Bradyrhizobium Diazoefficiens USDA110 Research Articles

Analysis of cultivar variability in arbuscular mycorrhizal responsiveness in soybean varieties

ABSTRACT In modern agricultural systems, high crop yields depend on the high input of orthophosphate-based fertilizers. Owing to the looming scarcity of rock phosphate in the near future, utilization of arbuscular mycorrhizal (AM) symbiosis for the efficient uptake of phosphorus is a necessary approach to maintain crop yield. Plant responsiveness to AM fungi is assessed based on the difference in biomass between plants with and without AM fungal inoculation. Soybean is a plant which establishes both mycorrhizal and rhizobial symbiosis. Therefore, it is difficult to eliminate the symbiotic effects of rhizobial nitrogen fixation during long-term cultivation until soybean harvest. In this study, Bradyrhizobium diazoefficiens USDA110 was inoculated at the same time as soybean seeds were sown, and soybean was grown under Rhizophagus irregularis inoculated and non-inoculated conditions to verify whether the mycorrhizal inoculation effect was observed at the yield level. In 2013, two soybean cultivars Tachinagaha and Williams 82, were grown at five levels of phosphate availability (0, 5, 10, 20, and 40 kg P 10a−1) in an open greenhouse in Tsukuba, Japan. Among the several growth indices analyzed, seed weight per plant was selected as a reliable indicator of plant response. Mycorrhizal inoculation showed a positive effect on soybean under 20 kg P 10a−1 phosphorus fertilization for Williams 82, while Tachinagaha showed almost no effect. This trend was replicated in 2014 and 2016, indicating that Tachinagaha has a lower AM responsiveness than Williams 82.

Soybean-mediated suppression of BjaI/BjaR1 quorum sensing in Bradyrhizobium diazoefficiens impacts symbiotic nitrogen fixation.

The acyl-homoserine lactones (AHLs)-mediated LuxI/LuxR quorum sensing (QS) system orchestrates diverse bacterial behaviors in response to changes in population density. The role of the BjaI/BjaR1 QS system in Bradyrhizobium diazoefficiens USDA 110, which shares homology with LuxI/LuxR, remains elusive during symbiotic interaction with soybean. Here this genetic system in wild-type (WT) bacteria residing inside nodules exhibited significantly reduced activity compared to free-living cells, potentially attributed to soybean-mediated suppression. The deletion mutant strain ΔbjaR1 showed significantly enhanced nodulation induction and nitrogen fixation ability. Nevertheless, its ultimate symbiotic outcome (plant dry weight) in soybeans was compromised. Furthermore, comparative analysis of the transcriptome, proteome, and promoter activity revealed that the inactivation of BjaR1 systematically activated and inhibited genomic modules associated with nodulation and nitrogen metabolism. The former appeared to be linked to a significant decrease in the expression of NodD2, a key cell-density-dependent repressor of nodulation genes, while the latter conferred bacterial growth and nitrogen fixation insensitivity to environmental nitrogen. In addition, BjaR1 exerted a positive influence on the transcription of multiple genes involved in a so-called central intermediate metabolism within the nodule. In conclusion, our findings highlight the crucial role of the BjaI/BjaR1 QS circuit in positively regulating bacterial nitrogen metabolism and emphasize the significance of the soybean-mediated suppression of this genetic system for promoting efficient symbiotic nitrogen fixation by B. diazoefficiens.IMPORTANCEThe present study demonstrates, for the first time, that the BjaI/BjaR1 QS system of Bradyrhizobium diazoefficiens has a significant impact on its nodulation and nitrogen fixation capability in soybean by positively regulating NodD2 expression and bacterial nitrogen metabolism. Moreover, it provides novel insights into the importance of suppressing the activity of this QS circuit by the soybean host plant in establishing an efficient mutual relationship between the two symbiotic partners. This research expands our understanding of legumes' role in modulating symbiotic nitrogen fixation through rhizobial QS-mediated metabolic functioning, thereby deepening our comprehension of symbiotic coevolution theory. In addition, these findings may hold great promise for developing quorum quenching technology in agriculture.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Independent Component Analysis Reveals the Transcriptional Regulatory Modules in Bradyrhizobium diazoefficiens USDA110.

