Selecting 27 Field-Grown Common Bean (Phaseolus vulgaris L.) Genotypes for Symbiotic Performance and Drought Tolerance at Malkerns Research Station, Eswatini ^†

Abstract

Legume crops, such as the common bean (Phaseolus vulgaris L.), are significant in many Sub-Saharan African (SSA) countries, including Eswatini, due to their numerous health benefits, including high protein, fiber, vitamins, and mineral content. Common beans are a staple food in many parts of the world and play a crucial role in nitrogen fixation, thereby improving soil fertility. A field experiment was conducted at Malkerns research station, Eswatini, using 27 common bean genotypes to assess their ability for N-fixation and water relations using the ¹⁵N and ¹³C natural abundance techniques. The data revealed significant differences among the common bean genotypes. Genotypes Cim-Rm-36 and Mwctz20a-Rm19 recorded an increase in plant growth by (6% and 5.74%), N content (5.69% and 5.97%) and greater C content (6.1% and 5.67%) while genotype Mwctz20a-Rm19 also showed an increase in N-fixation (155.73 kg.ha⁻¹). Genotype Mwctz20a-Rm-4 had the highest grain yield (1747.39 kg.ha⁻¹), while genotype Cim-Rm-14-Als61 had the highest N concentration (3.50%), indicating efficient N uptake. The genotypes with the lowest δ¹³C values (−27.38‰ to −28.06‰) suggested similar water use efficiency among the genotypes. The findings of this study revealed that common beans can make a significant contribution to N fertility under drought conditions. Genotypes Cim-Rm-36, Mwctz20a-Rm19, and Mwctz20a-Rm-4 showed desirable characteristics and can be good candidates for possible inclusion in breeding programs. These results have implications for improving common bean production in drought-prone areas and promoting sustainable agriculture practices.

1. Introduction

Common bean (Phaseolus vulgaris L.) is an important legume crop contributing to food security and nutritional well-being, particularly in sub-Saharan Africa [1]. It serves as a key source of protein, fiber, essential macro-micronutrients, carbohydrates, minerals, and nutrition for millions of people. In Eswatini, common bean is a vital crop, with over 80% of the country’s agricultural land dedicated to its production [2]. Common bean plays a crucial role in traditional cropping systems, contributing to nitrogen fixation and improving soil fertility [3]. Its significance extends beyond local consumption, as it boosts the national economy by serving as a key source of food and a valuable export commodity, generating income, creating jobs, and supporting livelihoods for smallholder farmers [4]. However, despite its importance, common bean production in Eswatini faces significant challenges. Soil fertility constraints, particularly nitrogen deficiency, and limited rainfall or irrigation, hinder production [5]. The use of chemical fertilizers in sub-Saharan Africa is limited due to high costs, environmental pollution, and unavailability of resources to poor rural farmers [6,7]. Climate change also poses a significant threat to global food security and agricultural productivity [8]. As a result, there is a pressing need to identify and promote climate-resilient agricultural practices that can mitigate these impacts [9]. Common bean can be grown in various environments; however, production is limited, particularly in regions with water scarcity. To address this challenge, 27 field-grown common bean genotypes were selected for symbiotic N performance, drought tolerance, and water use efficiency at Malkerns Research Station, Eswatini. A few studies have shown that common beans in Africa can meet their nitrogen (N) requirements through symbiotic fixation and accumulate carbon [10]. However, the actual amounts of N-fixed can vary due to several biotic and abiotic factors [11]. While several studies have evaluated symbiotic N contribution in common bean, the data collected were based on only a few genotypes [5,12,13]. In addition, common bean is not only resilient to harsh conditions like drought and poor soils but also boosts soil fertility by fixing nitrogen on nutrient-poor soils, ultimately enhancing crop yields and improving food security for local communities [10,14,15]. The most common interaction between legume plants and microorganisms, commonly referred to as rhizobia, is symbiotic with rhizobacteria and known for their capacity to fix nitrogen [10]. Nodules are generated by the symbiotic connection between rhizobia and legume roots, which utilize nitrogenase to fix atmospheric N₂. The interchange of nitrogen and carbon between the association nodulation root and its host plant is primarily responsible for controlling this relationship; the plant provides the bacteria with reduced carbohydrates, which the bacteria use as food and energy, and initiates the N₂ fixation process [16]. However, high temperatures, low soil fertility, acidity, and drought can limit the amount of nitrogen fixation that legumes can fix, and bacterial conditions also reduce the effectiveness of biological nitrogen fixation (BNF) [17]. Therefore, the aim of this study was to assess plant growth and symbiotic N nutrition, grain yield, C accumulation, and water use efficiency in 27 common bean genotypes grown under field conditions at Malkerns Research Station, Eswatini.

2. Materials and Methods

2.1. Site Description

A field experiment was conducted during the 2024 cropping season at the Malkerns Research Station, Eswatini, with reference GPS co-ordinates 26°33′18.33″ S; 31°10′10.29″ E, and elevation of 700 m above sea level. The area is classified as sub-tropical with an average annual rainfall ranging between 800 and 1000 mm and annual average temperatures ranging from 7 °C to 26.6 °C [18,19]. The soil type is categorized as Deep Red Loam under the Malkerns series soil classification [20]. The daily temperatures recorded during the experimental period (January 2024 to May 2024) ranged from 17.1 °C for minimum and 27.9 °C for maximum (

Table 1. Climatic data recorded during the experimental period at the Malkerns research station, Eswatini (January to May 2024).

