Co-inoculation of rhizobacteria promotes growth, yield, and nutrient contents in soybean and improves soil enzymes and nutrients under drought conditions

Drought stress is the major abiotic factor limiting crop production. Co-inoculating crops with nitrogen fixing bacteria and plant growth-promoting rhizobacteria (PGPR) improves plant growth and increases drought tolerance in arid or semiarid areas. Soybean is a major source of high-quality protein and oil for humans. It is susceptible to drought stress conditions. The co-inoculation of drought-stressed soybean with nodulating rhizobia and root-colonizing, PGPR improves the root and the shoot growth, formation of nodules, and nitrogen fixation capacity in soybean. The present study was aimed to observe if the co-inoculation of soybean (Glycine max L. (Merr.) nodulating with Bradyrhizobium japonicum USDA110 and PGPR Pseudomonas putida NUU8 can enhance drought tolerance, nodulation, plant growth, and nutrient uptake under drought conditions. The results of the study showed that co-inoculation with B. japonicum USDA110 and P. putida NUU8 gave more benefits in nodulation and growth of soybean compared to plants inoculated with B. japonicum USDA110 alone and uninoculated control. Under drought conditions, co-inoculation of B. japonicum USDA 110 and P. putida NUU8 significantly enhanced the root length by 56%, shoot length by 33%, root dry weight by 47%, shoot dry weight by 48%, and nodule number 17% compared to the control under drought-stressed. Co-inoculation with B. japonicum, USDA 110 and P. putida NUU8 significantly enhanced plant and soil nutrients and soil enzymes compared to control under normal and drought stress conditions. The synergistic use of B. japonicum USDA110 and P. putida NUU8 improves plant growth and nodulation of soybean under drought stress conditions. The results suggested that these strains could be used to formulate a consortium of biofertilizers for sustainable production of soybean under drought-stressed field conditions.

Experimental design. The effect of rhizobacteria on soybean growth was studied in pot experiments in a greenhouse at ZALF, Müncheberg, Germany, during July 2019. The experiments were carried out. All the experiments were carried out in a randomized block design with five replications. Experimental treatments included: T1-Control under normal water conditions T2-Control under drought stress conditions T3-Inoculation with B. japonicum USDA 110 under normal water conditions T4-Inoculation with B. japonicum USDA 110 drought stress conditions T5-Inoculation with P. putida NUU8 under normal water conditions T6-Inoculation with P. putida NUU8 under drought stress conditions T7-Co-inoculation with B. japonicum USDA 110 and P. putida NUU8 strains under normal water conditions T8-Co-inoculation with B. japonicum USDA 110 and P. putida NUU8 strains under drought stress conditions. Plants were grown in pots under greenhouse conditions at 24 °C during the day and 16 °C at night for 30 days. Normal water conditions (70% of the pot capacity) and drought stress conditions (40% of pot capacity) were maintained.

