Climate change and unfavorable abiotic factors in the recent era have adversely affected crop productivity and economic yields from agricultural land. Water scarcity, severe drought, and temperature extremes are reducing moisture levels in soil profiles, altering plant physiology, and reducing growth and yields (Deng et al., 2021). The worst heatwave due to global warming hit the water supply under threat while China and Europe face severe droughts in recent years (JRC, 2022; Global times, 2022). According to the United Nations Global Land Outlook report 2020–2022, Pakistan is one of 23 countries that have faced emergencies related to drought in the last 2 years (UNCCD, 2022).

Rice (Oryza sativa L.) as a paddy field crop is particularly susceptible to water scarcity. Due to insufficient, uneven, and unpredictable rainfall during the growing season, it is estimated that 50% of the world′s rice production will be decreased dramatically (Al-Zahrani et al., 2022). To alleviate global food insecurity, a large number of rice irrigations have to be replaced by different management strategies (Aalipour et al., 2020). Currently, strategies to enhance the plant′s ability to cope with drought stress include traditional breeding, genetic engineering of drought-tolerant transgenic plants, and the use of water-saving irrigation. Unfortunately, these strategies are very complex, time-consuming, and labor-intensive. Therefore, we need quick, reliable in situ methods that can be widely applied to enhance the ability of plants to produce more under limited irrigations (Kannepalli et al., 2020).

The use of plant growth-promoting bacteria (PGPB) is an effective approach to grow plants under water scarcity. Directly or indirectly, they can promote plant growth under biotic or abiotic stress environments (Fadiji et al., 2022). Known mechanisms of PGPB to improve plant growth and its survival in stressed environments, especially under drought conditions, include phytohormone production (IAA), phosphate (P) solubilization, exopolysaccharide (EPS) production, and siderophore production for sequestration of iron (Ilyas et al., 2020). In addition, PGPB is associated with the molecule′s catabolism, i.e., bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase production involved in signaling under stress. Ethylene content in plants is usually increased in stress; thus, inoculation with ACC deaminase-producing bacteria mitigates the effect of osmotic stress in plants by reducing the production of ethylene (Sagar et al., 2020).

Polyethylene glycol (PEG)-induced drought stress decreased the seed germination, vigor index, and seedling growth of rice as compared with well-irrigated seedlings (Delshadi et al., 2017). Most of the studies reported that inoculation of phosphate-solubilizing and indole acetic acid (IAA)-, 1-aminocyclopropane-1-carboxylate (ACC)-, and exopolysaccharide (EPS)-producing bacteria significantly improved percent seed germination and seedling vigor index of different plants, i.e., wheat, grapevine, and tomato under osmotic stress (Ilyas et al., 2020; Duan et al., 2021; Khairnar et al., 2022). PGPB are an important biological element with beneficial effects on plant biochemical and physiological properties, resulting in improved photosynthetic pigments, osmolyte production, leaf number, and relative water content (Fadiji et al., 2022). Wheat, maize, and rice plants inoculated with PGPB resulted in an improved root/shoot length, fresh/dry weights, leaf chlorophyll, membrane stability index (MSI), proline content, and upregulation in the antioxidants such as peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), polyphenol oxidase (PPO), and phenylalanine (PAL) to scavenge the production of reactive oxygen species (ROS) under drought conditions (Mathur et al., 2018; Li X et al., 2020; Inbaraj, 2021).

Solute accumulation is one of the most important mechanisms adopted by plants to withstand drought stress. PGPB confer tolerance to drought stress in plants by improving the plant′s ability to produce osmoprotectants like proline (Ngumbi and Kloepper, 2016). Bacterial communities not only alter the hormonal balance of the roots but also improve plant nutrient and water uptake, resulting in microbial colonization and symbiotic relationships (Nemeskeri et al., 2022). Nemeskeri et al. (2019) reported that an improvement in plant water uptake also reduced the plant temperature as compared with non-inoculated stressed plants as high plant temperature has an adverse impact on yield. Infrared thermography (IRTI) has appeared as an easy-to-use, non-destructive, and promising technique for measuring plant physiological status related to water availability (Kim et al., 2020).

Drought stress reduces the rice plant′s yield, whereas PGPB inoculation improved the plant traits such as height, panicles, plant weight, and yield grown even under drought stress (El-Mageed et al., 2022).

