Modulation of Growth and Antioxidative Defense Mechanism in Wheat (Triticum aestivum L.) Mediated by Plant-Beneficial Strain Pseudomonas veronii MR-15 under Drought Conditions

Abstract

Drought stress severely influences plants in various ways and is considered an alarming threat to sustainable crop production worldwide. However, plant-growth-promoting rhizobacteria (PGPRs) have the natural ability to tolerate drought and enable plants to induce stress resistance by altering critical metabolic pathways. In this study, we isolated and characterized a drought-tolerant rhizobacterium from the ground nut (Arachis hypogaea). Sequencing of the 16S rRNA gene traced its lineage to Pseudomonas veronii, named MR-15. The strain exhibited natural capabilities to solubilize phosphate, produce indole acetic acid, and grow a drought medium containing PEG (polyethylene glycol). The seeds of two wheat varieties (Triticum aestivum) inoculated with MR-15 were grown under drought and fully hydrated conditions and showed a significant increase in plant biomass, enhanced cellular antioxidant enzyme activity, and reduced reactive oxygen species. The MR-15 strain also significantly increased pigmentation and protein contents compared to plants raised from seeds grown without inoculation. These beneficial effects were consistent under drought stress conditions, indicating that MR-15 effectively alleviated wheat plants from drought-induced cellular oxidative damage. The findings suggest that MR-15 has the potential to serve as a biofertilizer, and further experiments should be conducted to explore its role in promoting plant growth and yield under drought conditions, particularly in semi-arid and arid zones. This is the first study reporting Pseudomonas veronii as a potential PGPR strain.

1. Introduction

Wheat, with over 700 million tons produced annually, is the second most significant food grain globally, following rice [1]. However, wheat crops in arid and semi-arid regions suffer substantial yield losses due to limited water availability [2]. Drought-induced osmotic and oxidative stresses alter plants’ physiological, biochemical, and molecular characteristics, consequently diminishing yields. The primary contributors to reduced crop growth and yields under drought include altered plant–water relations, reduced photosynthesis, cellular oxidative stress, membrane damage, and inhibited enzyme activity [3,4].

Developing drought-resistant varieties poses a substantial challenge for plant biologists. While genetic modification and transgenic methods are considered standard tools to address these challenges, their complexity, cost, time consumption, limited genetic diversity, ecological constraints, and social, ethical, and political issues pose significant hurdles [5,6,7].

In recent years, the potential use of biofertilizers has emerged as a notable adaptation against drought stress. Several bacterial genera play pivotal roles in soil ecosystems, regulating numerous biotic activities that diversify soil nutrients and ultimately enhance crop growth [8]. These biofertilizers stimulate nutrient transport, produce growth regulators, suppress phytopathogens, improve the soil structure, and detoxify the soil by degrading toxic compounds such as pesticides, xenobiotics, and plant hormones [9,10]. Notably, rhizobacteria, which colonize plant roots and the surrounding soil, represent a well-known type of biofertilizer due to their capacity to transform, solubilize, and mobilize nutrients from bulk soil [11,12].

Rhizobacteria are crucial in maintaining soil fertility by recycling nutrients [13]. Owing to their diverse roles in improving crop production, biological approaches have garnered attention from plant scientists for managing the plant nutrient system. Substantial research has explored the various roles of rhizobacteria in enhancing plant growth [10,14], stress tolerance [12,15], pesticide detoxification [9], the biological control of pathogens [16,17,18,19], phytohormone production [20], ammonia production, and nitrogenase activity [21,22]. Due to these beneficial effects, symbiotic and non-symbiotic rhizobacteria are used globally as bio-inoculants to enhance growth under severe environmental stress [23,24,25,26,27].

2. Materials and Methods

2.1. Bacterial Isolation

The MR-15 strain was obtained from the rhizospheric soil of groundnut during its growing season in the Talla Gang district of Chakwal. Following collection, the soil sample was stored at 4 °C for subsequent studies. Bacterial isolation was carried out using the serial dilution method proposed by Somasegaran and Hoben [28]. Specifically, 1 g of tightly adhered soil from the root was mixed with 9 mL of 0.85% (w/v) NaCl solution and subjected to further serial dilution. Then, 100 μL of dilution 5 was added to nutrient agar media to isolate the bacteria. The isolate selection from the numerous rhizospheric isolates was based on morphological parameters, including colony shape, color, texture, and motility differences.

