Morpho-Cultural Characterization, Physicochemical Activities, and Effect of Abiotic Factors on the Growth of Rhizobium sp. Isolated from Nodules of 18 Soybean (Glycine max (L.) Merr.) Varieties Farmed in Cameroon

Linda Manet^1,2*, Hippolyte Mouafo Tene¹, Eddy Léonard Ngonkeu Mangaptche^2,3, Onana Boyomo⁴, Ferdinand Lanvin Edoun Ebouel^1,5, Gabriel Medoua Nama¹, Aimé Didier Begoudé Boyogueno³

¹Centre for Food, Food Security and Nutrition Research, Institute of Medical Research and Medicinal Plant Studies (IMPM), Yaoundé, Cameroon.
²Department of Plant Biology, University of Yaoundé I, Yaoundé, Cameroon.
³Institute of Agricultural Research for Development (IRAD), Yaoundé, Cameroon.
⁴Department of Microbiology, University of Yaoundé I, Yaoundé, Cameroon.
⁵Department of Biochemistry, University of Yaoundé I, Yaoundé, Cameroon.

DOI: 10.4236/ojss.2025.156018                

Abstract

Acidic pH, high salinities, and extreme temperatures limit agricultural production in tropical soils, particularly in Cameroon. Rhizobia can improve crop growth and productivity in these soils, thanks to their ability to adapt to stressful conditions. In this study, nodules of 18 varieties of soybeans (Glycine max (L.) Merr.) were used to isolate rhizobia. The isolates were characterized and screened for their resistance to pH (2 to 6.8), salinity (NaCl 2% to 12%, w/v), and temperature (15˚C to 45˚C). Then, the physicochemical activity (catalase, proteins, total antioxidant capacity (TAC), ferrous reducing antioxidant power (FRAP), and malondialdehyde (MDA) of the isolates was assessed using referenced methods. The results showed that out of 108 isolates obtained, 73 were Rhizobium sp., endowed with variable pH, salinity, and temperature adaptabilities. The highest acid resistance (pH 2 to 4), salinity (4% to 12%), and extreme temperature (40˚C to 45˚C) were recorded with isolates 1M, 2M, 7M, and 5M. Isolate 10G2 showed the highest protein content (135.33 ± 5.65 µg/mL), while the isolates 1, 2G, 5P', and 13M scored the highest catalase activity (0.07 ± 0.00 µmol/mL/g prot). All the isolates demonstrated antioxidant activity, with the highest FRAP recorded by isolate 7M (298.46 ± 0.00 µg AAE/mL) and the highest TAC by isolate 4M1 (1335.93 ± 10.84 µg AAE/mL). They also presented the ability to inhibit oxidative stress through the inhibition of MDA production. The lowest MDA value (1.14 ± 0.05 µmol/L) was obtained with the isolate 11G3. Generally, the isolates with interesting adaptation to abiotic conditions and physicochemical activities were from soybean varieties TGX 2007-11 F, TGX 2001-12 F, TGX 1991-22 F, SC Sentinel, Panorama 3, Maksoy 2N, Panorama 237, and Panorama 2. These results highlight the potential of Rhizobium sp. isolated from soybean nodules in improving the crop productivity of tropical soils and suggest further characterization of these strains using genomic approaches.

Keywords

Soybean, Nodules, Rhizobium sp., Adaptation, Abiotic Stress, Physicochemical Activity

1. Introduction

Around one-third of tropical soils worldwide are acidic [1]. In Cameroon, over 80% of cultivated lands are acidic [2]. Acidic soils are characterized by ecosystems marked by the presence of limiting factors for most soil microorganisms [3]. Under these conditions, there is a great variability in the physico-chemical characteristics of soils as well as their microbial diversity. Humid tropical soils, considered favorable environments for productivity worldwide and particularly in Africa, can become unfavorable places for the survival of beneficial soil microorganisms due to soil acidity [4]. Besides acidity, some factors limited microorganism-plant interaction in tropical soils, including salinity and extreme temperatures [5]. These interacting abiotic factors reduce the growth and survival of beneficial soil microorganisms such as rhizobia [6]. Under such environmental conditions, the survival and persistence of rhizobia depend on their adaptive mechanisms to mitigate the adverse effects of abiotic stresses such as elevated temperatures, soil acidity, and salinity. These adaptations are essential for maintaining the population density and ensuring effective symbiotic interactions with leguminous plants [7]. Considering the rhizobium-legume symbiosis, the factors likely to hinder biological nitrogen fixation are not the absence of efficient rhizobia, but the ecological factors limiting their potential [8]. According to Manet et al. [9], various abiotic stresses, including survival in soils, competitiveness for nodulation, and symbiotic efficiency, can affect the dynamics of rhizobial populations and their functional roles in the rhizosphere. These abiotic factors cannot only interfere with nodulation but also influence nitrogen-fixing activity after symbiosis establishment [10]. Abd-Alla et al. [11] reported that nitrogenase, involved in nitrogen-fixing activity, is highly sensitive to variation in soil osmotic potential. Besides nitrogen fixation, abiotic stress also leads to the activation of defense systems and lipid peroxidation [12]. The reactive oxygen species (ROS) produced at high rates when plants are exposed to stress generate oxidative damage in plants that is reflected in their malondialdehyde (MDA) content, a by-product of lipid peroxidation that is one of the first nonspecific links in the overall stress response [13]. In the literature, it was highlighted that the total antioxidant capacity of plants is linked to the symbiotic nitrogen uptake activity in legume root nodules and depends on the efficiency of the rhizobium-legume symbiosis [14]. Antioxidants, catalase, osmolytes, stress proteins, and exopolysaccharides are produced by rhizobia to survive in harsh environments caused by abiotic stress [15]. Ben-Laouane et al. [16] mentioned that rhizobia can improve the plant’s tolerance to stress by inducing physical and chemical changes. Zhang et al. [17] reported that rhizobia can improve plant tolerance to low temperatures by affecting nitrogen metabolism and uptake. Rhizobia also play an important role in the management of oxidative stress by plants through the improvement of an antioxidant defense system, which includes an enzymatic system [18] and a non-enzymatic system [19].

The study of stress tolerance of rhizobia aims to find viable solutions for sustainable agriculture in the context of climate change and crop losses. To this end, researchers are mainly focused on legumes, which are the natural hosts for rhizobia that convert atmospheric nitrogen into plant-available nitrogen, thereby enhancing the nitrogen-fixing capacity of plants [20]. Indeed, an effective Rhizobium-legume symbiosis can ensure high yields and good nitrogen balance in depleted soils [21]. This requires an efficient Rhizobium capable of surviving in adverse environmental conditions and endowed with physicochemical activity, including inhibition of oxidative stress. For this purpose, nodules from soybean (Glycine max L. (Merr.) were chosen in this study as that oilseed crop is among the most common legumes grown in Africa [22], for its multiple food and industrial functions [23]. Also, owing to its efficient symbiosis, each year approximately 16.4 Tg of atmospheric nitrogen, constituting about 77% of the total nitrogen amount, is fixed by legumes [24]. In Africa, few research projects have been conducted on Rhizobium endowed with resistance to abiotic conditions and physicochemical activities. In Senegal and Tunisia, the resistance of Rhizobium strains to high temperatures (40˚C to 45˚C) and salinity (NaCl > 2%) was reported by Cacciari et al. [25]. In Cameroon, Ngo Nkot et al. [26] demonstrated that Rhizobium strains isolated from bambara groundnut can grow at low pH but might encounter difficulties at a NaCl concentration of 4%. Although the stress tolerance of rhizobia is the subject of numerous studies worldwide, knowledge remains limited concerning indigenous strains associated with soybean in poor, acidic tropical soils exposed to multiple abiotic stresses, particularly in Central Africa. In particular, the ability of these local strains to adapt to combined stress conditions (acidity, salinity, temperature) has not yet been systematically characterized. This study fills this gap by isolating and evaluating, for the first time in Cameroon, Rhizobium spp. strains according to their tolerance to abiotic stresses and their antioxidant responses, in view of identifying robust candidates for the development of bio-inoculants adapted to specific agroecological conditions. To our knowledge, there are few or no studies on the characteristics and Rhizobium profile of soybeans cultivated in acidic Cameroonian soils, as well as their physicochemical activities. It is in this context that the present study was designed. The objectives were to evaluate the tolerance to abiotic factors (acidity, salinity, and temperatures) of Rhizobium sp. cultures isolated from soybean varieties grown on acidic soils in the forest zone of Cameroon, and identify the physicochemical activity (antioxidant activity, inhibition of lipid peroxidation) of these isolates for select high-potential Rhizobium sp. isolates that could be good biofertilizer candidates.

2. Materials and Methods

2.1. Nodule Collection

The sampling site selected for this study is located in an experimental station of the Institute of Agricultural Research for Development (IRAD) at Nkolbisson (3˚51'N, 11˚27'E; altitude 769 m) in the Centre region of Cameroon. Root nodules were collected during March 2022 after complete pod filling with different soybean varieties originated from 7 countries as indicated in Table 1, and grown on an acidic soil (pH 4.10). Plant roots were carefully removed individually, wrapped in newspaper, and transferred to the laboratory, where detached nodules were stored at 4˚C. Root nodules were evaluated based on morphological characteristics, including shape, color, spatial arrangement on the roots, size, and microscopic features. Nodules exhibiting traits typically associated with active nitrogen fixation, such as a pink or reddish interior and a healthy, uniform structure, were selected for rhizobia isolation. Nodule size was classified according to the criteria of King and Purcell [27], where small nodules have a size ≤2.0 mm, medium with a size between 2.0 and 4.0 mm, and large with a size >4.0 mm.

2.2. Isolation of Rhizobia from Root Nodules

Rhizobia were isolated from root nodules following the method of Vincent [28] with some modifications. In the laboratory protocol, six healthy nodules showing a pink coloration due to the presence of leghemoglobin were collected from several soybean plants. The nodules were thoroughly washed with tap water to remove adhered soil particles and then separated from the roots. Healthy, intact root nodules were subjected to surface sterilization by immersion in a 3% calcium hypochlorite solution for 3 min, followed by three rinses with sterile distilled water. To confirm surface sterility, an aliquot of the final rinse water was plated on nutrient agar and incubated at 28 ˚C for 48 h. The absence of microbial growth indicated successful surface sterilization. After surface sterilization, the nodules were individually ground under aseptic conditions and streaked onto sterile Mannitol Yeast Extract Agar (YEMA) supplemented with Congo red, poured into Petri dishes. The Petri dishes were incubated for 7 days at 28˚C (Bioevopeak, China). After the incubation period, well-isolated colonies were picked up and purified by 3 successive streaking on a new sterile YEMA agar. Pure isolates were stored at −80˚C in sterile YEM broth containing 60% glycerol.

Table 1. Morphological and cultural characteristics of Rhizobium sp. isolates obtained from nodules of 18 soybean varieties grown on acidic soil of Cameroon.

Soybean variety names	Origin	Isolate growth rate (days)	Isolate diameter (µm)	Isolate structure	Mucus type	Appearance of isolates	Isolate shape	Varieties coding
TGX-1988-18F	DARS (MALAWI)	4	4	Thick	Viscous	Milky and sticky	irregular	14
S1180/5/54 (N)	SeedCo (ZIMBABWE)	3	4	Thick	Viscous	Whitish and sticky	roundness	15
TGX 2001-10 DM	IITA (ZAMBIA)	3	2	Thick	Viscous	Whitish and sticky	roundness	16
SC Sentinel	SeedCo (ZIMBABWE)	3	2	Thick	Viscous	Whitish and sticky	roundness	5
Songda	Savanna Agricultural Research Institute of Ghana (CSIR_SARI)	3	2	Thick	Viscous	Milky and sticky	roundness	8
TGX 2007-11 F	IITA (CAMEROON)	4	4	Thick	Viscous	Whitish and sticky	roundness	1
TGX 2001-12 F	IITA (CAMEROON)	3	2	Thick	Viscous	Milky	irregular	2
PANORAMA 1	Semillas Panorama SAS (Colombia)	3	2	Thick	Viscous	Milky	irregular	6
TGX 1991-22 F	DARS (MALAWI)	3	< 2	Thick	Very viscous	Milky	roundness	4
SC SIGNAL	SeedCo (ZIMBABWE)	3	< 2	Thick	Very viscous	Milky	roundness	9
MAKSOY 1N	U.Makerere (UGANDA)	3	< 2	Thick	Very viscous	Milky	roundness	18
PANORAMA 237	Semillas Panorama SAS (Colombia)	4	4	Thick	Viscous	Whitish	irregular	11
TGX-1989-60 F	DARS (MALAWI)	4	2	Thick	Viscous	Whitish	irregular	12
MAKSOY 2N	U.Makerere (UGANDA)	4	4	Thick	Viscous	Whitish	roundness	10
TGX 2010-3 F	IITA (CAMEROON)	4	4	Thick	Viscous	Whitish	irregular	3
PANORAMA 2	Semillas Panorama SAS (Colombia)	3	4	Thick	Viscous	Milky	roundness	13
PANORAMA 3	Semillas Panorama SAS (Colombia)	3	2	Thick	Very viscous	Milky	roundness	7
TGX 1835-10 E	IITA (CAMEROON)	3	4	Slightly thick	Viscous	Whitish	roundness	17

2.3. Cultural Characterization of Isolates

2.3.1. Morphology of Isolates

From frozen stock, each pure isolate was streaked on sterile YEMA followed by incubation at 28˚C for 7 days. After incubation, the morphology characteristics of the isolates (diameter, texture, color, appearance, and shape) were assessed.

2.3.2. Adaptation Tests under Stress Conditions

The tolerance of Rhizobium sp. isolates to various abiotic factors such as pH, temperature, and salinity was assessed. Morpho-cultural characteristics of the different isolates, including the growth speed and colonies’ color on YEMA supplemented with red Congo, and the formation of mucus, were used as criteria for selecting isolates that were tested in the different treatments.

Regarding salinity tolerance, 4 mL of sterile YEM broth supplemented with NaCl at concentrations ranging from 1% to 12% (w/v) was inoculated with a pure colony of each Rhizobium isolate. Control cultures were prepared by inoculating the broth without NaCl. All cultures were incubated at 28˚C for 7 days under shaking conditions at 120 rpm. Seven days was chosen as the optimal growth period of the selected isolates from preliminary experiments. Bacterial growth was assessed by measuring the optical density at 620 nm against the blank made from uninoculated broth treated under the same conditions (UV-Mini 1240, Shimadzu, Japan).

Concerning temperature, tubes containing 4 mL of sterile YEM broth were inoculated with pure colonies of the rhizobia, and the tubes were incubated at different temperatures ranging from 15˚C to 45˚C for 7 days. The rhizobial load was measured by reading the optical density of tubes at 620 nm.

To assess the effect of pH on the growth of rhizobia, 4 mL of sterile YEM broth was introduced into sterile tubes, and the pH of the broths was adjusted from 2 to 6.8 using 1N hydrochloric acid (HCl) and 1N sodium hydroxide (NaOH). The pH-adjusted media were then buffered with Citrate buffer to maintain a stable pH during bacterial growth. The cultures were incubated at 28˚C, and growth was measured by optical density at 620 nm.

2.4. Tolerance of the Isolates to Abiotic Stress Conditions

2.4.1. pH Tolerance

The ability of each Rhizobium sp. isolate to grow under acidic conditions was determined by inoculating each isolate into tubes containing YEM broth with pH adjusted to 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 6.8, followed by incubation for 7 days at 28˚C. The optical density of the tubes was read at 620 nm.

2.4.2. Salt Tolerance

The ability of the isolates to grow at different NaCl concentrations was assessed by inoculating each isolate into YEM broth containing 1%, 2%, 4%, 6%, 8%, 10%, and 12% NaCl. Cultures were incubated at 28 ˚C for 7 days under shaking conditions at 120 rpm to ensure adequate aeration. Bacterial growth was evaluated by measuring the optical density at 620 nm.

