Characterization of drought stress-mitigating Rhizobium from faba bean (Vicia faba L.) in the Chinese Qinghai-Tibet Plateau

Rhizobium-driven symbiotic nitrogen-fixation in legumes not only benefits the growth but also enhances the stress tolerance of plants. Isolating and characterizing efficient, drought-tolerant rhizobia is a central goal for improving crop yields in arid regions. Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium (‘QHCD11’) sampled from the root nodules of faba beans growing in an arid agricultural area in Qinghai-Tibet. We further tested the drought tolerance of the strain as well as of ‘Qingcan 14’ faba bean seedlings inoculated with it. Biochemical characterization involved bromothymol blue (BTB) tests, carbon metabolic profiling (Biolog GENIII), DNA–DNA hybridization (dDDH) assays, average nucleotide identity (ANI) analyses, and 16S rRNA sequencing. The result indicated that strain ‘QHCD11’ likely belongs to the Rhizobium indicum species. Drought stress tolerance was assessed by exposure to polyethylene glycol (PEG-6000) at concentrations of 0, 10, 15, and 20%. Increasing concentrations of PEG-6000 tended to result in decreased growth of ‘QHCD11’, although the strain performed better at 20% PEG 6000 than at 15%. Inoculation of drought-stressed faba bean seedlings with strain ‘QHCD11’ improved root vitality, chlorophyll content, antioxidant enzyme activities, and plant height. We suggest that inoculation of faba beans with ‘QHCD11’ is an environmentally sound strategy for mitigating crop drought stress in arid and semi-arid regions. In addition, the results presents here will benefit future studies into faba bean-rhizobia symbioses under drought stress.


