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Article

Water Availability Associated with Coinoculation with Growth-Promoting Rhizobacteria in Cowpea

by
Júlio José Nonato
1,
Tonny José Araújo da Silva
2,
Alessana Franciele Schlichting
2,
Luana Aparecida Menegaz Meneghetti
2,
Niclene Ponce Rodrigues de Oliveira
1,
Thiago Franco Duarte
2,
Salomão Lima Guimarães
2,
Marcio Koetz
2,
Ivis Andrei Campos e Silva
2,
Patrícia Ferreira da Silva
2,
Adriano Bicioni Pacheco
3 and
Edna Maria Bonfim-Silva
2,*
1
Programa de Pós-Graduação em Agricultura Tropical, Faculdade de Agronomia e Zootecnia, Universidade Federal de Mato Grosso, Cuiabá 78060-900, Brazil
2
Programa de Pós-Graduação em Engenharia Agrícola, Instituto de Ciências Agrárias e Tecnológicas, Universidade Federal de Rondonópolis, Rondonópolis 78736-900, Brazil
3
Programa de Pós-Graduação em Agronomia, Instituto de Ciências Agrárias, Universidade Federal Rural da Amazônia, Belém 66077-830, Brazil
*
Author to whom correspondence should be addressed.
Nitrogen 2025, 6(3), 74; https://doi.org/10.3390/nitrogen6030074
Submission received: 10 July 2025 / Revised: 26 August 2025 / Accepted: 27 August 2025 / Published: 29 August 2025
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Abstract

Soil water availability can become one of the decisive factors for crop production. The technology of coinoculation with plant growth-promoting bacteria capable of performing biological nitrogen fixation and producing plant hormones may be an alternative that minimizes the effects of variations in soil water availability. In this context, the objective was to evaluate the phytometric and productive characteristics of cowpea coinoculated with Azospirillum brasilense and Bradyrhizobium japonicum subjected to soil water availability stress. The experiment was carried out in a greenhouse in a completely randomized block design with four replications in a 4 × 4 factorial arrangement: not inoculated; inoculated with B. japonicum; and coinoculated with B. japonicum + A. brasilense and N fertilizer, associated with soil water tensions of 15, 30, 45, and 60 kPa. Statistically, the lowest soil water tension, 15 kPa, and the coinoculated and nitrogen fertilizer treatments resulted in greater development of plant height, stem diameter, and number of leaflets. The shoot dry mass was significantly different for only the soil water stress treatments, which showed a decrease in mass accumulation from 15 kPa to 50.22 kPa. Regarding the SPAD index, soil water tension showed a decreasing linear adjustment 24 days after plant emergence (DAEs), with the lowest value of 51.38 at a tension of 60 kPa. At 39 DAEs, the adjustment was polynomial, with the lowest tension index of 59.62 kPa, corresponding to 44.14. The treatments with the use of inoculants had a significant effect on the SPAD index, in which coinoculation with Bradyrhizobium and Azospirillum brasilense resulted in values equal to those of nitrogen fertilizer and greater than those of uninoculated treatments or those inoculated with Bradyrhizobium. Water tension influenced the total water consumption, and at a tension of 18.13 kPa, the lowest accumulation occurred, equivalent to 2.20 g of dry matter for each liter of irrigated water. Statistically, the lowest soil water tension, 15 kPa, resulted in higher numbers, lengths, and widths of pods. In relation to the length of pods, the uninoculated, inoculated with Bradyrhizobium, and coinoculated with Bradyrhizobium and A. brasilense treatments were superior to nitrogen fertilization. Coinoculation and nitrogen fertilization influenced phytometric characteristics. The productive characteristics of cowpea decreased as the soil water tension increased. These results highlight the importance of leveraging biological solutions, such as coinoculation, to mitigate the adverse effects of water stress on crop yields. In addition, by optimizing these practices, farmers ensure greater resilience in bean production, thereby guaranteeing food security in the face of changing environmental conditions.

