Exploring potential soybean bradyrhizobia from high trehalose-accumulating soybean genotypes for improved symbiotic effectiveness in soybean

Drought is the most important factor limiting the activity of rhizobia during N-fixation and plant growth. In the present study, we isolated Bradyrhizobium spp. from root nodules of higher trehalose-accumulating soybean genotypes and examined for moisture stress tolerance on a gradient of polyethylene glycol (PEG 6000) amended in yeast extract mannitol (YEM) broth. In addition, the bradyrhizobial strains were also evaluated for symbiotic effectiveness on soybean. Based on 16S rDNA gene sequences, four bradyrhizobial species were recovered from high trehalose-accumulating genotypes, i.e., two Bradyrhizobium liaoningense strains (accession number KX230053, KX230054) from EC 538828 and PK-472, respectively, one Bradyrhizobium daqingense (accession number KX230052) from PK-472, and one Bradyrhizobium kavangense (accession number MN197775) from Valder genotype having low trehalose. These strains, along with two native strains, viz., Bradyrhizobium japonicum (JF792425), Bradyrhizobium liaoningense (JF792426), and one commercial rhizobium, were studied for nodulation, leghaemoglobin, and N-fixation abilities on soybean under sterilized sand microcosm conditions in a completely randomized design. Among all the strains, D-4A (B. daqingense) followed by D-4B (B. liaoningense) was found to have significantly higher nodulation traits and acetylene reduction assay (ARA) activity when compared to other strains and commercial rhizobia. The bradyrhizobia isolates showed plant growth promotion traits such as indole acetic acid (IAA), exopolysaccharide (EPS), and siderophore production, phosphate-solubilizing potential, and proline accumulation. The novel species B. daqingense was reported for the first time from Indian soil and observed to be a potential candidate strain and should be evaluated for conferring drought tolerance in soybean under simulated stress conditions.


Introduction
Soybean [Glycine max (L.) Merrill] has received considerable attention in world oilseeds due to its high profitability and productivity and has a role in food uses besides maintaining soil health.Due to the high protein content (40%) and non-GMO status of the Indian soybean varieties, trading their by-products, such as de-oiled cake (DOC), makes more profitable especially in the European market (Pagano and Miransari 2016).India's average soybean productivity remains low compared to other parts of the world due to various abiotic and biotic stresses (Bharti et al. 2020).Among abiotic stresses, drought stress can decrease the soybean yield by up to 40% annually.Although the use of genetic engineering, breeding, and omics approaches is being advocated to combat stresses, however, the transmissibility of abiotic stress tolerance genes is very low as well as these approaches are time consuming (Dubey et al. 2019).Considering ecological sustainability and economics, harnessing the microbial potential to enhance soybean production could be a viable and feasible approach (Ma et al. 2019;Dubey et al. 2019).Symbiotic biological nitrogen fixation in soybean is mediated by slow-growing nitrogen-fixing rhizobia viz., Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense etc., and fast-growing rhizobia such as Sinorhizobium fredii (Salvagiotti et al. 2008;Ansari and Rao 2014;Collino et al. 2015).These bradyrhizobia imparts plant growth and drought stress alleviation in soybean through various mechanisms (Bharti et al. 2020).Indeed, the success of legume inoculation with Bradyrhizobium spp.depends on the effectiveness and survival of the strain on the seed.Approximately 95% of the Bradyrhizobium spp.population dies between inoculation and seed sowing (Roughley et al. 1993).Desiccation stress is a major factor in the rapid decline in Bradyrhizobium populations following seed application (Mary et al. 1994).Researchers have tried various carbon sources for better survivability of strains.For enhanced strain survival, researchers have evaluated a variety of carbon sources.Trehalose, however, might be a preferable choice because Bradyrhizobium spp.cannot utilize it as a carbon source and builds up in the cytoplasm for better survival.Hence, there is a need to have native nodule bradyrhizobia, which has high symbiotic efficiency and drought-tolerant ability.
Tr e h a l o s e ( α -D -g l u c o p y r a n o s yl -1 , 1 -α -Dglucopyranoside) is a non-reducing disaccharide that accumulates as an osmoprotectant in plants and in many soil microorganisms, like bacteria, fungi, and yeasts, under abiotic stresses as stress metabolite (Argüelles 2000;Kosar et al. 2019).Trehalose plays a role in the rehydration of cells, protects cell membranes and proteins, and maintains osmotic balance without altering cellular functions.Moreover, it acts as a reserve food material in vegetative as well as reproductive cells (Argüelles 2000).As a result, cells can survive under low moisture stress and high-temperature conditions.Bradyrhizobium japonicum and Bradyrhizobium elkanii, isolated from soybean root nodules and free-living states, synthesized trehalose by three mechanisms.Trehalose accumulation occurs, especially during the onset of the nodulation process in symbiosis.Similarly, the accumulation of trehalose in various plant parts like root, shoot, leaves, and nodules under various abiotic and biotic stress is well documented and reviewed by Fernandez et al. (2010).Overexpressing trehalose synthesizing gene in Rhizobium etli was found to enhance nodule formation, nitrogenase activity, and plant biomass in Phaseolus vulgaris under drought stress conditions (Suárez et al. 2008).However, it has not been established that high trehalose-accumulating soybean genotypes in nodule inhabit drought-tolerant Bradyrhizobium spp.Our study first aimed to determine whether various soybean genotypes accumulate trehalose differently.We next looked at the relationship between trehalose concentration in the nodule and the Bradyrhizobium isolates' ability to withstand drought.The second objective comprehends the native, culturable abundance of Bradyrhizobium spp.found inside the higher trehalose-accumulating soybean lines.
In addition, a microcosm trial using the most popular soybean cultivar JS 95-60 was conducted and evaluated the recovered drought-tolerant and plant growth-promoting Bradyrhizobium spp.for symbiotic efficiency.We hypothesized that trehalose accumulation would be higher in soybean genotypes that are drought-tolerant.Following inoculation, Bradyrhizobium spp.isolated from these genotypes may exhibit drought tolerance characteristics and nitrogen symbiotic effectiveness in terms of higher nodulation, leghaemoglobin, and acetylene reduction assay (ARA).

