Morphological and physiological properties of lupine nodule bacteria (Bradyrhizobium lupini) when grown on a typical urban soil.

In the conducted studies, the moorphological and physiological properties of nodule bacteria of lupine were studied. Lupine plants were grown under the conditions of a microfield experiment on a typical medium loamy urban soil. In the study, a pure culture of Bradyrhizobium lupini was isolated. Then, the morphological properties of nodule bacteria cells and the chemical composition of cell membranes of nodule bacteria were determined. The acid resistance and physiological properties of lupine nodule bacteria were also determined, as well as the ratio of Bradyrhizobium lupini to antibiotics. All studies were carried out according to generally accepted methods. The results of the research showed that during the cultivation of lupine on a typical urban soil, nodule bacteria Bradyrhizobium lupini were isolated, which can be characterized as gram-negative, non-spore-forming rods that do not exhibit amylolytic activity. It was revealed that the rhizobia of nodule bacteria are not acid-resistant. Nodule bacteria turned out to be the least resistant to polymyxin, then to levomycetin, and Bradyrhizobium lupini showed the greatest resistance to tetracycline.

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.

Synergistic N2-fixation and salt stress mitigation in soybean through dual inoculation of ACC deaminase-producing Pseudomonas and Bradyrhizobium.

We investigated the potential dual application of two Bradyrhizobium strains (B. diazoefficiens USDA110 and B. ottawaense SG09) and plant growth-promoting bacteria, PGPB (Pseudomonas spp.: OFT2 and OFT5), to improve nodulation and N2-fixation in soybean plants. The growth-promoting effects of dual inoculation were observed on plant growth, physiology, and nodulation of soybean under normal conditions compared with plants individually inoculated with either USDA110 or SG09. Both OFT2 and OFT5 promoted N2-fixation by 11% and 56%, respectively, when dual inoculation with USDA110 and by 76% and 81%, respectively, when dual inoculation with SG09. Salinity stress significantly reduces soybean growth, physiology, nutrient uptake, nodulation, and N2-fixation. However, these adverse effects were attenuated by the dual inoculation of PGPB and rhizobia depending on the combination of inoculants. In particular, dual inoculation of PGPB with SG09 was more effective in enhancing the salt tolerance of soybean by reducing salt-induced ethylene production and improving nutrient uptake. However, no such effect was observed with the combined inoculation of USDA110 and OFT5. An effective symbiotic association between SG09 and two Pseudomonas bacteria can be considered a beneficial approach to improving the symbiotic efficiency of nodulation and mitigating salinity stress in soybeans.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Natural variation of GmRj2/Rfg1 determines symbiont differentiation in soybean.

Symbiotic nitrogen fixation (SNF) provides much of the N utilized by leguminous plants throughout growth and development. Legumes may simultaneously establish symbiosis with different taxa of microbial symbionts. Yet, the mechanisms used to steer associations toward symbionts that are most propitious across variations in soil types remain mysterious. Here, we demonstrate that GmRj2/Rfg1 is responsible for regulating symbiosis with multiple taxa of soybean symbionts. In our experiments, the GmRj2/Rfg1SC haplotype favored association with Bradyrhizobia, which is mostly distributed in acid soils, whereas the GmRj2/Rfg1HH haplotype and knockout mutants of GmRj2/Rfg1SC associated equally with Bradyrhizobia and Sinorhizobium. Association between GmRj2/Rfg1 and NopP, furthermore, appeared to be involved in symbiont selection. Furthermore, geographic distribution analysis of 1,821 soybean accessions showed that GmRj2/Rfg1SC haplotypes were enriched in acidic soils where Bradyrhizobia were the dominant symbionts, whereas GmRj2/Rfg1HH haplotypes were most prevalent in alkaline soils dominated by Sinorhizobium, and neutral soils harbored no apparent predilections toward either haplotype. Taken together, our results suggest that GmRj2/Rfg1 regulates symbiosis with different symbionts and is a strong determinant of soybean adaptability across soil regions. As a consequence, the manipulation of the GmRj2/Rfg1 genotype or application of suitable symbionts according to the haplotype at the GmRj2/Rfg1 locus might be suitable strategies to explore for increasing soybean yield through the management of SNF.

