Biochar Amendment in Combination with Endophytic Bacteria Stimulates Photosynthetic Activity and Antioxidant Enzymes to Improve Soybean Yield Under Drought Stress

The beneficial effects of biochar (BC) and plant growth-promoting bacteria (PGPR) to increase crop yields under harsh environments have been widely reported. However, the knowledge on the synergistic effects of BC and PGPR to induce drought tolerance in oilseeds is scant. In the present study, the effects of cotton sticks derived BC together with PGPR strains (Paraburkholderia phytofirmans and Bacillus sp.) were examined in water-stressed soybean plants. Initially, the BC application rates (1% and 2%) for soybean were optimized in a pilot experiment (experiment I), and the most effective level (1%) was selected for further studies together with PGPR in the second experiment. The soybean plants were raised in pots under semi-controlled conditions and were subjected to drought stress (25–30% water holding capacity) at pod formation stage. In comparison with control, the drought-mediated decrease in photosynthetic pigments and gas exchange characteristics was markedly lower in BC and PGPR-treated plants. However, the maximum increase in photosynthetic (12–30%) and the enzymatic antioxidant activities (5–20%) was recorded in plants treated with BC + P. phytofirmans, whereas no further significant increase was observed in plants treated with combined BC and Bacillus sp. under water deficit conditions. Similarly, the combined BC and P. phytofirmans application considerably increased the grain yield (14%) under drought stress conditions. We conclude that the combined application of BC + P. phytofirmans could be utilized as an effective strategy to improve soybean yield in dry arid regions.