The dynamic adaptation of bacteria to environmental changes is achieved through the coordinated expression of many genes, which constitutes a transcriptional regulatory network (TRN). Bradyrhizobium diazoefficiens USDA110 is an important model strain for the study of symbiotic nitrogen fixation (SNF), and its SNF ability largely depends on the TRN. In this study, independent component analysis was applied to 226 high-quality gene expression profiles of B. diazoefficiens USDA110 microarray datasets, from which 64 iModulons were identified. Using these iModulons and their condition-specific activity levels, we (1) provided new insights into the connection between the FixLJ-FixK2-FixK1 regulatory cascade and quorum sensing, (2) discovered the independence of the FixLJ-FixK2-FixK1 and NifA/RpoN regulatory cascades in response to oxygen, (3) identified the FixLJ-FixK2 cascade as a mediator connecting the FixK2-2 iModulon and the Phenylalanine iModulon, (4) described the differential activation of iModulons in B. diazoefficiens USDA110 under different environmental conditions, and (5) proposed a notion of active-TRN based on the changes in iModulon activity to better illustrate the relationship between gene regulation and environmental condition. In sum, this research offered an iModulon-based TRN for B. diazoefficiens USDA110, which formed a foundation for comprehensively understanding the intricate transcriptional regulation during SNF.

Mixed Enterobacter and Klebsiella bacteria enhance soybean biological nitrogen fixation ability when combined with rhizobia inoculation

Biological nitrogen fixation is a key process for nitrogen geochemical cycling and plant‒microbe interactions. At present, little is known about the possible effects of nonnodulating microbes on rhizobia nodulation in soils. Here, we show that Enterobacter sp. Z1 and Klebsiella sp. Z2 significantly improved soybean growth and nitrogen fixation in the presence of Bradyrhizobium diazoefficiens USDA110. Soil metabolomic analysis showed that the application of mixed strains Z1 and Z2 significantly increased the levels of flavonoids, indoleacetic acid (IAA), salicylic acid (SA), and taurine in rhizosphere soil. The addition of IAA, SA, or taurine increased nitrogenase activity, nodule production, and chlorophyll content in soybean. Flavonoids were the determinant of the interaction between soybeans and rhizobia. Moreover, strains Z1 and Z2 produced an abundance of IAA and SA, thus increasing the expression of flavonoid synthesis-related genes and eventually increasing the accumulation of flavonoids. Both strains Z1 and Z2 exhibited two metabolic pathways of taurine, and they catalyzed taurine to produce acetyl-CoA, a key flavonoid synthesis substrate in soybeans. This study revealed the mechanism by which rhizosphere microorganisms mediated soybean-rhizobium interactions, thus promoting soybean nitrogen fixation.

Bradyrhizobium diazoefficiens USDA 110 displays plasticity in the attachment phenotype when grown in different soybean root exudate compounds.