	Temperature		Rainfall (mm)	Relative Humidity
	(°C)			(%)
2024	Max.	Min.		Max.	Min.
January	27.9	17.1	77.6	75	57
February	26.3	17.3	190.8	84	67
March	27.4	18.2	168.8	81	63
April	28.4	18.8	47.4	77	53
May	27.2	18.5	124.0	83	50
Mean	27.4	17.9	121.72	80	58

). The average rainfall recorded during the study period was 121.72 mm. The climatic data recorded at the Malkerns research station during the experimental period (temperature, relative humidity, and rainfall) are described in Table 1.

2.2. Experimental Design and Planting

The 27 common bean genotypes were planted in a randomized complete block design (RCBD) and replicated four times per genotype. The genotypes were obtained from the International Center for Tropical Agriculture (CIAT–Malawi). The experiment was conducted in early January 2024 and plowing of the field was done mechanically, and harrowing was done using a disk plow. The seeds were sown on a flat surface, with an inter-row spacing of 50 cm and an intra-row spacing of 10 cm. The plot size was measuring 13.5 m² (4.5 m × 3.0 m) and consisted of 4 rows with 20 plants per row. The experiment was conducted under natural rainfed conditions without irrigation or any chemical input. Two seeds were planted per hole and later thinned to one plant. About four to five weeks post-emergence, weeds were controlled manually using hand hoes.

2.3. Plant Sampling and Processing

Plant sampling for dry matter (DM) yield and isotopic analyses were done at an early pod-filling stage. Four plants were randomly sampled from the rows of each plot and separated into shoots and nodulated roots. The shoots were oven-dried at 65 °C (72 h) to a constant weight for shoot DM determination. The shoots were then ground to fine powder (0.85 mm sieve) and stored in vials for ¹⁵N and ¹³C analyses. Six non-leguminous weed species (namely, Cynodon dactylon, Bidens pilosa, Cyperus rotundus, Trifolium repens, Oxalis stricta, and Amaranthus retroflexus growing within the experimental plots were sampled as reference plants to determine the (%Ndfa) values of the common bean plants.

2.4. Measurement of Shoot N₂ Fixation and C Accumulation

2.4.1. ¹⁵N/¹⁴N Isotopic Analysis

The ¹⁵N and ¹⁴N isotopic analyses were done at the Stable Light Isotope at the University of Cape Town (UCT) laboratory in South Africa. About 2.0 to 2.5 milligrams of finely crushed plant material were weighed and placed into aluminum (Al) tin capsules for the purpose of analyzing ¹⁵N/¹⁴N and the N concentration (%N) values. This analysis was performed utilizing a Carlo Erba NA1500 elemental analyzer that is linked to a Finnigan MAT 252 mass spectrometer (Finnigan, MAT CombH, Bremen, Germany) through the Conflo II Open-Split Device. The δ¹⁵N values of the common bean plants were computed using the equation described by [21].

$$\mathrm{\delta }{}^{15}\mathrm{N}={\displaystyle \frac{({}^{15}\mathrm{N}/{}^{14}\mathrm{N}{)}_{\mathrm{sample}}-({}^{15}\mathrm{N}/{}^{14}\mathrm{N}{)}_{\mathrm{atm}}}{({}^{15}\mathrm{N}/{}^{14}\mathrm{N}{)}_{\mathrm{atm}}}}\times 1000$$

The ratio of the isotopes ¹⁵N and ¹⁴N in the sample is denoted as ¹⁵N/¹⁴N sample, while the atmospheric abundance ratio of nitrogen isotopes is denoted as ¹⁵N/¹⁴N atm.

2.4.2. Shoot N Content

Shoot %N was obtained directly from the mass spectrometer, and the shoot N content was calculated as the product of %N and shoot dry matter, as described by [22].

N content = %N shoot × dry mass shoot

2.4.3. The B-Value

The ¹⁵N natural abundance technique was used for N-fixation analyses. It is based on the principle that an effectively nodulated legume growing on a medium free from combined N (mineral N or organic N) is expected to be completely reliant upon symbiotic N₂ fixation for its growth, and hence, the isotopic composition of the legume would be expected to be similar to that of atmospheric N₂. A legume’s δ¹⁵N value, when grown in soil containing mineral N, is expected to resemble that of the soil mineral N [21]. Different non-legume plant species were collected from the experimental plots and used for the δ¹⁵N analyses and a B-value of −1.988‰ [21] was used for the calculations. The combined mean δ¹⁵N value of all the reference plant species sampled was +3.175.

2.4.4. Percent N Derived from Atmospheric Fixation

The proportion of N derived from the atmosphere (%Ndfa) was estimated as in [23]:

$$\%\mathrm{Ndfa}={\displaystyle \frac{\mathrm{\delta }{{}^{15}\mathrm{N}}_{\mathrm{ref}}-\mathrm{\delta }{{}^{15}\mathrm{N}}_{\mathrm{leg}}}{\mathrm{\delta }{{}^{15}\mathrm{N}}_{\mathrm{ref}}-\mathrm{Bvalue}}}\times 100$$

where δ¹⁵N_ref is the ¹⁵N natural abundance of the reference plant, δ¹⁵N_leg is the ¹⁵N natural abundance of the legume, and the B-value is the ¹⁵N natural abundance of common bean plants deriving all their N nutrition from N₂ fixation.

