Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

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.

The legume-rhizobia symbiosis can be supported on Mars soil simulants.

Legumes (soybeans, peas, lentils, etc.) play important roles in agriculture on Earth because of their food value and their ability to form a mutualistic beneficial association with rhizobia bacteria. In this association, the host plant benefits from atmospheric nitrogen fixation by rhizobia. The presence of nitrogen in the Mars atmosphere offers the possibility to take advantage of this important plant-microbe association. While some studies have shown that Mars soil simulants can support plant growth, none have investigated if these soils can support the legume-rhizobia symbiosis. In this study, we investigated the establishment of the legume-rhizobia symbiosis on different Mars soil simulants (different grades of the Mojave Mars Simulant (MMS)-1: Coarse, Fine, Unsorted, Superfine, and the MMS-2 simulant). We used the model legume, Medicago truncatula, and its symbiotic partners, Sinorhizobium meliloti and Sinorhizobium medicae, in these experiments. Our results show that root nodules could develop on M. truncatula roots when grown on these Mars soil simulants and were comparable to those formed on plants that were grown on sand. We also detected nifH (a reporter gene for nitrogen fixation) expression inside these nodules. Our results indicate that the different Mars soil simulants used in this study can support legume-rhizobia symbiosis. While the average number of lateral roots and nodule numbers were comparable on plants grown on the different soil simulants, total plant mass was higher in plants grown on MMS-2 soil than on MMS-1 soil and its variants. Our results imply that the chemical composition of the simulants is more critical than their grain size for plant mass. Based on these results, we recommend that the MMS-2 Superfine soil simulant is a better fit than the MMS-1 soil and it's variants for future studies. Our findings can serve as an excellent resource for future studies investigating beneficial plant-microbe associations for sustainable agriculture on Mars.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

Soil fertility interactions with Sinorhizobium-legume symbiosis in a simulated Martian regolith; effects on nitrogen content and plant health.

Due to increasing population growth and declining arable land on Earth, astroagriculture will be vital to terraform Martian regolith for settlement. Nodulating plants and their N-fixing symbionts may play a role in increasing Martian soil fertility. On Earth, clover (Melilotus officinalis) forms a symbiotic relationship with the N-fixing bacteria Sinorhizobium meliloti; clover has been previously grown in simulated regolith yet without bacterial inoculation. In this study, we inoculated clover with S. meliloti grown in potting soil and regolith to test the hypothesis that plants grown in regolith can form the same symbiotic associations as in soils and to determine if greater plant biomass occurs in the presence of S. meliloti regardless of growth media. We also examined soil NH4 concentrations to evaluate soil augmentation properties of nodulating plants and symbionts. Greater biomass occurred in inoculated compared to uninoculated groups; the inoculated average biomass in potting mix and regolith (2.23 and 0.29 g, respectively) was greater than the uninoculated group (0.11 and 0.01 g, respectively). However, no significant differences existed in NH4 composition between potting mix and regolith simulant. Linear regression analysis results showed that: i) symbiotic plant-bacteria relationships differed between regolith and potting mix, with plant biomass positively correlated to regolith-bacteria interactions; and, ii) NH4 production was limited to plant uptake yet the relationships in regolith and potting mix were similar. It is promising that plant-legume symbiosis is a possibility for Martian soil colonization.

Decreased coevolutionary potential and increased symbiont fecundity during the biological invasion of a legume-rhizobium mutualism.

Although most invasive species engage in mutualism, we know little about how mutualism evolves as partners colonize novel environments. Selection on cooperation and standing genetic variation for mutualism traits may differ between a mutualism's invaded and native ranges, which could alter cooperation and coevolutionary dynamics. To test for such differences, we compare mutualism traits between invaded- and native-range host-symbiont genotype combinations of the weedy legume, Medicago polymorpha, and its nitrogen-fixing rhizobium symbiont, Ensifer medicae, which have coinvaded North America. We find that mutualism benefits for plants are indistinguishable between invaded- and native-range symbioses. However, rhizobia gain greater fitness from invaded-range mutualisms than from native-range mutualisms, and this enhancement of symbiont fecundity could increase the mutualism's spread by increasing symbiont availability during plant colonization. Furthermore, mutualism traits in invaded-range symbioses show lower genetic variance and a simpler partitioning of genetic variance between host and symbiont sources, compared to native-range symbioses. This suggests that biological invasion has reduced mutualists' potential to respond to coevolutionary selection. Additionally, rhizobia bearing a locus (hrrP) that can enhance symbiotic fitness have more exploitative phenotypes in invaded-range than in native-range symbioses. These findings highlight the impacts of biological invasion on the evolution of mutualistic interactions.