2.1.1. Drought Tolerance Potential

To evaluate the potential for drought resistance, the isolated rhizospheric bacterial strain was grown at different levels (5%) of polyethylene glycol (PEG-6000) in Luria Broth (LB)-enriched media.

2.1.2. Culture Preservation

The purified bacterial isolate, grown in LB broth media at 27 °C for 24 h, was preserved in 20% glycerol at −80 °C after complete growth.

2.1.3. Phosphorus Solubilization

To assess the phosphorus solubilization ability of MR-15, which is one of the key features of PGPRs, a single purified bacterial colony was streaked on Pikovskaya agar using the method proposed by Pikovskaya [28]. After sealing the plate with parafilm, it was incubated at 27 °C for 48 h. The formation of a clear halo zone around the bacterial colony indicated phosphate solubilization. So, the strains exhibiting halo zones were selected as positive phosphate solubilizers. The diameter of the halo zone was measured after 48 h of incubation.

2.1.4. Phosphate Solubilization Index (PSI)

For PSI estimation, the bacterial isolate was grown on Pikovskaya’s media and incubated for 7 days at 28 °C. The PSI was determined using Pikovskaya’s formula [29].

SI = (Colony diameter (cm) + halo zone diameter (cm))/colony diameter (cm)

2.1.5. Indole-3-Acetic Acid Production

The ability of bacteria to produce indole-3-acetic acid (IAA) was investigated by inoculating the bacterial isolate into nutrient broth (100 mL) additionally supplemented with 100 mg L⁻¹ tryptophan in a 500 mL Erlenmeyer flask, followed by incubation at 28 ± 2 °C for 48 h on an orbital shaker at 150 rpm. After producing a well-grown culture, a 40 mL sample was collected in a Falcon tube and centrifuged at 13,000 rpm for 15 min to harvest the supernatant. Then, 80 μL of the supernatant was added to 160 μL of Salkovski reagent (150 mL of conc. H₂SO₄, 250 mL of distilled water, and 7.5 mL of 0.5 M FeCl₃). The mixture’s test tube was placed in the dark for 30 min. After 30 min, the development of a pink color indicated IAA synthesis by the isolate, and the observation was recorded at 535 nm on the spectrophotometer (Hitachi U-2100; Hitachi, Tokyo, Japan). The measurement was based on a standard curve drawn with a series of dilutions of IAA (Sigma Aldrich, Chemie GmBH, Munich, Germany).

2.1.6. Molecular Identification of Bacterial Isolates

The genomic DNA of the growth-promoting isolate having the inherent capability to decolorize azo dye and tolerate drought was isolated by following the Maniatis et al. [30] procedure and then further quantified using a Nanodrop spectrophotometer. The 16S rRNA gene was amplified using standard primers, including fD1 and rD1 [31]. The amplified PCR products were purified and sequenced by Macrogen, Seol, Republic of Korea. The Sequence Scanner software v1.0 software (Applied Biosystems, Foster City, CA, USA) was used for the analysis, and the sequencing results were compared with the GenBank database by using the NCBI (National Center for Biotechnology Information) BLASTn (Basic Local Alignment Search Tool) (https://blast.ncbi.nlm.nih.gov/Blast.cgi)

The final sequence of the isolate was deposited in GenBank to obtain the accession numbers.

2.1.7. Phylogenetic Analysis

The 16s rRNA sequence of the identified bacterial strain was subjected to BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for homology searching against the GenBank database of the National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed using the type strains of the identified strain(s) with the help of MEGA 7.0 software. For phylogenetic tree construction, the neighbor-joining (NJ) algorithm was used.

2.2. Field Experiment

2.2.1. Bacterial Culture, Soil Analysis, Field Site, and Seed Inoculation

After isolation and characterization, the bacterial strain MR-15 was used to inoculate the seeds of two wheat varieties. For the inoculum preparation, the bacterial colony was cultured in Luria Broth-based liquid medium in an Erlenmeyer flask (500 mL) and incubated in an orbital shaker for 24 h at 32 °C and 120 rpm until a concentration of 108 colony-forming units (CFU) mL⁻¹ was reached. The bacterium was then harvested by centrifugation at 8000 rpm for 10 min, and the pellet was washed with sterile distilled water. Further dilution after centrifugation to a concentration of 107 CFU mL⁻¹ was performed for the inoculation of seeds.