2.4.5. Amount of N-Fixed

The amount of fixed N (N-fixed) by the common bean plant was calculated as

N-fixed= %Ndfa × legume biomass N

where legume biomass N is the N content of the common bean shoots.

2.4.6. Soil N Uptake

The soil N uptake by the common bean plant was calculated as described by [21]:

Soil N uptake = (100 − %Ndfa) × legume biomass N

2.4.7. Measurements of ¹³C/¹²C and C Concentration

The ¹³C/¹²C isotopic analysis of bean shoots was performed at the Stable Light Isotope Laboratory, Department of Archaeology at the University of Cape Town, South Africa, as described for the ¹⁵N/¹⁴N isotopic analysis. Common bean shoot samples were analyzed to determine the ratio of ¹³C/¹²C and the C concentration (%C). The ¹³C natural abundance (or δ¹³C) was calculated as [24]:

$$\mathrm{\delta }{}^{13}\mathrm{C}={\displaystyle \frac{{\left(\raisebox{1ex}{${}^{13}\mathrm{C}$}\!\left/ \!\raisebox{-1ex}{${}^{12}\mathrm{C}$}\right.\right)}_{\mathrm{sample}}-{\left(\raisebox{1ex}{${}^{13}\mathrm{C}$}\!\left/ \!\raisebox{-1ex}{${}^{12}\mathrm{C}$}\right.\right)}_{\mathrm{standard}}}{{\left(\raisebox{1ex}{${}^{13}\mathrm{C}$}\!\left/ \!\raisebox{-1ex}{${}^{12}\mathrm{C}$}\right.\right)}_{\mathrm{standard}}}}\times 1000$$

where R_sample is the ¹³C/¹²C ratio of the sample and R_standard is the ¹³C/¹²C isotopic ratio of PDB, a universally accepted standard from the Belemnite Pee Dee limestone formation. The %C of each plant sample was obtained directly from the mass spectrometric analysis.

2.4.8. C Content

The C content of common bean shoots was calculated as the product of C concentration and shoot weight, where C concentration (%C) was obtained directly from the mass spectrometer:

2.5. Statistical Data Analysis

The data were subjected to analysis of variance (ANOVA) to compare means of the treatments using STATISTICA Software (version 10.1). The symbiotic N parameters and carbon accumulation parameters were analyzed using a one-way ANOVA to compare the performance of each genotype. Where treatment means were different, Duncan’s multiple range test (DMRT) was used to separate the means at p ≤ 0.05.

3. Results

3.1. Shoot δ¹⁵N Values of Reference Plants

Six non-legume reference plant species were sampled and analyzed to calculate the %Ndfa of the common bean genotypes. The δ¹⁵N values of the reference plants ranged from +2.43‰ to +3.96‰ with a combined mean of +3.175‰ (

Table 2. Reference plants used for estimating δ¹⁵N (‰).

Common Name	Scientific Name	δ15N‰
Star grass	Cynodon dactylon	+3.96
Black jack	Bidens pilosa	+3.49
Nutsedges	Cyperus rotundus	+3.48
broadleaf plants	Trifolium repens	+3.17
Oxalis	Oxalis stricta	+2.43
Amaranthus	Amaranthus retroflexus	+2.52
Average		+3.175

).

3.2. Plant Growth

A one-way ANOVA revealed a significant difference (p ≤ 0.001) in shoot dry matter yield among the 27 common bean genotypes planted at the Malkerns research station, Eswatini. The shoot dry matter yield ranged from 17.88 g.plant⁻¹ (genotype Mwctz20a-Rm19) to 47.43 g.plant⁻¹ (genotype Cim-Rm-36) (

Table 3. Plant growth and symbiotic performance of 27 common bean genotypes planted at Eswatini, Malkerns research station during 2024 cropping season.

