The Impact of Co-Inoculation with Bradyrhizobium japonicum and Azospirillum brasilense on Cowpea Symbiosis and Growth

Abstract

Biological nitrogen (N) fixation is a well-established practice in various legumes, such as soybeans. However, it has not been widely studied in cowpeas (Vigna unguiculata L. Walp). In this context, it is important to understand how the application of nitrogen-fixing bacteria, either alone or in association, can benefit the crop’s nitrogen demand. This study aimed to determine whether co-inoculation of Bradyrhizobium and Azospirillum favors nodulation and isoflavone production, and increases the nitrogen content, in cowpea crops. The experiment was set up using a randomized block design on two cowpea varieties, with seven treatments consisting of a control and the isolated application of Bradyrhizobium japonicum and Azospirillum brasilense, as well as different co-inoculation doses (75, 150, 225 and 300 mL per 50 kg of seed for each inoculant). There were four replications. Thirty days after emergence, the number of nodules and the dry masses of the nodules, roots and shoots of the plants were assessed. N content and isoflavone content in the fully developed third trifoliate leaf from the apex of the plants were also assessed. Statistical differences were observed between treatments for all analyzed variables, with higher values generally observed for co-inoculation treatments. Co-inoculation of B. japonicum and A. brasilense in cowpea seeds can be a viable and efficient practice. A dose of 75 mL of each inoculant favored nodule formation, root development and N content, as well as contributing to isoflavone production in the cowpea crop.

1. Introduction

The cowpea (Vigna unguiculata L. Walp.) is a legume native to Africa, with high production levels in the region. It is also widely cultivated in agroecological drought zones around the world, including Latin America and South Asia [1]. In recent years, the crop has become popular in several Brazilian regions due to the nutritional value of its grains. It is considered a source of vegetable protein, iron, zinc, carbohydrates, vitamins and amino acids, and is an important component of the diet of populations with lower purchasing power [2]. Also known as string beans or macassar beans, cowpeas are a crop of great socio-economic importance in the north-east of Brazil because they are the main source of vegetable protein for the population, especially the rural population [3].

The supply of high-quality, standardized products has attracted interest from agro-industries in other regions and helped open up new markets for the crop [4]. Cowpeas are moving away from being solely a subsistence crop towards becoming a viable agricultural product. However, low productivity in Brazil is mainly due to low production technology, particularly the inadequate supply of nutrients. In this context, nitrogen (N) is one of the most important agricultural inputs for cowpeas to achieve high productivity.

Bradyrhizobium [5] is one of the most studied genera of plant growth-promoting bacteria (PGPB), whose interaction with legumes is an example of an intensively studied symbiosis. The benefits of this symbiosis for agricultural sustainability are recognized due to the process of biological nitrogen fixation [6]. In contrast, Azospirillum are diazotrophic bacteria that can carry out biological nitrogen fixation (BNF) and promote root and aerial plant growth, primarily through their production of phytohormones such as auxins. They have gained prominence in association with certain grasses, although their use with legumes is well established [7].

When Azospirillum brasilense is used on legumes, the beneficial effect of its association with Rhizobium is largely due to its ability to produce phytohormones, which result in greater root system development and consequently the exploitation of a greater volume of soil [8]. Azospirillum also affects early nodulation by colonizing the roots before Rhizobium and producing flavonoids that attract Rhizobium [9], as well as increasing the number of nodules [10]. Isoflavones are a class of flavonoids that play an important role in the plant defense system, particularly in stressful situations [11,12].

In general, the role of isoflavones in plant nodulation is wide-ranging. They are involved in various stages of the process, including the chemotaxation of Rhizobium bacteria and nodule development through symbiosis with nitrogen-fixing bacteria [13,14]. Studies involving microorganisms and secondary compounds from the isoflavone class are therefore of great interest, particularly in view of the increasing demand for sustainable agricultural models.

It has been demonstrated that combining different microorganisms that interact synergistically can enhance inoculation efficiency and promote the growth and productivity of associated plants, although observing the soil and rhizosphere conditions in which the microorganisms are found is essential [15].

Based on this, the objective of the study was twofold: first, to verify whether co-inoculation with B. japonicum and A. brasilense could benefit cowpea crops and, second, to determine the recommended doses of each bacterium to favor nodulation, nitrogen content and isoflavones in the BRS Tumucumaque and BRS Novaera cultivars.

