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Article

Unlocking the Potential of Inoculation with Bradyrhizobium for Enhanced Growth and Symbiotic Responses in Soybean Varieties under Controlled Conditions

by
Haimanot Beruk
1,2,*,
Tarekegn Yoseph
2 and
Tewodros Ayalew
2
1
Hawassa Maize Research, Wondo Genet Agricultural Research Center, EIAR, Hawassa P.O. Box 1793, Ethiopia
2
College of Agriculture, Hawassa University, Hawassa P.O. Box 05, Ethiopia
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1280; https://doi.org/10.3390/agronomy14061280
Submission received: 27 April 2024 / Revised: 25 May 2024 / Accepted: 27 May 2024 / Published: 13 June 2024
(This article belongs to the Special Issue Advanced Technology for Climate Change Mitigation and Sustainable Management of the Agroecosystem)
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Abstract

:
Soybean is a crucial crop for sustainable agriculture development as it forms symbiotic relationships with rhizobia species. The effectiveness of inoculants in symbiosis, however, relies on the compatibility of the strain with a specific legume crop variety. This study assessed the symbiotic efficiency of eight Bradyrhizobium strains (SB-36, SB-37, SD-47, SD-50, SD-51, SD-53, SB-113, and SB-120) with five soybean varieties (Gishama, Awassa-95, Boshe, Hawassa-04, and Jalale) using sand culture. The experiment was arranged in a factorial, completely randomized design with three replicates. Data were collected on plant growth, and symbiotic effectiveness indices and subjected to statistical analysis using R software v4.3.1. The results revealed marked differences (p < 0.001) between the varieties, rhizobial strains, and their combined effects on all traits examined. The Jalale variety inoculated with Bradyrhizobium strains SB-113 and SD-53 produced the highest nodules per plant. When inoculated with SD-53, Awassa-95 demonstrated the highest relative symbiotic effectiveness [129.68%], closely followed by the Boshe variety [128.44%] when inoculated with the same strain. All strains exhibited high relative symbiotic effectiveness (>80%) with Awassa-95 and Boshe varieties. The highest absolute symbiotic effectiveness was observed in the Gishama variety inoculated with the SD-53 strain followed by Boshe and Awassa-95 varieties inoculated with this same strain. Notably, strain SD-53 demonstrated remarkable efficiency with the varieties Gishama, Boshe, and Awassa-95 based on both relative and absolute symbiotic effectiveness indices. Varieties inoculated with the SD-53 strain produced deeper green leaves. This study revealed the importance of Bradyrhizobium inoculation to improve soybean performance, for which the SD-53 strain performed best among the strains considered in the current experiment. Therefore, it is plausible to recommend inoculating soybeans with Bradyrhizobium strain SD-53 with prior field evaluation.

1. Introduction

Soybean (Glycine max L. Merr.) is an annual leguminous crop grown in Africa since 1896 [1], and it was first introduced to Ethiopia in the 1950s [2]. It is an important crop that provides essential nutrients for food and feed while also providing fiber and biodiesel [3]. Additionally, it is utilized as green manure to enhance soil fertility [4] owing to its ability to symbiotically fix atmospheric nitrogen (N) with various species of Bradyrhizobium, slow-growing rhizobia, and Sinorhizobium, fast-growing rhizobia [5]. Bradyrhizobia species are essential members of the soil microbiota, contributing immensely to soil fertility, plant growth, and nutrition, particularly evident in organic farming practices where they help reduce reliance on synthetic fertilizers and agrochemicals [6]. Establishing Bradyrhizobia infection and subsequent nodule development on leguminous plant roots fosters a mutualistic partnership that improves the plants’ capacity to absorb water and nutrients from the soil. This symbiosis is crucial for enhancing sustainable agricultural practices, making the Bradyrhizobial inoculation of legumes, including soybeans with effective bio-inoculants, a key factor [6,7].
The efficiency of the symbiosis differs with the type of rhizobia species, soybean cultivar, and biotic factors [8], suggesting that the contribution of inoculation also varies among varieties inoculated with the same strain. For instance, Agoyi et al. [9] tested 65 genotypes for their response to inoculation and reported significant differences in the number of nodules, effective nodules, and fresh and dry nodule weight among genotypes. Inoculation of soybean with Rhizobia significantly improved nodule number [10] and nodule dry weight [11]. Moreover, Argaw [12] reported that the response of soybean genotypes to inoculation with the same strain significantly affected nodule number and nodule dry weight under both field and greenhouse conditions. Egamberdiyeva et al. [13] observed significant positive effects of inoculation with Bradyrhizobium on growth and nodule number of soybeans. Szpunar-Krok et al. [14] reported an increase in the nodule number and dry weight, respectively, due to Bradyrhizobia inoculation of soybean cultivars.
Bradyrhizobium inoculation has been shown to promote nodulation, plant fresh weight, dry matter production and plant height of soybean varieties [15]. Miljaković et al. [16] reported an increase in plant height, shoot dry weight, nodule number and nodule dry weight, respectively, due to Bradyrhizobia inoculation of soybean. Inoculation of soybean with Rhizobia also significantly improved root dry weight [17]. An increase in root dry weight due to inoculation was reported by Miljaković et al. [16] and Desta et al. [18].
The rising expenses associated with fertilizers [19] and their detrimental effects on the environment have compelled the industry to explore alternative sources of plant nutrients [20]. Consequently, biological nitrogen fixation (BNF), a process in which atmospheric nitrogen is converted into organic forms by symbiotic and symbiotic microorganisms in the soil [21], has garnered significant attention. Symbiotic nitrogen fixation proves most beneficial for leguminous crops [22]. However, a specific relationship exists between the rhizobial strain and the plant varieties, necessitating compatibility for successful nodulation and nitrogen contribution [23]. Hence, this study aimed to assess the symbiotic efficacy of Bradyrhizobium strains on soybean varieties to determine the most effective combination for soybean cultivation in the field.