1. Introduction

The occurrence of rainfall variations has increased in agricultural production fields, and both an excess and a scarcity of water available in the soil may occur. In areas with cowpea production (Vigna unguiculata (L.) Walp.), irrigation is commonly used during the planting season.
Even though cowpea is considered a rustic crop, the occurrence of irregular rainfall can be the cause of low yields; in Brazil, up to three related crops are cultivated, with an average of 776.33 kg ha−1 in the 24/25 agricultural season [1].
Variations in water availability can cause changes in the morphology, physiology, and biochemical activities of plants [2], consequently affecting agricultural production and impacting food security with world population growth [3].
To minimize the effects of water variation and increase the average yield, inoculation with plant growth-promoting rhizobacteria (PGPR) represents a possible alternative, as some species have the capacity for biological nitrogen fixation (BNF) [4] and the production of plant hormones, such as indoleacetic acid (IAA) [5], an auxin that affects growth and cell elongation and is linked to the complex regulation of plant biosynthesis, (de)conjugation, and cell degradation [6]. The local distribution of auxin within crop tissues is directly related to the directional transport of this plant hormone between cells, thereby directly influencing root system development, such as root curvature and growth rate [6].
The BNF process consists of the conversion of atmospheric nitrogen into an organic form that can be assimilated by the plant. It occurs with the breakdown of N2 molecules and their conversion into ammonia [7]. In legumes, a tightly regulated signaling sequence leads to nodule organogenesis and efficient nitrogen fixation [8]. In addition, the conversion of atmospheric nitrogen into an organic form occurs through a symbiotic process between legumes and a group of bacteria (rhizobia) in the roots of these crops [8].
Studies carried out with the PGPR genus of Azospirillum reported the ability of these microorganisms to carry out both IAA production [4,9] and BNF [10], which are considered specific markers of IAA activity in the rhizosphere [11].
Experiments involving cosignaling in crops such as soybean (Glycine max L.) [12,13], chickpea (Cicer arietinum) [14], and cowpea [15,16] identified positive effects of the association of PGPRs with phytometric and productive characteristics. Studies are needed to determine the soil moisture that increases the efficiency of this coinoculation.
Considering the importance of soil-plant water relationships and microorganisms and their relationships, the objective of this study was to investigate the development and productive characteristics of cowpea coinoculated with plant growth-promoting rhizobacteria subjected to water availability stress.

2. Materials and Methods

2.1. Experimental Site and Design

The experiment was carried out in a greenhouse under controlled conditions of maximum temperature (mean 31.8 °C; SD 1.70), minimum temperature (mean 19.6 °C; SD 2.24), and air humidity (mean 51.4%; SD 2.63). Soil collected from the 0–0.2 m depth layer in the Cerrado area was used, and characterization of the chemical and granulometric characteristics was carried out: pH (CaCl2) = 4.0; O.M. = 21.2 g dm−3; P = 1.3 mg dm−3 (Mehlich−1); K = 33 mg dm−3; S = 2.0 mg dm−3; Ca = 0.4 cmolc dm−3; Mg = 0.2 cmolc dm−3; SB = 0.7 cmolc dm−3; CTC = 6.4 cmolc dm−3; V = 10.7%; sand = 445 g kg−1; silt = 100 g kg−1; and clay = 455 g kg−1.
The experimental design was randomized blocks, with 4 replications, in a 4 × 4 factorial arrangement, with soil water tensions of 15, 30, 45, and 60 kPa associated with the following treatments: (i) uninoculated, (ii) nitrogen fertilization, (iii) inoculation with Bradyrhizobium japonicum, and (iv) coinoculation with Bradyrhizobium japonicum + Azospirillum brasilense.
For soil corrections, the recommendations proposed by [17] were used in accordance with the results of the chemical analysis, with the acidity correction performed via the base saturation method and the application of calcitic limestone with the real total neutralization power equal to 99%. As indicated by [18], for chemical reactions to occur effectively, after the incorporation of limestone, the soil was moistened until it reached approximately 60% of the maximum water retention capacity, stored in plastic bags, sealed, and incubated in greenhouse conditions for a minimum of 20 days.
After the acidity neutralization process, the soil was separated into a volume of 5 dm−3 and fertilized with 110 mg dm−3 phosphorus (P2O5) using simple superphosphate as a source, 50 mg dm−3 potassium using potassium chloride (KCl) as a source, and 15 mg dm−3 commercial formulation FTE BR12 (1% sulfur, 1.8% boron, 0.85% copper, 2.0% manganese, and 9.0% zinc).
To compose each experimental unit, plastic pots filled with soil after fertilization were used. Five cowpea seeds, cv. BRS Guariba, were thinned seven days after emergence, and 2 plants remained per experimental unit.
Before planting, cowpea seeds were subjected to two disinfestations: first, they were submerged in 70% alcohol for 3 min, and then they were washed in distilled water. In the second step, they were submerged in 70% alcohol for one minute and, later, washed three times in distilled water and dried with paper towels.
The dried seeds were inoculated with commercial liquid inoculants of Bradyrhizobium japonicum (SEMIA 6462 and SEMIA 6463) at a concentration of 2 × 109 colony-forming units (CFUs) mL−1 at a dose of 30 mL kg seeds−1, and after a period of approximately 10 min, they were coinoculated with commercial liquid inoculants containing Azospirillum brasilense of the Ab-v5 and Ab-v6 strains at a concentration of 2 × 108 CFU mL−1 at a dose of 15 mL kg seed−1. In these treatments, nitrogen fertilizer was not applied.
In the treatment with only nitrogen fertilization, 50 mg dm−3 of nitrogen was applied. Nitrogen fertilization was carried out with urea diluted in water, and 50 mL was fertigated in each experimental unit on the same day of thinning.

