Effect of rhizobium inoculation on yield and some quality properties of fresh cowpea

Abstract

Biological nitrogen fixation is considered important to maintain agricultural sustainability. In this study, 2 different cowpea varieties were used consisting of 6 different treatments (seed inoculation, soil inoculation, nitrogen application, seed inoculation + nitrogen application, soil inoculation + nitrogen application, and control) to determine the effect of rhizobium inoculation on fresh cowpea yield and quality. The protein, total phenolic, β-carotene, chlorogenic acid, ascorbic acid, total and reducing sugar contents of fresh pods were investigated. Besides, parameters such as total fresh yield, pod width, pod length, and total chlorophyll in leaves were also investigated. As a result, it was determined that both rhizobium inoculation and nitrogen applications positively affected all parameters compared to the control treatment. Thus, rhizobium inoculation has the potential to be an alternative to nitrogen applications in fresh cowpea cultivation.

Keywords:

• fresh cowpea
• rhizobium
• inoculation
• yield
• quality

1. Introduction

Environmental needs, food security, and energy supply are all under threat as a result of the continued growth of the world’s human population and the depletion of natural energy resources. To meet the increasing population’s food demands, agricultural output must be raised (Kaari et al., 2023).

The demand for agricultural products must be met using sustainable and economical technology in order to keep up with the ongoing development of the human population. The use of chemical (inorganic or NPK) fertilizers has increased due to the limited nutrients in the soil and the desire to increase crop yields. However, this practice has led to nutrient shortages due to misuse and abuse (Osorio-Reyes et al., 2023), long-term changes in the ecology and physicochemical conditions of the soil (Mahapatra et al., 2022), and a hidden environmental and health problem worldwide (Osorio-Reyes et al., 2023).

Current soil management practices to increase food production depend highly on mineral fertilizers, which are costly and unsustainable (Iqbal et al., 2023). Increasing agricultural output is necessary, but the overuse of chemical fertilizers severely impacts the environment and causes a number of health concerns. Nowadays, environmentally friendly and sustainable methods are required to increase agricultural output and soil health. Therefore, using agriculturally significant microorganisms with suitable carrier materials, biofertilizers present safer, environmentally friendlier, and sustainable alternatives for improving crop output (Khan et al., 2023; Singh, 2022).

In order to improve crop health for sustainable agriculture, the use of biofertilizers can be very effective in restoring agricultural soil. This can help farmers improve their practices, improve the quality of their soil, and ultimately result in better plant growth (Chaudhary et al., 2022). Biofertilizers are living substances that comprise dormant or active microbe cells (bacteria, actinomycetes, fungi, algae) that solubilize or mobilize soil nutrients or fix atmospheric nitrogen while also secreting compounds that promote growth to increase yield and growth (Kandel et al., 2023). Microorganisms used as biofertilizers include nitrogen fixers such as Rhizobium Sp., Cyanobacteria, and Azotobacter chroococcum; potassium solubilizers such as Bacillus mucilaginous; phosphorus solubilizers such as Bacillus megaterium, Aspergillus fumigatus; Plant Growth Promoting Rhizobacteria (PGPR); Vesicular Arbuscular Mycorrhiza such as Glomus mosseae and sulfur oxidizers (Kumar et al., 2019).

Since ancient times, it has been understood that legumes have had a positive impact on increasing soil fertility (Kumar & Kumar, 2020). Because of the specialized structures, such as nodules produced by Rhizobium species, legumes compensate for mineral fertilizer by fixing nitrogen. According to the literature, legumes fix nitrogen as a result of Rhizobium inoculation at a rate of 50 to 300 kg NPK ha-1 year-1. Rhizobium performed as rhizobacteria that promoted plant growth in addition to nitrogen fixation, solubilized phosphates, produced growth hormones, and increased the growth and yield of non-legume plants due to its root colonization ability (Qureshi et al., 2019).

Cowpea is a multipurpose legume crop with combined nutritional, agronomic, environmental, and economic benefits. It has recently attracted more attention from consumers and researchers worldwide due to its exerted health beneficial properties, including anti-cancer, anti-hyperlipidemic, anti-diabetic, anti-inflammatory, and anti-hypertensive properties (Jayathilake et al., 2018). It increases soil fertility by adding a significant amount of N through N2 fixation (Ayalew & Yoseph, 2022). For millions of rural impoverished people in developing nations, it serves as a source of income and nutritional protein. Also, as a drought tolerant crop, it can be grown mostly under rainfed agricultural conditions (Melo et al., 2022).

Many studies show that the use of rhizobium as a biofertilizer (inoculant) is effective in cowpea. Kyei-Boahen et al. (2017) indicated that the use of inoculants can improve food safety by increasing grain yield and nutritional quality. da Silva Júnior et al. (2018) reported that inoculation increases nodule formation, biological nitrogen fixation, and grain yield. According to Nyaga and Njeru (2020), rhizobium inoculation significantly increased nodulation and shoot dry weight compared to the uninoculated controls. Also increased yields between 22.7–28.6%. Ayalew et al. (2021) found that due to inoculation the number of pods per plant, 100-seed weight, and seed yield of cowpea was significantly improved. Borges et al. (2023) have revealed that rhizobium inoculation improves the growth and nodulation of cowpea.

In comparison to conventional farming methods based on the use of chemical fertilizers, the use of bacterial biofertilizers is seen to be advantageous. It is also considered to be an environmentally sustainable alternative in relation to the latest trends in healthy consumption (Ayuso-Calles et al., 2020).

Here, we tested the hypotheses:

• The effects of inoculation methods on fresh cowpea yield and quality.

• The potential of rhizobium inoculation as an alternative to nitrogen application in cowpea cultivation and the effects of inoculation and nitrogen combinations on cowpea yield and quality.

