The bacterial isolates were collected from all six districts of the Kumaon region of Uttarakhand. The Kumaon is one of the two administrative provinces in Uttarakhand, and it encompasses six districts: Almora, Bageshwar, Champawat, Nainital, Udham Singh Nagar, and Pithoragarh (Figure 1). The Kumaon region has a geographical size of 21,313 km² and an elevation range of 223–3,669 m above MSL. Kumaon is ensconced in the highlands of North India, bordering Chinese Tibet and Nepal. It encompasses the Himalayan Mountain range and is one of India's most secluded, scarcely inhabited, and undeveloped areas, with a static agricultural economy. Along the elevational gradient, the Kumaon region is divided into three agro-climatic zones: (i) lower elevation (up to 1,200m), (ii) intermediate elevation (between 1,200 and 2,300 m), and (iii) higher elevations (above 2,300 m). This region is excellent for the growth of multiple varieties of plants owing to its diverse climate (subtropical to alpine), height, elevation, soil types, valleys, rivers, watersheds, and forest resources.

Soil samples were randomly obtained from the rhizosphere of diverse plants across six districts of the Kumaon region of Uttarakhand. Samples were taken from four separate places at each location. To generate the composite sample, five samples from each site were taken and mixed. The obtained soil samples were diluted serially for up to 10⁻⁶ dilutions and a 0.1 mL aliquot of this diluted soil solution was spread onto the Nutrient Agar (NA) plates. Microbial colonies began to develop on the plates during an overnight incubation at 28 ± 2°C. For further purifications, only colonies with clearly distinguishable morphologies were chosen. The pure culture was stored in a petri dish and maintained at 4°C for regular use. A solution of 20% glycerol was used to briefly store the pure isolates at −20°C.

A nutrient broth (NB) medium was prepared by amending polyethylene glycol (PEG 6000) with varying water potentials to test the drought endurance of isolated bacteria (−0.05, −0.15, −0.30, −0.49, and −0.73 MPa) (Sarma and Saikia, 2014; Gontia-Mishra et al., 2016). A 100 μL of each bacterial isolate having a concentration of 1 × 10⁷ colony-forming unit (CFU)/mL, calculated by taking optical density (OD) at 600 nm, was added to the test tubes containing 5 ml of NB. The OD at 600 nm was measured spectrophotometrically after overnight incubation in an orbital shaker (200 rpm) at 30°C. Under varying levels of PEG, the growth of bacterial cultures was evaluated. By growing the test isolates in the nutrient broth with varying pH i.e., 4, 6, 8, and 10 with either 1N HCl or NaOH their pH tolerance was determined (Küçük et al., 2006). Isolated cultures were evaluated for salinity endurance in nutrient broth (NB) supplemented with varying percentages of NaCl (0, 2.5, 5, 7.5, and 10%) (ben Romdhane et al., 2009). A 50 μl of each bacterial inoculum having culture turbidity of 1 × 10⁷ CFU/ml, accessed spectrophotometrically, was injected per 5 ml of media and overnight incubated in an orbital shaker (200 rpm) at 30°C. The absorbance at 600 nm was calculated with a UV-Vis spectrophotometer to quantify growth (Eppendorf) (Gontia-Mishra et al., 2016).

All the 27 isolates were tested for plant beneficial attributes e.g., indole acetic acid (IAA) generation, siderophore formation, ACC deaminase production, phosphate dissolution, HCN, and NH₃ generation, were evaluated as discussed by Bakker and Schippers (1987); Schwyn and Neilands (1987); Bric et al. (1991); Cappucino and Sherman (1992); Nautiyal et al. (2000); Penrose and Glick (2003), respectively. The Salkowski colorimetric test was used to identify and estimate IAA biosynthesis using Nutrient Broth supplemented with L-tryptophan (2 mgL⁻¹; Sigma-Aldrich, Milan, Italy). To analyze the potential of bacterial isolates to form siderophores, the isolates were spot inoculated on agar plates supplemented with Chrome-azurol S (CAS) dye, as defined by Schwyn and Neilands (1987). The appearance of an orange-yellow zone surrounding the colony after 5–7 days of incubation at 28°C revealed the siderophore synthesis by microbes. The presence of enzyme ACC deaminase (ACCd) in bacterial cultures was calculated by monitoring growth on nitrogen-free minimum medium (MM) agar (Dworkin and Foster, 1958) enriched with 3 mM ACC (Sigma-Aldrich) after incubating at 28°C in darkness for 5–7 days of incubation at, as defined by Jaemsaeng et al. (2018). 2 gL⁻¹ (NH₄)₂SO₄ enriched MM agar plates were employed for control. Phosphate solubilization was checked using a modified NBRIP medium defined by Nautiyal et al. (2000). The isolated strains were examined for exopolysaccharide (EPS) synthesis and biofilm production (Kavita et al., 2011). The indirect plant beneficial traits e.g., hydrogen cyanide (HCN) formation were confirmed as per (Bakker and Schippers, 1987).