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

Dual inoculation of alfalfa (Medicago sativa L.) with Funnelliformis mosseae and Sinorhizobium medicae can reduce Fusarium wilt.

AIMS: This study was designed to evaluate the biocontrol of the arbuscular mycorrhizal fungus (AMF) Funnelliformis mosseae and the rhizobium Sinorhizobium medicae on alfalfa (Medicago sativa) wilt caused by Fusarium oxysporum, a severe soil-borne fungal pathogen. METHODS AND RESULTS: The effects of co-inoculation of F. mosseae and S. medicae on alfalfa growth, nitrogen, phosphorus uptake and wilt caused by F. oxysporum were tested. Plant defence-related chemicals were measured to reveal the biochemical mechanism by which alfalfa responds to pathogen infection and how it is regulated by AMF and rhizobium. Pathogen infection caused typical yellowing of alfalfa leaflets and significantly reduced plant AMF colonization. AMF or rhizobium alone and the co-inoculation reduced the plant disease index by 83·2, 48·4 and 81·8% respectively. Inoculation with AMF or rhizobium alone increased the dry weight of alfalfa by more than 13 and 3 times respectively; it also increased plant chlorophyll content by 65·6 and 16·6% respectively. Co-inoculation of AMF and rhizobium induced the plant to accumulate more disease-related antioxidant enzymes, plant hydrolase and plant hormones, such as superoxide dismutase, ß-1,3-glucanase, chitinase, and phenylalanine ammonialyase, abscisic acid, ethylene and H2 O2 , under pathogen stress. CONCLUSIONS: Co-inoculation with F. mosseae and S. medicae offered complementarily improved alfalfa nutrient uptake and growth, which increased plant health. The co-inoculation of AMF and rhizobium regulated plant physiological and biochemical processes and induced plants to produce defence-related compounds, thus decreasing the severity of disease. The simultaneous application of F. mosseae and S. medicae is a potential biocontrol strategy to increase the systemic defence responses of alfalfa to Fusarium wilt. SIGNIFICANCE AND IMPACT OF THE STUDY: This research showed that complex plant-pathogen interactions are affected by rhizobium and AMF, providing insight into plant-microbiome interactions in the rhizosphere as well as the application of the microbiome in agriculture production.

Genomic insight into the origins and evolution of symbiosis genes in Phaseolus vulgaris microsymbionts.

BACKGROUND: Phaseolus vulgaris (common bean) microsymbionts belonging to the bacterial genera Rhizobium, Bradyrhizobium, and Ensifer (Sinorhizobium) have been isolated across the globe. Individual symbiosis genes (e.g., nodC) of these rhizobia can be different within each genus and among distinct genera. Little information is available about the symbiotic structure of indigenous Rhizobium strains nodulating introduced bean plants or the emergence of a symbiotic ability to associate with bean plants in Bradyrhizobium and Ensifer strains. Here, we sequenced the genomes of 29 representative bean microsymbionts (21 Rhizobium, four Ensifer, and four Bradyrhizobium) and compared them with closely related reference strains to estimate the origins of symbiosis genes among these Chinese bean microsymbionts. RESULTS: Comparative genomics demonstrated horizontal gene transfer exclusively at the plasmid level, leading to expanded diversity of bean-nodulating Rhizobium strains. Analysis of vertically transferred genes uncovered 191 (out of the 2654) single-copy core genes with phylogenies strictly consistent with the taxonomic status of bacterial species, but none were found on symbiosis plasmids. A common symbiotic region was wholly conserved within the Rhizobium genus yet different from those of the other two genera. A single strain of Ensifer and two Bradyrhizobium strains shared similar gene content with soybean microsymbionts in both chromosomes and symbiotic regions. CONCLUSIONS: The 19 native bean Rhizobium microsymbionts were assigned to four defined species and six putative novel species. The symbiosis genes of R. phaseoli, R. sophoriradicis, and R. esperanzae strains that originated from Mexican bean-nodulating strains were possibly introduced alongside bean seeds. R. anhuiense strains displayed distinct host ranges, indicating transition into bean microsymbionts. Among the six putative novel species exclusive to China, horizontal transfer of symbiosis genes suggested symbiosis with other indigenous legumes and loss of originally symbiotic regions or non-symbionts before the introduction of common bean into China. Genome data for Ensifer and Bradyrhizobium strains indicated symbiotic compatibility between microsymbionts of common bean and other hosts such as soybean.