Effects of nZnS vs. nZnO and ZnCl2 on mungbean [Vigna radiata (L.) R. Wilczek] plant and Bradyrhizobium symbiosis: A life cycle study.

Scarce of knowledge of using Zinc (Zn) nanoparticles (NPs) to augment plant growth, Zn availability to plants and its potential toxicity warrants more NPs-plant life cycle studies. The main objectives of this study were to compare nano zinc sulphide (nZnS) with nano zinc oxide (nZnO) and ionic Zn i.e., ZnCl2, as a source of Zn, as well as to establish physiological impact of NPs on growth, yield and symbiosis of mungbean [Vigna radiata (L.) R. Wilczek] plants at different concentrations (0, 0.01, 0.1, 1 and 10 mg kg-1 of soil). In this study, mungbean plants were grown for 60 days (life cycle study) in natural soil infested with Bradyrhizobium. Effects of Zn compounds (nZnS, nZnO and ZnCl2) on plant height, dry biomass, number of nodules per plant, yield and fruit agronomical parameters along with micronutrient assessment were determined. Impact of Zn compounds on Bradyrhizobium-mungbean symbiosis was also unravelled. Results showed that both the NPs, (nZnS and nZnO) were more effective than ZnCl2 in promoting growth and yield up to a critical concentration and above which phytotoxic effects were observed. Both the NPs were more effective than ZnCl2 at increasing fruit Zn content also. Whereas, nZnS treatment was found to be better than nZnO in improving overall plant growth. Bradyrhizobium-mungbean symbiosis was not affected at lower NPs concentrations, while higher concentration revealed toxicity by damaging bacterial morphology and nodule formation. There was no nano specific toxicity found while, ZnCl2 showed relatively more toxicity than both the NPs. The present investigation demonstrated the concept of nano-micronutrient as well as NPs phytotoxicity by understanding NPs-plant interactions in the soil environment.

Upstream Flanking Sequence Assists Folding of an RNA Thermometer.

Many heat shock genes in bacteria are regulated through a class of temperature-sensitive stem-loop (SL) RNAs called RNA thermometers (RNATs). One of the most widely studied RNATs is the Repression Of heat Shock Expression (ROSE) element associated with expression of heat shock proteins. Located in the 5'UTR, the RNAT contains one to three auxiliary hairpins upstream of it. Herein, we address roles of these upstream SLs in the folding and function of an RNAT. Bradyrhizobium japonicum is a nitrogen-fixing bacterium that experiences a wide range of temperatures in the soil and contains ROSE elements, each having multiple upstream SLs. The 5'UTR of the messenger (mRNA) for heat shock protein A (hspA) in B. japonicum has an intricate secondary structure containing three SLs upstream of the RNAT SL. While structure-function studies of the hspA RNAT itself have been reported, it has been unclear if these auxiliary SLs contribute to the temperature-sensing function of the ROSE elements. Herein, we show that the full length (FL) sequence has several melting transitions indicating that the ROSE element unfolds in a non-two-state manner. The upstream SLs are more stable than the RNAT itself, and a variant with disrupted base pairing in the SL immediately upstream of the RNAT has little influence on the melting of the RNAT. On the basis of these results and modeling of the co-transcriptional folding of the ROSE element, we propose that the upstream SLs function to stabilize the transcript and aid proper folding and dynamics of the RNAT.

Priming of rhizobial nodulation signaling in the mycosphere accelerates nodulation of legume hosts.

