Desert Soil Microbes as a Mineral Nutrient Acquisition Tool for Chickpea (Cicer arietinum L.) Productivity at Different Moisture Regimes

Drought is a major constraint in drylands for crop production. Plant associated microbes can help plants in acquisition of soil nutrients to enhance productivity in stressful conditions. The current study was designed to illuminate the effectiveness of desert rhizobacterial strains on growth and net-return of chickpeas grown in pots by using sandy loam soil of Thal Pakistan desert. A total of 125 rhizobacterial strains were isolated, out of which 72 strains were inoculated with chickpeas in the growth chamber for 75 days to screen most efficient isolates. Amongst all, six bacterial strains (two rhizobia and four plant growth promoting rhizobacterial strains) significantly enhanced nodulation and shoot-root length as compared to other treatments. These promising strains were morphologically and biochemically characterized and identified through 16sRNA sequencing. Then, eight consortia of the identified isolates were formulated to evaluate the growth and development of chickpea at three moisture levels (55%, 75% and 95% of field capacity) in a glass house experiment. The trend for best performing consortia in terms of growth and development of chickpea remained T2 at moisture level 1 > T7 at moisture level 2 > T4 at moisture level 3. The present study indicates the vital role of co-inoculated bacterial strains in growth enhancement of chickpea under low moisture availability. It is concluded from the results that the consortium T2 (Mesorhizobium ciceri RZ-11 + Bacillus subtilis RP-01 + Bacillus mojavensis RS-14) can perform best in drought conditions (55% field capacity) and T4 (Mesorhizobium ciceri RZ-11 + Enterobacter Cloacae RP-08 + Providencia vermicola RS-15) can be adopted in irrigated areas (95% field capacity) for maximum productivity of chickpea.


Introduction
In the recent era, climate change had adverse impacts on crop productivity and economic returns from agricultural lands. Water scarcity, drought severity and extreme temperatures have reduced moisture levels in soil profile that alter physiology of the crops resulting in low growth and yield [1]. Thus, for maintaining global food security, it is very important to focus on research to mitigate drought

Biochemical Characterization of Isolated Strains
Identified bacterial strains were biochemically characterized. Two strains (RZ-11 and RZ-22) showed positive results for ammonia production whereas others remained negative for this test. The appearance of yellowish colored zones around the colony indicated siderophore production. Isolates RP-01, RP-08, RS-14 and RS-15 were found positive for siderophore tests while the other two (RZ-11 and RZ-22) appeared negative. Regarding the hydrogen cyanide (HCN) test, all strains tested negative. The appearance of cherry red rings in the tube indicated positive signs for IAA production. Among the isolates, 2 strains (RP-08 and RS-15) were found strong positive, while RP-01 and RS-14 were observed as moderate IAA producers and Rhizobium isolates tested negative. However, for solubilization of tri-calcium phosphate, 2 strains (RP-01, RP-08) were found strongly positive with solubilization Intensity (200 and 190) and solubilization index % (3.00 and 2.90) whereas, RS-14 and RS-15 did not show P-solubilization activity. Moreover, 4 PGPR strains (RS-14, RS-15, RP-01, RP-08) revealed strong potential for the catalase and protease enzymes production along with ACC-deaminase and EPS activities. Similarly, 3 strains RP-01, RS-14 and RS-15 showed positive response for amylase production. (Table 2).

Growth Chamber Experiment for Screening of Isolates
Seventy-two isolated strains were evaluated in a trial conducted in growth chambers to screen out most efficient rhizobium and PGPR strains for different attributes of seedling growth ( Figure 3). Two isolates (RZ-11 and RZ-22) significantly affected the nodulation of chickpeas that produced 10.667 and 10.567 nodules per plant, respectively ( Figure 4). Among all tested isolates, two PSB strains (RP-01 and RP-08) significantly enhanced shoot length by 19.4 cm and 19.5 cm, respectively ( Figure 5). A similar trend was recorded in terms of root length (15.10 cm and 15.133 cm) by the same bacterial strains, respectively ( Figure 6). Two PGPR isolates (RS-14 and RS-15) were found most efficient for growth and development of the plant shoot and root in screening trials. Figure 7 shows maximum shoot length (22.067 cm) in the treatment where seeds were inoculated with RS-14 followed by RS-15 (21.6 cm). On the other hand, the highest root length (19.033 cm) was recorded for the treatment where chickpea seeds were inoculated with RS-15 followed by RS-14 (17.667 cm) ( Figure 8).