2.4.3. Temperature Tolerance

The ability of the isolates to grow at different incubation temperatures was evaluated by inoculating each of the 73 rhizobia isolates into YEM broth. The inoculated cultures were then incubated at 15˚C, 20˚C, 25˚C, 30˚C, 35˚C, 40˚C, and 45˚C for 7 days under shaking conditions at 120 rpm to ensure adequate aeration. Bacterial growth was assessed by measuring the optical density at 620 nm.

2.5. Physicochemical Activity of the Isolates

2.5.1. Extract Preparation

A well-isolated colony of each Rhizobium sp. was introduced into a conical flask containing 100 mL of sterile YEM broth. The inoculated tubes were incubated at 28˚C for 7 days. After incubation, the inoculated broths were centrifuged at 4000 rpm for 20 min (Rotofix 32A, Hettich, Germany), and the cell-free supernatants were collected and stored for testing.

2.5.2. Protein Content

The method of Lowry et al. [29] was used to assess the protein content of cell-free supernatants. Briefly, 1 mL of the supernatant was mixed with 5 mL of copper reactive solution (0.5 mL of CuSO₄ 1% (w/v), 0.5 mL of 2% sodium tartrate (w/v), and 50 mL of sodium carbonate 2% (w/v) in NaOH 0.1 M). After thoroughly mixing, the mixture was left at room temperature (25˚C ± 1˚C) for 10 min, and 0.5 mL of Folin-Ciocalteau was added. The mixture was incubated for 30 min at room temperature, and the absorbance was read at 750 nm against the blank. SAB at different concentrations was used as a standard to draw the calibration curve (r² = 0.94) that was used to calculate the protein content of the samples.

2.5.3. Antioxidant Activities

The antioxidant capacity of the cell free supernatants from the culture of Rhizobium sp. isolates in YEM broth at 28˚C for 7 days was assessed using 3 methods: the phosphomolybdenum method to evaluate total antioxidant capacity (TAC), the ferric reducing antioxidant power (FRAP) method to evaluate iron reduction power, and catalase.

Ferric Reducing Antioxidant Power (FRAP)

The method of Oyaizu [30] was used to assess the ferric-reducing power of Rhizobium sp. isolates. A volume of 1 mL of cell-free, was mixed with 2.5 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 mL of potassium ferrocyanide (1%) and incubated in a water bath at 50˚C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture, which was centrifuged at 650 rpm for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. Ascorbic acid was used as a positive control.

Determination of total antioxidant capacity

The total antioxidant activity of the extract was assessed by the formation of a phosphomolybdenum complex [31]. To this end, 0.2 mL of cell-free supernatant was added to 2 mL reagent solution (0.6 M H₂SO₄, 28 mM sodium phosphate, and 4 mM ammonium molybdate). Absorbance was measured at 765 nm after 60 min of boiling. Ascorbic acid was used as the standard, and total antioxidant capacity was expressed in milligrams of ascorbic acid equivalent (AAE) per gram of dry matter extract (mg AAE/gDM).

Catalase activity

The method of Sinha [32] used in this study was based on the fact that catalase present in the cell-free supernatant reduces hydrogen peroxide (H₂O₂) to water (H₂O) and oxygen (O₂). The remaining H₂O₂ molecules are bound by potassium dichromate to form a blue-green precipitate of unstable perchloric acid. That precipitate is then decomposed by heat treatment to form a green complex absorbing at 620 nm. Catalase activity was proportional to optical density and was determined by the calibration curve (r² = 0.94). The activity was expressed in mmoles of H₂O₂ decomposed per mg of protein.

2.5.4. Inhibition of Lipid Peroxidation

To assess the ability of rhizobia to inhibit lipid peroxidation due to ROS generated by stress conditions, the method described by Wilbur et al. [33] with slight modifications was used. In the protocol, 100 µL of cell-free supernatant was mixed with 250 µL of trichloroacetic acid 20% (w/v) and 400 µL of thiobarbituric acid 0.67% (w/v). The mixture was transferred into glass tubes and the tubes were sealed, heated at 100˚C for 15 min, and cooled in a water bath for 30 min. Then, the tubes were opened to allow gas evaporation, centrifuged (1500 rpm, 5 min), and the optical densities were read at 532 nm against a solution of NaCl 0.9% used as a blank. MDA concentration was expressed in mmol/mL of supernatant.

2.6. Statistical Analysis

All experiments were repeated at least three times, and the data obtained are presented as means ± standard deviations. The experimental design followed a completely randomized design, with three biological replicates per treatment. Data were submitted to analysis of variance (ANOVA) using Statgraphics Centurion XV version 16.1.18 (StatPoint Technologies, Inc., Virginia, USA), and comparisons between means were performed using Duncan’s multiple-range test. The significance level was set at 5%. Pearson correlation was used to assess the relationship between the 73 Rhizobium sp. isolates, abiotic factors, and biochemical tests. Principal component analysis (PCA) was performed to visualize the association between the different parameters analyzed with the 73 isolates using XLSTAT software version 2014.5.03 (Addinsoft, Inc., New York, USA).

3. Results

3.1. Nodule Characteristics

The nodules of the 18 soybean varieties were rounded or oval, as shown in Figure 1. These shapes varied from one soybean variety to another. They were large lobular shapes with a light-brown color (TGX 2007 11 F and Panorama 2 varieties) and globular clustered shapes with a brown color along the roots and small-sized absorbent hairs (Figure 1). These nodules were distributed in small numbers with lateral roots and small absorbent hairs. For Rhizobium sp. isolation, healthy, uninterrupted, and pink nodules were chosen, as these characteristics indicate their maturity and also that symbiosis had taken place.

Pictures were taken with an Olympus SZH10 SCOP PRO microscope, series 1S2219; magnification: 7X, width: 8.25 µm; pixel height: 8.25 µm; XY ratio: 1.00000; zoom 33%; image info: 5000 µm (Manet, 2022).

Figure 1. Rhizobium sp. root nodules of the 18 soybean varieties.

3.2. Characteristics of Rhizobium sp. Isolates

Nodulation tests were carried out on 108 Rhizobium sp. isolates from 18 soybean varieties, enabling 73 isolates to be selected for their ability to nodulate effectively. The remaining 35 isolates, although showing no nodulation ability, were kept in the lab for further studies. Gram staining revealed that all 73 isolates of Rhizobium sp. were Gram-negative. In all, the 73 Rhizobium sp. isolates obtained were studied for further experiments. They were fast-growing isolates, forming visible colonies on yeast extract mannitol agar (YEMA) within 72 to 96 h of culture (Table 1). The isolates ranged in diameter from 2 to 4 µm. They displayed a thick structure, with viscous mucus of a whitish, milky appearance. The colony morphology varied from round to irregular forms.

3.3. Effect of pH Variation on the growth of the 73 Rhizobium sp. Isolates

As shown in Table 2, many isolates showed their strongest growth at pH 5, with optical density values at 620 nm ranging from 0.90 ± 0.01 (isolate 6G2) to 1.57 ± 0.01 (isolate 3M'''), indicating a nearly 75% increase in growth between the lowest and highest performances at this pH. However, several isolates showed optimal growth at other pH levels, including pH 3 (16M, 16M3 and 17G1), pH 3.5 (7M, 15G, 15M3 and 16M3), pH 4 (7M, 12G2), pH 4.5 (isolate 1) and pH 6.8 (1G'', 1M, 6G2', 6G3, 7M', 14P3, 17G4 and 17M), where OD ranged from 0.78 to 1.52. This wide distribution of optimal pH conditions and the magnitude of growth differences, up to twofold between isolates depending on pH levels, suggests substantial physiological or ecological variation between strains, which may suggest specific adaptations to local soil conditions or host legume genotypes.

Table 2. Growth of Rhizobium sp. isolates at different pH levels in terms of optical density 620 nm.

Isolates

pH = 2

pH = 2.5

pH = 3

pH = 3.5

pH = 4

pH = 4.5

pH = 5

pH = 6.8

1

0.11 ± 0.00fghijklmnop

0.06 ± 0.00abcd

0.16 ± 0.00abcdefgh

0.32 ± 0.03cdef

0.52 ± 0.03ijklmnop

1.28 ± 0.02r

0.58 ± 0.00jklmn

0.58 ± 0.00mnopqrst

1G

0.07 ± 0.00abcdefg

0.07 ± 0.00abcd

0.06 ± 0.00ab

0.64 ± 0.00abcdefghijk

0.61 ± 0.00abcdef

0.19 ± 0.01abcdefg

0.67 ± 0.00mnopqrs

0.55 ± 0.03d

1G'

0.06 ± 0.00abcde

0.07 ± 0.00abcde

0.07 ± 0.00ab

0.35 ± 0.00defgh

0.51 ± 0.01hijklmnop

0.26 ± 0.02abcdefghijk

0.82 ± 0.00abcde

0.84 ± 0.01abc

1G''

0.08 ± 0.00abcdefghij

0.07 ± 0.02abcd

0.17 ± 0.01bcdefgh

0.41 ± 0.04efghijk

0.57 ± 0.04mnopqr

0.19 ± 0.03abcdefg

0.82 ± 0.02abcde

0.93 ± 0.02ab

1M

0.07 ± 0.00abcdef

0.06 ± 0.00abcd

0.06 ± 0.00ab

0.35 ± 0.00defghi

0.31 ± 0.01bcd

0.44 ± 0.02ijklmno

0.17 ± 0.00bc

1.00 ± 0.00 a

1P

0.15 ± 0.00abcdefghi

0.06 ± 0.00abcd

0.59 ± 0.00abcdef

0.71 ± 0.00abcde

0.76 ± 0.02abcd

0.17 ± 0.04abcde

0.95 ± 0.01abcd

0.83 ± 0.03abcd

2

0.10 ± 0.00bcdefghijkl

0.40 ± 0.1a

0.51 ± 0.05abcdefg

0.31 ± 0.12cdef

0.65 ± 0.01abcdef

0.46 ± 0.06jklmno

0.83 ± 0.02abcde

0.74 ± 0.00opqrstuv

2G

0.18 ± 0.00abcdef

0.06 ± 0.00ab

0.07 ± 0.01abc

0.74 ± 0.00abcde

0.57 ± 0.13mnopqr

0.14 ± 0.03abcd

1.15 ± 0.00abc

0.75 ± 0.01pqrstuvw

2M

0.11 ± 0.01fghijklmnop

0.06 ± 0.00ab

0.21 ± 0.05efghi

0.61 ± 0.01abcdefghij

0.49 ± 0.07ghijklmno

0.32 ± 0.12bcdefghijkl

1.01 ± 0.00ab

0.68 ± 0.02 jkl

2M'

0.18 ± 0.00abc

0.07 ± 0.01ab

0.21 ± 0.12gh

0.74 ± 0.23abcd

0.67 ± 0.16abcde

0.71 ± 0.63pq

1.11 ± 0.28abcd

0.80 ± 0.05abcdef

2P'C'

0.08 ± 0.00abcdefghijk

0.07 ± 0.00abcde

0.37 ± 0.06jklm

0.55 ± 0.02mnopqrstu

0.70 ± 0.07abcdef

0.77 ± 0.15q

1.21 ± 0.00ab

0.77 ± 0.00abcdef

3G

0.12 ± 0.01fghijklmnopq

0.08 ± 0.00abcdefg

0.33 ± 0.07ijkl

0.65 ± 0.01abcdefghij

0.83 ± 0.00ab

0.38 ± 0.00defghijklm

0.86 ± 0.02abcde

0.73 ± 0.00mnopqrstu

3M'

0.11 ± 0.00efghijklmn

0.16 ± 0.00ijk

0.43 ± 0.03lmnopqr

0.69 ± 0.01abcdef

0.51 ± 0.00hijklmnop

0.18 ± 0.04abcdefghi

0.64 ± 0.00lmnopqr

0.72 ± 0.00lmnopqrs

3M'''

0.08 ± 0.00abcdefghi

0.29 ± 0.00mn

0.53 ± 0.05abcdefg

0.48 ± 0.19jklmnop

0.52 ± 0.00ijklmnop

0.21 ± 0.00abcdefghi

1.57 ± 0.01a

0.60 ± 0.01ef

4M1

0.18 ± 0.00abcdef

0.06 ± 0.00ab

0.45 ± 0.00mnopqrs

0.39 ± 0.03efghij

0.57 ± 0.04mnopqr

0.14 ± 0.05abcd

0.86 ± 0.00abcde

0.71 ± 0.00klmnopq

4M3

0.08 ± 0.00abcdefgh

0.05 ± 0.00a

0.37 ± 0.00jklm

0.54 ± 0.07mnopqrst

0.84 ± 0.04ab

0.76 ± 0.08q

0.55 ± 0.10ijkl

0.77 ± 0.00abcdef

5G

0.13 ± 0.01klmnopqrs

0.06 ± 0.00ab

0.19 ± 0.01cdefgh

0.26 ± 0.00bcd

0.72 ± 0.01abcde

0.22 ± 0.00abcdefghi

1.25 ± 0.00a

0.76 ± 0.02rstuvw

5M

0.12 ± 0.01jklmnopqrs

0.05 ± 0.00a

0.64 ± 0.03abcde

0.45 ± 0.04ghijklm

0.78 ± 0.02abc

0.47 ± 0.18klmno

1.20 ± 0.01ab

0.76 ± 0.00stuvw

5M''

0.22 ± 0.00abcde

0.06 ± 0.00ab

0.75 ± 0.05abcd

0.71 ± 0.00abcde

0.72 ± 0.00abcde

0.42 ± 0.01ghijklmn

1.00 ± 0.00abc

0.86 ± 0.00ab

5M2'

0.07 ± 0.00abcdefg

0.06 ± 0.00ab

0.05 ± 0.00a

0.34 ± 0.03defg

0.32 ± 0.03cde

0.25 ± 0.00abcdefghijk

1.03 ± 0.00abc

0.66 ± 0.01hij

5P

0.06 ± 0.00abc

0.07 ± 0.00abcd

0.06 ± 0.00ab

0.46 ± 0.00hijklm

0.37 ± 0.06def

0.61 ± 0.07mnopq

0.59 ± 0.00klmno

0.89 ± 0.03ab

5P'

0.06 ± 0.00abcde

0.08 ± 0.00abcdefg

0.08 ± 0.00abcd

0.22 ± 0.00bc

0.77 ± 0.02abc

0.65 ± 0.01nopq

1.05 ± 0.02abcd

0.81 ± 0.00abcdefg

6G

0.10 ± 0.00defghijklmn

0.06 ± 0.00abcd

0.19 ± 0.18cdefgh

0.59 ± 0.00abcdefghij

0.55 ± 0.01lmnopq

0.26 ± 0.00abcdefghijk

1.13 ± 0.00abcd

0.76 ± 0.06rstuvw

6G'

0.09 ± 0.00bcdefghijkl

0.06 ± 0.00abc

0.47 ± 0.00mnopqrst

0.76 ± 0.00abc

0.75 ± 0.03abcde

0.13 ± 0.01 ab

1.04 ± 0.02abcd

0.77 ± 0.01abcdef

6G2

0.22 ± 0.02abcde

0.09 ± 0.01abcdefg

0.54 ± 0.03abcdefg

0.80 ± 0.00ab

0.53 ± 0.14jklmnop

0.15 ± 0.00abcd

0.90 ± 0.01abcd

0.72 ± 0.02mnopqrs

6G2'