The simultaneous symbiosis of leguminous plants with two root mutualists, endophytic fungi and rhizobia is common in nature, yet how two mutualists interact and co-exist before infecting plants and the concomitant effects on nodulation are less understood. Using a combination of metabolic analysis, fungal deletion mutants and comparative transcriptomics, we demonstrated that Bradyrhizobium and a facultatively biotrophic fungus, Phomopsis liquidambaris, interacted to stimulate fungal flavonoid production, and thereby primed Bradyrhizobial nodulation signaling, enhancing Bradyrhizobial responses to root exudates and leading to early nodulation of peanut (Arachis hypogaea), and such effects were compromised when disturbing fungal flavonoid biosynthesis. Stress sensitivity assays and reactive oxygen species (ROS) determination revealed that flavonoid production acted as a strategy to alleviate hyphal oxidative stress during P. liquidambaris-Bradyrhizobial interactions. By investigating the interactions between P. liquidambaris and a collection of 38 rhizobacteria, from distinct bacterial genera, we additionally showed that the flavonoid-ROS module contributed to the maintenance of fungal and bacterial co-existence, and fungal niche colonization under soil conditions. Our results demonstrate for the first time that rhizobial nodulation signaling can be primed by fungi before symbiosis with host plants and highlight the importance of flavonoid in tripartite interactions between legumes, beneficial fungi and rhizobia.

Nodulating white lupins take advantage of the reciprocal interplay between N and P nutritional responses.

The low bioavailability of nutrients, especially nitrogen (N) and phosphorus (P), is one of the most limiting factors for crop production. In this study, under N- and P-free nutrient solution (-N-P), nodulating white lupin plants developed some nodules and analogous cluster root structures characterized by different morphological, physiological, and molecular responses than those observed upon single nutrient deficiency (strong acidification of external media, a better nutritional status than -N+P and +N-P plants). The multi-elemental analysis highlighted that the concentrations of nutrients in white lupin plants were mainly affected by P availability. Gene-expression analyses provided evidence of interconnections between N and P nutritional pathways that are active to promote N and P balance in plants. The root exudome was mainly characterized by N availability in nutrient solution, and, in particular, the absence of N and P in the nutrient solution triggered a high release of phenolic compounds, nucleosides monophosphate and saponines by roots. These morphological, physiological, and molecular responses result from a close interplay between N and P nutritional pathways. They contribute to the good development of nodulating white lupin plants when grown on N- and P-free media. This study provides evidence that limited N and P availability in the nutrient solution can promote white lupin-Bradyrhizobium symbiosis, which is favourable for the sustainability of legume production.

Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume.

Bradyrhizobium is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two Bradyrhizobium strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. Bradyrhizobium localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of Bradyrhizobium biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. IMPORTANCE Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.

Comparative Analysis of Three Bradyrhizobium diazoefficiens Genomes Show Specific Mutations Acquired during Selection for a Higher Motility Phenotype and Adaption to Laboratory Conditions.

Microbial genomes are being extensively studied using next-generation sequencing technologies in order to understand the changes that occur under different selection regimes. In this work, the number and type of mutations that have occurred in three Bradyrhizobium diazoefficiens USDA 110T strains under laboratory conditions and during selection for a more motile phenotypic variant were analyzed. Most of the mutations found in both processes consisted of single nucleotide polymorphisms, single nucleotide deletions or insertions. In the case of adaptation to laboratory conditions, half of the changes occurred within intergenic regions, and around 80% were insertions. When the more motile phenotypic variant was evaluated, eight single nucleotide polymorphisms and an 11-bp deletion were found, although none of them was directly related to known motility or chemotaxis genes. Two mutants were constructed to evaluate the 11-bp deletion affecting the alpha subunit of 2-oxoacid:acceptor oxidoreductase (AAV28_RS30705-blr6743). The results showed that this single deletion was not responsible for the enhanced motility phenotype. IMPORTANCE The genetic and genomic changes that occur under laboratory conditions in Bradyrhizobium diazoefficiens genomes remain poorly studied. Only a few genome sequences of this important nitrogen-fixing species are available, and there are no genome-wide comparative analyses of related strains. In the present work, we sequenced and compared the genomes of strains derived from a parent strain, B. diazoefficiens USDA 110, that has undergone processes of repeated culture in the laboratory environment, or phenotypic selection toward antibiotic resistance and enhanced motility. Our results represent the first analysis in B. diazoefficiens that provides insights into the specific mutations that are acquired during these processes.