• The effect of inoculation on cowpea varieties.

Then, the objective of this study was to evaluate different rhizobium inoculation methods, nitrogen treatments, and their combinations on yield and biochemical characteristics of fresh cowpea.

2. Materials and methods

The research was carried out in the open field conditions (non-irrigated) of Isparta (Türkiye), during the period from April to November 2015 and 2016. The location’s geographic coordinates are 37° 49’ 13’’ N latitude and 30° 34’ 28” E longitude, and it is situated at an elevation of 982 m above sea level. The soil area has a sandy loam texture with a pH of 8.26, a lime content of 1.29%, an EC of 0.13 dS/m, and an organic matter content of 0.57%. Nitrogen, phosphorus, potassium, calcium, magnesium, iron, copper, manganese and zinc values in the soil are 952, 30.50, 973.47, 5258.9, 578.99, 4.34, 1.14, 16.7,0, and 1.30 ppm respectively. The meteorological data of the study are given in Figures 1(a,b).

Figure 1. Average temperature (°C) and average relative humidity (%) during the experimentation period [A]; and total precipitation (mm) during the experimentation period [B].

In the study, which was carried out in two varieties, Akkız and Karnıkara, 6 different treatments were included; rhizobium inoculation to seed, rhizobium inoculation to the soil, nitrogen fertilization only, nitrogen application with rhizobium inoculation to seed, nitrogen application with rhizobium inoculation to soil and no treatment control. No fertilization was applied except for the applications. The inoculation material [Rhizobium japonicum, 5 × 10⁵ cells/g] was provided by Ankara Soil, Fertilizer, and Water Resources Central Research Institute of the Ministry of Agriculture and Forestry.

2.1. Experimental design

The study was conducted with a triple factorial scheme 2 × 2× 6 (2 years x 2 cultivars x 6 treatments) according to a design of split-plot in randomized blocks, with three replications and 20 plants in each replication. The plots were composed as treatments and the subplots as cultivars. The seeds were sown in plots at a density of 50 × 25 cm.

Seed inoculation application was carried out as described in Kaya et al. (2002). Briefly, the surface of the seeds was completely wetted by adding 10% sucrose solution at the rate of 1% of their weight on the seeds to be planted in the plot. Then, the inoculant was added at the rate of 1% of the weight of the seeds in a shaded environment and mixed, then the seeds were homogeneously contaminated with the inoculant. Soil inoculation was carried out by applying 1% inoculant to the seeds sown on the seedbed and the seedbed was immediately covered with soil. For nitrogen application, 4 kg of pure nitrogen was applied per decare. Control is a parcel without any treatment.

2.2. Characteristics evaluated

The yield obtained from the plot (g/plot) was calculated as kg/da from the number of plants required per decare. Pod lengths and widths of 20 pod samples taken randomly from each plot were determined in cm and mm, respectively.

2.2.1. Protein content

Protein content was determined by using the Kjeldahl method after combustion, distillation and titration processes using the formula 0.1% H₂SO₄ x 6.25 = % crude protein content.

2.2.2. Total and reducing sugars

A 5 g sample of fresh cowpea pods was taken and homogenized with 20 ml of 95% ethyl alcohol for 2 minutes. The homogenized samples were incubated in a boiling water bath for 10 minutes. After cooling at room temperature, the samples were centrifuged at 8000 g for 15 minutes. The samples were filtered through filter paper and 20 ml of 80% ethyl alcohol was added. Total sugar content was determined by reading the total sugar content as described in Dubois et al. (1956) and reducing sugar content as described in Honda et al. (1980) using a spectrophotometer. Glucose at concentrations of 40, 80, 120, and 200 µg/ml was used as standard.

2.2.3. Total phenolic

For the extraction process, 5 grams of cowpea sample was taken and 10 ml of 95% ethanol was added to the sample and crushed in a homogenizer for 2.5 minutes. The crushed samples were boiled for 10 minutes and then centrifuged at 8000 rpm. The centrifuged samples were filtered through filter paper and 10 ml of 80% ethanol was added and boiled for another 10 minutes. After the boiling process was completed, the samples were made up to 100 ml with 80% ethanol. After these procedures, total phenol compounds were analyzed using Folin-Ciocalteu reagent as described in Coseteng and Lee (1987).

2.2.4. Chlorogenic acid

Phenolic compounds were extracted and readings were obtained at a wavelength of 370 nm in a spectrophotometer using chlorogenic acid as a standard as described by Coseteng and Lee (1987).

2.2.5. β-carotene

The samples were extracted using a mixture of acetone: hexane (4:6) and homogenized. The amounts of β-carotene were calculated by spectrophotometer at wavelengths of 663, 645, 505, and 453 nm as described in Nagata and Yamashita (1992) and the results were expressed as mg/100 g.

2.2.6. Ascorbic acid

A homogeneous mixture was obtained by adding 6% metaphosphoric acid solution equal to the sample weight. From this homogeneous mixture, 25 g of sample was taken and added to 100 ml of 3% metaphosphoric acid solution. The samples were shaken well and filtered through filter paper. Then, 10 ml of the filtered samples were taken and titrated with 2,6 dichlorophenolindophenol solution until the pink color was obtained. The amount of ascorbic acid was determined as mg/100 g ascorbic acid as stated in Cemeroglu (1992).

2.2.7. Determination of total chlorophyll in leaves

After the samples were kept in 96% ethanol, the absorption of chlorophyll extracts at 665 and 649 nm were measured spectrophotometrically. In order to convert the measurements into the amount of chlorophyll contained in the leaves, the formulas mentioned in Akçin (1980) were used. Total chlorophyll concentrations were expressed as μg/mg dry weight.