0.14 ± 0.01abcdefghi

0.08 ± 0.02abcdef

0.10 ± 0.01abcdef

0.56 ± 0.02mnopqrstuv

0.67 ± 0.01abcdefg

0.16 ± 0.02abcde

0.81 ± 0.00abcde

1.00 ± 0.00a

6G3

0.10 ± 0.00cdefghijklm

0.05 ± 0.01a

0.39 ± 0.01klmn

0.39 ± 0.00efghij

0.58 ± 0.00opqrs

0.37 ± 0.01cdefghijklm

0.68 ± 0.00mnopqrst

0.93 ± 0.01abc

6G4

0.12 ± 0.00ijklmnopqrs

0.06 ± 0.00abc

0.40 ± 0.00lmnop

0.59 ± 0.00abcdefghij

0.57 ± 0.04mnopqr

0.26 ± 0.22abcdefghijk

0.87 ± 0.00abcde

0.68 ± 0.01ijkl

7M

0.19 ± 0.00abcdef

0.19 ± 0.04jkl

0.50 ± 0.01abcdefgh

1.07 ± 0.04ab

1.38 ± 0.08a

0.20 ± 0.04abcdefgh

0.86 ± 0.03abcde

0.67 ± 0.00ijk

7M'

0.17 ± 0.00abcd

0.06 ± 0.00abcd

0.06 ± 0.00ab

0.63 ± 0.04abcdefghijk

0.71 ± 0.01abcde

0.27 ± 0.02abcdefghijkl

0.45 ± 0.09ghi

0.91 ± 0.02ab

8G

0.12 ± 0.01fghijklmnopq

0.07 ± 0.01abcde

0.37 ± 0.00jklm

0.57 ± 0.02opqrstuvwx

0.55 ± 0.02lmnopq

0.15 ± 0.01abcd

0.53 ± 0.01hijk

0.68 ± 0.02 jkl

8M'

0.26 ± 0.15ab

0.08 ± 0.00abcdef

0.55 ± 0.01abcdefg

0.46 ± 0.03ijklmno

0.24 ± 0.01bc

0.19 ± 0.00abcdefg

0.88 ± 0.01abcd

0.62 ± 0.03fg

8M''

0.09 ± 0.00bcdefghijkl

0.06 ± 0.00abcd

0.15 ± 0.00abcdefg

0.52 ± 0.01klmnopq

0.44 ± 0.08fghij

0.19 ± 0.00abcdefg

1.05 ± 0.02abcd

0.46 ± 0.01c

8P

0.10 ± 0.00cdefghijklmn

0.15 ± 0.00ghijk

0.09 ± 0.00abcde

0.65 ± 0.03abcdefghij

0.64 ± 0.05abcdef

0.40 ± 0.00efghijklm

0.92 ± 0.07abcd

0.73 ± 0.00mnopqrs

9G'

0.09 ± 0.00abcdefghijk

0.10 ± 0.00abcdefghi

0.19 ± 0.01defgh

0.57 ± 0.03nopqrstuvw

0.45 ± 0.02fghijk

0.21 ± 0.00abcdefghi

0.76 ± 0.02abcde

0.70 ± 0.00jklmn

9P

0.09 ± 0.00bcdefghijkl

0.06 ± 0.00ab

0.14 ± 0.01abcdefg

0.57 ± 0.05mnopqrstuvw

0.27 ± 0.00bc

0.40 ± 0.16efghijklm

0.61 ± 0.04klmnop

0.81 ± 0.00abcdefgh

10G

0.12 ± 0.01ghijklmnopq

0.08 ± 0.00abcdef

0.47 ± 0.01mnopqrst

0.55 ± 0.01mnopqrstu

0.28 ± 0.06bcd

0.19 ± 0.01abcdefg

0.47 ± 0.02ghi

0.72 ± 0.01lmnopqr

10G1

0.09 ± 0.00abcdefghijk

0.06 ± 0.00abcd

0.56 ± 0.04abcdef

0.53 ± 0.00lmnopqr

0.53 ± 0.00jklmnop

0.61 ± 0.06mnopq

0.86 ± 0.08abcde

0.64 ± 0.04ghi

10G2

0.09 ± 0.00bcdefghijkl

0.09 ± 0.00abcdefgh

0.65 ± 0.00abcde

0.46 ± 0.04hijklmn

0.43 ± 0.08fgh

0.41 ± 0.08fghijklm

0.71 ± 0.01pqrstu

0.51 ± 0.03c

11G1

0.09 ± 0.00abcdefghijkl

0.06 ± 0.00ab

0.07 ± 0.00abc

0.55 ± 0.09mnopqrstu

0.54 ± 0.00klmnop

0.14 ± 0.05abcd

0.78 ± 0.00abcde

0.78 ± 0.01abcdef

11G2

0.06 ± 0.00abcde

0.06 ± 0.00ab

0.22 ± 0.03fghi

0.54 ± 0.02mnopqrst

0.52 ± 0.00hijklmnop

0.14 ± 0.01abc

0.41 ± 0.07efg

0.95 ± 0.00 a

11G3

0.05 ± 0.00ab

0.09 ± 0.00abcdefg

0.39 ± 0.00klmn

0.19 ± 0.01 b

0.58 ± 0.02nopqrs

0.17 ± 0.00abcdef

0.55 ± 0.04ijkl

0.25 ± 0.01b

11G4

0.08 ± 0.00abcdefgh

0.08 ± 0.00abcdef

0.25 ± 0.04ghij

0.58 ± 0.02abcdefghijk

0.60 ± 0.02pqrst

0.13 ± 0.01ab

0.34 ± 0.02def

0.62 ± 0.00fgh

11G5

0.11 ± 0.00fghijklmnop

0.07 ± 0.00abcde

0.32 ± 0.02ijkl

0.65 ± 0.01abcdefghij

0.43 ± 0.08fghi

0.18 ± 0.01abcdef

0.80 ± 0.04abcde

0.83 ± 0.03abcd

12G

0.08 ± 0.00abcdefghijk

0.05 ± 0.00a

0.55 ± 0.04abcdefg

0.55 ± 0.01mnopqrstu

0.53 ± 0.02jklmnop

0.15 ± 0.01abcd

1.29 ± 0.03a

0.80 ± 0.01abcdefg

12G2

0.10 ± 0.01cdefghijklmn

0.06 ± 0.00abcd

0.51 ± 0.02abcdefg

0.46 ± 0.11ijklmno

1.04 ± 0.01a

0.18 ± 0.00abcdef

0.55 ± 0.02ijkl

0.78 ± 0.00abcdef

12G3

0.11 ± 0.00fghijklmn

0.08 ± 0.00abcdef

0.40 ± 0.00lmno

0.67 ± 0.02abcdefg

0.30 ± 0.00bcd

0.18 ± 0.03abcdefg

0.68 ± 0.12nopqrst

0.56 ± 0.01de

13M

0.14 ± 0.00abcdefghi

0.13 ± 0.06defghij

0.73 ± 0.00abc

0.48 ± 0.01jklmnop

0.72 ± 0.01abcde

0.17 ± 0.02abcde

0.44 ± 0.05fgh

0.70 ± 0.00klmno

13M2

0.21 ± 0.00abcdef

0.13 ± 0.02cdefghi

0.81 ± 0.03ab

0.53 ± 0.09lmnopqrs

0.22 ± 0.00 b

0.51 ± 0.00lmnop

0.58 ± 0.00jklmn

0.82 ± 0.02abcde

13M5

0.12 ± 0.00hijklmnopqr

0.06 ± 0.00abcd

0.69 ± 0.02 abc

0.40 ± 0.05efghij

0.57 ± 0.05mnopqr

0.33 ± 0.01bcdefghijkl

0.69 ± 0.04opqrst

0.77 ± 0.02abcde

14P2

0.06 ± 0.00abcde

0.04 ± 0.00a

0.41 ± 0.01lmnop

0.57 ± 0.02nopqrstuvw

0.46 ± 0.04fghijkl

0.17 ± 0.02abcdef

0.87 ± 0.01abcd

1.10 ± 0.01a

14P3

0.48 ± 0.01a

0.06 ± 0.00ab

0.60 ± 0.06abcde

0.65 ± 0.13abcdefghij

0.78 ± 0.01abc

0.18 ± 0.01abcdef

1.20 ± 0.07abc

0.75 ± 0.02rstuvw

14P4

0.28 ± 0.00 a

0.15 ± 0.18hijk

0.76 ± 0.01abc

0.75 ± 0.00abcd

0.77 ± 0.00abc

0.19 ± 0.03abcdefg

1.51 ± 0.05ab

0.69 ± 0.02jklm

15G

0.12 ± 0.00klmnopqrs

0.27 ± 0.01 mn

0.57 ± 0.06abcdefg

0.91 ± 0.13ab

0.75 ± 0.02abcd

0.67 ± 0.01opq

0.73 ± 0.01abcde

0.75 ± 0.03qrstuvw

15M1

0.16 ± 0.05abcdef

0.04 ± 0.00a

0.19 ± 0.03cdefgh

0.17 ± 0.00b

0.53 ± 0.00jklmnop

0.17 ± 0.00abcde

0.27 ± 0.00cd

0.81 ± 0.01abcdefgh

15M2

0.17 ± 0.00abcde

0.25 ± 0.02lm

0.67 ± 0.01abcd

0.60 ± 0.06abcdefghij

0.33 ± 0.01cde

0.26 ± 0.06abcdefghijk

0.62 ± 0.04klmnop

0.81 ± 0.02abcdef

15M3

0.22 ± 0.00abcd

0.14 ± 0.01fghijk

0.52 ± 0.02abcdefg

1.05 ± 0.02ab

0.54 ± 0.02klmnopq

0.34 ± 0.03bcdefghijkl

1.40 ± 0.02a

0.79 ± 0.01abcdef

15M4

0.11 ± 0.01fghijklmno

0.06 ± 0.01abcd

0.27 ± 0.04hijk

0.43 ± 0.06fghijkl

0.48 ± 0.05ghijklm

0.17 ± 0.02abcdef

0.16 ± 0.01b

0.85 ± 0.02 ab

16G

0.13 ± 0.01lmnopqrst

0.09 ± 0.00abcdefgh

0.87 ± 0.01a

0.69 ± 0.01abcdefg

0.57 ± 0.05mnopqr

0.23 ± 0.02abcdefghij

0.81 ± 0.03abcde

0.74 ± 0.01nopqrstu

16M

0.16 ± 0.01abcde

0.06 ± 0.00ab

1.24 ± 0.28a

0.64 ± 0.02abcdefghijk

0.71 ± 0.09abcde

0.22 ± 0.02abcdefghi

0.48 ± 0.07ghij

0.73 ± 0.01mnopqrst

16M1

0.17 ± 0.00abcde

0.05 ± 0.00a

0.56 ± 0.05abcdef

0.17 ± 0.03b

0.41 ± 0.16efg

0.21 ± 0.01abcdefghi

0.20 ± 0.00bc

0.59 ± 0.01def

16M2

0.16 ± 0.00abcdefg

0.08 ± 0.02abcdef

0.04 ± 0.00a

0.31 ± 0.03cde

0.60 ± 0.02abcdef

0.19 ± 0.00abcdefg

0.55 ± 0.01ijkl

0.71 ± 0.06klmnop

16M3

0.10 ± 0.00bcdefghijkl

0.10 ± 0.02abcdefghi

1.16 ± 0.00a

1.13 ± 0.23a

0.59 ± 0.08opqrst

0.18 ± 0.03abcdefg

0.62 ± 0.01klmnopq

0.77 ± 0.04abcde

17G

0.15 ± 0.00abcdefgh

0.12 ± 0.00bcdefghi

0.33 ± 0.01ijkl

0.66 ± 0.00abcdefghi

0.85 ± 0.07a

0.43 ± 0.05hijklmno

0.32 ± 0.02de

0.81 ± 0.01abcdefg

17G1

0.10 ± 0.00efghijklmn

0.13 ± 0.00efghijk

1.22 ± 0.00a

0.67 ± 0.04abcdefgh

0.56 ± 0.04lmnopq

0.15 ± 0.00abcd

0.73 ± 0.00abcdef

0.73 ± 0.01mnopqrs

17G2

0.13 ± 0.01klmnopqrs

0.32 ± 0.00 n

0.50 ± 0.04abcdefgh

0.70 ± 0.02abcde

0.78 ± 0.06abc

0.21 ± 0.03abcdefghi

1.42 ± 0.00a

0.78 ± 0.04abcdef

17G4

0.07 ± 0.00abcdef

0.06 ± 0.00ab

0.61 ± 0.00abcde

0.39 ± 0.01efghij

0.60 ± 0.00abcdef

0.37 ± 0.05bcdefghijkl

1.02 ± 0.00nopqrst

0.97 ± 0.04ab

17M

0.15 ± 0.00abcdefghi

0.08 ± 0.02abcdef

0.05 ± 0.00ab

0.97 ± 0.02ab

0.60 ± 0.01pqrst

0.14 ± 0.00abc

0.68 ± 0.05nopqrst

0.90 ± 0.00ab

17M2

0.23 ± 0.01ab

0.08 ± 0.02abcdefg

0.06 ± 0.00ab

0.96 ± 0.00ab

0.61 ± 0.01abcdef

0.18 ± 0.02abcdefg

0.58 ± 0.12jklm

0.72 ± 0.00lmnopqrs

18G

0.22 ± 0.00abc

0.20 ± 0.02kl

0.77 ± 0.01abc

0.84 ± 0.02ab

0.66 ± 0.04abcdefg

0.36 ± 0.03bcdefghijkl

1.27 ± 0.02a

0.78 ± 0.01abcdef

18M1

0.13 ± 0.01lmnopqrst

0.07 ± 0.01abcd

0.43 ± 0.02lmnopq

0.53 ± 0.01lmnopqrs

0.59 ± 0.00opqrs

0.35 ± 0.00bcdefghijkl

0.95 ± 0.01abcd

0.84 ± 0.01abc

18M2

0.06 ± 0.00abcd

0.05 ± 0.00a

0.37 ± 0.00jklm

0.68 ± 0.04abcdefg

0.49 ± 0.10ghijklmn

0.14 ± 0.00abc

0.76 ± 0.05abcde

0.81 ± 0.00abcde

Control without isolate

0.04 ± 0.00a

0.04 ± 0.00a

0.07 ± 0.00abc

0.05 ± 0.00a

0.05 ± 0.00a

0.04 ± 0.00a

0.04 ± 0.00a

0.04 ± 0.00a

Values with different superscript letters on the same column are significantly different at p ˂ 0.05.

3.4. Effect of Salinity Variation on the Growth of the 73 Rhizobium sp. Isolates

Generally, the optimal growth, measured by optical density at 620 nm, ranged from 0.44 ± 0.01 for isolate 17G4 to 1.05 ± 0.00 for isolate 5M at 1% NaCl, indicating more than two-fold difference in growth capacity under mild saline stress (Table 3). However, some isolates exhibited their highest growth at significantly higher salinity levels. For instance, isolate 6G reached 1.1 ± 0.01 at 2% NaCl, 10G2 exhibited a notable halotolerance with optimal growth values of 1.20 ± 0.04 at 6%, 1.39 ± 0.01 at 8%, and 1.19 ± 0.01 at 10% NaCl, maintaining over 90% of its maximum growth even at high salinity. In contrast, highly sensitive isolates such as 8P recorded only 0.08 ± 0.00 at 12% NaCl, underscoring a more than 14-fold difference in salt stress response compared to the most tolerant isolates.

Table 3. Growth of Rhizobium sp. isolates at different salinity levels in terms of optical density at 620 nm.

