Microbial inoculants alter resilience towards drought stress in wheat plants

Microbes play crucial roles in enhancing plant growth by forming symbiotic relationships, promoting nutrient uptake, and stimulating overall plant health in various habitats. The present study aimed to investigate the role of Piriformospora indica, arbuscular mycorrhiza fungi (AMF), and plant growth-promoting bacteria (PGPB) in alleviating drought stress in the Triticum aestivum HD-2967 cultivar. In a completely randomized design experiment, plants were subjected to different water regimes of 75% and 35% field capacity (FC) under greenhouse conditions. Under different water regimes, microbial inoculation significantly enhanced the morphological, physico-biochemical, and ultrastructural characteristics of the wheat plants. Plants inoculated with PGPB, P. indica, and AMF showed increased shoot and root length, shoot and root biomass, leaf area, photosynthetic rate, transpiration rate, stomatal conductance, and internal CO2 as compared to uninoculated plants under all water regimes. The PGPB, P. indica, and AMF-inoculated wheat plants accumulated higher content of glycine betaine, total sugars, trehalose, proline, putrescine, spermidine, carotenoids, proteins, α-tocopherol, and a decrease in lipid peroxidation, relative membrane permeability, and lipoxygenase enzyme activity as compared to uninoculated plants. Besides, microbes-inoculated wheat plants showed a higher level of antioxidant enzymes viz., superoxide dismutase, catalase, and ascorbate peroxidase than uninoculated plants. Microbial inoculation helped wheat plants to overcome water stress-induced deficiency of macro- (Ca2+, Mg2+, and K+) and micronutrient (Cu, Mn2+, Fe, and Zn2+), and reduced damage to the cell ultrastructure (plasma membrane and chloroplasts). Comparing the potential of microbial inoculants to increase growth and nutritional, biochemical, physiological, and ultrastructural changes, the PGPB-inoculated wheat plants showed greater drought resilience followed by AMF and P. indica inoculated plants.


Introduction
Drought is one of the major abiotic stresses that adversely affects agricultural production and threatens food security worldwide (Guo et al. 2020;Mirzabaev et al. 2023).A decline in rainfall and an increasing frequency of dry spells are contributing to drought stress (Jarrett et al. 2023).As a consequence, about 45% of agricultural fields are experiencing continuous drought conditions (Yadav et al. 2020) that eventually negatively affect plant growth by decreasing transpiration and photosynthesis rates (Naservafaei et al. 2023), osmolytes (Haghighi et al. 2023) and nutrient uptake (Shah et al. 2023).Under drought stress, plants face the problem of increased electrolyte leakage, lipid peroxidation, and reactive oxygen species (ROS) production (Yu et al. 2023).Nevertheless, prolonged drought may result in irreparable damage to plant growth, resulting in reduced crop yields.However, to overcome the problem, the plant needs to maintain better osmotic adjustment, antioxidant defense mechanisms, stomatal closure, and increased rootshoot ratios (López-Galiano et al. 2019).
In 2021, global wheat production was reported to be 770 million thousand tonnes; nevertheless, wheat productivity is vulnerable to drought stress, and climate change is projected to cause a 1.9% reduction in world wheat production by the mid-century (Pequeno et al. 2021).Various mitigation strategies have been developed to alleviate drought stress, including film farming (Mori 2015), drought-tolerant crops (Diatta et al. 2021), nanoparticles (Silva et al. 2023), super-absorbent hydrogels and biochar (Waqar et al. 2022).Besides being cost-intensive, most of these practices require the implementation of high technicalities.
Plant growth-promoting microorganisms have proven to be a preferred natural source to boost drought tolerance in plants to meet the growing demand for food (Ramakrishna et al. 2020).Soil microorganisms like arbuscular mycorrhizal fungi (AMF) (Wang et al. 2023;Liu et al. 2023a), Piriformospora indica (Azizi et al. 2021;Tsai et al. 2020) and PGPB viz.Azotobacter (Hashemi et al. 2022), Bacillus (Mahreen et al. 2023), Enterobacter (Jofre et al. 2023), and Rhizobium (Komatsu et al. 2023) can promote plant growth under drought conditions.These microbes can produce many bioactive compounds and have the ability to survive under drought stress (Tyagi et al. 2023).P. indica is a plant endophyte that enhances antioxidant capacity, activates induced systemic resistance, mobilizes nutrients, and regulates plant hormone levels during stress conditions.(Rajput et al. 2022;Aslani et al. 2023).AMF establishes mutualistic associations with more than 80% of land plants (van der Heijden et al. 2015).AMF hyphal networks proliferate several meters beyond the depletion zone and acquire soil nutrients and water (Tyagi et al. 2021), thereby boosting the nutrient uptake capacity of their partners.Besides, AMF maintains ion balance, stimulates antioxidant enzyme activity, and regulates water-holding capacities to combat drought stress (Israel et al. 2022;Bhardwaj et al. 2023;Fresno et al. 2023;Liu et al. 2023c).AMF induced favorable alterations in the root microenvironment, leading to drought tolerance by modulating root exudate constituents, glomalin content, phosphatase activity, and soil aggregate stability within the mycorrhizosphere (Cheng et al. 2022).The PGPB also benefits the plant's photosynthetic capacity, osmolyte accumulation, leaf area, and relative water content.(Fadiji et al. 2022).They could alleviate drought stress due to their ability to solubilize phosphate and produce phytohormone, exopolysaccharide, and siderophore (Ilyas et al. 2020;Mahreen et al. 2023;Liu et al. 2023b;Aslani et al. 2023).Inbaraj (2021) found that PGPB-inoculated wheat, maize, and rice plants improved biomass production, photosynthetic pigments, membrane integrity, proline content, and increased CAT and SOD activities to scavenge free radicals under drought stress.Furthermore, PGPB improves nutrient absorption as well as the water uptake capacity of plants growing under stress conditions (Nemeskeri et al. 2022).
So far, no studies have been conducted to compare the effectiveness of the PGPB consortium, AMF consortium, and P. indica in alleviating drought stress in crop plants.In fact, the influence of the PGPB consortium, AMF consortium, and P. indica on the antioxidant enzyme activity (SOD, CAT, and APX) and accumulation of non-enzymatic antioxidants (ascorbic acid, α-tocopherol, glutathione, and carotenoids) in host plants under drought conditions has been poorly studied (Rajput et al. 2022;Mahreen et al. 2023).Though PGPB, AMF, and P. indica inoculated plants under drought conditions have higher levels of osmolytes, reduced lipid peroxidation, and membrane integrity (Tyagi et al. 2023), no attempt has been made to link this to ultrastructural damage prevention.The role of macronutrients (Ca 2+ , Mg 2+ , K + , Na + ) and micronutrients (Cu, Fe, Mn 2+ , and Zn 2+ ) in plants under stress conditions has been studied by some researchers (Rahmani et al. 2023).However, not much attention has been paid to the role of the PGPB consortium, AMF consortium, and P. indica colonization in increasing nutrient uptake during drought stress.Membrane permeability has not been discussed in relation to nutritional imbalance.Taking this into account, the present study was undertaken to investigate the role of inoculation of PGPB (Azotobacter chroococcum, Enterobacter asburiae, Lactococcus lactis), AMF (Glomus intraradices, Glomus mosseae, and Scutellospora) and P. indica on the nutrient acquisition, antioxidant response, and ultrastructural changes in drought-stressed HD-2967 wheat cultivar.