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

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

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.

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.

Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) associated with legumes native to Canada possess rearranged symbiosis genes and numerous insertion sequences.

Six bacterial strains isolated from root nodules of soybean plants that had been inoculated with root-zone soil of legumes native to Canada were previously characterized and 1) placed in two novel lineages within the genus Bradyrhizobium and 2) assigned to symbiovar septentrionale. Here we verified the taxonomic status of these strains using genomic and phenotypic analyses. Phylogenetic analyses of five protein encoding partial gene sequences as well as 52 full length ribosome protein subunit gene sequences confirmed placement of the novel strains in two highly supported lineages distinct from named Bradyrhizobium species. The highest average nucleotide identity values of strains representing these two lineages relative to type strains of closest relatives were 90.7 and 92.3% which is well below the threshold value for bacterial species circumscription. The genomes of representative strains 1S1T, 162S2 and 66S1MBT have sizes of 10598256, 10733150 and 9032145 bp with DNA G+C contents of 63.5, 63.4 and 63.8 mol%, respectively. These strains possess between one and three plasmids based on copy number of plasmid replication and segregation (repABC) genes. Novel strains also possess numerous insertion sequences, and, relative to reference strain Bradyrhizobium diazoefficiens USDA110T, exhibit inversion and fragmentation of nodulation (nod) and nitrogen-fixation (nif) gene clusters. Phylogenetic analyses of nodC and nifH gene sequences confirmed placement of novel strains in a distinct lineage corresponding to symbiovar septentrionale. Data for morphological, physiological and symbiotic characteristics complement the sequence-based results. The data presented here support the description of two new species for which the names Bradyrhizobium septentrionale sp. nov. (sv. septentrionale) and Bradyrhizobium quebecense sp. nov. (sv. septentrionale) are proposed, with 1S1T (=LMG 29930T=HAMBI 3676T) and 66S1MBT (=LMG 31547T=HAMBI 3720T) as type strains, respectively.

Evolution of specialization in a plant-microbial mutualism is explained by the oscillation theory of speciation.

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

Identification of the metabolites regulated in soybean-Rhizobia symbiosis through solid phase microextraction coupled with LC-MS.

Legumes provide one of the uniquely nutrient-rich food sources to the population and are one of the primary field crops that play significant roles in agricultural sustainability. Inoculation with Bradyrhizobium japonicum is necessary for the high yield of leguminous crops, i.e. soybean. Nodulation of soybean by Bradyrhizobium japonicum is a complex process that is essential for cultivation of these legumes and external stress factors, such as draught and soil acidity, that influence the nodulation and crop yield. Alterations in the nodule metabolites are known to identify the type of stress that mitigates nodulation and lowers crop yield. Current techniques aimed at understanding the metabolic activities in the symbiont, such as in the case of metabolic regulations in varying nodule growth phases, rely on exhaustive techniques based on the removal of nodules or other plant tissue. Aiming to capture a more in-depth, accurate profile of this system without quenching the metabolic activity in the nodules, or removing the nodules, a workflow was prepared for the metabolite sampling through in vivo solid phase microextraction in thin film format (TF-SPME). This technique was followed by LC-QTOF-MS instrumental analysis with subsequent metabolite annotation and reference standard validation. Our approach is unique in terms of eliminating the effects that arise due to analyte partition coefficients. We show that the symbiont undergoes metabolic regulations throughout the cultivation period, displaying the efficacy of TF-SPME as a non-exhaustive sampling method that can be used as a tool to investigate the metabolic alterations in nodules. These alterations would potentially fingerprint the environmental effects on soybean yield.

