Improvement of yield parameters of cowpea (Vigna unguiculata) by rhizobia sp. isolated from Mucuna pruriens and Centrosema pubescens at Ile-Ife, Nigeria

Abstract

This research was conducted to isolate, authenticate, and assess the symbiotic effectiveness of cowpea nodulating rhizobia isolated from Centrosema pubescens and Mucuna pruriens at different locations in Ile-Ife, Nigeria with two varieties of cowpea (Ife BPC and Ife Brown). Thirteen Rhizobium and three Bradyrhizobium species were isolated and all of them significantly enhanced nodulation with Ife brown and Ife BPC cowpeas having 50% and 81.25% effective nodules formation, respectively. The inoculation of the cowpea with Bradyrhizobium sp (C7) increased the yields significantly with Ife brown recording 73.92 g, while Ife BPC had 58.14 g. The symbiotic relationship between the rhizobia species and the two varieties of cowpea increased the soil fertility with nitrogen concentration in the soil increasing to 84.28 mg/g for Ife brown and 55.89 mg/g for Ife BPC. All the sixteen rhizobia isolates were resistant to Carbendazim 12% + Mancozeb 63% W/P; 2,3 Dichlorovinyl dimethyl phosphate; Chlorpyriphos; Atrazine and 2,3 Dimethylamine. In contrast, four Rhizobium sp. were sensitive to Glyphosate at 14.4 mg/ml, while paraquat had inhibitory effect on 14 out of the 16 rhizobial species at 2.76 mg/ml. This study concluded that the rhizobia isolates improved the cowpea yield and also enriched the soil compared to the Nitrogen, Phoshorus and potassium (NPK) fertilized soil.

Mucuna pruriens, Centrocema pubescens, cowpea, Rhizobium and Bradyrhizobium species

Sustainability Statement

This research responds to the Sustainable Development Goal 2 and 15: Zero Hunger and Life on Land. Cowpea is a highly nutritious plant for humans and animals and also enhance soil fertility. However, its productivity is limited due to factors such as increase in population, climate change, drought, unrestrained usage of nitrogen fertilizers, and low soil nutrients, etc. This study provides significant information on the usage of Rhizobium and Bradyrhizobium sp. from wild legumes to improve yield of two varieties of cowpea (Ife BPC and Ife Brown). The symbiotic relationship between the cowpea and rhizobia species enhanced nitrogen fixation and enrich the soil with nutrients thereby encouraging viable agriculture and guarantee food protection.

Introduction

Improving the composition of nitrogen fixing bacteria in the soil is vital for some Sub-Saharan African Countries to achieve the sustainable development goals of the United Nations of zero hunger and sustainable food security and production. In most sub-Saharan African countries, farmers are faced with the challenge of low productivity in crops due to the low availability of the nitrogen in the soils, which is also the lowest among all the world regions. Intensive farming of the past 30 years without replenishing the soil nutrients has created a dire crisis where the soil is left with a deficit of approximately 22 kg of nitrogen, 2.5 kg of phosphorus, and 15 kg of potassium per hectare annually with an annual estimate of US$4 billions’ worth of fertilizer (Gilbert 2012).

Nitrogen is the most abundant gas in the air, with its biological fixation playing a substantial role in the nitrogen content of the soil and hence in plant growth and development (Dixon and Kahn 2004). Its deficiency in the soil and the crop adversely affects plant growth and yield in small-holder farms in Africa (Liu et al. 2010). Nitrogen in the soil is lost through microbial denitrification, soil erosion, leaching, chemical volatilization, and the removal of nitrogen containing crop residues from the land; therefore, it is the most limiting plant nutrient for crop production in West Africa (Nyoki and Ndakidemi 2018). Farmers commonly use synthetic fertilizers to improve the fertility of the soil, but the disadvantages of their application are numerous when applied over a long period. Some of these disadvantages include acidification of the soil, leaching, reduction in efficiency, and high dependence of soil on N2 (Vejan et al. 2021). There is a call for sustainable improvement in crop productivity in a way that is friendly to the environment, but the most and inexpensive one is using rhizobia inoculants which provide atmospheric nitrogen to the crop. (Vejan et al. 2021, Kyei-Boahen et al. 2023). Mucuna pruriens and Centrosema pubescens are leguminous plants belonging to the family of plants called Fabaceae commonly found in the tropical and sub-tropical forests (Thomas and Sumberg 1995, Lampariello et al. 2012). Mucuna pruriens or magic velvet is a medicinal plant that can increase phosphorus availability in the soil after applying rock phosphate (Vanlauwe et al. 2000). After processing by soaking in water, the plant is also used as a food crop in the eastern part of Nigeria to remove the L-DOPA (Kavitha and Thangamani 2014). Centrosema pubescens is cultivated as forage for livestock because of its intrinsically high protein, calcium, and potassium compared to other plants (Thomas and Sumberg 1995).

Legumes are cultivated principally for their seeds or pulses, which are protein sources for humans, livestock, and soil-enhancing green manure (Tharanathan and Mahadevamma 2003). Legumes are essential sources of protein, starch, oil, minerals, vitamins, and health-promoting compounds from the beginning of human history (Siddique et al. 2012). Agriculturally, legumes play an essential role in crop rotation due to their establishment of a symbiotic relationship with nitrogen-fixing bacteria, also known as plant growth-promoting rhizobacteria (PGPR) in structures called root nodules, therefore improving soil fertility (Peix et al. 2015). The improvement in soil fertility is achieved by fixing atmospheric nitrogen by a process called biological nitrogen fixation (BNF) (Soumare et al. 2020). This ability of legumes to use atmospheric nitrogen fixed through PGPR presents the possibility of improving crop yield without applying synthetic fertilizer (Van Heerwaarden et al. 2018).