2.2. Irrigation System

To measure the water tension in the soil, a tensiometer was installed in the center of each pot. Irrigation was carried out daily in the early morning, and the tension was read to calculate the volume of water to be applied. The calculation of the volume of water to be applied was determined with the aid of a characteristic curve of water retention in the soil carried out for the soil used, according to the theory [19] and adjusted in the computer program Soil Water Retention Curve (SWRC) [20], generating the following equation:
Ѳ v   =   0.030   +   0.736 − 0.030 [ 1   +   ( 2.2280 Ψ ) 3.5035 ] 0.1143
where Ѳv is the volumetric moisture (cm3 cm−3) and Ψ is the water tension in the soil (kPa).
To promote the establishment of the plants, the water supply was standardized until nine days after the emergence of the plants, after which the variation in water availability began.

2.3. Variables Analyzed

Metric evaluations of cowpea growth were performed at 24, 39, and 54 days after emergence (DAEs). The following parameters were evaluated: plant height, determined from the base of the stem, close to the ground, to the apex of the main branch with a graduated ruler; stem diameter, measured at the base of the stem, approximately 2 cm above the ground, with a digital caliper; and number of leaflets, determined by manually counting the number of expanded leaflets.
At 24 and 39 DAEs, the SPAD index was evaluated with the aid of a Minolta SPAD-502 electronic meter (Spectrum Technologies, Inc., Plainfield, IL, USA), with three random points in the central leaflet of the third expanded leaf in the main branch counting from top to bottom. The SPAD index was not evaluated at 54 DAEs because the plants were in the reproductive stage and had senescent leaves.
At 54 DAEs, the plants were harvested and separated into stems, leaves, branches, and pods and dried in a forced-air circulation oven at a temperature of 60–65 °C until they reached a constant weight, a procedure performed to determine the accumulation of shoot dry matter.
The number of pods per plastic pot was counted, and pod length was measured with the aid of a graduated ruler. With a digital caliper, we evaluated the width of the pods, adopting the center of the pod in the space between the grains as a standard.
The total water consumption of each experimental plot was the sum of the total irrigated water during the entire crop cycle. The water use efficiency (EUA) was obtained via the formula EUA (g L−1) = shoot dry mass (g)/total water consumption (L).

2.4. Statistical Analysis

The results were subjected to the Shapiro-Wilk normality test and analysis of variance. When bilateral analysis was significant, polynomial regression analysis (up to 10% probability) was performed to verify the fit of each treatment as a function of soil water stress. When it was not, polynomial regression for water availability isolated (up to 10% probability) and Tukey’s test for inoculation treatments (up to 10% probability). R software [21] with the statistical package ExpDes.pt [22] was used for the statistical analyses.

3. Results and Discussion

3.1. Plant Height

Plant height had an isolated effect on soil water tension and inoculation with plant growth-promoting bacteria. The lowest water tension in the soil (15 kPa) was associated with the greatest plant height, and in the first evaluation, at 24 days after emergence (DAEs), it was 13.69 cm (Figure 1A); at 39 DAEs, it was 23.54 cm (Figure 1B); and at 54 DAEs (Figure 1C), the greatest plant height was 33.90 cm. At 39 DAEs, there was a decreasing linear adjustment of the voltage from 15 to 60 kPa, resulting in a decrease of 14.86% (Figure 1B).
Water deficit in the soil is capable of reducing, by considerable amounts, the production of biomass by plants, as it reduces the assimilation of nutrients. In an attempt to maintain the accumulation of biomass, one of the responses of plants to water stress is hormonal [23], such as an increase in the concentration of abscisic acid [16], which can induce the osmotic adjustment of plant cells. This process consists of increasing the concentration of osmolytes [24] in the vacuole of the plant cell, with the aim of maintaining turgidity pressure so that the plants can continue physiological processes [25].
Among these osmolytes are amino acids, such as proline; carbohydrates, such as fructose; and ammonia compounds, such as glycinabetaine [26]. Thus, plant development and production are directly influenced by plant hormones, which are altered by drought stress [27]. This information justifies the data found in the study, in which lower soil water tension resulted in taller plants; i.e., beans grew taller in conditions of greater water availability for the crop.
With respect to inoculation, the height of cowpea plants was influenced at 24 days after emergence, with the treatment with nitrogen fertilizer resulting in the greatest height and the inoculation with Bradyrhizobium resulting in the lowest height (Table 1). Moreover, there were no differences in height among the treatments with nitrogen, uninoculated fertilizer and coinoculation with Bradyrhizobium and Azospirillum brasilense.
In a study evaluating the effects of inoculation with nitrogen-fixing bacteria and the use of nitrogen fertilizer on the growth of nitrogen fertilizers and cowpea, ref. [28] reported that inoculation resulted in higher growth rates than did fertilizer use. This result differs from that reported in the present study, as nitrogen fertilizer presented a better result than did inoculation with B. japonicum but was similar to the coinoculation treatment.
Inoculation is important in soils with low rhizobium persistence [29], but some soils may have native microbial activities that can maintain good plant development [30], and a strategy is interesting for reinoculation at different plant stages to maintain effective cell densities for nitrogen assimilation [31].
The results of this study on plant height as a function of soil water tension and plant growth-promoting bacteria show that lower soil water tension significantly promotes plant height, as it promotes a stronger root system, allowing greater nutrient absorption, in addition to improving water absorption efficiency. In addition, coinoculation strategies are effective at increasing cowpea development while maintaining sustainable agricultural practices, as they are an alternative to synthetic fertilizers.