Genotype	Shoot DM	N Concentration	δ15N	N Content	Ndfa	Amount N-Fixed	Soil N Uptake	Grain Yield
	g.plant−1	%	‰	mg.plant−1	%	kg.ha−1	kg.ha−1	kg.ha−1
M-S-15-D-F-1	17.88 ± 2.55 i	2.57 ± 0.16 f	0.35 ± 0.31 g–j	470.52 ± 78.43 g	54.71 ± 5.96 a–d	52.28 ± 8.71 g	8.87 ± 0.48 i	765.62 ± 111.53 k
M-S-16-A-1	18.37 ± 1.77 h–i	3.19 ± 0.09 a–d	0.40 ± 0.24 f–j	596.49 ± 72.52 e–g	53.73 ± 4.61 a–e	66.27 ± 8.06 e–g	12.98 ± 1.24 f–i	1045.57 ± 143.27 h–j
C-S-15-Z-1	21.40 ± 1.79 g–i	2.71 ± 0.16 e–f	1.45 ± 0.18 a–c	600.91 ± 77.05 e–g	33.34 ± 3.42 h–j	66.77 ± 8.56 e–g	19.09 ± 1.93 e–i	678.65 ± 81.85 k
M-S-15-D-E-22	22.03 ± 1.71 f–i	2.60 ± 0.12 f	0.59 ± 0.34 d–i	580.12 ± 60.98 f–g	50.11 ± 6.61 b–g	64.46 ± 6.78 f–g	13.04 ± 1.21–i	779.95 ± 104.06 k
M-S-15-D-10	22.05 ± 0.97 f–i	2.58 ± 0.012 f	0.09 ± 0.37 h–j	576.63 ± 46.68 f–g	59.77 ± 7.23 a–c	64.07 ± 5.19 f–g	10.44 ± 1.25 h–i	920.57 ± 87.46 i–k
M-S-16-A-2	24.66 ± 0.94 e–i	2.60 ± 0.15 f	0.55 ± 0.25 d–i	634.29 ± 26.21 e–g	50.82 ± 4.95 b–g	70.47 ± 2.91 e–g	15.55 ± 1.52 f–i	1089.01 ± 129.61 g–j
DAB 210	25.05 ± 1.31 e–i	2.82 ± 0.10 d–f	0.14 ± 0.39 h–j	702.29 ± 36.12 d–g	58.93 ± 7.50 a–c	78.03 ± 4.01 d–g	13.85 ± 2.12 f–i	1182.09 ± 171.28 e–i
DAB 174	25.29 ± 0.93 e–i	2.57 ± 0.17 f	0.48 ± 0.10 f–i	658.69 ± 56.86 e–g	52.25 ± 2.04 b–f	73.19 ± 6.32 e–g	15.47 ± 1.20 f–i	850.26 ± 99.87 j–k
PAN 9216	25.38 ± 1.93 e–i	2.72 ± 0.19 e–f	–0.202 ± 0.25 i–j	703.53 ± 85.56 d–g	65.48 ± 4.79 a–b	78.17 ± 9.17 d–g	11.60 ± 1.82 g–i	1042.97 ± 89.37 h–j
M-S-16-A-F-7	26.98 ± 3.87 d–i	2.70 ± 0.10 e–f	–0.373 ± 0.19 j	709.04 ± 86.00 d–g	68.81 ± 3.76 a	78.78 ± 9.56 d–g	11.63 ± 2.07 g–i	1407.55 ± 53.99 b–f
M-Rm-15-F-17	28.92 ± 1.32 d–i	3.04 ± 0.17 b–e	1.19 ± 0.26 b–f	863.70 ± 26.28 c–f	38.46 ± 5.04 e–i	95.97 ± 2.92 b–e	26.30 ± 1.88 c–g	1512.99 ± 55.78 a–d
M-RM-16-A-D-16	29.03 ± 4.17 d–i	2.97 ± 0.11 c–f	1.56 ± 0.09 a–c	885.95 ± 146.93 c–f	31.23 ± 1.88 h–j	98.44 ± 2.33 c–f	31.06 ± 5.77 b–e	1239.58 ± 44.78 d–h
NUA 45	29.13 ± 0.58 d–i	3.06 ± 0.16 b–e	0.55 ± 0.15 d–i	888.07 ± 42.90 c–f	50.92 ± 2.99 b–g	98.67 ± 4.76 c–e	21.51 ± 1.25 e–i	1379.95 ± 33.25 b–f
C-RM-14-A-C-2	29.68 ± 1.27 d–h	3.19 ± 0.08 a–d	1.30 ± 0.26 b–d	943.33 ± 30.72 b–f	36.26 ± 4.99 g–i	104.81 ± 3.41 b–f	30.44 ± 3.22 b–e	1335.93 ± 52.26 c–g
M-RM-F-18	32.42 ± 3.37 c–g	2.93 ± 0.07 c–f	2.18 ± 0.22 a	960.91 ± 12.44 b–e	19.13 ± 4.29 j	106.77 ± 3.05 b–e	39.08 ± 5.46 a–d	1428.38 ± 39.84 b–f
M-RM-16-A-D-7	33.12 ± 4.17 c–f	3.17 ± 0.24 a–d	1.36 ± 0.22 b–d	1114.37 ± 19.31 a–c	35.07 ± 4.27 g–i	123.82 ± 2.03 a–c	39.32 ± 8.24 a–d	1536.82 ± 32.31 a–c
M-RM-15-F-E-6	33.33 ± 2.33 c–f	2.57 ± 0.11 f	1.29 ± 0.22 b–d	843.004 ± 35.69 d–g	36.41 ± 4.29 g–i	93.67 ± 3.97 c–g	27.22 ± 2.82 c–f	1337.23 ± 77.45 c–g
M-RM-16-D-F-11	33.40 ± 3.68 c–f	3.14 ± 0.13 a–e	0.38 ± 0.19 f–j	1038.69 ± 10.64 a–d	54.15 ± 3.65 a–e	115.41 ± 1.63 a–d	24.68 ± 4.09 d–h	1627.60 ± 80.42 a–b
M-RM-15-F-6	33.94 ± 2.40 c–e	3.13 ± 0.11 a–e	1.57 ± 0.16 a–c	1054.02 ± 67.75 a–d	31.11 ± 3.09 h–j	117.11 ± 7.53 a–d	37.05 ± 3.65 a–d	1648.52 ± 55.03 a–b
Cim-Rm-14-Als61	34.38 ± 3.08 c–e	3.50 ± 0.12 a	1.48 ± 0.33 b–c	1210.95 ± 13.86 a–c	32.85 ± 6.49 h–j	134.55 ± 4.32 a–c	43.66 ± 8.44 a–b	1294.26 ± 74.04 d–h
C-RM-14-A-87	34.54 ± 7.14 c–e	3.32 ± 0.06 a–c	1.18 ± 0.14 b–f	1137.66 ± 24.38 a–c	38.75 ± 2.71 e–i	126.41 ± 3.26 a–c	33.87 ± 6.98 b–e	1152.34 ± 30.10 f–i
Mwctz20A-Rm-4	35.82 ± 3.61 b–e	3.06 ± 0.05 b–e	1.22 ± 0.10 b–e	1107.17 ± 12.91 a–c	37.86 ± 1.98 f–i	123.01 ± 1.65 a–c	33.85 ± 3.48 a–d	1747.39 ± 36.51 a
C-RM-14-A-C-27	37.38 ± 3.23 a–d	3.46 ± 0.16 a–b	1.79 ± 0.39 ab	1301.36 ± 14.36 a–b	26.63 ± 7.67 i–j	144.59 ± 5.93 a–b	50.58 ± 9.14 a	1458.33 ± 88.36 b–e
M-RM-15-D-A-21	42.54 ± 7.70 a–c	2.76 ± 0.08 d–f	0.62 ± 0.24 d–h	1153.43 ± 20.59 a–c	49.48 ± 4.65 c–g	128.16 ± 2.39 a–c	32.10 ± 7.49 b–e	1420.56 ± 103.03 b–f
Mwctz20a-Rm19	45.39 ± 3.31 a–b	3.12 ± 0.12 a–e	0.85 ± 0.18 c–h	1401.57 ± 85.86 a	45.07 ± 3.42 c–h	155.73 ± 9.54 a	37.95 ± 2.34 a–d	1347.66 ± 55.77 c–f
Cim-Rm-36	47.43 ± 6.01 a	2.79 ± 0.10 d–f	1.03 ± 0.19 b–g	1336.64 ± 18.36 a	41.56 ± 3.87 d–i	148.52 ± 20.48 a	41.58 ± 8.09 a–c	1433.59 ± 29.66 b–e
F-statistics	4.942 ***	4.77 ***	6.855 ***	5.643 ***	6.855 ***	5.643 ***	6.9827 ***	11.865 **