Introduction
The depletion of soil carbon resources and rising temperatures are the major threats to global crop yields. The negative effects of these challenging environmental factors are further aggravated by altered precipitation patterns that drastically impact agricultural production (Moussa et al. 2011;Rohbakhsh 2013). Soybean is regarded as a drought-sensitive crop. However, its water requirement is quite high compared to other crops of the Fabaceae family (Maleki et al. 2013). Water deficiency not only lowers the leaf water potential but also causes a decline in pod water potential and causes leaf abscission and premature shell dropping (Liu et al. 2004). It also causes the reductions in chlorophyll contents (Chl), stomatal conductance (g s ), and rate of transpiration (E) by 31-60 and 53-57% respectively (Hao et al. 2013;Mak et al. 2014). Limited water availability severely limits the germination rate of seeds, consequently decreasing the economic yield (Demirtas et al. 2010;Sadeghipour and Abbasi 2012;Li et al. 2013). A deficiency of water at flowering causes the highest reduction in numbers of seeds per plant (Maleki et al. 2013).
Several strategies have been reported to improve production in areas with low water availability. Recently, biochar application has been found effective in improving water use efficiency in many crops under limited water conditions. It is the organic product of biomass obtained by a process of pyrolysis in less or no oxygen conditions (Maia et al. 2011). The purpose of its production is to control climate change by the low release of CO 2 in the atmosphere. It also inhibits the emission of methane gas (Gwenzi et al. 2015). The soybean crop is sensitive to water shortage, but biochar increases the surface area to store water, thus making water available for a longer period (Laird et al. 2010;Karhu et al. 2011;Manzoor et al. 2022). Biochar has the property to retain the water for longer periods which helps the plant to perform their normal functions efficiently under a limited water supply (Mannan et al. 2016). Its addition in the soils of arid zones, where water shortage is the major issue, can promote crop growth and ultimately increase the capital of the farmers (Manzoor et al. 2022). A considerable increase in physical, biological, and chemical properties of soil amended with biochar leads to sustainable cultivation with an increase in production (Kookana et al. 2011). Biochar enhances the soil properties by increasing soil aeration and decreasing soil bulk density (Tayyab et al. 2018). The main purpose for its application is the carbon sequestration by converting CO 2 into a stable form that is available for a longer period (Santos et al. 2012). Biochar improves the availability of soil nutrients and makes them available for the plants and is a sustainable component making less impact on climate change (Elad et al. 2010). It improves N availability and water holding capacity of the soils due to its high surface area (Dong et al. 2015). It supports the health of the soil and reduces the leaching of artificially applied fertilizers, especially nitrogenous fertilizers (Adams et al. 2013). It elevates the growth of several crops including soybean by the long-term waterholding ability for plants, as it decreases the soil bulk density and increases the soil porosity (Tayyab et al. 2018). Biochar application improves yield attributes in soybean crop (Suppadit et al. 2012). The type, i.e., woody biochar, bamboo biochar, coir biochar, cornstalk biochar, and layer manure biochar (Chen et al. 2017), and rate (0, 1.5, 3 and 6 t ha −1 ) (Solaiman et al. 2010) of application of biochar also influence its effectiveness (Reibe et al. 2015). The soil amendment with biochar improves growth and yield attributes by associating with roots and microbes present in the rhizosphere (Egamberdieva et al. 2016). Moreover, it enhances the nutrient and water availability resulting in the better use of the inputs (Khan et al. 2012;2013).
Plant growth-promoting rhizobacteria (PGPR) considerably influence plant development. Seed inoculation with the PGPR can boost the availability of nutrients like nitrogen in the process of biological nitrogen fixation, and by the solubilization process, phosphorus accessibility is also triggered (Tagore et al. 2013;Argaw and Muleta 2018). The productivity of soybean and other pulses is increased by inoculation to a significant level (Murtaza et al. 2014). The treatment of seeds with bacterial and Rhizobium strains increases the quality and economic value of the product (Saleem et al. 2021). The inoculation of soybean with Pseudomonas solubilizes the inorganic P, makes the efficient fixation of atmospheric N, induces the production of hormones, and makes the trace elements available (Gull et al. 2004). The inoculation of seeds with Pseudomonas increases the availability of P for plants, which encourages the growth of plants to a significant level (Batool et al. 2021). Seed inoculation improves soil properties by increasing fertility, improving soil aeration, decreasing soil bulk density, and maintaining C:N ratio of the soil (O'Callaghan 2016). Li et al. (2019) suggested that biochar amendments improve the soil properties such as pH, water-retention capacity, and availability of macro-and micronutrients that serve as a growth promoter for soil microbes. However, the soil microbial activities are influenced by the nature of biochar, including its physical and chemical properties and soil conditions (Palansooriya et al. 2019). Recently, Bertola et al. (2019) showed that soil bacteria can successfully colonize biochar-amended soils and could be utilized as biochar carrier-mediated biofertilizers for increasing crop yields.
Bacillus species are among the most predominant plant growth-promoting bacteria. They can affect plant growth and internal physiological mechanism even under severe climatic conditions including drought stress. Yaish et al. (2015) reported that Bacillus sp. are involved in the secretion of 1-aminocyclopropane-1-carboxylate (ACC) deaminase which promotes the plant growth under water deficit conditions by regulating the activities of ROS. Also, Hassan (2017) demonstrated that Bacillus sp. inoculated biochar improves plant productivity and metabolism by regulating the nutrients uptake under dry conditions. Bacillus sp. colonization along with biochar application promotes water uptake (Marulanda et al. 2009) and also ensures the availability of nutrients to the roots of plants in dry soils (Armada et al. 2014). This property of Bacillus sp. inoculated biochar mitigates the negative effects on the physiological functions of plants under drought conditions.
The application of biochar with bacterial inoculation may be utilized as a promising and valuable approach to mitigate the negative impact of water deficiency in crop plants (Glodowska et al. 2017). Many studies have demonstrated the role of biochar in enhancing bacterial growth; however, very little information is available about the combined effects of biochar and bacterial inoculation on physiological and enzymatic processes of soybean under drought stress. Thus, the present study was conducted with the objectives to (i) investigate the effect of biochar on growth and physiological parameters of soybean under water deficit conditions, (ii) determine the role of bacterial inoculants in improving drought tolerance in soybean, and (iii) evaluate whether biochar application in combination with bacterial inoculants is effective to improve drought tolerance in soybean. We hypothesized that the inoculation of bacterial stains with biochar regulates physiological and antioxidant processes to mitigate drought stress in soybean.

Experimental Layout and Material
The study involved semi-controlled (wire-house) conditions to determine the biochar and PGPR effects on soybean. The wire-house experiments were conducted in a completely randomized design with three replications. All experiments were done at the experimental sites of MNS-University of Agriculture, Multan.
The seeds of soybean (cv. Faisal) were purchased from Ayub Agriculture Research Institute (AARI), Faisalabad, Pakistan. The selected cultivar is known for its ability to survive under harsh climate. It possesses the characteristics such as bold seed, high-temperature tolerance, short growing period, and high yield. To reach full maturity, it requires 90-100 days from the time of sowing.