3.2. Stem Diameter

There was an isolated effect of inoculation and soil water tension on stem diameter, with quadratic polynomial adjustment occurring in the three evaluations, with the smallest diameters occurring at stresses of 50.5, 54.42, and 57.5 kPa at 24, 39, and 54 DAEs, respectively (Figure 2A–C).
As with plant height, it can be seen from the stem diameter results that water stress impairs bean development. Thus, larger diameters were observed at lower soil water tension. This result can be explained by the fact that reductions in diameter due to increased water tension in the soil may be related to reductions in growth rates, as [32] reported that in conditions with water deficit, there is closure of the stomata and a reduction in transpiration, resulting in lower photosynthetic rates and, thus, reduced plant growth. Similar results were reported by [33], who subjected green mung bean (Vigna radiata L.) to water stress and reported decreases of up to 23.94% in the stem diameter at a 64 kPa soil water tension compared with the 4 kPa tension.
In the evaluations at 39 and 54 days after plant emergence, there was a significant effect of the inoculations alone. In the second evaluation period, at 39 DAEs, the results of the nitrogen fertilizer treatment were equal to those of the coinoculation treatment with Bradyrhizibium and Azospirillum brasilense and superior to those of the uninoculated treatment and that inoculated only with Bradyrhizobium (Table 2).
At 54 DAEs, the equality between the cowpea stem diameter values for the use of nitrogen fertilizer and coinoculation was maintained, although both were greater than those of the other treatments (Table 2). In an experiment in which the soybean cultivar Turbo (Glycine max L.) was subjected to nitrogen fertilizer, inoculation with Bradyrhizobium japonicum and coinoculation with B. japonicum + Azospirillum brasilense [5] resulted in a greater stem diameter under nitrogen fertilization.
The results highlight the importance of the interaction between water management and cowpea development. Efficient water use is essential, especially in regions susceptible to drought, as the results show that water stress directly affects bean growth. In addition, optimizing irrigation helps conserve water resources while maximizing the growth of crops such as cowpeas, thus contributing to agricultural sustainability and reducing the environmental impact associated with excessive water use.
On the other hand, there are positive effects on the stem diameter of cowpea from co-inoculation with Bradyrhizobium and Azospirillum brasilense, highlighting the importance of using growth-promoting bacteria in agricultural production. These results reinforce the potential of sustainable agricultural practices to optimize the use of inputs and increase bean development. Furthermore, the use of coinoculation is a viable alternative that can replace synthetic fertilizers such as nitrogen, contributing to responsible agriculture and reducing environmental impacts.

