Effects of Pseudomonas sp. OBA 2.4.1 on Growth and Tolerance to Cadmium Stress in Pisum sativum L.

Cadmium stress is a barrier to crop production, yield, quality, and sustainable agriculture. In the current study, we investigated the characteristics of bacterial strain Pseudomonas sp. OBA 2.4.1 under cadmium (CdCl2) stress and its influence on Cd stresses in pea (Pisum sativum L.) seedlings. It was revealed that strain OBA 2.4.1 is tolerant of up to 2 mM CdCl2, and seed treatment with the bacterium enhanced pea plant growth (length of seedlings) under 0.5 mM cadmium stress. This bacterial strain showed plant growth-promoting properties, including biofilm formation and siderophore activity. An important advantage of the studied strain OBA 2.4.1 is its ability to colonize the plant roots. Moreover, the inoculation with strain OBA 2.4.1 significantly reduced oxidative stress markers in pea seedlings under cadmium stress. These findings suggest that cadmium stress-tolerant strain OBA 2.4.1 could enhance pea plant growth by mitigating stress-caused damage, possibly providing a baseline and eco-friendly approach to address heavy metal stress for sustainable agriculture.


Introduction
Biotic and abiotic stresses affect plant growth, development, and crop yields. Heavy metals (HMs) adversely affect all processes occurring in plants and, in addition, are very toxic to the human body and can cause serious health problems for humans [1,2]. The rapid development of industry and manufacturing causes serious environmental pollution, especially in soil. HMs in the soil are absorbed by plant roots and accumulate in all plant tissues, seriously slowing down many physiological and molecular processes [3]. Excessive accumulation of Cd 2+ in plants leads to growth retardation, a decrease in the contents of chlorophyll and carotenoids, as well as a decline in leaf area and photosynthesis rate, along with reduced plant biomass and water content and increased protease activity [4]. Cd 2+ in plants can bind to proteins, causing denaturation and dysfunction, resulting in growth inhibition [5]. Cd can lead to the formation of reactive oxygen species (ROS) [1,6]. Sandalio et al. (2001) showed that Cd induces oxidative stress in Pisum sativum seedlings, promoting the accumulation of lipid peroxide and oxidized protein, and reducing catalase and superoxide dismutase (SOD) activity [7].
To mitigate the problems associated with HMs contamination, there is an urgent need for alternative environmentally friendly technologies, such as the use of plant growthpromoting microorganisms/rhizobacteria (PGPM/PGPR), for future use. This includes various taxonomic groups that have a wide range of beneficial properties for plants. Rhizospheric bacteria Pseudomonas sp. Belongs to PGPM [8,9]. These microorganisms actively
Nucleotide sequences were determined on an Applied Biosystems 3500 automatic sequencer (Applied Biosystems, Waltham, MA, USA) using the Big Dye Terminator v. 3.1. The analysis was carried out using the Lasergene software package (DNASTAR, Inc., Madison, WI, USA). Nucleotide sequences for comparative analysis were taken from the GenBank database (www.ncbi.nlm.nih.gov accessed on 28 December 2022). Computational analysis of DNA sequence fragments was performed using the Clustal W multiple alignment method in the Megalign Lasergene program (DNASTAR, Inc. Madison, WI, USA).
To obtain a fluorescently labeled strain of Pseudomonas sp. OBA 2.4.1, the pJNTur-boRFP vector was used [28]. Some of the root fragments were used to visually assess the colonization of the surface of plant root hairs by bacteria using an Axio Imager M1 fluorescent microscope (Carl Zeiss, Jena, Germany). For this, the roots were incubated together with labeled bacteria (10 6 CFU/mL) for 1 day.