Rhizobia are Gram-negative soil bacteria that can initiate an nitrogen-fixing endosymbiotic relationship with legumes. They live either in the soil or within the root nodules of the host plant, where they convert atmospheric nitrogen to ammonia and provide organic nitrogenous compounds to the plants (Masson-Boivin and Sachs 2018). The bacteria live in small outgrowths called nodules on the roots of the legumes alongside other non-PGPR (Martínez-Hidalgo and Hirsch 2017). The bacteria perform nitrogen fixation within these nodules, and the plant absorbs the ammonia. It has been estimated that rhizobia can fix about 50–300 kg/N/ha Thus their contribution to the nitrogen content of the soil can be pretty substantial (Singh et al. 2013). Undeniably, the application of PGPR in agriculture can reduce the dependence on chemical or synthetic fertilizer though not with its inherent challenges (Siddique et al. 2012, Nyoki and Ndakidemi 2018).

The acceptance of rhizobial inoculants as bio-fertilizers will encourage environmental and socioeconomic steadiness of farming and has been recommended as an option to enhance cowpea yields. Nonetheless, the selection of bacterial strains with numerous beneficial properties is vital to maximizing the effectiveness of the host plant (Soumare et al. 2020). Therefore, this study aims to isolate rhizobia sp. from M. pruriens and C. pubescens, authenticate and assess the symbiotic effectiveness of cowpea nodulating rhizobia with two varieties of cowpea (Ife BPC and Ife Brown) in order to explore their potential application in the improvement of crop yield. The effects of three different types of pesticides on the isolated rhizobia isolates were also evaluated.

Materials and methods

Study area and sample collection

The rhizobia used in this study were isolated from healthy nodules of two wild plants, namely C. pubescens and M. pruriens collected at different locations in Ile-Ife, Osun State, Nigeria (7.4905°N, 4.5521°E). The nodules were detached using sterile gloves into a sterile zip-lock bag, transported immediately to the laboratory for further analysis and culturing. The plant materials were taken to Obafemi Awolowo University Herbarium for identification.

Soil sampling and physico-chemical analysis

The nitrogen and phosphorus contents of the river sand used as growing medium were determined before and after cultivation in the greenhouse. With the aid of a sterile spatula, the test soil was scooped about 30 mm deep from the surface into a sterile McCartney bottle. It was transported into the laboratory for analysis. Nitrogen content of the soil was expressed as nitrate and phosphorous as phosphate. The nitrate content of the soil was determined using the ultraviolet-visible screening method. In contrast, the phosphate was determined by the vanado-molybdo phosphoric acid colorimetric method because of their high sensitivity, low detection limit, and low economic cost (Zui and Birks 2000, Moshoeshoe and Obuseng 2018).

Legume variety used

Two varieties of cowpea Ife BPC and Ife Brown, were obtained from the Institute of Agricultural Research and Training (IAR&T), Ibadan, Oyo State which is affiliated to Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria.

Isolation of Rhizobium from root nodules

The isolation of Rhizobium was performed using the method by Tounsi-Hammami et al. (2019) with minor modifications. The nodules were sieved, washed repeatedly with sterile water to remove adhering soil particles, and surfaced sterilized by immersing them into 95% ethanol for 5–10 s, 0.1% of acidified mercuric chloride (HgCl2) (Merck, Darmstadt, Germany) for 4–5 min, then repeatedly washed with sterile water and dipped into 70% ethanol followed by repeated washings with sterile water. The nodules were crushed in a small aliquot of sterile water; serially diluted, appropriate dilutions were spread on sterile yeast extract mannitol agar (YEMA) (Oxoid Ltd., Hampshire, UK) and incubated in the dark for 10 days at 28°C. Plates were checked periodically for the presence of large sticky colonies of bacteria. The isolated colony was purified on YEMA with Congo red; YEMA with bromothymol blue and glucose peptone agar (GPA); to distinguish colonies of Rhizobia from Agrobacterium. All the experiments were conducted using randomized complete blocks. Following standard microbiology procedures, the rhizobial isolates were identified based on morphological and biochemical characteristics.

Gnotobiotic experiment

The two varieties of cowpea (Vigna unguiculata); Ife BPC and Ife Brown were obtained from the IAR&T of the Obafemi Awolowo University located in Ibadan Oyo State, Nigeria. They were rinsed in 95% ethyl-alcohol for 10 s; hydrogen peroxide solution of 3% concentration for 3–5 min, followed by six rounds of sterile water to remove the trapped-air and waxy layer. Three percent concentration of hydrogen peroxide solution was added to the seeds in the Erlenmeyer flask in sufficient amount, and contents gently swirled for 3–5 min, after which the sterilant was drained off. The seeds were then rinsed six times with distilled water, aseptically placed in sterile petri dishes and refrigerated to prevent the cotyledons from falling apart. The seeds were pre-germinated in a wet sterile horticultural grade humus soil covered with aluminum foil and incubated at 28°C for 3 day until uniform early seed germination was observed. Then, the seedlings were selected and planted semi-aseptically into a 2 kg capacity pot filled with sterilized river sand.

The potting was done by weighing 2 kg of the sterile river sand into pre-sterilized plastic bowls with two drains at the bottom of the bowls and a plate to drain the water. Sterile Nitrogen-free nutrient solutions were added to the potting media and allowed to drain overnight. Pre-germinated seeds were then transplanted to the pots; these were later thinned to two plants per pot after two weeks of germination. A volume of 10 ml suspension of standardized (10⁸ cfu/ml) pure culture of actively growing Rhizobia isolates were inoculated into their corresponding pots after 1 week of germination. The various treatments were three consisting of non-inoculated cowpea plants (control), cowpea plants inoculated with synthetic fertilizers (1 g per pot), and cowpea plants inoculated with each of the Rhizobia isolates. Three replicates were established within each of these treatments with 108 total experimental units (Table 1). The cowpea plants were grown for 12 weeks in a greenhouse with an average minimum temperature of 25°C and an average maximum of 40°C (Tounsi-Hammami et al. 2019).

Table 1.

Treatment combinations of rhizobia isolates and cowpea varieties.