Bradyrhizobium diazoefficiens, a symbiotic nitrogen fixer for soybean, forms nodules after developing a symbiotic association with the root. For this association, bacteria need to move toward and attach to the root. These steps are mediated by the surface and phenotypic cell properties of bacteria and secreted root exudate compounds. Immense work has been carried out on nodule formation and nitrogen fixation, but little is known about the phenotype of these microorganisms under the influence of different root exudate chemical compounds (RECCs) or how this phenotype impacts the root attachment ability. To address this knowledge gap, we studied the impact of 12 different RECCs, one commonly used carbon source, and soil-extracted solubilized organic matter (SESOM) on attachment and attachment-related properties of B. diazoefficiens USDA110. We measured motility-related properties (swimming, swarming, chemotaxis, and flagellar expression), attachment-related properties (surface hydrophobicity, biofilm formation, and attachment to cellulose and soybean roots), and surface polysaccharide properties (colony morphology, exopolysaccharide quantification, lectin binding profile, and lipopolysaccharide profiling). We found that USDA 110 displays a high degree of surface phenotypic plasticity when grown on the various individual RECCs. Some of the RECCs played specific roles in modulating the motility and root attachment processes. Serine increased cell surface hydrophobicity and root and cellulose attachment, with no EPS formed. Gluconate and lactate increased EPS production and biofilm formation, while decreasing hydrophobicity and root attachment, and raffinose and gentisate promoted motility and chemotaxis. The results also indicated that the biofilm formation trait on hydrophilic surfaces (polystyrene) cannot be related to the attachment ability of Bradyrhizobium to the soybean root. Among the tested phenotypic properties, bacterial cell surface hydrophobicity was the one with a significant impact on root attachment ability. We conclude that USDA 110 displays surface plasticity properties and attachment phenotype determined by individual RECCs from the soybean. Conclusions made based on its behavior in standard carbon sources, such as arabinose or mannitol, do not hold for its behavior in soil.

Spontaneously Produced Lysogenic Phages Are an Important Component of the Soybean Bradyrhizobium Mobilome.

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.

Chemodiversity of soil organic matters determines biodegradation of polychlorinated biphenyls by a graphene oxide-assisted bacterial agent

A promising strategy for degrading persistent organic pollutants (POPs) in soil is amendment with nanomaterial-assisted functional bacteria. However, the influence of soil organic matter chemodiversity on the performance of nanomaterial-assisted bacterial agents remains unclear. Herein, different types of soil (Mollisol soil, MS; Ultisol soil, US; and Inceptisol soil, IS) were inoculated with a graphene oxide (GO)-assisted bacterial agent (Bradyrhizobium diazoefficiens USDA 110, B. diazoefficiens USDA 110) to investigate the association between soil organic matter chemodiversity and stimulation of polychlorinated biphenyl (PCB) degradation. Results indicated that the high-aromatic solid organic matter (SOM) inhibited PCB bioavailability, and lignin-dominant dissolved organic matter (DOM) with high biotransformation potential was a favored substrate for all PCB degraders, which led to no stimulation of PCB degradation in MS. Differently, high-aliphatic SOM in US and IS promoted PCB bioavailability. The high/low biotransformation potential of multiple DOM components (e.g., lignin, condensed hydrocarbon, unsaturated hydrocarbon, etc.) in US/IS further resulted to the enhanced PCB degradation by B. diazoefficiens USDA 110 (up to 30.34%) /all PCB degraders (up to 17.65%), respectively. Overall, the category and biotransformation potential of DOM components and the aromaticity of SOM collaboratively determine the stimulation of GO-assisted bacterial agent on PCB degradation.

Rhizobium Symbiotic Capacity Shapes Root-Associated Microbiomes in Soybean.

Root-microbiome interactions are of central importance for plant performance and yield. A distinctive feature of legumes is that they engage in symbiosis with N2-fixing rhizobia. If and how the rhizobial symbiotic capacity modulates root-associated microbiomes are still not yet well understood. We determined root-associated microbiomes of soybean inoculated with wild type (WT) or a noeI mutant of Bradyrhizobium diazoefficiens USDA 110 by amplicon sequencing. UPLC-MS/MS was used to analyze root exudates. The noeI gene is responsible for fucose-methylation of Nod factor secreted by USDA 110 WT strain. Soybean roots inoculated with the noeI mutant showed a significant decrease in nodulation and root-flavonoid exudation compared to roots inoculated with WT strain. The noeI mutant-inoculated roots exhibited strong changes in microbiome assembly in the rhizosphere and rhizoplane, including reduced diversity, changed co-occurrence interactions and a substantial depletion of root microbes. Root exudates and soil physiochemical properties were significantly correlated with microbial community shift in the rhizosphere between different rhizobial treatments. These results illustrate that rhizobial symbiotic capacity dramatically alters root-associated microbiomes, in which root exudation and edaphic patterns play a vital role. This study has important implications for understanding the evolution of plant-microbiome interactions.