Biofilm Formation on Inert Surfaces
Biofilms were examined on 24-well plastic plates (polystyrene) (Corning, Inc., USA). Bacteria were grown for 24 h in Lauria-Bertani (LB) medium at 28 • C and 140 rpm. The bacterial culture was diluted to 10 6 CFU/mL and 1 mL was transferred into the wells of the plate and incubated at 28 • C and 50 rpm for 7 days. To determine the relative biofilm density, the gentian violet staining method (Agat-Med, Russia) was used [29]. The optical density of the samples was measured using an Enspire Model 2300 Multilabel Microplate Reader (Perkin Elmer, Boston, MA, USA). The determination of the ability of the Pseudomonas sp. OBA 2.4.1 to mobilize inorganic phosphorus was tested on plates with Muromtsev's medium (glucose 10 g/L, asparagine 1 g/L, K 2 SO 4 0.2 g/L, MgSO 4 0.2 g/L, corn extract 0.02 g/L, agar 20 g/L; pH 6.8) containing insoluble phosphate. As a source of phosphorus, Ca 3 (PO 4 ) 2 was added to the medium at a concentration of 5 g/L [30]. A daily culture of bacteria was applied as a drop on the surface of an agar medium and incubated at a temperature of 28 • C.
CAS-blue agar was prepared as described by Schwyn (1986), with some modifications [31,32]. To prepare 100 mL of the medium, 6.5 g of chrome azurol S was dissolved in 5 mL of water and mixed with 1 mL of a solution containing 1 mM FeCl 3 and 10 mM HCl. After that, 4 mL of a solution containing 7.3 mg of HDTMA was added to the chrome azurol solution. The resulting mixture was autoclaved and added to the sterile LB medium. The bacteria were grown on the medium for 2 days. A change in color to yellow, orange, or pink revealed the release of siderophores.

Determination of H 2 O 2 Content
Hydrogen peroxide (H 2 O 2 ) levels were determined according to [33]. The samples of plant material were homogenized (1:5 weight/volume) in 0.05M sodium phosphate buffer pH 6.2. The supernatant was separated by centrifugation (Eppendorf ® Microcentrifuge 5415 R, Humburg, Germany, USA) at 15,000× g for 15 min. The concentration of H 2 O 2 in the supernatant was spectrophotometrically (SmartSpecTM Plus, Bio-Rad, Hercules, CA, USA) determined using xylenol orange in the presence of Fe 2+ at 560 nm. The H 2 O 2 was expressed as µmoL g -1 FW.

Determination of the Malondialdehyde (MDA) Content
For the measurement of lipid peroxidation in leaves, the thiobarbituric acid (TBA) test, which determines MDA as an end product of lipid peroxidation [34], was used. Plant material were ground in distilled water and then homogenized in 20% trichloroacetic acid (TCA). The homogenate was centrifuged at 10,000× g for 20 min, and 0.5 mL of the supernatant was added to 1 mL 0.5% (w/v) TBA in 20% TCA. The mixture was incubated in boiling water for 30 min, and the reaction was stopped by placing the reaction tubes in an ice bath. Then, the samples were centrifuged at 10,000× g for 5 min. Absorbance was spectrophotometrically (SmartSpecTM Plus, Bio-Rad, Hercules, CA, USA) measured at 532 nm. The amount of MDA-TBA complex (red pigment) was calculated from the extinction coefficient 155 mM −1 cm −1 . The MDA was expressed as nmoL g -1 FW. Pea seeds (Pisum sativum L., Kelvedonskoye miracle variety) were sterilized in 70% ethyl alcohol for 1 min and 10% sodium hypochlorite solution for 20 min [29]. Thereafter, the seeds were treated with Pseudomonas sp. OBA 2.4.1 (10 7 CFU/mL) for 30 min and germinated on filter paper with sterile water (control) and various concentrations of CdCl 2 (0.1, 0.2, 0.3, 0.4, and 0.5 mM) (stress) at 24 ± 1 • C in the dark for 7 days. Seven-day-old seedlings were taken to assess their shoot length. For the analysis, 50 seedlings were used in each variant in three independent biological replicates.