Overall, drought-tolerant rhizobacteria offer an eco-friendly and efficient approach to alleviate plant stress and enhance plant growth and yield under adverse climatic conditions. Therefore, in the present study, we focused on i) isolating, screening, and selecting drought-tolerant rhizobacteria and ii) evaluating efficient rhizobacteria for rice growth promotion under water stress conditions through the study of morpho-physiological and biochemical parameters. We hypothesized that i) drought-tolerant bacteria with plant growth promoting (PGP) traits can improve plant vigor and root architecture through phytohormone (IAA), osmolyte, and exopolysaccharide production, which ultimately increases the yield of rice under water stress conditions, and ii) integrated application of potential drought-tolerant bacteria obtained in this study and advanced non-destructive in situ handheld instruments, i.e., infrared thermal imaging (IRTI) and infrared gas analyzer (IRGA), with conventional methods may lead to the efficient selection and timely intervention of a tolerant variety to minimize yield loss from water scarcity. This integration of biological strategy with high-quality imagery and physiological processes will lead to a useful day-by-day comparison for the detection of drought stress, which is the need of the hour because conventional methods require a long time for the detection of drought conditions.

Water scarcity accompanied by decreased precipitation is one of the key obstacles to agricultural throughput globally and is expected to increase further, hence posing a major threat to future food security (Uzma et al., 2022). Pakistan is the sixth most drought-prone country in the world (UNCCD, 2022). In Pakistan, about 15 million hectares of cultivated area are affected by the aforementioned conditions resulting in less crop productivity and the situation is expected to worsen by the year 2025 (WHO, 2019). According to the Pakistan Rice Sector Report 2020, rice crop area decreased by 3.1% and crop production reduced to 3.3% due to a decrease in cultivated area, water shortage, and dry weather (Anser et al., 2020).

To cope with the adverse effects of global climate change, increasing population, and food insecurity, there is a dire need to develop and screen rice genotypes tolerant to water scarcity with high yield for sustainable rice production (Villalobos-López et al., 2022). Plant growth-promoting bacteria (PGPB) can mitigate the adverse effects of drought on crops (Barquero et al., 2022). Integration of sustainable approaches like plant-associated microbes and drought-tolerant varieties can enhance rice crop production under these alarming conditions of climate change (Kumar and Verma, 2019; Zhang et al., 2020; Shaffique et al., 2022).

Therefore, in the present study rhizospheric soil samples were collected from major rice-growing fields for the isolation of bacteria. The isolated bacterial population was screened for drought tolerance using 20% PEG-8000 and PGP traits (IAA production, P solubilization, organic acid production, EPS, siderophore, and ACC production) ( Table 1 ). Recent studies reported that IAA-, EPS-, and ACC-producing bacteria significantly improve the growth and yield of mung bean (Uzma et al., 2022), rice (Zhang et al., 2020), and tomato (Ghosh et al., 2019) under drought stress conditions. Bacteria with a combination of drought stress tolerance and multiple PGP determinants were reported as wheat growth-promoting under well-irrigated and stressed conditions (Zia et al., 2021). Danish and Zafar-ul-Hye (2019) also reported that maximum drought-tolerant strains show ACC deaminase production.

Santoyo et al. (2016) reported that PGPB can promote the activity of ACC deaminase, which directly or indirectly improves plant growth. PGPB can reduce the concentration of ethylene by consuming the ACC before its oxidation through ACC oxidases produced by the plant (Din et al., 2019). Drought-tolerant bacteria, i.e., E. aerogenes and B. thuringiensis, have been reported as higher phosphate-solubilizing bacteria with ACC deaminase activity (Timmusk et al., 2014; Zhang et al., 2020). Results of the present study showed maximum P solubilization (SI: 5.6, 356 µg ml^-1) by Brucella haematophila NM-4 ( Table 1 ). According to Uzma et al., 2022, Pseudomonas aeruginosa (MK513748) showed to have maximum P solubilizing capability (95 µg ml^-1) and solubilization index (SI: 3.3). The potential drought-tolerant rhizobacteria from the present study belonged to the genera Ochrobactrum (strains: NM-1), Brucella (strains: NM-3, NM-4), and Bacillus (strains: NM-2, NM-5, NM-6, NM-7, and NM-8). Ahmad et al., 2022a reported that Bacillus thuringiensis, Bacillus amyloliquefaciens, and Ochrobactrum pseudogrignonense can enhance plant growth of wheat, maize, and rice under water stress.