Selection of soybean lines based on high trehalose content in root nodules
Soybean genotypes were grown for 50 days in unsterilized soil under rainfed natural conditions.The experimental soil used was deep medium black soil/vertisols (Sarol series) taken from the experimental field of Microbiology Section, ICAR-Indian Institute of Soybean Research, Indore, India.The basic nutrient composition of the soil at the beginning of the experiment was as follows: soil pH 7.6; organic carbon 0.5%; available Olsen's phosphorus 6.22 mg/kg; mineral nitrogen 6.24 mg/kg; and DTPA extractable Zn 2.12 mg/kg.Root nodules were harvested at the flowering stage (50 days after sowing), and 100 mg was grounded into fine powder in liquid nitrogen.Samples were mixed with chilled isopropanol: acetonitrile: water (3:3:2) solvent.Here, 60 μL ribitol solutions were added as an internal standard, followed by centrifugation at 12,000 rpm for 10 min.The supernatant from each sample was collected and vacuum dried using a speed vacuum.Two-step chemical derivatization of the extracted metabolites was performed as previously described by Fiehn et al. (2008) with few modifications.Samples were dissolved in 50 μL methoxy-amine-hydrochloride in pyridine and incubated at 30 °C for 90 min and then derivatized by adding 100 μL N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) to trimethylsilylate the polar functional groups.The derivatized samples were analyzed using GCMS-QP2010 PLUS (Shimadzu, Japan) at the Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India.Trehalose was quantified by comparing its retention time with the trehalose standard.Other metabolites were identified by comparing retention time and mass spectra with the entries of NIST14 and Wiley 8 mass spectral libraries.The data was normalized in Excel using the ribitol as an internal standard.

Isolation of soybean rhizobia from high trehalose-accumulating soybean lines
The Bradyrhizobium spp. was isolated from the root nodules of drought-tolerant soybean lines as per the method described by Somasegaran and Hoben (1994).Soybean nodules were harvested at the flowering stage, and healthy pinkish intact nodules were selected.Nodules were detached from the root by keeping the root 0.5 cm in length on both sides of the nodule.Nodules were surface sterilized with 70% ethanol for 30 s, followed by 3% sodium hypochlorite (NaOCl) for 4 min.After that, nodules were rinsed six times with sterile distilled water to remove traces of NaOCl.Now, surface-sterilized nodules were crushed with a sterile glass rod, and the suspension was streaked on Congo red yeast extract mannitol agar (CRYEMA) plates and incubated at 28 °C for 10 days.Plates were carefully observed for colorless or white, mucoid, translucent, circular colonies and generation time (2-4 days for fast growers and 4-7 days for slow growers).The colonies were isolated and sub-cultured on CRYEMA plates and subsequently purified.

Evaluation for moisture stress tolerance under in vitro
The strains were evaluated under normal (UNST) and stress (ST) conditions in vitro in yeast extract mannitol broth (YEMB) (10 mL) supplemented with polyethylene glycol (PEG-6000) by the method of Sandhya et al. (2009).The stress levels of 0, − 0.14, − 0.49, and − 1.07 MPa were prepared in YEM broth with PEG 6000 corresponding to 0 %, 10%, 20%, and 30% concentrations in triplicates.Freshly grown culture (lag phase one loopful) was added to 10 mL of YEMB tubes and incubated on an incubator shaker at 28 °C for 4 to 7 days.The bacterial growth in each treatment/ PEG concentration was measured (optical density 600 nm) using a spectrophotometer (Shimadzu, Japan).