2. Materials and Methods

This work was carried out at the Experimental Campus of the Federal University of Mato Grosso do Sul (CPCS/UFMS) in Chapadão do Sul during the 2023/24 harvest season. The campus is located at an altitude of 790 m above sea level at the following geographical coordinates: Latitude: 18°41′33″ S″; Longitude: 52°40′45″ W″ of Greenwich. The predominant soil type is dystrophic red latosol, and the climate is humid tropical (Aw) according to the Köppen classification, with a rainy season in summer, a dry season in winter, and an average annual rainfall of 1850 mm. Figure 1 shows local rainfall and temperature data during the experimental period.

Soil analysis was carried out in the area before setting up the experiment (

Table 1. Soil analysis of the experimental area.

Depth cm	pH CaCl2	Al	Ca + Mg	P	K	SB	CTC	OM	V	m
		cmolc·dm−3		mg·dm−3		cmolc·dm−3		g·dm−3	%
0–20	5.0	0.07	4.10	21.9	99	4.35	8.4	26.4	51.5	1.6
20–40	4.7	0.09	2.00	5.1	83	2.21	6.9	19.8	32	3.9

).

Fertilization was carried out in accordance with the recommendations for cultivating cowpeas, with no nitrogen fertilization [16]. Liming was unnecessary, and minimum tillage was used for soil preparation. The crop was planted at the end of October, at the time of the first harvest. The experimental design used was a randomized block design with seven treatments and four replications. The plots consisted of five rows, each six meters long and spaced 0.45 m apart. The three central rows were considered the useful area, disregarding 0.5 m from each end. A density of 200,000 plants per hectare was used, with nine plants per meter.

The varieties used were BRS Tumucumaque and BRS Novaera, which were launched by Embrapa Meio-Norte and are recommended for the Midwest region. The treatments consisted of single and combined inoculations of B. japonicum (Gelfix 5^® SEMIA 5079 and SEMIA 5080—5 × 10⁹ CFU mL⁻¹) and A. brasilense (Masterfix L^® Abv5 and Abv6—5 × 10⁹ CFU mL⁻¹) in the seed treatment. The doses were applied to 50 kg of seed as follows: T1—control (no inoculation); T2 (Brady)—inoculation with B. japonicum (150 mL); T3 (Azo)—inoculation with A. brasilense (150 mL); T4 (B+A75)—B. japonicum + A. brasilense (75 mL + 75 mL); T5 (B+A150)—B. japonicum + A. brasilense (150 mL + 150 mL); T6 (B+A225)—B. japonicum + A. brasilense (225 mL + 225 mL); and T7 (B+A300)—B. japonicum + A. brasilense (300 mL + 300 mL). The choice of doses was based on the recommended dose (150 mg kg⁻¹) for legumes such as soybean because there is no recommendation for cowpea. So we used half the dose (75 mg kg⁻¹), one and a half doses (225 mg kg⁻¹) and double the dose (300 mg kg⁻¹).

The seeds were treated with Standak Top^® and Protreat^® to protect against pests and diseases that could hinder the crop’s establishment. For the experimental area, 3 kg of seeds was used for each treatment. Two hours before sowing, the seeds were placed in plastic bags, and the corresponding dose for each treatment was added. The bags were then shaken gently for two minutes to ensure the seeds were evenly distributed. The seeds were then distributed onto trays lined with germination paper and left to dry for one hour. The seeds were planted manually according to the layout of the treatments.

Thirty days after emergence (DAE), two meters of each plot’s border row were entered and twenty plants were carefully harvested and removed from the soil to preserve the root system as much as possible. The number of nodules (NN), nodule dry mass per nodule (NDM), dry root mass (RDM) and shoot dry mass (SDM) were then evaluated. The NN of each plant was counted, and the average was calculated. To determine the dry phytomass, the root systems, shoots and nodules of the plants were placed in paper bags and dried in a forced-air oven at 65 °C until they reached a constant mass. They were then weighed using a precision scale with a 0.0001 g capacity.

To determine the nitrogen content, the third fully developed trefoil leaf from the apex of the plant was harvested and washed in running water. It was then placed in solutions of 0.1% neutral detergent, 0.3% hydrochloric acid and deionized water [17]. The plant material was then dried in a forced air circulation oven at 65 ± 5 °C until a constant weight was obtained, after which the dry matter of the cowpea leaves was measured. After drying, the material was ground and weighed (0.1 g), following the Kjeldahl titration technique. It was then digested, distilled and titrated with sulphuric acid (H₂SO₄), after which the N content of the extracts obtained by sulphuric digestion was determined.