In the present study, selected drought-tolerant bacteria produced acetic acid, citric acid, gluconic acid, and succinic acid during P solubilization in NBRIP medium as detected by HPLC ( Table 1 ). The production of organic acids is one of the key mechanisms involved in P solubilization (Kapadia et al., 2022). Three most efficient drought-tolerant bacteria, i.e., Bacillus subtilis NM2, Brucella haematophila NM4, and Bacillus cereus NM-6, produced higher acetic acid, succinic acid, and gluconic acid ( Table 1 ). Bacillus sp. and Enterobacter sp. solubilized inorganic P and produced different organic acids such as gluconic acid, citric acid, lactic acid, propionic acid, and succinic acid (Sarkar et al., 2018; Mendes et al., 2020).

Multiple strains of the genus Bacillus showed a significant role in the alleviation of abiotic stresses in different crops due to their ability to produce IAA, EPS, and siderophore production (Chandra et al., 2018; Din et al., 2019). Concomitant to these findings, the present study showed maximum production of IAA (46.2 µg ml^-1) by Brucella haematophila NM-4 followed by Bacillus cereus NM-6 (37.5 µg ml^-1) and Bacillus subtilis NM-2 (31.5 µg ml^-1) ( Table 1 ). Abdelaal et al. (2021) reported that the growth hormone, i.e., IAA, produced by plant growth-promoting bacteria can enhance plant growth, especially the root architecture under stressed conditions. Bacillus subtilis NM-2 also showed the production of polysaccharide (fructose), detected by TLC. EPS-producing PGPB enhances soil aggregation and protects plants under water scarcity by retaining increased water potential around the root area (Ojuederie et al., 2019). These isolated bacteria also showed the production of iron-chelating compounds (i.e., siderophores) ( Figure 1 , Table 1 ).

The improved vigor index (1009.2-1100) was observed in response to consortium inoculation under PEG-8000-induced osmotic stress ( Table S2 ). Pseudomonas balearica RF-2 inoculation showed increased wheat seed germination and seedling vigor under 20% PEG-induced osmotic stress tolerance (Razzaq et al., 2017; Zia et al., 2021). Arun K et al. (2020) also reported that Bacillus megaterium PB50 with multifarious PGP determinants showed drought stress alleviating potency in rice plants. Inoculation of Pseudomonas fluorescence strains DR7 and DR11 stimulated the seed germination capability of foxtail millet from 13 to 141% over the non-treated seeds under the high level of osmotic stress (-0.5 MPa) (Niu et al., 2018).

Rhizoscanning of seedlings was also carried out to evaluate the effect of drought-tolerant PGPB on root architecture. Likewise, improved root rhizoscanning parameters like root surface area (3.46 cm²) and length per volume (36.5 cm m^-3) were found in consortium-inoculated osmotic stressed seedlings ( Figure 2 ; Table 2 ). Previous studies reported that Bacillus sp. 12D6 and Bacillus megaterium inoculation improved root architecture (root length and diameter), wheat and peanut adaptability, and nutrient uptake under water-stressed conditions (Jochum et al., 2019; Grover et al., 2021).

In planta evaluation of the drought-tolerant consortium (Brucella sp. NM-4, Bacillus subtilis NM-2, and Bacillus cereus NM-6) was further carried out in well-irrigated and water-stressed conditions in a pot experiment under net house conditions. The beneficial PGPR associated with the plant roots and tissues reduces abiotic stress on morphological, physiological, and biochemical mechanisms (Shahzad et al., 2013; Rolli et al., 2015). Dubey et al. (2019) reported that plant growth parameters, i.e., root/shoot length, plant fresh and dry weights, were reduced as the water stress increased. These findings supported our results as plant fresh and dry weights of inoculated plants improved as compared with the non-inoculated stressed plants ( Figure 7C ), whereas significant improvement in seedling growth parameters was observed in inoculated stressed plants as compared with the non-inoculated stressed plants (Ali et al., 2022).

