**1. Introduction**

Globally, the production rate of agriculture is far less than the estimated food requirement of the ever-increasing population and the gap will be widened over time [1] (GAP Report, 2018).

Agro-ecosystems are influenced by environmental and climatic conditions, farming techniques, and management practices. It is estimated that internationally, salinity affects 22% of the total cultivated and 33% of the total irrigated agricultural area, which is increasing at an alarming rate of 10% annually. Pakistan is also facing severe salinity issues and a total area of 6.30 million hectares is salt affected, out of which 1.89 million hectares is marked as saline [2].

Due to a higher concentration of sodium chloride (NaCl), plants growing in salt-affected soils suffer from both hyperosmotic and hyperionic effects. These stresses result in reduced water uptake; altered ion and mineral absorption rates; increased production of reactive oxygen species, causing disorganization of the cell membrane; and reduction of metabolic activities [3]. Halophytes adapt themselves to saline conditions by adjusting their physiological activities, maintaining their water balance by osmotic adjustments, producing compatible solutes, and modifying the antioxidant system [4]. Some plants overcome salinity stress through the production of osmolytes, particularly glycine betaine, proline, soluble sugars, and proteins [5].

Improvement in the crop yield of saline soils requires a multidimensional approach consisting of salt-tolerant varieties or amelioration by chemical neutralizers, but there is a dire need for eco-friendly sustainable approaches. Rhizobacteria, showing potential to improve plant growth, are termed as plant growth-promoting rhizobacteria (PGPR) [6]. PGPR have the potential to improve plant growth through various mechanisms, including better plant growth, the production of phytohormones, and amelioration of stresses [7]. Due to the natural coping mechanisms of PGPR, their inoculation can help the amelioration of various abiotic stresses in plants. PGPR inoculation can help to improve the growth and yield of crops, particularly in regions prone to drought and salt stress [8,9]. Natural halotolerant PGPR strains have better potential for the amelioration of salt stress in regional crops for sustainable yields. These native PGPR strains are well acclimated to indigenous conditions and the plant–microbe interactions can help the plants to tolerate stress [10].

In this study, native halotolerant PGPR strains were isolated from local saline soils, and their ability to promote plant growth when inoculated under salt stress was investigated. The objective of the present research was to focus on the evaluation of isolated bacterial strains to stimulate salinity tolerance and the promotion of wheat growth, as well as the identification and characterization of the candidate strain both bio-physiochemically and genetically. This study provides a basis to identify and characterize PGPR from natural saline conditions and testing their potential for improving salinity tolerance in wheat, the major staple crop across the world.

#### **2. Materials and Methods**

#### *2.1. Soil Sampling and Physicochemical Analysis*

The rhizospheric soil of four halophytes namely, *Abutilon bidentatum*, *Maytenus royleanus*, *Leptochloa fusca* (Kallar grass), and *Dedonia viscose*, was collected from a salt range of Pakistan (313–360 m.a.s.l; 32◦23–33◦00 north latitude and 71◦30–73◦30 east longitude). The rhizospheric soil was sieved and stored at 4 ◦C for future analysis. Rhizospheric soil was analyzed for pH and electrical conductivity (EC) [11], soil texture, macro and micronutrients [12], and available nutrients [13].

#### *2.2. Strain Isolation and HaloTolerance Assay*

Rhizobacteria were isolated from rhizospheric soil of *Abutilon bidentatum*, *Maytenus royleanus*, *Leptochloa fusca*, and *Dedonia viscose* by using the serial dilution and spread plate techniques [14]. The soil suspension was made by adding 1 g of soil in 9 mL of Milli-Q distilled water. An aliquot of soil suspension was inoculated on Luria-Bertani (LB) agar plates and incubated at 28 ± 2 ◦C for 48 h. The obtained colonies were purified by sub-culturing. The colony-forming unit (CFU) was calculated according to the formula given by [15]:

CFU/g = (colonies number × dilution factor/volume of inoculum).

Distinct bacterial colonies were examined for colony characteristics (shape, size, margin, elevation, appearance, texture, pigmentation, and optical properties) as well as for cellular characteristics (cell shape, gram testing) [16]. QTS-24 kits were used to determine the carbon/nitrogen (C/N) source utilization pattern of bacterial isolates. Isolated bacterial strains were tested for their halotolerance abilities by growing them in LB media supplemented with NaCl (2%, 4%, 6%, 8%, 10%, 15%) [16].