Growth Chamber Experiment for Screening of Isolates
Seventy-two isolated strains were evaluated in a trial conducted in growth chambers to screen out most efficient rhizobium and PGPR strains for different attributes of seedling growth ( Figure 3). Two isolates (RZ-11 and RZ-22) significantly affected the nodulation of chickpeas that produced 10.667 and 10.567 nodules per plant, respectively ( Figure 4). Among all tested isolates, two PSB strains (RP-01 and RP-08) significantly enhanced shoot length by 19.4 cm and 19.5 cm, respectively ( Figure  5). A similar trend was recorded in terms of root length (15.10 cm and 15.133 cm) by the same bacterial strains, respectively ( Figure 6). Two PGPR isolates (RS-14 and RS-15) were found most efficient for growth and development of the plant shoot and root in screening trials. Figure 7 shows maximum shoot length (22.067 cm) in the treatment where seeds were inoculated with RS-14 followed by RS-15 (21.6 cm). On the other hand, the highest root length (19.033 cm) was recorded for the treatment where chickpea seeds were inoculated with RS-15 followed by RS-14 (17.667 cm) ( Figure 8).

Growth Chamber Experiment for Screening of Isolates
Seventy-two isolated strains were evaluated in a trial conducted in growth chambers to screen out most efficient rhizobium and PGPR strains for different attributes of seedling growth ( Figure 3). Two isolates (RZ-11 and RZ-22) significantly affected the nodulation of chickpeas that produced 10.667 and 10.567 nodules per plant, respectively ( Figure 4). Among all tested isolates, two PSB strains (RP-01 and RP-08) significantly enhanced shoot length by 19.4 cm and 19.5 cm, respectively ( Figure  5). A similar trend was recorded in terms of root length (15.10 cm and 15.133 cm) by the same bacterial strains, respectively ( Figure 6). Two PGPR isolates (RS-14 and RS-15) were found most efficient for growth and development of the plant shoot and root in screening trials. Figure 7 shows maximum shoot length (22.067 cm) in the treatment where seeds were inoculated with RS-14 followed by RS-15 (21.6 cm). On the other hand, the highest root length (19.033 cm) was recorded for the treatment where chickpea seeds were inoculated with RS-15 followed by RS-14 (17.667 cm) ( Figure 8).

Physiological Attributes and Nutrient Acquisition of Chickpea
The data pertaining to proline content in leaves of chickpea (Table 5) show that the treatment T2 gave maximum proline content (4.7967 mg g −1 DW) at moisture level 1 (55% FC) followed by T7 (4.6067 mg g −1 DW) at same moisture level, while minimum proline contents (1.1533 mg g −1 DW) were determined from untreated plants at moisture level 3 (95% FC). Maximum grain N and protein contents (4.31% and 26.96%) were recorded for T 4 at moisture level 3 (95% FC) followed by 4.28% and 26.79% with application of T 7 at moisture level 2 (75% FC), respectively (Table 5). T 2 responded most effectively at moisture level 1 (55% FC) both for grain N and protein (4.19% and 26.17%) contents. Lower N and protein contents were observed in grains of untreated plants at all given moisture levels. Similarly, T 7 was the most promising consortium on moisture level 2, showing 0.35% P contents in grain, followed by T 4 at moisture level 3. T 2 was the best performing consortium among all treatments, including the control at moisture level 1 which resulted in 0.31% P in grains of chickpea. The data regarding nitrogen acquisition in chickpea shoot shows that the maximum nitrogen contents (1.66-1.65%) were recorded for treatments T 2 and T 4 at moisture level 1 (55% of field capacity). Similarly, T 7 appeared as the most efficient consortium with 1.65% N content in chickpea shoot at moisture level 2. Contrarily, T 9 (control) showed lowest N contents (1.31-1.32%) in shoot of chickpea at all moisture levels. The data pertaining to the P contents in chickpea shoot (Table 5) showed the highest P (0.34%) for T 4 at moisture level 3 followed by T 2 which showed 0.32% P at the same moisture level. Similarly, T 7 gave 0.30% P contents at moisture level 2. T 9 (control) was recorded as the lowest performing treatment, which showed 0.20-0.22% P contents in chickpea shoots at all given moisture regimes.