Statistical Analysis
All microbiological, molecular, biochemical, and physiological experiments were performed in three or more bioassays and three or four analytical tests. The arithmetic average values and confidence intervals calculated from the standard error are shown in the table and graphs (± SEM). Statistically significant differences between the mean values were evaluated using analysis of variance (ANOVA), followed by the Tukey test (p < 0.05).

Identification of the Strain OBA 2.4.1 and Its Main PGP Traits
The sequenced 16S rRNA sequence was deposited in GenBank (http://www.ncbi.nlm. nih.gov/genbank accessed on 28 December 2022) under the number OK039351 and in the rpoD gene under the number OM641958. When comparing fragments of the 16S rRNA and rpoD gene sequences with typical strains, it was found that Pseudomonas sp. OBA 2.4.1 is closest in homology to Pseudomonas fluorescens. This strain is described in detail in a previous study [27].
It was revealed that the strain Pseudomonas sp. OBA 2.4.1 is capable of forming biofilms on inert surfaces. In addition, siderophore activity was also observed ( Figure 1). lings were used in each variant in three independent biological r

Statistical Analysis
All microbiological, molecular, biochemical, and physiolo performed in three or more bioassays and three or four analyt average values and confidence intervals calculated from the stan the table and graphs (± SEM). Statistically significant difference ues were evaluated using analysis of variance (ANOVA), follow 0.05).

Identification of the strain OBA 2.4.1 and its Main PGP Traits
The sequenced 16S rRNA sequence was dep (http://www.ncbi.nlm.nih.gov/genbank) under the number OK gene under the number OM641958. When comparing fragmen rpoD gene sequences with typical strains, it was found that Pseu closest in homology to Pseudomonas fluorescens. This strain is des vious study [27].
It was revealed that the strain Pseudomonas sp. OBA 2.4.1 is films on inert surfaces. In addition, siderophore activity was also

Growth analysis of Pseudomonas sp. OBA 2.4.1 under cadmium s CdCl2)
The presence of Cd in the growth medium of the bacteria le growth of their colonies, especially in the presence of 1.5. and 2 growth analysis of the labelled bacterial colonies showed a simil The presence of Cd in the growth medium of the bacteria led to the inhibition of the growth of their colonies, especially in the presence of 1.5. and 2 mM Cd ( Figure 2A). The growth analysis of the labelled bacterial colonies showed a similar result ( Figure 2B).

Effect of Pseudomonas sp. OBA 2.4.1 strain on pea growth under Cd stress
It was found that the morphometric parameters showed significant visual differences ( Figure 3). Сd stress inhibits the germination and growth of pea plants ( Figure 3А). It was found that treatment with Pseudomonas sp. OBA 2.4.1 stimulated the growth of pea seeds; in the presence of Cd, a gradual deterioration in the growth and develop- It was found that treatment with Pseudomonas sp. OBA 2.4.1 stimulated the of pea seeds; in the presence of Cd, a gradual deterioration in the growth and d ment of seedlings was also observed ( Table 1). It was found that treatment with Pseudomonas sp. OBA 2.4.1 stimulated the growth of pea seeds; in the presence of Cd, a gradual deterioration in the growth and development of seedlings was also observed (Table 1). The results showed that Cd stress resulted in a more than twofold increase in H 2 O 2 content (Figure 4).

Effects of strain Pseudomonas sp. OBA 2.4.1 on the content H2O2 and M under Cd stress
The results showed that Cd stress resulted in a more than twofo content (Figure 4).   This result is accompanied by the same level of MDA accumulation ( Figure 5), but treatment with Pseudomonas sp. OBA 2.4.1 significantly reduced the damage caused by stress. Thus, the content of H 2 O 2 and MDA the outflow was higher (relative to the control) by 1.5 times and 1.6 times, respectively. At the same time, the treatment itself with Pseudomonas sp. OBA 2.4.1 did not affect the state of the membrane structures under normal conditions. This result is accompanied by the same level of MDA accumulation ( Figure 5), but treatment with Pseudomonas sp. OBA 2.4.1 significantly reduced the damage caused by stress. Thus, the content of H2O2 and MDA the outflow was higher (relative to the control) by 1.5 times and 1.6 times, respectively. At the same time, the treatment itself with Pseudomonas sp. OBA 2.4.1 did not affect the state of the membrane structures under normal conditions. .