2.3. Statistical evaluation of the data

The results obtained from the study were subjected to analysis of variance using Minitab (17) Inc. program. Differences between significant means were determined using Tukey test and indicated by different letters.

3. Results and discussion

3.1. Total fresh yield

The Table 1 showed that the effect of years and treatments on cowpea yield was significant, while the effect of cultivar was insignificant (P < 0.05). Fresh cowpea yield was 431.94 kg/da in the 1^st year and 453.31 kg/da in the 2^nd year of the experiment. The high yield in the 2^nd year can be explained by the fact that the average temperature and rainfall values were higher in the 2^nd year, especially considering that the experiment was carried out under arid conditions.

Table 1. Effects of treatments on cowpea total fresh yield, pod length and pod width

		Total fresh yield (kg/da)				Pod length (cm)				Pod width (mm)
Year	Treatments	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year
		Akkız	Karnıkara			Akkız	Karnıkara			Akkız	Karnıkara
1	Control	293.29	289.39	291.34	431.94 b*	12.06	12.94	12.50	13.31	5.42	6.17	5.79	6.50
	Seed inoculation	525.33	464.37	494.85		13.87	13.17	13.52		6.18	6.59	6.38
	Soil inoculation	468.26	459.01	463.64		14.83	13.61	14.22		6.09	6.33	6.21
	Nitrogen application	506.20	516.14	511.17		14.24	13.49	13.87		5.87	6.44	6.15
	Seed inoculation + Nitrogen	438.35	390.78	414.52		12.57	13.19	12.88		7.53	7.66	7.59
	Soil inoculation + Nitrogen	395.84	436.37	416.10		12.59	13.12	12.86		5.98	7.72	6.85
	Year x Cultivar Mean	437.86	426.01		453.31 a	13.36	13.25		13.49	6.18	6.82		6.53
2	Control	296.00	309.00	302.50		12.71	12.70	12.70		5.84	6.20	6.02
	Seed inoculation	531.33	475.00	503.17		13.65	13.36	13.51		6.09	6.68	6.39
	Soil inoculation	489.00	479.00	484.00		14.38	12.47	13.43		6.13	6.28	6.21
	Nitrogen application	511.33	488.00	499.67		15.08	13.74	14.41		5.72	6.50	6.11
	Seed inoculation + Nitrogen	458.00	443.00	450.50		13.25	14.21	13.73		7.48	7.76	7.62
	Soil inoculation + Nitrogen	466.00	494.00	480.00		13.22	13.06	13.14		6.05	7.60	6.83
	Year x Cultivar Mean	458.61	448.00	Treatment		13.72	13.26	Treatment		6.22	6.84	Treatment
MEAN	Control	294.65	299.20	296.92 c*	442.63	12.38	12.82	12.60 b*	13.40	5.63 d*	6.18 cd	5.90 c	6.52
	Seed inoculation	528.33	469.68	499.01 a		13.76	13.27	13.51 ab		6.13 cd	6.64 bc	6.38 bc
	Soil inoculation	478.63	469.00	473.82 ab		14.61	13.04	13.83 ab		6.11 cd	6.31 cd	6.21 c
	Nitrogen application	508.77	502.07	505.42 a		14.66	13.62	14.14 a		5.79 cd	6.47 cd	6.13 c
	Seed inoculation + Nitrogen	448.13	416.89	432.51 b		12.91	13.70	13.30 ab		7.50 ab	7.71 a	7.61 a
	Soil inoculation + Nitrogen	430.92	465.18	448.05 b		12.91	13.09	13.00 ab		6.02 cd	7.66 a	6.84 b
	Cultivar	448.24	437.01			13.54	13.25			6.20 b*	6.83 a

*Differences between means with different letters are significant at P<0.05 level

When the 2-year data of the study were evaluated together, the lowest fresh cowpea yields were observed in the control treatments of Akkız (294.65 kg/da) and Karnıkara (299.20 kg/da) varieties. The highest fresh cowpea yield was 528.33 kg/da in the seed inoculation treatment of the Akkız variety.

Significant differences in yield (296.92–505.42 kg/da) occurred among the treatments. The lowest yield value was obtained from the control treatment, while the highest yield values were obtained from the nitrogen and seed inoculation treatments. Nitrogen application increased the yield by 41.3%, while seed inoculation increased the yield by 40.5%. The significant yield response of plants to inoculation may be due to better nodule development than the control, which enhances more nitrogen fixation from the atmosphere and, as a result, improved yield formation (Habete & Buraka, 2016). Otieno et al. (2009) stated that inoculation increases grain legume production by increasing yield and nodulation. Similarly, different researchers (Adinurani et al., 2021; Agba et al., 2013; Athul et al., 2022; Das et al., 2018; Gedamu et al., 2021; Kellman, 2008; Razafintsalama et al., 2022; Rebika & Nongmaithem, 2019) reported that rhizobium inoculation increased the yield in their studies on different species. Also, Anjum et al. (2006) found that inoculation significantly increased yield and yield components in mung bean and reported that seed inoculation was more effective and gave better results than soil inoculation.

In addition to providing nitrogen to plants, the Rhizobium are effective in secreting phytohormones and siderophores and solubilizing insoluble phosphate. They also have the ability to elicit plant defense reactions against phytopathogens (Vargas et al., 2017). According to Singh et al. (2020), biofertilisers such as nitrogen fixers increase crop yields through better plant growth by protecting plants from stress and diseases. Malik and Sindhu (2011) reported that the promotion of plant growth after inoculation was attributed to the biosynthesis and secretion of IAA by Rhizobacteria. Researchers determined that both nodulating and non-nodulating Rhizobium leguminosarum strains produced indole-3-acetic acid (Wang et al., 1982) which promotes the growth of the plant and plays a crucial role in the production and expansion of root nodules. Also, rhizobium has the ability to protect plants from ethylene stress by using the plant ethylene precursor chemical 1-aminocyclopropane-1-carboxylate (ACC). The enzyme ACC deaminase mediates the breakdown of ACC to ammonia and -ketobutyrate, and this action lowers ethylene production in plants under stressful conditions. Rhizobium that produce ACC deaminase are better able to fix N2 and nodulate (Verma et al., 2020).