2. Materials and Methods

2.1. Sources of the Experimental Materials

Five soybean varieties were chosen for this lath house experiment, one early-maturing and four medium-maturing (Table 1). The Pawe Agricultural Research Center in Ethiopia provided the varieties, and a subsequent evaluation trial determined that they were superior under Boricha and Hawassa field conditions. On the other hand, eight Bradyrhizobia strains were used as inoculants (Table 2). These strains were obtained from the microbiology laboratory at Hawassa University isolated from Dore Bafano and Boricha districts using soybean varieties [23], and their genetic diversity and population composition were studied [24].

2.2. Bacterial Growth and Growth Medium

The yeast extract mannitol agar (YEMA) growth medium was prepared by combining 10 g of mannitol, 0.5 g of K2HPO4, 0.2 g of MgSO4.7H2O, 0.1 g of NaCl, and 0.5 g of yeast extract in 1 L of distilled water. To this mixture, 0.025 mL of Congo Red (CR) dye was added. The pH of the solution was adjusted to 6.8 using a 10% (0.1 N) solution of NaOH or HCl, with the help of a microprocessor pH meter. The medium was then sterilized by autoclaving at 121 °C and 15 lb/in2 for 15 min. After cooling to 55 °C, it was poured into sterile Petri dishes and solidified in a laminar hood flow.
A loopful of Bradyrhizobium strain suspension from the canned vials was streaked on the surface of the Petri dish containing YEMA and incubated at 28 ± 2 °C for 5–7 days [25]. The forming colonies were streaked several times on YEMA plates before being isolated as a pure strain. A loop containing a pure Bradyrhizobium strain was then inoculated into YEM broth and incubated. The YEM broth was formulated as YEMA except for agar and Congo red dye. The incubated strain was used for inoculation, while the remaining portion was kept in 50% glycerol.

2.3. Authentication Test and Seedling Management

Modified Leonard containers were prepared by combining two plastic cups to grow soybean seedlings to authenticate selected rhizobacterial strains. To support the seedlings, the upper containers (cup volume was 334.52 cm3) were filled with pre-treated river sand that had been washed in a quarter strength of 98% H2SO4 acid solution, and its pH had increased to nearly 7.0 after being washed with tap water. The bottom sets of containers were connected to the cups above them using cotton. The cotton absorbs water or nutrient solution from the bottom containers and transports it upward through capillary action to the upper containers. Soybean seed surfaces were sterilized with 70% ethanol for 10 s, followed by treatment with sodium hypochlorite for 3 min, and washed thoroughly six or more times with sterilized distilled water (Somasegaran and Hoben 1994 [25]). Sterile soybean seeds were used to start the growth process on aseptic tissue paper placed in sterilized Petri dishes. After pre-germinating, the soybean seedlings were sterilely transferred into the jars, ensuring that each jar contained only one seedling. The experimental treatments were set up in a factorial completely randomized design with three replications, leading to 50 treatment combinations (5 varieties × 10 (8 Bradyrhizobial strains inoculation and the negative and positive controls)) and 150 pots.
Using a micropipette, 1 mL of rhizobia strains from pure broth cultures in their logarithmic growth phases were inoculated into each seedling. The jars were then randomly arranged and allowed the seedlings to grow in the lath house at Hawassa University College of Agriculture. The seedlings were irrigated weekly at a rate of 100 mL per pot with sterile quarter-strength Jenson’s modified N-free nutrient solution and sterilized distilled water, as needed. The N-free solution was prepared using 1 g CaHPO4, 0.2 g KH2PO4, 0.2 g MgSO4.7H2O, 0.2 g NaCl, 0.1 g FeCl3, and 1 mL of other less-needed elements (2.86 g H3BO3, 2.03 g MnSO4.4H2O, 0.22 g ZnSO4.7H2O, 0.08 g CuSO4.5H2O, 0.14 g Na2MoO4.2H2O) to make up a stock solution in 1 L of distilled water. Each treatment included positive and negative controls. The positive control involved watering un-inoculated seedlings with a potassium nitrate solution (0.5 g KNO3 per 1 L of sterilized distilled water), while the negative control consisted of an N-free nutrient solution.