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

Medicago-Sinorhizobium-Ralstonia Co-infection Reveals Legume Nodules as Pathogen Confined Infection Sites Developing Weak Defenses.

Legumes have the capacity to develop root nodules hosting nitrogen-fixing bacteria, called rhizobia. For the plant, the benefit of the symbiosis is important in nitrogen-deprived conditions, but it requires hosting and feeding massive numbers of rhizobia. Recent studies suggest that innate immunity is reduced or suppressed within nodules [1-10]; this likely maintains viable rhizobial populations. To evaluate the potential consequences and risks associated with an altered immuni`ty in the symbiotic organ, we developed a tripartite system with the model legume Medicago truncatula [11, 12], its nodulating symbiont of the genus Sinorhizobium (syn. Ensifer) [13, 14], and the pathogenic soil-borne bacterium Ralstonia solanacearum [15-18]. We show that nodules are frequent infection sites where pathogen multiplication is comparable to that in the root tips and independent of nodule ability to fix nitrogen. Transcriptomic analyses indicate that, despite the presence of the hosted rhizobia, nodules are able to develop weak defense reactions against pathogenic R. solanacearum. Nodule defense response displays specificity compared to that activated in roots. In agreement with nodule innate immunity, optimal R. solanacearum growth requires pathogen virulence factors. Finally, our data indicate that the high susceptibility of nodules is counterbalanced by the existence of a diffusion barrier preventing pathogen spreading from nodules to the rest of the plant.

Inoculation with Efficient Nitrogen Fixing and Indoleacetic Acid Producing Bacterial Microsymbiont Enhance Tolerance of the Model Legume Medicago truncatula to Iron Deficiency.

The aim of this study was to assess the effect of symbiotic bacteria inoculation on the response of Medicago truncatula genotypes to iron deficiency. The present work was conducted on three Medicago truncatula genotypes: A17, TN8.20, and TN1.11. Three treatments were performed: control (C), direct Fe deficiency (DD), and induced Fe deficiency by bicarbonate (ID). Plants were nitrogen-fertilized (T) or inoculated with two bacterial strains: Sinorhizobium meliloti TII7 and Sinorhizobium medicae SII4. Biometric, physiological, and biochemical parameters were analyzed. Iron deficiency had a significant lowering effect on plant biomass and chlorophyll content in all Medicago truncatula genotypes. TN1.11 showed the highest lipid peroxidation and leakage of electrolyte under iron deficiency conditions, which suggest that TN1.11 was more affected than A17 and TN8.20 by Fe starvation. Iron deficiency affected symbiotic performance indices of all Medicago truncatula genotypes inoculated with both Sinorhizobium strains, mainly nodules number and biomass as well as nitrogen-fixing capacity. Nevertheless, inoculation with Sinorhizobium strains mitigates the negative effect of Fe deficiency on plant growth and oxidative stress compared to nitrogen-fertilized plants. The highest auxin producing strain, TII7, preserves relatively high growth and root biomass and length when inoculated to TN8.20 and A17. On the other hand, both TII7 and SII4 strains improve the performance of sensitive genotype TN1.11 through reduction of the negative effect of iron deficiency on chlorophyll and plant Fe content. The bacterial inoculation improved Fe-deficient plant response to oxidative stress via the induction of the activities of antioxidant enzymes.

The Multiple Faces of the Medicago-Sinorhizobium Symbiosis.

Medicago truncatula is able to perform a symbiotic association with Sinorhizobium spp. This interaction leads to the formation of a new root organ, the nodule, in which bacteria infect the host cells and fix atmospheric nitrogen for the plant benefit. Multiple and complex processes are essential for the success of this interaction from the recognition phase to nodule formation and functioning, and a wide range of plant host genes is required to orchestrate this phenomenon. Thanks to direct and reverse genetic as well as transcriptomic approaches, numerous genes involved in this symbiosis have been described and improve our understanding of this fantastic association. Herein we propose to update the recent molecular knowledge of how M. truncatula associates to its symbiotic partner Sinorhizobium spp.

Control of the ethylene signaling pathway prevents plant defenses during intracellular accommodation of the rhizobia.