3.2. Fresh pod length/width

The Table 1 shows no significant difference in pod length according to years, varieties, and treatment x variety interaction. But significant differences in the length of fresh cowpea pods occurred among the treatments. The longest pod length was obtained in the nitrogen treatment at 14.14 cm and the shortest pod length was obtained in the control group at 12.60 cm. Pod lengths increased between 3.17–12.2% in all other treatments compared to the control treatment.

According to Table 1 when the means of two-year pod width values were analyzed, it was found that the treatment x variety interaction was significant. While the lowest pod width value was obtained in the control treatment of the Akkız variety, the highest pod width values were determined in the seed inoculation + nitrogen application and soil inoculation + nitrogen application of the Karnıkara variety.

Table 1 shows that the effect of years on pod width was insignificant, while the effectiveness of treatments was significant. Pod width values varied between 5.90 and 7.61 mm according to the treatments. The highest pod width value was determined in the seed inoculation + nitrogen treatment, followed by the soil inoculation + nitrogen treatment. The treatments increased the width of fresh cowpea pods by 3.9–28.9% compared to the control treatment.

Both nitrogen and rhizobium inoculation treatments increased both the width and length of fresh cowpea pods. It is thought that this may be due to the positive effect of nitrogen provided to the plant as a result of nitrogen fertilization and rhizobium inoculation. Indeed, Carranca et al. (2018) reported that nitrogen is a very important element for plant growth and development and has a positive effect on fruit size, while Kebede (2021) reported that rhizobium inoculation promotes the growth and development of plants by increasing the bio-fixation of atmospheric nitrogen. These findings led to the conclusion that rhizobium inoculation could replace nitrogen application. To support these findings Naseri Rad et al. (2014) in Pinto beans, Akman (2017) in beans, Ayalew et al. (2021) in cowpea, Hannan et al. (2022) in mungbean reported that rhizobium inoculation increased pod length, Noufal et al. (2018) reported that pod width increased as a result of rhizobium inoculation in pea. In addition, Biswas et al. (2020) reported that both nitrogen and rhizobium treatments increased French Bean pod length and pod width. Nyoki and Ndakidemi (2014) indicated that the better plant growth parameters in the inoculated treatments compared to the control treatment were due to the fact that Bradyrhizobium japonicum was effective in fixing nitrogen, the building block of plant proteins that determine the structure and size of plant tissues.

The effect of cultivars which was found significant at P < 0.05 level on cowpea pod width is given in the Table 1. The highest value was found in the Karnıkara variety (6.83 mm), while the lowest value was found in the Akkız variety (6.20 mm). The higher pod width value in the Karnıkara variety may be related to genetically engineered traits of the variety. In agreement with this, some studies have reported that there is a large variation in pod width among cowpea cultivars (Ashinie et al., 2020; Molosiwa & Makwala, 2020; Nwofia, 2012; Peksen, 2004).

3.3. Protein content in fresh pods

According to Table 2 it is seen that the effects of varieties and treatment x variety interaction on the protein content in cowpea were insignificant.

Table 2. Effects of treatments on protein and total phenolic in fresh cowpea pods and total chlorophyll in leaves

		Protein (%)				Total chlorophyll (μg/mg kuru ağırlık)				Total phenolic (mg/100 g)
Year	Treatments	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year
		Akkız	Karnıkara			Akkız	Karnıkara			Akkız	Karnıkara
1	Control	29.34	28.88	29.11	30.21 b*	6.01 e*	6.15 de	6.08	6.99 b*	150.89	186.96	168.92	190.05 b*
	Seed inoculation	30.22	30.97	30.60		6.97 a-e	7.32 abc	7.14		154.08	201.50	177.79
	Soil inoculation	30.56	30.03	30.30		7.72 ab	6.42 cde	7.07		171.20	234.08	202.64
	Nitrogen application	30.16	31.19	30.67		7.87 a	7.38 abc	7.63		155.29	229.08	192.18
	Seed inoculation + Nitrogen	30.75	30.34	30.55		6.40 cde	7.63 ab	7.02		193.62	226.05	209.83
	Soil inoculation + Nitrogen	30.38	29.69	30.03		6.64 b-e	7.35 abc	7.00		167.11	210.74	188.92
	Year x Cultivar Mean	30.23	30.13		32.65 a	6.93	7.04		7.19 a	165.36	214.73		214.20 a
2	Control	31.78	31.24	31.51		6.09 de*	6.43 cde	6.26		167.71	200.44	184.08
	Seed inoculation	32.18	31.97	32.08		7.29 abc	7.57 ab	7.43		174.53	239.53	207.03
	Soil inoculation	33.62	33.05	33.33		7.10 a-d	7.00 a-e	7.05		180.59	257.11	218.85
	Nitrogen application	33.64	32.25	32.95		7.29 abc	7.71 ab	7.50		204.68	245.89	225.29
	Seed inoculation + Nitrogen	33.08	33.64	33.36		7.13 a-d	7.75 a	7.44		205.29	250.59	227.94
	Soil inoculation + Nitrogen	33.27	32.12	32.69		7.17 a-d	7.79 a	7.48		202.26	241.80	222.03
	Year x Cultivar Mean	32.93	32.38	Treatment		7.01	7.37	Treatment		189.18	239.23	Treatment
MEAN	Control	30.56	30.06	30.31 b*	31.43	6.05 f*	6.29 ef	6.17 c	7.09	159.30	193.70	176.50 b*	202.13
	Seed inoculation	31.20	31.47	31.34 a		7.13 a-d	7.45 abc	7.29 ab		164.30	220.52	192.41 ab
	Soil inoculation	32.09	31.54	31.82 a		7.41 abc	6.71 def	7.06 b		175.89	245.59	210.74 a
	Nitrogen application	31.90	31.72	31.81 a		7.58 ab	7.54 ab	7.56 a		179.99	237.49	208.74 a
	Seed inoculation + Nitrogen	31.92	31.99	31.95 a		6.77 cde	7.69 a	7.23 ab		199.46	238.32	218.89 a
	Soil inoculation + Nitrogen	31.82	30.90	31.36 a		6.90 b-e	7.57 ab	7.24 ab		184.68	226.27	205.48 ab
	Cultivar	31.58	31.28			6.97 b*	7.21 a			177.27 b*	226.98 a