Isolates	NaCl 1%	NaCl 2%	NaCl 4%	NaCl 6%	NaCl 8%	NaCl 10%	NaCl 12%
1	0.60 ± 0.02ghij	0.55 ± 0.03ijklm	0.45 ± 0.01jklmnopq	0.37 ± 0.02defghijk	0.13 ± 0.01abcdefg	0.17 ± 0.00abcdefghijklm	0.39 ± 0.00pqr
1G	0.61 ± 0.04hijk	0.65 ± 0.04abcd	0.50 ± 0.02abcd	0.59 ± 0.01abcd	0.26 ± 0.02ghijklmn	0.18 ± 0.01bcdefghijklmn	0.27 ± 0.03klmnop
1G'	0.53 ± 0.02cd	0.50 ± 0.00ef	0.44 ± 0.02ghijklmn	0.43 ± 0.05jklmnopqr	0.18 ± 0.00abcdefghijk	0.34 ± 0.02rs	0.23 ± 0.01ghijklmno
1G''	0.67 ± 0.02nopqrst	0.55 ± 0.05ijklm	0.41 ± 0.01defg	0.38 ± 0.01efghijklm	0.42 ± 0.00opqrs	0.53 ± 0.01ab	0.71 ± 0.05abc
1M	0.89 ± 0.04ab	0.76 ± 0.03ab	0.67 ± 0.00abc	0.54 ± 0.02abcdefgh	0.34 ± 0.0 3lmnop	0.31 ± 0.02pqrs	0.27 ± 0.04lmnop
1P	0.63 ± 0.02ijklmn	0.55 ± 0.01hijkl	0.42 ± 0.00efghijkl	0.44 ± 0.02lmnopqrs	0.13 ± 0.01abcdefg	0.33 ± 0.00qrs	0.24 ± 0.04ijklmno
2	0.67 ± 0.03mnopqr	0.58 ± 0.04lmnopq	0.43 ± 0.00fghijklm	0.47 ± 0.02opqrstuv	0.22 ± 0.01cdefghijklm	0.57 ± 0.01ab	0.28 ± 0.04lmnop
2G	0.74 ± 0.01abcdef	0.64 ± 0.01abcd	0.46 ± 0.00lmnopqrst	0.45 ± 0.00nopqrstu	0.59 ± 0.02a	0.64 ± 0.02abc	0.21 ± 0.02defghijklmn
2M	0.62 ± 0.01hijkl	0.57 ± 0.02klmno	0.49 ± 0.02abcdef	0.54 ± 0.06abcdefgh	0.11 ± 0.00abcd	0.22 ± 0.00ghijklmno	0.39 ± 0.04pqr
2M'	0.77 ± 0.05ab	0.71 ± 0.05ab	0.62 ± 0.08abc	0.57 ± 0.06abcde	0.72 ± 0.04a	0.86 ± 0.24a	0.79 ± 0.05ab
2P'C'	0.89 ± 0.10ab	0.71 ± 0.00abc	0.45 ± 0.03hijklmnop	0.40 ± 0.02ghijklmno	0.18 ± 0.00abcdefghijk	1.03 ± 0.00a	0.24 ± 0.00hijklmno
3G	0.81 ± 0.02ab	0.61 ± 0.00abcdef	0.4 ± 0.01efghij	0.43 ± 0.05jklmnopqr	0.22 ± 0.05cdefghijklm	0.18 ± 0.01bcdefghijklmn	0.31 ± 0.03mnopq
3M'	0.69 ± 0.01abcdefgh	0.67 ± 0.01abcd	0.46 ± 0.01lmnopqrs	0.38 ± 0.02efghijkl	0.15 ± 0.01abcdefgh	0.37 ± 0.05st	0.20 ± 0.01abcdefghijklm
3M'''	0.86 ± 0.02ab	0.64 ± 0.01abcd	0.42 ± 0.01efghijk	0.36 ± 0.05cdefgh	0.44 ± 0.05pqrs	0.13 ± 0.00abcdefg	0.13 ± 0.02abcdefghi
4M1	0.57 ± 0.02defgh	0.56 ± 0.04jklmn	0.47 ± 0.00mnopqrstu	0.46 ± 0.01nopqrstu	0.08 ± 0.00ab	0.19 ± 0.01defghijklmn	0.08 ± 0.01a
4M3	0.67 ± 0.01mnopqrs	0.64 ± 0.03abcd	0.52 ± 0.02abcd	0.58 ± 0.09abcdef	0.44 ± 0.07pqrs	0.61 ± 0.01abc	0.78 ± 0.01ab
5G	0.92 ± 0.05a	0.86 ± 0.04a	0.46 ± 0.01lmnopqrs	0.42 ± 0.04ijklmnopq	0.25 ± 0.00efghijklmn	0.25 ± 0.05ijklmnopq	0.21 ± 0.01cdefghijklmn
5M	1.05 ± 0.00a	0.90 ± 0.00a	0.58 ± 0.02abc	0.39 ± 0.06efghijklm	0.20 ± 0.00bcdefghijkl	0.16 ± 0.01abcdefghij	0.18 ± 0.01abcdefghijkl
5M''	0.61 ± 0.00hijk	0.56 ± 0.02ijklmn	0.34 ± 0.01a	0.33 ± 0.10bcde	0.54 ± 0.02ab	0.36 ± 0.00st	0.16 ± 0.00abcdefghijkl
5M2'	0.76 ± 0.03abcd	0.79 ± 0.04a	0.74 ± 0.01a	0.42 ± 0.00hijklmnop	0.30 ± 0.04ijklmno	0.51 ± 0.00ab	0.17 ± 0.03abcdefghijkl
5P	0.76 ± 0.02abcd	0.5 ± 0.04ef	0.39 ± 0.01bcde	0.37 ± 0.04defghijk	0.20 ± 0.00bcdefghijkl	0.21 ± 0.01fghijklmno	0.13 ± 0.00abcdefghi
5P'	0.59 ± 0.00fghi	0.48 ± 0.01de	0.41 ± 0.01efgh	0.38 ± 0.01efghijklm	0.13 ± 0.01abcdefg	0.11 ± 0.01abcde	0.13 ± 0.00abcdefghi
6G	0.67 ± 0.01mnopqrst	1.11 ± 0.01a	0.42 ± 0.02efghi	0.36 ± 0.05cdefgh	0.35 ± 0.02mnopq	0.85 ± 0.02a	0.25 ± 0.04ijklmno
6G'	0.56 ± 0.02cdefg	0.50 ± 0.00efg	0.45 ± 0.01ijklmnopq	0.50 ± 0.00abcdefgh	0.17 ± 0.00abcdefghij	0.22 ± 0.02ghijklmnop	0.16 ± 0.01abcdefghijkl
6G2	0.55 ± 0.02cdefg	0.55 ± 0.00hijklm	0.46 ± 0.00lmnopqrs	0.29 ± 0.02b	0.16 ± 0.00abcdefghi	0.14 ± 0.00abcdefgh	0.27 ± 0.24klmnop
6G2'	0.67 ± 0.01mnopqrst	0.64 ± 0.00abcd	0.36 ± 0.01ab	0.33 ± 0.02bcdef	0.22 ± 0.01bcdefghijklm	0.20 ± 0.02fghijklmno	0.10 ± 0.00abcdef
6G3	0.67 ± 0.03mnopqrs	0.59 ± 0.01lmnop	0.48 ± 0.01abcdefghi	0.41 ± 0.00hijklmno	0.31 ± 0.02jklmnop	0.85 ± 0.00a	0.66 ± 0.05ab
6G4	0.77 ± 0.03abc	0.57 ± 0.05klmno	0.42 ± 0.01efghij	0.31 ± 0.00bcd	0.21 ± 0.07bcdefghijklm	0.27 ± 0.00nopqr	0.34 ± 0.01opq
7M	0.72 ± 0.07abcdefg	0.61 ± 0.00abcdef	0.61 ± 0.00abc	0.87 ± 0.00a	0.75 ± 0.00a	0.96 ± 0.00a	0.58 ± 0.04st
7M'	0.77 ± 0.02abcd	0.65 ± 0.01abcd	0.39 ± 0.01bcde	0.46 ± 0.04nopqrstu	0.13 ± 0.01abcdefg	0.22 ± 0.08ghijklmnop	0.18 ± 0.07abcdefghijkl
8G	0.65 ± 0.00klmnop	0.42 ± 0.00c	0.50 ± 0.02abcdef	0.40 ± 0.02fghijklmn	0.23 ± 0.02cdefghijklmn	0.17 ± 0.00abcdefghijklm	0.11 ± 0.00abcdefg
8M'	0.57 ± 0.00defgh	0.54 ± 0.00ghijk	0.48 ± 0.01abcdefgh	0.58 ± 0.01abcde	0.14 ± 0.00abcdefg	0.17 ± 0.00abcdefghijkl	0.10 ± 0.00abcdef
8M''	0.59 ± 0.02ghi	0.44 ± 0.04 cd	0.61 ± 0.00abc	0.68 ± 0.04a	0.49 ± 0.02abcd	0.79 ± 0.01 abc	0.59 ± 0.02 abc
8P	0.87 ± 0.00ab	0.92 ± 0.00a	0.44 ± 0.00hijklmno	0.32 ± 0.03bcde	0.21 ± 0.01bcdefghijkl	0.17 ± 0.01abcdefghijkl	0.08 ± 0.00ab
9G'	0.73 ± 0.00abcdef	0.68 ± 0.00abc	0.49 ± 0.00abcdefg	0.53 ± 0.07abcdefgh	0.12 ± 0.00abcde	0.08 ± 0.00a	0.10 ± 0.01abcde
9P	0.51 ± 0.03c	0.58 ± 0.00lmnop	0.59 ± 0.03abc	0.58 ± 0.01abcdef	0.55 ± 0.01ab	0.79 ± 0.08ab	0.33 ± 0.03nopq
10G	0.73 ± 0.02abcdefg	0.55 ± 0.03hijkl	0.44 ± 0.00ghijklmn	0.45 ± 0.03nopqrstu	0.12 ± 0.00abcdef	0.16 ± 0.01abcdefghijk	0.10 ± 0.00abcde
10G1	0.70 ± 0.03abcdefg	0.71 ± 0.01abc	0.55 ± 0.03abcd	0.43 ± 0.02jklmnopq	0.32 ± 0.01klmnop	0.65 ± 0.04abc	0.42 ± 0.05qr
10G2	0.63 ± 0.01ijklm	0.22 ± 0.02a	0.67 ± 0.01 ab	1.20 ± 0.04a	1.30 ± 0.04a	1.19 ± 0.01a	1.17 ± 0.07a
11G1	0.42 ± 0.00b	0.42 ± 0.00c	0.48 ± 0.01abcdefg	0.52 ± 0.05abcdefg	0.13 ± 0.02abcdefg	0.19 ± 0.00defghijklmn	0.09 ± 0.00abcde
11G2	0.54 ± 0.01cde	0.51 ± 0.01efgh	0.46 ± 0.01klmnopqr	0.57 ± 0.00abcdefg	0.09 ± 0.01abc	0.09 ± 0.00ab	0.08 ± 0.01abc
11G3	0.65 ± 0.00klmnop	0.57 ± 0.04jklmn	0.40 ± 0.00cdef	0.60 ± 0.05abc	0.26 ± 0.01fghijklmn	0.17 ± 0.03abcdefghijklm	0.47 ± 0.04rs
11G4	0.66 ± 0.00lmnopqr	0.62 ± 0.00abcde	0.53 ± 0.00abcd	0.45 ± 0.03nopqrstu	0.18 ± 0.02abcdefghijk	0.16 ± 0.00abcdefghijk	0.21 ± 0.00bcdefghijklmn
11G5	0.68 ± 0.06abcdefg	0.68 ± 0.00abc	0.70 ± 0.00ab	0.69 ± 0.00a	0.77 ± 0.03a	0.96 ± 0.03 a	0.83 ± 0.03a
12G	0.75 ± 0.00abcde	0.57 ± 0.00jklmn	0.52 ± 0.00abcd	0.47 ± 0.02opqrstuv	0.24 ± 0.00defghijklmn	0.12 ± 0.01abcdef	0.14 ± 0.01abcdefghijk
12G2	0.63 ± 0.00ijklmno	0.59 ± 0.01abcdef	0.36 ± 0.01abc	0.30 ± 0.01bc	0.50 ± 0.05abc	0.18 ± 0.00cdefghijklmn	0.09 ± 0.01abcde
12G3	0.45 ± 0.00b	0.32 ± 0.00b	0.44 ± 0.02fghijklm	0.46 ± 0.00nopqrstu	0.26 ± 0.02ghijklmn	0.47 ± 0.02a	0.61 ± 0.06 ab
13M	0.54 ± 0.01cdef	0.53 ± 0.00fghij	0.49 ± 0.01abcdefg	0.52 ± 0.04abcdefg	0.19 ± 0.05abcdefghijk	0.25 ± 0.01jklmnopqr	0.14 ± 0.00abcdefghij
13M2	0.61 ± 0.04hijk	0.60 ± 0.00abcdef	0.65 ± 0.01abc	0.61 ± 0.05ab	0.28 ± 0.01hijklmno	0.09 ± 0.01abc	0.17 ± 0.01abcdefghijkl
13M5	0.72 ± 0.01abcdefg	0.79 ± 0.00a	0.68 ± 0.01ab	0.60 ± 0.07abc	1.39 ± 0.01a	0.46 ± 0.2a	0.48 ± 0.00rs
14P2	0.67 ± 0.00mnopqrs	0.63 ± 0.00abcde	0.63 ± 0.02abc	0.63 ± 0.05ab	0.75 ± 0.03a	0.57 ± 0.03ab	0.70 ± 0.03abc
14P3	0.61 ± 0.02hijk	0.55 ± 0.01hijklm	0.64 ± 0.00abcd	0.46 ± 0.01nopqrstu	0.37 ± 0.05nopqr	0.70 ± 0.01abc	0.21 ± 0.01cdefghijklmn
14P4	0.59 ± 0.03ghi	0.65 ± 0.02abcd	0.56 ± 0.00abc	0.50 ± 0.04abcdefgh	0.31 ± 0.04jklmnop	0.15 ± 0.00abcdefgh	0.43 ± 0.04qr
15G	0.68 ± 0.00nopqrst	0.63 ± 0.02abcd	0.64 ± 0.02abcd	0.54 ± 0.05abcdefghi	0.41 ± 0.04opqrs	0.73 ± 0.03abc	0.28 ± 0.03lmnop
15M1	0.60 ± 0.01ghij	0.53 ± 0.00fghjij	0.45 ± 0.01hijklmnop	0.49 ± 0.03qrstuvwx	0.16 ± 0.01abcdefgh	0.22 ± 0.00ghijklmnop	0.11 ± 0.00abcdefg
15M2	0.59 ± 0.00ghij	0.58 ± 0.00lmnop	0.52 ± 0.00abcd	0.54 ± 0.02abcdefghi	0.11 ± 0.00abcd	0.20 ± 0.00fghijklmno	0.17 ± 0.00abcdefghijkl
15M3	0.71 ± 0.00abcdefg	0.63 ± 0.01abcde	0.56 ± 0.05abcd	0.48 ± 0.05pqrstuvw	0.11 ± 0.00abcd	0.19 ± 0.01defghijklmn	0.12 ± 0.01abcdefgh
15M4	0.86 ± 0.06a	0.75 ± 0.02ab	0.47 ± 0.00mnopqrstu	0.32 ± 0.03bcde	0.11 ± 0.01abcd	0.29 ± 0.00opqrs	0.25 ± 0.04ijklmno
16G	0.64 ± 0.01jklmnop	0.64 ± 0.02abcde	0.51 ± 0.02abcd	0.45 ± 0.03mnopqrst	0.14 ± 0.07abcdefgh	0.10 ± 0.01abcd	0.18 ± 0.00abcdefghijkl
16M	0.52 ± 0.03c	0.51 ± 0.01efghi	0.48 ± 0.00abcdefghi	0.44 ± 0.00klmnopqrs	0.24 ± 0.01defghijklmn	0.24 ± 0.01ijklmnopq	0.24 ± 0.01hijklmno
16M1	0.65 ± 0.00klmnopq	0.71 ± 0.00abc	0.63 ± 0.01abc	0.41 ± 0.00hijklmno	0.15 ± 0.00abcdefgh	0.22 ± 0.01ghijklmno	0.09 ± 0.00abcd
16M2	0.57 ± 0.01defgh	0.63 ± 0.01abcde	0.50 ± 0.00abcde	0.43 ± 0.01jklmnopqr	0.14 ± 0.01abcdefg	0.26 ± 0.00lmnopqr	0.10 ± 0.00abcdef
16M3	0.73 ± 0.00abcdefg	0.61 ± 0.04abcdef	0.37 ± 0.00abcd	0.36 ± 0.02cdefghi	0.12 ± 0.02abcdef	0.20 ± 0.00efghijklmno	0.42 ± 0.55qr
17G	0.87 ± 0.00a	0.67 ± 0.02abcd	0.46 ± 0.00jklmnopqr	0.63 ± 0.00ab	0.20 ± 0.00bcdefghijk	0.22 ± 0.00ghijklmnop	0.22 ± 0.00fghijklmno
17G1	0.68 ± 0.00abcdefgh	0.59 ± 0.03lmnopq	0.59 ± 0.01ab	0.51 ± 0.03abcdefg	0.18 ± 0.00abcdefghijk	0.27 ± 0.01mnopqr	0.22 ± 0.00efghijklmno
17G2	0.61 ± 0.04hijkl	0.72 ± 0.02abc	0.42 ± 0.00efghijkl	0.40 ± 0.00fghijklmn	0.31 ± 0.03jklmnop	0.26 ± 0.00lmnopqr	0.16 ± 0.01abcdefghijkl
17G4	0.44 ± 0.01b	0.42 ± 0.07c	0.47 ± 0.03mnopqrstu	0.34 ± 0.06bcdefg	0.11 ± 0.01abcde	0.45 ± 0.01ab	0.18 ± 0.00abcdefghijkl
17M	0.57 ± 0.01defgh	0.64 ± 0.01abc	0.72 ± 0.00ab	0.55 ± 0.00abcdefgh	0.23 ± 0.00cdefghijklm	0.26 ± 0.01klmnopqr	0.12 ± 0.00abcdefgh
17M2	0.6 ± 0.01ghij	0.50 ± 0.00efg	0.50 ± 0.02abcd	0.37 ± 0.01defghij	0.34 ± 0.04lmnop	0.19 ± 0.00defghijklmn	0.18 ± 0.01abcdefghijkl
18G	0.51 ± 0.01c	0.54 ± 0.00ghijk	0.48 ± 0.03abcdefghi	0.56 ± 0.04abcdefg	0.37 ± 0.02nopqr	0.83 ± 0.03a	0.41 ± 0.03qr
18M1	0.59 ± 0.01efghi	0.53 ± 0.00fghijk	0.47 ± 0.01abcdefghi	0.52 ± 0.07abcdefgh	0.24 ± 0.03defghijklmn	0.15 ± 0.03abcdefghi	0.13 ± 0.00abcdefghij
18M2	0.52 ± 0.00cd	0.45 ± 0.00cd	0.50 ± 0.00abcd	0.58 ± 0.01abcdef	0.27 ± 0.04ghijklmn	0.23 ± 0.01hijklmnop	0.25 ± 0.05jklmno
Control without isolate	0.27 ± 0.00a	0.59 ± 0.00lmnopq	0.46 ± 0.02lmnopqrs	0.07 ± 0.00a	0.06 ± 0.00a	0.19 ± 0.24defghijklmn	0.10 ± 0.00abcdef