3.3. Number of Leaflets

In the first evaluation (24 DAEs), only the water tension in the soil influenced the number of leaflets (Figure 3A); however, there was an interaction between the water tension in the soil and the rhizobacteria in the second evaluation (39 DAEs) of the plants (Figure 3B).
The lowest number of leaflets, approximately six, was observed at 52.49 kPa at 24 DAEs (Figure 3A). The reduction in the number of leaflets with increasing water tension may be linked to plant mechanisms due to water deficit, which consists of limiting water consumption to maintain the hydration of plant tissues [34]. Among these mechanisms are a reduction in the number of leaves emitted and an increase in leaf abscission [35].
Results close to those of the present study were observed for green mung bean subjected to water stress in experiments carried out by [5,36], which identified significant reductions in the phytometric variables of plants under water limitation in the soil compared with those of plants under soil conditions without water restriction.
In the interaction, at 39 DAEs, the lowest number of leaflets was found for the treatment inoculated with Bradyrhizobium, approximately 16 at 48.05 kPa, and the highest number of leaflets occurred in the treatments with nitrogen fertilization and not inoculated, approximately 18 for soil water tensions of 50.46 and 45.26 kPa, respectively. In the coinoculation of Bradyrhizibium and Azospirillum brasilense, the number of leaflets was approximately 17 at a tension of 48.01 kPa (Figure 3B).
The soil microbiome is not homogeneous [37], so we can agree that the same number of leaflets found for the uninoculated treatment and the use of nitrogen fertilizer is due to the presence of native soil microorganisms and their interactions. An example of an interaction between bacterial species was reported by [38], who reported that cellulose in the soil is degraded by Cellulomonas, providing carbon to Azospirillum, which fixes atmospheric nitrogen.
Ref. [39] used Bacillus amyloliquefaciens and Azospirillum brasilense in wheat subjected to water stress and suggested that the joint action of these microorganisms provided tolerance to water deficit in the soil through various mechanisms, such as morphological, physiological, and metabolic changes in plants.

3.4. Shoot Dry Mass

The shoot dry mass was significantly different for only the soil water stress treatments, with second-order polynomial regression, in which there was a decrease in the accumulation of mass from 15 kPa to 50.22 kPa (Figure 4). After this tension, there was an increase of 5.73% up to a voltage of 60 kPa.
This decrease may be explained by the decrease in tension to 50.22 kPa; when enough water is available in the soil for the plants, uptake by the roots is favored, which increases the translocation of solutes and nutrients, as well as cell growth, which may result in increased accumulation of shoot dry mass.
In an experiment with cowpea cv. IPA-206, ref. [40] reported a reduction in shoot dry matter accumulation as the degree of soil water stress increased. The increase of 5.73% may be related to the release of exudates by the roots because, according to the authors of [41], to tolerate the scarcity of water, it occurs with the exudation of osmolytes in the rhizosphere to maintain the cellular structure.
This strategy is similar to that of microorganisms, which secrete polysaccharides [42], which can lead to the association of both exudates in the rhizosphere and increase cell membrane integrity and protein stability, protecting against cell damage [43]. In an experiment with corn subjected to water deficit and inoculated with drought-tolerant BPCP species, ref. [44] reported an increase in the concentration of osmolytes in seedlings.

3.5. SPAD Index

The result of the isolated factor for soil water tension also occurred with the SPAD index at 24 days after plant emergence, in which it presented a decreasing linear adjustment with the lowest value of 51.38 at the 60 kPa tension (Figure 5A). For the evaluation that occurred at 39 DAE, the adjustment was polynomial, with the lowest tension index of 59.62 kPa, corresponding to 44.14 (Figure 5B). At 54 DAE, there was no evaluation of the SPAD index due to senescence of the diagnostic sheets in the 15 and 30 kPa treatments.
The SPAD index is the quantification of the chlorophyll index, which is determined by the luminous flux transmitted by the leaf through wavelengths of different absorbances, indirectly indicating the nitrogen in the plant [45]. Environmental factors, such as soil water availability, can affect the formation of chlorophylls [46], which can be observed in this experiment, since the values of chlorophyll indices decrease as the water tension in the soil increases.
One of the strategies used by vegetables to circumvent the effect of water deficit on the soil is to maintain the water potential in the leaves. Observations have been reported for cowpea, which, even under conditions of continuous scarcity in soil, was able to maintain the water potential in the leaves [47] and relative water content [48]. Similar results were also observed for common bean (Phaseolus vulgaris L.) [49].
The treatments with the use of inoculants had a significant effect on the SPAD index, in which, at 24 days after plant emergence, coinoculation with Bradyrhizobium and Azospirillum brasilense resulted in values equal to those of the nitrogen fertilizer treatment and greater than those not inoculated or inoculated with Bradyrhizobium (Table 3).
However, the similarity between coinoculation and nitrogen fertilization was maintained; however, the inoculated treatment resulted in the same levels as the nitrogen fertilizer and the uninoculated control (Table 3).
Owing to the relationship between the SPAD index and the nitrogen present in the leaves, the effect of the inoculations was due to the ability of the bacteria used to fix atmospheric nitrogen [4]. Furthermore, as soil water tension increases, chlorophyll levels decrease, highlighting the importance of irrigation management to improve the chlorophyll index of cowpeas.
In addition, the results found in relation to treatments with coinoculation of Bradyrhizobium and Azospirillum brasilense show that biological nitrogen fixation is an effective alternative to synthetic fertilizers, and is a sustainable practice that contributes to more responsible agriculture.