*Differences between means with different letters are significant at P<0.05 level.

The effects of the years which were found to be significant on the protein content in pods are given in Table 2. The mean protein content increased from 30.21% in the first year to 32.65% in the second year.

The protein content was found to be significantly affected by the treatments, as shown in Table 2. All treatments except the control treatment increased the protein content in cowpea pods. Alam et al. (2015) reported that high levels of nitrogen fixation increased the production of protein molecules that contribute to yield characteristics. With similar thoughts, Dhillon et al. (2022) notified that the efficient development of a symbiotic relationship results in higher nitrogen fixation and protein accumulation. In addition, studies have shown that rhizobium inoculation significantly increased the mean seed/pod protein content compared to uninoculated plants (Ahmed, 2013; Özsoy Altunkaynak & Ceyhan, 2018; Shome et al., 2022; Türkmen et al., 2016; Yousaf et al., 2019).

3.4. Total chlorophyll in leaves

When the Table 2 is examined, it is seen that the effect of treatment x variety interaction on total chlorophyll content in the 1^st and 2^nd year of the study is significant (P < 0.05). The highest chlorophyll value was obtained in the nitrogen application of Akkız in the 1^st year of the study. In the second year, the seed inoculation + nitrogen and soil inoculation + nitrogen treatments produced the highest chlorophyll values. The control treatment of the Akkız variety had the lowest chlorophyll values in both years.

When the 2 years of total chlorophyll values were evaluated together, it was found that the treatment x variety interaction was significant. The lowest value was found in the control treatment of the Akkız cultivar (6.05 μg/mg dry weight), while the highest value was obtained with 7.69 μg/mg dry weight in the seed inoculation + nitrogen treatment of the Karnıkara cultivar.

Significant differences in total chlorophyll values occurred among the cultivars. Table 2 shows that the total chlorophyll content of the Karnıkara variety (7.21 μg/mg dry weight) is higher than the total chlorophyll content of the Akkız variety (6.97 μg/mg dry weight).

The effect of years on total chlorophyll content in cowpea leaves was found significant at P < 0.05 level. In the 1^st year, the total chlorophyll value was 6.99 μg/mg dry weight, while this value increased to 7.19 μg/mg dry weight in the 2^nd year.

When the Table 2 is examined, it is seen that the treatments were significant on chlorophyll values at P < 0.05 level. The mean total chlorophyll content of the treatments varied between 6.17 μg/mg dry weight (control treatment) and 7.56 μg/mg dry weight (nitrogen treatment). The treatments increased the total chlorophyll content by 14.4–22.5% compared to the control group.

In support of our findings, studies conducted by different researchers found that rhizobium inoculation increases the total chlorophyll content in chickpea (Bejandi et al., 2012; Özbağ, 2013), in cowpea (Arumugam et al., 2010), in alfalfa (Duan et al., 2022), in Arabidopsis (Sujkowska-Rybkowska et al., 2022), in Vicia faba (Mowafy et al., 2022), and in French bean (Athul et al., 2022).

According to the results they obtained from their studies Mfilinge et al. (2014) reported that rhizobium inoculation considerably increased the bush bean leaf chlorophyll (Chl) concentration by 19% and 44% in the field experiment and 40% and 98% in a glasshouse experiment. They state that these increases in inoculated treatments could be attributed to rhizobium inoculants’ more effective biological nitrogen fixation, which improved nitrogen availability to the plants and, as a result, increased the total chlorophyll contents of the legumes’ leaves.

In general, as seen in Table 2, it is thought that the increase in total chlorophyll values in all other treatments compared to the control group is due to nitrogen, which has an important place in the structure of chlorophyll and which is available to the plant as a result of rhizobium inoculation and nitrogen treatments (Müftüoğlu & Demirer, 1998). Nitrogen plays an important role in synthesizing active substances such as protein, nucleic acids, hormones, chlorophyll, vitamins and various metabolic enzymes that have important functions such as the processes of photosynthesis, respiration, and carbohydrate and signal transport in plants (Krapp, 2015). Zhang et al. (2021) expressed that with the increase in nitrogen supply, plants’ nitrogen and chlorophyll contents increased. To support this, Fathi (2022) reported that there is a positive correlation between N content and chlorophyll content in plants. Higher levels of N in leaves are linked to higher levels of chlorophyll, higher levels of chloroplast activity, and consequently higher levels of photosynthetic production. Increasing leaf photosynthetic rate results in increased crop yield (Shen et al., 2020). Furthermore, higher chlorophyll content is an important factor for improving crop yields (Yan et al., 2021).