2.4. Data Collection

The Bradyrhizobium strains were assessed initially for their effectiveness by qualitatively observing the seedlings’ leaf color and vegetative growth 45 days after planting. The leaves were sorted into categories based on their color: dark green, green, pale green, and yellow. After that, the young plants were removed from the sand and divided into shoots (consisting of leaves and stems), roots, and nodules. The nodules were carefully separated from the roots and placed on a sieve to ensure thorough cleaning and removal of excess water. After that, the nodules were counted and the number per plant was recorded. Next, the nodules and shoots were oven-dried at 70 °C for 48 h to measure their dry weight. Plant height was measured from plants collected for nodulation and shoot biomass assessments.

2.5. Determination of Symbiotic Effectiveness Indices of Bradyrhizobia Strains

Relative symbiotic effectiveness (RSE%) was calculated by comparing the inoculated plant to the plant that received N-fertilizer (positive control), following the procedures outlined by Purcino et al. [26].
% R S E = I n o c u l a t e d   s h o o t   d r y   m a t t e r   w e i g h t N   f e r t i l i z e d   s h o o t   d r y   m a t t e r   w e i g h t × 100
Nitrogen-fixing efficiency is classified as highly effective, effective, poorly effective, and ineffective with relative symbiotic effectiveness values of >80%, 50–80%, 35–50%, and < 35%, respectively [26].
Absolute Symbiotic effectiveness percentage (ASE%) was estimated by contrasting the inoculated plant to the negative control, which was not inoculated or fertilized, using the technique described by Dos et al. [27].
A S E % = I n o c u l a t e d   s h o o t   d r y   m a t t e r   w e i g h t − t h e   w e i g h t   o f   S h o o t   d r y   m a t t e r   w i t h o u t   N W e i g h t   o f   S h o o t   d r y   m a t t e r   w i t h o u t   N × 100

2.6. Statistical Data Analysis

Before performing an analysis of variance, the assumptions were verified, and nodule number and dry weight were adjusted using the square root transformation, while shoot and root dry weight were adjusted using the appropriate Box and Cox [28] variance stabilizing transformation. Then, the data were analyzed using R software v4.3.1 [29]. If significant treatment effects were found, the least significant difference (LSD) was used to compare differences between treatment means at a 5% significance level. The relationships between the parameters were examined by analyzing Pearson’s correlation using R software.

3. Results

3.1. Growth, Nodulation, and Leaf Color Response of Soybean Varieties to Inoculation

3.1.1. Plant Height

Inoculation, variety, and their interaction had a highly significant (p < 0.001) effect on plant height at 6 weeks after planting (Table 3). The tallest plant was recorded for the soybean variety Jalale under the positive control, followed by the Hawassa-04 variety inoculated with SB-36, SD-51, and SD-50, while the shortest plant height was observed for the Boshe variety inoculated with SB-36 (Table 4). Plant height correlated positively with both relative and absolute symbiotic effectiveness (Figure 1).

3.1.2. Nodule Number and Dry Weight Plant−1

Table 3 shows significant differences (p < 0.001) in nodule number and dry weight across varieties, Bradyrhizobium strains, and their interaction. Nodules were consistently observed on the roots of all inoculated varieties; however, the efficacy of nodulation varied depending on the specific Bradyrhizobium strain and soybean variety used. Among the tested varieties, Jalale displayed the highest values for these parameters when inoculated with strains SB-113 and SD-53, followed by Awassa-95 inoculated with strain SD-53 (Table 4). The number of nodules exhibited a positive and significant correlation with nodule dry weight, shoot dry weight, and absolute symbiotic effectiveness. While nodule dry weight showed a positive and significant correlation with shoot dry weight, relative symbiotic effectiveness, and absolute symbiotic effectiveness.

3.1.3. Shoot and Root Dry Weight

The dry weight of soybean shoots and roots varied notably between varieties, Bradyrhizobium strains, and their combined effects (Table 3). The Jalale variety had the highest shoot dry weight (2.08 g plant−1) in the positive control treatment, followed by Awassa-95 inoculated with SD-53 and SB-113. However, the lowest (0.82 g plant−1) was found in Hawassa-04 inoculated with SB-37 or SB-37 plus a negative control (Table 4). In the varieties Jalale and Gishama, the mean shoot dry weight was higher in the positive control (N-treated plants) than in the rhizobia-inoculated varieties. The dry weight of the soybeans’ roots showed similar patterns to the shoot biomass for the significance of the interaction effect, with the highest value observed in Awassa-95 inoculated with SB-37 and the lowest value in the Gishama variety inoculated with the SD-53 strain (Table 4). Shoot dry weight positively and significantly correlated with nodule number and dry weight, root dry weight, and relative and absolute symbiotic effectiveness (Figure 1).

3.1.4. Leaf Color

The color of leaves can be a valuable indicator for evaluating the efficacy of rhizobial inoculants in legumes. Upon comparing the plant’s phenotype after being inoculated with different strains of Bradyrhizobium and the negative control, it became apparent that certain strains were successful (displaying deep and light green leaves), while the negative control proved to be ineffective (resulting in yellow and light green leaves) in establishing a symbiotic relationship with soybean varieties (Figure 2). Inoculation of soybean varieties with the SD-53 strain showed more deep green leaves over other inoculant strains and also form positive control (Figure 2).

3.2. Symbiotic Effectiveness of Bradyrhizobia Strains

The two indices used in this study to determine the symbiotic effectiveness are relative symbiotic effectiveness (RSE) and absolute symbiotic effectiveness (ASE).