Plant material, microbial inoculum, and soil composition
HD-2967 wheat cultivar (Triticum aestivum var.Pusa Borlaug), a double dwarf variety, was procured from the Department of Genetics, Indian Agricultural Research Institute (IARI), New Delhi, India.The seeds were surface sterilized by treating them with 4% sodium hypochlorite for 2 min and then washed with distilled water.Five seeds were sown at a depth of 3 cm in pots containing 4 kg of sterile soil.AMF consortium and PGPB consortium were procured from the Department of Microbiology, IARI, New Delhi, India, and P. indica inoculum were acquired from AIMT, Amity University, Noida, India.An amount of 20 g PGPB inoculum (A.chroococcum, E. asburiae, and L. lactis present in equal ratio), 0.5 g P. indica, and 50 g AMF inoculum (G.intraradices, G. mosseae, and Scutellospora sp.present in equal proportion) was mixed with the soil at a depth of 3 cm at the time of seeding in different treatments.The same amount of sterile sand was mixed with soil in control.For the pot experiment the recommended dose of fertilizer was viz: nitrogen (N) as urea: 187.5 kg ha −1 , diammonium phosphate (P): 75 kg ha −1 , and muriate of potash as (K): 75 kg ha −1 .The soil used in the experiment was collected from the fields of Alipur village, New Delhi, and was autoclaved at 1 3 121°C and 15 psi for 30 min.The soil properties were determined by the Department of Soil Sciences and Agricultural Chemistry, IARI, Delhi, India, and the results revealed the following nutrient levels: Available N: 196 mg g −1 , Available P: 49.8 mg g −1 , and Available K + 492 mg g −1 .

Experimental design, growth condition, and water stress treatment
A pot experiment was conducted in the botanical garden of Swami Shraddhanand College, University of Delhi, India.During the experimental period, the temperature ranged between 23 and 30 °C and relative humidity between 55 and 65%.The experiment was laid out in a completely randomized block design with two factors: (i) microbe status (non-microbial control, inoculation with P. indica, inoculation with PGPB, and inoculation with AMF) and (ii) two levels of water stress (75% and 35% of soil field capacity).Therefore, 8 treatments (4×2) with three replicates each were used.Prior to the water stress treatment, plants were allowed to grow for 20 days to allow microbes to colonize.The plants were irrigated with different water regimes (75% and 35% of soil field capacity) weekly for 2 months.After 80 days of sowing, the entire plant was uprooted manually (Supplementary material; Fig. S1).

Estimation of AMF and P. indica colonisation
The roots of wheat plants inoculated with AMF and P. indica were thoroughly washed with distilled water.For the study of percentage colonization, samples were softened in 10% KOH for 15 min.Further, the samples were treated with 1 N HCl for 15 min.The roots were then stained with 0.02% trypan blue for 12 h (Phillips and Hayman 1970).The excess stain from the roots was removed using lactophenol, and the roots were observed under a microscope.Microbe colonization was determined by the presence of arbuscules, hyphae, and vesicles in the root cortex.

Estimation of plant growth parameters
Shoot and root lengths were measured using a ruler.To measure shoot and root biomass the samples were oven dried for 3 days at 75 °C till constant weight was obtained and based on the dried weight of the samples, biomass was calculated.The leaf area was measured using a CI-202 laser area meter.

Elemental analysis of tissue
The dried leaf powder sample (100 mg) was digested with 3 ml nitric acid and 5 ml hydrogen peroxide in a microwave digestion unit (Microwave Reaction system, Multiwave PRO).After cooling, the digested sample was transferred volumetric flask and the final volume was made to 25 ml with Milli Q water.The element standards were prepared in 2% HNO 3 solution.The standard concentrations ranged from 0.1-10 ppm for each element.The standards and samples were run through Inductively coupled plasma mass spectrometry (ICP-MS) instrument (Agilent Technologies 7700 series ICPMS) and the data were analyzed with Mass Hunter workstation software.

Estimation of physiological changes
Measurements of leaf physiological parameters viz.photosynthetic rate, transpiration rate, stomatal conductance, and internal CO 2 were done using LI-6400XT Portable photosynthesis system between 8:00 am and 12:00 pm.All measurements were taken using a 2 cm 2 leaf chamber having a red-blue light-emitting diode light source at 1200 µmol m −2 s −1 photosynthetic active radiation and 400 ppm CO 2 .A light curve was prepared to determine the light saturation rate (Bidalia et al. 2018).

Photosynthetic pigments
The content of photosynthetic pigments in plant leaves was estimated according to the method of Hiscox and Isradtom (1979) and Caesar et al. (2018).