The experiment was conducted between November 25 (temperature ranges between 25–27 °C) and April 25 in the experimental field located in BARI (Barani Agriculture Research Institute), Chakwal, Pakistan. The soil was classified as typical sandy clay loam in texture. The detailed physiochemical analysis of the soil is given in

Table 1. Soil profile of BARI field, Chakwal.

Parameters	Value
Textural class	Sandy clay loam
pH	8.1
Bulk density (g cm−3)	1.49
Electrical conductivity (dS m−1)	0.62
Organic matter (%)	0.58
Available P (mg kg−1)	6.7
Extractable K (mg kg−1)	128
Total nitrogen (%)	0.03

. The seeds of two Triticum aestivum varieties, Sehar-06 (V1) and GA-2002 (V2), were sterilized by using ethanol (70%) for 2 min and then sodium hypochlorite (5%) for 5 min and subsequently washed with distilled water three times. The inoculum was applied to sterilized seeds; control seeds were divided into two categories. Non-primed seeds (NP) were sown directly into the soil, but hydro-primed seeds (HP) were sown after soaking the seeds in water for 1 h. Nine seeds were sown in each block. The field experiment was designed as a Randomized Complete Block Design (RCBD) with three replicates. To maintain the stress conditions, the soil field capacity was determined, and according to the treatment, water was applied. One group was irrigated at 60% field capacity, while the second group was irrigated at normal field capacity (100%).

2.2.2. Germination Attributes

Germinating seeds were counted daily after sowing until the total and uniform germination of seeds was attained. The following aspects of germination indicate the extent to which PGPRs affect the germination potential:

• Germination Energy (GE);
• Mean Emergence Time (MET);
• Days to 50% Emergence (E₅₀);
• Coefficient of uniformity of emergence (CUE);
• Final germination percentage (FG%);
• Germination Index (GI).

2.2.3. Morphological Attributes

The fresh/dry weight, the length of the root shoot, the number of leaves, and the flag leaf area of the plants were measured immediately after the harvest. The weight of the shoot was measured directly, while the root weight was measured after removing the soil particles by washing under running tap water and blotting. The shoot/root length and leaf area were measured in centimeters with the help of a long wood scale. The shoot and root samples were then wrapped in paper bags to measure the dry mass. To dry these plant samples, 60-degree temperatures were set in the oven for a week to dry the root and shoot samples completely.

2.2.4. Photosynthetic Attributes

The growth and yield of any plant are directly related to its photosynthetic ability, which in turn is dependent on plant pigmentation. The estimation of photosynthetic pigments in the leaves, such as chlorophyll a (Chl. a), chlorophyll b (Chl. b), total chlorophyll (T.Chl.), and carotenoid contents, were estimated by using the Arnon (1949) [32] method. Briefly, 0.1 g of fresh leaf material was ground thoroughly in 80% acetone (10 mL) and filtered. At wavelengths of 663, 645, and 480 nm, the absorbance of the filtrate was measured spectrophotometrically.

The formulas given below were used for the estimation of photosynthetic pigments.

Chl. a = [12.7(A 663) − 2.69 (A 645)] × V/1000 × W

Chl. b = [22.9 (A 645) − 4.68 (A 663)] × V/1000 × W

Total Chl. = [20.2 (A 645) − 8.02 (A 663)] × V/1000 × W

V = volume of the extract (mL)

W = weight of the fresh leaf tissue (g)

However, for the estimation of leaf carotenoid contents, the formula given by Kirk and Allen (1965) was used [33].

Carotenoids (mg mL⁻¹) = A.car/Em 100% × 100

A Car (carotenoid) = (OD 480) + 0.114 (OD 663) − 0.638(OD 645)

Em (Emission) = Em 100% = 2500

2.2.5. Antioxidant Enzymes

To evaluate the tolerance potential of the wheat plants inoculated with the MR-15 strain, the activities of different antioxidant enzymes, such as POD, SOD, and CAT, were determined by following the recommended methods.

The peroxide dismutase (POD) activity was estimated using the method developed by Chance and Maehly [34]. In test tubes, 0.1 mL of guaiacol solution, 2.7 mL of phosphate buffer, and 0.1 mL of enzyme extract were added and mixed well. The absorbance at 470 nm was noted with a spectrophotometer every 20 s up to 120 s. The change in absorbance of 0.01 units per minute defines one unit of POD activity. The unit enzyme/m protein was used to express the specific enzyme activity.

