Synergistic Interaction between Symbiotic N2 Fixing Bacteria and Bacillus strains to Improve Growth, Physiological Parameters, Antioxidant Enzymes and Ni Accumulation in Faba Bean Plants (Vicia faba) under Nickel Stress

Several activities in the agriculture sector lead to the accumulation of Nickel (Ni) in soil. Therefore, effective and economical ways to reduce soil bioavailability of Ni must be identified. Five isolates of Rhizobium leguminosarum biovar Viceae (ICARDA 441, ICARDA 36, ICARDA 39, TAL–1148, and ARC–207) and three bacterial strains (Bacillus subtilis, B. circulance, and B. coagulans) were evaluated for tolerance and biosorption of different levels of Ni (0, 20, 40, 60, and 80 mg L−1). Pot experiments were conducted during the 2019/2020 and 2020/2021 seasons using four inoculation treatments (inoculation with the most tolerant Rhizobium (TAL–1148), inoculation with the most tolerant Rhizobium (TAL–1148) + B. subtilis, inoculation with the most tolerant Rhizobium (TAL–1148) + B. circulance, and inoculation with the most tolerant Rhizobium (TAL–1148) + B. coagulans) under different levels of Ni (0, 200, 400, and 600 mg kg−1), and their effects on growth, physiological characteristics, antioxidant enzymes, and Ni accumulation in faba bean plants (Vicia faba C.V. Nobaria 1) were determined. The results showed that Rhizobium (TAL–1148) and B. subtilis were the most tolerant of Ni. In pot trials, inoculation with the most tolerant Rhizobium TAL–1148 + B. subtilis treatment was shown to be more effective in terms of growth parameters (dry weight of plant, plant height, number of nodules, and N2 content), and this was reflected in physiological characteristics and antioxidant enzymes under 600 mg kg−1 Ni compared to the other treatments in the 2019/2020 season. In the second season, 2020/2021, a similar pattern was observed. Additionally, lower concentrations of Ni were found in faba bean plants (roots and shoots). Therefore, a combination of the most tolerant Rhizobium (TAL–1148) + B. subtilis treatment might be used to reduce Ni toxicity.


Introduction
Faba bean (Vicia faba L.) is one of the most important leguminous crops grown in Asia and the Mediterranean region [1]. It is high in protein (25-30%) and carbohydrates (55-60%), which contributes to its placement among the popular annually produced grain crops for use among humans and domestic animals [2]. Green seeds are utilized in fresh vegetable salads during vegetative growth, while dry seeds are used in prepared food, and the entire plant can be fed to farm animals [3]. Due to a lack of domestic production, Egypt is one of the leading importers of faba bean [1].
Some stresses, such as heavy metal contamination, have an impact on faba bean productivity. In addition, human, agricultural, and industrial activities all contribute to metal contamination of soils [4]. As a result of these processes, mineral residues accumulate

Assessment of Different Rhizobium Isolates and Bacillus Strains for Ni Tolerance
When cultivated in a Yeast Extract Mannitol Broth medium (YEMB) for Rhizobium isolates and Nutrient Broth (NB) medium supplemented with varying doses of Ni (0, 10, 20, 40, 60, and 80 mg L −1 ), the growth patterns of Rhizobium leguminosarum bv. Viceae isolates (ICARDA 441, ICARDA 36, ICARDA 39, TAL-1148, and ARC-207) and Bacillus strains (B. subtilis, B. circulance, and B. coagulans) showed substantial change after 72 h. The optical density (OD540) of different bacteria decreased with increasing Ni concentration when compared to normal growth (no Ni). Compared to the other bacteria under study, the TAL-1148 isolate and B. subtilis were the most tolerant of higher applied Ni concentrations, showing good abilities to grow on YEMB and NB medium supplemented with 80 mg L −1 Ni, achieving 2.53 and 0.84 log numbers, respectively (Table 1).

Biosorption of Ni by Different Rhizobium Isolates and Bacillus Strains
To gain insight into the biosorption of different amounts of Ni by the studied Rhizobium leguminosarum bv. Viceae isolates (ICARDA 441, ICARDA 36, ICARDA 39, TAL-1148, and ARC-207) and Bacillus strains (B. subtilis, B. circulance, and B. coagulans), we used an atomic absorption spectrophotometer to quantify it in a supernatant ( Figure 1). The biosorption of the TAL-1148 isolate was the highest among the five isolates evaluated, with 38.83 mg L −1 at 80 mg L −1 . Biosorption of Ni was shown to be significantly higher at all other concentrations examined as compared to the lower concentration of Ni (10 mg L −1 ) and increased with increasing concentration. In comparison to other Ni concentrations, biosorption of 37.99, 33.74, and 30.84 mg L −1 were found for B. subtilis, B. circulance, and B. coagulans at 80 mg L −1 , respectively. Herein, biosorption of Ni by the studied Rhizobium isolates followed the descending order of TAL-1148 > ICARDA 441 > ICARDA 39 > ICARDA 36 > ARC-207, and for Bacillus strains followed the descending order of B. subtilis > B. circulance > B. coagulans ( Figure 1).