Osmolytes
To determine glycine betaine content, a 100 mg leaf sample was crushed in 1000 µl Milli Q water.The homogenate was centrifuged for 20 min at 20 °C at 12,000 rpm (Eppendorf, Centrifuge 5810 R).To the extract 2 N sulphuric acid was added in the ratio of 1:1.Aliquot (500 µl) was cooled for 1 h, added cold 200 µl KI-I 2 reagent and stored at 4 °C for 16 h.The mixture was centrifuged at 10,000 rpm for 15 min at 2 °C and the supernatant was discarded to isolate the periodide crystals.To dissolve the periodide crystals, 9 ml of 1,2-dichloroethane was added and then vortexed.An absorbance measurement at 365 nm was performed after 2 h (Eppendorf UV-Vis Spectrophotometer, BioSpectrometer basic model) (Grieve and Grattan 1983;Abbas et al. 2014) The Anthrone method was used to estimate leaf total sugar content following the protocol of Sadasivam and Manickam (2008).
For trehalose estimation, the leaves (40 mg) were crushed in liquid nitrogen with the help of a mortar and pestle.The sample was boiled in 2 ml of ethanol in a water bath for a few minutes.Ethanol was then evaporated using nitrogen gas and the residue was dissolved in 5 ml of the mobile phase (acetonitrile: H 2 O (70:30)) of the High-performance liquid chromatography (HPLC).The content was centrifuged at 10,000 rpm for 10 min and filtered through a 0.2 µm syringe filter.The filtrate was heated in boiling water for 60 min.The pH was adjusted to 7.0.The solution was evaporated and the residue was dissolved in the mobile phase.The extract was analyzed by HPLC system (Agilent 1200 infinity series), using a monosaccharide column (C-8, 150 mm × 4.6 mm × 5 µm) at a flow rate of 0.5 ml/min, 20 µl injection volume, and the elute was detected by refractory index detector (Hewlett-Packard 1037A).By comparing the chromatogram with a standard trehalose concentration, the trehalose content was calculated (Ferreira et al. 1997;Liu et al. 2009).
The quantification of proline content was carried out using the procedure described by Bates et al. (1973).
To determine the concentrations of polyamines viz.putrescine, spermidine, and spermine in dried leaf samples methods of Marcé et al. (1995) and Harada et al. (2019) were followed with some modifications.A dried leaf sample (100 mg) was extracted in 4 ml 5% cold perchloric acid (Minocha et al. 1994).The extract was centrifuged at 20,000 g for 30 min and the supernatant was stored at − 20 °C until dansylation.For dansylation, 200 µl perchloric acid extract was mixed with 40 µl 0.05 M diamino heptane (internal standard).Next 200 µl of Na 2 CO 3 and 400 µl of dansyl chloride were added.The resultant mixture was vortexed and incubated in the dark overnight.Then 100 µl of proline solution (100 mg ml −1 water) was added and incubated in the dark for 30 min.To the dansylated polyamines 500 µl toluene was added and vortexed for a few seconds.The 400 µl of the organic phase was aspirated, evaporated by nitrogen gas, and dissolved in 800 µl acetonitrile.The sample was filtered using a 0.22 µm syringe filter and 50 µl was injected into the HPLC system (Agilent 1200 infinity series) at a flow rate of 1 ml/min.Polyamines were separated on an ORO Chem C-18, 150 mm × 4.6 mm × 5 µm column and detected by Hewlett Packard 1046A Programmable Fluorescence detector (excitation at 340 nm, emission at 500 nm).The polyamines were eluted with acetonitrile and 10 mM 1-Heptanesulphonic acid.

Protein
The analytical Kjeldahl method (EN ISO 20483: (2006), AN 300) for determining protein was conducted by estimating total nitrogen using a FOSS nitrogen analyzer (FOSS Kjeltec 8200).To 1 g of dried leaf sample 7 g K 2 SO 4 , 0.8 g CuSO 4 × 5H 2 O, and 12 ml concentrated H 2 SO 4 were added.The mixture was digested at 420 °C for 60 min with a digestor (FOSS Labtec ™ Line DT208 Digestor) attached to a scrubber (Labquest Borosil KSC 01) to neutralize harmful fumes.The solution was left to cool for 15 min and then diluted cooled digest with 80 ml H 2 O.To the diluted digest 50 ml of 40% NaOH was added and to the receiver flask, 30 ml of 4% boric acid having methyl red and bromocresol green dye was added.The content was distilled for 5 min with FOSS fully automated distillation unit.A reagent blank was performed before each batch of samples.The solution in the receiving conical flask was titrated with 0.1 M of HCl, the titre was measured and the percentage of nitrogen content was calculated T = Sample titration, B = Blank titration, N = Normality of titrant.F = 6.25 for cereal.

α-Tocopherol
The quantification of α-tocopherol content in the samples was conducted using the methodology outlined by Sadasivam and Manickam (2008).

Membrane damage
The malondialdehyde content (MDA) of leaves was used to determine the level of lipid peroxidation (Heath and Packer 1968).The leaf tissue (0.5 g) was crushed in 10 ml of 0.1% trichloroacetic acid (TCA).Homogenate was centrifuged at 12,000 rpm for 15 min (Eppendorf, Centrifuge 5810 R).A solution of 0.5% thiobarbituric acid (TBA) prepared in 20% TCA was added to 2 ml supernatant.After heating for 30 min at 95 °C, the mixture was cooled.For 10 min, the mixture was centrifuged at 10,000 rpm.Supernatant absorbance was measured at 532 nm.Calculation of MDA content was done using its extinction coefficient.
To determine relative permeability twenty-five leaf discs with a diameter of 5 mm were cut out and placed in a test tube containing 25 ml Milli Q water.The electrical conductivity of the solution was measured after 1 h of incubation using a conductivity meter (HACH analyzer, HQ440d multi).After boiling the leaf discs for 30 min, the solutions were cooled and conductivity was measured again (Zwiazek and Blake 1991).
LOX activity assessment followed the experimental protocol established by Doderer et al. (1992).

Antioxidant enzymes
Leaf tissue (500 mg) was homogenized in a 0.1 M phosphate buffer (pH 7.5) solution containing 0.5 mM EDTA.After centrifugation at 12,000 rpm for 15 min at 4 °C, the resulting extract was utilized for enzyme activity measurements.SOD activity was determined as per the protocol of Dhindsa et al. (1981), CAT activity was measured following the procedure of Teranishi et al. (1974), and APX activity was determined using the method outlined by Nakano and Asada (1981).
After rinsing with 0.1 M phosphate buffer (pH 7.0), secondary fixation was performed using 2% osmium tetroxide.Dehydration, resin embedding, and ultrathin sectioning were done.Copper grid-mounted sections were stained with uranyl acetate.Stained sections were washed with 50% alcohol, followed by distilled water.Finally, the sections were stained using lead citrate, washed with 1 N NaOH, and dried.Ultrastructural changes in mesophyll cell plasma membrane and chloroplast were examined using a transmission electron microscope (Thermoscientific, Talos).

Statistical analysis
The data were analyzed using IBM SPSS Statistics 21.A two-way ANOVA was conducted with drought and microbes as independent factors.Post hoc Duncan's test was used to determine the significance of differences between treatments (P < 0.05).Multivariate regression analysis was conducted to examine the relationship between plant physiological traits and growth parameters.

