*2.8. Plant Inoculation*

A pot experiment was conducted in the greenhouse of the Botany Department, PMAS-AAUR, Rawalpindi. A complete randomized design was applied with three replications. Each selected halotolerant strain was grown overnight in LB media. To obtain a cell pellet, the supernatant was discarded after centrifugation at 3000 rpm for 3 min. The cell pellet was washed three times with autoclaved water and the absorbance was recorded with a spectrophotometer at 600 nm to obtain the desired concentration, i.e., 107 CFU. Ten sterilized seeds were sown in each pot (containing 10 kg of soil) in the greenhouse with the day 10 h/14 h night at a temperature of 21/15 ◦C. Soil moisture was maintained at 15 ± 1%. Four strains and their consortium were evaluated under two treatment controls and 150 mM NaCl stress. The salt level was maintained with EC of 4.0 dS m−<sup>1</sup> (first irrigation) or 8.5 dS m−<sup>1</sup> (second irrigation). Plants were harvested after 45 days of sowing. Fresh and dry biomass was recorded. Leaf area was measured with the help of a leaf area meter. All the samples were collected in zipper bags and stored at −20 ◦C freezer for further biochemical assays. The percent of water content was determined by measuring the ratio between the fresh and dry weight of the upper fully developed leaf by using the following formula [27]:

$$\text{RWC} = [\text{FW} - \text{DNV}] \text{[TW} - \text{DW]} \times 100 \tag{1}$$

#### *2.9. Electrolyte Leakage (%)*

Electrolyte leakage was determined by the method of Srairam [28]. Leaf discs weighing 0.1 g were heated in 10 mL of distilled water for 30 min at 40 ◦C and the electrical conductivity (C1) was recorded. The same discs were then heated at 100 ◦C and again electrical conductivity (C2) was recorded. Whereas, calculations were done by the following formula:

$$\text{MSI} = \left[1 - \text{(C1/C2)}\right] \times 100\tag{2}$$

#### *2.10. Chlorophyll and Carotenoid Content*

Leaf chlorophyll a, b, total chlorophyll, and carotenoid contents were estimated by the method of Arnon [29]. Fresh leaves (0.5 g) were ground in 10 mL of 80% acetone. The readings of the filtrate were measured at 470 nm, 663 nm, and 645 nm. Calculations were done by the following equations:

$$\text{Chla (mg/g)} = \left[ 12.7 \text{A}\_{663} - 2.69 \text{A}\_{645} \right] \left( \upsilon / w \right) \tag{3}$$

$$\text{Chllb (mg/g)} = \left[ 22.9 \text{A}\_{645} - 4.68 \text{A}\_{663} \right] \left( \upsilon / w \right) \tag{4}$$

$$\text{Total chlorophyll} \left(\text{mg/g}\right) = \left[\left(20.2 \text{A}\_{645} + 8.02 \text{A}\_{663}\right) \text{v/}w\right] \tag{5}$$

$$\text{Caroteroids content(mg/g)} = (1000 \text{ A}\_{470} - 1.8 \text{ Chl}\_{\text{a}} - 85.02 \text{ Chl}\_{\text{b}}) / 198 \tag{6}$$

where A is the optical density at a specific wavelength.

#### *2.11. Proline Content*

Proline contents were determined by following the protocols of Bates [30]. Fresh leaves (0.5 g) were homogenized with 10 mL of sulfosalicylic acid (3.0%). The solution was filtered, and the filtrate was mixed with equal amounts of glacial acetic acid and ninhydrin reagent. The mixture was heated for 1 h in a water bath at 90 ◦C and the reaction was stopped by transferring the mixture to ice. Toluene (1 mL) was added to the mixture and the solution was mixed and the solution separated into two layers. The upper layer was isolated in separate test tubes and the reading was measured at 520 nm. Proline was determined as follows:

$$\text{Proline} = \text{(Reading of sample} \times \text{Diluted concentration} \times \text{K value)/material weight} \tag{7}$$

#### *2.12. Total Soluble Sugar and Amino Acid*

Soluble sugars were estimated after the method of Dubois et al. [31]. Ground plant tissue (0.1 g) was mixed with 3 mL of 80% methanol. The solution was heated in a water bath for 30 min at 70 ◦C. An equal volume of extract (0.5 mL) and 5% phenol was mixed with concentrated sulphuric acid (1.5 mL) and was again incubated in the dark for 30 min. The absorbance of the sample was checked at 490 nm and the calculations were done by applying the following formula:

$$\text{Sugar } (\mu \text{g/mL}) = \text{Absorbance of sample} \times \text{Dilution factor} \times \text{K value} \tag{8}$$

Fresh tissue in grams.