Plant growth and productivity are the results of well-regulated photosynthesis, functioning in the leaf and important interlinked physiological responses (Lefe et al., 2017). In the present study, growth inhibition in water-deficit plants might be linked with the rate of photosynthesis, which resulted in stunted plant growth and development. However, consortium inoculation showed an improved plant growth and capability to maintain a higher rate of photosynthesis, the concentration of intracellular CO₂, transpiration rate, and stomatal conductance as compared with the non-inoculated stressed plants ( Table 3 ). Batool et al. (2020) stated that stomatal conductance, rate of transpiration, photosynthesis, and intracellular CO₂ are interconnected such that under drought stress downregulation in the efficiency of one decreased that of another. Zhang et al., 2022 reported that Bacillus pumilus inoculation improved the photosynthetic characteristics (stomatal conductance, photosynthetic and transpiration rates) of G. uralensis plants grown under drought stress.

The present study showed stimulation (50%) of relative water content (RWC) in V2-inoculated plants as compared with non-inoculated stressed plants ( Table 5 ). Abbasi et al. (2020) reported that Streptomyces strains IT25 and C-2012 not only suppressed the signals of stress in tomato plants but also improved plant water status in the form of RWC. Nemeskeri et al. (2022) stated that inoculation of Pseudomonas sp. MUS04 in tomato plants improved the water uptake and also decreased the plant temperature as compared with non-inoculated stressed plants. These findings are congruent to the present study, as a percent decrease at 15 DAS was observed in inoculated rice genotype V2, i.e., 6% as compared with non-inoculated stressed plants ( Table 4 ).

Ali et al. (2022) reported that inoculation of Bacillus strains improved the chlorophyll content in drought and salt-stressed rice plants as compared with the non-inoculated stressed plants. However, in the present study in plants inoculated with the drought-tolerant consortium, an increased amount of chlorophyll content was observed ( Table S4, S6 ). This increase in chlorophyll content might be the source of improved plant growth under water-deficit conditions (Nadeem et al., 2010; Ahmad et al., 2013; Kang et al., 2014). The present study showed that inoculation with the drought-tolerant plant growth-promoting consortium stimulated stressed plants to accumulate proline content with 122%, 114%, and 85.8% increased rates in three rice genotypes, i.e., V2, V1, and V3, respectively, as compared with non-inoculated stressed plants ( Table 4 ). Wu et al. (2014) stated that inoculated plants synthesized and accumulated a significant amount of proline, a compatible solute, to develop osmotic adjustment under water stress. Accumulation of osmoprotectant (proline) is one of the key cellular adaptations that plants undergo for osmotic adjustment during water scarcity (Zouari et al., 2019). Shaffique et al. (2022) reported that PGPB increases the proline content in plants, which minimizes dehydration loss, protects membrane proteins, and maintains cellular turgidity caused by water stress. Dastogeer et al. (2020) reported that the proline content and photosynthetic rate of plants are closely linked, which is consistent with our results, whereas consortium inoculation increased proline content in all tested genotypes under water scarcity, confirming the beneficial role of proline in maintaining photosynthetic activity (Ahmad et al., 2022a).

Increased electrolyte leakage (EL) damages the plant cell membrane under abiotic stress (Gangadhar et al., 2016). The present study showed that the PGPB consortium improved the membrane stability index by 62% of V2 under water stress as compared with the non-inoculated stress plants ( Table 4 ). Uzma et al. (2022) reported that plant growth-promoting bacterial inoculation increased the membrane stability of plants under water-stressed conditions as compared with the non-inoculated stressed plants.

In the current study, we found that the consortium-inoculated three rice genotypes (V2, V3, and V1) showed increased accumulation of antioxidants (SOD, CAT, PAL, PPO, and POD) variably under water stress as compared with non-inoculated stressed plants ( Table 4 ). Al-Zahrani et al. (2022) reported that plant inoculation with PGPB often reduces the overproduction of ROS by producing more ROS-scavenging enzymes. Antioxidant enzyme production is an effective mechanism, i.e., microbe-induced systemic tolerance (MIST), which confers tolerance to drought (Fadiji et al., 2022). Singh et al. (2020) reported that microbial inoculation activated the antioxidant enzymes PAL and PPO in plants to alleviate oxidative stress and crop yield as compared with the non-inoculated plants.

Consortium inoculation significantly improved the yield and yield-related attributes of all the tested genotypes under well-irrigated treatment (IC) as compared with their well-irrigated controls ( Table 5 ). Ilyas et al. (2020) reported that PGPB improves root architecture that helps to enhance plants’ water and nutrient uptake efficiency, resulting in low plant temperature and better growth and yield under water scarcity. Inoculation significantly increased the grain weight (26%) of V2 under water scarcity as compared with non-inoculated water-stressed plants ( Table 5 ). Raheem et al. (2018) reported that plant inoculation with drought-tolerant plant growth-promoting bacteria improved the shoot length, the number of grains, and the total yield as compared with their respective control (non-inoculated) stressed plants.