Values with different superscript letters on the same column are significantly different at p ˂ 0.05.

3.5. Effect of Temperature Variation on the Growth of the 73 Rhizobium sp. Isolates

As shown in Table 4, most isolates showed better growth at 30˚C, with OD values ranging from 0.50 ± 0.06 for isolate 5P to 0.84 ± 0.17 for isolate 2M, representing a 68% increase between the lowest and highest performance at this temperature. However, several isolates showed significantly (p < 0.05) high growth at other temperatures. Isolates 2M (1.06 ± 0.00) and 4M1 (1.12 ± 0.02) reached optimal

Table 4. Growth of Rhizobium sp. isolates at different temperatures in terms of optical density 620 nm

Isolates	15˚C	20˚C	25˚C	30˚C	35˚C	40˚C	45˚C
1	0.33 ± 0.00b	0.53 ± 0.01abcd	0.57 ± 0.01cdefg	0.75 ± 0.01abcdefghi	0.76 ± 0.00abcde	0.81 ± 0.00abcde	0.28 ± 0.00cdef
1G	0.80 ± 0.01abcdef	0.85 ± 0.02ab	0.62 ± 0.00jklmnop	0.77 ± 0.03abcdefghi	0.82 ± 0.00abcd	0.78 ± 0.00abcdef	0.72 ± 0.00ab
1G'	0.69 ± 0.00nopqrstu	0.55 ± 0.02abcde	0.60 ± 0.01ghijkl	0.74 ± 0.00abcdefghi	0.28 ± 0.03abcd	0.61 ± 0.03kl	0.28 ± 0.00bcde
1G''	0.76 ± 0.04abcde	0.36 ± 0.01bcdefg	0.63 ± 0.01lmnopqr	0.66 ± 0.00ijklmnopqr	0.20 ± 0.00 a	0.77 ± 0.03abcdefg	0.64 ± 0.02abcdefg
1M	0.85 ± 0.04abc	0.68 ± 0.00abc	0.69 ± 0.00abcdefg	0.78 ± 0.01abcdefg	0.70 ± 0.04abcde	0.44 ± 0.04ghij	0.21 ± 0.00a
1P	0.68 ± 0.00lmnopqr	0.40 ± 0.00efghijkl	0.62 ± 0.03jklmnop	0.67 ± 0.00ijklmnopqrs	0.76 ± 0.02abcd	0.26 ± 0.00abc	0.64 ± 0.01abcdefgh
2	0.75 ± 0.01abcdefgh	0.38 ± 0.00cdefghij	0.71 ± 0.01abcdef	0.72 ± 0.00nopqrstuvwx	0.50 ± 0.07hijklmno	0.78 ± 0.00abcdef	0.72 ± 0.00ab
2G	0.68 ± 0.00lmnopqr	0.60 ± 0.02abcd	0.60 ± 0.00fghijkl	0.66 ± 0.00ijklmnopqr	0.69 ± 0.02abcde	0.74 ± 0.02abcdef	0.64 ± 0.02abcdefg
2M	0.15 ± 0.00a	1.06 ± 0.00a	0.56 ± 0.00bcdef	0.77 ± 0.00abcdefgh	1.20 ± 0.02a	0.36 ± 0.00cdefghi	0.67 ± 0.01abc
2M'	0.60 ± 0.15hi	0.45 ± 0.01lmn	0.60 ± 0.05ghijkl	0.84 ± 0.17abc	0.38 ± 0.25efg	0.74 ± 0.03nopq	0.47 ± 0.09lm
2P'C'	0.70 ± 0.00abcdefghijk	0.35 ± 0.02bcdefg	0.76 ± 0.00a	0.67 ± 0.06jklmnopqrs	0.71 ± 0.01abcdef	0.87 ± 0.01ab	0.56 ± 0.02qrst
3G	0.82 ± 0.01abcdef	0.49 ± 0.02mno	0.67 ± 0.00abcdefghij	0.66 ± 0.09ijklmnopqr	0.74 ± 0.00abcdef	0.75 ± 0.02abcdef	0.26 ± 0.01bcd
3M'	0.61 ± 0.00hijk	0.33 ± 0.03bcd	0.58 ± 0.01cdefgh	0.41 ± 0.18a	0.41 ± 0.05fghi	0.81 ± 0.03abcde	0.26 ± 0.00bcd
3M'''	0.33 ± 0.02b	0.45 ± 0.02klmn	0.61 ± 0.04ghijklmn	0.61 ± 0.02defghijk	0.66 ± 0.03abcdefg	1.20 ± 0.03a	0.66 ± 0.02abcdef
4M1	0.70 ± 0.00nopqrstuvw	1.12 ± 0.02a	0.63 ± 0.02klmnopqr	0.65 ± 0.00hijklmnopq	0.42 ± 0.07fghij	0.36 ± 0.07cdefghi	0.34 ± 0.00hi
4M3	0.45 ± 0.02c	0.70 ± 0.00ab	0.61 ± 0.00hijklmn	0.77 ± 0.03abcdefghi	0.77 ± 0.01abcd	0.85 ± 0.04abcde	0.65 ± 0.02abcdefg
5G	0.43 ± 0.03c	0.62 ± 0.00abcd	0.68 ± 0.00abcdefgh	0.83 ± 0.01abcde	0.68 ± 0.01abcde	0.78 ± 0.02abcdef	0.36 ± 0.00ij
5M	0.75 ± 0.01abcdefg	0.41 ± 0.01efghijkl	0.58 ± 0.01defgh	0.68 ± 0.00jklmnopqrstu	0.52 ± 0.03ijklmnop	0.87 ± 0.00abc	0.31 ± 0.00efgh
5M''	0.73 ± 0.00abcdefghij	0.70 ± 0.01ab	0.66 ± 0.03opqrstuv	0.71 ± 0.00mnopqrstuvwx	0.42 ± 0.04fghij	0.83 ± 0.01abcde	0.38 ± 0.00jk
5M2'	0.74 ± 0.02abcdefgh	0.65 ± 0.00abc	0.66 ± 0.00abcdefghij	0.64 ± 0.02ghijklmnop	1.17 ± 0.05a	0.85 ± 0.01abcde	0.68 ± 0.01ab
5P	0.55 ± 0.00fgh	0.33 ± 0.01bcd	0.67 ± 0.00abcdefghi	0.50 ± 0.06bc	0.22 ± 0.01a	0.78 ± 0.01abcdef	0.75 ± 0.02a
5P'	0.74 ± 0.04abcdefghi	0.30 ± 0.00ab	0.55 ± 0.01abcde	0.69 ± 0.00klmnopqrstuv	0.52 ± 0.02hijklmnop	0.72 ± 0.04lmnop	0.73 ± 0.01a
6G	0.74 ± 0.00abcdefghi	0.33 ± 0.01bcd	0.69 ± 0.00abcdefgh	0.66 ± 0.00ijklmnopqrs	0.61 ± 0.01abcdef	0.30 ± 0.02abcdef	0.28 ± 0.00cdef
6G'	0.76 ± 0.05abcde	0.63 ± 0.00abc	0.68 ± 0.02abcdefghi	0.73 ± 0.05abcdefghi	0.65 ± 0.00abcdef	0.26 ± 0.01abc	0.63 ± 0.00abcdef
6G2	0.84 ± 0.01abcde	0.35 ± 0.00bcdef	0.64 ± 0.01lmnopqrs	0.72 ± 0.00abcdefghijk	0.72 ± 0.02abcdef	0.26 ± 0.00abc	0.25 ± 0.01bc
6G2'	0.62 ± 0.01ijkl	0.69 ± 0.00 abc	0.71 ± 0.00abcdef	0.69 ± 0.03klmnopqrstuv	0.64 ± 0.01abcdef	0.22 ± 0.00ab	0.34 ± 0.00hi
6G3	0.84 ± 0.00abcd	0.35 ± 0.00bcdefg	0.70 ± 0.00abcdefg	0.90 ± 0.00a	0.29 ± 0.02abcde	0.27 ± 0.04abcd	0.30 ± 0.00def
6G4	0.52 ± 0.02ef	0.36 ± 0.00bcdefghi	0.67 ± 0.02abcdefghi	0.73 ± 0.00abcdefghi	0.49 ± 0.02hijklm	0.33 ± 0.01abcdefg	0.67 ± 0.01abcd
7M	0.53 ± 0.03efg	0.56 ± 0.00abcde	0.68 ± 0.01abcdefgh	0.65 ± 0.00hijklmnopq	0.26 ± 0.00abc	0.41 ± 0.03fghij	0.57 ± 0.02rstu
7M'	0.67 ± 0.00lmnopq	0.36 ± 0.00bcdefgh	0.73 ± 0.00abc	0.64 ± 0.01ghijklmno	0.76 ± 0.00abcd	0.35 ± 0.00bcdefgh	0.46 ± 0.01lm
8G	0.69 ± 0.01nopqrst	0.35 ± 0.00bcdefg	0.54 ± 0.04abc	0.59 ± 0.12defghi	0.25 ± 0.05abc	0.25 ± 0.01abc	0.29 ± 0.01def
8M'	0.62 ± 0.00ijklm	0.41 ± 0.03ghijkl	0.59 ± 0.01fghij	0.65 ± 0.01hijklmnopq	0.25 ± 0.03abc	0.27 ± 0.00abcd	0.34 ± 0.00ghi
8M''	0.57 ± 0.00fghi	0.36 ± 0.02bcdefghi	0.63 ± 0.02klmnopqr	0.59 ± 0.01defghi	0.34 ± 0.06bcdef	0.41 ± 0.04fghij	0.58 ± 0.00stuvw
8P	0.77 ± 0.02abcd	0.39 ± 0.02defghijk	0.72 ± 0.00abcde	0.72 ± 0.00abcdefghijk	0.62 ± 0.02abcdef	1.08 ± 0.01a	0.48 ± 0.00mn
9G'	0.54 ± 0.02efg	0.39 ± 0.02defghijk	0.70 ± 0.00abcdefg	0.66 ± 0.01ijklmnopqr	0.75 ± 0.01abcde	0.66 ± 0.02lmno	0.44 ± 0.01l
9P	0.76 ± 0.02abcde	0.34 ± 0.00bcde	0.61 ± 0.00ghijklm	0.68 ± 0.00jklmnopqrst	0.38 ± 0.01defg	0.49 ± 0.11jk	0.67 ± 0.01abc
10G	0.66 ± 0.00jklmno	0.60 ± 0.04abcde	0.74 ± 0.00 ab	0.84 ± 0.02abcd	0.41 ± 0.00fgh	0.82 ± 0.01abcde	0.40 ± 0.00k
10G1	0.69 ± 0.00nopqrstuv	0.40 ± 0.08efghijkl	0.58 ± 0.00cdefgh	0.70 ± 0.00klmnopqrstuv	1.23 ± 0.04a	0.30 ± 0.00abcdef	0.68 ± 0.01ab
10G2	0.79 ± 0.01abcde	0.67 ± 0.00abc	0.61 ± 0.00ghijklmn	0.48 ± 0.00ab	0.57 ± 0.05lmnopqr	0.31 ± 0.00abcdef	0.56 ± 0.01pqrst
11G1	0.32 ± 0.01b	0.26 ± 0.00a	0.70 ± 0.02abcdef	0.61 ± 0.00defghijkl	0.61 ± 0.09abcdefg	0.76 ± 0.00abcdefg	0.30 ± 0.00efgh
11G2	0.86 ± 0.00 a	0.32 ± 0.04abc	0.64 ± 0.00lmnopqrs	0.66 ± 0.00ijklmnopqr	0.36 ± 0.03cdef	0.34 ± 0.00bcdefgh	0.34 ± 0.01ghi
11G3	0.66 ± 0.03jklmno	0.59 ± 0.00abcde	0.52 ± 0.01a	0.64 ± 0.00fghijklmno	0.26 ± 0.01abc	0.31 ± 0.03abcdef	0.48 ± 0.01lm
11G4	0.85 ± 0.02ab	0.60 ± 0.01abcd	0.63 ± 0.00lmnopqrs	0.73 ± 0.01abcdefghij	0.68 ± 0.01abcdef	0.28 ± 0.00abcdef	0.63 ± 0.00abcde
11G5	0.75 ± 0.00abcdefgh	0.65 ± 0.00 abc	0.59 ± 0.11fghijk	0.80 ± 0.02abcdef	0.72 ± 0.02abcdef	0.65 ± 0.01lmn	0.62 ± 0.00abcdef
12G	0.44 ± 0.02c	0.91 ± 0.00a	0.69 ± 0.00abcdefgh	0.82 ± 0.01abcdef	0.66 ± 0.03abcdefg	0.95 ± 0.00a	0.30 ± 0.02defg
12G2	0.33 ± 0.00b	0.69 ± 0.00abc	0.66 ± 0.00abcdefghij	0.87 ± 0.03ab	0.63 ± 0.00abcdef	0.87 ± 0.02abc	0.56 ± 0.00pqrst
12G3	0.49 ± 0.04 cde	0.53 ± 0.01opq	0.63 ± 0.01jklmnopq	0.56 ± 0.07bcdefg	0.23 ± 0.01ab	0.72 ± 0.01lmnop	0.28 ± 0.00bcde
13M	0.75 ± 0.02abcdef	0.58 ± 0.01abcde	0.66 ± 0.00abcdefghij	0.70 ± 0.00lmnopqrstuvw	0.42 ± 0.16fghij	0.35 ± 0.06bcdefgh	0.38 ± 0.00jk
13M2	0.72 ± 0.01abcdefghij	0.58 ± 0.04abcde	0.54 ± 0.03abcd	0.76 ± 0.05abcdefghi	0.74 ± 0.04abcde	0.49 ± 0.34 jk	0.30 ± 0.02efgh
13M5	0.79 ± 0.00abcdef	0.34 ± 0.01bcde	0.66 ± 0.02opqrstuvw	0.73 ± 0.00abcdefghi	0.84 ± 0.00ab	0.23 ± 0.00ab	0.24 ± 0.01 ab
14P2	0.65 ± 0.03jklmn	0.35 ± 0.00bcdefg	0.71 ± 0.01abcdef	0.77 ± 0.00abcdefgh	0.41 ± 0.00fghi	0.31 ± 0.00abcdef	0.66 ± 0.00abcde
14P3	0.46 ± 0.00cd	0.37 ± 0.03cdefghij	0.70 ± 0.01abcdefgh	0.61 ± 0.00defghijk	0.69 ± 0.10abcde	0.62 ± 0.06 lm	0.63 ± 0.00abcdef
14P4	0.67 ± 0.03klmnop	0.82 ± 0.02a	0.73 ± 0.03abcd	0.66 ± 0.01ijklmnopqr	0.73 ± 0.05abcdef	0.76 ± 0.04abcdefg	0.62 ± 0.01abcdef
15G	0.52 ± 0.02def	0.50 ± 0.06nop	0.61 ± 0.00hijklmn	0.63 ± 0.05efghijklm	0.50 ± 0.13hijklmn	0.78 ± 0.01abcdef	0.59 ± 0.02tuvwx
15M1	0.51 ± 0.08def	0.33 ± 0.00bcd	0.66 ± 0.01abcdefghijk	0.68 ± 0.00jklmnopqrst	0.83 ± 0.07abc	0.36 ± 0.00cdefghi	0.56 ± 0.02qrst
15M2	0.75 ± 0.05abcdef	0.37 ± 0.01cdefghi	0.62 ± 0.05ijklmno	0.55 ± 0.13bcdef	0.88 ± 0.01a	0.46 ± 0.15hij	0.60 ± 0.00abcdef
15M3	0.78 ± 0.00abcde	0.35 ± 0.01bcdefg	0.66 ± 0.00opqrstuvw	0.62 ± 0.00defghijklm	0.68 ± 0.02abcdef	0.71 ± 0.01lmnop	0.68 ± 0.00ab
15M4	0.66 ± 0.00jklmn	0.55 ± 0.07abcde	0.53 ± 0.01ab	0.78 ± 0.03abcdefg	0.83 ± 0.01abcd	0.23 ± 0.00ab	0.55 ± 0.00opqrs
16G	0.60 ± 0.00hij	0.31 ± 0.00abc	0.63 ± 0.01klmnopqr	0.63 ± 0.02fghijklmn	0.73 ± 0.01abcdef	0.26 ± 0.01abc	0.56 ± 0.02pqrst
16M	0.51 ± 0.06def	0.35 ± 0.00bcdefg	0.64 ± 0.02lmnopqrst	0.57 ± 0.01cdefgh	0.55 ± 0.02klmnopq	0.21 ± 0.01a	0.61 ± 0.00abcdef
16M1	0.72 ± 0.02abcdefghijk	0.41 ± 0.05fghijkl	0.52 ± 0.02a	0.66 ± 0.03ijklmnopqr	0.76 ± 0.00abcd	0.39 ± 0.02defghij	0.63 ± 0.01abcdef
16M2	0.76 ± 0.01abcde	0.35 ± 0.00bcdef	0.72 ± 0.01abcde	0.60 ± 0.11defghij	0.77 ± 0.03abcd	0.48 ± 0.02jk	0.35 ± 0.01ij
16M3	0.75 ± 0.01abcde	0.35 ± 0.00bcdefg	0.64 ± 0.00mnopqrstu	0.71 ± 0.00mnopqrstuvwx	0.61 ± 0.09abcdef	0.31 ± 0.01abcdef	0.53 ± 0.02opqr
17G	0.76 ± 0.01abcde	0.44 ± 0.00jklmn	0.65 ± 0.00nopqrstu	0.71 ± 0.00mnopqrstuvwx	0.88 ± 0.09a	0.66 ± 0.03lmno	0.58 ± 0.00stuvw
17G1	0.68 ± 0.00mnopqrs	0.43 ± 0.01ijklm	0.61 ± 0.00hijklmn	0.57 ± 0.00cdefgh	0.61 ± 0.07mnopqrs	0.30 ± 0.00abcdef	0.56 ± 0.01pqrst
17G2	0.31 ± 0.01b	0.42 ± 0.01hijklm	0.61 ± 0.01hijklmn	0.74 ± 0.00abcdefghi	0.25 ± 0.01abc	0.81 ± 0.00abcde	0.57 ± 0.01stuv
17G4	0.73 ± 0.03abcdefghij	0.48 ± 0.07mno	0.58 ± 0.00efghi	0.71 ± 0.04nopqrstuvwx	0.55 ± 0.17lmnopq	0.29 ± 0.00abcdef	0.56 ± 0.03pqrst
17M	0.86 ± 0.01a	0.63 ± 0.01 abc	0.67 ± 0.02abcdefghi	0.70 ± 0.02mnopqrstuvwx	0.44 ± 0.04fghijk	0.47 ± 0.03ij	0.51 ± 0.00no
17M2	0.59 ± 0.00ghi	0.74 ± 0.00 a	0.64 ± 0.01mnopqrstu	0.54 ± 0.05bcd	0.53 ± 0.02jklmnop	0.28 ± 0.01abcde	0.52 ± 0.00op
18G	0.29 ± 0.03b	0.59 ± 0.03abcdef	0.64 ± 0.01lmnopqrs	0.89 ± 0.00a	0.48 ± 0.14ghijkl	0.86 ± 0.02abcd	0.32 ± 0.01fgh
18M1	0.65 ± 0.03jklmn	0.35 ± 0.00bcdefg	0.61 ± 0.01ghijklm	0.54 ± 0.03bcde	0.82 ± 0.04abcd	0.67 ± 0.02lmno	0.59 ± 0.00abcdef
18M2	0.76 ± 0.04abcd	0.41 ± 0.00fghijkl	0.61 ± 0.00hijklmn	0.66 ± 0.00ijklmnopqr	0.21 ± 0.00a	0.40 ± 0.08efghij	0.53 ± 0.00opq
Control without isolate	0.16 ± 0.00a	0.71 ± 0.02ab	0.63 ± 0.01lmnopqr	0.64 ± 0.00fghijklmno	0.74 ± 0.03abcde	0.66 ± 0.01lmno	0.25 ± 0.00b