To isolate the isoflavones, 50 mg of dried and ground plant leaves was placed in a 2 mL Eppendorf tube containing 1.5 mL of 70% methanol supplemented with 0.1% acetic acid. The mixture was briefly vortexed and then incubated in an ultrasonic bath for 2 h. After sonication, the samples were centrifuged at 5000 rpm for 15 min. The resulting supernatant was passed through a 0.2 µm syringe filter and transferred to 1.5 mL vials for analysis by ultra-performance liquid chromatography (UPLC). A volume of 10 µL from each sample was injected into the UPLC system in triplicate [18].

Isoflavone separation and quantification were performed using a Waters Acquity 1100 Series UPLC system equipped with an automatic sample injector. Chromatographic separation was achieved using a reverse-phase HSS C18 column (1.8 µm particle size, 2.1 mm × 100 mm), preceded by a matching guard column (1.8 µm, 2.1 mm × 5 mm). A binary linear gradient system was employed with the following mobile phases: Solvent A consisted of Milli-Q water with 0.1% acetic acid, and Solvent B comprised acetonitrile with 0.1% acetic acid. The gradient profile was set as follows: from 0 to 9 min, 99% A and 1% B; from 9 to 9.1 min, 41.2% A and 58.8% B; from 9.1 to 11 min, 100% B; returning to initial conditions (99% A and 1% B) at 11 min and maintained until 15 min, completing the run time [18]. The mobile phase flow rate was 0.289 mL/min, and the column temperature was maintained at 30 °C throughout the analysis.

A Waters photodiode array detector, set to 254 nm, was used to identify the isoflavones. Commercially purchased standards of daidzein (D1), daidzin (D2) and genistin (G1) were solubilized in 70% methanol and used to detect the isoflavones at the following concentrations: 0.000125, 0.0002, 0.0005, 0.001, 0.01 and 0.02 mg/mL. The qualitative and quantitative identity of each peak was confirmed by comparing its retention time and UV spectrum with those of the individual standards using the standard addition method.

All the solvents used in the chromatographic analysis were HPLC grade. Before use, they were vacuum filtered through a 0.2 µm pore membrane, then degassed in a vacuum system using ultrasound. The water used was distilled, ultra-purified in a Milli-Q system, and degassed.

The varieties were analyzed separately. To verify normality and homoscedasticity, the data were analyzed using the Shapiro–Wilk and Levene tests, respectively. The data were then submitted to analysis of variance, and the means were compared using the Scott–Knott test at a 5% significance level. Multivariate principal component analysis was performed on the data, using R software, version 3.5.1 [19].

3. Results

3.1. Quantification of Number of Nodules, Nodules Dry Mass and Root Dry Mass

The inoculation and co-inoculation treatments had a significant effect on NN, NDM, RDM and SDM for both varieties. The B+A75 treatment provided greater NN for both varieties, although it did not differ from B+A225 for BRS Novaera (Figure 2A,B). For NDM (Figure 2C,D) and RDM (Figure 2E,F), the B+A75, B+A150 and B+A225 treatments performed better for the BRS Tumucumaque variety, while the B+A75 and B+A225 treatments were statistically superior for the BRS Novaera variety.

3.2. Determination of Shoot Dry Mass and Nitrogen Content

For the BRS Tumucumaque variety, the B+A75, B+A150 and B+A225 treatments produced statistically higher results for SDM. For the BRS Novaera variety, however, the Bradyrhizobium treatments B+A75 and B+A225 produced better results (Figure 3A,B). For N content (Figure 3C,D), the B+A75, B+A150 and B+A225 treatments performed best for the BRS Tumucumaque variety, while the B+A75 and B+A225 treatments showed higher N values for the BRS Novaera variety.

3.3. Quantification of Daidzein, Daidzin, Genistin and Total Isoflavones

In the BRS Tumucumaque variety, treatment B+A75 produced the best results for daidzein (Figure 4A), daidzin (Figure 4C), genistin (Figure 4E) and total isoflavones (Figure 4G). In the BRS Novaera variety, the best performing treatments for daidzein were B+A75, B+A225 and B+A300 (Figure 4B); for daidzin, it was B+A75 (Figure 4D); for genistin, it was B+A75 and B+A150 (Figure 4F); and for total isoflavones, it was B+A75 (Figure 4H).