Microbial colonization
AMF and P. indica successfully colonized the roots of wheat plants at 75% and 35% FC.Plant roots not inoculated with AMF and P. indica did not show microbial colonization (Supplementary material; Fig. S2).Different levels of water regime significantly affected the percent root colonization.
A gradual decline in microbial colonization was observed with increasing water stress levels in wheat plants (Table 1).3).On comparing the potential of microbes to increase growth parameters, PGPB-inoculated plants showed higher drought resistance as compared to AMF and P. indica-inoculated plants.
Similarly, for the drought treatment, the results showed that LA significantly predicted PN (F = 14.54, p < 0.05).This implies that changes in LA had a direct impact on the PN.The R 2 in this case was found to be 0.91, demonstrating that the model accounted for 91% of the variance in PN.Additionally, for the drought treatment, the results showed that shoot dry weight (SDW) significantly predicted E (F = 30.63,p < 0.04).This implies that changes in LA had a direct impact on the E. The R 2 in this case was found to be 0.95, demonstrating that the model accounted for 95% of the variance in E.

Photosynthetic pigment content
Microbial-inoculated wheat plants exhibited significantly higher levels of photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids, compared to uninoculated plants at 75% and 35% FC (Fig. 2).At 35% FC, PGPB-inoculated plants displayed the highest increase in chlorophyll a (391.71%),followed by AMF (361.93%) and P. indica (260.19%)compared to uninoculated plants.PGPB also showed the highest increase in chlorophyll b (60.70%) at 35% FC, followed by AMF (35.23%) and P. indica (29.48%).Total chlorophyll content Fig. 1 Effects of drought and microbial inoculation in wheat on A photosynthetic rate; B stomatal conductance; C transpiration rate; and D internal CO 2 .Values represent averages across replicates.Different letters represent significant differences at P < 0.05 was highest in PGPB-inoculated plants (253.79%)compared to AMF (226.16%) and P. indica (163.79%) at 35% FC.Carotenoid content was also notably higher in PGPB (50%), AMF (41.30%), and P. indica (28.74%) inoculated plants compared to uninoculated plants at 35% FC.Two-way ANOVA revealed significant independent effects of drought, P. indica, PGPB, AMF, and interactions of drought × P. indica, drought × PGPB, and drought × AMF on chlorophyll a and total chlorophyll content (Table 3).Therefore, PGPB exhibited superior efficiency in alleviating drought stress in wheat plants compared to AMF and P. indica in terms of physiological response.

Protein
Water stress significantly increased protein content in both microbial inoculated and uninoculated wheat plants.However, under water stress, microbial-inoculated plants Values represent averages across replicates.Different letters represent significant differences at P < 0.05 possessed significantly higher levels of protein than uninoculated plants (Fig. 4A).As compared with uninoculated wheat, PGPB, AMF, and P. indica inoculated plants produced 112.38%, 91.95%, and 47.97% more protein respectively at 35% FC.Thus, at all water regimes, PGPB has a greater ability to alleviate drought stress than P. indica and AMF in wheat plants.Water stress and microbial inoculation significantly enhanced protein content.

α-Tocopherol content
Water stress significantly increased α-tocopherol content in both microbial inoculated and uninoculated wheat plants.However, under water stress, microbial-inoculated plants possessed significantly higher levels of α-tocopherol than uninoculated plants (Fig. 4B).As compared with uninoculated wheat, PGPB, AMF, and P. indica inoculated plants produced 41.03%, 17.15%, and 9.20% more α-tocopherol respectively at 35% FC.Thus, at all water regimes, PGPB has a greater ability to alleviate drought stress than P. indica

Membrane damage
Exposure to water stress significantly increased MDA content, relative permeability, and LOX activity in both microbially inoculated and uninoculated wheat plants.However, microbially inoculated plants exhibited significantly lower MDA content, relative permeability, and LOX activity at all water regimes.PGPB-inoculated plants showed the greatest reduction in MDA content (29.74%), followed by AMF (22.12%) and P. indica (17.21%) at 35% FC (Fig. 5A).At 35% FC, PGPB, AMF, and P. indica-inoculated plants displayed 29.74%, 12.09%, and 9.4% lower relative permeability, respectively, compared to uninoculated plants (Fig. 5B).PGPB-inoculated plants exhibited lower LOX activity than AMF and P. indica at all water regimes (Fig. 5C).Twoway ANOVA confirmed the significant independent effects of drought, P. indica, PGPB, and AMF, and interactions of drought × P. indica, and drought × PGPB on membrane damage (Table 3).Hence, PGPB demonstrated greater efficiency in alleviating drought stress in terms of membrane damage compared to P. indica and AMF in wheat plants.

Antioxidant enzymes activity
Under water stress and well-watered conditions, microbial colonization enhanced SOD activity in wheat plants (Fig. 6A).Water stress increased SOD activity in microbial-and uninoculated plants.At 35% FC, PGPB inoculation raised SOD activity by 8.87% followed by AMF inoculation which improved it by 6.7% while P. indica inoculation increased it by 3.10% compared to uninoculated plants.At 35% FC, PGPB and P. indica inoculated plants displayed increased CAT activity.Conversely, AMF inoculated plants exhibited decreased CAT activity in response to increased water stress.Plants inoculated with PGPB and P. indica displayed CAT activity increase of 37.20% and 21.54% as opposed to uninoculated plants respectively at 35% FC (Fig. 6B).Microbial-inoculated plants displayed higher APX activity than uninoculated plants under all water regimes.At each level of water stress, P. indica inoculated plants exhibited significantly higher APX activity than PGPB and AMF-inoculated plants.At all water regimes, PGPB inoculated plants displayed 1.3-fold higher APX activity than uninoculated plants.Plants inoculated with AMF had APX activities 1.2 and 1.1-fold higher than those uninoculated plants at 75% and 35% FC, respectively.At 75% and 35% FC, P. indica inoculated plants showed 1.4-fold higher APX activity than uninoculated plants (Fig. 6C).

Cell ultrastructural studies
At all levels of water regimes, the electron micrographs showed qualitative differences between microbial inoculated and uninoculated plants.

Chloroplast
Figure 7 shows changes in the chloroplast ultrastructure in microbial-inoculated and uninoculated wheat leaves under water stress.Microbial inoculated and uninoculated wheat plants displayed chloroplasts with distinct thylakoids and grana surrounded by double-layered membranes under well-watered conditions (75% FC) (Fig. 7A-D).However, PGPB inoculated plants exhibited a greater number of thylakoids and granum than AMF and P. indica inoculated plants.Consequently, at 35% FC, the chloroplast in uninoculated plants was characterized by disorganized thylakoids and grana and the accumulation of a large number of smaller-size plastoglobules (Fig. 7E).Contrary to this, microbial-inoculated plants contained intact thylakoids, disorganized grana, and fewer but larger size plastoglobules (Fig. 7F-H).As compared to AMF and P. indica, plants inoculated with PGPB showed more intact thylakoids and fewer but large-size plastoglobules at severe drought conditions.