The standard curve was prepared for glucose solution, which was used for the determination of the amount of sugar, expressed in mg g−<sup>1</sup> fw<sup>−</sup>1.

The Ninhydrin method was used for the determination of free amino acids [32]. Leaf extract (1 mL) was mixed with the same volume of 0.2 M citrate buffer (pH-5) and 80% ethanol, and 2 mL of the ninhydrin reagent. The absorbance of the reaction mixture was taken to 570 nm. Amino acids were computed with the equation:

```
Amino acids = Absorption × volume × Diluted concentration/Sample weight × 1000.
```
The amino acid, leucine, was used for preparing the standard curve, and results were expressed in mg of amino acid per g of dry tissue.

#### *2.13. Total Protein Content*

The concentration of protein was quantified by the Bradford assay [33]. Bovine serum albumin was used as a standard. Proteins were extracted by dissolving 0.2 g of leaf samples in 4 mL of sodium phosphate buffer (pH 7), and 0.5 mL of the extract was mixed with 3 mL of Comassive bio red dye. The optical density of the solution was measured at 595 nm. Protein was determined by:

Protein = Reading of extract × Diluted concentration × value of K/sample weight (9)

#### *2.14. Antioxidant Enzyme Assay*

Enzyme extract was prepared by grinding one gram of leaf in liquid nitrogen. The obtained powder was added in 10 mL of 50 mM phosphate buffer (pH 7.0) and was mixed with 1 mM Ethylene Diamine Tetra Acetic acid (EDTA) and 1% polyvinylpyrrolidone (PVP). The whole mixture was centrifuged at 13,000× *g* for 20 min at 4 ◦C. The supernatant was used for the enzyme assay.

The catalase (CAT) content was estimated by observing the degradation of H2O2 at 240 nm [34]. Catalase activity (U mg protein<sup>−</sup>1) was calculated from the molar absorption coefficient of 40 mm<sup>−</sup>1cm<sup>−</sup>1for H2O2. Peroxidase dismutase (POD) was determined by following the procedure of Rao [35]. The reaction mixture consisted of 10 μL of crude enzyme extract, 20 μL of 100 mM guaiacol, 10 μL of 100 mM H2O2, and 160 μL of 50 mM sodium acetate (pH 5.0). Absorbance was recorded at 450 nm.

Superoxide dismutase (SOD) activity was done by using the procedure of Giannopolitis and Ries [36]. The composition of the reaction mixture was 50 mM sodium phosphate buffer (pH 7.8), 0.1 M tris-HCL, 14 mM methionine, 1.05 mM riboflavin, 0.03% TritonX-100, 50 mM nitroblue tetrazolium chloride (NBT), 100 mM EDTA, and 20 μL enzyme extracts. After adding riboflavin, the glass tubes were illuminated for 5 min, and reactions were stopped by turning off lights. The absorbance was recorded at 560 nm.

#### *2.15. Statistical Analysis*

Three replicates were used for the mean and standard deviation values of the data. The obtained data were further analyzed by Duncan's multiple range tests using MSTAT-C version 1.4.2. The correlation coefficient of the data was done using the software Statistix version 8.1. Mean values were compared by the least significant difference (LSD) at *p* ≤ 0.05 [37]. The heatmap for the correlation coefficient was prepared by using web tool clustvis (https://biit.cs.ut.ee/clustvis/).

#### **3. Results**

#### *3.1. Soil Analysis*

Analysis of the rhizospheric soil samples of all four plants showed the soil was sandy clay loam with an EC range of 0.76–0.85 dSm<sup>−</sup>1, pH in the range of 7.99–8.12, high Na/K ratio, and a low concentration of nutrients (Table 1).



**Table1.**Physiochemicalpropertiesoftherhizospheresoilandrhizobacterialpopulation.

#### *3.2. Isolation and Screening of Salt-Tolerant PGPR Strains*

A total of 50 isolates were obtained from the rhizospheric soil of four halophytic plants. Among all isolates, 90% of colonies were round, creamy, and had entire margins (Supplementary Materials Table S1). Further, 78% of isolates were Gram-negative and rod-shaped (Supplementary Materials Table S2).

In the halotolerant assay, 70% of strains were able to grow up to 6%, 20% strains showed tolerance at 10%, while four strains SR1, SR2, SR3, and SR4 were able to grow at 15% NaCl (Supplementary Materials Table S2). These four strains also showed positive results for phosphorous solubilization, hydrogen cyanide, and siderophore production (Supplementary Materials Table S3).

