Effect of Rhizobium spp. Inoculation on Growth and Grain Yield of Cowpea (Vigna unguiculata) Under Greenhouse and Ex Vitro Conditions

1. Introduction

Nitrogen is an essential element for plant growth because it forms part of amino acids, proteins, nucleotides, nucleic acids, chlorophyll, and numerous other compounds involved in plant metabolism. Although molecular nitrogen (N₂) is abundant in the atmosphere, it cannot be directly assimilated by most plants. Biological nitrogen fixation therefore plays an essential ecological and agricultural role by transforming atmospheric nitrogen into biologically available forms (Baca et al., 2000; Paredes, 2013).

Among nitrogen-fixing microorganisms, bacteria associated with legumes are especially important. Species traditionally classified within Rhizobium establish symbiotic interactions with members of the Fabaceae and stimulate the development of root nodules where biological nitrogen fixation occurs (Lloret & Martínez-Romero, 2005; Nápoles et al., 2007; Villanueva Tarazona & Quintana Díaz, 2012).

The interaction begins through molecular signaling between the plant and bacterial symbiont. Flavonoids and other compounds released by legume roots activate bacterial nodulation genes, stimulating infection of root hairs and subsequent nodule development (Dusha et al., 1986; Nápoles et al., 2007; Kawaharada et al., 2015; Nápoles García et al., 2016). Within functional nodules, nitrogenase catalyzes the reduction of atmospheric nitrogen, producing nitrogen compounds that can ultimately be incorporated into plant metabolism (Laane et al., 1978; Wang et al., 2001).

The effectiveness of this symbiotic process can be influenced by environmental factors. Soil moisture, temperature, pH, nutrient availability, and other physicochemical conditions can alter microbial activity and the establishment of effective nodulation (Ramos & Zúñiga, 2008; Rachwal et al., 2015).

Modern agricultural production commonly relies on inorganic fertilizers containing nitrogen, phosphorus, and potassium to increase crop productivity. Although fertilizer applications can increase yield, excessive or continuous use can modify soil conditions, microbial populations, nutrient cycling, and the biological processes associated with soil fertility (Pérez et al., 2008; Ramos Vásquez & Zúñiga Dávila, 2008; Mendoza Labrador & Bonilla Buitrago, 2014).

Continuous dependence on inorganic fertilizers may therefore have both economic and environmental consequences. Nutrients that are not absorbed by the crop can remain in the soil or be transported into surrounding ecosystems, potentially affecting soil and water quality. These considerations have increased interest in agricultural systems that integrate ecological processes with crop production.

Agroecological approaches seek to combine agricultural productivity with the conservation and sustainable management of ecological resources. Biological inoculation, organic amendments, and manipulation of beneficial soil microorganisms are among the strategies evaluated as alternatives or complements to conventional fertilization systems (Restrepo et al., 2000; Sivila de Cary & Angulo, 2006; Dibut et al., 2009).

Cowpea (Vigna unguiculata (L.) Walp.) is particularly relevant for this purpose. The species is cultivated extensively throughout tropical and subtropical regions and is characterized by its adaptability to warm climates, relatively low-fertility soils, and periods of reduced water availability (Beaver & Flores, 2005; Hernández González et al., 2012).

Cowpea is also capable of establishing effective symbiotic relationships with nitrogen-fixing bacteria. Previous investigations have demonstrated positive responses of V. unguiculata to rhizobial inoculation, including improvements in plant development, biological nitrogen fixation, and nutrient acquisition (Mayz et al., 2010; Mendoza Labrador & Bonilla Buitrago, 2014; Nyoki & Ndakidemi, 2014).

Nitrogen management can nevertheless influence cowpea productivity. Abayomi et al. (2008) reported responses of cowpea genotypes to NPK fertilization, while Apáez Barrios et al. (2013) demonstrated that nitrogen and phosphorus management can influence agronomic efficiency and crop production.

The nutritional status of the plant may also modify symbiotic nodulation. When nitrogen is readily available in the soil, the physiological advantage of maintaining an energetically expensive nitrogen-fixing symbiosis may decrease. Consequently, evaluating fertilization and bacterial inoculation simultaneously is important when developing sustainable nutrient-management strategies.

Cowpea also faces biological limitations. The cowpea aphid, Aphis craccivora, can adversely affect plant development and yield (Annan et al., 1997; Obopile & Ositile, 2010; Pava & Sepúlveda-Cano, 2015). This factor is particularly relevant when evaluating plants under semi-controlled or natural environmental conditions.

The objective of the present study was therefore to determine the effects of Rhizobium spp. inoculation, inorganic fertilization, and their combination on nodulation, biomass production, pod production, grain yield, and seed weight of cowpea under greenhouse and ex vitro conditions.