Introduction
Nitrogen (N) is arguably one of the most important in all of the essential plant nutrients.Globally, the nitrogen cycle transforms approximately 3 × 10 9 tons of gaseous N 2 into plantavailable forms annually.Importantly, approximately 60% of the total N conversion results from biochemical activities (Hardarson, 2003;Mabrouk and Belhadj, 2010).Rhizobium, a genus of Gram-negative soil bacteria, can develop symbiotic relationships with leguminous plants in the form of root nodules, they fix atmospheric nitrogen (Remigi et al., 2016;Stefan et al., 2018).As one of the most important contributors to total biological nitrogen fixation, Rhizobium-legume Li et al. 10.3389/fmicb.2023.1212996Frontiers in Microbiology 02 frontiersin.orgsymbioses contribute more than 50% of the nitrogen available in agricultural soils (Herridge et al., 2008).Not only does this symbiotic relationship enhance nitrogen fixation capacity, but it also improves legume productivity and yield (Jida and Assefa, 2012;Laranjo et al., 2014;Alinia et al., 2022).Rhizobium collected from the nodules and roots of legumes may be promising biofertilizers for use in improving crop growth, nodulation, nitrogen fixation, and resistance to stress and disease.Several studies indicate that naturally-occurring populations of rhizobia from certain geographic regions exhibit greater nodulation competency than many commercial strains (Gaci et al., 2021;Tuan Vo et al., 2021).The isolation and characterization of native rhizobial populations is likely to provide valuable biological resources for the enhancement of crop productivity (Humaira and Bano, 2011).Several studies have subjected native rhizobial populations isolated from legume crops to molecular identification through the use of genomic sequence-based digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI) analysis, and 16S rRNA sequencing (Deng et al., 2022;Zaw et al., 2022).It is clear from these studies that phylogenetic analysis is a stable and dependable method for the description and classification of species.
Unfavorable environmental conditions can compromise the legume-Rhizobium relationship, including drought and excessively high temperatures.Because of this, it is crucial to study legume-Rhizobium symbioses under such conditions.Studies suggest that host plants are often less tolerant of environmental stress than their microbial partners (Jebara et al., 2001;Mnasri et al., 2007;Vriezen et al., 2007).Inoculating seeds with Rhizobium has been shown to enhance the environmental stress tolerance of plants (Franzini et al., 2013;Zeenat et al., 2021).Such increased resistance likely results from a combination of decreased generation of reactive oxygen species (ROS), improvement of leaf water status, facilitation of water uptake, mitigation of ionic imbalance, and more efficient nutrient acquisition.Furthermore, utilizing highly efficient and stress-tolerant rhizobial inocula can further enhance the yield of legume crops in unfavorable environments (Peoples et al., 2009;Denton et al., 2017;Kanonge-Mafaune et al., 2018).Rhizobia appear to boost the stress resilience and growth of host plants through several mechanisms, including inducing the biosynthesis of osmolytes such as ectoines, polyamines, proline, and glycine betaine to guard cells from osmotic stress-and desiccation-related damage; promoting the activity of both enzymatic and non-enzymatic antioxidants; and increasing photosynthetic activity and biomass accumulation (Reina-Bueno et al., 2012;Wdowiak-Wróbel et al., 2013;Lunn et al., 2014;Ding, 2022).Taken together, it is clear that rhizobia can support plant health in unfavorable conditions.This ability may be crucial for enhancing legume cultivation on marginal land and in semi-arid and arid regions.
Qinghai is situated in northwestern China, in the northeastern section of the Qinghai-Tibet Plateau, and has an average altitude of more than 3,000 m.Seventy percent of the agricultural production of Qinghai occurs in semi-arid and arid regions (Li et al., 2018).Faba bean (Vicia faba L.; Fabaceae) has proven adaptable to the varied climates of this region, and is utilized as a grain, vegetable, and fodder crop.In addition, this leguminous crop enhances the sustainability of local cropping systems by contributing nitrogen to agricultural fields through biological fixation (Amede et al., 1999;Khan et al., 2007).At present, faba bean is the primary legume crop grown across the Qinghai-Tibet Plateau, and is a major contributor to economic and ecological development in the region.Unfortunately, faba bean is considerably less drought tolerant than other legumes such as chickpea, pea, and common bean (McDonald and Paulsen, 1997;Amede and Schubert, 2003;Sharma et al., 2017).
Here, we sought to determine the phylogenetic status and symbiotic efficacy of the Rhizobium strain 'QHCD11' , which was isolated from an arid agricultural area in Qinghai-Tibet.We analyzed the morphological, physiological, biochemical, and genetic characteristics of Rhizobium strain 'QHCD11' , as well as assessed its ability to colonize and enhance the drought resistance and growth of faba beans.The results of this study help to illuminate the complex relationship between rhizobia and legumes under stressful environmental conditions.We suggest that Rhizobium strain 'QHCD11' may be further developed into a useful inoculant for improving the drought tolerance of faba beans.

Root nodule
The root nodule was obtained from an arid agriculture area in Haidong City, Minhe County, Qinghai Province, China (36.0607°N, 102.4520°E).The field soil pH was 8.1, with 16.65 g kg −1 organic matter (OM), 1.20 g kg −1 total N, 82 mg kg −1 alkaline N, 2.11 g kg −1 total phosphorus (P; P 2 O 5 ), 24.6 mg kg −1 available P, 20.65 g kg −1 total potassium (K; K 2 O), 178 mg kg −1 available K, and 0.62 g kg −1 total salt.A single healthy plant at flowering stage was uprooted along with nodules and transported to the laboratory.The nodules was chose to isolate of red color (leghemoglobins).

Isolation of rhizobial cultures
Faba bean root nodules were isolated in accordance with the method of Vincent (1970).The Rhizobium isolate was grown on yeast mannitol agar (YMA) plates at 28°C.The pure culture was preserved in 10% (v/v) glycerol stock for subsequent analyses.All morphological, biochemical, and molecular studies were carried out as described previously (Li et al., 2021).The reference Rhizobium leguminosarum strain ' ACCC15854' was obtained from the Agriculture Culture Collection of China (ACCC; Beijing, China).

