Selection of high trehalose accumulating soybean lines
The role of trehalose in abiotic stress tolerance and Rhizobium-legume symbiosis was nicely reviewed by Sharma et al. (2020). It has been hypothesized that the higher trehalose content in the soybean genotypes nodule would harbour 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, 07 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 (Fig. 1 and Fig. 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 (60.73 ± 0.22µg/g nodules) was analyzed 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 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 of the lines did not show high trehalose and drought tolerance.
Figure 1 and Fig. 2
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 as well as during the nodule senescence stages (Streeter 1980, 1987; Müller et al. 1994). A drought-tolerant genotype symbiotic with Bradyrhizobium spp. SEMIA 6144 accumulated a higher amount of trehalose in peanut nodules as a principal metabolite 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 enzyme under drought stress tolerance (Zacarías et al. 2004). However, higher concentration of trehalose depends on the rhizobial strain present inside the nodules (Streeter and Gomez 2006). The role of trehalose in host-induced stress tolerance was reported particularly during early stages of 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 feba in Morocco showed that the trehalose accumulation and osmotolerance of rhizobia are highly correlated (Benidire et al. 2018). Trehalose plays a vital role in the symbiotic tripartite interaction of Bradyrhizobium, arbuscular mycorrhizal fungi, and legumes and also helps in combating 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 trehalose accumulating soybean genotypes belongs to native soils of Central India. Furthermore, trehalose biosynthesis in Bradyrhizobium provides higher nodulation in the wild strain as compared to 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 has drought tolerance capabilities. Based on generation time (after 96 h of growth on CRYEMA), all the isolates recovered from these lines showed extra slowing behaviour and were found to be moisture tolerant through various tests (Table 1,2, 3 and Fig. 2 &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 and 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 18:1ω7c/18:1ω6c as dominant fatty acid (more than 75%) in their cellular membrane. 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, 18:1 ω7c/ω9t/ω9c/ω12t. However, due to the lack of a specific FAME profile of bradyrhizobial strains in the MIDI library software, further characterization at the species level could not be done. 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 genes sequencing analysis and were properly placed into respective bradyrhizobial groups (Van Berkum and Fuhrmann 2001). The FAME analysis of bradyrhizobial strains isolated from peanut had 16:1 ω5c as a distinguishing lipid and helped in clustering the strains into three groups. Contrary to this, 16:1ω5c 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 & KX230054) were recovered from EC 538828, PK-472 genotypes, respectively and B. daqingense (KX230052) from PK-472 which has higher trehalose content in their root nodules. Whereas, B. kavengense (MN197775) recovered from Valder genotypes which had lower trehalose content in their nodules. The sequences of all these strains have been submitted to NCBI (Supplementary Table 1&2, Fig. 3). One of the rhizobial isolate D 4A i.e., B. daqingense, isolated from soybean variety PK-472 was found to be novel 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, in our study, we obtained 18:1w7c/18:1w6c as the dominant fatty acid in this strain. Interestingly, the soybean line (PK 472) inhabiting B. daqingense strain was found to be drought-tolerant based on relative water content and other physiological traits.
Figure 3.
Biochemical And Functional Characterization Of Bradyrhizobial Isolates
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, 04 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; Marinkovi 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, where Rhizobium spp. NBRI 2505 Sesbania isolated from Sesbania aculeata showed tolerance of up to 45% PEG, whereas drought-sensitive mutants were unable to 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 micro environment of the nodular tissue (Vauclare et al. 2013). However, in our study, we 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 which were found drought-tolerant were also showed higher IAA production, higher nodulation, ARA activity, and nodule nitrogen content even at higher PEG concentrations (Uma et al. 2013).
Table 1
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, the IAA production of all the strains was statistically at par, and it ranged 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 both the reference strains than in the rest of the strains. 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, out of 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).