Glycine max NNL1 restricts symbiotic compatibility with widely distributed bradyrhizobia via root hair infection.

Symbiosis between soybean (Glycine max) and rhizobia is essential for efficient nitrogen fixation. Rhizobial effectors secreted through the type-III secretion system are key for mediating the interactions between plants and rhizobia, but the molecular mechanism remains largely unknown. Here, our genome-wide association study for nodule number identified G. max Nodule Number Locus 1 (GmNNL1), which encodes a new R protein. GmNNL1 directly interacts with the nodulation outer protein P (NopP) effector from Bradyrhizobium USDA110 to trigger immunity and inhibit nodulation through root hair infection. The insertion of a 179 bp short interspersed nuclear element (SINE)-like transposon into GmNNL1 leads to the loss of function of GmNNL1, enabling bradyrhizobia to successfully nodulate soybeans through the root hair infection route and enhancing nitrogen fixation. Our findings provide important insights into the coevolution of soybean-bradyrhizobia compatibility and offer a way to design new legume-rhizobia interactions for efficient symbiotic nitrogen fixation.

Soybean tolerance to drought depends on the associated Bradyrhizobium strain.

We evaluated the effect of three different Bradyrhizobium strains inoculated in two soybean genotypes (R01-581F, drought-tolerant, and NA5858RR, drought-sensitive) submitted to drought in two trials conducted simultaneously under greenhouse. The strains (SEMIA 587, SEMIA 5019 (both B. elkanii), and SEMIA 5080 (B. diazoefficiens)) were inoculated individually in each genotype and then submitted to water restriction (or kept well-watered, control) between 45 and 62 days after emergence. No deep changes in plant physiological variables were observed under the moderate water restriction imposed during the first 10 days. Nevertheless, photosynthesis and transpiration decreased after the severe water restriction imposed for further 7 days. Water restriction reduced growth (- 30%) and the number of nodules (- 47% and - 58% for R01-581F and NA5858RR, respectively) of both genotypes, with a negative effect on N-metabolism. The genotype R01-581F inoculated with SEMIA 5019 strain had higher photosynthetic rates compared with NA5858RR, regardless of the Bradyrhizobium strain. On average, R01-581F showed better performance under drought than NA5858RR, with higher number of nodules (51 vs. 38 nodules per plant, respectively) and less accumulation of ureides in petioles (15 µmol g-1 vs. 34 µmol g-1, respectively). Moreover, plants inoculated with SEMIA 5080 had higher glutamine synthetase activity under severe water restriction, especially in the drought-tolerant R01-518F, suggesting maintenance of N metabolism under drought. The Bradyrhizobium strain affects the host plant responses to drought in which the strain SEMIA 5080 improves the drought tolerance of R01-518F genotype.

Bradyrhizobium sp. strain ORS278 promotes rice growth and its quorum sensing system is required for optimal root colonization.

Many Gram-negative bacteria communicate by using homoserine lactones (HSLs) as quorum sensing (QS) signals in a cell density-dependent manner. In addition to fatty acyl-HSL (acyl-HSL) signals, certain strains, most of them associated with plants, produce non-canonical aryl-HSLs such as cinnamoyl-HSL. However, the role of aryl-HSL in endophytic associations remained elusive. Bradyrhizobium sp. strain ORS278 possesses a LuxI-LuxR type QS system and produces cinnamoyl-HSL as a QS signal. Here, we report that strain ORS278 promotes growth of domesticated rice (Oryza sativa). QS mutants unable to produce cinnamoyl-HSL exhibited reduced plant-growth promoting activity in comparison to the parent strain ORS278. Likewise, the QS mutants were impaired in their ability to colonize rice roots. These findings suggest that genes controlled by cinnamoyl-HSL play an important role in the association between rice and ORS278. However, biofilm production was not visibly altered in these mutants. In conclusion, our study highlights the importance of aryl-HSLs in endophytic plant-bacteria interactions.