BTB test of Rhizobium 'QHCD11'
To determine the alkali and acid production of the pure culture isolates, they were streaked onto fresh YMA plates (pH 6.8) containing 30 μg L −1 bromothymol blue (BTB) (Sharma et al., 2010).In a slight alteration of the original method, all plates were incubated for 3-5 d at 28°C, and the plates were evaluated each day.When acidic compounds are produced, the plate color changes from green to yellow, and when alkaline compounds are produced, the plate color changes from green to blue.To evaluate the biochemical and metabolic profile of Rhizobium strain 'QHCD11' , we studied the biochemical parameters associated with carbon metabolism.A Biolog GEN III MIDI system (BIOLOG, CA, United States) was used to test whether Rhizobium strain 'QHCD11' could utilize 95 different carbon sources, according to the manufacturer's instructions.Briefly, the isolate was cultured for 24 h in YM broth.The cells were then purified twice by 3 min of centrifugation at 8000 rpm.Subsequently, the cells were resuspended in sterilized water and adjusted to 10 8 cells ml −1 .Both experimental and control samples were prepared according to the manufacturer's standard protocol.Positive (+) tests were indicated by a color change to purple, and a negative (−) tests were indicated by a lack of color change.

Genomic characteristics of Rhizobium 'QHCD11'
After the removal of connector sequences, the sequencing data were assembled to construct contigs and scaffolds using A5-MiSeq and SPAdes.The assemblies were evaluated and compared, and base correction was performed, using Pilon.Subsequent to filtration, PGCGAP was utilized to assemble the high-quality paired-end reads (Liu et al., 2021).Genomic reannotation was performed with Prokka (Seemann, 2014).Using the draft genome, the RAST server was utilized to determine the G + C content (Brettin et al., 2015).Both the ANI and dDDH were utilized to determine the taxonomic status of the novel isolate.JSpecies WS 2 was used to obtain ANI values and the Genome-to-Genome Distance Calculator (GGDC) 3 was used to obtain dDDH values.

Inoculation of faba beans with Rhizobium
Faba bean'Qingcan 14' seeds were provided by the Qinghai Academy of Agriculture and Forestry Science (Xining City, Qinghai Provence, China).The surfaces of healthy, uniform seeds were sterilized using 1% sodium hypochlorite and washed at least three times with sterilized water.The seeds were subsequently germinated in darkness at 28°C in a petri dish carrying wet filter paper.The stored strain QHCD11 with 10% glycerol was streaked onto Yeast Mannitol Agar (YMA) plates and incubated at 28°C for 3 to 5 days until good growth was observed.A standard curve describing the relationship between cell number and optical density at 600 nm (OD 600nm ), was developed for strain QHCD11 to enable the application of a standard CFU mL −1 across experiments.Moreover, population counts (CFU mL −1 ) were determined by dilution plating.One milliliter of rhizobial suspension (≈10 8 CFU mL −1 ) was used to inoculate each test tube containing 20 mL of YMB media.When the radicle reached 0.5-1.0cm, seedlings were soaked in a Rhizobium standard suspension (10 8 CFU mL −1 ) for 30 min and then planted in sterile containers containing quartz sand.To the base of each 5-day-old seedling was added an additional 2 mL of inoculum (10 8 CFU mL −1 ).Each week, seedlings were provided with 20 mL of sterilized water and 20 mL of N-free McKnight's nutrient solution.The experiment treatments consisted of four different treatments, no inoculation plants under well-watered conditions (NN-CK), no inoculation drought-stressed plants (NN-DS), inoculation plants under well-watered conditions (NA-CK), and inoculation plants under drought stress (NA-DS).Three replicates and three pots per replicate were designated for each treatment, making a total of 36 pots.All plants were grown in a greenhouse with natural daylight from 9 March to 9 Jun.The average day/night temperature was 26/20°C, air relative humidity was 70-80%.The plants were irrigated with distilled water every 1-2 days, and supplied with ½-strength Hoagland's nutrient solution every 30 days (approximately 200 mL per pot).Drought stress was applied during faba bean flowerings stage and was subjected to withdrawing water for 3 days.Physiological index measurements were performed on plants after being subjected to drought stress for 3 days.As a positive control, additional seedlings were inoculated with the R. leguminosarum reference strain ' ACCC15854' , while uninoculated seedlings were used as negative controls.All treatments were arranged in a randomized block design, and included uninoculated seedlings under well-watered conditions (NN-CK), uninoculated seedlings under drought conditions (NN-DS), inoculated seedlings under well-watered conditions (NA-CK), and inoculated seedlings under drought conditions (NA-DS).Each treatment consisted of three replicates, each replicate consisted of three pots, and a total of 36 pots were used for the experiment.All seedlings were cultivated in a climatecontrolled chamber with 70-80% relative humidity and an average night/day temperature of 20/25°C.All morphological, physiological,