Identification of an Exopolysaccharide Biosynthesis Gene in Bradyrhizobium diazoefficiens USDA110.

Exopolysaccharides (EPS) play critical roles in rhizobium-plant interactions. However, the EPS biosynthesis pathway in Bradyrhizobium diazoefficiens USDA110 remains elusive. Here we used transposon (Tn) mutagenesis with the aim to identify genetic elements required for EPS biosynthesis in B. diazoefficiens USDA110. Phenotypic screening of Tn5 insertion mutants grown on agar plates led to the identification of a mutant with a transposon insertion site in the blr2358 gene. This gene is predicted to encode a phosphor-glycosyltransferase that transfers a phosphosugar onto a polyprenol phosphate substrate. The disruption of the blr2358 gene resulted in defective EPS synthesis. Accordingly, the blr2358 mutant showed a reduced capacity to induce nodules and stimulate the growth of soybean plants. Glycosyltransferase genes related to blr2358 were found to be well conserved and widely distributed among strains of the Bradyrhizobium genus. In conclusion, our study resulted in identification of a gene involved in EPS biosynthesis and highlights the importance of EPS in the symbiotic interaction between USDA110 and soybeans.

The Bradyrhizobium diazoefficiens type III effector NopE modulates the regulation of plant hormones towards nodulation in Vigna radiata

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata, including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Application of biofertilizer containing Bacillus pumillus TUAT1 on soybean without inhibiting infection by Bradyrhizobium diazoefficiens USDA110

ABSTRACT Application of bio-fertilizer with the ability to promote root development in legume plants could be beneficial, and it is vital to find an inoculation method that does not inhibit symbiosis between rhizobium and plants. However, co-inoculation of rhizobia with different microorganisms on legumes generally inhibits interdependency with rhizobia. The present study was conducted to determine the ideal inoculation method of the bio-fertilizer ‘Yume-bio’ containing Bacillus pumilus TUAT1, which has plant growth-promoting activity, without inhibiting rhizobial infection of soybean. Soybean plants were inoculated with Bradyrhizobium diazoefficiens USDA110 during the time of sowing seeds. Then, two types of ‘Yume-bio’ applications were conducted: simultaneous inoculation with the rhizobia (SI) and 1 week after inoculation the rhizobia (I). No inoculation (NI) was used as the control. Four weeks after sowing, biomass dry weight (shoot and root) and nitrogenase activity based on acetylene reduction assay (ARA) both, per plant and nodule weight increased significantly in ‘I’ treatment compared to those in ‘NI.’ However, there were no significant differences between ‘SI’ and ‘NI’ for shoot biomass and ARA per plant. Nodule numbers decreased in ‘SI’ as compared to ‘NI,’ while they were not different between ‘I’ and ‘NI.’ This study suggests that simultaneous inoculation of ‘Yume-bio’ and rhizobia inhibits nodule development, while inoculation of ‘Yume-bio’ 1 week after inoculation of rhizobia is ideal for promoting soybean growth without inhibiting rhizobium infection in soybean.

Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110spc4

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

Enhancing the Efficiency of Soybean Inoculant for Nodulation under Multi-Environmental Stress Conditions.