Antioxidant Enzyme Activity
The activities of catalase (CAT), ascorbate peroxidase (APX), and polyphenol oxidase (PPO) were considerably altered as a result of bacterial inoculation treatments and Ni stress, as shown in Table 3. At 60 days after planting, varying amounts of Ni stress increased the amount of antioxidant enzyme activity in faba bean leaves compared to the control (Table 3).

Antioxidant Enzyme Activity
The activities of catalase (CAT), ascorbate peroxidase (APX), and polyphenol oxidase (PPO) were considerably altered as a result of bacterial inoculation treatments and Ni stress, as shown in Table 3. At 60 days after planting, varying amounts of Ni stress increased the amount of antioxidant enzyme activity in faba bean leaves compared to the control (Table 3).
Under different bacterial inoculation treatments, T4 treatment (inoculation with Rhizobium TAL-1148 + B. subtilis) efficiently increased the CAT content by 32.13 and 32.75 µM H 2 O 2 g −1 FW min −1 , APX content by 503.79 and 523.79 µM H 2 O 2 g −1 FW min −1 , and PPO content by 1.07 and 1.14 µM tetra-guaiacol g −1 FW min −1 during the first growing season (2019/2020), and the second growing season (2020/2021), respectively (Table 3). For diverse applications of bacterial inoculation treatments under Ni stress conditions, the results showed the following descending order: T4 > T2 > T3 > T1. Table 4 shows that faba bean plants treated with bacterial inoculation had reduced Ni levels and accumulation in their tissues. In comparison to the 600 mg kg −1 Ni stress concentration, the T4 < T2 < T3 < T1 treatments attained 47.70, 50.90, 56.67, 97.71 µg g −1 for root contents and 24.28, 29.90, 38.67, 77.37 µg g −1 for shoot contents at 60 days after sowing in the 2019/2020 season, respectively. In the 2020/2021 season, a similar trend was observed.  These findings clearly suggest that treating faba bean plants with Rhizobium TAL-1148 + B. subtilis had a better effect than the other bacterial inoculation treatments due to the fact that the Ni content was lower. Under varied doses of Ni stress, the bioconcentration factor (BCF) and translocation factor (TF) of faba bean plants revealed that the application of Rhizobium TAL-1148 + B. subtilis (T4) considerably reduced the accumulation of Ni in plant tissues compared to the control treatment T1 (Table 4).

Assessment of Different Rhizobium Isolates and Bacillus Strains for Ni Tolerance
These differences in response shown by the studied strains might be due to differences in their inherent tolerance capacities, supported by active Ni efflux mechanisms to avoid dangerous intracellular Ni levels [19].
Several investigations have found that heavy metals, notably Ni, have a negative impact on symbiotic N fixation; for example, from the nodules of pea and lentil plants cultivated in polluted fields, Ni-tolerant Rhizobium strains (RP5 and RL9) were isolated and showed great tolerance to 350 and 500 mg mL −1 of Ni [20]. At the lowest dose of 0.2 mM, Rhizobium strains L9 and L19 showed better resistance to Ni than Mesorhizobium L42 and L50 [21]. In addition, in vitro, the rhizobium HGR-4 isolated from horse gram root nodules could tolerate 1000 mg g −1 Ni [14]. On the other hand, among the bacteria tested (B. thuringiensis 002, B. fortis 162, B. subtilis 174, and B. farraginis 354), B. subtilis 174 had the highest Ni tolerance, growing in conditions containing Ni at a concentration of 400 mg L −1 [13].

Biosorption of Ni by Different Rhizobium Isolates and Bacillus Strains
For the concentrations examined, the biosorption of Ni by different bacterial strains was significantly increased. The reason for this specific behavior is due to the smaller ionic radius of Ni (0.69 Å). In addition, bacteria can also accumulate metal in their cell walls, as well as protein polyphosphate complexes, polysaccharides, and complex forms with carboxyl groups of peptidoglycans [22]. Tobin et al. [23] hypothesized that molecules with a smaller ionic radius sorb more quickly. Biosorption of Ni has been well supported by previous findings based on ionic radius [24,25]. As a result, the aforementioned strains could be employed as potential heavy metal immobilizers in polluted soils. Ajmal et al. [26] reported that the bacterial strain Citrobacter werkmanii (WWN1) showed maximum net removal of 87% of Ni from an aqueous solution, followed by Enterobacter cloacae (JWM6), which showed 86% net removal of Ni, in a comparison with other studied strains.