3.2.1. Relative Symbiotic Effectiveness

The evaluation of RSE was conducted by comparing the dry shoot biomass of inoculated plants to that of nitrogen-fertilized control plants. The calculated RSE values varied from 55.77% (in the case of the Jalale variety inoculated with strains SD-51 and SB-37) to 129.68% (for the Awassa-95 variety inoculated with strain SD-53) (Figure 3). Notably, the Awassa-95 and Boshe varieties exhibited higher RSE (>80%) across all strains, underscoring the effectiveness of these strains with these specific varieties. Among the strains, five were classified as highly effective and three as effective (with RSE ranging from 50% to 80%) for the Hawassa-04 variety. For the Gishama variety, all of the strains were effective, while the SD-53 strain exhibited high effectiveness. Conversely, for the Jalale variety, all of the tested strains were categorized as effective.
A comparative analysis was conducted among strains based on their origin or site of isolation, revealing a significant difference in RSE between the strains isolated from Dore Bafano (92.85%) and strains isolated from Boricha (82.99%) (Figure 4). Notably, Figure 4 illustrates higher variability in RSE at both sites, with the median 97.64% for the strains isolated from Dore Bafano and 71.23% for the strains isolated from Boricha.
The four strains isolated from Dore Bafano (SD-47, SD-50, SD-51, and SD-53), one strain (SD-53) demonstrated high effectiveness across all varieties except for Jalale (where it was effective) (Figure 4). A weak positive correlation observed between the RSE and both nodule number (r = 0.14) and nodule dry weight (r = 0.22).

3.2.2. Absolute Symbiotic Effectiveness

The absolute symbiotic effectiveness (ASE) percentages were determined by comparing the performance of inoculated plants with a negative control comprising un-inoculated and unfertilized plants. Results varied from 0% for the Jalale variety inoculated with SB-37 and SD-51 to a high ASE of 58.24% for the Gishama variety inoculated with SD-53 (Figure 5). There is a positive and statistically significant relationship between ASE and several other variables, including nodule number, nodule dry weight, shoot dry weight, relative symbiotic effectiveness, and plant height (Figure 1).

4. Discussion

4.1. Growth, Nodulation, and Leaf Color Response of Soybean Varieties to Inoculation

4.1.1. Plant Height

In this study variations were observed among the introduced strains on the growth performance of the soybean crop which explains their effectiveness in supplying nitrogen to the plants, consequently enhancing crop vegetative growth compared to the negative control, irrespective of the soybean variety considered in the experiment. Moreover, the discrepancies among the inoculants in affecting the crop performance illustrate their differences in nitrogen fixing efficiency. The enhanced plant height of the soybean varieties with specific strain, as opposed to the negative control, emphasizes the importance of utilizing effective bio-inoculants for improving crop growth. Horácio et al. [30] reported that rhizobia inoculation significantly raised the height of common bean plants. Similarly, Argaw [12] observed an increase in soybean plant height due to inoculation with Bradyrhizobium strains. Miljaković et al. [16] reported 15 % increase in plant height in soybean crop due to Bradyrhizobia inoculation. Desta et al. [18] also found a 75 to 150% increase in soybean plant height due to inoculation. In contrast, Roriz et al. [31] found that indigenous soybean isolates excelled over introduced strains in promoting plant growth and enhancing iron uptake.
On top of these, a positive correlation coefficient of 0.28 for relative symbiotic effectiveness and 0.25 for absolute symbiotic effectiveness (Figure 1) with plant height suggests a moderate positive relationship between plant height and the inoculants effectiveness indices. These results could imply that as symbiotic effectiveness increases, so does the plant height.

4.1.2. Nodule Number and Dry Weight Plant−1

Our result Showed significant differences (p < 0.001) in nodule number and dry weight across the tested soybean varieties, Bradyrhizobium strains, and their interaction. Other studies by Argaw [12] and Księżak and Bojarszczuk [32] support these findings, showing the significant impact of inoculation, and genotype x inoculation interaction on nodulation performances of soybean genotypes under both field and greenhouse conditions. Similarly, Desta et al. [18] emphasized the significant importance of rhizobial inoculation to enhance the number and dry weight of soybean nodules. Inoculation of Jalale with strains SB-113 and SD-53, and Awassa-95 with strain SD-53 demonstrated the highest nodulation performance (Table 4). This indicates that the efficacy of nodulation varied depending on the specific Bradyrhizobium strain and soybean variety used. This finding aligns with the research performed by Beyan et al. [23], highlighting the diverse responses of different Bradyrhizobium strains and genotypes to nodulation performance. Szpunar-Krok et al. [14] reported an increase by 439 to 800% and 450 to 514% in the nodule number and dry weight, respectively, due to Bradyrhizobia inoculation in soybean cultivars. Furthermore, other researchers have highlighted the beneficial effect of rhizobial inoculation on the number of nodules and dry mass of various leguminous plants [33,34,35]. The tested varieties also displayed different response for the nodule dry weight when inoculated with different strains. The differences in nodule dry weights per plant among inoculated plants can be attributed to variations in nodule quantity and size.
Notably, a strong positive correlation (r = 0.74) was observed between nodule number and nodule dry weight (Figure 1), consistent with previous findings by Agoyi et al. [9], who reported a significantly high correlation (r = 0.72) between these two variables. Rhizobial strains with higher infective capacity and effectiveness produced more and heavier nodules than those with lower effectiveness. This underscores the competitive advantage of highly effective rhizobial strains, enabling successful formation of nodule with soybean roots.