Treatment	Experimental content
VAR + M1	SS + M1 + VAR seed
VAR + M2	SS + M2 + VAR seed
VAR + M3	SS + M3 + VAR seed
VAR + M4	SS + M4 + VAR seed
VAR + M5	SS + M5 + VAR seed
VAR + M6	SS + M6 + VAR seed
VAR + M7	SS + M7 + VAR seed
VAR + M8	SS + M8 + VAR seed
VAR + C1	SS + C1 + VAR seed
VAR + C2	SS + C2 + VAR seed
VAR + C3	SS + C3 + VAR seed
VAR + C4	SS + C4 + VAR seed
VAR + C5	SS + C5 + VAR seed
VAR + C6	SS + C6 + VAR seed
VAR + C7	SS + C7 + VAR seed
VAR + C8	SS + C8 + VAR seed
**VAR + Synthetic	SS + NPK (1 g/pot) + VAR seed
***VAR + Neg	SS + VAR seed

*SS, sterile soil; VAR, variety of seed (Ife BPC and Ife Brown), MI—M8: isolates of rhizobia from root nodules of Mucuna spp. C1–C8: isolates of rhizobia from root nodules of Centrosema spp. NPK fertilizer (15–15-15 combination, NAFCON, Nigeria) was added 4 weeks after planting using the side-dress application method.

**Positive control. ***Negative control.

Nodulation and symbiotic efficiency and harvesting of plant materials

After 12 weeks of planting, the plants were carefully uprooted so that no nodules were left in the potting material. The nodules were separated, collected, and counted. The abundance of the nodule were scored using the methods of Yates et al. (2016).

Five nodules per plant were incised using a sharp razor blade to check the interior color of the nodules. Shoot and nodule materials were oven-dried at 70°C for 48 h until a constant dried weight was attained and recorded. Symbiotic effective index (EI) of the isolates is a measure of how favorably the isolates competed with the positive control (NPK fertilizer). This was expressed as shoot biomass of inoculated plant divided by shoot biomass of the positive control plant:

$$\begin{eqnarray} &&\textit{Effective}\ \textit{index}\ \left( {EI} \right)\\ &&\quad= \frac{{{\rm{shoot\ biomass\ of\ inoculated\ plant}}}}{{{\rm{shoot\ biomass\ of\ the\ positive\ control\ plant}}:}} \times 100. \end{eqnarray}$$

The symbiotic EI of the isolated rhizobia species were categorized as follows according to Beck et al. (1993) and Purcino et al. (2000):

$$\begin{eqnarray} \begin{array}{@{}*{3}{l}@{}} {{\rm{Highly}}\,{\rm{effective}}}& = &{ > 80{\rm{\,and}}\,{\rm{above}}}\\ {{\rm{Effective}}}& = &{50 - 80}\\ {{\rm{Lowly}}\,{\rm{effective}}}& = &{35 - 49}\\ {{\rm{Ineffective}}}& = &{{\rm{<}}\,35}. \end{array} \end{eqnarray}$$

The grain yield of plants was measured as count of the seeds per pod, and the gross weight of the seeds were expressed in grams. The nitrogen and phosphorus content of the soil after the greenhouse experiments were determined and expressed as nitrate and phosphate.

In-vitro pesticides susceptibility testing of the isolates

A susceptibility test was carried out on the Rhizobia isolates to determine the effects of the pesticides on the isolated PGR. The pesticides tested against the isolated Rhizobia strains were prepared according to manufacturers’ specifications (Table 2). The test was carried out using the methods of Drouin et al. (2010). Sterile discs of 5 mm diameter of filter paper were soaked in medical bottles containing the prepared concentrations of the pesticides. The discs were then packed into sterile glass Petri dishes and were oven-dried at 25°C overnight. With the aid of sterile swab sticks, sterile plates of YEMA supplemented with Congo red dye were swab with standardized culture (10⁸ cfu/ml) of the isolates in yeast extract mannitol broth. A sterile forceps was used to place four discs of filtered paper from different concentrations on the YEMA plates, and a sterile control disc soaked in the solvent was placed at the center of the plate. The plates were left on the laboratory bench for 2 h to ensure proper diffusion of the pesticides, after which the plates were incubated at 28°C for 4 day. Plates with zones equal to or greater than 20 mm were recorded as sensitive to the tested pesticides.

Table 2.

Treatments used for in vitro pesticide susceptibility testing.

Active Ingredients	Concentrations (mg/ml)	Solvents
Herbicides
Glyphosate (480 mg/l)	14.40, 7.20, 4.80, 3.60	Distilled water
Paraquat dichloride (276 g/l)	2.76, 1.84	Distilled water
2,4–D—dimethylamine (720 g/l)	14.40, 7.20	Distilled water
Atrazine (500 mg/l)	4.00	Methanol
Fungicides
Carbendazim 12% and Mancozeb 63% W/P (750 g/kg)	0.75, 0.50, 0.37	Distilled water
Imidacloprid 20% + Metalaxyl—M 20% + Tebuconazole 2% (420 g/Kg)	0.75	Methanol
Insecticides
2,3–Dichlorovinyl dimethyl phosphate (1000 g/l)	50.00, 23.00, 15.00	Distilled water
Chlorpyriphos (480 g/l)	4.80, 3.60	Methanol

Statistics

All experiments were performed in triplicate. Analysis of variance was done in GraphPad Prism (version 8.0.2) using Dunnette multiple comparison hypothesis testing to determine significant differences between the treatments (P ≤ 0.05).

Results

Physico-chemical analysis of soil

The chemical analysis of the river soil used as the potting medium revealed the pH as 8.8, nitrogen as 4.21 mg/g, and phosphorus as 4.20 mg/g and without any indigenous Rhizobium spp. population. The concentrations of nitrogen and phosphorus are approximately the same in the soil with the nitrogen concentration slightly higher than the phosphorus concentration.

Isolation and identification of rhizobia isolates

A total of sixteen (16) bacteria were isolated from the root nodules of Mucuna and Centrosema. Eight were isolated from Mucuna (M1–M8) and Eight from Centrosema (C1–C8). All the Rhizobial isolates (100%) were circular, convex, regular, opaque, and Gram-negative short rods under the microscope, while 88% were white and 12% were creamy. The isolates were motile, positive for oxidase, catalase, and citrate production. All the eight isolated bacteria from Mucuna were yellow (acidic) on bromothymol-blue + YEMA, indicating that they were fast growers (Rhizobium), while out of the eight isolated bacteria from Centrosema, five (63%) were Rhizobium sp and three (37%) were blue (alkaline) on bromothymol-blue + YEMA, indicating that they were slow growers (Bradyrhizobium). On the GPA containing 1.6% bromocresol purple, all the isolates showed a negative reaction which distinguished them from Agrobacterium species (Table 3).