Principal component analysis (PCA) confirmed the positive correlation between drought-tolerant bacterial inoculation with grain yield, photosynthetic rate, chlorophyll content, transpiration rate, sub-stomatal conductance, reference CO₂, and IR temperature across inoculated varieties ( Figure 9 ). No parameter was found to have a negative effect on drought-tolerant bacterial inoculation. Regression analysis further validated the positive correlation between yield, photosynthetic rate, IR temperature, and POD in inoculated rice plants ( Figure 9 ).

PGPB used multiple direct and indirect mechanisms, i.e., IAA production, phosphate solubilization, EPS production, ACC deaminase activity, and siderophore production for the alleviation of drought stress and plant growth promotion. Through these mechanisms, rhizobacteria enhance the water-retaining capability of the soil and improve photosynthetic characteristics, water uptake, and nutrient uptake by plants through alteration in root architecture, which in return reduces the plants’ temperature, alleviates ethylene stress in plants, and enhances cellular turgor and osmotic adjustment through the accumulation of osmoprotectant (proline) under water-deficit conditions. Hence, as a result of this, the beneficial interaction between drought-tolerant PGPB and plants improved the crop yield under water scarcity.

In Pakistan, Basmati rice with aromatic long grain has significant importance among rice traders, millers, and consumers. It covers more than 80% of the total Basmati area in the major rice-growing province Punjab, but it is susceptible to water stress (Sabar et al., 2019). The current scenario of the climatic shift resulted in uncertainty for rice growers due to water shortage. In the present study, consortium inoculation showed its supporting role to sustain the yield of rice genotypes NIBGE-DT02 and Super Basmati under water scarcity. The integration of drought-tolerant bioinoculant and genotype (NIBGE-DT02) will lead to improved water stress tolerance concomitant with the increased yield under alarming conditions of climate change. Based on the promising results of the designed consortium, whole-genome sequencing of the promising drought-tolerant plant growth-promoting bacteria (Bacillus subtilis NM-2, Brucella haematophilum NM-4, and Bacillus cereus NM-6) was performed (unpublished data) and is currently being annotated and studied for genes and multiple pathways involved in drought tolerance. This will allow more insight into the genetics behind the synergism between the drought-tolerant bacteria and pants to alleviate and survive under severe water stress.

Exploiting the beneficial consortium as bioinoculant and drought-tolerant rice genotypes can efficaciously enhance crop productivity under water scarcity. Novel strains Bacillus subtilis NM-2, Brucella haematophilum NM-4, and Bacillus cereus NM-6 that can produce EPS, IAA, siderophore, ACC deaminase, and organic acids and solubilize P were drought-tolerated up to -0.5 MPa (20% PEG-8000). The developed consortium showed the efficient ability to alleviate drought stress in the rice genotype with significantly improved grain yield. Furthermore, bacterial inoculation will be investigated to explore the real-time expression of drought-responsive genes in rice plants under osmotic stress. Hence, in the future, integrated application of these potential drought-tolerant bacteria along with drought-tolerant rice genotypes and advanced non-destructive, handheld robust instruments, i.e., infrared thermal imaging (IRTI) and infrared gas analyzer (IRGA), with conventional methods will be used for the efficient selection and timely prevention of yield loss from water scarcity.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

NM did the overall execution of the experiment, analytical work, collection of IRGA data, infrared thermal images and software analysis, collection of data after morpho-physiological and biochemical analysis of leaves, organization of resulting data, and writing up and revision of manuscript. MAr and SYa contributed to the planning, designing and finalization of basic idea of experiments and overall supervision during analytical work, revised and finalized the manuscript. MAs did the arrangement and provision of rice seeds, contributed in study basic idea and planning of net house experiment and help in data collection. MY and KE performed data analysis and reviewed the manuscript. SYo helped in plant antioxidant analysis and data execution. M-u-R and SZ provide the Infrared Gas Analyzer and helped in the data collection and analysis. IA helped in the sequencing of 16S rRNA gene amplification. AI helped in FISH analysis and SK helped exopolysaccharide detection. All authors contributed to the article and approved the submitted version.