Parameters of Growth
Rhizospheric bacteria have the ability to reduce/detoxify heavy metal stress through a variety of methods, such as metal ions outside the cell, biostimulation, bioaugmentation, metal reduction, and biosorption [27]. Improved plant development in metal-contaminated soils has been attributed to a bacterial biosorption/bioaccumulation mechanism with plant growth-promoting characteristics [28]. Metal accumulation in root nodules may be aided by rhizobial nodulation of the host plants. Additionally, different processes of precipitation, chelation, immobilization, and biosorption might lower metal toxicity when microbes remain in the rhizosphere [29]. Heavy metals such as Ni have a significant impact on plant nodulation growth parameters [30], and excessive Ni has been found to have negative effects on microorganisms, particularly rhizobia, and therefore on nodule formation in various leguminous species [31]. At 100 mg kg −1 Ni, more nodules were detected in Vigna cylindrica, while the production of root nodules was substantially hampered in Vigna mungo and Vigna radiata [32]. A phytotoxic effect was observed at 580 mg Ni/kg soil, which dramatically reduced the number of lentil nodules [20].

Photosynthetic Pigments
Nickel almost completely destroys the photosynthetic apparatus/machinery, i.e., mesophyll cells and epidermal tissues [33], and reduces chlorophyll content (chlorophyll a, b, total chlorophyll) at all levels [34,35]. Furthermore, Ni affects the structure of thylakoid membranes and grana, lowering the size of grana and increasing the frequency of nonappressed lamellae [36]. However, higher levels of nutrients and organic matter in the rhizosphere could explain the rise in chlorophyll and carotenoids in faba bean leaves by bacterial inoculation [37,38]. Several studies have shown that bacterial inoculation accelerates the production of photosynthetic pigments in stressed plants [13,[39][40][41].

Activity of Antioxidant Enzymes
Plants enhance the activity of antioxidant enzymes in their main state in response to abiotic challenges, such as heavy metal stress; this is dependent on plant stress sensitivity as a first line of defense against high antioxidant ROS concentrations [42,43]. According to our findings, antioxidant enzymatic defense systems appear to play a key part in faba bean plant Ni toxicity. This defense can be activated at the transcriptional level, and at the enzymatic activity can help the plant adapt to Ni toxicity. A similar trend was observed in rye [44], Lemna polyrhiza [45], Helianthus annus [13], and lettuce [42].

Nickel Content
Irrespective of Ni concentrations, the data showed that treating faba bean plants with Rhizobium TAL-1148 + B. subtilis had a better effect than other bacterial inoculation treatments due to the fact that the Ni content was lower. Under varied conditions of Ni stress, the bioconcentration factor (BCF) and translocation factor (TF) of faba bean plants revealed that the application of Rhizobium TAL-1148 + B. subtilis (T4) considerably reduced the accumulation of Ni in plant tissues compared to the control treatment, T1 (Table 4).
Hence, the increase in Ni content in the roots of faba bean plants is due to biosorption of Ni by Rhizobium + B. subtilis. Based on these findings, it appears that biosorption by bacterial inoculation is responsible for the change of Ni into insoluble forms [14,42]. Reduced Ni levels in plant organs could be attributed to RL9 strain's adsorption/desorption, according to research by the authors of [20,46]. The bioinoculant strains lowered Ni levels in the organs of inoculated plants cultivated in soils polluted with various metals [18,21].

Assessment of Different Rhizobium Isolates and Bacillus Strains for Ni Tolerance
Nickel chloride (NiCl 2 .6H 2 O, Merck, Germany) was used to prepare a 1000 mg L −1 stock solution. In a shaker, 50 mL of YEMB and NB + 1 mL (10 8 CFU mL −1 ) of fresh cultures of different strains and different levels of Ni (0, 10, 20, 40, 60, and 80 mg L −1 ) were built up and shaken at 150 rpm at 30 • C, then incubated for three days. Using a UV-Visible spectrophotometer (model 6705, Jenway, UK), the growth of bacteria was measured using optical density (OD) at 540 nm in five repetitions. A sterile uninoculated YEMB medium for Rhizobium isolates and NB media for Bacillus strains served as blanks.