Quantitative bacterial indole acetic acid (IAA) assay
IAA quantitative estimation was measured as per the protocol described in Bric et al. (1991).Bradyrhizobium cultures were grown in YEM amended with filter-sterilized precursors L-tryptophan at 100 μg/mL, and without L-tryptophan, both the conditions evaluated, under the amendment with 25% PEG6000 and without for 7 days at 28 °C with 200 rpm.After 7 days, culture broths were centrifuged at 10,000 rpm for 10 min.Then, 2 mL supernatant was mixed with three drops of orthophosphoric acid and 4 mL of Salkowski's reagent (2% 0.5 M ferric chloride in 35% perchloric acid solution).This solution was placed in the darkroom for 30 min at 28 °C until the pink color developed.The absorbance was taken at 530 nm against non-inoculated broth using the standard curve of IAA (0-100 μg/mL).The IAA content was shown as a microgram of IAA per milligram protein.

Isolation of extracellular polysaccharides
Bradyrhizobium cultures were grown in a 250-mL flask containing 100 mL of YEM medium with 25% PEG 6000 and kept in an incubator shaker at 28 °C for 7 days.The Bradyrhizobium cultures were harvested by centrifugation and pelleting at 10,000 rpm for 15 min.Further supernatant was taken in a fresh tube, and about three volumes of chilled 95% (vol/vol) were added and kept at − 20 °C overnight for precipitating exopolysaccharides (EPS).Finally, the EPS was pelleted by centrifugation at 10,000 rpm for 15 min and dried at 70 °C for 2 days, and powdered EPS was weighed into an Eppendorf tube (Ohno et al. 2000).

Proline content
Proline accumulation in Bradyrhizobium cultures was measured as per the method of Bates et al. (1973).A 1 mL of bacterial culture was added to 10 mL of 3% aqueous sulfosalicylic acid and amended with 25% PEG 6000 and without and filtered through Whatman filter paper.Further, 2 mL of the mixture was added with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid in a test tube.This mixture was placed at 100 °C in a water bath for 1 h, followed by the reaction being inhibited by keeping it ice.Finally, 4 mL of toluene was mixed and vortexed for 15 to 20 s.The chromophore was aspirated, and absorbance of the toluene phase was measured in a UV-spectrophotometer at 540 nm against blank.

Phosphorus solubilization and siderophore detection assay
The Bradyrhizobium cultures were determined for phosphate-solubilizing ability using Pikovskaya's medium (Pikovskaya 1948) under normal (UNST) and stress (ST) conditions.Stress condition was imposed in broth using 25% PEG 6000.The cultures were inoculated in Pikovskaya's broth supplemented with insoluble calcium phosphate.It was further incubated at 28 °C on an incubator shaker.After 7 days of incubation, broth containing bradyrhizobial cells was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken in a 50-mL volumetric flask.About 10 mL of supernatant was mixed with 10 mL of ammonium molybdate and 0.25 mL of freshly made SnCl 2 , and the final volume was made to 50 mL with distilled water.The absorbance was taken at 660 nm against a blank without a bacterial culture.
The quantitative assay of siderophore was carried out using the method of Schwyn and Neilands (1987).The chrome azurol S (CAS) dye was prepared as follows: solution A: 60.6 mg of chrome azurol in 50 mL distilled water; solution B: 10 mL of 1 mM FeCl 3 .6H 2 O in 10 mM HCl; and solution C: 72.9 mg of CTAB in 40 mL distilled water.Solutions A and B were mixed first and then mixed with solution C, resulting in a dark blue-colored CAS dye mixture.The Bradyrhizobium strains were grown in an iron-free succinate medium with the amendment of 25% PEG 6000 at 28 °C for 180 rpm on a shaking incubator for 7 days.After incubation, the medium was centrifuged at 10,000 rpm for 10 min.Cell-free supernatant mixed with 0.5 mL CAS solution and absorbance at 630 nm was recorded after 20 min of incubation against 0.5 mL CAS solution.A standard curve of OD vs. siderophore concentration was plotted.

S rDNA sequencing
The genomic DNA and PCR amplification were done as per the methods described by Ansari and Rao (2014).Bacterial cells were grown in YEM broth (5 mL) for 5 days shaking at 180 rpm to get the desired OD value of 0.6-0.8 at 600 nm.Total genomic DNA was isolated using Invitrogen Pure Link TM genomic DNA mini kit manual instructions.The quantity and purity of DNA were checked in the Denovix DS-11+ spectrophotometer.The DNA was amplified using universal primers 27 F and 1492 R. The PCR Cocktail mix consisted of DNA template 2.00 μL, forward and reversed primer 0.6 μL each, dNTPs (25 mM) 1.5 μL, Taq polymerase 1.0 U, nuclease-free water 17.65 μL, and 10 × PCR buffer with MgCl2 2.5 μL.The DNA amplification was carried out by Gene Pro thermal cycler.The cycle has initial denaturation at 94 °C for 4 min, followed by 35 cycles of amplification (94 °C for 30 s, 52 °C for 45 s, and 72 °C for 1 min) and a final extension at 72 °C for 7 min.The PCR product was resolved in 1.5% agarose gel in 1× TAE buffer stained with ethidium bromide solution (0.5 mg/mL).DNA sequencing of the amplified products (Sanger method) was carried out, and the DNA sequences were subjected to NCBI-BLAST analysis, and the right match of strains was obtained.