Table 3.

Biochemical characterization of the rhizobia isolates from Mucuna and Centrosema plants.

Isolates codes	Gram stain	Growth on GPA	Growth on BBT—YEMA	Acid Prod.	Oxi	Cat	Glu	Lac	Gas	H2S	Mot	Cit	MR	VP	Ind	Sta	2% NaCl	Probable Identity
M1	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	–	–	+	–	–	+++	Rhizobium sp.
M2	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	–	+	–	–	+++	Rhizobium sp.
M3	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	+	+	–	–	++	Rhizobium sp.
M4	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	+	+	–	–	++	Rhizobium sp.
M5	- SR	Neg	Yellow	+	+	+	+	+	+	–	+	+	+	–	–	–	+	Rhizobium sp.
M6	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	+	+	–	+	+++	Rhizobium sp.
M7	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	+	+	–	–	+	Rhizobium sp.
M8	- SR	Neg	Yellow	+	+	+	+	–	–	–	+	+	+	+	–	–	++	Rhizobium sp.
C1	- SR	Neg	Yellow	+	+	+	+	+	–	–	+	+	+	+	–	–	+	Rhizobium sp.
C2	- SR	Neg	Blue	–	+	+	–	–	–	–	+	+	+	+	–	–	+	Bradyrhizobium sp.
C3	- SR	Neg	Yellow	+	+	+	–	–	–	–	+	+	+	+	–	–	+	Rhizobium sp.
C4	- SR	Neg	Yellow	+	+	+	+	+	+	–	+	+	+	+	–	–	++	Rhizobium sp.
C5	- SR	Neg	Yellow	+	+	+	+	–	–	–	+	+	+	+	–	–	++	Rhizobium sp.
C6	- SR	Neg	Yellow	+	+	+	+	–	+	–	+	+	–	–	–	–	+++	Rhizobium sp.
C7	- SR	Neg	Blue	–	+	+	+	+	+	–	+	+	–	+	–	+	+++	Bradyrhizobium sp.
C8	- SR	Neg	Blue	–	+	+	+	–	+	–	+	+	–	+	–	–	++	Bradyrhizobium sp.

Key:—SR, Gram negative short rods; Oxi, oxidase; Cat, catalase; Lac, lactose; Glu, glucose; Gas, gas production, Cit, citrate utilization; MR, methyl red; VP, Voges–Proskauer; Ind, indole production; Sta, starch hydrolysis; + (Positive); and − (Negative).

Nodulation and symbiotic efficiency

Generally, nodules were not observed in negative control but the positive control had few nodules which are green indicating that they are poor nitrogen fixers. All the nodules observed in Ife BPC were pink indicating healthy and effective good fixers of nitrogen; however, in Ife brown, the nodule are pink with the exception of Rhizobium sp (M2 and M3) with green nodules which indicated that they are poor nitrogen fixers.

The number of nodules observed in the Ife BPC cowpea ranged from 13.00 ± 2.83 to 98.50 ± 2.02, while Ife Brown cowpea ranged from 0.71 ± 0.11 to 131.50 ± 2.12. The highest nodules produced in Ife BPC was by the Rhizobium sp. (M4) followed by the Bradyrhizobium sp. (C2 and C8), while the least nodules were produced by the Rhizobium sp, (M2 and C1). The highest nodules produced in Ife Brown cowpea was by the Bradyrhizobium sp. (C7) (Table 4). The nodulation observed in the Ife BPC cowpea ranged from moderate to extremely abundant. The Rhizobium sp with isolate code (M1, M2, and C1) had moderate nodules which is the same with the synthetic treatment with NPK fertilizer, while the others had adequate to extremely abundant nodulation. The three Bradyrhizobium sp (C2, C7, and C8) had very abundant and extremely abundant nodulation. The nodulation observed in the Ife brown cowpea also alternated between moderate to extremely abundant with two of the Bradyrhizobium sp (C7 and C8) showing an extremely abundant nodulation (Table 4).

Table 4.

Nodule numbers and dry weight of Nodules of the two species of cow-peas and soybeans.

Isolate codes	Nodule number/plant Ife BPC cowpea	Nodule internal color	Dry weight (g/plant)	Symbiotic effectiveness	Nodule Nos/plant Ife Brown cowpea	Nodule internal color	Dry weight (g/plant)	Symbiotic effectiveness
M1	17.50 ± 3.54	Pink	1.40 ± 0.18	HE	14.50 ± 3.54	Pink	1.09 ± 0.18	E
M2	13.00 ± 4.24	Pink	1.04 ± 0.21	HE	14.00 ± 1.41	Green	1.05 ± 0.07	LE
M3	25.00 ± 21.21	Pink	2.23 ± 1.06	HE	17.00 ± 2.83	Green	1.28 ± 0.14	LE
M4	98.50 ± 12.02	Pink	8.77 ± 0.06	HE	14.00 ± 5.66	Pink	1.05 ± 0.28	E
M5	78.00 ± 11.31	Pink	6.94 ± 0.57	HE	9.50 ± 2.12	Pink	0.71 ± 0.11	E
M6	85.00 ± 7.07	Pink	7.57 ± 0.35	HE	13.00 ± 1.41	Pink	0.98 ± 0.07	E
M7	60.50 ± 0.71	Pink	5.38 ± 0.04	HE	17.00 ± 1.41	Pink	1.28 ± 0.07	E
M8	57.50 ± 10.61	Pink	5.12 ± 0.53	HE	32.00 ± 2.83	Pink	2.40 ± 0.14	E
C1	13.00 ± 2.83	Pink	1.04 ± 0.14	E	28.50 ± 4.95	Pink	2.57 ± 0.25	HE
C2	88.50 ± 4.95	Pink	7.88 ± 0.25	HE	41.00 ± 1.41	Pink	3.69 ± 0.07	HE
C3	22.50 ± 3.54	Pink	2.00 ± 0.18	HE	65.00 ± 1.41	Pink	5.85 ± 0.07	HE
C4	57.50 ± 10.61	Pink	5.12 ± 0.53	HE	36.00 ± 2.83	Pink	3.24 ± 0.14	HE
C5	72.50 ± 3.54	Pink	6.45 ± 0.18	HE	54.00 ± 5.66	Pink	4.86 ± 0.28	HE
C6	64.00 ± 5.66	Pink	5.70 ± 0.28	HE	94.00 ± 4.24	Pink	8.46 ± 0.21	HE
C7	67.00 ± 2.83	Pink	5.96 ± 0.14	HE	131.50 ± 2.12	Pink	11.84 ± 0.11	HE
C8	88.00 ± 4.24	Pink	7.83 ± 0.21	HE	118.00 ± 4.24	Pink	10.62 ± 0.21	HE
SYN	0.00	–	0.00		0.00	–	0.00
Neg	–	–	–		–	–	–
	P = 0.0001 R = 0.7545				P = 0.001 R = 0.7218