4.1.3. Shoot and Root Dry Weight

Shoot dry weight varies among inoculated varieties, with Awassa-95, Gishama, Jalale, and Boshe having higher values when inoculated with strain SD-53. Inoculating the SD-53 strain to these varieties significantly increased shoot dry weight, with 52%, 58%, 24%, and 56%, for Awassa-95, Gishama, Jalale, and Boshe, respectively, when compared to un-inoculated and unfertilized control plants (Table 4). It was realized that plants that were able to form efficient nodules produced more shoot dry biomass than the negative control group, which did not produce any nodules. According to Argaw [12], inoculating soybean varieties with Bradyrhizobium significantly influenced the buildup of shoot dry weight. Miljaković et al. [16] reported 34% increase in soybean shoot dry weight due to Bradyrhizobia inoculation. Similarly, Abera et al. [36] reported a 24–46% increase in soybean shoot dry weight following Bradyrhizobial inoculation. Furthermore, in a greenhouse study, Temesgen [37] found that soybean plants treated with fast and slow-growing local inoculants had higher shoot dry weights than non-inoculated plants.
Compared to the Jalale and Gishama varieties that were Bradyrhizobia-inoculated, the positive control (plants treated with N) had a greater mean shoot dry weight. This difference could be attributed to the application of KNO3, which was used to boost plant growth and biomass production. Correspondingly, Ahmad et al. [38] found that the application of KNO3 under waterlogged conditions improved maize growth characteristics. Desta et al. [18] also found the highest shoot dry weight from the N-treated plants compared to the rhizobia-inoculated soybean crop.
Positive correlation observed between shoot dry weight and root dry weight, nodule number, and nodule dry weight (Figure 1), indicating strains with higher fitness and nodule dry weight provide a greater benefit to their host plants. According to He et al. [39], there was a positive and significant correlation between N2 fixation and the dry weight of the nodules and the entire plant.
Inoculation increased the root dry weight of the varieties with specificity to the strains. Similarly, 26 and 100 % increase in root dry weight due to inoculation was reported by Miljaković et al. [16] and Desta et al. [18], respectively. In the Boshe and Jalale varieties 75% (6 strains out of 8 inoculated strains), 50% (4 strains out of 8 inoculated strains) improved the root dry weight compared to the negative control, while in Gishama, Hawassa-04, and Awassa-95, 37.5, 25, and 12.5% of inoculant strains improved the root dry weight, respectively. Samudin and Kuswantoro [17] and Szpunar-Krok et al. [14] observed a significant increase in the root dry weight with inoculation. In this study, only 40% of the strains (only 16 out of 40 inoculation treatments) improved root dry weight, while 60% did not provide a positive response; this led to a negative and significant correlation of this parameter with the number of nodules (Figure 1).

4.1.4. Leaf Color

In this study specific strains were effective showing deep and light green leaves in soybean (Figure 2), in addition to color differences, plant stand performance effectively distinguished between the control and inoculated plants. The negative control pots exhibit poor crop performance and limited growth, which is consistent with the findings of Ayalew and Yoseph [35] on cowpea plants. Their findings showed that plants treated with Bradyrhizobium strains had healthy green leaves, while untreated plants had pale yellow leaves. Gwata et al. [40] reported that chlorotic plants with yellow leaves (ineffective nodulation and no nitrogen fixation) were visually distinguishable from the vigorous plants with dark green leaves (effective nodulation and nitrogen fixation). These color changes in plants vary according to the decrease or increase in the chlorophyll amount [41], which is a reliable index of physiological plant condition [42]. Rhizobium inoculation considerably increased the bush bean leaf chlorophyll concentration by 19% and 44% in a field experiment and 40% and 98% in a glasshouse experiment [43].
Inoculation of soybean varieties with the SD-53 strain showed more deep green leaves over other inoculant strains (Figure 2). Since the plants were grown in a nitrogen-free medium, the available nitrogen indicated by the dark green leaves was derived from the N2-fixation process [44]. Higher nodule number, dry weight and shoot dry weight associated with inoculating this strain indicated the greater effectiveness of this strain. Inoculation of soybean varieties with the SD-53 strain also showed deeper green than the positive control (Figure 2).

4.2. Symbiotic Effectiveness of Bradyrhizobia Strains

The evaluation of rhizobia–host relationships often rely on two key factors: nodulation capacity (infectivity) and nitrogen fixation capability (symbiotic effectiveness), as highlighted by Osei et al. [45]. Optimizing these symbiotic interactions between legumes and rhizobia is essential for enhancing biological nitrogen fixation and agricultural productivity. In environments with restricted mineral nitrogen availability, one commonly utilized method to assess nitrogen fixation efficiency is by measuring shoot dry weight accumulation [46].