The determination of superoxide dismutase was carried out by using the method proposed by Beauchamp and Fridovich [35]. The principle of photoreduction of nitroblue tetrazolium (NBT) was followed. First, 100 microliters of methionine, along with 560 microliters of phosphate buffer, 50 microliters of riboflavin, 100 microliters of enzyme extract, and NBT, was taken for the preparation of the reaction mixture. After shaking, the mixture was placed under 15 W light lamps for 10 min. The reaction soon began under the light. After a period of 10 min, the absorbance was measured at 560 nm by using a spectrophotometer.

The catalase activity was measured using the method proposed by Chance and Maehly [34]. Freshly frozen leaves (0.2 g) were ground, and 5 mL of 50 mM phosphate buffer at pH 7.8 was added. The temperature was maintained by placing the mortar and pestle on ice. After thorough grinding, the mixture was centrifuged at 14,000 rpm for 10 min at 4 °C. The reaction mixture was prepared by adding 0.1 mL of the supernatant and 0.3 mL of the reaction mixture of H₂O₂ (30% H₂O₂ mixed with 200 mL of phosphate buffer at pH 7.0). The absorbance of this reaction mixture was recorded at 240 nm (at intervals of 20 s) for 120 s.

2.2.6. Non-Enzymatic Molecules with Antioxidant Function

The approach developed by Mukherjee and Choudhuri [36] was followed to determine the ascorbic acid content in the leaves. Briefly, 0.5 g of fresh leaf material was homogenized in 6% trichloroacetic acid (TCA). The supernatant was obtained after centrifugation at 10,000 rpm for 10 min. Then, 2 mL of the supernatant was mixed with 0.5 mL of DTC reagent (dinitro phenyl hydrazine, thiourea, copper sulfate). After adding ice-cold sulfuric acid, the optical density was measured spectrophotometrically at 520 nm.

The method given by Julkenen-Titto [37] was followed to determine phenolics. First, 0.5 g of fresh leaf material was ground in 10 mL of ethanol (80%). After centrifugation, 0.25 mL of the supernatant was mixed with 1.25 mL of Folin–Ciocalteu phenol reagent, and then 1.25 mL of sodium carbonate was added to the mixture. The absorbance was measured at 750 nm with a spectrophotometer.

The total flavonoid content was measured using the aluminum chloride colorimetric method [38]. First, 0.5 g of fresh plant sample was ground by adding 5 mL of 80% methanol. After thorough grinding, the reaction mixture was prepared by adding 1 mL of sample to 0.3 mL of NaNO₂ (5%), 0.3 mL of 10% AlCl₃ (10%), and 2 mL of NaOH (1M). After the preparation of the reaction mixture, the absorbance at 510 nm was taken with a spectrophotometer.

Total anthocyanins were determined using the Hodge and Nozzolillo method [39]. With this method, 0.5 g of leaf material was ground thoroughly using a pestle and mortar, and 5 mL of phosphate buffer (K₂HPO₄ + KH₂PO₄, pH 7.8) was added. The pestle and mortar temperature was maintained by using ice during grinding. After grinding, centrifugation at 10,000 rpm at 4 °C was carried out, and the supernatant was carefully stored in the refrigerator. The absorbance of the supernatant was directly measured at 600 nm with a spectrophotometer using phosphate buffer as a blank.

2.2.7. Total Soluble Protein, Total Soluble Sugars, and Membrane Permeability

The total soluble protein content was measured by following the Bradford [40] method. First, 0.5 g of leaf material was thoroughly ground in 10 mL of 50 mM phosphate buffer (pH 7.8) in a mortar with a pestle, and the temperature was maintained using liquid nitrogen. The ground leaf material was centrifuged at 10,000 rpm for 10 min, and the supernatant was obtained. Then, 100 µL of the extract and 2 mL of Bradford reagent were mixed in clean test tubes, and after 15–20 min, the absorbance was measured at 595 nm with a spectrophotometer.

Total soluble sugars were determined by following the method of Yoshida et al. (1971) [41]. In the first step, 0.1 mL of plant sample was taken in the test tube, and 3 mL of anthrone reagent was added. After being vortexed gently, the reaction mixture was heated at 95 °C for 15 min. The absorbance of the samples was measured at 625 nm with a spectrophotometer.