Measurement of plant growth parameters and plant nutrients. Soybean plants were harvested
from pots after 30 days of germination. The measurement of seed germination rate (%), root length (cm), shoot length (cm), root dry weight (mg/g), shoot dry weight (mg/g), and the number of nodules per plant was measured.
For the estimation of plant nutrients, such as nitrogen, phosphorus, potassium, magnesium, sodium, and calcium, 1 g of crushed plant tissue was added in phosphate buffer (pH 6.7), and these nutrients were measured spectrophotometrically (iCAP 6300 Duo, Thermo Fischer Scientific Inc., Waltham, MA, USA) 41 www.nature.com/scientificreports/ and phosphorus contents of root and shoot were determined from dried plant biomass. For nitrogen estimation, one g of dried leaf biomass was digested in 10 mL concentrated H 2 SO 4 and 5.0 g catalyst mixture in a digestion tube. The digested and cooled mixture distillate and the distillate was titrated with H 2 SO 4 . The mixture that did not contain leaf biomass served as a control. Total nitrogen was calculated from the blank and sample titer reading 43 . The P content of plant biomass was first extracted with 0.5 N NaHCO 3 buffer (pH 8.5) followed by treatment with ascorbic acid. The intensity of the blue color produced was measured at 540 nm. The amount of P from plant biomass was calculated from the standard curve of P 44 . For the estimation of potassium content of the plant, 5 g of the plant biomass was added in 25 mL of ammonium acetate, shaken for 5 min, and filtered. The amount of potassium from the filtrate was measured according to Upadhyay and Sahu 45 . To estimate Na, Mg, and Ca, one g of plant extract was mixed with 80 mL of 0.5 N HCl and incubated for 5 min at 25 °C and filtered. The amount of Na, Mg, and Ca from the filtrate was estimated according to Sahawat 46 . Analysis of soil nutrient. The root soil (10 g) of each experimental pot was air-dried and shaken with 100 mL ammonium acetate buffer (0.5 M) for 30 min to displace the adhered nutrients and minerals. Soil organic carbon (SOC), nitrogen, phosphorus, and potassium contents of soil were determined according to the method of Sims 34 . This method mixed 1.0 g of soil with 10 mL of 1 N K 2 Cr 2 O 7 and 20 mL of concentrated H 2 SO 4 . This suspension was mixed thoroughly and diluted to 200 mL of distilled water, followed by the addition of 10 mL each of H 3 PO 4 and sodium fluoride. The resulting solution was used to estimate N, P, and K 47 . Blank (without soil) served as control.
Estimation of soil enzymes. The acid and alkaline phosphomonoesterase activities of soil were assayed according to the method of Tabatabai and Bremner 48 . For this, 0.5 g of moist soil was mixed in 2 mL of modified universal buffer (pH 6.5 for the acid phosphatase and pH 11 for the alkaline phosphatase) and 0.5 mL of p-nitrophenyl phosphate (PNP) substrate solution (0.05 M). The change in the color of solution due to p-nitrophenol (p-NP) production due to acid and alkaline phosphomonoesterase activities was measured at 400 nm, and the amount of p-NP was calculated from a p-NP calibration curve. Solution without soil served as the control. One unit of phosphomonoesterase activity was defined as the amount of enzyme required to liberate 1 mM of p-NP (product) from 1 kg of dried soil at 37 °C per 1 h 48 .
Protease activity was assayed according to the method of Ladd and Butler 49 . For this, 0.5 g of soil was added in 2.5 mL of 0.2 M phosphate buffer (pH of 7.0) and 0.5 mL of 0.03 M N-benzoyl-l-arginine amide (BAA) substrate solution. The amount of ammonium released during the reaction was measured at 690 nm. One unit of protease activity was defined as the amount of enzyme ammonium equivalents released from BAA per minute.

Statistical analyses.
All the experiments were performed in five replicates, and the mean values of five replicates were considered. The data were statistically analyzed by one-way analysis of variance (ANOVA) and multiple comparisons of HSD employing the Tukey test with Stat View Software (SAS Institute, Cary, NC, USA, 1998). The significance of the effect of various treatments on plant growth parameters, plant nutrients, and soil nutrients was determined by the magnitude of the p-value (p < 0.05 < 0.001).