Statistical analysis
Statistically significant differences were determined using the standard error (SE) of at least three replicates with SPSS 17.0 (SPSS, IL, United States).

Characterization of Rhizobium isolated from faba bean plants
The isolate was purified by repeated plate streaking, and cultured on YMA plates at 28°C for 3 d in darkness.Morphologically, the isolates were described as 3-5 mm diameter, mucilaginous, opaque, shiny white colonies with a wet surface, which produced copious amounts of sticky exopolysaccharides.Microscopically, the bacteria were Gram-negative, rot-shaped, and red in color, which matches the general description of Rhizobium.The YMA-BTB-based acid/alkali production test indicated that the isolate was producing acidic compounds (green to yellow plate color transition) in medium.These result suggested that the isolate was a species of Rhizobium.

Phylogenetic analysis and genomic characteristics of the Rhizobium isolate
The complete nucleotide sequence (1,477 bp) of the 16S rRNA gene was evaluated.An EzBioCloud sequence search revealed that strain 'QHCD11' shared 99.93% similarity were the Rhizobium type strains R. leguminosarum 'USDA 2370' T , R. anhuiense 'CCBAU 23252' T , R. laguerreae 'FB206' T , R. ruizarguesonis 'UMP1133' T , and R. indicum 'JKLM12A2' T (Table 1).The constructed NJ phylogenetic tree indicated that Rhizobium 'QHCD11' formed a phyletic group with R. changzhiense 'WYCCWR 11279' T (MH778807) (Figure 1).Our results were largely in agreement with previous studies suggesting that variation between Rhizobial strains may be more common at the species level, with diversity observed genus-wide.

Drought stress tolerance of the Rhizobium isolate
The drought tolerance of Rhizobium 'QHCD11' was evaluated by culturing the cells in YMA liquid culture media containing 0, 10, 15%, or 20% PEG-6000.The growth of the isolate, as determined spectrophotometrically based on OD 600nm , was compared with R. leguminosarum reference strain ' ACCC15854' (Table 4).Increasing concentrations of PEG 6000 tended to result in decreased population size and optical density.Interestingly, Rhizobium 'QHCD11' performed better at 20% PEG 6000 than at 15% PEG 6000 (Figure 4), with the turbidity rising from 63.53 to 68.63%.Overall, Rhizobium 'QHCD11' was found to have superior drought tolerance.Research suggests that drought tolerant Rhizobium can improved the drought tolerance of host plants, so Rhizobium 'QHCD11' was selected for further studies of inoculation efficiency and growth improvement in faba bean under drought stress.