For the estimation of H₂O₂ content, the method described by Velikova [42] was followed. Leaf samples were thoroughly ground in TCA, the mixture was centrifuged at 10,000 rpm, and then 0.1 mL of the supernatant was mixed with 1 mL of KI. The absorbance of the resultant solution was taken at 390 nm.

The Carmak and Horst [43] method was used to estimate the amount of Malondialdehyde (MDA) in the leaves. Thiobarbituric acid (TBA) was made in TCA to measure MDA. A leaf sample weighing 0.5 g was ground in 0.1% trichloroacetic acid. The supernatant was collected after centrifuging the reaction mixture for ten minutes at 12,000 rpm. Then, 0.5 mL of TBA solution, 1 mL of 20% TCA, and 1 mL of the supernatant were combined. After 30 min of heating the reaction mixture at 100 °C, the absorbance was measured at 450, 532, and 600 nm using a spectrophotometer. MDA was calculated using an absorbance coefficient of roughly 155,000 nmol mol⁻¹ nmol mol⁻¹.

Malondialdehyde (nmol mL⁻¹) = {(A532-A600)/155,000}106

2.2.8. Yield Attributes

Different yield attributes were determined to evaluate MR-15’s influence on plant productivity under normal and stressed conditions. Yield attributes, including spike length, no. of spikes per plant, no. of spikelets per plant, no. of tillers per plant, no. of grains per spike, no. of grains per plant, no. of grains per spikelet, 100-grain mass, total grain mass/plant, and total plant yield, were recorded.

2.3. Statistical Analysis

Using the CoStat computer program (Version 6.303, PMB 320, Monterey, CA, USA), an ANOVA of the data for all variables was calculated. At a probability level of 5%, the LSD values were calculated [44]. XLSTAT software (Version 19.4; Addinsoft, New York, NY, USA) was also used to determine the Pearson Product Moment Correlation Coefficient (‘r’), which is a measure of the linear relationship between two independent variables.

3. Results

3.1. Physico-Chemical Identification and Characterization of Bacterial Strain MR-15

MR-15 was isolated from the rhizosphere of peanut in water-deficient soil located in the Tallagang district of Chakwal, Pakistan. Morphologically, it appeared as a short, rod-shaped, Gram-negative bacterium, displaying round, small, smooth, milky white, and creamy colonies on culture media (

Table 2. Morphological analysis and identification of bacterial isolate from rhizosphere of ground nut by 16S rRNA gene sequence analysis.

Isolate Code	Colony Morphology on LB		Cell Morphology
MR-15	Small, smooth, creamy		Short rod
Isolate code	Base pair sequence from 16S rRNA gene	Closest GenBank match	% identity	Strain identified as	GenBank accession No.
MR-15	1594	Pseudomonas xanthomarina strain SBT1	98.88	Pseudomonas veronii	OL851707

). The 16S rRNA gene sequence analysis, spanning 1595 base pairs, using the BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) technique revealed a 98.88% sequence homology with the genus Pseudomonas and several strains within the same species. Based on this homology, the strain was identified as Pseudomonas veronii (OL851707).

3.1.1. Screening of Drought Tolerance Potential

The MR-15 strain was grown in a nutrient broth culture supplemented with 5% PEG to assess its drought tolerance potential. The strain exhibited robust growth even under PEG-induced drought conditions, as depicted in Figure 1. Rhizospheric isolates other than MR-15 were unable to grow on 5% PEG-enriched media.

3.1.2. Phosphate Solubilization by MR-15

Insoluble phosphate was utilized in the culture medium to assess phosphate solubilization. MR-15 exhibited a clear halo zone on Pikovskaya agar (Supplementary Figure S1). The phosphate solubilization index measured approximately 2.5 cm. A halo zone surrounding the bacterial colony indicated the solubilization of insoluble phosphates, leading to the production of soluble phosphates and organic acids in the medium (

Table 3. Estimation of phosphate solubilization index and indole acetic acid.

Sr. No.	Strain Label	Source	PSI	IAA ppm
1	MR-15	Rhizosphere	2.5	13.31955

) [45].

Indole Acetic Acid Production

The capacity for IAA production by MR-15 was determined using the Salkowski reagent. A distinct pink color developed and intensified over 30 min (Supplementary Figure S2). The optical density was measured via spectrophotometric analysis. MR-15 exhibited a sharp pink color, with an IAA concentration of 13.32 ppm (Table 3).