4.2.1. Relative Symbiotic Effectiveness (RSE)

Based on the effectiveness percentage, the strains varied from effective to highly effective. Similarly, Desta et al. [18] reported a symbiotic effectiveness range of 70–96%. Moreover, various ranges of RSE in leguminous plants have been documented by different researchers. For instance, in field pea, highly effective strains accounted for 36.4%, while 62% were effective [47]. In common beans, over 85% of strains were highly effective [48]. Likewise, Temesgen [37] observed that soybean infecting isolates from Ethiopian soils demonstrated either effectiveness or high effectiveness, indicating the capability of native rhizobia to establish highly effective symbiosis. This study suggests the presence of highly effective soybean-nodulating rhizobia in Ethiopian soils, offering the potential to select isolates suitable for abundant and effective nodulation, thus serving as bio-fertilizers. However, the variation in RSE among genotypes with the same strain underscores the importance of considering both host genotype and rhizobia strains’ effects. This observation is supported by the findings of Temesgen and Assefa [5], who reported higher symbiotic effectiveness in the Jalale and Cheri varieties compared to Ethio-Yugslovia inoculated with similar strains.
The median RSE for the strains isolated from Dore Bafano (97.64) exceeded that of the strains isolated from Boricha (71.23), indicating that 50% of the strain from Dore Bafano exhibited symbiotic effectiveness greater than 97.64% (Figure 4), thus classifying it as highly effective strain. Among the four strains isolated from Dore Bafano (SD-47, SD-50, SD-51, and SD-53) strain SD-53 demonstrated high effectiveness with the 4 varieties (name the varieties). Consequently, this indigenous strain holds a promise as a viable inoculant source for soybean production.
A positive and weak correlation observed for relative symbiotic effectiveness with nodule number (r = 0.14) and nodule dry weight (r = 0.22) indicates, not all strains that formed a higher nodule number and dry weight shows the highest effectiveness. For instance, inoculation of the Jalale variety with SB-113 provides the highest nodule number (40 nodule plant−1) and nodule dry weight (0.090 gm plant−1) (Table 4), which is significantly higher than the other treatments in the experiment, but had 68% relative symbiotic effectiveness, which is rated as effective (Figure 4). In contrast to this, inoculation of the Boshe variety with SB-36 and SB-37 provides a lower nodule number (1.67 nodules plant−1) and dry weight (0.002 and 0.005 gm plant−1 respectively) than most of the treatments in the experiment but had highly effective symbiotic effectiveness (89.91 and 107.95%, respectively). This could be attributed to the fact that nodule number and nodule dry weight include nonfunctional nodules that may not be as valid indicators of nitrogen fixation as shoot dry weight. This suggests that apart from nodule number and nodule dry weight, other nodule factors such as effective nodule number and dry weight may be more important parameters in estimating the amount of nitrogen fixed and play a crucial role in influencing the amount of total accumulated dry matter. However, the positive association of relative symbiotic effectiveness with most of the parameters confirms the dependence of soybean effectiveness on these parameters.

4.2.2. Absolute Symbiotic Effectiveness (ASE)

In this study, the highest ASE was consistently observed in plants inoculated with the SD-53 strain, which was particularly evident in the Gishama, Boshe and Awassa-95 varieties. This underscores the remarkable effectiveness of the SD-53 strain with Gishama, Boshe, and Awassa-95 compared to the other strains, and varieties. This result is further supported by the observed shoot dry weight and leaf coloration performances, as detailed in Table 4 and Figure 2, respectively.
A positive and statistically significant relationship observed for ASE with nodule number, nodule dry weight, shoot dry weight, relative symbiotic effectiveness, and plant height suggests that as the absolute symbiotic effectiveness increases, there is a corresponding increase in nodule number, nodule dry weight, shoot dry weight, relative symbiotic effectiveness, and plant height. This correlation implies that stronger symbiotic relationships between soybean and nitrogen-fixing bacteria lead to higher nodule counts, greater nodule and shoot dry weights, enhanced relative symbiotic effectiveness, and increased plant height.

5. Conclusions

The experiment aimed to assess the symbiotic effectiveness of Bradyrhizobium strain inoculation across various soybean varieties. Five soybean varieties, eight Bradyrhizobium strains, and two controls (positive and negative) were tested in sand-filled pots. The results revealed significant impacts of Bradyrhizobium inoculation, soybean varieties, and their interaction with growth, biomass accumulation, and nodulation performances. Relative Symbiotic Efficiency (RSE) varied from effective to highly effective across the Bradyrhizobium strains and correlated positively and significantly with plant height, nodule number, nodule dry weight, and shoot and root dry weight. Similarly, ASE positively and significantly correlated with nodule number, nodule dry weight, shoot dry weight, relative symbiotic effectiveness, and plant height. Notably, strain SD-53 demonstrated remarkable effectiveness with Gishama, Boshe, and Awassa-95 varieties based on both RSE and ASE, supported by observed shoot dry weight and leaf coloration. The finding suggests the potential use of the SD-53 Bradyrhizobia strain for enhancing sustainable soybean production.