Plasma membrane
Under well-watered (75% FC) conditions the plasma membrane of both microbial inoculated and uninoculated wheat plants closely adhered to the cell wall (Fig. 8A-D).As a result of water stress, the plasma membrane detached from the cell wall, forming an apoplastic space.In response to increased water stress, the space between the plasma membrane and the cell wall became larger.Plasma membrane detachment, however, was less pronounced in microbialinoculated plants, resulting in a smaller cell wall-plasma membrane gap.At all levels of water stress, PGPB-inoculated plants showed comparatively less plasma membrane separation from the cell wall than AMF and P. indica.Furthermore, at severe water stress, invaginations of the plasma membrane led to the formation of vesicles in the cytoplasm.Compared with their microbial-inoculated counterparts, uninoculated plants displayed less vesicle formation (Fig. 8F-H) at 35% FC.

Discussion
In this study, AMF and P. indica colonization in wheat roots decreased at 35% FC, consistent with previous research showing reduced hyphal growth and mycorrhizal colonization under drought stress (Ostadi et al. 2022).Reduced colonization led to decreased shoot and root mass, indicating the dependence of beneficial effects on root colonization.Both microbially-inoculated plants and uninoculated plants under drought stress showed reduced plant growth.This finding may be explained by reduced nutrient uptake and photosynthesis in the plant due to drought stress (Ullah and Farooq 2022).However, drought-stressed wheat plants inoculated with PGPB, AMF, and P. indica exhibited greater shoots and root biomass levels than uninoculated plants.The results of our study are in agreement with earlier reports on AMF colonization in Dalbergia sissoo (Bhardwaj et al. 2023), and Commiphora myrrha (Birhane et al. 2023).Based on the results of a study by Ghabooli and Kaboosi (2022), inoculating Solanum lycopersicum with P. indica under water stress conditions enhanced root and shoot biomass and root volume.Miranda et al. (2023) reported that Zopfiella erostrate significantly enhanced the biomass of wheat plants under drought stress.We found that microbial-inoculated plants had higher leaf area than uninoculated plants under drought stress as has also been reported by Oliveira et al. (2022) in the case of AMF-inoculated soybean plants had increased leaf area as compared to uninoculated plants.The improved nutrient uptake (Rajput et al. 2022), increased osmolyte accumulation and antioxidant activity, and decreased relative permeability led to improved drought tolerance and increased plant growth in microbial-inoculated plants (Tyagi et al. 2023).On comparing the potential of microbes to increase growth parameters, PGPB inoculated plants showed higher drought resistance as compared to AMF and P. indicainoculated plants.
The nutrient contents of both microbial inoculated and uninoculated wheat plants decreased at 35% FC.This is because of the decreased root development, and less water availability and a meagre absorption of essential nutrients during drought condition.However, at all levels of drought stress, microbial inoculation provided better nutrient acquisition than uninoculated plants.It is believed that the increased absorption surface of extraradical hyphae is responsible for enhancing nutrient status in AMF and P. indica inoculated plants (Ortiz et al. 2015).Nutrients play a vital role in plant growth and development.Calcium is essential for membrane integrity, cell wall stability, and ion transport (de Bang et al. 2021).Drought induces Ca 2+ deficiency in plants.During drought, Na + replaces Ca 2+ in the membranes and cell walls, reducing cell turgidity and hydraulic conductivity.Present study showed that microbial-inoculated plants were better at absorbing Ca 2+ ions which is in line with the research findings of Devarajan et al. (2021).
K + maintains osmotic and membrane potential (Sardans and Peñuelas 2021) and regulates the synthesis of proteins, sugars, and enzyme activity (Hasanuzzaman et al. 2018).We found that drought treatment reduced K + levels both in inoculated and uninoculated plants.As Na + and K + have similar physiological properties, they are inevitable competitors.Consequently, cytoplasmic Na + competes with K + for binding sites, inhibiting metabolic processes that rely on K + .However, microbial inoculated plants showed higher uptake of K + ions than uninoculated plants.According to present findings, higher osmolyte accumulation, protein synthesis, and antioxidant enzyme activity can be attributed to high K + accumulation (Hasanuzzaman et al. 2018) in microbialinoculated plants under drought stress.
Magnesium is an essential component of chlorophyll, and it is required for photosynthesis and protein synthesis (Kleczkowski and Igamberdiev 2021).It was reported that drought stress reduced chlorophyll content and photosynthesis in plants, but PGPB and P. indica inoculation increased these parameters (Inbaraj 2021).Similarly, drought reduced Mg 2+ uptake in wheat, but Azospirillum alleviated this effect.We found that drought and microbial inoculation significantly affected Mg 2+ concentration in plant tissues.This may be attributed to the low availability of Ca 2+ under drought stress.Ca 2+ is a strong competitor of Mg 2+ (Pandey et al. 2021) and has more affinity for plasma membrane sites than Mg 2+ .In our study, higher chlorophyll content, membrane integrity, and photosynthesis can also be correlated with increased Mg 2+ content in microbial-inoculated plants under drought stress.8 Transmission electron micrographs of the cell wall and plasma membrane in mesophyll cells of A control plant at 75% FC; plasma membrane closely appressed to the cell wall B P. indica inoculated plant at 75% FC; well-preserved plasma membrane in close association with the cell wall C PGPB inoculated plant 75% FC; plasma membrane closely appressed to the cell wall D AMF inoculated plant at 75% FC; well-preserved plasma membrane in close association with the cell wall E control plant subjected to 35% FC stress; tremen-dous increase in the apoplastic space between the plasma membrane and cell wall F P. indica inoculated plant subjected to 35% FC stress; a slight increase in the space between the cell wall and plasma membrane G PGPB inoculated plant subjected to 35% FC stress; plasma membrane structure still intact H AMF inoculated plant subjected to 35% FC stress; plasma membrane slightly detached from the cell wall.The values in the bracket denote the level of water regimes imposed on plants.cw cell wall, pm plasma membrane, v vesicle There was a significant increase in Na + content in wheat plants in both microbial-inoculated and uninoculated wheat plants under drought stress.However, uninoculated wheat plants had higher Na + contents compared to PGPB, AMF, and P. indica which implies that microbes control Na + uptake.AMF excludes Na + by discriminating its uptake from soil or plant transfer.The findings indicate that microbes influence Na + translocation to the aerial parts.Through exopolysaccharide binding to sodium ions in the soil, PGPB can confer drought tolerance to plants by decreasing sodium absorption from the soil and increasing nutrient uptake by roots (Bhagat et al. 2021).Low Na + content found in AMFtreated roots can be attributed to the ability of the fungi to retain Na + in intraradical hyphae or compartmentalize it in root cell vacuoles.The mycorrhizal symbiosis enhanced plant growth and physiological process, thereby diluting these ions in tissues.
Unfortunately, most plant nutrient studies under drought stress have paid little attention to micronutrients, therefore, in this study, drought stress and microbial inoculation were investigated to reveal their impact on micronutrient uptake.Micronutrients are essential for plant growth and development.Zn 2+ plays a crucial role in the synthesis of proteins, chlorophyll, and enzymes (Kumar et al. 2021).Chlorophyll synthesis and chloroplast structure and function require Fe (Kumar et al. 2021).In plant cells, copper plays a vital role in photosynthesis, respiration, redox reactions, and the maintenance of cell wall structure (Kumar 2015).In photosystem II, Mn 2+ contributes to the structure of the water-splitting system which provides electrons for photosynthesis.In plants, drought reduces micronutrient uptake.We observed that at 35% FC, the concentrations of Cu, Fe, Mn 2+ , and Zn 2+ in microbially inoculated and uninoculated wheat plants decreased.This is because Cu, Fe, and Zn 2+ are less soluble and mobile in soil solutions during drought conditions.Under drought stress, copper, zinc, and iron levels decrease, and a depletion zone forms around the roots.Because of this, uninoculated plants have limited access to these nutrients.In addition, decreases in Cu, Zn 2+ , Mn 2+ , and Fe in microbial-inoculated plants may be due to decrease hyphal growth during drought.However, at different water regimes, microbial inoculation exhibited better micronutrient acquisition than uninoculated plants.In our study increase in Zn 2+ content maintained the cell membrane integrity as it influenced the lipid composition of membrane of wheat plants inoculated with microbes under drought conditions.In our study, higher chlorophyll content and photosynthesis can also be correlated with increased micronutrient content in microbial-inoculated plants under drought stress.