3.1.3. Phylogenetic Analysis

The identification of the bacterial isolate via 16S rRNA sequence analysis indicated its classification within the Pseudomonas genus. The strain’s 16S rRNA sequence was aligned with closely related bacterial sequences retrieved from the NCBI database (Figure 2). A phylogenetic tree was constructed using Mega version 7 through maximum-likelihood analysis [46].

3.2. Germination Response of Wheat to Inoculation with MR-15

Different germination parameters showed that non-stressed seeds had a higher germination ratio than drought-stressed seeds, whereas a considerable decrease in all of the germination attributes was observed (Figure 3). The application of MR-15 as an inoculating agent increased all of the germination parameters under water stress conditions. The germination index significantly increased in normal (41%) and stress (51%) conditions; the coefficient of uniformity of emergence (CUE) was increased by up to 8% under normal conditions and 4% under stress. However, MET increased under normal conditions by up to 12%. Similarly, E50 improved by up to 17% under normal conditions and 15% under stress in V1, but the opposite was true for V2 under stress. FG% increased by between 10 and 30% under normal conditions in both varieties, but it increased by 25% under stress in V2. Similarly, the inoculated seeds of V2 showed a significant increase in G.E. under normal (50%) and stressful (33%) conditions.

3.3. Growth Response of Wheat to Inoculation with MR-15

The data presented in Figure 4 illustrate the plants’ growth response. The application of MR-15 notably increased the shoot length, exhibiting a significant rise in V2 under both normal (40%) and stress (33%) conditions. The most significant increase in shoot fresh weight was observed in V1 under stress conditions. Both fresh and dry masses exhibited a significant increase, up to 72%. Regarding root fresh (43%) and dry weights (85% and 22%), a notable increase was observed in seeds inoculated with MR-15, particularly in V1 under normal and stress conditions. The leaf area also expanded significantly (36%) under stress conditions, while the increase in leaf number in V2 was non-significant (13%).

3.4. Photosynthetic Pigment Response of Wheat to Inoculation with MR-15

MR-15 had a substantial impact on all three photosynthetic pigments. The positive effect on chlorophyll a was more pronounced in V1 under normal conditions (133%) and in V2 under stress conditions (80%) (Figure 5). Similarly, there was a highly significant increase (160%) in chlorophyll b in V2 under normal conditions, whereas this increase was non-significant under stress. Total chlorophyll content followed a similar pattern, with the maximum impact observed in V2 under both normal (75%) and stress (55%) conditions for inoculated seeds.

3.5. Response of Wheat Antioxidant Enzymes to the Inoculation with MR-15

Under normal conditions without MR-15 inoculation, plants exhibited increased catalase activity. However, drought stress notably reduced catalase activity. Inoculation with MR-15 conferred resistance to water stress, resulting in a 52% increase compared to un-inoculated plants. Notably, V1 (Sehar-06) showed a highly significant difference from the control plants, while in V2 (GA-2002), this increase was non-significant at 26% (Figure 6).

Regarding peroxidase (POD) activity, both varieties showed a significant difference compared to non-primed plants, with a POD enhancement of 30% under normal conditions. However, under stress, MR-15-primed plants exhibited increased POD activity (43%) compared to non-primed plants, with a more pronounced effect observed in V2 than in V1.

In the absence of MR-15, 60% water field capacity (WFC) induced a significant reduction in superoxide dismutase (SOD) activity. Conversely, under drought stress, MR-15 significantly increased SOD activity by up to 91%. Under normal conditions, an 80% increase in SOD activity was recorded.

3.6. Non-Enzymatic Molecules with Antioxidant Function

Non-primed plants exhibited a phenolic content of up to 2.6 mg g⁻¹ F.wt. Inoculation with MR-15 significantly increased total phenolics in both varieties under normal conditions (100% and 60%), with the most significant increase recorded in V2 (66%) under drought stress. However, a notable difference was found in flavonoid accumulation, where V1 showed a highly significant increase (65%) compared to V2 under normal conditions (Figure 7).