It was hypothesized that inoculation with Rhizobium spp. would improve the agronomic performance of cowpea relative to uninoculated plants and that inorganic fertilizer application could modify the effectiveness of the plant–Rhizobium symbiotic association.

2. Materials and Methods

2.1. Study Site and Experimental Conditions

The investigation was conducted at the Pontifical Catholic University of Puerto Rico, Ponce Campus, Puerto Rico.

The research was divided into three principal experimental stages: microbiological laboratory procedures, greenhouse experiments, and cultivation under natural environmental conditions designated in the original study as the ex vitro experiment.

The experimental period extended from August 2016 through August 2017.

Laboratory procedures were performed in the microbiology facilities of the university. Greenhouse experiments were conducted using controlled irrigation, while the ex vitro experiment was established outdoors to provide natural conditions of light and environmental exposure.

The ex vitro experimental approach was adapted from methodologies used for the establishment of cowpea plants outside conventional in vitro environments (Mellor et al., 2012).

2.2. Experimental Design

Four experimental groups were established:

Group 1 – Control: Plants not inoculated with Rhizobium spp. and without inorganic fertilizer.

Group 2 – Fertilizer: Plants not inoculated with Rhizobium spp. and receiving inorganic fertilizer.

Group 3 – Fertilizer + Rhizobium: Plants inoculated with Rhizobium spp. and receiving inorganic fertilizer.

Group 4 – Rhizobium: Plants inoculated with Rhizobium spp. without inorganic fertilizer.

A total of 400 plants were included throughout the experimental study. Experimental units were distributed among greenhouse and ex vitro conditions using an interspersed block arrangement.

2.3. Isolation and Preparation of Rhizobium spp.

Active root nodules were obtained from cowpea plants and processed for bacterial isolation.

Root nodules were surface-disinfected before isolation using procedures based on previously reported rhizobial isolation techniques (Villanueva Tarazona & Quintana Díaz, 2012).

Yeast Mannitol Agar (YEMA) was used for bacterial isolation. Plates were incubated at 30°C for six days. The use of YEMA and subsequent culture characterization is consistent with procedures commonly employed for isolation and biochemical characterization of rhizobia (Niste et al., 2015).

Colonies obtained from the isolation procedure were transferred to Yeast Mannitol Broth (YEMB). The bacterial cultures were subsequently used for preparation of the inoculum.

2.4. Seed Inoculation

Cowpea seeds of the cultivar Gorda were used. This cultivar was developed and characterized for cultivation in Puerto Rico (Beaver & Flores, 2005).

Seeds assigned to inoculated treatments were exposed to 100 mL of bacterial inoculum for 1 h and 45 min before planting.

Seeds assigned to the non-inoculated treatments received distilled water rather than bacterial inoculum.

The use of bacterial inoculation during the early stages of seed germination was intended to facilitate contact between the developing root system and rhizobial microorganisms. Cowpea germination and early development under controlled conditions have previously been described by Díaz et al. (2007).

2.5. Plant Establishment

Plants were established using a commercial Pro-Mix germination substrate.

Greenhouse plants were maintained under controlled irrigation and illumination conditions. Ex vitro plants were maintained under natural environmental conditions with controlled irrigation.

The use of containers under ex vitro conditions provided an experimental approximation of field cultivation while maintaining control over individual experimental units.

2.6. Fertilization

Plants assigned to fertilizer treatments received inorganic fertilizer according to the experimental protocol established in the original study.

Fertilizer calculations were based on crop density and nutrient application procedures adapted from Escalante Estrada et al. (2006).

The purpose of the fertilizer treatments was to compare conventional inorganic nutrient supplementation with biological nitrogen acquisition resulting from rhizobial inoculation.

2.7. Evaluation of Nodulation

Root systems were evaluated for the presence of active nodules. Functional nodules were identified principally by their red or pink internal coloration associated with active symbiotic structures.

Nodule development has been widely used as an indicator of successful rhizobial infection and biological nitrogen fixation in legumes (Nápoles et al., 2007; Idris Hassen et al., 2014).

2.8. Plant Growth and Biomass

Plant height and basal diameter were recorded.

Total plant biomass was determined using fresh and dry biomass measurements. Plant material was dried at approximately 60°C for 20 days to determine dry biomass.

Dry biomass procedures followed general approaches previously applied to plant biomass assessment (Fonseca et al., 2009).

The treatment effectiveness index was determined by comparing biomass production under each treatment with that of the untreated control.

Previous investigations have used biomass measurements to evaluate growth responses associated with microbial inoculation and beneficial rhizosphere microorganisms (Santillana et al., 2005; Neyra Vazallo et al., 2013).