The results of the principal component analysis (PCA) are shown in the graph, where the direction and length of the arrows indicate the variable loadings, i.e., how each variable contributes to the principal components. A variable with a high loading on a specific component is strongly correlated with that component. This shows which variables significantly impact data variations. The vectors of NN, NDM, N, RDM and SDM were closer to the B+A75, B+A150 and B+A225 treatments, indicating a strong relationship between these treatments and the analyzed variables, unlike the treatments with B. japonicum, A. brasilense, B+A300 and the control, which occupy opposite quadrants with no relation to the BRS Tumucumaque dataset (Figure 4A). For BRS Novaera, N, NDM, NN and SDM had vectors close to the B+A75 treatment, indicating that this treatment was superior for the analyzed variables; however, MSR was arranged in the lower quadrant, closer to the B+A225 treatment (Figure 4B).

Conversely, the treatments involving B. japonicum, A. brasilense, B+A150 and B+A300, as well as the control, exhibited vectors at opposite ends, suggesting an inverse relationship within the dataset (Figure 4B). Notably, the percentage of variation explained by the first two components accounted for over 90% of the total variation in the data (Figure 4A,B).

PCA condenses information from multiple original variables into a smaller number of components while preserving as much of the variation in the data as possible. This makes it easier to visualize and interpret high-dimensional data sets. In the PCA for isoflavones, the vectors for D1, D2, G1 and Isotot in the BRS Tumucumaque variety were close to treatment B+A75. This indicates that this treatment was most closely related to the analyzed variables. Treatments B+A150 and B+A300 showed little proximity to the variables analyzed, being scattered in the lower quadrant. The same was true of treatments involving B. japonicum, A. brasilense, B+A225 and the control; these were in opposite quadrants to B+A75 (see Figure 5A).

For the BRS Novaera variety, the D1 and D2 variables, as well as the Isotot, were close to the B+A75 treatment vector, while the G1 variable showed a similar distance for the B+A150 and B+A300 treatments. Finally, treatments involving isolated bacteria (B. japonicum and A. brasilense) as well as B+A225 and the control were positioned in opposite quadrants (Figure 5B), indicating a negative relationship with the presented variables. As in Figure 4A,B, the percentage of variation explained by each component is notable, with the first two components together accounting for over 90% of the total variation in the data.

4. Discussion

Nitrogen (N) plays a fundamental role in plant metabolism and is one of the most essential elements for plants. It is involved in the synthesis of nucleic acids, proteins, and other nitrogenous compounds. N-fixing bacteria, such as B. japonicum and Azospirillum, are therefore valuable allies in the cultivation of legumes such as soybeans and cowpeas [20,21]. The nitrogen content may be associated with the number of nodules and the vegetative growth of the aerial and root parts, given that they have greater dry phytomass. This was observed in the B+A75 treatment (Figure 2 and Figure 3). Similar data was found by [22] when Bradyrhizobium was used as an N-fixing bacterium in cowpea cultivation. Ref. [23] emphasized the importance of using bio-inputs in agriculture due to their ability to maximize various plant characteristics, such as root and nodule growth, thereby improving nutrient absorption and utilization from the soil. Soybeans exhibited enhanced nodulation performance, resulting in a 35% increase in yield and reduced greenhouse gas emissions through co-inoculation [24]. These results are consistent with those of the present study, in which nodulation doubled in the B+A75 treatment compared to the B. japonicum treatment alone for the BRS Tumucumaque variety, and increased by 25% for the BRS Novaera variety (Figure 2).

Several studies currently report co-inoculation as an advantageous technique. One study found a significant increase in plant growth and production parameters in the common bean crop through co-inoculation with Bradyrhizobium and Rhizobium tropici strains [25]. The associated use of Bacillus subtilis in cowpea genotypes with acidic water from an abandoned gold mine in Africa resulted in a significant increase in nodulation, as well as in the fresh and dry root mass, demonstrating that the effects of applying microorganisms are not limited to nutritional parameters, but can also tend to be beneficial in extreme situations, such as contaminated soils [26].

Isoflavones are related to nodulation because they are secreted by the roots, establishing an association with the rhizobia and helping to form nodules by activating their biosynthesis [27,28]. This may be related to treatment B+A75, in which a higher number of nodules and isoflavone content were obtained (Figure 2, Figure 4, Figure 5 and Figure 6). Inoculation and co-inoculation are complementary tools for cultivating established cowpea varieties on the market when aiming to increase the quantity and quality of secondary metabolites from the isoflavone group and their antioxidant properties.