Seeds of late sown wheat cultivar VL-892 were procured from VPKAS Hawalbagh, Uttarakhand, India. Robust seeds were hand-picked and surface disinfected by immersing them in 70% ethanol for 3 min and then by dipping them in 0.2 % (v/v) HgCl₂. The seeds were rinsed five times in deionized water to eliminate residual ethanol. For each bacterial isolate, a 0.8 OD bacterial solution was prepared in which sterilized seeds were soaked for around 60 min. Seeds serving as control were kept in 0.5% (w/v) saline solution and the immersed seeds were laid on Petri plates coated with damp sterilized filter paper and cultured in triplicate at 25°C and under dark conditions. After incubating for 72 h, the germination percent, root, and shoot length were calculated, and an average score was determined for ten seedlings. For pot assay, cultures displaying greater impacts on seedlings (in terms of root/shoot or vitality index) were selected. In the current work, the bacterial cultures were chosen depending on their tolerance to drought, and various plant-beneficial traits e.g., ACCd synthesis, siderophore formation, IAA production, P and Zn dissolution, HCN production, and seed germination rate. Based on the above-mentioned properties, four isolates namely RR1, ASC1, AFS3, and NG4 were selected. Discrete PGPR isolates were chosen for their high drought endurance, and potential to promote root proliferation and vitality index.

The surface-sterilized, untreated (control), and PGPR-treated seeds of wheat cultivar VL-892 were grown in separate 12″ × 9″ pots, filled with three kilograms of sterile soil, sand, and peat. The pot assay experiment involved 10 treatments as follows:

The experiment was done in triplicate and ten plants were maintained per pot. Seven days old PGPR-treated seedlings were again supplemented with 1% bacterial solution (~10⁵ CFU ml⁻¹), while untreated control plants were given an equal amount of MS media. Both the PGPR-treated and control plants were watered every alternate day with 70% of field capacity for 3 weeks. Further, these plants were subjected to 7 days of drought stress by not providing water. The non-stressed plants were watered normally. For drought recovery purposes, plants were watered again subsequently for 3 days. Water-stressed saplings without any PGPR treatment were taken as the negative control, while regularly watered plants were taken as the positive control. The saplings were treated with PGPR strains RR1, ASC1, NG4, and AFS3 to evaluate their growth promotion potential in both non-stressed (watered every alternate day) and drought-stressed (watered after 7 days) conditions.

After the 7 days of induced drought and 3 days of drought recovery, the growth characteristics of each plant, e.g., root length, shoot length, fresh and dry weight of root and shoot were measured. To calculate the total dry weight (DW) plants were oven dried at 70°C for 72 h. The fraction of root adhering soil to root tissue (RAS/RT) was calculated by Sandhya et al. (2009). The relative water content (RWC) was determined according to Zhang and Blumwald (2001).

For PCR amplification of the 16S ribosomal DNA, the primer sets 27f (5′AGAGTTTGATCCTGGCTCAG-3′) and 1378r (5′-CGGTGTGTACAAGGCCCGGGAACG-3′) targeting the rDNA of about 1,500 bp were utilized (Gontia-Mishra et al., 2016). The PCR reaction steps were early denaturation for 5 min at 94°C, afterward 35 rounds for 1 min at 55°C, extension for 3 min at 72°C, and final extension for 10 min at 72°C. The 16S rRNA gene sequence was obtained by Biologia Pvt. Ltd. (India) via genomic sequencing of the PCR sample. The basic sequence alignment BLAST Program was used to compare the sequences, scanned against the database given on the National Center for Biotechnology Information's website (http://www.ncbi.nlm.nih.gov/BLAST). MEGA version 6 was used to perform a phylogenetic assessment of the 16S rRNA gene sequences (Tamura et al., 2013). The neighbor-joining technique (Saitou and Nei, 1987) was used to construct the phylogenetic tree.

The data from all 10 different wheat saplings was computed. All the experiments were carried out in triplicates and the results were shown as mean ± SD. The results for, bacterial drought tolerance and bacterial treatments were examined by one-way ANOVA using GraphPad Prism (version 5) software. To calculate the significant differences between the means bacterial treatment under control and stress conditions. The critical difference (C.D.) values were calculated at the p = 0.05 level. The significantly different mean values are indicated by different letters.

A total of twenty-seven bacteria were isolated from the rhizosphere of Finger millet, Linseed, Wheat, Rice, Potato, Red kidney bean, Maize, Sugarcane, Nettle grass, Barley, Tomato, Chili, Horse gram, Black soybeans, and, Hemp from all the six districts of Kumaon region of Uttarakhand, India (Table 1).