Without MR-15 application, both non-primed and hydro-primed plants exhibited ascorbic acid contents between 1.3 and 1.6 µg/g F.wt. Drought stress decreased ascorbic acid in both types of plants by 33% and 30%. In contrast, MR-15-inoculated plants showed an 87.5% increase compared to control plants, with only a 6.5% decrease in ascorbic acid under drought stress. The most significant difference was observed in V2, while V1 showed a modest improvement (18%) only under normal conditions.

Total flavonoid content significantly increased in V2 under both conditions. Control plants exhibited a higher phenolic concentration under normal conditions than under stress, but MR-15 significantly increased (66%) under normal and drought stress conditions (75%).

Plants grown at 100% WFC with MR-15 exhibited higher carotenoid content (1.42 mg/g F.wt) than plants at 60% without MR-15 inoculation (0.21–0.42 mg/g F.wt). The difference between MR-15-treated and untreated plants at 60% WFC and 100% WFC was 140% and 150% for V1 and V2.

Without MR-15 application, decreases of 53% (V1) and 42.8% (V2) in anthocyanin content under stress were observed. However, plants treated with MR-15 at 100% WFC exhibited concentrations of 2.1 (V1) and 2.2 (V2) mg/g F.wt, representing a 200% increase in both varieties. Similarly, MR-15-treated plants at 60% WFC exhibited anthocyanin concentrations of 1.27 and 1.5 mg/g F.wt., nearly three times higher than those of their non-primed counterparts.

3.7. Membrane Permeability Response of Wheat to Inoculation with MR-15

In the absence of MR-15, MDA increased more under stress compared to normal conditions; this was observed in both varieties(Figure 8). V1 showed higher accumulation in non-primed (50%) and hydro-primed plants (80%), while V2 showed lower MDA accumulation in non-primed (12.9%) and hydro-primed (25%) plants. The application of MR-15 significantly reduced MDA accumulation in both varieties (27% and 43%).

H₂O₂ levels were 3.2–3.1 µg/g F. wt. in V1 (N.P and H.P) at 100% WFC, increasing significantly to 4.33–6.46 µg/g F. wt. at 60% WFC. V2 showed a similar trend under normal conditions (2.7–3.4 µg/g F. wt. at 100%) and a significant increase (4.4–4.5 µg/g F. wt.) at 60% WFC. MR-15 application reduced H₂O₂ levels in both varieties (40%, 30%) at 100% WFC.

Without MR-15 application, 100% WFC increased total protein accumulation in V1 (2.64–3.1 mg/g F. wt.) and V2 (3.5–3.9 mg/g F. wt.), while reduced levels of WFC (60%) significantly decreased the protein concentration in both varieties (V1: 1.5–2.4 mg/g F. wt.; V2: 2.1–2.7 mg/g F. wt.) in both types of plants. Applying 100% WFC and MR-15 significantly improved both varieties’ total soluble protein content. However, the combined application of 60% WFC and MR-15 showed less protein accumulation in both varieties, although still significantly higher than in non-inoculated plants (V1: 73%; V2: 90%).

Plants grown at 100% WFC (non- and hydro-primed) exhibited higher total sugar content compared to those grown at 60% WFC in V1 (2.4–2.6 µg/g F. wt.) and V2 (2.26–2.48 µg/g F. wt.). MR-15 treatment increased total sugars at 100% and 60% WFC in both varieties. V1 exhibited a non-significant increase (12%), while V2 showed a significantly higher level of total sugars at both 100% (54%) and 60% WFC (45%).

3.8. Yield Response of Wheat to Inoculation with MR-15

A decrease in the number of tillers (3.3–2.6) was observed with reduced water field capacity (100–60%) (Figure 9). MR-15 inoculation significantly increased the number of tillers per plant (4–3.6) in both varieties. Without MR-15 application, the spike length was reduced by 8% and 11% under normal and stressful conditions. Inoculation with MR-15 increased the spike length by 9% in V1, but this improvement was non-significant in V2. Non-primed and hydro-primed control plants exhibited 2.3 and 2.6 spikes per plant, respectively. However, at 60% WFC, V1 showed a maximum of 3.6 spikes. MR-15 priming enhanced spike numbers, but this increase was non-significant compared to the control plants.

Under a reduced water supply, both varieties showed a reduction in the 100-grain mass when MR-15 was not applied. The maximum 100-grain mass was achieved by V2 in non-primed (5.7 g) and hydro-primed plants (5.2 g) at 100% WFC. At 60% WFC, a significant reduction in the 100-grain mass was observed in both varieties. MR-15 significantly increased the 100-grain mass in both varieties under normal (46% and 12%) and stress (60%, 14%) conditions.