Fatty acid methyl esters
Isolates were characterized for fatty acid methyl esters (FAME) using Sherlock's microbial identification system, MIDI, DE, Inc., USA (Sasser 1990).All the isolates were streaked in quadrant (YEMA) for 4-6 days at 28 °C.Cultures were harvested after good growth, preferably from the third quadrant with a 4 mm loop, and placed in a clean 13 × 100 mm culture tube.Add 1 mL of saponification agent (45 g NaOH in 150 mL methanol and 150 mL distilled water), vortexed for 5-10 s, and incubate at 100 °C for 5 min, followed by re-vortexed for 5-10 s and re-incubated at 100 °C for 25 min.The second step was methylation, in which reagent 2 (2 mL: 325 mL 6.0 N HCl and 275 mL methanol) was added, vortexed for 5-10 s, and incubated at 80 °C for 10 min.After methylation, the extraction was performed by adding reagent 3 (1.25 mL: 200 mL hexane and 200 mL methyl tert-butyl ether (MTBE)), and tubes were capped and kept in the tumbler for 10 min.Finally, base washing was performed with reagent 4 (3 mL: 10.8 g NaOH dissolved in 900 mL distilled water).The top 2/3 phase was removed and placed in a vial for analysis.The analysis was performed in a gas chromatograph (Agilent Technologies model 7890A) through a 25 m × 0.2 mm phenylmethyl silicone-fused silica capillary column with a flame ionization detector (FID).The peaks were identified based on MIDI's Sherlock microbial identification system (MIS) library and compared with sample peaks via pattern recognition (Sasser 1990).

Symbiotic effectiveness of Bradyrhizobium isolates
All bacterial cultures were grown in yeast extract mannitol (YEM) broth at 30 °C at 180 rpm for 7 days.The bacterial cells were recovered by centrifugation at 8000 rpm for 10 min.The pellet was washed twice with sterile distilled water.A bacterial suspension of 1 OD600 nm was prepared in phosphate-buffered saline (PBS), and used 500 μL was inoculated at the time of seed sowing around the seed in the pots.
The symbiotic effectiveness of bradyrhizobial isolates on soybean (cultivar JS 95-60) was carried out in sterilized sand 1 3 (121 °C at 15 psi for 1 h) under microcosm conditions at ICAR-IISR, Indore, India.The pots (polypropylene make, 700 g capacity) were used and placed in the net house (temperature range 28 °C day and 22 °C night).The experiment comprised of bradyrhizobial inoculation treatments, i.e. japonicum), replicated 6 times and were laid out as per a completely randomized design (CRD).Plants were grown for 50 days.Plants were watered regularly, and standard cultural practices were followed.Nodulation parameters were measured and recorded, viz., nodule number, dry nodule weight, leghaemoglobin content, and nitrogenase activity (ARA) parameters.

Determination of leghaemoglobin content and nitrogenase activity in root nodules
Leghaemoglobin content in root nodules was determined by the cyanmethemoglobin method described by Wilson and Reisenauer (1963).As per protocol, Drabkin's reagent was prepared by mixing one vial of Drabkin's reagent and 30% Brij L23 solution in 1000 mL water.Further, one gram of nodules was crushed in a 3 mL Drabkin's solution.Further, the mixture was centrifuged at 500×g and 4 °C for 15 min.The supernatant was re-extracted twice, and volume was made up to 10 mL by adding Drabkin's reagent.Absorbance was measured at 540 nm against Drabkin's solution in UV-visible spectrophotometer (UV-1800) against control without nodules.
The nitrogenase activity in root nodules was measured using acetylene reduction assay (ARA) as per the method described by Hardey et al. (1968).The nitrogen fixation potential of Bradyrhizobium isolates was measured in terms of conversion of acetylene to ethylene by gas chromatography (Simadzu 2014 using FID with Rt-U-bond Restek fused silica plot column-thickness 20 mm, diameter 0.53 mm, and 30 m in length) coupled flame ionization detector (GC-FID).One gram of fresh nodules was washed with sterile water and placed in 60-mL tubes, and then, 10% of the air was replaced with an equal volume of acetylene gas (commercial grade) sealed with rubber septa and incubated for 30 min in the dark at room temperature.After incubation, 1 mL volume of ethylene was injected into the inlet valve (injector temp.170 °C, detector temperature 180 °C; initial oven temp.40 °C hold for 5 min, a ramp with 30 °C/min to 170 °C hold with 3 min, run time 12.3 min, and clean the column at 180 °C) using hydrogen as a carrier gas and nitrogen as a makeup gas.

Chlorophyll content
Chlorophyll content in fresh leaves was determined colorimetrically as per the method of Hiscox and Israelstam (1979).A fresh leaf sample of 25 mg was placed in a 5 mL DMSO and then kept at 65 °C for 5 h.Further, the absorbance was taken as blank at 645 nm and 663 nm against DMSO.The chlorophyll content was expressed as mg g −1 FW.

Statistical analysis
The data were analyzed by analysis of variance (ANOVA) using COSTAT.The differences among the treatment means were compared using LSD by Duncan's multiple range test (DMRT) at a 1% level of significance.