3.5. Total phenolic in fresh pods

The Table 2 shows that the effects of years, cultivars and treatments on total phenolic content were significant (P < 0.05). While the total phenolic value of fresh cowpea was 190.05 mg/100 g in the first year of the study, this value increased to 214.20 mg/100 g in the second year. In addition, the Karnıkara cowpea variety was found to have higher total phenolic content than the Akkız cowpea variety.

In the study, total phenolic values of rhizobium inoculation and nitrogen treatments ranging from 176.50 to 218.89 mg/100 g were positively affected compared to the control treatment. It was observed that all treatments increased the total phenolic matter content by 9.01–24.02% compared to the control treatment. The increase in total phenolic content in response to inoculation has been reported by other researchers such as Charitha Devi and Reddy (2002), Mishra et al. (2006), Couto et al. (2011), Farfour et al. (2015), Ayuso-Calles et al. (2020), Makgato et al. (2020) and Jiménez-Gómez et al. (2021).

3.6. β-carotene in fresh pods

When the 2-year data of the study were evaluated together, it was determined that the effect of treatment x variety interaction on β-Carotene values was significant (Table 3). The lowest values were found in the control treatments of Karnıkara (8.35 mg/100 g) and Akkız (8.62 mg/100 g) varieties, while the highest values were obtained from seed inoculation + nitrogen application (12.19 mg/100 g) and soil inoculation + nitrogen application (11.87 mg/100 g) of Karnıkara variety.

Table 3. Effects of treatments on β-carotene, chlorogenıc acid and ascorbic acid in fresh cowpea pods

		β-Carotene (mg/100 g)				Chlorogenic acid (mg/g)				Ascorbic acid (mg/100 g)
Year	Treatments	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year
		Akkız	Karnıkara			Akkız	Karnıkara			Akkız	Karnıkara
1	Control	8.21	8.15	8.18	9.36 b*	0.31	0.39	0.35	0.46	16.97	17.25	17.11 e*	21.21 b*
	Seed inoculation	8.69	9.51	9.10		0.38	0.46	0.42		21.79	23.36	22.58 bcd
	Soil inoculation	8.55	8.24	8.39		0.40	0.59	0.50		18.72	24.58	21.65 cd
	Nitrogen application	9.60	10.33	9.96		0.42	0.55	0.48		19.71	21.95	20.83 d
	Seed inoculation + Nitrogen	9.52	11.55	10.54		0.49	0.56	0.53		20.77	21.63	21.20 d
	Soil inoculation + Nitrogen	8.71	11.29	10.00		0.42	0.55	0.49		23.01	24.64	23.82 b
	Year x Cultivar Mean	8.88	9.84		10.31 a	0.40	0.52		0.48	20.16 d*	22.23 b		22.66 a
2	Control	9.03	8.54	8.79		0.34	0.43	0.39		17.72	19.78	18.75 e*
	Seed inoculation	9.56	9.47	9.51		0.40	0.48	0.44		24.86	22.83	26.85 a
	Soil inoculation	9.40	9.77	9.59		0.43	0.63	0.53		19.31	24.03	21.67 cd
	Nitrogen application	10.18	11.93	11.05		0.45	0.57	0.51		21.73	25.95	23.84 b
	Seed inoculation + Nitrogen	10.97	12.83	11.90		0.51	0.58	0.54		21.15	21.82	21.49 d
	Soil inoculation + Nitrogen	9.58	12.45	11.01		0.43	0.55	0.49		21.76	26.02	23.39 bc
	Year x Cultivar Mean	9.79	10.83	Treatment		0.43	0.54	Treatment		21.09 c	24.24 a	Treatment
MEAN	Control	8.62 c	8.35 c	8.48 c*	9.84	0.33	0.41	0.37 c*	0.47	17.34 g	18.51 g	17.93 c*	21.93
	Seed inoculation	9.12 bc	9.49 bc	9.31 bc		0.39	0.47	0.43 bc		23.33 bcd	26.10 a	24.71 a
	Soil inoculation	8.97 bc	9.01 bc	9.00 c		0.42	0.61	0.51 a		19.01 fg	24.30 ab	21.66 b
	Nitrogen application	9.89 abc	11.13 ab	10.51 ab		0.43	0.56	0.50 ab		20.72 ef	23.95 bc	22.34 b
	Seed inoculation + Nitrogen	10.25 abc	12.19 a	11.22 a		0.50	0.57	0.53 a		20.96 e	21.73 de	21.34 b
	Soil inoculation + Nitrogen	9.14 bc	11.87 a	10.50 ab		0.43	0.55	0.49 ab		22.38 cde	24.83 ab	23.61 a
	Cultivar	9.33 b*	10.34 a			0.42 b*	0.53 a			20.62 b*	23.24 a

*Differences between means with different letters are significant at P<0.05 level

Varieties were significantly affected the β-Carotene values of cowpea pods. The Table 3 shows that the β-Carotene value of the Karnıkara variety (10.34 mg/100 g) is higher than the β-Carotene value of the Akkız variety (9.33 mg/100 g).

The effects of years, which were found to be significant, on the β-Carotene value in fresh cowpea pods are given in Table 3. While β-Carotene value was 9.36 mg/100 g in the 1^st year of the experiment, it was determined as 10.31 mg/100 g in the 2^nd year.

According to the Table 3, it is seen that the effect of the treatments on β-Carotene is significant. The β-Carotene values of the treatments ranged between 8.48 mg/100 g (control treatment) and 11.22 mg/100 g (seed inoculation + nitrogen treatment). Compared to the control treatment, other treatments increased β-carotene content by 9.8–32.3%. Similarly, Sujkowska-Rybkowska et al. (2022) stated in their study on Arabidopsis that rhizobium inoculation increased the carotene content. Also, Mowafy et al. (2022) reported that carotenoid content in Vicia faba plants increased as a result of rhizobium inoculation.