Preparation of Biochar and Inoculum
Cotton sticks collected from the university farm were used to prepare biochar treatments. These sticks were burnt in the Kon Tiki kiln with controlled conditions of low oxygen to decrease the less release of CO 2 at 500 °C. After preparation, the biochar was spread for drying to make it suitable for application. The physicochemical properties of biochar product were analyzed in the Eurofins research laboratory (Hamburg, Germany) and are reported in our previous study .
Two bacterial strains viz. Paraburkholderia phytofirmans (PsJN, accession number NR 103,337) and Bacillus sp. (Y14, accession number KM 652421) were collected from the Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Pakistan (for details, please see Saleem et al. 2021), and were left for growth in the Tryptone Soy Broth media after sterilization. After 3 days, this growth media was kept in an incubator for 24 h. The growth of bacteria in the media was observed by naked eye. The density of this media was further tested at 400 nm using a double-beam spectrophotometer (Shmadzu 35, China). The observed optical densities were 2.90 and 2.70 for P. phytofirmans and Bacillus sp., respectively.
The randomly selected healthy and physically pure seeds were dipped into the inoculum for 30 min and were later used for sowing. The inoculation rate was 5 ml of liquid inoculum per 100 seeds.

Pot Experiment-I
The experiment consisted of two factors, water stress levels (normal irrigation and drought stress) and the biochar levels (0%, 1%, and 2%). The total numbers of experimental units were 18. For optimizing the biochar dose, pots (25 cm diameter and 15 cm depth) were filled with 10 kg of calcareous soil (sandy loam, pH 7.8, organic matter 0.8%, available NPK: 91:8.4:112 mg kg −1 ). Biochar at 1 and 2% W/W of soil was thoroughly mixed in the soil as the soil amendment. The water-holding capacity of biochar was calculated to maintain the soil moisture content. The pots were irrigated three times a week, and the moisture level was determined using a moisture meter. Drought was applied to one set of pots by no watering (moisture level reduced to 25-30% WHC), while other pots were watered regularly and kept at 65-70% WHC. To apply recommended NPK fertilizer (25:25:50 kg ha −1 , 2.0 g of urea, 2.44 g of DAP, and 3.75 g MOP) were thoroughly mixed in soil before pot filling. Randomly selected six seeds treated with inoculum were sown in the individual pots. After the establishment of seedlings, thinning was done in each pot to maintain four healthy seedlings per pot. The plants were harvested after 5 weeks at the V 3 stage of growth of soybean to record growth and biomass attributes.

Pot Experiment-II
The experiment comprised of three factors including water stress levels (normal irrigation and drought stress), inoculants (no inoculation, P. phytofirmans, and Bacillus sp.), and the optimized biochar level (1%) from previous study, i.e., pot experiment-I. Randomly selected pure and healthy six seeds of soybean (cv. Faisal) inoculated with the strains were sown in each pot. The recommended doses of N and P, including the optimized biochar rate (1%) from pot experiment-I, were mixed in the soil before pot filling as described in 2.3. After 2 weeks of sowing, thinning was done to retain two healthy seedlings for each pot. The experimental units comprised of 30 pots, which were irrigated at the optimum moisture level of the soil for 8 weeks. Afterwards, the soil moisture content of pots containing water-stressed plants was reduced to 25-30% WHC, while other pots were watered regularly to maintain soil moisture levels at 65-70% WHC. After about 2 weeks, on the appearance of drought stress symptoms, the youngest mature leaves were selected from each treatment to record pigments, gas exchange, and antioxidant activity. Then, the water-stressed plants were rewatered to bring their soil moisture levels at 65-70% WHC till harvesting. The plants were harvested at physiological maturity to obtain data regarding yield attributes.

Determination of Growth Attributes
For the determination of growth attributes viz. fresh weight (FW), dry weight (DW), shoot length (SL), and root length (RL), two seedlings from each pot were randomly selected and carefully removed to store in the plastic bags. An electric balance (OHAUS-GT400, USA) was used to measure FW and expressed in grams. Later, these seedlings were kept in an oven (LEEC-F2, Uk) at 65 °C dried for 48 h to measure the DW. A stainless steel meter scale (DUX) was used to measure RL and SL and expressed in centimeters.