Selection of high trehalose-accumulating soybean lines
The role of trehalose in abiotic stress tolerance and Rhizobium-legume symbiosis was reviewed by Sharma et al. (2020).It has been hypothesized that the higher trehalose content in the nodules of soybean genotypes would harbor drought-tolerant bradyrhizobia and, therefore, would enhance the nodulation process.
Trehalose content in soybean nodules varied significantly among the soybean lines.Out of 21 lines evaluated, 7 lines, viz., JS 90-41, Young, NRC 7, Jackson, NRC 2, NRC 37, and PK 472, showed significantly higher trehalose and relative water content than the rest of the lines (Figs. 1 and 2 and Supplementary Table 1).The trehalose content in nodules of these lines ranges from 51.6 to 60.73 μg/g nodules.The highest content (60.73 ± 0.22 μg/g nodules) was recorded in JS 90-41, whereas JS 335 showed the lowest (9.53 ± 0.07 μg/g nodules).When compared to the magnitude of drought tolerance observed based on relative water content (RWC) (Supplementary Table 1) in the lines with the trehalose content, it was found that, by and large, the high content of trehalose was related to tolerance to drought.However, it cannot be generalized as some lines did not show high trehalose and drought tolerance.
The previous researchers reported that trehalose is the key carbohydrate compound accumulated in soybean nodules, and its concentration increases more quickly than in other parts in the first few days of biological nitrogen fixation and during the nodule senescence stages (Streeter 1980(Streeter , 1987;;Müller et al. 1994).A drought-tolerant genotype in symbiosis with Bradyrhizobium spp.SEMIA 6144 accumulated a higher amount of trehalose in peanut nodules as a principal metabolite that was found to protect plants against drought (Furlan et al. 2017).In common beans infected with high trehalose-producing native rhizobia, Rhizobium spp., NGR234 showed a higher accumulation of trehalose and was involved in protecting nitrogenase enzymes under drought stress tolerance (Zacarías et al. 2004).However, a higher trehalose concentration depends on the rhizobial strain inside the nodules (Streeter and Gomez 2006).The role of trehalose in host-induced stress tolerance was reported particularly during the early stages of the symbiotic association between Sinorhizobium meliloti and S. medicae with Medicago sativa plants in which the trehalose utilizer mutant thuB bacteria showed higher nodule formation (Ampomah et al. 2008).The rhizobia isolated from Vicia faba in Morocco showed that rhizobia's trehalose accumulation and osmotolerance are highly correlated (Benidire et al. 2018).Trehalose plays a vital role in the symbiotic tripartite interaction of  Trehalose (μg/g fresh nodules)

Soybean lines
Bradyrhizobium, arbuscular mycorrhizal fungi, and legumes and helps combat abiotic stresses (Sharma et al. 2020).So, it is evident that trehalose has a role in abiotic stress tolerance.
The indigenous Bradyrhizobium has higher symbiotic performance and competitiveness than the non-indigenous strains (Thies et al. 1991).This supports that the current bradyrhizobial isolates of various high trehaloseaccumulating soybean genotypes belong to native soils of Central India.Furthermore, trehalose biosynthesis in Bradyrhizobium provides higher nodulation in the wild strain than mutants of the trehalose biosynthesis pathways gene (Sugawara et al. 2010).Another study showed that two Bradyrhizobium japonicum strains, 163 and 366, isolated from Argentina soils, had higher nodulation, nodule biomass, and leghaemoglobin content showing that native bradyrhizobia strains were better than high-quality commercial inoculants due to their higher competitiveness (López et al. 2018).
Our results also coincide that high trehalose-accumulating soybean lines in their nodule have drought tolerance capabilities.Based on generation time (after 96 h of growth on CRYEMA), all isolates recovered from these lines showed extra slowing behavior.They were found to be moisture-tolerant through various tests (Tables 1, 2, 3, and Figs. 2 and 3).