Biosorption of Ni by Different Rhizobium Isolates and Bacillus Strains
Experiments with initial concentrations of 0, 10, 20, 40, 60, and 80 mg L −1 were performed with 10 8 CFU mL −1 of fresh cultures (30 • C and 150 rpm for 3 days) to evaluate the effect of varying Ni concentrations on biosorption by the different Rhizobium isolates and Bacillus strains. The bacterial cultures were then centrifuged for 10 min at 5000 rpm, with the supernatant filtered (5 mL) and examined with an Atomic Absorption Spectrophotometer (AAS PerkinElmer 3300). YEMB and NB broth, with the treated level of Ni and without inoculum, was used as a blank. The discrepancies between the first and last concentrations suggested that the studied bacteria absorbed Ni, and the experiment was repeated 3 times [49].

Pot Trial
Sandy soil was washed three times with 0.1 M HCl, then several times with distilled water to remove other minerals, sterilized twice for 4 h at 1.5 par and 121 • C, then mixed with different Ni concentrations and left for 2 weeks, after which 8 kg was placed into a polyethylene bag (22 cm in diameter and 35 cm in height) under greenhouse conditions during the 2019/2020 and 2020/2021 seasons [43]. With six repetitions, the experiment was performed according to a split-plot design. The main plots were the Ni pollution treatments (0, 200, 400, and 600 mg kg −1 ), while the inoculation treatments were subplots. There were four treatments in the subplots: (1) inoculation with Rhizobium (TAL-1148, control), (2) inoculation with Rhizobium (TAL-1148) + B. circulance, (3) inoculation with Rhizobium (TAL-1148) + B. coagulans, and (4) inoculation with Rhizobium (TAL-1148) + B. subtilis.

Trait Measurements
At 60 days following sowing, five healthy plants per treatment were uprooted, and growth parameters (dry weight (g plan −1 ), plant height (cm plant −1 ), number of nodules, and N 2 %) were measured. Plant dry weight was determined using an electronic scale, and the N 2 % was determined using the micro-Kejeldahl method, as previously described by [51]. Physiological properties (photosynthetic pigments, carotenoids, total soluble sugars), antioxidant enzyme activity (catalase (CAT), ascorbate peroxidase (APX), and poly phenol oxidase (PPO)), and Ni content in the roots and shoots of the faba bean plants were also studied.

Photosynthetic Pigments
To determine total chlorophyll and carotenoid contents, leaf samples (0.1 g) from each treatment were pulverized and extracted in 5 mL of acetone (80%), as described by [52]. The extract was detected at 663 nm, 645 nm, and 470 nm after centrifugation (13,000× g for 10 min). Carotenoid and total chlorophyll contents were calculated and expressed as mg g −1 FW. Following the protocol outlined in [53], total soluble sugars was determined. Leaf samples (0.5 g) from each treatment were homogenized in 5 mL ethanol (80%), then put in a water bath (80 • C for 30 min). After centrifugation (10,000× g for 10 min), the extract was collected and a UV spectrophotometer (Model 6705) was used to determine concentrations at 620 nm, based on a glucose standard curve and expressed as mg g −1 FW.

Determination of Ni in the Roots and Shoots of the Faba Bean Plants
Plant roots and shoots were cleaned with distilled water, dried in an oven (70 • C for 24 h), and then ground in a stainless-steel blender, according to [57]. Then, 0.5 g of the ground samples was mixed with 4.0 mL HNO 3 and 1.0 mL HClO 4 and digested at 230 • C, then filtered to produce a clear solution. Flame atomic absorption spectroscopy was used to determine the overall concentration of Ni (AAS PerkinElmer 3300).

Bioconcentration and Translocation Factors
For each plant component (roots and shoots), the Ni content efficiency of faba bean plants was measured. According to [58,59], the following equations were used to calculate the bioconcentration factor (BCF) and translocation factor (TF): BCF = Concentration of Ni in roots Concentration of Ni in test soil TF = Concentration of Ni in shoots Concentration of Ni in roots

Statistical Analysis
Using CoStat software, the data were statistically evaluated using the analysis of variance (ANOVA) procedure (Pack-age 6.45, CoHort, USA). DMRT was used to compare the differences between the means at p < 0.01 and p < 0.05 [60]. The data are presented as means ± SDs.

Conclusions
The effects of several bacterial inoculations on Ni accumulation in faba bean plants grown in various levels of Ni-contaminated soil were studied. Inoculation with the most tolerant Rhizobium TAL-1148 + B. subtilis treatment was more effective in terms of growth parameters (dry weight of plant, plant height, number of nodules, and N 2 content), as evidenced by physiological characteristics and antioxidant enzymes in soil treated with 600 mg kg −1 Ni compared to the other treatments. As a result, during the two growing seasons, the treatment combining the most tolerant Rhizobium (TAL-1148) with B. subtilis could be utilized as an option to reduce Ni toxicity.