Isolates Survival in Rhizospheric Soil
The data regarding the isolate population in rhizospheric soil (Figure 9) shows that the maximum Colony Forming Unit (2.45 × 10 8 ) value was recorded for the treatment T 2 at moisture level 3 followed by T 7 with 2.36 × 10 8 at the same moisture level. T 2 and T 7 showed 1.67 × 10 8 and 1.36 × 10 8 , respectively, at moisture level 1. T 9 (control) showed zero population of the tested consortia isolates at all given moisture levels.

Isolates Survival in Rhizospheric Soil
The data regarding the isolate population in rhizospheric soil (Figure 9) shows that the maximum Colony Forming Unit (2.45 × 10 8 ) value was recorded for the treatment T2 at moisture level 3 followed by T7 with 2.36 × 10 8 at the same moisture level. T2 and T7 showed 1.67 × 10 8 and 1.36 × 10 8 , respectively, at moisture level 1. T9 (control) showed zero population of the tested consortia isolates at all given moisture levels.

Post-Harvest Soil Nutrient Status
The data regarding the nitrogen percentage in post-harvest soil ( Figure 10) shows that the highest N (0.016%) was recorded for the treatments T4, T5 and T7 at moisture level 3 followed by T2 with 0.0157% N in post-harvest soil at moisture level 1. Similarly, T7 gave 0.0157% N at moisture level 1 and 2. T9 (control) was recorded as the lowest performing treatment, which showed 0.012% N in post-harvest soil, at all given moisture levels. The data pertaining to phosphorus percentage in postharvest soil ( Figure 11) shows that the maximum phosphorus (4.5 ppm) was recorded for both treatments T2 and T7 at moisture level 1 (55% of field capacity). T7 was recorded as the most efficient consortium at moisture level 3 (4.166 ppm post-harvest soil P). T9 (control) showed the lowest P contents (3.00 ppm) in post-harvest soil at all moisture levels.

Post-Harvest Soil Nutrient Status
The data regarding the nitrogen percentage in post-harvest soil ( Figure 10) shows that the highest N (0.016%) was recorded for the treatments T 4 , T 5 and T 7 at moisture level 3 followed by T 2 with 0.0157% N in post-harvest soil at moisture level 1. Similarly, T 7 gave 0.0157% N at moisture level 1 and 2. T 9 (control) was recorded as the lowest performing treatment, which showed 0.012% N in post-harvest soil, at all given moisture levels. The data pertaining to phosphorus percentage in post-harvest soil ( Figure 11) shows that the maximum phosphorus (4.5 ppm) was recorded for both treatments T 2 and T 7 at moisture level 1 (55% of field capacity). T 7 was recorded as the most efficient consortium at moisture level 3 (4.166 ppm post-harvest soil P). T 9 (control) showed the lowest P contents (3.00 ppm) in post-harvest soil at all moisture levels.