Characterization of rhizobial strains based on fatty acid methyl esters (FAME) and 16S rRNA sequencing
In this study, we have isolated four bacterial isolates from the higher trehalose-accumulating soybean nodule lines.They were characterized biochemically through FAME and further identified through 16S rDNA-based approach.Based on the FAME profile of rhizobial strains analyzed through Sherlock microbial identification software (MIDI, Inc., DE, USA) indicates that all the strains had 18:1w7c/18:1w6c as the dominant fatty acid (more than 75%) in their cellular membrane indicating the presence of Bradyrhizobium strains.Other lipids present in lesser amounts were 16:1 ω5c, 16:0, 16:1 ω5c, 16:1 ω7c, 19:0 cyclo ω8c, 18:0; 18:1, and 18:1 ω7c/ω9t/ω9c/ω12t.However, further characterization at the species level could not be done due to the lack of a specific FAME profile of bradyrhizobial strains in the MIDI library software.A similar study on FAME characterization of 382 bradyrhizobial accessions was conducted by Joglekar et al. (2020), which showed the presence of lipids such as 16:0, 16:1 ω5c, 16:1 ω7c, 19:0 cyclo ω8c, unknown 18:0, and 18:1 ω7c/ω9t/ω9c/ω12t into their cellular membrane.These were classified into three groupings, namely, FAME cluster X (B.japonicum), Y (B.diazoefficiens), and Z (B.elkanii).Similarly, nine soybean Bradyrhizobium strains whose serogroups affinities were not established based on FAME were analyzed and showed concordant with 16S rRNA gene sequencing analysis and were properly placed into respective bradyrhizobial groups (Van Berkum and Fuhrmann 2001).The FAME analysis of bradyrhizobial strains isolated from peanuts had 16:1 ω5c as a distinguishing lipid and helped cluster the strains into three groups.Contrary to this, 16:1w5c has been reported in the cellular profile of gram-negative bacteria and AM fungi (Zelles 1997).Therefore, the strains were further characterized and identified through 16S rDNA gene sequencing.
Based on 16S rDNA sequencing, two B. liaoningense (KX230053 and KX230054) were recovered from EC 538828 and PK-472 genotypes, respectively, and B. daqingense (KX230052) from PK-472, which has higher trehalose content in their root nodules.In contrast, B. kavengense (MN197775) recovered from Valder genotypes with lower trehalose content in their nodules.The sequences of all these strains have been submitted to NCBI (Supplementary Tables 1 and 2, Fig. 3).One of the rhizobial isolates, D 4A, i.e., B. daqingense, isolated from soybean variety PK-472, was found novel and reported for the first time from the Indian soil.This bacterium was first reported from China and got similar predominant unique fatty acids 15:0 iso and summed feature 5 (18: 2ω6, 9c, and/or 18:0 anteiso) (Wang et al. 2013).However, our study obtained 18:1w7c/18:1w6c as the dominant fatty acid in this strain.Interestingly, the soybean line (PK 472) inhabiting B.
daqingense strain was drought-tolerant based on relative water content and other physiological traits.