Inoculation on faba bean growth under drought stress
Inoculation of plants with beneficial microbes has been shown to ameliorate abiotic stress (Egamberdieva et al., 2016).Here, we studied the biochemical, physiological, and morphological effects of inoculation with Rhizobium 'QHCD11' on 'Qingcan 14' faba bean seedlings subjected to drought stress.The results were compared with an uninoculated negative control and a positive control inoculated with R. leguminosarum reference strain ' ACCC15854' (Table 5).
Physiological analyses revealed that drought stress not only hindered the biosynthesis of chlorophyll, but also accelerated the decomposition of existing chlorophyll.Rhizobium inoculation improved the SPAD value under both well-watered (+32.31%) and drought-stressed (+41.69%)conditions.Leaf RWC is an indicator of water status and reflects the balance between water supply and transpiration rate.Inoculation with Rhizobium 'QHCD11' significantly increased RWC by 81.05% under drought-stressed conditions and 48.05% under well-watered conditions.
Drought stress increased the proline content of faba bean seedlings, with Rhizobium 'QHCD11'-inoculated seedlings exhibiting the greatest increase in proline.Compared to uninoculated seedlings, rhizobial inoculation resulted in a 38.14% increase in proline content under drought-stressed conditions.Furthermore, drought stress upregulated the activity of the enzymatic antioxidant SOD compared to well-watered conditions.Compared to un-inoculated seedlings, rhizobial inoculation increased SOD activity by 27.26% under well-watered conditions and a 33.76% increase in SOD activity under drought-stressed conditions.In water-limited environments, root health and vitality are predictors of survival (Zhang et al., 2022;Yu et al., 2023).The TTC test is an indicator of plant vitality broadly.Inoculated seedlings had significantly higher root vitality than uninoculated seedlings, especially Rhizobium 'QHCD11' -inoculated seedlings.Drought stress reduced root vitality, which was ameliorated with rhizobial inoculation.Increased root viability in inoculated plants is likely to have contributed to the increased drought tolerance of inoculated faba bean seedlings.

Discussion
Crop yield can be severely impacted by abiotic stress, such as high temperatures, alkalinity, high salt, and drought.Both naturally occurring and inoculated microbes have proven efficacious in allaying abiotic stress in crops and other plants.Specifically, Rhizobium inoculants enhance plant abiotic stress resistance by decreasing the generation of ROS, improving leaf water status, facilitating water uptake, and maintaining ionic balance (Thrall et al., 2008).Native rhizobia collected from targeted host plants and coexisting leguminous plants can serve as resources for the development of new and improved crop inocula.Here, we characterized a rhizobial isolate collected from the roots of a faba bean plant growing in the arid Haidong City region in Qinghai Province, China.The isolate, 'QHCD11' , exhibited a fast growth rate, acid production, and a morphological appearance in line with other members of the genus Rhizobium.
The 'QHCD11' 16S rRNA sequence was 1,477 bp long, and the draft genome was 7.50 Mbp in size, with a G + C content of 60.9%.Analysis of sequence identity indicated that the strain shared 99.93% similarity with R. laguerreae 'FB206' T , R. leguminosarum 'USDA 2370' T , R. anhuiense 'CCBAU 23252' T , R. ruizarguesonis 'UMP1133' T , and R. indicum 'JKLM12A2' T .NJ phylogenetic analysis revealed that 'QHCD11' formed a phyletic group with R. changzhiense 'WYCCWR 11279' T (MH778807).Furthermore, phylogenomic analysis indicated that 'QHCD11' clustered with R. indicum 'MCC3961' T .Our results were largely in agreement with previous studies suggesting that variation between Rhizobial strains may be more common at the species level, with diversity observed genus-wide.The ANI comparison value of strain 'QHCD11' to the closely-related R. indicum 'FA23' T was 95.62%, which is greater than the ≥95% threshold to discriminate between different species of microbes (Chun et al., 2018).
Based on these results, strain 'QHCD11' was preliminarily identified as belonging to the fast-growing genus Rhizobium.
Leguminous are important components of natural ecosystems and agricultural systems, and their microsymbionts of legumes have been intensively characterized (Han et al., 2010;Furseth et al., 2012;Zaw et al., 2021).Abiotic stresses are stress conditions to plants arising from the environment, it not only affected plants but also affect microbes.Many studies have shown that strains with drought tolerance can be screened out in arid environment, and inoculation of rhizobia can improve the growth of crops under abiotic stress conditions, increases  crop yield and stress resistance (Enebe and Babalola, 2018;Bouhnik et al., 2022).Strain QHCD11 was identified by 16 s rRNA and whole genome sequencing as Rhizobium indicum species.Rahi et al. (2020) study found that Rhizobium indicum sp.nov., were characterized using 16S rRNA, atpD and recA genes, they were isolated from root nodules of pea (Pisum sativum) cultivated in the Indian trans-Himalayas.Earlier studies have reported that Rhizobium confers defence, enhances nodule number and promotes growth biomarkers in mung bean and Vicia faba (Ahmad et al., 2013;Benidire et al., 2017) under salt stress.Hussain et al.
(2016) found a significant increase in the photosynthesis of maize plant as a result of inoculation with Rhizobium phaseoli under drought stress and confer tolerance to abiotic stresses.In this study, we isolated a strain from root nodueles of faba bean growing in the arid Qinghai province of China.We evaluated the drought tolerance of strain 'QHCD11' subjecting the microbial culture to different concentrations of PEG-6000, and comparing the results to the reference R. leguminosarum strain ' ACCC15854' .It shown that strain 'QHCD11' exhibited better drought tolerance in higher PEG osmotic solution and better than ' ACCC15854' .Our findings are in line with the results of Bouhnik et al.
(2022), who found the ability of the rhizobial strains isolated from the arid zones to grow in high concentrations of PEG-6000 could probably be due to adaptation to their original soils conditions.Some researchers point out that inoculation of seeds with Rhizobium improved abiotic stress tolerance, it is attributed to produce light molecular weight organic solutes such as glycine betaine, proline, polyamines and ectoines, and these solutes protect the plant cells by stabilizing the structure and conformation of proteins as well as cell membranes from water stress, desiccation; increase photosynthetic and biomass productivity and also play role in the enhancement of the activity of various enzymatic and non-enzymatic antioxidants (Santos and Costa, 2002;Yurgel et al., 2013).In this study, we found that inoculation with strain  Neighbor-joining (NJ) phylogenetic analysis of Rhizobium 'QHCD11' whole genome sequence.Scale = 0.01 nucleotide substitutions per position.The outgroup was R. esperanzae 'CNPSo668' T .
'QHCD11' initiates a response in various physiological activities like improves leaf water status, decreases the generation of reactive oxygen species, increase photosynthetic, enhance of the activity of various enzymatic antioxidants.Rhizobium inoculation improved the SPAD value under both drought-stressed and well-watered conditions.Furthermore, rhizobial inoculation can maintain leaf RWC by reducing dehydration-related cell damage, such as by increasing the proline content, as shown here.The fatty acid contents of Rhizobium 'QHCD11'.Here, found that inoculation with strain 'QHCD11' resulted in increased SOD resulting in reduced oxidative stress and oxidative damage.And the same time, inoculated seedlings exhibited the best growth of all experimental groups under adequate water supply condition.