The positive effect of inoculation on β-Carotene can be explained by the nitrogen it provides. According to their studies, the researchers found that nitrogen increased the beta carotene content in parsley (Chenard et al., 2005), in cabbage (Kopsell et al., 2007) and in lettuce (Shamsullah et al., 2023).

3.7. Chlorogenic acid in fresh pods

According to the Table 3, the effects of treatment x variety interactions and years on chlorogenic acid in cowpea were insignificant. On the other hand, it was determined that the effect of cultivars on chlorogenic acid in fresh cowpea was significant. β-Carotene value is higher in the Karnıkara variety (0.53 mg/g) than Akkız variety (0.42 mg/g).

Table 3 shows that the effect of the treatments on chlorogenic acid was found to be significant at P < 0.05 level. All treatments except the control treatment increased chlorogenic acid values between 16.2% and 43.2%. Charitha Devi and Reddy (2002), Zamani et al. (2022) and Namazi et al. (2022) reported that chlorogenic acid values increased as a result of rhizobium inoculation in their studies.

The increase in chlorogenic acid in fresh pods could be attributed to several factors. One of theses is the positive correlation between chlorogenic acid and chlorophyll. Sheen (1973) reported that chlorophyll deficiency resulted in a decrease in chlorogenic acid content.

This situation can also be explained by light intensity. The results we obtained from the treatments other than the control show that the chlorophyll content increased (Table 2). With the increase in chlorophyll content, plants can absorb more light energy from the sun. According to Ma et al. (2020) the increase in light intensity enhances the chlorogenic acid content. Another factor is nitrogen. Some researchers have reported that the chlorogenic acid content increases depending on the nitrogen content (Allahdadi & Raei, 2017; Liu et al., 2010).

Chlorogenic acid has an important role in human health due to iits ts main pharmacological effects such as antioxidant, antibacterial, anti-inflammatory, hypoglycemic, antiviral, lipid-lowering, anticardiovascular, antimutagenic, anticancer, and immunomodulatory (Miao & Xiang, 2020). So the data show that rhizobium inoculation positively affects not only yield but also pod quality in fresh cowpea.

3.8. Ascorbic acid in fresh pods

The effect of the year x variety interaction on ascorbic acid in fresh cowpea pods was found significant (P < 0.05) (Table 3). The highest ascorbic acid value (24.24 mg/100 g) was obtained in the Karnıkara variety in the 2^nd year of the study, while the lowest ascorbic acid value (20.16 mg/100 g) was determined in the Akkız variety in the 1^st year of the study.

The interaction of the year x treatment, which was found to be significant on ascorbic acid in fresh cowpea pods, is given in Table 3. The highest ascorbic acid value (26.85 mg/100 g) was determined in the 2^nd year of the study in the seed inoculation application. The lowest ascorbic acid values (17.11 and 18.75 mg/100 g) were obtained in the control treatments of both years of the study.

When the data of the 2 years of the study were evaluated together, it was determined that the effect of treatment x variety interaction on ascorbic acid values was significant. The highest value was found in the seed inoculation treatment of Karnıkara variety (26.10 mg/100 g), while the lowest values were obtained from the control treatments of Akkız (17.34 mg/100 g) and Karnıkara (18.51 mg/100 g) varieties.

As seen in Table 3 the effect of years on ascorbic acid was found significant at P < 0.05 level. While the ascorbic acid value was obtained as 21.21 mg/100 g in the first year, this value increased to 22.66 mg/100 g in the second year.

The effect of cultivars on ascorbic acid value in fresh cowpea pods was found significant. When Table 3 is examined, it is seen that the ascorbic acid value of the Karnıkara variety (23.24 mg/100 g) is higher than the ascorbic acid value of the Akkız variety (20.62 mg/100 g).

These results are linked to the characteristics of the cultivar. Similar variations were determined by Chavan et al. (2013) in their study of 12 cowpea genotypes. Devi et al. (2015) determined that ascorbic acid contents differed depending on the varieties in their study in which they investigated the related changes in sprouting characteristics and nutritional quality in three cowpea varieties. Variations in the functional qualities of cowpea could influence the potential usage of the crop (Asare et al., 2013).

Significant differences in ascorbic acid values occurred among the treatments. When the mean values were analyzed, the lowest value was obtained in the control treatment with 17.93 mg/100 g, and the highest values were obtained in the seed inoculation (24.71 mg/100 g) and soil inoculation + nitrogen treatments.

It was determined that all treatments increased the ascorbic acid content in fresh cowpea pods between 19.02% and 38% compared to the control treatment. Similar to these findings, ascorbic acid contents of plants inoculated with rhizobium were reported to be higher than those of non-inoculated plants by different researchers who conducted studies in pea (Singh et al., 2012); Noufal et al. (2018), French bean (Das et al., 2018), strawberry (Flores-Félix et al., 2018) and wheat (Maslennikova et al., 2022). The increase in ascorbic acid may be due to nitrogen. In support of this, Kumar et al. (2017) reported that increasing nitrogen levels increased ascorbic acid content in fresh okra pods. In addition, according to Lee and Kader (2000), light intensity and ascorbic acid content are related. Higher light intensity causes an increase in ascorbic acid content in plant tissues. They also stated that the ascorbic acid content of many products can be increased by low irrigation. The data we obtained as a result of our study carried out in the period of high light intensity and under non-irrigated conditions are in accordance with these reports.