The development of rhizobial inoculants with increased resistance to abiotic stress is critical to mitigating the challenges related to climate change. This study aims at developing a soybean stress-tolerant Bradyrhizobium inoculant to be used under the mixed stress conditions of acidity, high temperature, and drought. Six isolates of Bradyrhizobium with high symbiotic performance on soybean were tested to determine their growth or survival abilities under in vitro conditions. The representative stress-tolerant Bradyrhizobium isolates 184, 188, and 194 were selected to test their ability to promote soybean growth under stress conditions compared to the type strain Bradyrhizobium diazoefficiens USDA110. The plant experiment indicated that isolate 194 performed better in symbiosis with soybean than other Bradyrhizobium strains under stress conditions. Based on the stress tolerance index, soybeans inoculated with isolate 194 showed a high growth performance and significantly better nodulation competition ability than USDA110 under several stress conditions. Interestingly, supplementation of sucrose in the culture medium significantly enhances the survival of the isolate and leads to improved plant biomass under various stress conditions. Analysis of the intra-cellular sugars of isolate 194 supplemented with sucrose showed the accumulation of compatible solutes, such as trehalose and glycerol, that may act as osmoprotectants. This study indicates that inoculation of stress-tolerant Bradyrhizobium together with sucrose supplementation in a medium could enhance bacterial survival and symbiosis efficiency under stress conditions. Although it can be applied for inoculant production, this strategy requires validation of its performance in field conditions before adopting this technology.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency.

Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Sulfur application enhances secretion of organic acids by soybean roots and solubilization of phosphorus in rhizosphere

ABSTRACT Nitrogen (N) fixation by rhizobia plays an important role in N assimilation of soybean (Glycine max). Nitrogenase, the enzyme responsible for N fixation in rhizobia, contains a large amount of sulfur (S) molecules and requires phosphorus (P) in adenosine triphosphate. Thus, S and P are essential for plant growth and N fixation by rhizobia. However, P in soils forms insoluble PO4 3 – salts with metals. Plant roots solubilize these PO4 3- salts by secreting organic acids and absorb solubilized PO4 3-. In this study, the effects of S application on P solubilization by soybean roots and the involvement of rhizobia were evaluated. Soybean plants inoculated with Bradyrhizobium diazoefficiens USDA110 were grown in vermiculite, and Ca3(PO4)2 was added as the sole P source. A hydroponic solution supplemented with three S concentrations (0, 20, and 1000 µM) was applied, and plants were grown for 5 weeks. Root fresh weight, the soil-plant analysis development (SPAD) values, and shoot and root S concentrations increased significantly with S application, but root nodule numbers and nitrogenase activity were not significantly affected. Organic acid secretion by roots was drastically increased by S application, and shoot P concentration and content also increased significantly. These results indicated that S application can promote P solubilization by increasing the secretion of organic acids by roots and increase in N fixation is not involved in this promotion at least in early vegetative growth period.

The inhibitory mechanism of natural soil colloids on the biodegradation of polychlorinated biphenyls by a degrading bacterium

In spite of extensive studies of soil model components, the role of natural soil colloids in the biodegradation of organic pollutants remain poorly understood. Accordingly, the present study selected Mollisol colloids (MCs) and Ultisol colloids (UCs) to investigate their effects on the biodegradation of 3, 3′, 4, 4′-tetrachlorobiphenyl (PCB77) by Bradyrhizobium diazoefficiens USDA 110. Results demonstrated that both natural soil colloids significantly decreased the biodegradation of PCB77, which partly resulted from the significant decrease in the bioaccessibility of PCB77. Furthermore, the activity of Bradyrhizobium diazoefficiens USDA 110 was remarkably inhibited under the exposure to the two types of soil colloids, which was mainly ascribed to the inhibition of cell reproduction but not the lethal effect of reactive oxygen species. The calculated results from Ex-DLVO theory further indicated that the repulsion between UCs and biodegrading bacteria retarded the effective contact of cells with adsorbed PCB77 from UCs, resulting in the decline of the rate of cell reproduction. In general, the inhibition of MCs was limited to PCB77 bioaccessibility, whereas the negative effect of UCs was controlled by PCB77 bioaccessibility and the effective contact of cells with colloids. This study could provide implication for the enhancement of microbial remediation in contaminated soil.