Values with different superscript letters on the same column are significantly different at p < 0.05.

growth at 20˚C, values which are 112% and 124% higher, respectively, than the lowest OD observed at this temperature (0.30 ± 0.00 for 5P). Similarly, isolates 10G1 and 3M reached maximum growth at 35˚C (1.23 ± 0.04) and 40˚C (1.20 ± 0.03), demonstrating the high thermotolerance of these strains. At 15˚C, growth varied significantly (p < 0.05), with the 1M isolate showing the highest OD (0.85 ± 0.04), more than five times that of the 2M isolate (0.15 ± 0.00). Even at the stressful temperature of 45˚C, the 5P isolate maintained relatively high growth (0.75 ± 0.02), which was more than three times higher than the lowest value recorded at this temperature (0.21 ± 0.00 for 1M). These substantial differences in growth responses illustrate the high thermal adaptability between isolates and suggest significant physiological variation probably shaped by environmental pressures.

3.6. Distribution of the Rhizobium sp. Isolates According to Abiotic Factors (pH, Salinity, and Temperature)

A synthesis was carried out to identify isolates that simultaneously tolerate extreme pH, salinity, and temperature conditions, and the results are depicted. The isolates were classified into two groups according to these criteria. The first group includes growth at pH 2, NaCl 12%, and a temperature of 45˚C. The isolates belonging to this group were from TGX 2007 11 F, TGX 2001 12 F, SC Sentinel, Pan 1, Songda, Maksoy 2 N Pan 237, Pan 2, TGX 1988 18 F, S1180/15/54, TGX 1835-10 E, and Maksoy 1N. This means these varieties can be cultivated in highly acidic soils with high salinity and under high environmental temperatures. The second group includes growth at pH 2.5, NaCl 10%, and a temperature of 40˚C. It contains isolates from TGX 2007 11 F, TGX 2001 12 F, TGX 2010 3F, TGX 1991 22F, SC Sentinel, Pan 1, Pan 3, Songda, SC Signal, Maksoy 2 N, Pan 237, TGX 1989 60 F, TGX 1988 18 F, S1180/15/54, TGX 1835-10 E, and Maksoy 1N. These varieties can also be cultivated in hard environmental conditions. However, the isolates able to grow at high salinities (NaCl 12%) and low temperatures (between 15 and 20˚C) were from soybean varieties TGX 2007 11 F, Pan 1, Songda, SC Signal, Maksoy 2 N, Pan 237, Pan 2, TGX 1988 18 F, and TGX 1835-10 E.

3.7. Physicochemical Activities of the 73 Rhizobium sp. Isolates

Some physicochemical activities of the isolates, including antioxidant activity and inhibition of lipid peroxidation, were assessed in this study. Before that, the protein content of the isolates was determined. The presence of intracellular proteins in Rhizobium sp. induces the expression of nodulin genes that trigger the differentiation of cortical cells responsible for nodule formation, which are connected to the plant’s conducting vessels, thus ensuring the system’s energy supply. The protein content of the rhizobia cell-free supernatants is presented in Table 5. The isolates cell free supernatants showing the highest total protein concentrations were 10G2 (135.33  ±  5.65 µg/mL), 3M''' (122.22  ±  13.19 µg/mL), 5G (102.33  ±  19.01 µg/mL), 13M2 (110.88  ±  10.68 µg/mL), 15M1 (112.44  ±  18.54 µg/mL), 15M3 (109.88  ±  11.78 µg/mL), and 16G (106.33  ±  20.89 µg/mL). Lower protein concentrations were observed in isolates 1G (73.00  ±  2.67 µg/mL), 2M' (80.77  ±  0.47 µg/mL), 4M3 (82.44  ±  4.39 µg/mL), 5M2 (77.44  ±  6.12 µg/mL), 6G4 (82.55  ±  3.29 µg/mL), 12G3 (89.33  ±  0.62 µg/mL), and 13M (82.11  ±  6.44 µg/mL).

As different mechanisms may be involved in the antioxidant activity of the isolates, 3 methods were used: FRAP, TAC, and catalase. In the case of FRAP, the reducing power of ferrous ions varied according to the isolate. Regarding FRAP, isolate 7M from Pan 3 showed the highest ferrous ions reducing power (298.46 ± 0.00 µg AAE/mL). It was followed by isolates 15M2 (260.0 ± 0.00 µg AAE/mL) from S1180/15/54, 8P (225.76 ± 5.98 µg AAE/mL) from Songda, 6G (219.23 ± 17.94 0 µg AAE/mL) from Pan 1, and 4M3 (205.57 ± 17.13 0 µg AAE/mL) from TGX 1991 22F. Weak ferrous ions reducing power were observed with some isolates, including 11G3 (75.0 ± 2.17 µg AAE/mL) from Pan 237, followed by 6G3 (70.19 ± 2.44 µg AAE/mL) from Pan 1, 16M2 (69.23 ± 6.52 µg AAE/mL) from TGX 2001-10 DM, 8M'' (64, 61 ± 5.43 µg AAE/mL ) from Songda, 1P (50.76 ± 1.63 µg AAE/mL) from TGX 2007 11 F, isolate 2G (46.92 ± 13.05 µg AAE/mL) from TGX 2001 12 F, 5M2 (43.65 ± 2.99 µg AAE/mL) from SC Sentinel, 15M4 (33.84 ± 4.35 µg AAE/mL) from S1180/15/54, and 14P2 from TGX 1988 18 F (14.61 ± 8.70 µg AAE/mL).

Concerning the total antioxidant capacity, the highest activities were obtained with isolates 4M1 (1335.93 ± 10.84 µg AAE/mL), 6G2' (1307.6 ± 9.42 µg AAE/mL), 10G2 (1194.6 ± 2.35 µg AAE/mL), 10G (1051.0 ± 9.33 µg AAE/mL), 7M (1033.93 ± 33.46 µg AAE/mL), and 5M (1033.93 ± 33.46 µg AAE/mL). These highly active isolates were from soybean varieties TGX 1991 22F (4M1), SC Sentinel (5M), Pan 1 (6G2'), Pan 3 (7M), and Maksoy 2N (10G2). Another antioxidant mechanism assessed in this study is through the production of catalase, an enzyme that inhibits the oxidation process by degrading the ROS called hydrogen peroxide. The results obtained showed that the catalase activity varies from one isolate to another. The highest catalase activity (0.07 ± 0.00) was recorded with isolates 1, 2G, 5P', and 13M, while the lowest ones (0.02 ± 0.00) were observed with isolate 10G2.

MDA is a stress marker in plants, and high MDA levels cause oxidative damage in plants, inhibiting their growth. In this study, the ability of isolates to inhibit MDA production was assessed. Generally, MDA values varied significantly (p ˂ 0.05) from one isolate to another, depending on the soybean variety (Table 5). The isolates endowed with high inhibition of lipid peroxidation (low MDA values) were 4M1 (1.66 ± 0.11 µmoL/L) from TGX 1991 22F; 5P' (1.13 ± 0.13 µmoL/mL) from SC Sentinel; 8G (1.34 ± 0.12 µmoL/L) from Songda; 9P (1.70 ± 0.12 µmoL/L) from SC Signal; 10G1 (1.46 ± 0.01 µmoL/L) from Maksoy 2N; 11G3 (1.14 ± 0.05 µmoL/L) from Pan 237; 12G3 (1.42 ± 0.01 µmoL/L) from TGX 1989 60 F; 13M (51.48 ± 0.02 µmoL/L), and 14P4 (1.40 ± 0.04 µmoL/L) from TGX 1988 18 F. The low inhibition of lipid peroxidation (high MDA values) was recorded with isolates 16G (3.25 ± 2.07 µmoL/L) from TGX 2001-10 DM; 15M1 (2.74 ± 1.08 µmoL/L), 15M2 (2.85 ± 1.07 µmoL/L), and 15M3 (2.71 ± 0.69 µmoL/L) from S1180/15/54.

Generally, the isolates with the highest values of MDA, TAC, FRAP, protein, and catalase were from soybean varieties TGX 2007 11 F, TGX 2001 12 F, TGX 2010 3F, TGX 1991 22F, SC Sentinel, Pan 1, Pan 3, Songda, Maksoy 2 N, Pan 2, S1180/15/54, TGX 2001-10 DM, and TGX 1835-10 E.