Author Contributions

H.B.: conceptualized and finalized the research methodology, conducted and managed the experiment, collected and analyzed the data, wrote the original manuscript, and revised the manuscript. T.Y.: reviewed and edited the manuscript, and supervision. T.A.: initiated the concept, funding acquisition, project administration, and reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Hawassa University through a thematic research project on ‘Soybean Popularization, Agronomic and Nutritional Evaluation, and Product Development for Agro-processing Industry Linkage in the Sidama Regional State, Ethiopia’. The APC is covered by “Climate Change Effects on Food Security (CLIFOOD)”, a project supported by the DAAD with funds from the Federal Ministry for Economic Cooperation and Development (BMZ).

Data Availability Statement

All data gathered for this study are analyzed and included in this published article. For further information, please contact the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 01280 g001
Figure 1. Correlation coefficients among plant height, nodule number, nodule, shoot and root dry weight, and relative and absolute symbiotic effectiveness.
Figure 1. Correlation coefficients among plant height, nodule number, nodule, shoot and root dry weight, and relative and absolute symbiotic effectiveness.
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Figure 2. Leaf color of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia, in 2023.
Figure 2. Leaf color of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia, in 2023.
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Figure 3. Relative symbiotic efficiency of the soybean-modulating Bradyrhizobia strains. The horizontal lines indicate the borders of the rates for highly effective (>80%), effective (50–80%), poorly effective (35–50%) and ineffective (<35%).
Figure 3. Relative symbiotic efficiency of the soybean-modulating Bradyrhizobia strains. The horizontal lines indicate the borders of the rates for highly effective (>80%), effective (50–80%), poorly effective (35–50%) and ineffective (<35%).
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Figure 4. Group comparison of relative symbiotic effectiveness of the Bradyrhizobial strains based on their isolation site. Boxplots with different letters are significantly different, at p < 0.05 (*).
Figure 4. Group comparison of relative symbiotic effectiveness of the Bradyrhizobial strains based on their isolation site. Boxplots with different letters are significantly different, at p < 0.05 (*).
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Figure 5. Absolute symbiotic efficiency of the soybean-modulating Bradyrhizobia strains.
Figure 5. Absolute symbiotic efficiency of the soybean-modulating Bradyrhizobia strains.
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Table 1. List of varieties that were used for the experiment.
Table 1. List of varieties that were used for the experiment.
VarietySourceBreeder/MaintainerAltitude (m.a.s.l.)Year—Released/RegisteredMaturity Group
Awassa-95 (G 2261)PARCAwARc/SARI520–18002005Early
Boshe (IAC-13-1)PARCBARI/OARI1200–19002008Medium
Gishama (PR-143-(26))PARCPARC520–18002010Medium
Hawassa-04 (AGS-7-1)PARCAwARc/SARINA2012Medium
JallalePARCBARC/OARI1300–18502003Medium
Note: PARC = Pawe Agricultural Research Center, BARC = Bako Agricultural Research Center, OARI = Oromia Agricultural Research Institute, AwARC = Awassa Agricultural Research Center, SARI = South Agricultural Research Center, NA = Not available.
Table 2. Bradyrhizobia strains used as inoculants: their origin and soybean varieties.
Table 2. Bradyrhizobia strains used as inoculants: their origin and soybean varieties.
Origin of Strains (Locations)StrainsVarieties Used for Isolation
Boricha SB-36Wegayen
SB-37Wegayen
Dore BafanoSD-47TGX-3326-44
SD-50TGX-3326-44
SD-51Gishama
SD-53Gishama
BorichaSB-113Awassa-95
SB-120Awassa-04
Source: Jaiswal et al. [24] and Beyan et al. [23].
Table 3. Analysis of variance for growth and nodulation parameters of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia during 2023.
Table 3. Analysis of variance for growth and nodulation parameters of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia during 2023.
Mean SquareTraits