Measurement of Photosynthetic Pigments
The leaf chlorophyll content (Chl) was measured using fresh leaf tissue. The collected leaf tissue (0.2 g) was grounded and dipped overnight in 20 ml of 80% solution of acetone. Later, the solution was centrifuged (15,000 × g; 10 min), and the supernatant was used to record the observations at 645 and 663 nm (Arnon 1949), which were later used to calculate leaf Chl a and Chl b content using the following formulae: where V is the volume of sample extract, and W is the weight of the sample.

Gas Exchange Measurements
To measure the gas exchange attributes, a portable open-flow gas exchange system viz. CIRAS-3 (PP systems, Amesbury, USA) was used. The observations were recorded from a fully expanded upper leaf early in the morning (9:00-11:00 am). The leaf chamber was adjusted following the reports of Shehzad et al. (2020).

Assay of Antioxidant Enzymes Activity
Frozen leaf tissue (0.5 g) was thawed and then completely mixed together with mortar and pestle in 5 ml extraction buffer (50 mM Na 2 HPO 4 pH 7.0 and 1 mM dithiothreitol). The sample mixture was then centrifuged (20,000 × g; 15 min) at 4 °C, and the supernatant was used to estimate the activity of superoxide dismutase (SOD), guaiacol peroxidase (GPX), and catalase (CAT) using a UV-Vis spectrophotometer (Shmadzu 35, PR China). The SOD activity was determined according to the method of Van Rossum et al. (1997), whereas GPX and CAT activities were analyzed following the reports of Urbanek et al. (1991) and Aebi (1984), respectively.

Estimation of Yield and Yield Attributes
Randomly selected 10 plants from each treatment were used to estimate yield attributes. The grain weight per plant (GY) was calculated manually from each treatment. The 1000grain weight (GW) was measured by collecting thousand grains from each treatment and weighing them using an electric balance (OHAU GT400, USA).

Statistical Analysis
The statistical analyses were performed using STATISTIX 8.1. The probability level was maintained at 5% to compare treatment means with post-hoc Tukey's test using the ANOVA (analysis of variance) technique.

Chlorophyll Pigments
Multivariate analysis showed a marked (P ≤ 0.001) reduction in Chl a (15%) and Chl b (4%) content in the leaves of soybean compared to control (well-watered conditions) (Suppl. Table 2   considerably (P ≤ 0.001) influenced the Chl a of soybean seedlings (Fig. 2a); however, treatments showed a non-significant (P > 0.05) difference for Chl b (Fig. 2b). The highest increase (16%) in Chl a was observed with seed inoculation of P. phytofirmans with comparison to control (Fig. 2a).

Gas Exchange Characteristics
Multivariate analysis showed a marked (P ≤ 0.01) reduction in A (9%), E (4%), gs (16%), and Ci (5%) of soybean plants by drought in comparison to control (well-watered conditions). Treatment application considerably (P ≤ 0.001) influenced these variables. The highest increase in A (18%) was observed by 1% biochar + P. phytofirmans, whereas the control treatment gave the lowest A (Suppl. Table 2). The highest increase in E (12%), gs (30%), and Ci (4%) was recorded by treatment of plants with 1% biochar + Bacillus sp. with respect to control (Suppl. Table 3). The interactive effect of D × T showed a marked (P ≤ 0.05) difference for g s of the soybean plants (Fig. 3a−c). The highest (17.33 mmol H 2 O m −2 s −1 ) g s was noted in the plants grown with 1% biochar + Bacillus sp., whereas the lowest (14.00 mmol H 2 O m −2 s −1 ) was observed with no biochar or microbial inoculation (Fig. 3a−c).

Yield and Yield Components
Exposure to drought markedly (P ≤ 0.001) influenced GW and GY of soybean crop, and a significant reduction of 15% and 13%, respectively, was observed in drought-stressed crop in comparison to control (Suppl. Table 3). The application of biochar or inoculation with bacterial strains considerably (P ≤ 0.01) increased (3%) the grain weight of soybean crop compared to control (Suppl. Table 3). The highest increase (14%) in GY was observed by the application of treatment 1% biochar + P. phytofirmans as compared to control (Fig. 5b). The interactive effect of D × T showed considerable (P ≤ 0.05) for GW of the soybean. The maximum (76.7 g) GW under drought was obtained by the application of treatment with 1% biochar + P. phytofirmans, whereas the minimum (72.83 g) was noted with the treatment of P. phytofirmans (Fig. 5a).