Table 5. Protein content, antioxidant activity, and malondialdehyde content of 73 Rhizobium sp. isolates

Isolates	MDA (µmoL/L)	TAC (µg AAE/mL)	FRAP (µg AAE/mL)	Proteins (µg/mL)	Catalase (µmol/mL/g prot)
1	1.82 ± 0.20abcdefghijk	640.26 ± 19.79abc	85.19 ± 2.44efghij	85.0 ± 2.67abcdefghi	0.07 ± 0.00ab
1G	1.37 ± 0.06abc	396.6 ± 8.95opqrs	117.5 ± 1.35lmnopqrst	73.0 ± 2.67a	0.05 ± 0.00mnopqrst
1G'	1.49 ± 0.13abcdef	247.26 ± 15.55ghijkl	131.15 ± 7.07abcdefg	99.11 ± 2.51ijklmnopqrstu	0.05 ± 0.00abcdefgh
1G''	1.53 ± 0.05abcdefgh	248.6 ± 7.07ghijkl	105.0 ± 10.33ijklmnopq	95.44 ± 5.49defghijklmno	0.04 ± 0.00bcdefghi
1M	1.84 ± 0.01abcdefghijk	324.6 ± 6.12opqrs	142.30 ± 5.98abcde	88.33 ± 7.07bcdefghijk	0.06 ± 0.00abcdefg
1P	1.58 ± 0.05abcdefghi	251.26 ± 10.84ghijkl	50.76 ± 1.63bcd	87.66 ± 9.27abcdefghijk	0.06 ± 0.00abcdefg
2	1.37 ± 0.00abc	58.26 ± 0a	162.5 ± 2.44abcdefg	96.22 ± 3.77efghijklmnop	0.03 ± 0.00abcd
2G	1.32 ± 1.10abc	323.6 ± 18.85klmnop	46.92 ± 13.05bc	89.22 ± 3.61bcdefghijk	0.07 ± 0.00a
2M	1.74 ± 0.07abcdefghijk	585.13 ± 0.65abc	190.19 ± 6.79 ab	76.33 ± 9.89ab	0.05 ± 0.00klmnopqr
2M'	1.35 ± 0.12abc	592.6 ± 4.24abc	97.69 ± 7.61hijklmn	80.77 ± 0.47abcd	0.06 ± 0.00abcdefghij

Continued

2P'C'	1.64 ± 0.13abcdefghij	385.6 ± 15.08opqrs	136.15 ± 31.54abcde	88.55 ± 2.04bcdefghijk	0.06 ± 0.00abcdef
3G	1.77 ± 0.01abcdefghijk	339.6 ± 65.05lmnopq	124.42 ± 6.25pqrstuv	96.88 ± 0.31fghijklmnopqr	0.06 ± 0.00abcdefgh
3M'	1.52 ± 0.34abcdefg	538.26 ± 32.99abc	173.84 ± 11.96abcd	94.11 ± 2.04defghijklmn	0.05 ± 0.00pqrstuv
3M'''	2.01 ± 0.02cdefghijklm	521.53 ± 1.97abcd	98.26 ± 13.87hijklmno	122.22 ± 13.19wx	0.04 ± 0.00bcdefgh
4M1	1.66 ± 0.11abcdefghij	1335.93 ± 10.84a	86.92 ± 2.17fghij	99.77 ± 5.02ijklmnopqrstu	0.06 ± 0.00abcdefg
4M3	1.86 ± 0.00bcdefghijk	385.26 ± 268.22opqrs	205.57 ± 17.13ab	82.44 ± 4.39abcdef	0.06 ± 0.00abcdefg
5G	1.66 ± 0.27abcdefghij	289.93 ± 16.49ijklmn	130.76 ± 2.71abcdefg	102.33 ± 19.01klmnopqrstuv	0.05 ± 0.00jklmnop
5M	1.55 ± 0.25abcdefgh	1033.93 ± 33.46a	151.53 ± 20.12abcdefg	97.55 ± 1.88ghijklmnopqrs	0.05 ± 0.01opqrstu
5M''	1.48 ± 0.16abcde	434.26 ± 30.16abcde	137.88 ± 6.79abcde	95.44 ± 7.07defghijklmno	0.05 ± 0.00nopqrstu
5M2	1.58 ± 0.06abcdefgh	496.8 ± 10.55abcd	43.65 ± 2.99bc	77.44 ± 6.12abc	0.05 ± 0.00klmnopqr
5P	1.54 ± 0.01abcdefgh	72.26 ± 10.37a	179.42 ± 7.34abcd	98.88 ± 0.62hijklmnopqrstu	0.03 ± 0.00bcdefgh
5P'	1.13 ± 0.13a	242.93 ± 1.88ghijk	125.0 ± 9.24pqrstuv	85.33 ± 5.97abcdefghi	0.07 ± 0.00abc
6G	1.58 ± 0.11abcdefgh	450.6 ± 9.89abcde	219.23 ± 17.94ab	87.77 ± 2.82abcdefghijk	0.05 ± 0.00qrstuvw
6G'	1.83 ± 0.08abcdefghijk	695.6 ± 0ab	106.15 ± 13.59jklmnopq	91.11 ± 4.39bcdefghijkl	0.06 ± 0.00abcd
6G2	2.41 ± 0.01klmn	275.26 ± 31.58ghijklm	191.15 ± 47.86ab	100.55 ± 10.21jklmnopqrstu	0.06 ± 0.00abcdefgh
6G2'	1.53 ± 0.08abcdefgh	1307.6 ± 9.42a	144.80 ± 17.13abcde	88.55 ± 4.87bcdefghijk	0.05 ± 0.00lmnopqrs
6G3	1.95 ± 0.09cdefghijkl	863.6 ± 36.76ab	70.19 ± 2.44def	98.66 ± 6.59hijklmnopqrstu	0.05 ± 0.00abcdefghi
6G4	1.62 ± 0.04abcdefghi	227.93 ± 81.55ghij	121.34 ± 13.87opqrstu	82.55 ± 3.29abcdef	0.06 ± 0.00abcde
7M	1.74 ± 0.21abcdefghijk	1033.93 ± 33.46a	298.46 ± 0a	92.55 ± 9.89defghijklmn	0.05 ± 0.00pqrstu
7M'	1.99 ± 0.05cdefghijklm	357.93 ± 22.15mnopqr	167.30 ± 14.14abcdef	92.11 ± 5.81cdefghijklmn	0.06 ± 0.00abcdefg
8G	1.34 ± 0.12abc	376.26 ± 21.68nopqrs	96.34 ± 8.43ghijklm	95.11 ± 2.51defghijklmno	0.06 ± 0.00abcdefghi
8M'	1.43 ± 0.19abcd	844.6 ± 21.21abc	147.11 ± 9.51abcde	96.55 ± 6.44fghijklmnop	0.05 ± 0.00opqrstu
8M''	1.60 ± 0.09abcdefghi	359.6 ± 15.05mnopqrs	64.61 ± 5.43cde	111.77 ± 7.22rstuvw	0.03 ± 0.00abcd
8P	1.51 ± 0.04abcdefg	676.93 ± 13.19abcd	225.76 ± 5.98 ab	97.88 ± 0.47ghijklmnopqrst	0.06 ± 0.00abcdefghi
9G'	1.83 ± 0.07abcdefghijk	690.93 ± 28.28 ab	88.46 ± 15.23fghij	94.77 ± 0.15defghijklmn	0.06 ± 0.00abcdefghij
9P	1.70 ± 0.12abcdefghijk	933.6 ± 57.51 abc	124.03 ± 10.06pqrstuv	88.55 ± 5.81bcdefghijk	0.06 ± 0.00abcdefghi
10G	1.63 ± 0.19abcdefghij	1051.0 ± 9.33a	175.0 ± 16.86abcd	93.88 ± 1.41defghijklmn	0.06 ± 0.00abcdefgh
10G1	1.46 ± 0.01abcde	218.93 ± 158.32efghij	147.69 ± 13.59abcdef	94.88 ± 1.57defghijklmn	0.04 ± 0.00fghijklm
10G2	1.68 ± 0.31abcdefghijk	1194.6 ± 2.35a	168.65 ± 1.90abcde	135.33 ± 5.65x	0.02 ± 0.00a
11G1	1.42 ± 0.23abcd	380.6 ± 10.84nopqrs	110.0 ± 7.61klmnopqr	96.88 ± 4.71fghijklmnopqr	0.06 ± 0.00abcdefghij
11G2	1.67 ± 0.29abcdefghij	768.26 ± 1.88abc	165.96 ± 12.23abcdefg	90.33 ± 1.09bcdefghijk	0.06 ± 0.00abcdefgh
11G3	1.14 ± 0.05ab	359.6 ± 20.74mnopqrs	75.0 ± 2.17efg	113.55 ± 5.34uvw	0.03 ± 0.00ab
11G4	1.52 ± 0.22abcdefg	542.26 ± 20.74abc	187.30 ± 6.52abc	86.44 ± 5.97abcdefghij	0.05 ± 0.00nopqrstu
11G5	1.56 ± 0.04abcdefgh	68.93 ± 0a	183.84 ± 9.79abc	91.33 ± 0.62cdefghijkl	0.04 ± 0.00ghijklmn
12G	1.79 ± 0.08abcdefghijk	451.6 ± 0.94abcde	173.65 ± 3.53abcd	93.33 ± 0.31defghijklmn	0.06 ± 0.00abcdefghij
12G2	1.57 ± 0.07abcdefgh	181.6 ± 5.65bcdefg	159.03 ± 4.62abcdefg	100.55 ± 4.87jklmnopqrstu	0.03 ± 0.00bcdefg
12G3	1.42 ± 0.01abcd	963.6 ± 27.34abc	90.0 ± 11.42fghijk	89.33 ± 0.62bcdefghijk	0.06 ± 0.00abcdefgh
13M	2.14 ± 0.10defghijklmn	891.93 ± 1.41abc	114.80 ± 10.60klmnopqrs	82.11 ± 6.44abcdef	0.07 ± 0.00ab
13M2	1.73 ± 0.01abcdefghijk	206.6 ± 1.41defghi	82.5 ± 1.35efgh	110.88 ± 10.68pqrstuvw	0.04 ± 0.00cdefghij
13M5	1.48 ± 0.02abcde	434.26 ± 40.54abcde	166.92 ± 5.98abcdef	91.0 ± 0.15bcdefghijkl	0.04 ± 0.00efghijkl
14P2	1.52 ± 0.23abcdefg	909.26 ± 8.01ab	14.61 ± 8.70a	85.44 ± 8.95abcdefghi	0.05 ± 0.00jklmnop
14P3	1.74 ± 0.19abcdefghijk	412.93 ± 21.68pqrst	114.61 ± 5.43klmnopqr	111.55 ± 1.25qrstuvw	0.04 ± 0.00bcdefghi
14P4	1.40 ± 0.04abc	126.26 ± 27.34abcde	136.73 ± 8.97abcd	91.55 ± 6.59cdefghijklm	0.04 ± 0.00ijklmno
15G	1.91 ± 0.25cdefghijkl	191.6 ± 30.16cdefgh	116.92 ± 4.89lmnopqrst	94.0 ± 0.94defghijklmn	0.04 ± 0.00ghijklm
15M1	2.74 ± 1.08no	800.26 ± 4.71abc	178.26 ± 0.81abcd	112.44 ± 18.54stuvw	0.03 ± 0.00abc
15M2	2.85 ± 1.07no	88.6 ± 4.24ab	260.0 ± 0a	105.77 ± 11.94lmnopqrstuv	0.05 ± 0.00jklmnopq
15M3	2.71 ± 0.69mno	103.26 ± 38.18abc	131.73 ± 1.35abcdef	109.88 ± 11.78opqrstuvw	0.05 ± 0.00jklmnop
15M4	2.17 ± 0.06efghijklmn	722.26 ± 7.54abc	33.84 ± 4.35ab	96.22 ± 5.65efghijklmnop	0.04 ± 0.00bcdefgh
16G	3.25 ± 2.07o	892.26 ± 19.79abc	132.69 ± 7.61abcdef	106.33 ± 20.89mnopqrstuv	0.03 ± 0.00bcdef
16M	2.25 ± 0.03hijklmn	435.26 ± 6.12abcd	238.07 ± 2.17a	93.77 ± 15.08defghijklmn	0.04 ± 0.00fghijklm
16M1	2.21 ± 0.52fghijklmn	239.93 ± 31.58ghijk	119.23 ± 1.08nopqrst	107.0 ± 3.61nopqrstuv	0.03 ± 0.00abc
16M2	2.31 ± 0.00ijklmn	51.93 ± 52.32a	69.23 ± 6.52def	83.33 ± 2.82abcdefg	0.03 ± 0.00abcd
16M3	2.35 ± 0.48jklmn	131.26 ± 15.55abcdef	104.23 ± 0ijklmnopq	91.22 ± 12.09bcdefghijkl	0.04 ± 0.00efghijkl
17G	1.94 ± 0.49defghijkl	368.26 ± 23.57mnopqrs	85.38 ± 1.08efghij	96.77 ± 19.09fghijklmnopq	0.04 ± 0.00ghijklmn
17G1	1.82 ± 0.22abcdefghijk	686.26 ± 12.25abc	172.11 ± 0.81abcde	84.0 ± 0.62abcdefgh	0.05 ± 0.00jklmnop
17G2	1.98 ± 0.05cdefghijkl	280.26 ± 31.11hijklm	167.88 ± 0.27abcdef	109.88 ± 4.55opqrstuvw	0.03 ± 0.00abcde
17G4	2.62 ± 0.26lmno	855.26 ± 173.94abc	118.65 ± 1.35mnopqrst	94.55 ± 1.72defghijklmn	0.04 ± 0.00hijklmn
17M	2.23 ± 0.03ghijklmn	306.6 ± 2.37jklmno	102.5 ± 6.79hijklmnop	102.33 ± 2.98klmnopqrstuv	0.03 ± 0.00bcdefg
17M2	1.85 ± 0.11abcdefghijk	280.26 ± 16.72hijklm	94.42 ± 5.71ghijkl	116.22 ± 4.39vw	0.03 ± 0.00abc
18G	1.53 ± 0.67abcdefgh	223.93 ± 12.72fghij	134.42 ± 0.81abcdef	81.33 ± 2.82abcde	0.06 ± 0.00abcdefgh
18M	1.84 ± 0.15abcdefghijk	1023.93 ± 9.89ab	141.15 ± 0abcde	112.77 ± 3.61tuvw	0.03 ± 0.00abc
18M2	1.60 ± 0.6abcdefghi	116.93 ± 6.59abcd	113.07 ± 2.17klmnopqr	96.66 ± 15.08fghijklmnopq	0.04 ± 0.00defghijk
Control without isolate	1.4 ± 0.09abc	453.93 ± 8.01abcd	84.42 ± 5.71efghi	91.11 ± 0.94bcdefghijkl	0.06 ± 0.00abcdefg

TAC = total antioxidant capacity; FRAP = ferrous reducing antioxidant power; MDA = malondialdehyde; Values with different superscript letters on the same column are significantly different at p ˂ 0.05.