LEGEND: LE, lowly effective; E, effective; and HE, highly effective.

The symbiotic effectiveness between the isolated bacteria and the two varieties of cowpeas showed an improved effectiveness when compared with the fertilizer applied treatment. Almost all the isolated bacteria had highly effective symbiotic effectiveness with the Ife BPC cowpea except for Rhizobium sp. (C1) which was effective. Six out of eight Rhizobial sp from M. pruriens had effective symbiotic relationship with Ife Brown cowpea, while all the isolated Rhizobium and Bradyrhizobium species from C. pubescens had highly effective symbiotic relationship. In Ife brown cowpea, nodules number and effectiveness were significantly affected by inoculation (P = 0.001). Ife BPC cowpea nodules number and effectiveness were also affected by inoculation (P = 0.007).

Nodule dry weight

The nodule dry weight recorded by Ife BPC cowpea ranged from 1.04 g/plant to 8.77 g/plant. The highest was observed in isolate codes M4, while the lowest were observed in isolate codes M2 and C1 isolated from M. pruriens. In the isolated bacteria from C. pubescens, isolate Bradyrhizobium sp (C2) had the highest, while Rhizobium sp. (C1) had the lowest nodule dry weight. However, the nodule dry weight observed for Ife Brown cowpea ranged between 0.71 g/plant and 11.84 g/plant with isolate code C7 identified as Bradyrhizobium sp having the highest nodule dry weight while the lowest was in M5 (Rhizobium sp). The variation in the nodule dry weight per plant may be attributed to the size and number of the nodules (Table 4).

Grain yield and plant biomass

Ife BPC cowpea was significantly (P = 0.01) affected by inoculation, Bradyrhizobium sp (C7) produced the highest grain yield of 58.14 g/plant, and isolate Rhizobium sp. (C1) produces 6.88 g/plant, which was the most negligible grain value. The grain yield of inoculated Bradyrhizobium sp. (C7) and Rhizobium sp. (C8) competed favorably with the positive control (NPK fertilized-plant). The Ife Brown cowpea was significantly (P = 0.0003) affected by inoculation with the highest grain yield of 73.92 g/plant recorded by Bradyrhizobium sp. (C7), while the lowest was in Rhizobium sp. (M4) with a negligible yield of 5.40 g/plant. The NPK (synthetic treatment) had a grain yield of 51.52 g/plant, while the uninoculated control (Neg) had a yield of 7.36 g/plant (Fig. 1). The grain yield of inoculated Bradyrhizobium sp. (C7 and C8) competed favorably with the positive control (NPK fertilized-plant) (Fig. 1).

Figure 1.

Grain yield of the two varieties of cowpea (g/plant).

The highest plant biomass for Ife BPC was 4.66 g from the Bradyrhizobium sp. (C7), while the lowest was 1.28 g from Rhizobium sp. (C1) which was lower than the plant biomass from the un-inoculated treatment. The plant biomass from NPK treatment was also higher than two Rhizobium sp. (M1 and M2) but lower than the remaining isolated bacteria (Fig. 2). In the case of Ife Brown cowpea, the highest plant biomass of 10.56 g was recorded in the Bradyrhizobium sp. (C7), while the lowest was 1.38 g from Rhizobium sp. (M2). All the isolated Rhizobium sp. from Mucana pruriens produced lower plant biomass than the NPK treatment samples, while the Rhizobium and Bradyrhizobium species from C. pubscens produce a better plant biomass than the NPK treatment samples (Fig. 2). The interaction between nodules effectiveness and plant biomass was significant (P = 0.007) for Ife brown variety of cowpea and Ife BPC cowpea at (P = 0.0241).

Figure 2.

The plant biomass yield of inoculation of (a) Ife Brown (b) Ife BPC.

Elemental composition of the soil after planting

Elemental parameters tested for in the soil after before and during harvesting were N₂ and P contents expressed in the form of nitrate and phosphate. The treatments applied to the plants shows various elemental concentration which were significantly different from inoculated plants, to NPK treated plants, and non-inoculated plants. The highest nitrogen concentration of 84.28 mg/g in the soil after harvesting was observed in treatment containing Ife Brown cowpea and Bradyrhizobium sp. (C7), while the highest nitrogen concentration of 55.89 mg/g was recorded in the treatment containing Ife BPC and Rhizobium sp. (M4) (Fig. 3).

Figure 3.

The nitrogen concentration of the different varieties of Ife BPC and Ife Brown after planting.

A significant increase in nitrogen concentration at P < 0.0001 was observed in all the soil inoculated with Ife Brown cowpea and each of the rhizobial isolates compared with the NPK treatment. In Ife BPC cowpea variety, Tukey’s multiple comparisons test revealed that there is significant difference (P < 0.0004) in the nitrogen concentration of the soil after planting between the inoculated vs. the negative control and there is also significant difference between the inoculated and positive control (NPK fertilizer). However, only three soil containing the rhizobial isolates (M4, M5, and M6) and Ife BPC cowpea contain nitrogen concentration higher than the positive control (NPK fertilizer) (Fig. 3).