The drought endurance of these PGPR isolates was tested by culturing them in varying concentrations of PEG 6000 in NB medium, and nine of them showed luxuriant growth in 15% PEG. Among these nine, only five demonstrated growth in 25% PEG. Lastly, only four isolates exhibited growth up to 35% PEG (Figure 2). These isolates were further tested for pH tolerance, and five of them demonstrated tolerance in NB medium up to pH 4.5 and only two demonstrated tolerance up to pH 9.5 (Figure 3). However, salt tolerance remains lacking in all the selected isolates.

All twenty-seven isolates were analyzed for IAA generation, phosphate solubilization, and ACCd activity among other direct PGP characteristics. Out of 27 isolates, 18 isolates were IAA producers. Isolate NG 4 registered the highest IAA production (0.9 μg/ml of culture medium). Similarly, except for nine isolates (ASB5, RR5, RR7, NG1, JKK2, JKK3, PPS3, AP1, and AP3), all the other isolates showed in vitro phosphate solubilization. Isolate ASC2 showed maximum phosphate solubilization (0.83 μg/ml). The PGPR isolates were assessed for ACC deaminase activity and except for 5, all 22 isolates exhibited this trait. The PGPR isolates were also tested for many indirect PGP attributes such as NH3 production, siderophore production, HCN production, and biofilm formation. Except for 5, all other PGPR isolates exhibited NH3 production whereas all of the isolates registered HCN production (Figure 4). Twenty-one isolates showed siderophore production whereas only 16 isolates showed biofilm/EPS secretion. The direct and indirect PGP traits of the PGPR isolates are shown in Table 2.

To benefit plants, all 27 bacterial isolates were screened for plant promotion characteristics. Four best bacterial isolates were selected for the pot experiment, out of which three isolates namely ASC1, RR1, and AFS3 were the highest producers of siderophores, due to an orange halo around the colony. Similarly, the IAA production potential was found highest for these four isolates NG4, RR1, AFS3, and ASC1, respectively. In addition to the other PGPR traits, these 4 were also able to solubilize insoluble forms of phosphorus on the NBRIP plate as well. All four isolates also exhibited ACC deaminase activity. The PGPR isolates were also found positive for many indirect PGP attributes such as NH3 production, EPS production, and HCN production. The highest ammonia production was shown by the bacterial isolate NG4. Similarly, the greatest EPS and HCN production was recorded for isolate ASC1. Bacterial strains isolated from the rhizosphere of red kidney bean and hemp i.e., RR1 and ASC1, respectively, were able to tolerate drought for up to 35% PEG. Additionally, isolates AFS3 and ASC1 demonstrated remarkable pH tolerance with a range of 4.5–9.5. Based on variable plant growth-promoting traits AFS3, ASC1, NG4, and RR1, were selected for application as individual test subjects.

A 1,500 bp region of the the16S rRNA gene was purified and sequenced. All 4 bacterial strains were assigned to two distinct phyla based on their 16S rRNA gene sequences, including γ-proteobacteria, and Firmicutes. Out of the four test isolates, two strains belonged to the genus Bacillus, these were RR1 (Bacillus velezensis), and NG4 (Bacillus cereus), one strain belonged to the genus Pseudomonas, which was ASC1 (Pseudomonas baetica) and one strain belonged to the genus Staphylococcus, AFS3 (Staphylococcus pasteuri). A phylogenetic tree was built using 16S rDNA data to show the relationship between the tested isolates and related bacteria (Figure 5). Despite the two isolates belonging to the same genus as Bacillus sp., they exhibited sufficient variation in plant growth-promoting characteristics along with diverse seed vitality metrics. Owing to their stress adaption and nutrient solubilization potential, these PGPRs are highly effective at reducing stressful situations.

The final isolates AFS3, ASC1, NG4, and RR1 were chosen based on their PGP characteristics and capacity to endure high pH and higher concentrations of PEG in an NB medium. These isolates were analyzed for drought stress tolerance for seven consecutive days along with 3 days of the recovery phase. Plant root length, shoot length, and fresh and dry weights were calculated in PGPR inoculated and un-inoculated pots under normal and drought-stressed conditions (Table 3).

During normal circumstances, ASC1-primed wheat seedlings developed considerably longer shoots, and roots, over uninoculated control and AFS3, NG4, and RR1 seedlings, and their fresh and dry weight of shoot and root were also greater. Drought suffered, control plants greatly reduced in root length and shoot fresh and dry weights relative to PGPR primed plants. Under drought stress conditions, PGPR treatment was found to be beneficial in elevating the growth of wheat seedlings. PGPR-infected plants recovered from drought stress more effectively regarding the following growth parameters such as shoot length, root length, and shoot and root fresh and dry weights. However, the recovery of uninoculated plants under drought stress was minimal. A prominent upsurge in fresh and dry weight of root and shoot was observed with all bacterial treatments in comparison to the un-inoculated control. Thus, it was observed that isolate ASC1 emerged as the most effective PGPR for boosting plant growth under drought conditions and recovery.