Conclusion
In the present study, we characterized a fast-growing Rhizobium isolate ('QHCD11') which can utilize a range of carbon sources.Based on phylogenetic, chemotaxonomic, genotypic, and phenotypic analyses, the 'QHCD11' strain putatively belongs to the R. indicum 'MCC 3961' T species.Strain 'QHCD11' exhibited superior drought tolerance, and inoculation with this strain conferred drought tolerance and growth enhancement to faba bean seedlings.We suggest that Rhizobium strain 'QHCD11' may be further developed into a useful inoculant for improving the drought tolerance of faba beans and, potentially, other leguminous.These data help to illuminate the complex relationship between rhizobia and legumes under stressful environmental conditions.

TABLE 1
16S rRNA gene sequence comparative analysis.

TABLE 3 dDDH
analysis (%).The meaning of the bold values is which the dDDH value of Rhizobium 'QHCD11' was 69.7%, indicating that it is a species of R. indicum 'MCC 3961'T in table.

TABLE 5
Morphophysiological indices of inoculated and uninoculated faba bean seedlings under control and drought-stressed conditions., SPAD values; RWC, Relative water content; PRO, Proline content; SOD, Superoxide dismutase activity; TTC, Triphenyltetrazolium chloride test; CK, Control (well-watered); DS, Drought stress (PEG-6000); NA, Inoculated; NN, Non-inoculated.Within the same column, different lowercase letters indicate significant differences according to the ANOVA test followed by the post-hoc Fisher test (LSD) with the 95% confidence interval. SPAD

TABLE 4
Turbidity (OD 600nm ) of the rhizobium strains under simulated drought conditions.