Phosphorus concentration in the soil after planting of Ife Brown cowpea was significantly (P < 0.0001) affected by inoculation. It was noted that the soil phosphorus concentration of Ife BPC cowpea in the positive control (5.00 mg/g), negative control (5.54 mg/g) and the inoculated treatments increased compared to the pre-planting concentration of 4.40 mg/g. The Rhizobium sp (M8) had the highest concentration of soil phosphorus concentrations 6.60 mg/g. The results recorded for the Ife brown cowpea showed that the soil phosphorus concentration in the negative treatment (9.28 mg/g) was higher than that of the positive control (4.01 mg/g) and the Rhizobium sp. (M1) inoculated treatment (8. 60 mg/g) (Fig. 4).

Figure 4.

The phosphorus concentration of the different varieties of Ife BPC and Ife Brown after planting.

In vitro pesticide susceptibility

The isolates used for this study were subjected to different groups of pesticides namely 4 herbicide, 2 fungicides, and 2 insecticides as shown in Table 5. The isolates showed different susceptibility and resistance patterns to the pesticides.

Table 5.

Pesticides susceptibility pattern for all the rhizobia Isolates from M. pruriens and C. pubescens.

Glyphosate (480 mg/ml).					Paraquat dichloride		2,4–Dimethylamine		Atrazine	Carbendazim 12% and Mancozeb 63% W/P			Imidacloprid 20% + Metalaxyl 20% + Tebuconazole 2%	2,3 dichlorovinyl dimethyl phosphate			Chlorpyriphos
Isolate	Concentration (mg/ml)
	14.4	7.2	4.8	3.6	2.76	1.89	14.4	7.2	4	0.75	0.5	0.37	0.75	50	23	15	4.8	3.6
M1	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
M2	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R	R
M3	S	S	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
M4	S	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R	R	R
M5	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
M6	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
M7	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
M8	S	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
C1	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R	R	R
C2	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R	R	R
C3	R	R	R	R	S	R	R	R	R	R	R	R	R	R	R	R	R	R
C4	S	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
C5	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
C6	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
C7	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R
C8	R	R	R	R	S	S	R	R	R	R	R	R	R	R	R	R	R	R

*S, sensitive, R, resistance, **Control (distilled water) except for atrazine, Imidacloprid 20% + Metalaxyl 20% +Tebuconazole 2 and Chlorpyriphos and which is methanol.

All the 16 isolated Rhizobium and Bradyrhizobium species were resistant to Glyphosate at 3.6 and 4.8 mg/ml; increase in concentration resulted in Rhizobium sp (M3) being sensitive to the glyphosate. Furthermore, more Rhizobium sp. (M3, M4, M8, and C4) became sensitive to glyphosate at 14.4 mg/ml.

In general, 11 out of the 16 rhizobial species were sensitive to 1.86 mg/ml of paraquat dichloride except Rhizobium sp. M2, M4, C1, C3, and Bradyrhizobium sp. (C2). When the concetration of paraquat dichloride was increased to 2.76 mg/ml, the number of rhizobia species sensitive to the herbicide increased to 15 except for Rhizobium sp (M2) that retained their resistant profile (Table 5). It was observed that all the isolates were resistant to 2,4–dimethylamine at the concentrations of 7.2 mg/ml and 14.4 mg/ml; and Atrazine at 4.0 mg/ml.

The two fungicides used in this study also showed that all the isolates were resistant to the various concentration of Carbendazim 12% + Mancozeb 63% W/P and Imidacloprid 20% + Metalaxyl 20% + Tebuconazole 2%. The same results as the fungicides were also recorded for the two insecticides used in this study with all the isolates exhibiting resistance to 2,3 Dichlorovinyl dimethyl phosphate and Chlorpyriphos.

Discussion

Inadequate nitrogen concentration in the soil is one of the biggest challenges that some developing countries face with regard to increasing crop cultivation to a sustainable level. The nitrogen fixing synergy between legumes and rhizobia plays an essential part in providing adequate nitrogen for legumes and non-leguminous produces (Ondieki et al. 2017). In this study, the isolated bacteria belonged to the genus Rhizobium and Bradyrhizobium sp. Eight strains of Rhizobium were recovered from the root nodules of M. pruriens, while in C. pubescens, five Rhizobia species and three Bradyrhizobium sp. were recovered, which is similar to report by Zahran (2001) where the genera Rhizobium, Bradyrhizobium, Mesorhizobium, and Sinorhizobium were isolated from nodules of wild legumes in Egyptian soils. Furthermore, species of Rhizobium and Bradyrhizobium have been identified in cowpea in Botswana and Mozambique (Pule-Meulenberg et al. 2010, Simbine et al. 2021). According to Muindi et al. (2021), several Rhizobium species exhibited symbiotic relationship with cowpea in semiarid areas in Kenya, while Kebede et al. (2020) isolated cowpea nodulating rhizobia in Ethiopia identified as Rhizobium and Bradyrhizobium species.

Generally, the Rhizobium sp. (81%) were more prevalent than the Bradyrhizobium (slow growers) species which corroborated report by Ondieki et al. (2017) that 55% of the isolated rhizobial bacteria from agricultural soils of lower Eastern Kenya were fast growers. A research conducted by Paudyal and Gupta (2017) also reported that 100% of the rhizobia isolated from root nodules of M. pruriens were fast growers; Ennacheril and Mahesh (2019) isolated 100% fast growers rhizobial bacteria from C. viriginianum. Some other researchers had contrary view about this, a report by Kebede et al. (2020) stated that the Bradyrhizobia (slow growers) were the most frequently isolated bacteria from the soils of major cowpea producing areas in Ethiopia. The accelerated development typical of rhizobia has been establish as an adaptation of rhizobia flourishing in dry and semiarid areas which aids in their rapid multiplication over a brief period of time (Borges et al. 2010).

