ojss Open Journal of Soil Science 2162-5360 2162-5379 Scientific Research Publishing 10.4236/ojss.2025.156018 ojss-143507 Articles Earth Environmental Sciences 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 Tene 1 Eddy Léonard Ngonkeu Mangaptche 2 3 Onana Boyomo 4 Ferdinand Lanvin Edoun Ebouel 1 5 Gabriel Medoua Nama 1 Aimé Didier Begoudé Boyogueno 3 aCentre for Food, Food Security and Nutrition Research, Institute of Medical Research and Medicinal Plant Studies (IMPM), Yaoundé, Cameroon aDepartment of Plant Biology, University of Yaoundé I, Yaoundé, Cameroon aInstitute of Agricultural Research for Development (IRAD), Yaoundé, Cameroon aDepartment of Microbiology, University of Yaoundé I, Yaoundé, Cameroon aDepartment of Biochemistry, University of Yaoundé I, Yaoundé, Cameroon 05 06 2025 15 06 408 444 25, April 2025 21, April 2025 21, June 2025 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ 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. 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.

<xref ref-type="bibr" rid="scirp.143507-"></xref>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

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.

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

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.

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.

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

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.

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.

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.

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.

<xref ref-type="bibr" rid="scirp.143507-"></xref>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.

<xref ref-type="bibr" rid="scirp.143507-"></xref>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

<xref ref-type="bibr" rid="scirp.143507-"></xref>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.

<xref ref-type="bibr" rid="scirp.143507-"></xref>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.

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