3.9. Total and reducing sugars in fresh pods

When the two-year means of total sugar values of cowpea fresh pods are analyzed in Table 4, it is seen that the treatment x variety interaction is significant. Total sugar values were found to vary between 3.30–4.80 mg/g. The lowest value was obtained in the control treatment of the Akkız variety. On the other hand, the highest total sugar values were obtained in the nitrogen and soil inoculation + nitrogen treatments in the Karnıkara variety and in the seed inoculation treatment in the Akkız variety.

Table 4. Effects of treatments on total and reducing sugar in fresh cowpea pods

		Total Sugar (mg/g)				Reducing Sugar (mg/g)
Year	Treatments	Cultivar		Year x Treatment Mean	Year	Cultivar		Year x Treatment Mean	Year
		Akkız	Karnıkara			Akkız	Karnıkara
1	Control	3.27	3.56	3.41	4.18	0.56	0.64	0.60	0.75
	Seed inoculation	4.65	3.95	4.30		0.71	0.70	0.70
	Soil inoculation	4.20	3.83	4.01		0.61	0.66	0.63
	Nitrogen application	4.75	4.78	4.77		0.83	0.70	0.77
	Seed inoculation + Nitrogen	4.45	3.61	4.03		0.72	0.85	0.79
	Soil inoculation + Nitrogen	4.34	4.74	4.54		1.01	0.96	0.98
	Year x Cultivar Mean	4.28	4.08		4.18	0.74	0.75		0.74
2	Control	3.32	3.60	3.46		0.51	0.60	0.56
	Seed inoculation	4.92	4.03	4.48		0.67	0.66	0.67
	Soil inoculation	4.11	4.02	4.06		0.63	0.68	0.65
	Nitrogen application	4.51	4.81	4.66		0.94	0.65	0.80
	Seed inoculation + Nitrogen	4.49	3.37	3.93		0.76	0.92	0.84
	Soil inoculation + Nitrogen	4.14	4.84	4.49		0.91	0.99	0.95
	Year x Cultivar Mean	4.25	4.11	Treatment		0.74	0.75	Treatment
Mean	Control	3.30 d	3.58 bcd	3.44 c*	4.18	0.54 d	0.62 cd	0.58 e*	0.75
	Seed inoculation	4.79 a	3.99 a-d	4.39 ab		0.69 cd	0.68 cd	0.69 cd
	Soil inoculation	4.15 a-d	3.92 a-d	4.04 abc		0.62 cd	0.67 cd	0.64 de
	Nitrogen application	4.63 ab	4.80 a	4.71 a		0.89 ab	0.68 cd	0.78 bc
	Seed inoculation + Nitrogen	4.47 abc	3.49 cd	3.98 bc		0.74 bc	0.89 ab	0.81 b
	Soil inoculation + Nitrogen	4.24 a-d	4.80 a	4.51 ab		0.96 a	0.98 a	0.97 a
	Cultivar	4.26	4.10			0.74	0.75

*Differences between means with different letters are significant at P<0.05 level

While the effect of cultivars and years on the total sugar content of fresh cowpea pods was found insignificant, the effect of the treatments was found significant (P < 0.05). According to the Table 4, the lowest total sugar value was found in the control treatment at 3.44 mg/g, and the highest total sugar value was found in the nitrogen treatment at 4.71 mg/g.

The effects of rhizobium inoculation, nitrogen application, their combinations, and control treatments on reducing sugar content are given in Table 4. The effects of treatments on reducing sugar content in cowpea were found to be significant, while the effects of cultivars and years were found to be insignificant. Reducing sugar values varied between 0.58–0.97 mg/g among the treatments. The lowest reducing sugar content was observed in the control treatment with 0.58 mg/g, while the highest reducing sugar content was observed in the soil inoculation + nitrogen treatment with 0.97 mg/g.

According to the mean values of the 2 years of the study, it is seen that the treatment x variety interaction on reducing sugar content is significant. The lowest reducing sugar content in cowpea was obtained in the control treatment of the Akkız variety with 0.54 mg/g, while the highest values were obtained in the soil inoculation + nitrogen treatment of the Karnıkara variety with 0.98 mg/g and in the soil inoculation + nitrogen treatment of the Akkız variety with 0.96 mg/g. As a result of the analysis of fresh cowpea pods, it was observed that all other treatment groups increased total sugar content by 15.7–36.9% and reducing sugar content by 10.3–67.2% compared to the control group.

Amine-Khodja et al. (2022) suggested that soluble sugars are important as osmolytes, and their role in plants’ metabolism and in maintaining plant development under abiotic stress conditions. Du et al. (2020) reported that drought stress increased the contents of soluble sugar. The fact that our study was carried out under non-irrigated conditions may have stressed the plants and thus caused an increase in sugar content.

According to Makgato et al. (2020), the use of rhizobium inoculation increased ferric, reducing antioxidant power assay and accumulation of soluble sugars, primarily fructose, and sucrose. Also, their findings suggested that rhizobium inoculation at various levels can induce and enhance the biosynthesis of soluble sugars. This may point to an improvement in the development of soluble sugars’ metabolic activities under various conditions. Liu et al. (2019) reported that rhizobia nodulation enhanced the accumulation of soluble sugars in plants. Abbas et al. (2018) found that the rhizobium inoculation increased the total soluble sugars of Vicia faba shoot.

4. Conclusions

The current study investigated on the effects of rhizobium inoculation on yield and some biochemical traits of fresh cowpea cultivation. Rhizobium inoculation and nitrogen treatments improved all growth parameters compared to the control. Besides, rhizobium inoculation increased some bioactive compounds in fresh pods. Thus, rhizobium inoculation practices especially seed inoculation can be an alternative to nitrogen applications.

Acknowledgements

This study is derived from a master’s thesis and was financially supported by the Süleyman Demirel University Scientific Research Projects Coordination Unit (4830-YL1-16).

Disclosure statement

No potential conflict of interest was reported by the author(s).