Means followed by dissimilar letters are significantly different (p ≤ 0.05). Where ** p ≤ 0.01, *** p ≤ 0.001.

). Three of the genotypes produced greater shoot biomass: Cim-Rm-36 (47.43 g.plant⁻¹), Mwctz20a-Rm19 (45.39 g.plant⁻¹), and M-Rm-15-D-A-21 (42.54 g.plant⁻¹). Nine other genotypes recorded values within 30 g.plant⁻¹, and 13 genotypes recorded values within 20 g.plant⁻¹. The remaining genotypes showed the least shoot biomass of 17.88 g.plant⁻¹ (M-S-15-D-F-1) and 18.37 g.plant⁻¹ (M-S-16-A-1).

3.3. N Concentration and N Content

The N concentration and N content showed significant differences. Genotypes Cim-Rm-14-Als61 (3.50%) and C-Rm-14-A-C-27 (3.46%) exhibited the highest N concentration (Table 3). Twelve out of 27 genotypes recorded N concentration of approximately 3%, while the remaining 15 genotypes recorded N concentration of approximately 2%. The levels of N concentration for genotypes M-S-15-D-F-1, DAB 174, and M-Rm-15-F-E-6 were the same (2.57%).

The N content of the 27 common bean genotypes ranged from 470.52 mg.plant⁻¹ (M-S-15-D-F-1) to 1401.57 mg.plant⁻¹ (Mwctz20a-Rm19). Ten other genotypes accumulated N content between 1000 and 1410 mg.plant⁻¹, and 17 genotypes recorded values below 1000 mg.plant⁻¹. Genotype M-S-15-D-F-1 recorded the lowest N concentration (2.57%) and N content (470.52 mg.plant⁻¹) (Table 3).

3.4. δ¹⁵N and %Ndfa Values of Common Bean Plants

The δ¹⁵N and %Ndfa of the common bean genotypes planted at Malkerns research station varied from −0.373‰ (M-S-16-A-F-7) to 2.18‰ (M-Rm-F-18) (Table 3). One genotype recorded the highest δ¹⁵N value of 2.18‰, followed by 12 other genotypes with values ranging approximately above 1‰. Eleven genotypes had δ¹⁵N values below 1‰, and the remaining two genotypes recorded values of −0.373‰ (M-S-16-A-F-7) and −0.202‰ (PAN 9216). These genotypes with the lowest δ¹⁵N values derived the most N from the atmosphere (%Ndfa), with the highest values of 68.81% and 65.48%, respectively. In contrast, the genotypes with the highest δ¹⁵N values recorded the lowest %Ndfa, such as M-Rm-F-18 (19.13%) and C-Rm-14-A-C-27 (26.63%). Eleven out of the 27 common bean genotypes derived over 50% or more N nutrition from the atmosphere.

3.5. Amount of N-Fixed

The amount of N-fixed revealed significant differences among the 27 common bean genotypes. The amount of N-fixed ranged from 52.28 kg.ha⁻¹ (M-S-15-D-F-1) to 155.73 kg.ha⁻¹ (M-Rm-15-F-19). Twelve out of the 27 common bean genotypes recorded N-fixed above 100 kg.ha⁻¹, while the remaining 15 genotypes recorded values below 100 kg.ha⁻¹ (Table 3). Only genotype M-S-15-D-F-1 fixed a N value below 60 kg.ha⁻¹, resulting from a lower shoot dry matter yield (17.88 g.plant⁻¹).