Discussion
In this study, we isolated 125 bacterial strains from collected samples of nodules, rhizoplane and rhizospheric soil of chickpea from the Thal desert of Punjab, Pakistan during a severe drought spell. Out of all isolated strains, 72 were selected for screening. Most efficient strains were biochemically tested, and RZ-11 and RZ-22 (Mesorhizobium ciceri) produced ammonia. Similar findings were given by [23] who showed that some soil microbes produce ammonia and enhanced crop growth attributes. Two PGPR isolates RS-14 (Bacillus mojavensis) and RS-15 (Providencia vermicola) were evaluated as IAA producing bacterial strains. Several microbes produce active auxin as IAA, which is a plant growth promoter [24,25]. Two PGPR isolates RP-01 (Bacillus subtilis) and RP-08 (Enterobacter cloacae) were IAA producers and the most efficient phosphate solubilizers. The results are also in agreement with the findings of [26], who observed the maximum phosphate solubilizing ability for Enterobacter sp. The appearance of hallo zones surrounding the microbial colonies could be due to the synthesis of organic acid with low molecular weight, or due to polysaccharides and phosphatase production by phosphate solubilizing microbes [27]. In our study, most promising strains (RZ-11, RZ-22, RP-01, RP-08, RS-14, RS-15) produced exopolysaccharides (EPS), phytohormones, 1-aminocyclopropane-1-1 carboxilate (ACC) deaminase and helped the chickpea in acquisition of drought tolerance at 55% field capacity. Similar studies were conducted by [28,29] on the mechanism to induce drought tolerance in wheat and chickpea grown on dry lands. They highlighted the role of microorganisms to manage abiotic and biotic stress by producing indole acetic acid (IAA) and ACC-deaminase to reduce ethylene levels of in roots.
In the growth chamber assay for isolates' screening, nodulation plant −1 was increased over 100% by rhizobium strains (RZ-11, RZ-22) as compared to un-inoculated plants of chickpea grown in pre-autoclaved soil. Similar results were found by [30,31] who reported that Mesorhizobium ciceri inoculation increased significantly the nodulation of chickpea plants through symbiotic relationship. These results are in conformity with the results of [32] who recorded increase in chickpea nodulation by seed inoculation with Mesorhizobium ciceri. PGPRs having P-solubilizing activity (RP-01 and RP-08) showed significant enhancement in chickpea shoot and root length by 54.4% and 54.9% over the un-inoculated control. Similar findings on phosphate solubilizing bacteria were given by [33], who noted that PSB enhances shoot and root length of chickpea plants. The root and shoot length may be increased due to increased availability of phosphorus thanks to PSBs activity, as phosphorus has an important role in root development and cell division. All plant growth promoting isolates showed significant improvement in seedling shoot length, between 6.39% and 45.82%, as compared to the control; two PGPR isolates, RS-14 and RS-15, having phytohormonal activity, increased chickpea shoot length 42.73% and 45.82%, respectively, in comparison to the untreated control. Moreover, PGPR strains (RS-01 to RS-24) showed a significantly positive response in chickpea seedlings root length (from 0.55% to 56.87%) as compared to the control. However, the maximum root length of chickpea was statistically increased 58.6% and 47% by RS-15 and RS-14, respectively, over the control without inoculation. These results are in conformity with the findings of [34] who reported that phytohormone production is the main character of shoot-root increasing strains in chickpea plants. Our experimental results are also supported by findings of Marasco et al. [35], who showed that plant-root system increases up to 40% higher in PGPRs treated plants as compared to untreated controls.
Six most efficient strains were used to make 8 different prolific consortia that were evaluated in pot experiment to study their efficiency on growth and yield attributes of chickpea variety Bhkkar-2011 at 3 moisture regimes (55%, 75% and 95% of field capacity). T 2 consortium (Mesorhizobium ciceri RZ-11 + Bacillus subtilis RP-01 + Bacillus mojavensis RS-14) was found most effective for chickpea growth and yield attributes at moisture level 1, which was maintained on 55% of field capacity. The treatment showed 44% increase in plant height, 29% in number of pods pot −1 , 19.8% in root length, 29% in 100 grain weight, 46.9% in biological yield pot −1 , 50% in economic yield pot −1 compared to the untreated control at moisture level 1 (55% of FC). Increments in the yield and yield attributes of chickpea might be due to ammonia production by rhizobium, and IAA production, ACC deaminase and PSB activity of PGPRs from the consortia in the applied treatment. Our results are supported by [36] who took samples of Rhizospheric soil and root nodules from a selected chickpea field and revealed that grain yield and yield attributes were increased by inoculation with Rhizobium strains in comparison with the un-inoculated treatment. The physiological attributes of chickpea as grain N and protein content were statistically increased by 7.6% over the control, with application of T 2 at moisture level 1 (55% FC) and by 10.12% (grain N) with T 4 at moisture level 3 (95% FC). T 2 resulted in postharvest soil N and P increases of 30% and 50%, respectively, in comparison with the un-inoculated treatment at 55% of field capacity. Moreover, in the present study, the calculated proline content of chickpea leaves showed an increase at moisture level 1 (55% of FC) by inoculation with T 2 -consortium having ACCD activity, and IAA production. These results are in accordance with findings of Mandhurendra [37]. Statistically higher grain yield (35.49%) over that of untreated chickpea plants was obtained with T 7 treatment (Mesorhizobium ciceri RZ-22 + Enterobacter Cloacae RP-08 + Providencia vermicola RS-15) at moisture level 2 (75% of FC) and with T 2 (Mesorhizobium ciceri RZ-11 + Bacillus subtilis RP-01 + Bacillus mojavensis RS-14) (50% higher grain yield) at moisture level 1 (55% of FC). T 4 (Mesorhizobium ciceri RZ-11 + Enterobacter Cloacae RP-08 + Providencia vermicola RS-15) had 27.53% better economic yield at moisture level 3 (95% of FC) as compared to uninoculated chickpea plants. Hence, microbial combinations inT 2 and T 7 could be used to make effective biofertilizers for chickpea growing areas under rainfed conditions to help plants to cope with drought spells.