Total grains per plant were significantly reduced by stress in both varieties (12% and 39%). MR-15 application exhibited a significant increase in total grains at 100% WFC (66%) and 60% WFC (64%). Overall, the total plant yield was significantly boosted by MR-15 application, increasing by up to 200% under normal conditions and 150% under stress conditions.

4. Discussion

The present study documented a significant decrease in all growth attributes [47] alongside reduced leaf photosynthetic pigments. Similar decreases in plant growth and photosynthetic pigments due to drought have been previously reported in various crops, including soybean [48], maize [49], and cucumber [50]. Drought stress also significantly influences the leaf total phenolic content, although the increase or decrease varies among plant species and cultivars [51].

Various adaptations are being explored to mitigate the harmful effects of abiotic stress with the prioritization of environmental sustainability in plant science. The utilization of bio-inoculants, particularly soil microbes, is advocated as a superior alternative to fertilizers and pesticides. Plant-growth-promoting rhizobacteria (PGPRs) are a vital soil component among these. Under diverse abiotic stresses, PGPRs have demonstrated efficacy in enhancing growth and nutrient uptake [52,53,54]. The priming agent MR-15 notably enhanced shoot length and shoot fresh and dry weights under stress conditions. Moreover, this PGPR strain increased the shoot length and induced resistance to drought stress. Similar findings have been reported in prior studies involving Ocimum basilicum L. [55], maize [56], canola plants [57], and cotton [58] under various abiotic stresses, including drought, oxidative, and salt stresses.

The application of different PGPR strains has been linked to increased root and shoot growth in various plants under drought stress, such as Helianthus annuus, potato, and cucumber, as documented by several researchers [59,60,61]. It is suggested that plant-growth-promoting rhizobacteria inherently enhance rhizospheric colonization, significantly increasing the crop yield [62].

Our genome sequence analysis of this PGPR strain revealed a direct relation to Pseudomonas. Previous studies have also shown a correlation between root development and indole-3-acetic acid (IAA) secretion by PGPRs. IAA promotes root surface area, growth, development, and metabolism [63]. Hence, the observed increase in root development in both wheat varieties might be attributed to IAA secretion by the studied PGPR. MR-15 exhibited the highest concentration of IAA, supporting a positive relationship between IAA and root growth, as noted in other studies [64].

Oxidative stress intensity correlates with the cellular Malondialdehyde (MDA) concentration [65]. Elevated MDA levels trigger physiological disturbances within cells, including increased membrane permeability, reduced chlorophyll content, the degradation of macromolecules like proteins and nucleic acids, nutrient remobilization, and stunted crop growth [66,67]. During drought stress, non-treated plants exhibited increased MDA and reactive oxygen species (ROS) contents (Figure 7), indicating the reduced antioxidant capacity of wheat plants. Conversely, wheat plants primed with the MR-15 strain displayed lower MDA and H₂O₂ contents than non-primed plants, highlighting an enhanced ability to scavenge oxidants under drought stress.

Plant enzymes like SOD, POD, and CAT play pivotal roles in removing the reactive oxygen species generated during metabolism, maintaining a balance between their production and removal, and preventing oxidative damage [65].

5. Conclusions

The comprehensive impact of drought stress was evident, adversely affecting growth, yield, pigmentation, and antioxidant levels. However, the positive influence of the Pseudomonas veronii (MR-15) strain was notable in mitigating drought stress and effectively sustaining wheat growth and associated functions. Although many Pseudomonas strains have been reported as PGPRs, the current study documented the PGPR characteristics of Pseudomonas veronii for the first time. The application of Pseudomonas veronii (MR-15) led to increases in the germination rate, growth parameters, photosynthetic and non-photosynthetic pigments, the activities of antioxidant enzymes, and non-enzymatic molecules with antioxidant function, ultimately bolstering yield attributes. Pseudomonas veronii (MR-15) enhanced drought stress tolerance across both wheat varieties. Its inherent capacity to produce indole-3-acetic acid (IAA) and solubilize phosphate may underlie its positive impact on plants. Considering this perspective, the application of MR-15 (Pseudomonas veronii) strain isolates emerges as a promising approach to enhancing the growth and yield of wheat under drought stress conditions.