Site-directed mutagenesis of Bradyrhizobium diazoefficiens USDA 110 aroA improves bacterial growth and competitiveness for soybean nodulation in the presence of glyphosate.

The in vitro growth of Bradyrhizobium diazoefficiens USDA 110 strain is inhibited by glyphosate. The herbicide affects 5-enolpyruvylshikimic acid-3-phosphate synthase, a key enzyme for aromatic aminoacid synthesis. In this study, site-directed mutagenesis was used to change only two nucleotides of the coding region of phosphoenolpyruvate binding site. This change improved the in vitro growth of B. diazoefficiens USDA 110 in the presence of glyphosate, without affecting its normal growth in the absence of the herbicide. Plant co-inoculation experiments demonstrated a better competitiveness of the glyphosate-resistant strain for soybean nodulation in the presence of glyphosate.

Involvement of a Novel TetR-Like Regulator (BdtR) of Bradyrhizobium diazoefficiens in the Efflux of Isoflavonoid Genistein

A wide variety of leguminous plant-released (iso)flavonoids, such as genistein, are potential inducers of the nodulation (nod) genes of endosymbiotic rhizobia for the production of Nod factors, which are vital signaling molecules for triggering the symbiotic process. However, these (iso)flavonoids are generally thought to be toxic to the bacterial partner to varying degrees. Here, a novel TetR-like regulator gene of the soybean symbiont Bradyrhizobium diazoefficiens USDA110, bdtR (systematic designation blr7023), was characterized. It was found to be rapidly and preferentially induced by genistein, and its mutation resulted in significantly increased expression of the neighboring bll7019-bll7021 genes, encoding a multidrug resistance efflux pump system, in the absence of this isoflavonoid. Then, the transcriptional start site of BdtR was determined, and it was revealed that BdtR acted as a transcriptional repressor of the above efflux system through the binding of an AT-rich operator, which could be completely prevented by genistein. In addition, the ΔbdtR deletion mutant strain showed higher accumulation of extracellular genistein and became less susceptible to the isoflavonoid. In contrast, the inactivation of BdtR led to the significantly decreased induction of a nodulation gene (nodY) independent of the expression of nodD1 and nodW and to much weaker nodulation competitiveness. Taken together, the results show that BdtR plays an early sensing role in maintaining the intracellular homeostasis of genistein, helping to alleviate its toxic effect on this bacterium by negatively regulating neighboring genes encoding an efflux pump system while being essentially required for nodule occupancy competitiveness.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Symbiotic performance of indigenous soybean bradyrhizobia from the Philippines with soybean (Glycine max [L.] Merill) cultivars harboring different Rj genotypes

This report aimed to evaluate the ability of the dominant indigenous strains of bradyrhizobia obtained from different locations in the Philippines in terms of their symbiosis, N-fixation and nodulation with various soybean cultivars harboring different Rj genotypes. This was done to select the most efficient and effective strain that can be used as an inoculant under the Philippines’ local condition. Two soybean cultivars from the Philippines and three cultivars from Japan and Brazil were used and inoculated with 12 indigenous strains and the reference strain Bradyrhizobium diazoefficiens USDA110. Culture pots were grown inside a growth chamber for four weeks with 33 °C for 16 h as daytime and 28 °C for 8 h as night time. All the strains formed nodules on all soybean cultivars, except for B. elkanii BO-4, which only formed nodules on Philippines’ cultivars. Among the indigenous strains, B. elkanii IS-2 is significantly the most efficient and effective N-fixer than the other strains for the Philippines’ cultivars. In contrast, B. diazoefficiens SK-5 was found to be the most efficient and effective N-fixer for cultivars from Japan and Brazil. Additionally, a positive correlation was observed between the symbiotic efficiency and the nitrogenase activity indicating an efficient N-fixation by the indigenous strains. Thus, we were able to identify the most promising indigenous bradyrhizobia that could be used as an inoculant to increase the soybean yield in the Philippines and provided insights on soybean inoculation.