Study Area and Sample Collection
Sample collection was done from different major chickpea producing tracts of Thal desert, Pakistan during cropping season 2017-2018. Roots of five healthy plants having nodules, rhizospheric soil (RS) and rhizoplane soils (RP) were uprooted from sand dunes of each location during drought spell (soil moisture ≈ 7%).

Isolation of Rhizobium and PGPRs
The rhizobium strains were isolated from pink colored nodules of collected roots of chickpea plants. A milky suspension was obtained after dissolving the crushed nodules in 5 mL distilled water. A droplet of milky suspension (100 µL) was shifted to Yeast Mannitol Agar (YMA) plates [38], and colonies were grown on plates after incubation at 28 ± 2 • C for 7 days. PGPR were isolated from rhizoplane (RP) and rhizospheric soil (RS) of collected roots by using serial dilution technique on Pikovskaya agar and Luria Bertani medium [39], respectively. The colonies were incubated at 28 ± 2 • C for 24 to 48 h.
A total of 125 rhizobacterial strains were isolated from root nodules (RZ), rhizospheric (RS) and rhizoplane (RP) soil of chickpea plants. Among them, 72 were repeatedly streaked on respective mediums in petri plates to obtain pure colonies for a screening process in growth chambers.

Morpho-Physiological Characterization of Isolated Strains
Colony morphologies of Rhizobium and PGPR isolates were observed by spreading pure colonies on respective media for their shape, margin and color [40]. Further strains were characterized as gram positive or gram negative via gram staining protocol [41].

Biochemical Characterization of Isolates
Bacterial strains were cultured in Luria Bertani (LB) broth with tryptophan to test the Indole Acetic Acid (IAA) production that was confirmed by adding 5 drops of Kovac's reagent directly to the respective tubes containing bacterial isolates [42]. However, isolates were evaluated for ammonia production by the method of Dinesh [43]. Bacterial strains were also screened for siderophore and hydrogen cyanide (HCN) production by using the methods adopted by [44] and [45], respectively. Phosphate solubilization efficiency and index of isolates were determined by the method of Macfaddin [46].

PSE =
Colony diameter + Halozone diameter Colony diameter (1) Bacterial strains were tested for catalase production using the procedure given by [47]. For this purpose, a loop having fresh bacterial strains was placed on glass slides. Hydrogen peroxide (H 2 O 2 ) was added on each spot of isolates, and production of bubbles was counted as a positive sign of catalase enzyme. For the amylase test, iodine solution was added on developed bacterial colonies to observe the formation of clear halo zones that indicated amylase production [48]. Skimmed milk agar medium (SKM) was used to observe the protease production for isolates by adopting the method described in [49]. For determination of ACC deaminase enzyme activity, bacterial strains were grown in Tryptic soy broth (TSB) for 24 h and centrifuged at 3000 g. Then, a loop of the bacterial strains suspended in Tryptic Soy Broth (TSB) media was shifted onto sterile DF (Dworkin and Foster) salt media containing ACCs as a single source of nitrogen Afterwards, the plates with the bacterial strains were incubated for 3 days at 28 • C and checked for colony growth [50]. ACC-deaminase activity was quantified as nmol α-ketobutyrate mg protein −1 h −1 using a spectrophotometer at 540 nm wavelength [51]. Exopolysaccharide (EPS) producing bacterial strains were characterized by streaking them on American Type Culture Collection (ATCC) medium no.14 and incubated for 3 days at 28 • C. Consequently, the bacterial colonies showing slimes around them were characterized as EPS producing strains [52].