2.9. Pod Production and Grain Yield

The total number of pods produced within each treatment was recorded.

Grain yield was determined from the total weight of harvested cowpea seeds.

The relationship between pod number and grain yield was also evaluated.

Yield responses in cowpea can be affected by genotype, fertilization, environmental conditions, and nutrient availability (Abayomi et al., 2008; Fagwalawa & Yakasai, 2013).

2.10. One-Hundred-Seed Weight

Seed size was evaluated using the weight of 100 seeds (P100G).

The categories established in the original experimental protocol were:

• Large seeds: >25.00 g per 100 seeds

• Medium seeds: 18.00–25.00 g

• Small seeds: <18.00 g

2.11. Harvest Index

Harvest index was calculated as the relationship between grain yield and total plant biomass:

Harvest Index (%) = (grain yield / total biomass) × 100

This parameter was used as an indicator of the proportion of plant biomass allocated to harvested grain.

Relationships between vegetative biomass and seed production have previously been evaluated in grain-legume cropping systems (Morales Rosales et al., 2007).

2.12. Agronomic Nitrogen Efficiency

Agronomic nitrogen efficiency was calculated to evaluate the relationship between nutrient availability and grain production.

Nitrogen-use efficiency provides an important indicator for comparing agricultural nutrient-management strategies (Apáez Barrios et al., 2013).

2.13. Statistical Analysis

Descriptive analyses and tests of data distribution were performed before comparisons among experimental treatments.

Analysis of variance (ANOVA) was used to evaluate differences among the four experimental groups. Tukey's multiple comparison procedure was subsequently used to evaluate differences among treatments.

Linear regression and correlation analyses were used to evaluate relationships among selected agronomic variables, including plant dimensions, biomass, pod number, and grain yield.

Statistical significance was evaluated using a 95% confidence level.

3. Results

3.1. Nodulation

Substantial differences in active root nodulation were observed between inoculated treatments.

Plants receiving inorganic fertilizer and Rhizobium spp. inoculation produced markedly fewer active nodules than plants inoculated with Rhizobium spp. without inorganic fertilizer.

The fertilizer + Rhizobium treatment produced 15 active nodules, whereas the Rhizobium-only treatment produced 2,533 active nodules during the corresponding greenhouse assessment.

This marked difference indicates that fertilizer availability was associated with a considerable reduction in nodulation.

3.2. Biomass Production

Treatment significantly influenced plant biomass.

Under ex vitro conditions, fresh biomass was:

• Control: 2.74 kg

• Fertilizer: 4.44 kg

• Fertilizer + Rhizobium: 5.91 kg

• Rhizobium: 6.13 kg

Relative to the control, the treatment effectiveness index for fresh biomass was approximately:

• Fertilizer: 62%

• Fertilizer + Rhizobium: 115%

• Rhizobium: 123%

Dry biomass was:

• Control: 0.511 kg

• Fertilizer: 0.684 kg

• Fertilizer + Rhizobium: 0.710 kg

• Rhizobium: 0.899 kg

The corresponding increases in dry biomass relative to the control were approximately:

• Fertilizer: 8%

• Fertilizer + Rhizobium: 38%

• Rhizobium: 75%

Thus, inoculation with Rhizobium spp. without inorganic fertilizer produced the greatest overall biomass response.

3.3. Pod Production

Pod production differed among treatments.

Under ex vitro conditions, mean pod production was:

A positive relationship was observed between pod number and grain yield.

3.4. Grain Yield

Grain yield also increased in response to the experimental treatments.

Mean grain yield was:

Statistical comparison using Tukey's procedure indicated significant differences between treatments and the control.

3.5. One-Hundred-Seed Weight

The 100-seed weight increased progressively across the experimental treatments:

3.6. Harvest Index

Harvest index showed a different pattern from total biomass production.

The harvest indices were approximately:

• Control: 49%

• Fertilizer: 37%

• Fertilizer + Rhizobium: 32%

• Rhizobium: 31%

The lower harvest index associated with high-biomass treatments reflects a greater proportional allocation of plant production to vegetative biomass.

Similar reductions in harvest index associated with increased nitrogen availability and vegetative biomass have previously been described (Morales Rosales et al., 2007; Apáez Barrios et al., 2013).

3.7. Agronomic Nitrogen Efficiency

Agronomic nitrogen efficiency differed among treatments:

4. Discussion

The results demonstrate that biological inoculation with Rhizobium spp. can substantially influence cowpea development and productivity.

The most striking response occurred in active nodulation. Plants inoculated exclusively with Rhizobium spp. showed markedly greater nodule production than plants simultaneously receiving Rhizobium spp. and inorganic fertilizer.