Discussion
Soybean is a water-sensitive crop, and soil water deficits at critical growth stages incur significant yield losses that may exceed 25% (Gebre and Earl, 2020

Well-watered Drought
Sub-stomatal conductance (μmol mol -1 ) Treatments d. Fig. 3 Effects of the combined application of biochar and bacterial strains on leaf a photosynthetic rate, b transpiration rate, c stomatal conductance, and d sub-stomatal conductance of soybean under well-watered and water deficit conditions. Different alphabets show a significant difference among the treatments tested water-holding capacity of biochar positively influence soil physicochemical characteristics that help to increase crop growth and productivity, especially under water deficit conditions (Paneque et al. 2016). Similarly, microbial inoculants mitigate the damaging effects of drought through increased production of antioxidants, exopolysaccharides, and enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Naveed et al. 2014;Shaffique et al. 2022). However, the studies related to the combined application of microbial inoculants and biochar are scant, and only few researches  have explored the interactive effects, for instance by Ahmad et al. (2020) in Zea mays.
In the present study, we initially optimized the biochar application rates for improving soybean growth under drought stress. A marked effect of different biochar rates on soybean shoot and root growth suggested the dosedependent effects of biochar on early soybean growth. Plant roots perform an important function of maintaining the water potential and nutrient availability required for optimal plant growth. While the drought stress significantly reduced the RL of soybean seedlings, a marked increment in RFW and RDW was observed by the application of 1% biochar (Fig. 1). Previously, Liu et al. (2021) reported that biochar application has a positive impact on root morphology and growth. They were of the view that biochar-mediated increase in root biomass could be due to its ability to reduce bulk density of top soil by 10-12%, consequently improving the soil porosity that enables the extension in root systems. Along with enhancing the proportion of organic carbon in calcareous soil, biochar also increases the availability of nutrients, i.e., nitrogen (N), phosphorus (P), and potassium (K) to the plant roots which ultimately improves the root growth (Amin and Mihoub 2021). According to Zhang et al. (2019), biochar has the characteristic of maintaining the roots' vitality during the booting stage, which ensures maximum accumulation of nutrients to increase grain yield. Similarly, Ducey et al. (2013) concluded that biochar presence in soil can provide living space for microorganisms which ultimately improves soil health thus providing an appropriate environment for better root growth. In addition to the provision of raw material and environment for a root redox reaction, biochar also has the ability to absorb maximum toxic substances within soil, produced as a result of continuous conventional cropping pattern thus ensuring maximum root growth (Gong et al. 2019).
Our results showed a significant decline in soybean shoot biomass under water deficit conditions (Fig. 1); however, the application of 1% biochar considerably increased the shoot biomass in water-stressed seedlings. This drought-induced reduction in SL, SFW, and SDW could be due to loss of turgidity causing a reduction of cell division and expansion ultimately resulting decline in growth and productivity (Hussain et al. 2009). Biochar application stimulates plant growth by making the micro-climatic conditions favorable to the plant (Akhtar et al. 2014;Mihoub et al. 2019). Previously, Zimmerman (2010) also concluded that the application of 1% biochar to the soybean under water stress shows a positive response and relatively increases SL, SFW, and SDW that could be due to the potential of biochar in improving soil physical properties ultimately boost up crop growth. Similar findings were observed by Berihun et al. (2017), showing better crop growth by increasing shoot biomass under drought conditions supplemented with biochar. Meanwhile, the ratio of above and below-the-ground biomass considerably increased by application of biochar due to improvement in the soil water-holding  properties, as suggested earlier by Karhu et al. (2011) andMihoub et al. (2022). P. phytofirmans and Bacillus sp. are well-reported for their beneficial effects under dry conditions due to their role in the production of IAA and siderophore (Minaxi et al., 2012). Biochar act as a supporting material for extending the shelf life to enhance the activities of microbes in soil and protect them from harsh climatic conditions including drought stress. Tripti et al. (2017) showed biochar as appropriate supporting material for P. phytofirmans, and maximum plant growth and physiological functions of tomato were observed including Chl and A where biochar was applied in addition with the inoculation of P. phytofirmans. Moreover, Gagne-Bourque et al. (2016), after conducting a trial on timothy (Phleum pratense L.) grown under drought stress, revealed that Bacillus sp. stimulates the formation of Chl a , Chl b , and carotenoids in addition with the production of endogenous amino acids which is due to the production of metabolites thus in return increasing photosynthesis rate by minimizing the oxidative stress. Abideen