Discussion
For successful colonization of plant roots, the bacteria must be highly competitive. For example, the ability to form biofilms has a positive effect on the survival strategy of bacteria. There are studies showing that when P. aeruginosa bacteria formed a biofilm, they became more resistant to HMs ions (Cu 2+, Pb 2+ , Zn 2+ ) compared to single bacteria, since polymer compounds bind metal ions, preventing them from entering the biofilm [34,35]. The studied strain of Pseudomonas sp. OBA 2.4.1 also has the ability to form a biofilm, which characterizes it as a PGPM with good competitiveness. Another indicator of PGPM quality was the discovery of siderophore activity (Figure 1), since phosphate mobilization and siderophore activity are also traits of PGPM. Phosphate mobilization helps plants absorb phosphorus, and the secreted siderophores inhibit the growth of pathogenic fungi by reducing the amount of iron available to them in the soil. The ability to produce siderophores is another important feature of PGPM involved in plant growth stimulation [36,37]. Siderophores are low molecular weight (<1000 Da) molecules with a high specificity and affinity for a chelate or Fe 3+ bond. They play an important role in stimulating plant growth, increasing resistance, and protecting against pathogens [38]. OBA 2.4.1 showed good growth on medium with cadmium stress at concentrations up to 1 mM (Figure 2A). Further, with an increase in Cd, the growth of bacteria was