Screening of rhizobial strains for tolerance to drought stress
When strains were exposed to stress (simulated through a gradient of PEG 6000 under in vitro), out of all, 4 strains, viz., B. daqingense-D-4A, and two strains of B. liaoningense, D-1C, D-4 B, and reference strain IND-1 were found to be tolerant to PEG 6000 stress (up to 30%) (Table 1).However, one reference strain, i.e., IND-1, showed tolerance at 20% PEG 6000 stress which was isolated earlier from popular soybean variety JS 93-05 (Sharma et al. 2012).Many researchers tested soybean bradyrhizobia for drought tolerance in PEG osmoticum and showed that different bradyrhizobial species differed in their drought tolerance potential (El-Nahrawy and Yassin, 2020; Kibido et al. 2020;Marinković et al. 2013).Previously, the rhizobial strains isolated from the cluster bean were also screened in vitro for drought and heat tolerance in PEG osmoticum (Dhull and Gera 2017).In other studies, Rhizobium spp.NBRI 2505 Sesbania isolated from Sesbania aculeate showed tolerance up to 45% PEG, whereas its drought-sensitive mutants could not tolerate the PEG stress and showed ineffective symbiosis with the host (Rehman and Nautiyal 2002).It was suggested that the osmotolerance in the rhizobia isolated from Acacia plants was due to the accumulation of trehalose for drought tolerance (Essendoubi et al. 2007).Similarly, the Bradyrhizobium japonicum 110 isolated from nodule accumulated trehalose and many other osmolytes to maintain osmolarity with the microenvironment of the nodular tissue (Vauclare et al. 2013).However, our study did not quantify the trehalose in the bradyrhizobial cultures (Essendoubi et al. 2007).In an earlier study, Bradyrhizobium strains were screened for drought tolerance on PEG, and strains that were found drought-tolerant also showed higher IAA production, higher nodulation, ARA activity, and nodule nitrogen content even at higher PEG concentrations (Uma et al. 2013).
The IAA production profile of different strains under stress (PEG concentration 25%) and normal conditions revealed that the magnitude of response of strains to IAA production to stress was varied.Under normal conditions, all strains' IAA production was statistically at par, ranging from 15.55 to 19.28 μg/mL.The highest production was obtained in strain D-4B (19.28 ± 1.77), whereas the lowest was observed in D-1C (9.5 ± 0.08).Under stress conditions, the IAA production was significantly higher in the reference strains than in the rest.The interaction effects of stress with strains analyzed through two-way AVOVA showed that irrespective of strains, the IAA production has declined significantly due to stress than under normal conditions (Table 2).On the other hand, regardless of stress, all strains except D-1C showed higher IAA production, but their differences were non-significant.Moreover, the IAA production with tryptophan amendments was non-significant in both stressed and unstressed conditions in all the tested strains (Table 2).
It has been reported that rhizobial strains isolated from black gram or Sesbania plants produced IAA, siderophore, HCN, and phosphorus solubilization as plant growth-promoting mechanisms (Satyanandam et al. 2021;Sridevi and Mallaiah 2007).
The exopolysaccharide (EPS) production of experimental isolates ranged from 166.33 to 221.16 mg/mL, and the mean production was non-significant across both tested conditions.The highest production was observed in reference strain IND-1, followed by D-4A under both stressed and unstressed conditions (Table 3).Through two-way ANOVA, the strain × condition interaction for EPS production was non-significant.Exopolysaccharides play a crucial role in Rhizobium and legume symbiosis and stress tolerance (Acosta-Jurado et al. 2021;Ali and Orf 2022).The reference strain Bradyrhizobium japonicum (IND-1) was isolated from the popular soybean variety JS 93-05 grown in the Malwa region, Central India, and had capabilities of enhanced nodulation (Sharma et al. 2010;Sharma et al. 2012;Sharma et al. 2016).The inoculation of chickpea Rhizobium ciceri Ca 181 strain produced hydroxamate type of siderophore and was found to be higher in terms of symbiotic potentials like nodule number, biomass, ARA, and total plant nitrogen in chickpea (Dhul et al. 1998).
The Bradyrhizobium diazoefficiens USDA110 has an EPS synthesis gene (exo genes), and the deletion of these genes in mutants hampers the nodulation processes (Xu et al. 2021).
The phosphate solubilization ranged from 65.25 to103.10μg/mL across different strains.The highest production was observed in the case of D-1C, followed by D-4A under unstressed conditions, and significantly lower production was reported in IND-1 under stressed conditions.Moreover, the two-way ANOVA interaction showed that the strain × condition effect was highly significant.The soybean B. japonicum USDA110 produced 69.56 μg/mL of PO 4 3-by dissolving tricalcium phosphate (Hemachandra et al. 2021).The siderophore production of experimental strains ranged from 13.88 to 40.41 μg/mL across both tested conditions.The highest production was reported in reference strain IND-1 (40.41 ± 10.23 μg/mL) during stress, whereas other strains showed significantly lower production.However, during normal conditions, D-4A showed the highest production (32.36 ± 0.23 μg/mL)  4).Additionally, the interaction between strains × condition for siderophore production was highly significant.In the previous study, under normal conditions, the highest siderophore production was observed in the Bradyrhizobium japonicum NCIM 2746, which produced citrate and catecholate type of siderophore and thereby promoted soybean growth, nodulations, and chlorophyll content (Khandelwal et al. 2002).Similarly, Bradyrhizobium japonicum SEMIA 5079 and Bradyrhizobium diazoefficiens SEMIA 5080 strains showed higher siderophore production due to genes producing siderophore of citrate and catecholate type (Argüelles 2000).
Proline accumulation in the experimental strains ranged from 0.39 to 2.22 μg/mL across both conditions.Significantly higher production was observed with reference strain (IND-1) followed by D-11A than the other strains (Table 4).Our study showed that all the bradyrhizobial cultures showed positive for IAA, siderophore, phosphorus solubilization, proline, and EPS production (Tables 2,  3, and 4).Similar results were also obtained by Valdez et al. (2016), where cowpea Bradyrhizobium strains Rc-458-01, Rc-352-01, and Rc-391-01isolated from chickpea nodules were found to produce IAA, siderophore, and ACC-deaminase enzymes.However, many studies showed that B. japonicum strains were negative or weak for siderophore production and phosphorus solubilization (Marinković et al. 2013;Boiero et al. 2007).Hence, the ability to produce IAA and siderophore in the strains depends on the bacterial strain.It may be concluded that indigenous strains are better competitors than commercial inoculants and show higher nodule occupancy and symbiotic effectiveness.Therefore, native strain Bradyrhizobium spp.LSBR-3 was found to solubilize insoluble tri-calcium phosphate IAA, showed higher siderophore production, and had higher plant biomass, chlorophyll content, nodule biomass, and leghaemoglobin content upon inoculation (Kumawat et al. 2019).

Nodule number and nodule biomass
The inoculation of D-1C showed a significantly (p < 0.01) higher nodule number per plant (26.78 ± 1.35) than the plants inoculated with other strains.However, the plants inoculated with commercial strain showed the lowest nodule number.The nodule biomass value ranged from 104.63 to 139.03 mg/g.When compared to nodule biomass, significantly higher nodule biomass was reported in plants inoculated with D-4B (138.15 ± 0.31 mg/plant) and D-11A (139.03 ± 2.48 mg/plant) over the other strains (Table 5).
Many studies on the role of bradyrhizobia on soybean nodulation have been conducted and showed higher nodulation and saving of fertilizer inputs even under drought conditions (Sheteiwy et al. 2021;Zilli et al. 2021;Purwani et al. 2021).Similarly, a combination of B. elkanii BLY3-8, B. japonicum SAY3-7, and Streptomycetes gave higher nodule numbers, nodule biomass, and ARA activities in soybean Japanese and Myanmar variety compared to uninoculated (Htwe et al. 2019).