Table 2
It has been reported that rhizobial strains isolated from black gram or Sesbania plants produced IAA, siderophore, phosphorus solubilization, and HCN production as plant growth-promoting substances (Satyanandam et al. 2021; Sridevi & 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. 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, it was found 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 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).
Table 3
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 key role of EPS in soybean nodulation was validated in Bradyrhizobium japonicum 634b and Bradyrhizobium japonicum 631 (Melnykova 2019). Similarly, higher production of EPS in soybean rhizobia i.e., Bradyrhizobium japonicum (ARC 517) was reported by Ali and Orf (2022).
The phosphate solubilization ranged from 65.25 to103.10 µg/mL across different strains and a 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 strain × condition effect was highly significant. The soybean B. japonicum USDA110 produced 69.56 µg/mL of PO4 -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. 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) as compared to other strains (Table 4). Additionally, the interaction between strains × condition for siderophore production was highly significant. In the previous study, under the 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 presence of genes producing siderophore of citrate and catecholate type (Argüelles 2000).
Table 4
Proline accumulation in the experimental strains ranged from 0.39 to 2.22 µg/mL across both conditions. Significantly highere production was observed with reference strain (IND-1) followed by D-11A than the other strains (Table 4). Our study showed all the bradyrhizobial cultures showed positive for IAA, siderophore, phosphorus solubilization, proline, and EPS production (Table 2, 3, 4). Similar results were also obtained by Valdez et al., (2016) where cowpea Bradyrhizobium strain Rc-458-01, Rc-352-01, and Rc-391-01isolated from chickpea nodules were found to produce IAA, siderophore, and ACC-deaminase enzymes. However number of studies showed that B. japonicum strains were found to be negative or week for siderophore production and phosphorus solubilisation (Marinkovi et al. 2013; Boiero et al. 2007). Hence the ability for production of IAA and siderophore in the strains depends on bacterial strain and may be concluded that indigenous strains are better competitors than commercial inoculants and showed higher nodule occupancy and symbiotic effectiveness. Therefore native strain Bradyrhizobium spp. LSBR-3 was found to solubilize insoluble tri-calcium phosphate IAA, and showed higher production of siderophore and had higher plant biomass, chlorophyll content, nodule biomass, and leghaemoglobin content upon inoculation (Kumawat et al. 2022).
Evaluation Of Soybean Rhizobia For Higher Nodulation And N-fixation Abilities
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 role of bradyrhizobia on soybean nodulation have been conducted and showed higher nodulation, 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 number, nodule biomass, and ARA activities in soybean Japanese and Myanmar variety compared to uninoculated (Htwe et al. 2019).
Meta-analysis studies (2009–2020) showed that Bradyrhizobium japonicum and Azospirillum brasilense had higher nodule numbers (5.4%) and biomass (10.6%). However, we have not used any combinations of Bradyrhizobium with any PGPR (Barbosa et al. 2022). Soybean crops inoculated with compatible bradyrhizobia in the soil of soybean-grown fields and without grown soybean had greater nodule number, nodule biomass, and leghaemoglobin content (Halwani et al. 2021).
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 were similar to both the reference strains (Table 5). The ARA activities ranged from 13.86 to 151.33 nmoles /g nodule dry weight/hr among all the tested strains. Significantly highest ARA was obtained in both the reference strain IND-2 (151.33 ± 0.01nmoles /g nodule dry weight/hr) and IND-1 (125.55 ± 10.84nmoles /g nodule dry weight/hr) as 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/hr) (Table 5).
Table 5
Different Bradyrhizobium strains differs 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, were 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 and 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 (some 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 ± 0.10mg/g) followed by D-4A (1.40 ± 0.08mg/g), D-4B (1.38 ± 0.04mg/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 have also been reported by (Kühling et al. 2018; 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 trehalose accumulating lines could be an important aspect to understand and decipher further at molecular level. The potential novel rhizobial candidate strain D 4A (B. daqingense) identified in the study can be attempted for bio-formulation and utilization at field level.