#### *3.3. E*ff*ect of Bacterial Isolates on Germination of Wheat*

Salt stress resulted in a considerable reduction in the germination parameters of the wheat seeds. Under salt-stressed conditions, the seedling vigor index and germination index showed a 12.5% and 31% decrease compared to the control. Though most of the strains showed a significant increase in seed germination, four strains SR1, SR2, SR3, and SR4 showed prominent results (14.28%, 35%, 42%, and 55%), respectively, as compared to the non-inoculated control under the salt stress condition (Supplementary Materials Table S4).

#### *3.4. Identification of Isolates*

Initially, the four strains were identified based on the C/N source utilization pattern (Supplementary Materials Table S5). Molecular identification of the screened halotolerant strains was done based on 16S rRNA sequences and on the comparison of the 1500-bp sequence of 16S rRNA gene subjected to BLAST to confirm the relatedness with other bacterial strains. The isolate SR1 (1485 base pair) was closely related (98% nucleotide identity) to sequences of bacteria annotated as *Bacillus* strain JQ 926435 in the GenBank database. The sequence of SR2 (1480 base pairs) was 99% identical to *Azospirillum brasilense* DQ 288686.1, SR3 (1482 base pairs), and 96% identical to strain *Azospirillum lipoferum* accession no. M. 5906.1. Furthermore, the isolated strain SR4 showed a 99% homology with *Pseudomonas stutzeri* JQ 926435. The accession numbers of the identified strains were obtained from NCBI and are given in Table 2.


**Table 2.** Molecular identification of the isolates based on partial 16S rDNA analysis.

Further phylogenetic analysis of the identified bacteria was conducted in MEGA4 software to determine their affiliation [38]. The evolutionary history was inferred using the maximum parsimony method [26]. The results are shown in Supplementary Materials Figures S1–S4.

#### *3.5. Production of Phytohormones*

Based on the halotolerance assays, PGP traits, and germination assay results, four isolates were selected for further analysis. All the halotolerant PGPR strains showed the production of phytohormones in liquid culture (Figure 1). Halotolerant PGPR strains were able to produce IAA (0.5–2.1 μg mL<sup>−</sup>1), gibberellic acid (1.5–2.5 μg mL<sup>−</sup>1), CK (0.39–0.64 μg mL<sup>−</sup>1), and ABA (1.9–3.4 <sup>μ</sup>g mL<sup>−</sup>1). The PGPR strains SR2 and SR3 produced higher concentrations of phytohormones than those of SR1 and SR4; however, the bacterial consortium produced maximum concentrations of IAA (2.1 μg mL<sup>−</sup>1), gibberellic acid (2.5 μg mL<sup>−</sup>1), CK (0.64 μg mL<sup>−</sup>1), and ABA (3.4 μg mL<sup>−</sup>1).

**Figure 1.** Production of phytohormones (Indole Acetic Acid (IAA), Gibberellic Acid (GA), Cytokinin (CK), and Abscisic Acid (ABA) by PGPR strains and their consortium in culture media. (SR1: Inocualted with *Bacillus* sp; SR2: Inocualted with *Azospirillum brasilense*; SR3: Inocualted with *Azospirillum lipoferum*; SR4: Inocualted with *Pseudomonas stutzeri*; Consortium is a combination of all four strains *Bacillus* sp, *Azospirillum brasilense, Azospirillum lipoferum, Pseudomonas stutzeri*). This data displays the means and standard deviation (*n* = 3). Different letters show significant differences between treatments (*p* < 0.05).

#### *3.6. Production of Compatible Solutes*

A considerable amount of proline was produced by all the screened halotolerant strains when subjected to different salinity levels. Production of proline by SR2 and SR3 was the highest in the 10% saline condition than the control. The maximum amount of proline (12.1 μg mg<sup>−</sup>1) was produced by the bacterial consortium, which was 23% greater than SR2 and SR3. For the carbohydrate contents, a significant amount of soluble sugars was recorded by all the strains (Figure 2). The production of soluble sugars was more pronounced at different salinity levels than the control. The bacterial strains SR2 and SR3 produced a greater amount of (89–111 <sup>μ</sup>g mg<sup>−</sup>1) soluble sugar as compared to the control, but the consortium of bacterial isolates recorded the maximum values at 10% NaCl (222 μg mg<sup>−</sup>1) (Figure 3).

**Figure 2.** Production of proline by PGPR strains and their consortium in culture media supplemented with different concentrations of NaCl (2%, 4%, 6%, 8%, and 10%). The treatment details are the same as in Table 3. This data displays the means and standard deviation (*n* = 3). Different letters show significant differences between treatments (*p* < 0.05).

**Figure 3.** Production of total soluble sugar by PGPR strains and their consortium in culture media supplemented with different concentrations of NaCl (2%, 4%, 6%, 8%, and 10%). The treatment details are the same as in Table 3. This data displays the means and standard deviation (*n* = 3). Different letters show significant differences between treatments (*p* < 0.05).