3.6. Soil N Uptake

The common bean genotypes differed markedly in their levels of soil N uptake. The soil N uptake ranged from 8.87 kg.ha⁻¹ (M-S-15-D-F-1) to 50.58 kg.ha⁻¹ (C-Rm-14-A-C-27) (Table 3). Three out of the 27 genotypes took about 40 kg.ha⁻¹ (i.e., C-Rm-14-A-C-27, Cim-Rm-14-Als61, and Cim-Rm-36) from the soil, while nine other genotypes recorded values within 30 kg.ha⁻¹. Four genotypes recorded values within 20 kg.ha⁻¹, and the remaining 11 genotypes recorded values below 20 kg.ha⁻¹.

3.7. Grain Yield

The grain yield of the common bean genotypes revealed a significant difference, ranging from 678.65 kg.ha⁻¹ (C-S-15-Z-1) to 1747.39 kg.ha⁻¹ (Mwctz20a-Rm-4). Twenty-two out of the 27 common bean genotypes recorded grain yields ranging from 1000 kg.ha⁻¹ to 1700 kg.ha⁻¹. In contrast, the remaining five genotypes produced the lowest grain yields: C-S-15-Z-1 (678.65 kg.ha⁻¹), M-S-15-D-F-1 (765.62 kg.ha⁻¹), M-S-15-D-E-22 (779.95 kg.ha⁻¹), DAB 174 (850.26 kg.ha⁻¹), and M-S-15-D-10 (920.57 kg.ha⁻¹) (Table 3).

3.8. Carbon Concentration and C Content

The carbon (C) concentrations and carbon content values showed significant differences among the 27 common bean genotypes. The carbon (C) concentrations ranged from 39.08% (M-RM-15-F-E-6) to 41.15% (PAN 9216) (

Table 4. Comparison of dry matter yield, %C, C content, C/N ratio and δ¹³C of common bean genotypes planted in the field of Malkerns research station, Eswatini.

Genotype	Shoot DM	C Concentration	C Content	δ13C	C:N Ratio
	g.plant−1	%	mg.plant−1	‰	g.g−1
M-S-15-D-F-1	17.88 ± 2.55 i	40.21 ± 0.30 a–e	715.75 ± 98.56 i	−29.84 ± 0.22 a	15.67 ± 0.92 a–c
M-S-16-A-1	18.38 ± 1.77 h–i	39.66 ± 0.35 d–f	724.91 ± 64.31 i	−21.64 ± 6.29 a	15.49 ± 1.36 a–d
C-S-15-Z-1	21.4 ± 1.79 g–i	39.63 ± 0.16 d–f	848.04 ± 71.09 h–i	−22.28 ± 6.29 a	13.93 ± 0.46 c–g
M-S-15-D-E-22	22.03 ± 1.71 f–i	39.44 ± 0.37 d–f	870.20 ± 70.07 g–i	−28.68 ± 0.18 a	16.45 ± 1.12 a
M-S-15-D-10	22.05 ± 0.96 f–i	40.97 ± 0.19 a–b	903.25 ± 39.35 f–i	−29.07 ± 0.19 a	14.35 ± 0.24 a–f
M-S-16-A-2	24.67 ± 0.94 e–i	40.18 ± 0.22 a–f	991.74 ± 39.75 e–i	−28.78 ± 0.29 a	14.68 ± 0.76 a–e
DAB 210	25.05 ± 1.31 e–i	40.48 ± 0.48 a–d	1016.12 ± 59.19 d–i	−28.18 ± 0.36 a	14.96 ± 1.09 a–e
DAB 174	25.29 ± 0.93 e–i	40.24 ± 0.25 a–e	1019.12 ± 41.72 d–i	−28.77 ± 0.12 a	15.09 ± 0.67 a–e
PAN 9216	25.38 ± 1.93 e–i	41.15 ± 0.63 a	1048.88 ± 86.86 d–i	−29.29 ± 0.10 a	14.89 ± 0.68 a–e
M-S-16-A-F-7	26.98 ± 3.87 d–i	39.48 ± 0.77 d–f	1070.54 ± 160.86 d–i	−28.87 ± 0.21 a	16.17 ± 1.12 a–b
M-Rm-15-F-17	28.91 ± 1.32 d–i	39.61 ± 0.19 d–f	1145.34 ± 52.72 d–i	−28.50 ± 0.35 a	13.38 ± 0.80 c–g
M-RM-16-A-D-16	29.03 ± 4.17 d–i	39.59 ± 0.19 d–f	1149.02 ± 164.66 d–i	−27.98 ± 0.32 a	13.46 ± 0.54 c–g
NUA 45	29.13 ± 0.58 d–i	39.79 ± 0.19 c–f	1158.66 ± 21.34 d–i	−27.51 ± 0.16 a	13.39 ± 0.74 c–g
C-RM-14-A-C-2	29.68 ± 1.27 d–g	40.29 ± 0.33 a–e	1197.60 ± 57.09 d–h	−22.29 ± 6.27 a	12.68 ± 0.37 e–g
M-RM-F-18	32.42 ± 3.37 c–g	39.32 ± 0.28 e–f	1277.97 ± 136.82 d–h	−28.86 ± 0.15 a	13.52 ± 0.36 c–g
M-RM-16-A-D-7	33.12 ± 4.72 c–f	40.06 ± 0.22 a–f	1325.73 ± 188.57 c–g	−28.26 ± 0.24 a	13.34 ± 1.19 c–g
M-RM-15-F-E-6	33.33 ± 2.33 c–f	39.08 ± 0.28 f	1303.55 ± 93.56 d–h	−28.98 ± 0.09 a	15.37 ± 0.68 a–d
M-RM-16-D-F-11	33.40 ± 3.68 c–f	39.70 ± 0.24 c–f	1326.31 ± 145.51 c–g	−22.43 ± 6.30 a	12.84 ± 0.52 e–g
M-RM-15-F-6	33.94 ± 2.40 c–e	39.77 ± 0.16 c–f	1349.27 ± 94.61 c–f	−27.74 ± 0.27 a	12.84 ± 0.46 e–g
Cim-Rm-14-Als61	34.38 ± 3.09 c–e	40.13 ± 0.27 a–f	1377.29 ± 120.78 b–f	−27.38 ± 0.27 a	11.54 ± 0.39 g
C-RM-14-A-87	34.55 ± 7.15 c–e	40.01 ± 0.27 b–f	1385.68 ± 287.15 b–e	−27.22 ± 0.21 a	12.11 ± 0.21 f–g
Mwctz20a-Rm-4	35.83 ± 3.61 b–e	39.99 ± 0.29 b–f	1428.96 ± 139.97 b–e	−28.42 ± 0.14 a	13.09 ± 0.25 d–g
C-RM-14-A-C-27	37.37 ± 3.23 a–d	39.68 ± 0.32 d–f	1482.79 ± 127.70 b–d	−28.07 ± 0.21 a	11.66 ± 0.58 g
M-RM-15-D-A-21	42.54 ± 7.70 a–c	40.81 ± 0.29 a–c	1736.79 ± 315.89 a–c	−29.42 ± 0.09 a	12.77 ± 0.49 e–g
Mwctz20a-Rm19	45.40 ± 3.31 a–b	39.44 ± 0.14 d–f	1791.09 ± 131.02 a–b	−27.99 ± 0.19 a	12.77 ± 0.49 e–g
Cim-Rm-36	47.43 ± 6.01 a	40.53 ± 0.26 a–d	1927.07 ± 249.11 a	−28.52 ± 0.25 a	14.65 ± 0.48 a–e
F-statistics	4.942 ***	2.5 ***	4.883 ***	0.943 ns	3.564 ***