Measurement of plant growth parameters.
Drought stress conditions affected the seed germination in soybean ( Fig. 1) compared to the normal water conditions. Application of rhizobacteria enhanced seed germination under drought conditions and normal conditions compared to control under drought and normal conditions, respectively. Inoculation of B. japonicum USDA 110 alone increased the seed germination by 12.5% under drought conditions and by 10.0% under normal conditions over the control. Co-inoculation of B. japonicum USDA 110 and P. putida strain NUU 8 significantly improved the seed germination under drought stress and normal water conditions. Under drought conditions, co-inoculation of B. japonicum USDA 110 and P. putida strain NUU 8 enhanced the seed germination by 16.2% and 13% under drought and normal conditions compared to the control under drought and normal conditions, respectively (Fig. 1a).
The rhizobacterial inoculation significantly improved the growth of the soybean plant under normal and drought stress conditions. Inoculation of B. japonicum USDA 110 alone significantly enhanced the root length by 30% (Fig. 1b), shoot length by 36% (Fig. 1c), root dry weight by 33% (Fig. 1d), and shoot dry weight by 26% (Fig. 1e), as compared to the control under normal water conditions. Inoculation of B. japonicum USDA 110 under drought stress conditions significantly increased the root length by 29% (Fig. 1b), shoot length by 22% (Fig. 1c), root dry weight by 28% (Fig. 1d), and shoot dry weight by 22% (Fig. 1e) over the control under drought and normal conditions, respectively. Whereas the co-inoculation with B. japonicum USDA 110 and P. putida strains NUU8 significantly increased the root length, shoot length, root dry weight, shoot dry weight, and nodule number compared to the control under normal and drought conditions. A 59% and 56% increase in root length (Fig. 1b), 43% and 33% increase in shoot length (Fig. 1c), 53% and 47% rise in root dry weight (Fig. 1d), 48% and 46% improvement in shoot dry weight (Fig. 1e) and 29% and 27% rise in nodule number (Fig. 1f) was evident over the control under normal condition and drought stress, respectively.

Measurement of plant nutrient contents.
Analysis of nutrients in a soybean plant revealed that single inoculation of B. japonicum USDA 110 significantly increased N content by 29% and 28%, P content by 15% and 12%, K content by 32 and 28%, Mg content by 12%, and 9.0%, Na content by 50% and 43% and Ca content by 13% and 11% respectively as compared to control under normal condition and drought stress conditions respectively. A single inoculation of P. putida NUU8 also exhibited a substantial increase in the nutrient contents. It Analysis of soil nutrient contents. Analysis of soil nutrient contents revealed significant improvement in soil N, P, and K content due to rhizobacterial inoculation compared to control (Table 2). Inoculation with B. japonicum USDA 110 alone significantly increased total N content by 16% and 12%, P content by 18% and 16%, and K content by 16% and 14%, respectively, compared to the control under normal conditions and drought conditions respectively. In comparison, single inoculation with P. Putida NUU8 increased total N content by www.nature.com/scientificreports/ 13% and 11%, P content by 16% and 13%, and K content by 13% and 11% compared to the control under normal conditions and drought conditions, respectively. However, the highest N, P, and K values were observed in soil amended co-inoculation with B. japonicum USDA 110 and P. putida NUU8 treatment under normal and drought stress conditions. The co-inoculation significantly increased the total N content by 20% and 23%, P content by 14% and 12%, and K content by 48% and 30%, respectively, compared to the control under conditions and drought stress conditions, respectively ( Table 2).

Analysis of soil enzyme activities.
Data regarding soil enzymes showed that rhizobacteria treatments improved the protease, acid, and alkaline phosphomonoesterase activities in both conditions (Table 3). A single inoculation of B. japonicum USDA 110 significantly increased the protease, acid, and alkaline phosphomonoesterase compared to the control under both conditions. However, the co-inoculation of B. japonicum USDA www.nature.com/scientificreports/ 110 and P. Putida NUU8 significantly improved the activities of these enzymes under both conditions (Table 3). Co-inoculation of soybean with B. japonicum USDA 110 and P. putida NUU8 strains significantly enhanced protease activity by 19%, acid phosphomonoesterase activity by 10%, and acid phosphomonoesterase and alkaline phosphomonoesterase activity by 27% over the control under normal conditions. Co-inoculation with B. japonicum USDA 110 and P. putida NUU8 under drought stress conditions significantly increased the protease activity by 32%, acid phosphomonoesterase by 27%, and alkaline phosphomonoesterase by 19% over the control (Table 3).