3.6. Total Water Consumption and Water Use Efficiency

The total water consumption of cowpea changed as a function of variations in soil water tension, with a decreasing linear adjustment according to the increase in stress (Figure 6A). To estimate the effectiveness of plants in converting irrigated water into dry matter accumulation, the variable water use efficiency is used. This variable was influenced by the water tension in the soil, and at a tension of 18.13 kPa, the lowest accumulation occurred, equivalent to 2.20 g of dry matter for each liter of irrigated water (Figure 6B).
The constant reduction in total water consumption as water availability decreases (at higher voltages) may be related to stomatal closure; under drought conditions, endogenous levels of the plant hormone abscisic acid increase so that closure of the stomata occurs, thus reducing the loss of water from plants to the atmosphere [50].
A result divergent from that reported in the present study [51], which identified a reduction in the efficiency of water use for cowpea subjected to irrigation depths when in soil conditions with low water availability. This divergence can be explained by the greater accumulation of dry matter that occurred at the 15 and 30 kPa tensions (Figure 4), which was associated with the high water consumption during the cycle (Figure 6A), resulting in a low efficiency.
The greater efficiency in the use of water in conditions of greater deficiency in terms of the availability of water in the soil, 60 kPa, may be related to the adaptive strategy of acclimatization, which, according to [52], consists of controlling stomatal opening so that the assimilation of CO2 remains superior to the loss of water via the stomata. With the increase in water use efficiency, there were also higher grain yields in cowpeas and corn [53].

3.7. Number, Length, and Width of Pods

The number of pods significantly differed in isolation for the water tension with quadratic adjustment, with the minimum point occurring at 57.63 kPa (Figure 7A). In terms of the length of the pods, a significant effect was also observed for the isolated factor with respect to the soil water tension (Figure 7B) and the treatments with inoculations (Table 4).
An interaction effect was observed between the inoculation factors and the water tension in the soil on the width of the pods. The use of nitrogen fertilizer showed a second-order polynomial adjustment, with a minimum point of 0.33 mm at a tension of 53.67 kPa (Figure 7C). Adjustments for the treatments not inoculated, inoculated with Bradyrhizobium, and coinoculated with Bradyrhizobium and A. brasilense decreased (Figure 7C).
The opening of the stomata results in the loss of water to the atmosphere and the assimilation of CO2, indispensable processes for the production of carbohydrates by plants [54]. This finding corroborates the finding [55] that the reduction in cowpea yield due to water deficit was due to the regulation of the opening and closing of the stomata and was associated with a reduction in the yield of cowpea.
The greater number and length of pods as a function of the lower water tension in the soil are similar to those reported in [56], who studied cowpea associated with water depth and reported that the highest results for the productive components were found at depths equivalent to 100% of ETo (reference evapotranspiration). A reduction in soil water availability can have negative impacts on crop production. Ref. [57] studied the BRS Guariba and BRS Paraguaçu cowpea cultivars under water deficit and reported yield decreases in soil water availability of less than 54%.
The reduction due to the lower water availability in the pod width in the inoculated and coinoculated treatments can be explained by the difficulty in communication between the roots and the nitrogen-fixing symbiont bacteria [58]. In addition to impairing communication, abortion of the nodules formed can also occur [59].
The treatments with the inoculations significantly influenced, in isolation, the length of the pods, in which the uninoculated, inoculated with Bradyrhizobium and coinoculated with Bradyrhizobium and A. brasilense were superior to nitrogen fertilization (Table 4).
The shorter length of the pods in the treatment with nitrogen fertilizer may be associated with the influence of nitrogen on vegetative growth and may postpone the beginning of the reproductive phase. The theory that corresponds to the highest results is plant height (Table 1) and stem diameter (Table 2).
In an experiment involving the association of Rhizobium tropici based inoculants with industrial residue and biofertilizer, no differences were identified in the length of cowpea pods cv. IPA-207 compared with the absolute control [60].
Thus, observing the analyzed variables, it is clear that the coinoculated treatment provided excellent development in the bean crop, as did the nitrogen fertilizer, in terms of both phytometric and productive characteristics. Furthermore, regarding soil water tension, the lower tension provided the greater development of the bean plants, making more water available for the bean crop. However, regarding water use efficiency, the higher tension provided greater efficiency.
The study of the relationship between soil water stress and cowpea development emphasizes the complex interactions that influence the phytometric and productive characteristics of plants. In addition, the significant interaction between inoculation treatments and stresses, particularly in pod width, which was reduced under lower water availability, shows that water deficit is a limiting factor and impairs the root function of cowpeas, reducing communication and nutrient absorption by symbionts [58].
Understanding these dynamics is essential to determine irrigation strategies that increase water efficiency and, consequently, cowpea productivity. The research reinforces that future studies relating to physiological processes are important, seeking more sustainable agriculture, in addition to practical applications to increase the resilience and yield of cowpeas in response to regions with water deficit.