Growth Chamber Experiment for Screening of Isolates
A plastic jar experiment in a growth chamber was carried out in the laboratory of Land Resources Research Institute (LRRI), National Agriculture Research Centre, Islamabad to evaluate the effect of isolates, 24 Rhizobium (RZ-01 to RZ-24) and 48 Plant Growth Promoting Rhizobacteria) on growth attributes of chickpea seedling. The chosen PGPRs were isolated from rhizoplane soil (RP-01 to RP-24) and from rhizospheric soil (RS-01 to RS-24) to screen out 2 best strains from each category. The jars were sterilized with 20% sodium hypochlorite solution and filled with pre-autoclaved sandy soil (at 120 • C for 90 min). In each jar, three surface sterilized seeds of variety Bhakker-2011 inoculated with respective isolates were sown. Uninoculated seeds were sown in the jar which was designated as control treatment. These jars were kept in the growth chambers in a complete randomized design. The data regarding nodulation per plant, shoot length and root length of all treated plants were recorded. Among the isolates, two best Rhizobium and four PGPRs were chosen on the basis of their effectiveness on growth attributes of chickpea. The chosen isolates were molecularly characterized and identified for further experimentation to evaluate their consortium effects on the productivity of chickpeas at different moisture regimes.

Molecular Characterization of Selected Strains
The most efficient strains of rhizobium (RZ-11, RZ-22) and PGPRs (RP-01, RP-08, RS-14, RS-15) from the previous screening experiment were molecularly identified by amplification and sequencing of 16sRNA gene. Purified colonies of the most efficient bacterial strains were plucked and mixed with 20 µL Tris-EDTA buffers in Polymerase Chain Reaction (PCR) strips. This mixture was placed in a PCR apparatus (Thermal Cycler PCR PEQSTAR, Munich, Germany) for 10 min at 95 • C to extract the template DNA, which was collected in the supernatant after centrifugation. DNA amplification was carried in the same apparatus using 2 µL of forward and reverse universal primers as 9F (5 -GAGTTGATCCTGGCTCAG-3 ) and 1510R (5 -GGCTACCTTGTTACGA-3 ), respectively, 25 µL TAKARA Pre-mix Ex-Taq, 20 µL PCR water and 1 µL of DNA template. The amplified PCR products were sent to Macrogen, Seoul Korea, for sequencing, and strains were identified using the EzBioCloud server Macrogen, Seoul Korea. All sequences were submitted to gene bank for allotment of accession numbers.

Compatibility of Isolates for Consortia
Three bacterial strains in consortium were grown jointly on nutrient agar medium (3.0 g yeast extract, 5.0 g peptone and 20.0 g agar L −1 ) to test their compatibility. For that purpose, isolates were refreshed overnight on 25 mL of Nutrient broth (NB) medium and inocula of 2 µL, containing 10 6 bacterial cells of a distinct isolate were inoculated 1 cm apart from other isolate on nutrient agar (NA) medium in one petri plate. Such petri plates in triplicate were incubated at 30 • C for 72 h. The inhibiting effect of isolates was examined visually using culture images and close ups of the overlapping areas of expanding colonies as adopted by [53].
Moreover, ACC-deaminase activity, IAA production and phosphate solubilization activity of consortia were quantified using the methods as discussed in Section 2.4. Glass house experiment at different moisture regimes.
A pot experiment was conducted under control conditions at Plant Genomic Research Institute (PGRI), NARC Islamabad to evaluate the efficacy of characterized Rhizobium and PGPRs on growth and yield attributes of chickpea in comparison to the untreated control. For the experiment, the sandy loam soil was collected from the Thal desert and passed through a sieve of 2 mm diameter. The pots with equal sizes were filled with 8 kg autoclaved soil. Three moisture regimes (55%, 75% and 95% of field capacity) were calculated by determining the porosity, bulk density and volume of pots with the help of formula in [54,55], and the moisture was maintained with the help of Time Domain Reflectometer (TDR) used during the experiment.
Three surface sterilized seeds of Bhakker-2011 (both inoculated and uninoculated) were sown in respective pots, and the experiment was followed with all the three germinated seeds. Experiment with three replicates of each treatment was conducted in CRD (Completely Randomized Design) under controlled conditions. Pure cultures of rhizobium strains and Plant Growth Promoting Rhizobacteria (PGPR) and the PGPRs having phosphate solubilizing activity were inoculated individually in 50 mL NB and further incubated overnight at 30 • C, 150 rpm. The 24 h old cultures of each isolate were shifted into a sterile 50 mL tube and centrifuged at 2150 g for 3 min. The resulting pellets were washed 5 times with sterile distilled water [53]. The resulting washed pellets of each isolate were resuspended in sterile water, and the optical density was measured at 550 nm and adjusted to a concentration of 10 6 CFU mL −1 . Strain suspensions were combined in equal amounts (i.e., equal cfu mL −1 ) to prepare the respective consortium for each treatment. Bacterial strain consortia (Table 6) were tested for their efficacy to improve chickpea productivity with following set of treatments:

Seed Inoculation with Consortia
The chickpea seeds were washed with sterile water and rinsed with 70% ethanol. Seeds were then immersed in 6.5% sodium hypochlorite and agitated for 20 min. Afterwards, seeds were washed eight times with sterile water under sterile conditions. The washed seeds were soaked in respective consortia for 60 min. The seeds for the untreated control were soaked in sterile water (Molina Romero et al.) [56].

Isolate Survival in Rhizospheric Soil
Survival of the inoculated insolates was determined from rhizospheric soil of the chickpea plants 60 days after seed inoculation through plate count method. Calculations were performed on the basis of serial 10 folds dilution in duplicate, using the pour plate method, replicated samples of 1 g soil and an appropriate dilution [57]. Each value is presented as an average of three individual plate counts of the colonies of the PGPR isolates within a consortium. Petri dishes (90 mm diameter) contained 25 mL of Nutrient Agar (NA) medium, and plates were incubated at 28-30 • C. Colony forming units (CFU) were recorded after 48 h, and the average number per g oven dry weight of soil was calculated as CFU = Bacterial plate count × Dilution factor Oven dry weight o f soil

Soil Properties
Three composite samples were collected from the pile of pre-sowing soil for physico-chemical analysis; soil texture [58], organic matter [59] total phosphorus [60], total nitrogen [61], extractable phosphorus and extractable potassium [62], and soil pH (1:5 soil-water) were determined following the methodology described by [63]. The mean of the initial soil replicated data is mentioned in Table 7. The post-harvest soil properties (Total Nitrogen and Extractable phosphorus) were determined by adopting the methods described in [61,62], respectively. Proline contents in 130 days old leaves were determined following the method described in [64]. Samples were weighed, and proline was extracted in sulphosalicylic acid and evaluated using the ninhydrin reagent. Two layers were obtained in separating funnels during the estimation process, and the upper pink layer was selected for quantification using a spectrophotometer at 520 nm wavelength. Seed protein contents were analyzed following the method described in [61].

Statistical Analysis
The data regarding biological and economic yield attributes of chickpea grown in jars and pots under growth room chambers, and glass house conditions were analyzed statistically by adopting a Complete Randomized Design (CRD). The means were separated with Least Significance Difference (LSD), and the comparison of the treatment means was done through LSD test [65].

Conclusions
Extreme drought events are expected to be one of the main challenges for agriculture and a threat to global food security. Explo/ration and utilization of desert microbes to cope with the issue of drought through experimentation on desert soil is a valid idea being adopted in the present study. Here, a series of experiments revealed growth promotion as well as substantial nodulation in chickpea that enhanced its grain yield under drought stress. This approach indicates the vital role of isolated strains to be utilized as bio-fertilizers under drought spell in the Thal desert, a main chickpea producing area in Pakistan, which is the 4th largest chickpea producing country. In this study, we found that the consortium T 2 (Mesorhizobium ciceri RZ-11 + Bacillus subtilis RP-01 + Bacillus mojavensis RS- 14) can perform best in drought conditions (55% field capacity), and T 4 (Mesorhizobium ciceri RZ-11 + Enteroabacter Cloacae RP-08 + Providencia vermicola RS-15) can be adopted in irrigated areas (95% field capacity) for maximum productivity of chickpea.