Leghaemoglobin content and acetylene reduction assay
The leghaemoglobin content was significantly highest in D-4B (7.00 ± 0.42 mg/g), and D-11A (6.38 ± 0.41 mg/g) showed higher leghaemoglobin content compared to commercial (4.07 ± 0.44 mg/g) and reference strain IND-1.Leghaemoglobin content in the case of D-1 C and D-4 A was similar to both the reference strains (Table 5).The ARA activities ranged from 13.86 to 151.33 nmoles/g nodule dry weight/h among all the tested strains.Significantly highest ARA was obtained in the reference strain IND-2 (151.33 ± 0.01nmoles/g nodule dry weight/h) and IND-1 (125.55±10.84nmoles/g nodule dry weight/h) compared to other bacterial treatments.The lowest ARA value was observed in the case of commercial strain (49.51 ± 3.93 nmoles/g nodule dry weight/h) (Table 5).Different Bradyrhizobium strains differ in their symbiotic nitrogen potential with soybean crops under drought stress (Marinković et al. 2019).The symbiotic performance, viz., nodule number and nitrogen fixation of indigenous Bradyrhizobium yuanmingense, was higher than B. japonicum (Appunu et al. 2015).One hundred bradyrhizobial isolates were obtained from cowpea nodules, and they found that symbiotic effectiveness varies with the type of isolates, and native isolates were better than standard strains (Fening and Danso 2002).Another report of the inoculation of either commercial or native bradyrhizobial in promiscuous soybean varieties enhanced nodulation, nodule weight, and higher nitrogen fixation than native strains (Thuita et al. 2012).Our study showed that the native bradyrhizobial strains showed higher symbiotic performance, such as nodule number, biomass, ARA, and leghaemoglobin content than the commercial strains (Table 5) since all isolates were recovered from the root nodules compatible with host plants.
The inoculation of biofix and legume fix commercial Bradyrhizobium formulation in soybean enhanced the nodule number, dry nodule weight, and grain yield (1.5-folds) over the control in a field experiment (Ulzen et al. 2016).We also found that commercial Bradyrhizobium-treated plants have higher symbiotic parameters than uninoculated control.However, the nodulation response was comparatively higher (e.g., D-4 A showed significantly higher) when inoculated with experimental native strains.Similar reports of increased nodule number, biomass, grain yield, and seed protein content were also reported from China and other countries in soybean upon inoculation with soybean rhizobia (Yang et al. 2018).

Chlorophyll content
The leaf chlorophyll content ranged from 0.46 to 1.41 mg/g among all the inoculation treatments.Significantly higher content was observed in plants inoculated with IND-2 (1.41 ± 0.10 mg/g) followed by D-4A (1.40 ± 0.08 mg/g) and D-4B (1.38 ± 0.04 mg/g) than the plants inoculated with commercial rhizobia (Table 5).The uninoculated plants had the lowest chlorophyll content in their leaves.The increased content of chlorophyll in Bradyrhizobium japonicum-inoculated soybean plants has also been reported by Kühling et al. (2018) and Sheteiwy et al. (2021).
In summary, the present study highlights the role played by trehalose in identifying soybean lines for drought stress mitigation.The isolation and characterization of soybean rhizobia with moisture stress abilities from high trehaloseaccumulating lines could be important to understand and decipher further at the molecular level.The potential novel rhizobial candidate strain D 4A (B.daqingense) identified in the study can be attempted for bioformulation and utilization at the field level.

Fig. 1
Fig. 1 Heat map indicating the relative concentration of trehalose in 21 soybean genotypes; dark-red blocks indicated through arrows showing a high concentration of trehalose in root nodules

Fig. 3
Fig. 3 Phylogenetic tree based on the 16S rDNA sequences showing the relationships of the native Bradyrhizobium strains isolated from the different soybean genotypes.The tree was constructed by the

Table 1
Data are means of three replications ± standard error of mean.Means followed by same alphabet do not differ significantly at p < 0.01 (*p < 0.05, **p < 0.01, and ***p < 0.001, ns means non-significant) by least significant difference (LSD) of Duncan's multiple range test (DMRT)

Table 3
Exopolysaccharide production and phosphorus solubilization potential of bradyrhizobial strains under normal and simulated stressed conditions.
Data are means of three replications ± standard error of mean.Means followed by same alphabet do not differ significantly at p < 0.01 (*p < 0.05, **p < 0.01, and ***p < 0.001, ns means non-significant) by least significant difference (LSD) of Duncan's multiple range test (DMRT)

Table 4
Siderophore production and proline accumulation of bradyrhizobial strains under normal and simulated stressed conditions.Data are means of three replications ± standard error of mean.Means followed by same alphabet do not differ significantly at p < 0.01 (*p < 0.05, **p < 0.01, and ***p < 0.001, ns means non-significant) by least significant difference (LSD) of Duncan's multiple range test (DMRT)

Table 5
Evaluation of bradyrhizobial strains for nodulation parameters and acetylene reductase activity under microcosm conditions ARA , acetylene reduction assay; LegH, leghaemoglobin.Data are means of three replications ± standard error of means.Means followed by same alphabet do not differ significantly at p < 0.01 by least significant difference (LSD) of Duncan's multiple range test (DMRT)