Discussion
For successful colonization of plant roots, the bacteria must be highly competitive. For example, the ability to form biofilms has a positive effect on the survival strategy of bacteria. There are studies showing that when P. aeruginosa bacteria formed a biofilm, they became more resistant to HMs ions (Cu 2+, Pb 2+ , Zn 2+ ) compared to single bacteria, since polymer compounds bind metal ions, preventing them from entering the biofilm [34,35]. The studied strain of Pseudomonas sp. OBA 2.4.1 also has the ability to form a biofilm, which characterizes it as a PGPM with good competitiveness. Another indicator of PGPM quality was the discovery of siderophore activity (Figure 1), since phosphate mobilization and siderophore activity are also traits of PGPM. Phosphate mobilization helps plants absorb phosphorus, and the secreted siderophores inhibit the growth of pathogenic fungi by reducing the amount of iron available to them in the soil. The ability to produce siderophores is another important feature of PGPM involved in plant growth stimulation [36,37]. Siderophores are low molecular weight (<1000 Da) molecules with a high specificity and affinity for a chelate or Fe 3+ bond. They play an important role in stimulating plant growth, increasing resistance, and protecting against pathogens [38]. OBA 2.4.1 showed good growth on medium with cadmium stress at concentrations up to 1 mM (Figure 2A). Further, with an increase in Cd, the growth of bacteria was markedly inhibited. Similar growth was also observed in labeled bacteria ( Figure 2B). This fact shows that labeled bacteria do not lose their resistance to cadmium stress.
It is well known that the antioxidant system plays a fundamental role in maintaining the redox homeostasis of plants under stress [1]. As expected, the presence of Cd in the growth medium led to the development of oxidative stress [1,39], which was accompanied by the depletion of glutathione (GSH) and ascorbate (AsA) pools, as well as the stressinduced activation of GR and APX. The over-accumulation of ROS led to the excessive synthesis of MDA and an increase in the permeability of the membrane structures. An excess of MDA, the end product of lipid peroxidation, and a depletion of GSH, which is a fundamental molecule regulating mitosis [33,39], led to the inhibition of plant growth under stress [32].
An important indicator for assessing the prospects for the use of bacteria is the assessment their influence on plant growth parameters. In this work, it was found that the strain Pseudomonas sp. OBA 2.4.1 stimulated pea seed growth ( Figure 3). The data obtained showed that inoculation of pea seeds with OBA 2.4.1 had a positive effect on the length of the seedlings, which may indicate an increase in plant resistance to cadmium stress at the initial stage of plant growth. Using labeled bacteria, we visually confirmed that they colonize seedling root hairs well. Due to their properties, bacteria have a beneficial effect on growth under cadmium stress.
When growing seeds under cadmium stress at low concentrations of Cd (0.1, 0.2 mM), an improvement in the growth of seedlings was observed. Further, an increase in the concentration of Cd led to the inhibition of seedlings growth. A similar effect is observed in many studies regarding cadmium stress. For example, Jalil et al. (1994) found that a low concentration of Cd 2+ can promote the growth of durum wheat, but at the same time, at a relatively high concentration, wheat growth was inhibited, and Cd resistance varied in different varieties [40]. Yang et al. (2005) found that when the concentration of Cd 2+ was 0-1 mg/kg, the height of the vine also increased, indicating a beneficial effect of Cd 2+ on vine growth [41]. It is also worth noting that with an increase in the growth period under cadmium stress, plant growth is also inhibited; as shown in the study by Liu (2004), the height of corn seedlings under Cd 2+ treatment is significantly reduced. In sorghum plants, and low concentrations of Cd 2+ (≤25 mg/kg) stimulated an increase in plant height, which may be associated with their certain resistance to Cd, but high levels of Cd 2+ inhibited the height growth of plants of the sorghum genus [42]. Thus, it is possible that lower concentrations of Cd 2+ under growing conditions can stimulate plant growth to a certain extent, while higher concentrations suppressed their growth [43].
Many studies show that PGPM treatment has a positive effect on plants, even in the presence of HMs in the medium. For example, alfalfa Medicago sativa L. plants treated with PGPR and grown in the presence of Cu, Pb, and Zn increased shoot length by 22-77% and shoot biomass by up to 220% compared to untreated plants [44,45]. Treatment of Atriplex halimus and Arthrocnemum macrostachyum plants growing in the presence of HM showed an improvement in their morphometric parameters compared to the untreated controls. This may be due to the fact that microorganisms improve plant nutrition by dissolving phosphates, iron, and nitrogen fixation; in addition, they can stimulate plant growth by secreting auxins [46][47][48][49][50][51].
Thus, plant resistance to the toxic effect of Cd may be due to more efficient root growth due to the positive effect of substances released by microorganisms and a decrease in the concentration and accumulation of HMs in the plant root system. The predominant accumulation of Cd in the roots compared to its accumulation in the aboveground plant organs is determined by the barrier functions of the plant root system in relation to toxic HMs [52]. In our opinion, the ability of bacteria to colonize the surface of the roots makes an important contribution to reducing the toxicity of Cd in the environment.

Conclusions
The present study showed that seed inoculation with Pseudomonas sp. OBA 2.4.1, isolated from the rhizosphere of the Oxytropis baschkiriensis, had a growth-promoting and protective effect on pea plants under Cd stress. Treatment with OBA 2.4.1 resulted in a 2.6fold increase in seedling length compared to the untreated plants. The bacterium exhibits PGP properties, including biofilm formation and siderophore production. Moreover, the strain OBA 2.4.1 contributed to the reduction of oxidative stress caused by Cd. The level of hydrogen peroxide and MDA is significantly lower than in untreated stressed plants. In addition, bacteria are able to colonize the roots of pea plants. This ability of bacteria also positively affects the growth and development of plants under stress. The evaluation of the current study suggests that the strain Pseudomonas sp. OBA 2.4.1, can potentially be used as a promising alternative and an environmentally friendly approach to facilitating pea growth and stress tolerance under cadmium stress. However, further field experiments are required to evaluate its full potential for mitigating heavy metals-caused stress in plants.