Photosynthesis is integral to plant growth and is, therefore, an important indicator of a plant's potential for growth and drought tolerance (Liang et al. 2019).In response to drought stress, stomatal closure decreases gas exchange and internal CO 2 levels, resulting in a decrease in photosynthetic pigment production, thereby reducing the photosynthetic rate (Liang et al. 2019), which was further confirmed in the present study.Moreover, the transpiration rate decreases during drought leading to reduced nutrient and water uptake.In contrast, drought-stressed wheat plants inoculated with PGPB, AMF, and P. indica had an increase in photosynthetic rate, stomatal conductance, internal CO 2 , and transpiration rate.Our results are in agreement with previous reports on PGPB inoculation improved plant photosynthetic efficiency by increasing the CO 2 assimilation rate, stomatal conductance, and transpiration rate (Vishnupradeep et al. 2022), and reducing structural damages to the photosynthetic apparatus by enhancing osmolyte accumulation in plants.Additionally, the results indicated that microbial inoculation significantly increased the carotenoid content.The antioxidant property of carotenoids protects the photosynthetic apparatus against free radical damage, inhibits lipid peroxidation, stabilizes cell membranes, and plays a critical role in light-harvesting complex assembly (Farooq et al. 2009).Increased photosynthesis rates are a result of higher chlorophyll content and leaf area.
As light-absorbing pigments, chlorophylls and carotenoids are crucial to photosynthesis.Drought decreases chlorophyll and carotenoid content (Khazaei et al. 2020), which is consistent with present findings.It was observed that PGPB, AMF, and P. indica inoculated plants had higher chlorophyll a and chlorophyll b levels, total chlorophyll and carotenoid levels than uninoculated plants.This suggests that microbial inoculation reduces the impact of drought on wheat.In a similar study, an AMF substantially increased the chlorophyll and carotenoid content of Cupressus arizonica (Aalipour et al. 2023).A study found that maize plants colonized with PGPB had more chlorophyll and carotenoid content (Vishnupradeep et al. 2022).Present findings are in line with earlier studies suggesting higher photosynthetic pigments are probably a consequence of antioxidant accumulation, protecting the chloroplast structure, and increasing the solubilization and bioavailability of organic minerals, such as Mg (Khan et al. 2019).Our results coincide with the findings that Bacillus strains increase chlorophyll levels in maize due to increases in nutrient balance (Moreno-Galván et al. 2020).
Drought stress is associated with a significant accumulation of osmolytes, including carbohydrates, amino acids, quaternary ammonium compounds, and polyamines, which play a critical role in maintaining cell turgor pressure and stabilizing membranes, enzymes, and proteins (Sharma et al. 2019).Furthermore, osmolytes control ROS levels, provide energy for stress management, help repair processes, and support growth (Silva et al. 2018).As a highly stable molecule, trehalose is resistant to high temperatures and acids; it can prevent protein aggregation and degradation during droughts (Jalili et al. 2009).Proline is an important secondary amino acid that has a major role in osmotic regulation and scavenging ROS under drought stress (Adejumo et al. 2021).We observed significantly higher osmolyte accumulation in both microbial-inoculated (PGPB, AMF, and P. indica) and uninoculated wheat plants.However, microbial-inoculated plants had a greater amplitude of osmolyte increases than uninoculated plants.These results are consistent with a previous study that demonstrated increased proline and sugar content in maize inoculated with the microbial consortium under drought stress (Hashem et al. 2016).Acinetobacter calcoaceticus and Penicillium sp.effectively ameliorated drought-induced stress in foxtail millet by increasing glycine betaine, proline, and sugar accumulation (Kour et al. 2020) by maintaining cell turgidity and membrane integrity thereby protecting the ultrastructure of chloroplast and plasma membrane.According to Liu et al. 2022, rhizobium symbiosis enhances drought tolerance in Astragalus sinicus through the production of putrescine, spermidine, and spermine.Based on these results, microbialinoculated plants are more efficient at osmotic adjustment than their uninoculated counterparts.However, PGPB outpaces AMF and P. indica in osmotic adjustment in wheat plants under drought stress.
Proteins are organic molecules that form the structural and functional units of a cell.These compounds improve chlorophyll content, protect chloroplast structure, activate ROS scavenging, and enhance osmolyte synthesis (Riyazuddin et al. 2022).In our study, there was a remarkable difference in protein content in microbial inoculated and uninoculated plants under different water regimes.Plants inoculated with PGPB, P. indica, and AMF had higher protein content.In microbial-inoculated plants, higher protein content is associated with higher photosynthetic rates, chlorophyll contents, membrane integrity, and osmolyte accumulation.Similarly, Boutasknit et al. (2020) found that drought stress could be mitigated by AMF-colonized carob plants with higher levels of protein.According to this study, inoculating wheat plants with PGPB, P. indica, and AMF enhanced drought tolerance through protein regulation.
Since α-tocopherol is an antioxidant, it inhibits ROS generation during photosynthesis (mainly 1 O 2 and OH•) and reduces lipid peroxidation (Kim et al. 2019).Wheat plants inoculated with PGPB, P. indica, and AMF have a high level of α-tocopherol, which indicates that α-tocopherol is a free radical scavenger as well as a modulator of lipid peroxidation during abiotic stress (Kim et al. 2019).This study substantiates the findings of Fresno et al. (2023) that higher levels of α-tocopherol in AMF-colonized Trifolium repens plants mitigate drought stress.Based on the current study, inoculating wheat plants with PGPB, P. indica, and AMF enhanced drought tolerance by regulating α-tocopherol content, thus maintaining a high level of photosynthetic efficiency and chlorophyll content and reducing ion leakage.
A key physiological indicator of drought in plants is malondialdehyde, a by-product of membrane lipid peroxidation (Yasmeen et al. 2022).Lipid peroxidation alters cell membrane ultrastructure and permeability.Additionally, the LOX enzyme converts lipids into hydroperoxyl fatty acids and releases free radicals that damage cells.Prevention of lipid peroxidation and relative permeability are key factors in drought tolerance (Kour et al. 2020).An increase in lipid peroxidation, relative membrane permeability, and LOX activity was observed in wheat plants inoculated with microbes (PBPB, AMF, and P. indica) and uninoculated plants at 35% FC.However, the amplitude of the increase in lipid peroxidation and electrolyte leakage in microbialinoculated plants was lower than that of uninoculated plants, which is in accordance with earlier reports (Begum et al. 2022).The reduced LOX activity in microbial-inoculated plants is inconsistent with a previous report in which the co-inoculation of AMF and PGPB resulted in an increase in LOX activity in tobacco plants (Begum et al. 2022).The microbial inoculated plant maintained a lower MDA concentration and relative membrane permeability by regulating adaptive responses such as the synthesis of non-enzymatic molecules (carotenoids, proline, and α-tocopherol) and enzymatic antioxidants (CAT, APX, and SOD).These findings are similar to previous findings that PGPB-inoculated plants showed improved drought stress tolerance by maintaining a high level of carotenoids, proline, and tocopherol (Timmusk et al. 2014), as well as CAT, APX, and SOD activity (Chiappero et al. 2019), thereby reducing ROS accumulation.