For example, some studies show that isoflavones from the daidzein group play a significant role in nodulation factors, such as promoting bacterial growth in isolated cases in vitro and inducing gene expression to promote the growth of B. japonicum bacterial cells [29,30]. In this case, the similar trend observed in the principal component analysis of the B+A75 isoflavone treatment, as shown in Figure 5 and Figure 6, is justifiable. Other studies also emphasize the importance of researching the nutritional aspects of micronutrients such as nickel and beneficial elements such as selenium in increasing isoflavone production, particularly daidzein [31,32]. This reinforces the need for a more complex exploration of soil analysis, particularly for crops that benefit from biological nitrogen fixation, such as cowpea, to produce more resilient plants in the field. When using the isoflavone genistin in soybean plants to relieve salt stress, an increase in the number of nodules and shoot dry mass of the crop was found with B. japonicum [33]. This may be associated with the transcription of genes related to the biosynthesis of isoflavones, as seen in this study. Here, NN and SDM values were higher when genistin content increased, with a similar trend observed for both (see Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6). In Sophora flavescens plants, Ref. [34] identified that nodulation biosynthetic pathways enrich the biosynthesis of metabolites such as flavonoids, particularly isoflavones. This is a relevant factor that adds to the exploration of studies in Fabaceae family plants such as cowpea due to their nodulation capacity.

When it comes to secondary metabolites, isoflavones are becoming increasingly important in research due to their potential as food supplements for improving human and animal health, which determines the optimization of these compounds in plants [35]. Ref. [36] demonstrated that genetics considerably influences the isoflavone content of cowpea crops, with varieties containing higher levels of these compounds being identified due to the pigmentation of the seed coat. A similar observation was made in this study: the isoflavone content of the BRS Tumucumaque variety was much higher than that of the BRS Novaera variety, demonstrating the significant influence of genetics on the production of secondary plant compounds (Figure 4C).

The fact that the highest amount of isoflavones was associated with the B+A75 treatment is probably due to greater nodule production and root development in the crop. These compounds are produced in the nodule infection zone, and rhizobacteria are involved in producing phytohormones such as auxins [37,38]. This relationship is supported by the mechanism of isoflavones in auxin transport, whereby binding to transport proteins of this hormone increases auxin transport when nodules appear on plant roots [14].

The plant rhizosphere is home to various types of bacteria, some of which are pathogenic and some of which are beneficial. However, this environment can become competitive for the survival of these organisms, with various means of intra- and interspecific confrontation being employed [39]. Thus, the B+A75 co-inoculation possibly guaranteed a sufficient number of GPBP cells to minimize competition between them. In the B+A300 treatment, the values of most variables decreased, which can be attributed to competition arising from the large number of bacterial colonies. Furthermore, Ref. [5] found that co-inoculation with B. japonicum and A. brasilense also benefited soybean crop development by increasing production at lower doses.

Some studies have explored the potential of cowpea crops and shown the beneficial effects of microbial associations. The cowpeas showed a significant response to the co-inoculation of Bacillus, Brevibacillus and Paenibacillus strains with Bradyrhizobium sp. [40]. Co-inoculation of Bradyrhizobium and Bacillus subtilis increased nodulation in cowpea. The symbiotic efficiency of the joint colonization of cowpea by Paenibacillus and Bradyrhizobium strains using different inoculation methods was found to be variable [41].

There is a growing demand for sustainable agricultural practices. Reducing the use of inorganic fertilizers, particularly nitrogen fertilizers, has emerged as an alternative way of mitigating greenhouse gas emissions on farms.

5. Conclusions

In light of the above, this experiment demonstrated that the co-inoculation of B. japonicum and A. brasilense in cowpea seeds can be a viable and efficient practice at various dosage levels. A dose of 75 mL of each inoculant increased the number of nodules and root development, as well as the N content. It also contributed to the production of isoflavones in cowpea crops of the BRS Tumucumaque and BRS Novaera varieties. Similarly, a dose of 225 mL of each inoculant favored nodulation factors and the morphological aspects of roots and shoots; however, consistency was not obtained for the evaluated isoflavone classes. Finally, treatments using solitary bacteria were inferior to the others due to the variations observed, confirming the benefits of co-inoculation. However, to confirm the hypothesis of this study, future studies are suggested that cover more years of cultivation, other environments and other cowpea cultivars.