The prospective of the symbiotic interaction in performance of the leguminous plant relies on the effectiveness of the rhizobia to fix nitrogen into ammonia. According to Stajkovic (2011), Rhizobia strains applied as biofertilizer on legumes is the most efficient methods to boost legume production. In addition, the relationship between Rhizobia sp. and legume which resulted into nodulation may lead to various realizable outcomes for nitrogen fixation ranging from absent to increased nitrogen fixation (Terpolilli et al. 2008). Therefore, determination of infectivity, nodulation and symbiotic effectiveness of autochthonous rhizobial community is a crucial factor for the choice of isolates for inoculum output. A significant increase in the nodule numbers was noted in cowpeas inoculated with the Rhizobium and Bradyrhizobium strains isolated from wild legumes in comparison with the un-inoculated control which was corroborated by studies conducted by Yusif et al. (2016) and Boddey et al. (2017). The lack of nodules in un-inoculated Ife BPC cowpea, and in the un-inoculated and NPK supplemented standard in Ife Brown cowpea, portrayed the deficiency of external contamination as stated by Mwenda et al. (2019). The Bradyrhizobium sp. exhibited high nodulation with the two varieties of cowpea, especially with the Ife Brown cowpea and according to Nyaga and Njeru (2020), the probability of the relationship beween Rhizobia and legume being cooperative and beneficial to the host is resolute on the strength of the rhizobia isolates used. The spectacular action of Bradyrhizobium and Rhizobium species in enhancing nodulation in cowpea could connote that these strains were more harmonious with the Ife BPC and Ife Brown cowpea than the other species. It could imply effective colonization of the Ife BPC and Ife Brown cowpea by the rhizobial strains, which might result to satisfactory nitrogen fixed in the soil for the two varieties of cowpea to use for improvement.

The symbiotic efficiency index varied among the isolated Rhizobium and Bradyrhizobium sp.; and the cowpeas owing to host specificity, the same observation was reported by Girija et al. (2020). The symbiotic efficiency is associated with the rate of nitrogen fixation and is considered highly effective if the value is of >80%. All the Bradyrhizobium sp. had a highly effective symbiotic efficiency with Ife BPC and Ife Brown cowpea which implied that the isolates can survive and adequately colonize the rhizosphere of the two varieties of cowpea (Barret et al. 2011); and also emphasized the capability of isolates to be developed into an economical inoculant for cowpea. A research by Kyei-Boahen et al. (2017) corroborated this result whereby cowpea plants inoculated with Bradyrhizobium sp. strain USDA 3456 had high symbiotic efficiency. There is a rising agreement in the written material that Bradyrhizobium is a sundry bacterial set that nodulate a broad of host legumes in Africa (Paudyal and Gupta 2017), hence more research needed to be conducted on legume nodulation by Bradyrhizobia in Africa.

The highest nodule dry weight (11.84 g/plant) was observed with the inoculation of Ife brown cowpea with Bradyrhizobium sp (C7) followed by Ife BPC cowpea inoculated with Rhizobium sp. (M4) with nodule dry weight (8.77 g/plant). It was noted that inoculation of the cowpea with the Rhizobia strains resulted into a significantly higher nodule dry weight compared to the positive and negative control treatments which corroborated the research by El-Wakeil and El-Sebai (2007). Voisin et al. (2003) demonstrated that, increment in nodule dry weight was connected with increased symbiotic efficiency during nodule maturation. According to Graham et al. (2004) the dry weight of the nodule is a good index for symbiotic efficiency, hence it could be used as a crucial instruments in strain assessment. The variation in the nodule dry weight per plant observed in the inoculated Ife BPC and Ife Brown cowpea could be as a result of the magnitude and amount of the nodules. A corresponding effect of inoculation on the dry weight of nodules per plant has also been documented by Nyoki and Ndokidemi (2014).

The highest grain yield recorded in the Ife BPC and Ife Brown cowpea inoculated with Bradyrhizobium sp. (C7) showed that it outperformed un-inoculated and NPK fertilizer treatments. Some of the isolated Rhizobium species also possess this trait which could be connected to their ability to infect and form a positive association with cowpea by fixing the ntirogen in the soil for the cowpea to utilize for improved grain yield. This is in agreement with Kyei-Boahen et al. (2017) who stated inoculation of legume with the appropriate species of rhizobia and application of nitrogen enhanced proceeds of legumes over the un-inoculated control. According to Desta et al. (2015) and Sameh et al. (2017), the usage of effective rhizobia strain can significantly enhanced grain yield of beans as reported in this study. Despite the high grains yield reported in this study, some of the rhizobia species did not produce good yield and their performance ware significantly lower than the NPK treatment. A similar result was reported by Zerihun and Abera (2014) on low grain yield of faba beans inoculated with rhizobia isolates. Furthermore, it has been documented that cowpea is a promiscuous legume which nodulate numerous rhizobia species, including the ineffective strains leading to undesirable outcome to rhizobia inoculation (Kanonge-Mafaune et al. 2018).

A substantial increase was observed in the plant biomass of Ife BPC and Ife brown cowpea inoculated with Bradyrhizobium sp (C7) when compared with the uninoculated and NPK supplemented treatments. This observation is in harmony with the research of El-Azeem et al. (2007) which stated that inoculation of Rhizobium strain led to a significant increase in the biomass of faba bean which could be from the extra provision of nitrogen through the extraordinary BNF by the rhizobia species. It is also supposed to be eco-friendly practices used for improvement of N fixation resulted in increased shoot growth, number of pods, and grain yield of faba bean (Siczek and Lipiec 2016).

Nitrogen fixation, is the transformation of N2 to ammonia by Rhizobia in root node of leguminous plants which plays an crucial function in the world nitrogen cycle and farming (Fujita et al. 2014). Someof the Rhizobium and Bradyrhizobium species isolated in this study were able to increase the concentration of nitrogen and phosphorus in the soil planted with Ife BPC and Ife brown cowpea which does not have direct correlation to the grain and biomass yield. However, it may be increase the yield of other crops planted in the soil after harvesting especially during crop rotation. The quantity of nitrogen fixed in the various rhizobia species varies with the two varieties of cowpea, similar result was reported by Abdul-Aziz (2013). The bright potential of the Bradyrhizobium and Rhizobium species as an option to inorganic fertilizer was noted which corroborated research by Tena et al. (2016). Guo et al. (2010) noted that inoculation of legumes with rhizobia sp. may impact on growth by enhancing the node production and nitrogen fixation. The phosphorus in the soil assist the rhizobia species to fix more nitrogen in the soil which may lead to increment in yield and growth of cowpea. According to Kyei-Boahen et al. (2017), phosphorus is a significant component of ATP in legumes which play an important function in change of energy in plants. Hence, the inoculation of cowpea with the proper strains of rhizobia as biofertilizer might increase the yield.