During abiotic stress, antioxidant enzymes (CAT, SOD, and APX) actively protect the plant cell from oxidative bursts and assist in scavenging ROS during adverse environmental conditions (Ru et al. 2023).The SOD enzyme plays a critical role in protecting the plant against oxidative stress by converting O2• − to H 2 O 2 .In order to detoxify H 2 O 2 , it must be converted into H 2 O. SOD produces H 2 O 2 that is scavenged by CAT and attenuates oxidative stress on the cell.In our study, the antioxidant enzyme activity of uninoculated and inoculated plants differed significantly.This result is consistent with an earlier study demonstrating that microbial inoculation improved plant growth under drought stress because of the enhanced activity of the ROSscavenging antioxidant enzyme (Tyagi et al. 2023).In the study conducted by Ma et al. (2022), it was observed that AMF triggers a reduced oxidative burst in drought-stressed walnut plants by stimulating the activation of antioxidant defense systems.Thus, microbial inoculation reduces lipid peroxidation and membrane permeability in plants by increasing antioxidant enzyme activity (Tyagi et al. 2023).Earlier studies have found that microbial consortium-treated maize exhibited increased antioxidant enzyme activity in stress conditions (Abbas et al. 2020), which lends weight to our study.Tyagi et al. (2023) reported that deploying a microbial consortium of Serendipita indica, Rhizophagus intraradices, and Azotobacter chroococcum boosts drought tolerance in maize by enhancing the activity of antioxidant enzymes.CAT activity in AMF-inoculated plants decreased at 35% FC, which is consistent with previous findings that PGPB reduced CAT activity during drought stress in maize (Siddique et al. 2022).The current study proposes the use of PGPB, AMF, and P. indica to enhance antioxidant defense mechanisms in wheat and reduce drought stress.Future agriculture practices may benefit from the use of microbes as a bio-inoculant for promoting plant growth.
The chloroplast is the most susceptible cell organelle to drought stress (Gunasekera and Ratnasekera 2023).The present study illustrated that the ultrastructure of both microbially-inoculated and uninoculated wheat plants was affected by drought stress.At 35% FC, the thylakoid lumen swelled, resulting in the destacking of grana.A number of studies have shown that osmotic stress causes thylakoid swelling and membrane damage (Aldesuquy et al. 2018).Microbially inoculated plants, however, seem to have less chloroplast damage than uninoculated plants.This may be attributed to more accumulation of osmolytes (glycine betaine, polyamines, and sugars) in microbial-inoculated plants.More accumulation of osmolytes prevents osmotic collapse, while polyamines inhibit membrane degradation by interacting with negatively charged membrane sites in grana and stroma thylakoids (Ali et al. 2020).
Microbial-inoculated wheat plants showed reduced chloroplast membrane damage, possibly due to larger plastoglobule sizes that prevented oxidative damage.It has been reported that plastoglobules increase in size during drought (Zhu et al. 2021).Plastoglobules are lipoprotein bodies present in the chloroplast and function as sites for α-tocopherol synthesis under oxidative stress (Brehelin et al. 2007).In our study, microbial-inoculated plants had higher levels of α-tocopherols than uninoculated plants.α-tocopherols have been reported to protect membrane lipids and photosystem II against photooxidation.In microbial-inoculated plants, plastoglobule accumulation may be attributed to higher polyamine levels (Shu et al. 2015).Thus, in this study, microbialinoculated plants had reduced damage to the ultrastructure of chloroplast due to high levels of osmolytes and large plastoglobules that prevented oxidative damage.
Cell membrane stability in plants is considered a reliable indicator of drought tolerance (Abobatta 2019).Electron micrographs showed that plasma membranes disintegrated under drought stress.At 35% FC, the distance between the plasma membrane and cell wall increased.A similar finding was reported for Ormosia hosiei seedlings (Liu and Wei 2021).The disintegration of membranes occurs due to the peroxidation of polyunsaturated fatty acids by free radicals (Zhang et al. 2021) and the shrinkage of protoplasm during drought conditions.Compared to uninoculated plants, however, microbial-inoculated plants showed less membrane damage and protoplasm shrinkage.This statement is further strengthened by the accumulation of osmolytes (glucose, trehalose, proline, glycine betaine, and polyamines), which maintain cell turgor pressure and stabilize membranes, enzymes, and proteins (Sharma et al. 2019).We found lower levels of electrolyte leakage, lipid peroxidation, and LOX activity in microbial-inoculated plants, which play a significant role in membrane protection.This is because microbialinoculated plants acquire more non-enzymatic antioxidant molecules (proline, α-tocopherols, and polyamines), enzymatic antioxidants (SOD, CAT, and APX), and nutrients (calcium and potassium) compared with uninoculated plants.
Proline is an effective ROS scavenger, protecting cells from damage by oxidative stress under a stressful environment (Ashraf and Foolad 2007).Moreover, α-tocopherol protects host tissue from lipid peroxidation and maintains the integrity of their membrane by scavenging free radicals (Niki 2021).Polyamines provide stability to membranes (Wang et al. 2022).In adverse environmental conditions, antioxidant enzymes (CAT, SOD, and APX) scavenge ROS and protect membranes from oxidative stress (Ru et al. 2023).By forming intermolecular links, calcium maintains the structural integrity of plant membranes, stabilizes cell walls, and regulates ion transport and selectivity (de Bang et al. 2021).Potassium plays a vital role in plants, including osmoregulation and membrane potential (Sardans and Peñuelas 2021).So, microbial inoculation reduces membrane damage through osmolyte, nutrient, and antioxidantmediated reductions of lipid peroxidation in plants (Tyagi et al. 2023).Vesicle formation was more in microbial inoculated as compared to uninoculated plants.Vesicle formation increases under stress conditions (Karim et al. 2014).They increase the membrane surface area, which facilitates ion transport and storage, thus protecting the cells from ionic imbalance.Thus, more vesicle formation observed in this study indicates a better ability of microbial-inoculated plants to tolerate droughts.
In conclusion, the results of this study confirm that the PGPB consortium, AMF consortium, and P. indica inoculation significantly improved wheat performance under drought-stress conditions.Our findings demonstrate that microbial inoculation protects wheat plants from drought stress by enhancing physiological processes and osmolyte accumulation, leading to better growth and biomass than uninoculated plants.The higher osmolyte accumulation ensures osmotic adjustment and scavenges free radicals more effectively.Additionally, microbial colonization maintains favorable levels of macronutrients and micronutrients than uninoculated plants, thereby mitigating drought-induced ionic imbalances.PGPB consortium, AMF consortium, and P. indica inoculation reduces lipid peroxidation, membrane permeability, and LOX activity and increase antioxidant enzymes (SOD, CAT, and APX) activity and accumulation of antioxidant metabolites (α-tocopherol, proline, carotenoids, polyamines) in wheat plants.Microbial-inoculated plants, therefore, have a better antioxidant defense system for preventing oxidative damage, which makes them more drought resistant.Colonization with the PGPB consortium, AMF consortium, and P. indica mitigates ultrastructural alterations caused by drought-induced ion toxicity, osmotic stress, and oxidative stress.Plants inoculated with microbes suffer less ultrastructural damage as a result of improved osmolyte accumulation, nutrient acquisition, and antioxidant defense system.The preservation of ultrastructure in microbial-inoculated plants indicates enhanced drought tolerance.According to this study, the PGPB consortium has greater potential for improving wheat yield and tolerance to drought stress than the AMF consortium and P. indica, making it an excellent choice for agricultural practice.

