native Bradyrhizobium strains doubled EFI in all INIA-432 treatments and in at least 50% of CAR-9 treatments, highlighting the impact of host genotype on symbiotic effectiveness (Mohammed et al., 2020).
       
Similarly, EFV was higher in CAR-9, where all inoculated treatments-except Bradyrhizobium sp. 1.1-were highly effective in BNF (Table 2). Notably, B. yuanmingense strains 8.1.1, 19.3 and 20.1 performed well in both varieties. In addition, native rhizobia in control treatments formed few, poorly located nodules due to late, inefficient infections. Although, consortium inoculation reduced nodulation compared to single strains, B. diversitatis 25.1 enhanced nodule dry weight (NDW) in both varieties (Table 3). These differences may reflect limitations in the nitrogen-fixing capacity of the selected genotypes (Graham et al., 2004).




       
At harvest, all agronomic yield parameters-except N-G% and K-G%-were higher in inoculated treatments than in the non-inoculated control without nitrogen fertilization. Inoculation with Bradyrhizobium yuanmingense 20.1 increased the NPP by 39.2% in CAR-9 and 64.03% in INIA-432. Likewise, Bradyrhizobium sp. 12.3 improved the NGP by 25.2% (CAR-9) and 86.3% (INIA-432). Strains 12.3, 19.3 and 20.1 showed consistent effects in increasing the GWP across both cowpea varieties. In INIA-432, strain 20.1 outperformed both the inoculant consortium and the uninoculated control, increasing GWP by 98.2% (CAR-9) and 118.7% (INIA-432). These results highlight the superior symbiotic efficiency of strain 20.1 under nitrogen-deficient conditions (Table 4).


       
Pearson correlation coefficients (Table 5) revealed significant positive relationships between vegetative and yield parameters in both cowpea varieties. Strong correlations were observed between SDW and RDW (0.98***), as well as between SNA and EFV (0.95***). SNA also showed a highly significant correlation with GWP in INIA-432 (0.94***) and CAR-9 (0.75***), confirming its relevance as an indicator of symbiotic efficiency (Araújo et al., 2017; Kyei-Boahen et al., 2017).


       
In contrast, the correlation between SDW and NDW was weak in INIA-432 and non-significant in CAR-9, likely due to soil limitations such as texture and low phosphorus availability (Rocha et al., 2021). The P-G% was strongly correlated with vegetative traits-SDW, RDW, SNA, EFV-and moderately correlated with yield components-NGP and GWP. Phosphorus is essential for photosynthesis, ATP synthesis and nitrogen fixation, emphasizing its critical role in promoting effective legume-rhizobia symbiosis and cowpea productivity (Araújo et al., 2017; Mobeena et al., 2025).
       
B. yuanmingense is a widespread symbiont of cultivated cowpea across Asia, Africa and South America (Valdez et al., 2016; Leite et al., 2018; Ndungu et al., 2018; Girija et al., 2020). Its high nitrogen-fixing efficiency, as demonstrated in strains like BR3267 (Leite et al., 2018). In our study, B. yuanmingense strains 19.3 and 20.1, along with the undefined species Bradyrhizobium sp. 12.3, showed strong symbiotic performance in both cowpea varieties. These strains, isolated from arid Peruvian regions, possess key plant growth-promoting traits such as auxin and siderophore production, phosphate solubilization and stress tolerance (Valdez-Nuñez et al., 2025). They represent promising inoculant candidates for arid agroecosystems, but require field validation across diverse environments and farmer-oriented implementation strategies.

The strains studied enhanced the agronomic performance of both commercial cowpea varieties. However, the Vaina Verde CAR-9 variety demonstrated greater symbiotic potential for BNF, particularly in terms of EFV and GWP. Bradyrhizobium yuanmingense strains 19.3 and 20.1, along with Bradyrhizobium sp. 12.3, were selected for both cowpea varieties due to their ability to promote SNA and GWP. These strains outperformed currently recommended inoculants, making them promising candidates for the formulation and production of strain-specific inoculants adapted to the arid coast of Peru.

We acknowledge Angel Valladolid Chiroque for providing the cowpea varieties for the tests and professor José Carlos Rojas García of the Universidad Nacional de San Martín-Peru, for the interpretation of the soil analysis and the fertilization calculations of this assay. We thank the Universidad Nacional de Barranca for the funding provided for the publication of this manuscript.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Funding
 
This work was supported by the Universidad Nacional de Barranca (R.C.O. N° 629-2022-UNAB and R.C.O. N°153-2024-UNAB).

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.