Discussion
Drought stress has adverse effects on seed germination and growth in various plants. Several researchers 2,3,29 reported a decrease in the germination rate in legumes crops by drought stress. The negative impacts of drought on seed germination, plant growth, nodulation, and soybean yield have been reported 2,3 . Mafakheri et al. 17 reported a 73% decrease in soybean yield under drought stress conditions. PGPR strains like Bradyrhizobium sp. and Pseudomonas sp. improve drought tolerance and plant growth by modifying root architecture and the secretion of siderophore, phytohormones, and EPS 20,21,24 . Gholami et al. 50 reported improved germination and growth in soybean due to the synergistic effect of co-inoculation of B. japonicum and P. putida. Inoculation with PGPR improves plant growth, development, nodulation, and yield of different crops 24,30,33,35 . Co-inoculation of Rhizobium sp. and other PGPR in bean and chickpea enhance nodulation, plant growth, and nutrient uptake 36,43 .
Co-inoculation of Rhizobium tropici CIAT 899 and P. polymyxa DSM36 significantly increase plant growth and nodulation in common bean compared to inoculation with Rhizobium sp. alone under drought-stressed conditions 51 . Tewari and Arora 52 reported a 50% increase in germination due to the inoculation with EPS producing Pseudomonas aeruginosa PF23 under stress. A wide variety of PGPR have been reported to produce EPS 53 , and they help crop plants in better root colonization 52 , better seed germination, and stress tolerance 55 . They enhance water retention by maintaining the diffusion of organic carbon sources 54 . Vardharajula et al. 56 observed that Bacillus sp. synthesized osmolytes and antioxidants that facilitate plant growth under drought stress conditions. The synthesis of phytohormones by bacterial strains is another mechanism that imparts stress tolerance in plants 29,57 . Drought stress also adversely affects plant nutrient uptake such as N, P, K, Ca, and Mg 20 . Several studies reported that drought stress reduces the concentration of N, K, and P in plant tissue and declines nutrient uptake from soil 58,59 . Drought stress is known to significantly decrease N content in cowpea 60 . He and Dijkstra 58 reported that drought stress conditions significantly decline N and P in plant tissues. Results of the present study shows that co-inoculation with B. japonicum USDA 110 and P. putida strains NUU8 significantly increased the N content, P content, and K content compared to the control under drought conditions. PGPR is known to colonize the plant's rhizosphere, adhere to the root surface, and maintain moisture content 25,[61][62][63] . This makes stable aggregates that help in nutrient absorption in plants 52 .
Drought stress exhibits adverse effects on soil nutrient availability, soil nutrient adsorption, and soil enzyme activities. Hinsinger et al. 64 reported that drought-stressed conditions significantly decrease the soil nutrients such as N, P, K, and microelements such as B, Fe, Mn, and Zn. Drought-stressed in the soil is known to decrease enzyme activities 65 . The decrease in soil enzyme activities observed in this study is in agreement with the decrease in P available forms in the drought-stressed conditions 41 . The enhancement in soil enzymes such as protease, acid phosphomonoesterase, and alkaline phosphomonoesterase due to rhizobial inoculation has been observed by Fall et al. 66 and Jabborova et al. 67 . Nitrogen fixing symbionts, alone or in combination with other rhizobacteria have been reported to improve growth, nutrient uptake and root architecture in soybean as well as to improve the resistance in soybean and other plants 46,47,[68][69][70][71][72][73][74][75][76] .

Conclusions
The application of PGPR exerts beneficial effects on plant growth and nodulation in soybean through increased uptake of nutrients such as N, P, and K in soil under normal and drought stress conditions. Inoculation with single strains of PGPR, i.e., B. japonicum USDA 110, improve soybean growth; however, co-inoculation of B. japonicum USDA 110 and P. putida NUU8 improves more growth, nutrient contents in soybean and soil, and activities of soil protease and acid and alkaline monophosphoeserase, as compared to the single inoculation and control under drought condition. Thus the combination of B. japonicum USDA 110 and P. putida NUU8 can serve as an effective and sustainable approach for improving the growth, nutrient contents, and enzyme activities in soybean and soil under drought-stressed conditions.

Data availability
Permissions were obtained to collect the Soybean (Glycine max L. Merr.) seeds from Leibniz Centre for Agricultural Landscape Research (ZALF), Müncheberg, Germany. Experimental research and field studies on plants were in accordance with the guidelines of ZALF, Müncheberg, Germany.