et al. (2020) demonstrated the correlated alterations in photosynthetic rate (A), stomatal (g s ), and sub-stomatal conductance (C i ) in the vicinity of 0.75% additional biochar, regarded as carboxylation efficiency optimization to boost biomass output under water deficit conditions. The greater water availability in the presence of biochar allows for a slight rise in g s and a rise in A. Increased soil biochar concentration of up to 2.5% improves water availability and hence g s . Batool et al. (2020) stated that inhibited growth in drought-stressed plants might be attributed to a slower rate of photosynthesis, resulting in reduced cell expansion and development. Plants treated with PGPR, on the other hand, were able to retain greater g s , A, C i , and E in their leaves than plants not treated with PGPR, as in our study, indicating maintained plant health and growth. A drop in Chl during drought stress hastened a fall in chloroplast photochemical activity, which may be responsible for the decrease in photosynthesis, causing a decline in photosynthetic energy (ATP) consumption in the Calvin cycle due to reduced electron transport rate (Izanloo et al. 2008).
Adequate chlorophyll availability in plants might enhance overall photosynthetic efficiency and regulate g s under the less water availability because almost half of the plant's green portion actively works for light collection to drive the process of photosynthesis (Ji et al. 2010). Hence, A is correlated with the total amount of pigments (Chl a and Chl b ) in the leaf area, for which the rhizobacteria application to the plants may be a key point. In addition, g s in relation with the plant water status in terms of leaf relative water status, photosynthetic activity, and working of electron transport chain is directly linked with the PGPRs (Wright et al. 2004). Biochar along with PGPR enhances Chl, thus improving the efficiency of a photosynthetic phenomenon, indicating that Chl are a sign of stress resistance as reported by Nadeem et al. (2017). Crop productivity is closely dependent on net photosynthesis that contributes 90-95% to crop yields (Lefe et al. 2017). The physiological processes (A, g s , C i , E and Chl) are happening within leaves of plant parallel; therefore, the efficiency of one will definitely regulate other processes under water deficit conditions (Meng et al. 2016). Previously, it was reported that biochar application improved the crop health and productivity by increasing the photosynthesis rate and also enhances water use efficiency (Akhtar et al. 2014;Baronti et al. 2014). Xu et al. (2015) also observed a significant increment in leaf A in peanut plants grown in soil amended with biochar under water deficit conditions. Our study showed the negative impact of water deficit conditions on the overall growth and productivity of soybean by limiting the A and other physiological processes including g s , Ci, E, and Chl. However, the application of biochar along with plant growth-promoting rhizobacteria mitigates the negative impact of water stress by maintaining activities at a cellular level. Biochar application along with rhizobacteria showed a remarkable increase in A, E, and g s . Leaf E is directly linked with plant biomass and an indicator of overall crop productivity; in our study, the reduction in leaf E under drought may be due to the yellowing and wilting of leaves under water stress, also reported by Limousin et al. (2009).
Drought stress severely affects plant internal mechanism by generating reactive oxygen species (ROS) causing damage to cells. Plants respond to ROS through various biochemical regulation such as accumulating several protective osmolytes, proteins, secondary metabolites, and anti-oxidants including catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (GPX) which have the ability to scavenge ROS under water stress (Hosseini et al. 2018). The significance of antioxidant enzymes in the drought and dehydration tolerance mechanisms of soybean is well documented (Vasconcelos et al. 2009). Our study also showed a significant increase in antioxidant enzymes when soybean plants were exposed to water-deficit conditions. However, the treatment of biochar sole and in combination with microbial inoculation (P. phytofirmans and Bacillus sp.) reduced the GPX activity. Our results are in conformity with the report of Mahajan et al. (2005) who showed that the plants treated with microbes had lower antioxidant enzyme levels than plants without microbes, indicating an increased reactive oxygen species scavenging potential of microbes under dry circumstances. Previously, the positive impact of biochar ) and microbial inoculation (Batool et al. 2021) on antioxidant enzyme activities under water deficit conditions has been reported. Reduction in water stress by microbes can be due to their close association with the roots of plants thus making the favorable conditions for plant roots for up taking maximum moisture from soil so that plant maintains its ROS equilibrium for better growth and development. Our results are in accordance with Li et al. (2020) who observed higher SOD and CAT activity in drought-stressed plants; however, inoculation with microbes decreases the stress environment in the root zone area making the water more available in dry conditions. In another study,  reported the beneficial effects of biochar by scavenging the ROS activity in Areca catechu L. seedlings under water deficit conditions. Wang et al. (2015) showed the beneficial effects of biochar addition on microbial activities in soil. They observed a significant increase in the soil enzyme activities which decreased the antioxidants ratio due to the scavenging of the ROS by the symbiotic relationship between microbes and the biochar. Interestingly, a previous study by Lioussanne et al. (2010) supported the fact that biochar application is directly linked with soil organic matter and microbial population hence maintaining the proportion of CAT and SOD when water is not sufficient in the root zone for optimal plant growth.