4. Conclusions

Coinoculation with Bradyrhizobium japonicum and Azospirillum brasilense can effectively improve the phytometric characteristics of beans to levels comparable to those obtained through traditional nitrogen fertilization. These results show a viable alter-native to synthetic fertilizers, aligning with sustainable practices aimed at food security and responsible agriculture, reducing environmental impacts.
Regarding soil water tension, the most promising results were found specifically at a tension of 15 kPa, indicating a need to prioritize management practices that maintain higher soil moisture. In this context, the results obtained suggest that the integration of biological inoculants in cowpea cultivation can help producers of this crop to increase bean productivity, especially in regions with water scarcity problems.
Furthermore, there is a need for future studies to expand the investigation to other bean varieties, in addition to evaluating the long-term impacts of this coinoculation practice in different environments, and to explore interactions with combinations of moisture levels and soil types, contributing to further optimizing sustainable agricultural practices.

Author Contributions

J.J.N.: conceptualization, methodology, data curation, software, and writing—original draft. T.J.A.d.S. and E.M.B.-S.: conceptualization, resources, writing—review and editing, and project administration. A.F.S., L.A.M.M., N.P.R.d.O., P.F.d.S. and I.A.C.e.S.: formal analysis, data curation, investigation, writing—original draft, and writing—review and editing. T.F.D., S.L.G., M.K. and A.B.P.: data curation, investigation, validation, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

The authors would like to thank the Federal University of Rondonópolis, CAPES, and CNPq.

Conflicts of Interest

The manuscript data are original, and the manuscript has not been submitted to any other publication, either in whole or in part, or in any other format or language. The authors have no conflicts of interest to declare.