Plant Height (cm)Number of NodulesShoot Dry Weight (gm plant−1)Root Dry Weight (gm plant−1)Nodule Dry Weight (gm plant−1)
Variety (V)493.77 ***3.56 ***0.09 ***0.93 ***0.02 ***
Rhizobial strains (R)356.83 ***40.42 ***0.01 ***0.15 ***0.10 ***
V × R316.97 ***3.75 ***0.004 ***0.05 ***0.01 ***
Error29.147.950.00030.0030.002
CV9.6516.871.734.8438.08
LSD8.744.570.130.090.03
LSD: least significant difference, CV: coefficient of variance, ***: significant at p < 0.001.
Table 4. Mean values for growth and nodulation performance of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia in 2023.
Table 4. Mean values for growth and nodulation performance of Bradyrhizobium-inoculated soybean varieties at Hawassa, Ethiopia in 2023.
VarietiesStrainPlant Height (cm)Nodule Number Plant−1Nodule Dry Weight (g)Root Dry Weight (g)Shoot Dry Weight (g)
Gishama−ve control40.67 st0.00 m0.000 j0.11 r–x0.91 xy
+ve control74.67 a–c0.00 m0.000 j0.29 c–h1.76 cd
SB-3664.00 d–h1.33 lm0.006 ij0.09 t–x1.18 n–r
SB-3749.33 l–s1.00 lm0.003 ij0.14 t–x1.10 r–v
SD-4744.67 o–t4.00 j–m0.011 g–j0.13 p–x1.12 q–t
SD-5049.67 m–t1.67 lm0.005 ij0.08 v–x1.04 t–w
SD-5152.33 k–q11.67 gh0.081 ab0.18 i–s1.29 l–o
SD-5343.67 q–t9.67 g–i0.010 g–j0.05 x1.44 g–j
SB-11343.67 q–t11.00 gh0.012 f–j0.07 wx1.03 t–w
SB-12047.67 m–s6.33 i–k0.006 ij0.09 u–x1.04 s–w
Awassa–95−ve control44.33 p–t0.00 m0.000 j0.33 a–d1.24 m–q
+ve control44.67 o–t0.00 m0.000 j0.23 f–n1.46 f–i
SB-3646.33 m–t21.33 cd0.041 c–g0.23 g–o1.53 e–h
SB-3749.33 l–s2.33 k–m0.002 ij0.41 a1.57 ef
SD-4754.00 k–n4.67 j–l0.015 f–j0.26 d–k1.65 c–e
SD-5053.00 k–p4.00 j–m0.004 ij0.30 b–g1.64 de
SD-5157.00 h–l2.33 k–m0.004 ij0.19 i–r1.55 e–g
SD-5363.33 d–j24.67 c0.089 a0.18 k–u1.89 b
SB-11358.67 g–k11.00 gh0.056 b–e0.15 n–w1.76 c
SB-12055.00 i–m13.67 fg0.056 b–e0.33 a–e1.70 cd
Boshe−ve control53.33 k–o0.00 m0.000 j0.17 k–u0.90 vw
+ve control45.00 o–t0.00 m0.000 j0.14 p–x1.09 r–w
SB-3637.67 t1.67 lm0.002 ij0.14 o–x0.98 wx
SB-3765.33 d–h1.67 lm0.005 ij0.23 g–o1.18 o–r
SD-4759.33 e–k23.00 c0.060 a–d0.20 h–p1.25 m–p
SD-5068.00 b–e7.33 h–j0.042 c–f0.33 a–d1.31 k–m
SD-5171.33 a–d4.33 j–m0.005 ij0.24 e–m1.32 k–m
SD-5366.33 c–g24.00 c0.064 a–c0.18 j–t1.40 i–l
SB-11354.00 k–n17.00 d–f0.054 b–e0.16 l–v1.04 t–w
SB-12043.33 r–t4.67 j–l0.037 c–h0.19 i–r1.10 r–w
Hawassa–04−ve control63.33 d–j0.00 m0.000 j0.20 h–q0.82 y
+ve control45.50 n–t0.00 m0.000 j0.37 a–c1.06 r–w
SB-3676.00 ab1.33 lm0.006 ij0.17 l–v0.82 y
SB-3749.50 l–r1.00 lm0.003 ij0.11 r–x0.82 y
SD-4754.00 k–n3.33 j–m0.028 e–j0.27 d–i0.98 v–x
SD-5074.50 a–c2.67 k–m0.003 ij0.18 i–s1.11 r–u
SD-5175.00 a–c2.67 k–m0.002 ij0.24 e–l1.01 u–x
SD-5363.67 d–i20.33 c–e0.038 c–h0.09 s–x1.03 t–w
SB-11358.67 g–k24.67 c0.058 b–e0.14 n–w0.98 wx
SB-12045.33 n–t16.67 ef0.031 d–i0.11 q–x0.83 y
Jalale−ve control47.67 m–s0.00 m0.000 j0.21 h–p1.16 p–s
+ve control79 a0.00 m0.000 j0.32 a–f2.08 a
SB-3654.00 k–n2.00 k–m0.012 f–j0.39 ab1.33 j–m
SB-3759.00 f–k4.00 j–m0.010 h–j0.19 i–r1.16 p–s
SD-4751.67 k–r1.00 lm0.004 ij0.38 ab1.16 p–r
SD-5059.33 e–k1.00 lm0.008 h–j0.27 d–j1.30 l–n
SD-5154.67 j–m2.00 k–m0.006 ij0.30 b–g1.16 p–s
SD-5367.67 b–f33.00 b0.079 ab0.16 l–v1.44 g–j
SB-11370.67 a–d40.00 a0.090 a0.17 k–u1.42 h–k
SB-12045.67 n–t23.33 c0.055 b–e0.15 m–w1.16 q–r
Means in the column followed by different letters are significant at p < 0.001.
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Beruk, H.; Yoseph, T.; Ayalew, T. Unlocking the Potential of Inoculation with Bradyrhizobium for Enhanced Growth and Symbiotic Responses in Soybean Varieties under Controlled Conditions. Agronomy 2024, 14, 1280. https://doi.org/10.3390/agronomy14061280

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Beruk H, Yoseph T, Ayalew T. Unlocking the Potential of Inoculation with Bradyrhizobium for Enhanced Growth and Symbiotic Responses in Soybean Varieties under Controlled Conditions. Agronomy. 2024; 14(6):1280. https://doi.org/10.3390/agronomy14061280

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Beruk, Haimanot, Tarekegn Yoseph, and Tewodros Ayalew. 2024. "Unlocking the Potential of Inoculation with Bradyrhizobium for Enhanced Growth and Symbiotic Responses in Soybean Varieties under Controlled Conditions" Agronomy 14, no. 6: 1280. https://doi.org/10.3390/agronomy14061280

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