Water stress severely impacts soybean growth and development thus causing a decline in overall yield. However, the inoculation of soybean with rhizobacteria combined with biochar application mitigates the deleterious effects of water deficit. Interestingly, biochar + Bacillus sp. treatment significantly decreased the 1000-grain weight but improved the grain weight per plant with respect to control under wellwatered conditions (Fig. 5a, b). This could be attributed to a decline in sink capacity or delayed maturity, resulting in small grain size. The effects of biochar and Bacillus sp. on delayed maturation and low 1000-grain weight are not common, and very little or no information is available for such effects. In this study, a severe decline in 1000-grain weight and grain yield was observed under drought conditions. However, soybean plants treated with biochar alone or in combination with inoculated rhizobacteria (P. phytofirmans and Bacillus sp.) showed a marked increment yield attributes. Previously, Jahan et al. (2018) also reported a significant effect of biochar in the mitigation of drought-induced damages in soybean. The decline in yield attributes of soybean under water stress could be associated to the reduction in transport and photo-assimilation of carbon (Muller et al. 2011). Our findings related to the decline in soybean yield under water stress and the positive effects of rhizobacteria and biochar are concurrent with the reports of Major et al. (2010) who showed yield increment in maize supplemented with biochar under water deficit conditions. Also, Danish et al. (2019), in an experiment involving co-inoculation of PGPR and biochar, concluded that the application of PGPR plus biochar is a better approach toward mitigating the adverse effects of drought stress on crop growth and yield. Correa et al. (2009) revealed that soybean inoculation with Bacillus sp. along with biochar as a carrier acts as a protective agent against severe drought stress environment. Mitigation of drought impact may be due to the property of Bacillus sp. to colonize around roots of plant, thus enhancing growth and increasing tolerance against water deficit environment. Furthermore, Tripti et al. (2017) stated that the survival rate of P. phytofirmans and Bacillus sp. increased when inoculated along with biochar thus longer survival of microbes along with biochar stimulates overall growth and fruit formation of tomato. This increment in yield attributes by microbes and biochar might be due to biochar properties of making most of the soil nutrients available to the plant so that roots can uptake the maximum ratio of these nutrients and utilize in biomass formation. Besides the imperative P. phytofiramans and Bacillus sp. role, more water and nutrient holding capacity, ion exchange property, and high surface area of biochar make it an efficient modification in plants that ensures the maximum uptake and utilization of water and nutrients in plants (Lehmann et al. 2006;Paetsch et al. 2018). Meanwhile, Saxena et al. (2013) after conducting a field trial reported that water stress severely affects crop yield, but the treatment of Bacillus sp. along with biochar increases the root-shoot ratio, number of pods, and economical yield of Phaseolus vulgaris. The positive correlation between P. phytofirmans, Bacillus sp., and biochar is due to the porous structure of biochar that supports more proliferation of these bacterial strains by proper aeration thus making sure more availability and absorption of nutrients also more tolerance of strains against various harsh environmental conditions including drought stress (Sangeetha 2012).

Conclusion
Drought stress is one of the most drastic abiotic stresses affecting normal plant growth and production all over the world. In the present study, the water deficiency considerably affected soybean growth and yield. The combined application of 1% biochar and seed inoculation with P. phytofirmans was found most effective to improve soybean yield under water deficit conditions. The drought tolerance of soybean plants was found associated to the maintenance of chlorophyll pigments, increase in the activity of photosynthetic apparatus, and regulation of antioxidant machinery, consequently increasing the final grain yield. Our findings extend the understanding of the importance of biochar inoculation with a suitable bacterial strain (P. phytofirmans) and demonstrated how combined biochar and P. phytofirmans application could be exploited for increasing soybean yield in dry arid regions. Hence, future studies aimed at evaluating the field performance of such applications are suggested to enhance crop yields in dry agricultural systems.
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