Pesticides protect the plants by curtailing diseases caused by pests and also keep the pest away but may also harm the symbiotic microflora, plant maturation, and yield of plants (Gupta et al. 2014). The utilization of pesticides in cultivation of plants has led to accumulation of their residues which act as a constrain for Rhizobium inoculation. As reported by Naylor (1996), herbicide is the most predominant pesticide added in farm lands, followed by numerous types of insecticides.

The effect of the two fungicides (Carbendazim 12% + Mancozab (63%); Imidacloprid 20% + Metalaxyl—M 20% + Tebuconazole 2%) on the Rhizobium and Bradyrhizobium strains varied based on the strain and the concentration; however, all the sixteen Rhizobium and Bradyrhizobium strains were resistant to the two fungicides. Hashem et al. (1997) reported that the sensitivity of the Rhizobium and Bradyrhizobium strains to fungicides varied based on concentration. Several researchers have also established that some fungicides pose less noticeable toxicity to the rhizobia species and are capable of enduring the effects of the fungicides, while others reported the sensitivity of Rhizobia species to some fungicides which may alter their biological process and reproduction and therefore disrupt the native soil microbial balance (Odeyemi and Alexander 1977, Heinonen-Tanski and Turkki 1987, Hamuda 2020). Gauri et al. (2011) reported that the fungicides used at manufacturers’ specific concentrations has no inhibitory effect on the tested Rhizobium isolates.

The resistance of all the 16 Rhizobium and Bradyrhizobium strains to the various concentrations of insecticides (2,3–dichlorovinyl dimethyl phosphate and chlorpyriphos) observed in this study was also corroborated by Drouin et al. (2010). Based on the interaction between the Rhizobium and Bradyrhizobium species and the four herbicides (glyphosate, paraquat, 2–4 dimethyamine and atrazine) used in this study, all the isolated rhizobia species were resistant to the effects of 2–4 dimethyamine at 7.2 mg/ml and 14.4 mg/ml; and atrazine at 4.0 mg/ml. However, 75% of the isolated rhizobia species were sensitive to paraquat, this increased to 88% at a concentration of 2.74 mg/ml, which is similar to reports of Sabeen and Tarranum (2013), that Rhizobium sp. were sensitive to paraquat. It is important to note that glyphosate did not show any inhibitory effect on the rhizobia species at low concentration of 3.6 and 4.8 mg/ml, but with increase in concentrations four of the Rhizobium sp became sensitive. Atrsaw et al. (2023) reported a similar occurrence whereby the rhizobia species became sensitive to glyphosate with an increase in concentration. Singh et al. (2020), stated that the dangerous constituent of glyphosate affect rhizobia species accountable for nitrogen fixation in the soil when applied at higher concentrations. Generally, it has been established that glyphosate impair microbial growth in the soil (Zhang et al. 2021).

In conclusion, eight Rhizobium sp. were isolated from the wild legume M. pruriens, while five Rhizobium and three Bradyrhizobium species were isolated from C. pubescens. The isolates were authenticated and confirmed to be genuine rhizobia which infected and formed nodules with two varieties of cowpea (Ife BPC and Ife Brown). Specifically, the Bradyrhizobium sp (C2, C8, and C7 ) exhibited the highest nodulation in Ife BPC in decreasing order, while in Ife Brown, it was C7, C8, and C6 based on dry weight. Regarding the grain weight, Rhizobium sp (M7 and C6) and Bradyrhizobium sp. (C7) had the highest grain yield with Ife BPC, while in Ife Brown, it was Bradyrhizobium sp. (C6 and C7) and Rhizobium sp. (C6). Therefore, it is suggested that farmers could use cost-effective rhizobia strains to enhance yield of cowpea after conducting field trials to decipher their stability under various environmental situation. The Rhizobium and Bradyrhizobium species isolated in this study were able to increase the concentration of nitrogen and phosphorus in the soil planted with Ife BPC and Ife brown cowpea. All the sixteen rhizobia species were resistant to the two fungcides (Carbendazim 12% + Mancozab (63%); Imidacloprid 20% + Metalaxyl—M 20% + Tebuconazole 2%) and insecticides (2,3–dichlorovinyl dimethyl phosphate and chlorpyriphos) used in this study. In addition, two of the herbicides (2–4 dimethyamine and atrazine) could not inhibit the growth of the 16 rhizobia species; however, the other two (glyphosate and paraquat) showed deleterious action on the some of the isolates. The sensitivity of the rhizobia species to the pesticides showed that increase in concentration of the pesticides led to increase in sensitivity of isolated species. The high pesticides resistance recorded in the study might be attributed to exposure of the wild legumes to some of these pesticides. It is recommended to analyze these rhizobia isolates with advanced molecular techniques. The rhizobia isolates should be assessed under field conditions for their pesticide sensitivity, as well as their influence on yield (and nodulation) in cowpea cultivation in order to gain a better insight into their potential for agricultural uses.

Author contributions

Olasupo O. Adeyemi (Data curation [equal], Formal analysis [equal], Investigation [equal], Software [equal], Writing – original draft [equal], Writing – review & editing [equal]), Yetunde M. Feruke-Bello (Conceptualization [equal], Supervision [equal], Writing – original draft [equal], Writing – review & editing [equal]), and Olu Odeyemi (Conceptualization [equal], Supervision [equal], Writing – original draft [equal], Writing – review & editing [equal])

Conflict of interest

None declared.

Funding

None declared.

Data availability

The datasets generated and analyzed during the current study are available in the manuscript. Upon a reasonable request, the corresponding author can provide any other information.