3.8. Principal Component Analysis

Principal component analysis (PCA) was used to explore associations between 73 Rhizobium sp. isolates, abiotic stress factors, protein content, and physicochemical activities. Figure 2 illustrates the distribution of variables along the F1 × F2 axis, revealing four distinct groups with shared phenotypic and biochemical traits. Group 1 comprised 24 isolates, characterized by catalase activity and growth under conditions of moderate salinity (2% NaCl) and mesophilic temperatures (20-35˚C). Although catalase activity indicates a potential response to oxidative stress, the observed lack of tolerance to more extreme conditions, such as elevated salinity or temperature, suggests a limited overall adaptability to abiotic stress. These isolates can thrive in moderately stressed environments, but may be less competitive in more difficult soils. Group 2, located opposite group 1 on the PCA graph, included 15 isolates able to grow under high acid (pH 2 - 6.5) and salinity (up to 10% NaCl) conditions. This combination of acid and salt tolerance indicates robust adaptation mechanisms, probably involving efficient osmotic and pH homeostasis. These isolates are promising candidates for the development of bioinoculants in degraded acidic and saline soils, such as those found in various agroecological zones of Cameroon and sub-Saharan Africa. Group 3 consisted of 10 psychrotolerant isolates, able to grow at 15˚C and under stress conditions such

Figure 2. Principal component analysis showing the distribution of the 73 Rhizobium sp. isolates, the resistance of these isolates to abiotic parameters (pH, salinity, temperature), and their physicochemical activities on the F1 × F2 axis system.

as low pH (pH 3) and high salinity (12% NaCl). They also displayed high protein production and high total antioxidant activity. The clustering of these features suggests a potential synergistic response to abiotic stress, where protein biosynthesis may contribute to boosting antioxidant defenses. This group could harbor cold-adapted, stress-responsive enzymes or protective proteins, which would be useful for crops grown at high altitudes or in temperate tropical regions. Group 4 was defined by the presence of malondialdehyde (MDA), a marker of lipid peroxidation, and comprised 24 isolates. The association of these isolates with high levels of MDA suggests their involvement in oxidative stress responses, potentially linked to lipid membrane remodeling or reactive oxygen species scavenging. These isolates could enhance stress-induced cellular damage in host plants.

4. Discussion

Cameroonian soils are known for their acidic nature, which hampers the cultivation and productivity of several crops, including soybeans [9]. Amongst strategies for that issue, Rhizobium sp. appears as a good alternative. The microorganisms that can tolerate hard environmental conditions act in symbiosis with plants and help these latter to resist changes in abiotic parameters and the associated stress. In this context, the present study was designed to isolate and evaluate bacterial strains potentially belonging to the Rhizobium genus, capable of withstanding various abiotic stresses and endowed with physico-chemical activities favorable for plant growth under unfavorable conditions. The bacterial isolates were obtained from soybean root nodules. A total of 73 presumptive Rhizobium sp. isolates were obtained from nodules of 18 varieties of soybean farmed in acidic soil in Cameroon.

Acidity was the first abiotic parameter for which the tolerance of isolates was screened. Indeed, acidity affects nodulation during symbiosis through the excretion of isoflavonoids from soybean roots [34]. It also alters cell membrane permeability and damages roots, leading to reduced water and nutrient uptake, and jeopardizing the survival of soil micro-organisms [35]. According to Ferguson et al. [36], soil acidity alone is responsible for significant losses in the global production of legumes. Hence, it appears interesting to identify Rhizobium sp., which can resist to acidic conditions. In this study, the growth abilities of Rhizobium sp. isolates showed significant resilience across the pH range from 2 to 4. Some isolates showed a good ability to grow under a pH lower than 4. The most interesting acid-tolerant isolates were 14P3, 2, 17G2, 16G, 15M3, and 7M. This resistance to low pH can be explained by the ability of these isolates to produce extracellular polysaccharides or organic compounds in the cells. A similar observation was noticed in Argentina by Muglia et al. [37]. The results of this study suggest the capacity of isolates 14P3, 2, 17G2, 16G, 15M3, and 7M to establish a symbiotic relationship with soybean even in an acidic environment, and boost their productivity. It is in direct line with findings of Nkot et al. [38], who demonstrated that acid-tolerant strains of rhizobia can successfully establish under acid environmental conditions, symbiosis with the legume Vigna unguiculata, and improve its nitrogen uptake and productivity.

Besides acidity, temperature was an important abiotic parameter assessed in this study. The rhizobia isolates displayed various growth rates at the different temperatures tested. This can be related to the fact that rhizobia are ubiquitous bacteria that thrive in subarctic, temperate, and tropical regions and therefore respond to different environmental conditions [39]. However, most of the isolates showed optimum growth at temperatures between 25˚C and 30˚C. 52% of isolates were unable to grow at 15˚C, and 62% at 40˚C and more. A few proportions of isolates displayed optimal growth at low temperatures, with 1M and 4 M1 showing the optimal growth at 15˚C and 20˚C, respectively. This result shows that they can be suitable for soils of temperate regions. However, the negative effects of low temperature such a reduction of rhizobia competitiveness, a reduction of the synthesis and secretion of the Nod factor, inhibition of the symbiotic process, and restriction of nodule development [40]. The limited growth of isolates at 40˚C and above may be explained by an alteration in key processes of rhizobia symbiosis, including bacterial adhesion, nodule initiation, and nitrogenase activity. Recent studies show that high temperatures disrupt these steps, reducing the efficiency of nitrogen fixation [41]. High temperature depletes photosynthetic activity, decreases absorbing hair formation, reduces nodulation sites, and alters bacterial adhesion to absorbing hairs [42] [43]. However, there are some isolates (15M3, 10G1, 9P, 5P, 5P’, 2, and 1G) for which optimum growth was at 45˚C. The growth at high temperatures recorded with these isolates can arise from their ability to synthesize heat shock proteins [44]. It can also be explained by the origin of the isolates. Rhizobia isolated from subtropical regions like the sampling site of this study are known to be resistant to high temperatures [45]. According to Zhang et al. [46], isolates that are tolerant to stress, particularly high temperatures, can maintain efficient nitrogen fixation under unfavorable abiotic conditions. That resilience also promotes a constant supply of nitrogen by rhizobia, thus compensating for the low natural fertility of these soils [47]. Hence, there is a need for further investigations on the effect of inoculating these thermotolerant isolates (15M3, 10G1, 9P, 5P, 5P', 2, and 1G) on soybean seeds.

The last abiotic parameter assessed in this study was salinity. Salt stress impairs crop growth and yield by reducing photosynthetic activity through reduced gas exchange, altering morphological development, disrupting membrane functions, and affecting antioxidant activities [48]. Hence, identifying rhizobia endowed with resistance to salinity can be beneficial for protecting leguminous crops through the regulation of oxidative stress, balancing of phytohormone levels, and secretion of osmolytes [49] [50]. In this study, the isolates showed variable growth in the presence of different NaCl concentrations. This variation could be attributed to the difference in metabolic activities leading to the production of compounds associated with salinity resistance as reported by Mhadhbi et al. [51] and Del Cerro et al. [52]. That property is very important for plant-rhizobia symbiosis. In fact, during salinity stress, the plant can survive if the rhizobia with which they are in symbiosis persist in soils and keep their ability to colonize, infect, produce nodulation factors, and fix nitrogen. At the high NaCl concentration of 12%, the optimum growth was recorded with isolate 10G2. The tolerance of isolate 10G2 to high NaCl concentration can be explained by its ability to produce and accumulate osmoprotectants, such as proline, thus helping to limit the deleterious effects of osmotic stress [50] [53]. In addition, some Rhizobium sp. strains have demonstrated their ability to mitigate the ionic toxicity of NaCl through the production of exopolysaccharides or the regulation of ionic homeostasis. This dual action can promote the establishment and functioning of the soybean-Rhizobium symbiosis under salinity conditions, by promoting the initiation of nodulation and sustaining nitrogen fixation activity. The observed resistance of isolate 10G2 to high NaCl concentrations can be helpful for the soybean-Rhizobium symbiosis. It helps the plant during nodulation initiation and improves its nitrogen-fixing capacity under high salinity stress. Khan et al. [54] showed that inoculation of soybeans with halotolerant isolates improved the growth, biomass, and chlorophyll content of seeds farmed on a salty soil. These strains promote ionic homeostasis by increasing K⁺ uptake and limiting Na⁺ accumulation, while stimulating antioxidant activity. They also induce the expression of genes associated with salt tolerance, such as GmST1 and GmLAX3, contributing to improved physiological resilience. Furthermore, Shahid et al. [55] showed that some halotolerant strains contribute to the resilience of legumes by producing phytohormones that promote growth and nodulation, thus improving the ecological integration of plants in constrained environments while maintaining nitrogen fixation.

The production of proteinaceous compounds is amongst the resistance mechanisms of rhizobia to abiotic stress conditions. According to Boominathan and Doran [56], under edaphic stress, enzymes are secreted in rhizobia for their protection. In this study, the isolates have demonstrated the ability to grow and survive under extreme conditions of pH, temperature, and salinity.

The protein content of isolates was assessed and revealed significant variability between strains. Such variations are commonly observed among microbial isolates and may reflect differences in metabolic activity, stress tolerance mechanisms, or adaptation to environmental conditions. Up-regulation of genes encoding for proteins and lipopolysaccharides (LPS) in Rhizobium sp. exposed to a temperature of 43˚C was reported by Nandal et al. [57]. Studies by Gomes et al. [58] in Brazil showed that Rhizobium tropici strain PRF81, when exposed to heat stress, increased the synthesis of proteins such as bacterioferritin and thioredoxin, associated with oxidative stress responses. The highest protein values, recorded in this study with isolate 10G2 (135.33 ± 5.65 µg/mL), followed by 3M''' (122.22 ± 13.19 µg/mL) and 5G (102.33 ± 19.01 µg/mL), suggest that these isolates might be suitable for application in soybean production under stress conditions.

The proteins produced in response to stress conditions generally have different roles. Some are involved in the constitution of cell structure, while others directly act on the compounds generated by stress conditions. The main compounds generated by stress conditions are free radicals and reactive oxygen species (ROS) that affect cell function, leading to oxidative damage and ultimate plant death [59]. One of the proteins involved in the antioxidant mechanism is catalase. That enzyme plays an essential role in trapping ROS and thus preventing oxidative damage [60]. The catalase activity of the different isolates was assessed in this study. All the isolates were positive with activity ranging from 1033.93 ± 33.46 to 1335.93 ± 10.84 µg AAE/mL. The variation in catalase activity can be explained by the difference in the metabolic mechanism of synthesis of that enzyme, which occurs in the peroxisomes. Indeed, the synthesis of catalase in the peroxisome is induced by the substrate, and varies according to the microorganism, the type of cell, and the environmental conditions [61]. The catalase activity recorded in this study with rhizobia isolates suggests that they can be used in soybean production to maintain the plant’s pro-oxidant-antioxidant balance and ensure good functioning of the symbiotic relationship under stressful conditions. Kots et al. [14] highlighted that catalase activity is linked to the symbiotic nitrogen uptake activity in legume root nodules and depends on the efficiency of the Rhizobium-legume symbiosis.

Besides catalase activity, other antioxidant mechanisms of the rhizobia isolates were assessed through the ferric reducing power and total antioxidant capacity. The results obtained showed that all isolates were positive for TAC and FRAP activities, with values that vary significantly from one isolate to another. The highest TAC values were obtained with 4M1 (1335.93 ± 10.84 µg AAE/mL), 6G2' (1307.6 ± 9.42 µg AAE/mL), 10G2 (1194.6 ± 2.35 µg AAE/mL), 10G (1051.0 ± 9.33 µg AAE/mL), 7M, and 5M (1033.93 ± 33.46 µg AAE/mL). A similar trend was observed with the FRAP activity of the isolates. This means that the isolates can protect plants against stress due to an imbalance in favor of free radicals and pro-oxidants. Hence, they might be suitable for Cameroonian soils, for which soybean plants are exposed to several abiotic stress factors.

Lipids play an important role in signal transduction in plant response to abiotic stress [62]. When plants are stressed, the free radicals and ROS generated can affect membrane properties by oxidizing membrane lipids. In response to lipid oxidation, plants increase their content of some compounds, such as MDA. MDA represents the main marker used to assess lipid oxidation [63]. The accumulation of MDA in cells after exposure to stress was highlighted by Thounaojim et al. [64]. In the present study, the MDA levels of the isolates were measured. Isolates 16G (3.25 ± 2.07 µmoL/L), 15M1 (2.74 ± 1.08 µmoL/L), 15M2 (2.85 ± 1.07 µmoL/L) and 15M3 (2.71 ± 0.69 µmoL/L) recorded the highest MDA levels. These rhizobia isolates may exhibit a relatively low antioxidant activity as suggested by their high levels of MDA, which are indicative of oxidative stress and reduced antioxidant capacity [65]. While the other isolates scored significantly reduced MDA contents. The significant variation in the MDA contents can be explained by the variability in the production of antioxidant metabolites by the isolates. That property is very important for soybeans as rhizobia isolates can regulate membrane lipid peroxidation and thus, protect plants from severe oxidative damage [66].

Considering the overall growth and biological activities, Santos et al. [67] in their studies in Brazil noticed that under a high salinity environment, halotolerant strains of Bradyrhizobium stimulate Vigna unguiculata resistance to stress through the production of antioxidants, including catalase. Manassila et al. [68] highlighted that the acid tolerance ability of Bradyrhizobium sp. DASA01007, isolated from acidic soils in Thailand, might promote an efficient symbiosis in acidic environments. In the present study, we identified rhizobia strains endowed with acid tolerance, salt tolerance, high synthesis of proteins, high ability to inhibit lipid oxidation, and noticeable antioxidant activities. Thus, suggesting their unique ability and potential to promote through symbiosis, the growth of legumes, particularly soybean, under stress conditions.

The results obtained in this study open up prospects for identifying the most promising isolates and assessing their effects on agronomic performance parameters of soybean. By identifying the most efficient strains for the production of commercial inoculants, we will not only improve the efficiency of fermentation processes but also select strains better suited to various biotechnological applications, such as biomass and biofertilizer production. In agronomic terms, this research contributes to enhancing soil fertility by providing crucial information on which strains are most effective under specific conditions. This in-depth understanding can guide the choice of the best options for crops while supporting sustainable agricultural practices. By promoting the use of adapted strains, we could also reduce dependence on chemical fertilizers, thus promoting more environmentally friendly agriculture.

5. Conclusion

In this study, 73 presumptive Rhizobium sp. isolates were obtained from 18 varieties of soybeans. The majority of these isolates demonstrated the ability to grow and survive in extreme conditions of acidity, salinity, and temperature. The adaptation was adapted differently from one isolate to another according to the various environmental stresses encountered. The isolates displayed antioxidant activities through different mechanisms, thanks to the ability to produce proteins endowed with antioxidant activity, like catalase. They also demonstrated the ability to inhibit lipid peroxidation through the inhibition of MDA production. The combination of the antioxidant potential of Rhizobium sp. isolates and resistance to abiotic factors suggests that they might be suitable as inocula for large-scale production of soybeans in acidic soils. This study enabled morphological and physiological characterization of Rhizobium sp. isolates. For that, there is a need for further studies on the exploration of their taxonomic diversity using molecular tools. The evaluation of Rhizobium strains is of crucial importance for their adaptation to specific environments. By examining the behavior of these strains under a variety of conditions (such as soil types and climates), we can fine-tune inoculation strategies to suit local realities. Furthermore, selecting strains capable of withstanding extreme conditions, such as drought and salinity, can significantly boost crop productivity in times of environmental stress. Finally, exploring the interactions between different Rhizobium strains and other microorganisms opens the way to strengthening synergies, thereby improving the efficiency of cultivation practices. This integrative approach could therefore play a key role in optimizing cropping systems and agricultural sustainability.

Data Availability

Data available on request from the authors.

Authors’ Contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by LM, HM, EL, OB, FL, GM, and AD. The first draft of the manuscript was written by LM and all authors read and approved the final manuscript.

Acknowledgements

The authors thank the Director General of the Institute of Agricultural Research for Development (IRAD) for permission to conduct this research at the experimental plots of IRAD and for providing facilities for the completion of this work. They also recognize the cooperation of Mr. Akem Michael Ndingwan involved in monitoring on-site trials and the provision of soybean varieties in the study.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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