#### *2.3. Plant Growth-Promoting (PGP) Traits*

All the bacterial isolates were evaluated for their PGP characteristics. Phosphorous (P) solubilization was done by spot inoculating overnight grown cultures onto pikovaskaya's agar (Sigma) containing tri-calcium phosphate as an insoluble P source [17]. The colonies, which produced clearing zones in the pikovaskaya's agar plates, were considered positive for phosphorous solubilization. Total solubilized phosphate was measured by using the phosphomolybdate blue color method [18]. Modified pikovaskaya's broth medium was inoculated with each strain and incubated at 30 ◦C for 5 days. The cultures were centrifuged at 6000 rpm for 15 min. The supernatant (500 μL) was mixed with 40 μL of 2,4-dinitrophenol, after which 20 μL of dilute sulfuric acid were added, followed by 5 mL of chromogenic reagent, and the volume was diluted to 50 mL using sterilized water and absorbance was recorded at 680 nm. Siderophore production was done by spot inoculation on chrome azurol S (CAS) media as described by Schwyn and Neilands [19]. Bacterial strains were spot inoculated on petri plates containing CAS media. An uninoculated plate was taken as the control. After inoculation, plates were incubated at 28 ◦C for 5–7 days and observed for the formation of an orange zone around the bacterial colonies. Bacterial isolates were tested for hydrogen cyanide production through the method of Lorck [20]. Bacterial strains were streaked on nutrient agar medium (pre-soaked in 0.5% picric acid and 2% sodium carbonate *w*/*v*), supplemented with glycine (4.4 g/L). Plates were sealed with parafilm paper and incubated at 30 ◦C for 4 days. The appearance of an orange or red color indicates the production of hydrogen cyanide.

### *2.4. Germination Experiment*

This experiment was carried in the Plant Physiology Laboratory of PMAS-Arid Agriculture University. Seeds of the wheat variety (Galaxy 2013) obtained from the National Agricultural Research Centre, Islamabad were surface sterilized by treatment with sodium hypochlorite (1%) solution for 5 min. After, seeds were successively washed with distilled water. All the isolated strains were tested for germination attributes. Sterilized seeds of wheat were placed in pre-soaked filter paper in Petri dishes. NaCl solution (50 mM, 100 mM, 150 mM, 200 mM) was given instead of normal water. The germination experiment was carried out under laboratory conditions with an average photoperiod of 10 h day/14 h night at 24 ◦C. The germination percentage, seedling vigor index, and promptness index were measured for each treatment [21]. Four strains were selected for further analysis, based upon their efficacy in the germination experiment and were labeled as SR1, SR2, SR3, and SR4.

#### *2.5. Production of Osmolytes*

To analyze proline and total soluble sugars, the supernatant of PGPR grown in LB broth supplemented with NaCl concentrations (0%, 2%, 4%, 6%, 8%, and 10%) were analyzed as described by Upadhyay et al. [22]. For the estimation of the proline contents, centrifugation of the culture broth was done at 1000× *g* for 10 min and the supernatant was used for estimation. Total soluble sugar (TSS) was estimated by mixing 1 mL of supernatant with 4 mL of anthrone reagent, the mixture was later boiled in a water bath for 8 min. After rapid cooling, the optical density was measured at 630 nm, and the amount of TSS was calculated from a standard curve.

### *2.6. Phytohormone Production*

The ability of four selected halotolerant strains to produce phytohormones (IAA, GA, CK, ABA) in the culture media was measured by the method of Tien et al. [23]. The extraction of hormones was

done by centrifugation of bacterial cultures at 10,000 rpm for 15 min. For adjustment of the pH (2.8), 1 N HCl was used. In the next step, an equal volume of ethyl acetate was used for hormone extraction. The resulting solution was evaporated at 35 ◦C and the end residue was mixed in 1500 μL of methanol. Finally, the samples were run on High Performance Liquid Chromatography (HPLC) (Agilent 1100), which had a C18 column (39 × 300 mm) and a UV detector. For standardization of HPLC, pure grade chemicals of the hormones IAA, CK, GA, and ABA (Sigma Chemical Co., St. Louis, MO, USA) were dissolved in HPLC-grade methanol and were used. The wavelength used for the detection was as follows: IAA at 280 nm; and GA, CK, and ABA at 254 nm. The phytohormone content of LB media, without inoculum, was used to normalize the data.

#### *2.7. 16S rRNA Gene Sequence and Phylogenetic Analysis*

DNA was extracted from pure LB broth cultures as described by Chen and Kuo [24]. Amplification of genomic DNA of isolated strains was done as described by Weisburg et al. [25]. The PCR was carried out for amplification of the 16S rRNA gene with universal nucleotide sequence forward primer (fd1) AGAGTTTGATCCTGGCTCAG, and reverse primer (rd1) (AAGGAGGTGATCCAGCC). DNA was purified and sequenced on an automated sequencer by gel purification kits (JET quick, Gel Extraction Spin Kit, GENOMED). The strains were identified by using a nearly complete sequence of the 16s rRNA gene on (BLAST) NCBI by comparing sequence homology with other strains. The maximum parsimony method was used for the analysis of evolutionary linkages [26].