References

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Figure 1. Plant height at 24 (A), 39 (B), and 54 (C) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; Ph = plant height; *** significant at a 0.1% probability.
Figure 1. Plant height at 24 (A), 39 (B), and 54 (C) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; Ph = plant height; *** significant at a 0.1% probability.
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Figure 2. Stem diameter at 24 (A), 39 (B), and 54 (C) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; SD = stem diameter; *** significant at a 0.1% probability.
Figure 2. Stem diameter at 24 (A), 39 (B), and 54 (C) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; SD = stem diameter; *** significant at a 0.1% probability.
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Figure 3. Number of leaflets at 24 (A) and 39 (B) days after the emergence of cowpea plants subjected to soil water tension and coinoculation with plant growth-promoting bacteria. Ψ = soil water tension; NL = number of leaflets; *, ***significant at 5 and 0.1% probabilities, respectively.
Figure 3. Number of leaflets at 24 (A) and 39 (B) days after the emergence of cowpea plants subjected to soil water tension and coinoculation with plant growth-promoting bacteria. Ψ = soil water tension; NL = number of leaflets; *, ***significant at 5 and 0.1% probabilities, respectively.
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Figure 4. Shoot dry mass (g) of cowpea subjected to water stress in the soil and coinoculated with plant growth-promoting bacteria. SDM = shoot dry mass, Ψ = soil water tension. *** significant at a 0.1% probability.
Figure 4. Shoot dry mass (g) of cowpea subjected to water stress in the soil and coinoculated with plant growth-promoting bacteria. SDM = shoot dry mass, Ψ = soil water tension. *** significant at a 0.1% probability.
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Figure 5. SPAD index at 24 (A) and 39 (B) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; SPAD = SPAD index; *, *** significant at 5 and 0.1% probability, respectively.
Figure 5. SPAD index at 24 (A) and 39 (B) days after the emergence of cowpea plants subjected to soil water stress. Ψ = soil water tension; SPAD = SPAD index; *, *** significant at 5 and 0.1% probability, respectively.
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Figure 6. Total water consumption (A) and water use efficiency (B) of cowpea subjected to soil water stress. Ψ = soil water tension; TWC = total water consumption; WUE = water use efficiency; *** significant at a 0.1% probability.
Figure 6. Total water consumption (A) and water use efficiency (B) of cowpea subjected to soil water stress. Ψ = soil water tension; TWC = total water consumption; WUE = water use efficiency; *** significant at a 0.1% probability.
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Figure 7. Number of pods (A), pod length (B), and pod width (C) of cowpea subjected to soil water stress and coinoculation with plant growth-promoting bacteria. Ψ = soil water tension; NP = number of pods; LP = length of pods; WP = width of pods; **, *** significant at a 1% and 0.1% probability, respectively.
Figure 7. Number of pods (A), pod length (B), and pod width (C) of cowpea subjected to soil water stress and coinoculation with plant growth-promoting bacteria. Ψ = soil water tension; NP = number of pods; LP = length of pods; WP = width of pods; **, *** significant at a 1% and 0.1% probability, respectively.
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Table 1. Height of cowpea plants at 24 days after emergence coinoculated with plant growth-promoting bacteria.
Table 1. Height of cowpea plants at 24 days after emergence coinoculated with plant growth-promoting bacteria.
TreatmentPlant Height at 24 DAEs (cm)
Not Inoculated8.68 ab
Inoculated7.84 b
Coinoculated8.65 ab
Nitrogen Fertilizer9.56 a
Average8.68
Averages followed by equal letters in the column do not differ from each other according to Tukey’s test at the level of 10% statistical probability. DAEs = days after plant emergence.
Table 2. Cowpea stem diameter at 39 and 54 days after emergence after coinoculation with plant growth-promoting bacteria.
Table 2. Cowpea stem diameter at 39 and 54 days after emergence after coinoculation with plant growth-promoting bacteria.
TreatmentStem Diameter at 39 DAEs (mm)Stem Diameter at 54 DAEs (mm)
Not Inoculated3.50 b3.03 b
Inoculated3.47 b2.96 b
Coinoculated3.69 ab3.26 a
Nitrogen Fertilizer3.85 a3.31 a
Average3.633.14
Averages followed by equal letters in the column do not differ from each other according to Tukey’s test at the level of 10% statistical probability. DAEs = days after plant emergence.
Table 3. SPAD index of cowpea plants coinoculated with plant growth-promoting bacteria at 24 and 39 days after emergence.
Table 3. SPAD index of cowpea plants coinoculated with plant growth-promoting bacteria at 24 and 39 days after emergence.
TreatmentSPAD Index at 24 DAEsSPAD Index at 39 DAEs
Not Inoculated52.10 b47.36 c
Inoculated51.61 b49.44 bc
Coinoculated55.44 a54.91 a
Nitrogen Fertilizer56.82 a53.15 ab
Average53.9951.22
Averages followed by equal letters in the column do not differ from each other according to Tukey’s test at the level of 10% statistical probability. DAEs = days after plant emergence.
Table 4. Length of pods coinoculated with plant growth-promoting bacteria.
Table 4. Length of pods coinoculated with plant growth-promoting bacteria.
TreatmentLength of Pods (cm)
Not Inoculated7.48 a
Inoculated6.86 a
Coinoculated7.47 a
Nitrogen Fertilizer5.78 b
Average6.9
Averages followed by equal letters in the column do not differ from each other according to Tukey’s test at the level of 10% statistical probability.
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Nonato, J.J.; da Silva, T.J.A.; Schlichting, A.F.; Meneghetti, L.A.M.; de Oliveira, N.P.R.; Duarte, T.F.; Guimarães, S.L.; Koetz, M.; Campos e Silva, I.A.; da Silva, P.F.; et al. Water Availability Associated with Coinoculation with Growth-Promoting Rhizobacteria in Cowpea. Nitrogen 2025, 6, 74. https://doi.org/10.3390/nitrogen6030074

AMA Style

Nonato JJ, da Silva TJA, Schlichting AF, Meneghetti LAM, de Oliveira NPR, Duarte TF, Guimarães SL, Koetz M, Campos e Silva IA, da Silva PF, et al. Water Availability Associated with Coinoculation with Growth-Promoting Rhizobacteria in Cowpea. Nitrogen. 2025; 6(3):74. https://doi.org/10.3390/nitrogen6030074

Chicago/Turabian Style

Nonato, Júlio José, Tonny José Araújo da Silva, Alessana Franciele Schlichting, Luana Aparecida Menegaz Meneghetti, Niclene Ponce Rodrigues de Oliveira, Thiago Franco Duarte, Salomão Lima Guimarães, Marcio Koetz, Ivis Andrei Campos e Silva, Patrícia Ferreira da Silva, and et al. 2025. "Water Availability Associated with Coinoculation with Growth-Promoting Rhizobacteria in Cowpea" Nitrogen 6, no. 3: 74. https://doi.org/10.3390/nitrogen6030074

APA Style

Nonato, J. J., da Silva, T. J. A., Schlichting, A. F., Meneghetti, L. A. M., de Oliveira, N. P. R., Duarte, T. F., Guimarães, S. L., Koetz, M., Campos e Silva, I. A., da Silva, P. F., Pacheco, A. B., & Bonfim-Silva, E. M. (2025). Water Availability Associated with Coinoculation with Growth-Promoting Rhizobacteria in Cowpea. Nitrogen, 6(3), 74. https://doi.org/10.3390/nitrogen6030074

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