Fig. 2
Fig. 2 Effects of drought and microbial inoculation in wheat on the content of A chlorophyll a; B chlorophyll b; C total chlorophyll; and D carotenoid.Values represent averages across replicates.Different letters represent significant differences at P < 0.05

Fig. 3 Fig. 4
Fig. 3 Effects of drought and microbial inoculation in wheat on the content of A glycine betaine; B total sugar; C trehalose; D proline; E putrescine; and F spermidine.Values represent averages across replicates.Different letters represent significant differences at P < 0.05

Fig. 5 Fig. 6
Fig. 5 Effects of drought and microbial inoculation in wheat on A MDA content; B relative permeability; and C LOX activity.Values represent averages across replicates.Different letters represent significant differences at P < 0.055

Fig. 7
Fig. 7Transmission electron micrographs of chloroplast in mesophyll cells of A control plant at 75% FC; distinct thylakoids and grana surrounded by a well-defined double-layered membrane B P. indica inoculated plant at 75% FC; well-developed thylakoids and properly stacked grana surrounded by a double membrane C PGPB inoculated plant at 75% FC; distinct thylakoids and well-developed grana surrounded double-layered membrane D AMF inoculated plant at 75% FC; properly stacked grana in the stroma E the control plant was subjected to 35% FC; disorganized thylakoids and grana.Accumula-

Fig.
Fig. 8 Transmission electron micrographs of the cell wall and plasma membrane in mesophyll cells of A control plant at 75% FC; plasma membrane closely appressed to the cell wall B P. indica inoculated plant at 75% FC; well-preserved plasma membrane in close association with the cell wall C PGPB inoculated plant 75% FC; plasma membrane closely appressed to the cell wall D AMF inoculated plant at 75% FC; well-preserved plasma membrane in close association with the cell wall E control plant subjected to 35% FC stress; tremen-

Table 1
InfluenceGrowth parameters of microbial inoculated and uninoculated wheat plants at two different water regimes are shown in Table1.At all levels of water regimes, the microbialinoculated wheat plants performed significantly better in terms of growth response compared to uninoculated plants.Under severe drought conditions (35% FC), PGPB inoculation increased the shoot and root biomass by 222.6% and 109.20%,respectively, followed by AMF inoculation which improved it by 188.1% and 85.89% respectively, while P. indica inoculation increased it by 65.6% and 72.19%, respectively, compared to uninoculated plants.The leaf area of wheat plants was significantly improved with inoculation with PGPB (64.87%) followed by AMF (57.58%) and P. indica (49.18%) in comparison to non-inoculated wheat plants.Two-way ANOVA demonstrated a significant inde- of drought stress and microbe inoculations on percentage microbe colonization, leaf area, and biomass of shoot and root in wheat plantsValues represent averages across replicates.Letters within a column represent significant differences at P < 0.05 ns not significant ***P < 0.001, **P < 0.01, *P < 0.05 pendent effect of drought, P. indica, PGPB, AMF, and interaction of drought × P. indica, drought × PGPB, and drought × AMF on leaf area (Table

Table 2
Influence of water stress and microbial inoculation on the content of macronutrients and micronutrients in leaves of the wheat plants Values represent the mean of replicates.Different letters within a column indicate significant differences at P < 0.05 P. indica, PGPB, and AMF on physiological parameters (Table3), indicating PGPB as the most efficient microbe in mitigating drought stress across all water regimes in wheat plants compared to AMF and P. indica.