Nodulation represents a highly regulated biological process involving chemical recognition between bacteria and the host plant (Nápoles et al., 2007; Kawaharada et al., 2015; Nápoles García et al., 2016). Once an effective association is established, rhizobial bacteria differentiate within plant tissues and participate in nitrogen fixation (Lloret & Martínez-Romero, 2005; Villanueva Tarazona & Quintana Díaz, 2012).

The reduced nodulation observed in fertilized plants suggests that nutrient availability influenced the establishment or maintenance of the symbiotic interaction. Similar research has demonstrated that plant nutritional status and environmental conditions can influence rhizobial activity and nitrogen fixation (Ramos & Zúñiga, 2008; Mendoza Labrador & Bonilla Buitrago, 2014).

The response of biomass provides further evidence of the agronomic effect of inoculation. Fresh biomass increased by 123% and dry biomass by 75% in the Rhizobium-only treatment compared with the control.

Beneficial rhizobial interactions have previously been associated with enhanced plant growth and nutrient acquisition (Santillana et al., 2005; Mayz et al., 2010; Neyra Vazallo et al., 2013).

Nyoki and Ndakidemi (2014) similarly reported improved macronutrient uptake in cowpea following inoculation with nitrogen-fixing bacteria, supporting the hypothesis that effective symbiosis can enhance plant nutritional status.

The greatest number of pods was also obtained from plants inoculated exclusively with Rhizobium spp. Pod number was positively associated with grain production, indicating that treatment-induced improvements in reproductive development contributed directly to crop productivity.

Previous studies have demonstrated that nitrogen availability influences cowpea growth and yield components (Abayomi et al., 2008), while environmental factors such as light intensity can also affect cowpea grain production (Fagwalawa & Yakasai, 2013).

Seed weight provided another important indicator of treatment response. The control produced a mean 100-seed weight of 18.05 g, whereas the Rhizobium-only treatment reached 25.07 g.

The cultivar Gorda used in this study was previously characterized as a cowpea cultivar suitable for Puerto Rican agricultural conditions (Beaver & Flores, 2005). The observed response indicates that inoculation may contribute to improved seed development in this cultivar.

The highest agronomic nitrogen efficiency was also observed in the Rhizobium-only treatment. Nitrogen-use efficiency is an important parameter when considering the economic and environmental sustainability of nutrient-management practices (Apáez Barrios et al., 2013).

The lower efficiency observed when inorganic fertilizer and Rhizobium spp. were combined suggests that simply combining biological and conventional nutrient sources does not necessarily produce an additive response.

This finding is especially relevant to sustainable agriculture because biological nitrogen fixation could potentially reduce dependence on repeated inorganic nitrogen applications.

Agroecological practices seek to maintain agricultural productivity by increasing reliance on ecological processes and minimizing unnecessary external inputs (Restrepo et al., 2000; Sivila de Cary & Angulo, 2006). From this perspective, promoting effective legume–rhizobia interactions may contribute to nutrient cycling and long-term soil management.

Another important observation was the presence of aphids during the greenhouse experiment. Aphis craccivora is a recognized pest of cowpea capable of reducing plant productivity (Annan et al., 1997; Obopile & Ositile, 2010; Pava & Sepúlveda-Cano, 2015).

The original experiment observed differences in plant survival among treatments during pest pressure. However, additional controlled studies would be necessary before establishing a causal relationship between Rhizobium inoculation and aphid resistance.

The results should therefore be interpreted within the conditions of the present study. Additional replicated field experiments using different soils, environmental conditions, cowpea cultivars, and characterized bacterial strains would be valuable to determine whether the observed responses are reproducible at larger agricultural scales.

5. Conclusions

The experimental results demonstrate that inoculation with Rhizobium spp. influenced several important agronomic characteristics of cowpea (Vigna unguiculata).

Plants inoculated without inorganic fertilizer developed substantially greater active nodulation and exhibited the greatest fresh and dry biomass production.

The Rhizobium-only treatment also produced the greatest number of pods, the greatest 100-seed weight, and the highest calculated agronomic nitrogen efficiency.

The combination of inorganic fertilizer and Rhizobium spp. produced greater grain yield than the untreated control; however, it was associated with substantially reduced nodulation compared with bacterial inoculation alone.

These results support the potential application of Rhizobium spp. inoculation as an agroecological strategy for improving cowpea production while potentially reducing dependence on inorganic nitrogen fertilization.

Further experiments should evaluate this response under field-scale conditions, characterize the bacterial strain at species or molecular level, quantify biological nitrogen fixation directly, determine soil nutrient dynamics, and evaluate the economic feasibility of inoculation compared with conventional fertilization.