Means followed by dissimilar letters are significantly different (p ≤ 0.05). Where *** p ≤ 0.001 and ns means not significant.

). Twelve out of 27 common bean genotypes recorded the highest carbon concentration of approximately 40%, and the remaining 15 genotypes recorded carbon concentrations of approximately 39%.

The carbon content values ranged from 715.75 mg.plant⁻¹ (M-S-15-D-F-1) to 1927.07 mg.plant⁻¹ (Cim-Rm-36). Twenty out of 27 common bean genotypes recorded the highest carbon content, ranging from 1000 mg.plant⁻¹ to 1927.07 mg.plant⁻¹, due to their greater biomass. Meanwhile, the remaining seven genotypes displayed the lowest carbon content, below 1000 mg.plant⁻¹.

3.9. Shoot C:N Ratio

The C/N ratio recorded significant differences among the 27 common bean genotypes (Table 4). All genotypes recorded C/N ratios above 10, with the highest genotype being 16.45 g.g⁻¹ (M-S-15-D-E-22) and the lowest being 11.54 g.g⁻¹ (Cim-Rm-14-Als61).

3.10. δ¹³C Values of Common Bean Plants

The δ¹³C values showed non-significant differences (p > 0.05) among all 27 common bean genotypes, ranging from −29.8‰ to −21.6‰. Four out of 27 common bean genotypes recorded the highest δ¹³C values (least negative) M-S-16-A-1 (−21.6‰), C-S-15-Z-1 (−22.3‰), C-RM-14-A-C-2 (−22.3‰), and M-RM-16-D-F-11 (−22.4‰). The lowest δ¹³C values were recorded in genotypes M-S-15-D-F-1 (−29.8‰) and M-S-15-D-10 (−29.1‰), indicating their potentially lower water-use efficiency. Nineteen of the 27 common bean genotypes recorded similar shoot δ¹³C values, ranging from −28.9‰ to −27.2‰. Furthermore, four out of the 27 common bean genotypes (M-S-16-A-1, C-S-15-Z-1, C-RM-14-A-C-2, and M-RM-16-D-F-1) exhibited higher δ¹³C values and hence were considered to be low water-use efficient genotypes (Table 4).

4. Discussion

The study revealed three out of 27 genotypes, namely Cim-Rm-36 (47.43 g.plant⁻¹), Mwctz20a-Rm19 (45.39 g.plant⁻¹), and M-Rm-15-D-A-21 (42.54 g.plant⁻¹), exhibited greater shoot biomass above 40 g.plant⁻¹ and higher N concentration, N content, as well as a greater amount of N-fixed (Table 3). These genotypes with the highest symbiotic N parameters also recorded the highest grain yield Mwctz20a-Rm-4 (1747.39 kg.ha⁻¹), M-Rm-15-F-6 (1648.52 kg.ha⁻¹), M-Rm-16-D-F-11 (1627.60 kg.ha⁻¹), C-Rm-14-A-C-27 (1458.33 kg.ha⁻¹), Cim-Rm-36 (1433.59 kg.ha⁻¹), M-Rm-15-D-A-21 (1420.56 kg.ha⁻¹), and Mwctz20a-Rm19 (1347.66 kg.ha⁻¹). [5] reported an increase in shoot biomass above 42 g.plant⁻¹ and grain yield of 202,000 kg.ha⁻¹ by common bean plants when treated with phosphorus. Moreover, [25] revealed that the greater biomass accumulation by common bean with high levels of atmospheric N₂ fixation tends to exhibit greater grain yield. In contrast, the genotypes M-S-15-D-F-1 and M-S-16-A-1 performed poorly in terms of plant growth, N content, and amount of N-fixed among the 27 common bean genotypes. However, the poor performance could be attributed to environmental factors such as biotic and abiotic factors compared to other common bean genotypes [14,15,26,27] (Table 3). The grain yield showed significant variation among the 27 genotypes, ranging from 678.65 kg.ha⁻¹ (C-S-15-Z-1) to 1747.39 kg.ha⁻¹ (Mwctz20a-Rm-4). The top-performing genotypes for grain yield were Mwctz20a-Rm-4 (1747.39 kg.ha⁻¹) followed by M-Rm-15-F-6 (1648 kg.ha⁻¹). The δ¹⁵N values ranged from −0.373‰ to 2.18‰. Two out of the 27 genotypes show lower δ¹⁵N values for M-S-16-A-F-7 (−0.373‰) and PAN 9216 (−0.202‰). These genotypes with the lowest δ¹⁵N values derived most of their nitrogen from the atmospheric N₂ fixation, with %Ndfa values of 68.81% and 65.48%, respectively. These genotypes are highly efficient and good nitrogen fixers, since they are able to capture more than 60% of their N requirement from the atmosphere and take up less N from the soil. These findings are consistent with previous studies [11,28] that reported lower δ¹⁵N values have capacity to fix nitrogen compared to non-fixing legumes. The genotypes with low value δ¹⁵N suggest a strong reliance on atmospheric nitrogen fixation [29]. Eleven out of 27 common bean genotypes could derive 50% or more of their N nutrition from the atmosphere. The findings are consistent with [28,29,30,31] which identified different genotypes with high biological nitrogen fixation (BNF), capable of deriving at least 50% of their nitrogen from the atmosphere.

The carbon concentration and carbon content in common bean plants ranged from (39.08% to 41.15%) and (715.75 to 1927.07 mg.plant⁻¹). Twenty out of 27 common bean genotypes had the highest carbon content ranging from (1000–1927.07 mg.plant⁻¹) due to greater shoot biomass, while 7 other genotypes recorded carbon content below (1000 mg.plant⁻¹). Genotypes with the highest C content also recorded the highest shoot biomass and vice versa, which is consistent with previous studies [32,33]. The C:N ratio revealed significant variations among the 27 common bean genotypes, ranging from 11.54 g.g⁻¹ in genotype Cim-Rm-14-Als61 to 16.45 g.g⁻¹ in genotype M-S-15-D-E-22 (Table 4). The positive relationship between C:N ratio and grain yield suggests that genotypes with optimal C:N ratios tend to have higher grain yields. This has been observed in genotype Cim-Rm-36, which obtained a grain yield of 1433 kg.ha⁻¹ and C:N ratio of 14.65 g.g⁻¹ [24]. The stable carbon isotope composition in shoots revealed that a higher δ¹³C value (less negative) signifies greater water use efficiency (WUE), whereas a lower δ¹³C value (more negative) indicates lower WUE [24]. Four out of 27 common bean genotypes were found to have water use efficiency with (δ¹³C) values of less negative for genotypes M-S-16-A-1 (−21.64‰), C-S-15-Z-1 (−22.28‰), C-Rm-14-A-C-2 (−22.29‰), and M-Rm-16-D-F-11 (−22.43‰). These results are consistent with the study by [34], showing common bean genotypes with improved water use efficiency (WUE) identified through various studies. The δ¹³C values showed a strong link with WUE, indicating that genotypes with higher δ¹³C values (less negative) tend to have higher WUE. This is consistent with previous studies [24,35], who reported that δ¹³C values can be used as an indicator of WUE in plants. Recent studies have also confirmed the positive link between δ¹³C values and WUE in crops such as soybean [36]. Even though the exact mechanisms of water use efficiency by the genotypes were not determined; nevertheless, plant traits related to root morphology or root architecture often play key roles in the adaptation of plants to water-stressed environments [37].

5. Conclusions

In conclusion, this study reveals significant variation in symbiotic parameters among common bean plants and successfully identifies genotypes that exhibit superior performance in shoot biomass, grain yield, N-fixation, and WUE. Genotypes Cim-Rm-36, Mwctz20a-Rm19, and M-Rm-15-D-A-21 exhibited greater shoot biomass and grain yield. In addition, M-S-16-A-F-7 and PAN 9216 showed high N-fixation efficiencies. Genotypes with optimal C:N ratios tended to exhibit increased grain yields, highlighting the importance of this parameter in determining crop productivity. The δ¹³C values of the common bean genotypes ranged from (−27.38‰ to −28.06‰) which is an indication that the genotypes have similar water requirements. These findings suggest that these genotypes possess significant potential for improving common bean production and promoting sustainable agricultural productivity.