Massive intracellular populations of symbiotic bacteria, referred to as rhizobia, are housed in legume root nodules. Little is known about the mechanisms preventing the development of defense in these organs although genes such as SymCRK and DNF2 of the model legume Medicago truncatula are required for this control after rhizobial internalization in host nodule cells. Here we investigated the molecular basis of the symbiotic control of immunity. Proteomic analysis was performed to compare functional (wild-type) and defending nodules (symCRK). Based on the results, the control of plant immunity during the functional step of the symbiosis was further investigated by biochemical and pharmacological approaches as well as by transcript and histology analysis. Ethylene was identified as a potential signal inducing plant defenses in symCRK nodules. Involvement of this phytohormone in symCRK and dnf2-developed defenses and in the death of intracellular rhizobia was confirmed. This negative effect of ethylene depended on the M. truncatula sickle gene and was also observed in the legume Lotus japonicus. Together, these data indicate that prevention of ethylene-triggered defenses is crucial for the persistence of endosymbiosis and that the DNF2 and SymCRK genes are required for this process.

Adaptive evolution of rhizobial symbiotic compatibility mediated by co-evolved insertion sequences.

Mutualism between bacteria and eukaryotes has essential roles in the history of life, but the evolution of their compatibility is poorly understood. Here we show that different Sinorhizobium strains can form either nitrogen-fixing nodules or uninfected pseudonodules on certain cultivated soybeans, while being all effective microsymbionts of some wild soybeans. However, a few well-infected nodules can be found on a commercial soybean using inocula containing a mixed pool of Tn5 insertion mutants derived from an incompatible strain. Reverse genetics and genome sequencing of compatible mutants demonstrated that inactivation of T3SS (type three secretion system) accounted for this phenotypic change. These mutations in the T3SS gene cluster were dominated by parallel transpositions of insertion sequences (ISs) other than the introduced Tn5. This genetic and phenotypic change can also be achieved in an experimental evolution scenario on a laboratory time scale using incompatible wild-type strains as inocula. The ISs acting in the adaptive evolution of Sinorhizobium strains exhibit broader phyletic and replicon distributions than other ISs, and prefer target sequences of low GC% content, a characteristic feature of symbiosis plasmid where T3SS genes are located. These findings suggest an important role of co-evolved ISs in the adaptive evolution of rhizobial compatibility.

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance.

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342mM NaCl as well as 127 or 99mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44°C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.

Retrieved 16S rRNA and nifH sequences reveal co-dominance of Bradyrhizobium and Ensifer (Sinorhizobium) strains in field-collected root nodules of the promiscuous host Vigna radiata (L.) R. Wilczek.

In the present study, the relative distribution of endophytic rhizobia in field-collected root nodules of the promiscuous host mung bean was investigated by sequencing of 16S ribosomal RNA (rRNA) and nifH genes, amplified directly from the nodule DNA. Co-dominance of the genera Bradyrhizobium and Ensifer was indicated by 32.05 and 35.84% of the total retrieved 16S rRNA sequences, respectively, and the sequences of genera Mesorhizobium and Rhizobium comprised only 0.06 and 2.06% of the recovered sequences, respectively. Sequences amplified from rhizosphere soil DNA indicated that only a minor fraction originated from Bradyrhizobium and Ensifer strains, comprising about 0.46 and 0.67% of the total retrieved sequences, respectively. 16S rRNA gene sequencing has also identified the presence of several non-rhizobial endophytes from phyla Proteobacteria, Actinobacteria, Bacteroides, and Firmicutes. The nifH sequences obtained from nodules also confirmed the co-dominance of Bradyrhizobium (39.21%) and Ensifer (59.23%) strains. The nifH sequences of the genus Rhizobium were absent, and those of genus Mesorhizobium comprised only a minor fraction of the sequences recovered from the nodules and rhizosphere soil samples. Two bacterial isolates, identified by 16S rRNA gene sequence analysis as Bradyrhizobium strain Vr51 and Ensifer strain Vr38, successfully nodulated the original host (mung bean) plants. Co-dominance of Bradyrhizobium and Ensifer strains in the nodules of mung bean indicates the potential role of the host plant in selecting specific endophytic rhizobial populations. Furthermore, successful nodulation of mung bean by the isolates showed that strains of both the genera Bradyrhizobium and Ensifer can be used for production of inoculum.

Management of the soybean cyst nematode Heterodera glycines with combinations of different rhizobacterial strains on soybean.

Soybean cyst nematode (SCN) is the most damaging soybean pest worldwide. To improve soybean resistance to SCN, we employed a soybean seed-coating strategy through combination of three rhizobacterial strains, including Bacillus simple, B. megaterium and Sinarhizobium fredii at various ratios. We found seed coating by such rhizobacterial strains at a ratio of 3:1:1 (thereafter called SN101) produced the highest germination rate and the mortality of J2 of nematodes. Then, the role of soybean seed coating by SN101 in nematode control was evaluated under both greenhouse and two field conditions in Northeast China in 2013 and 2014. Our results showed that SN101 treatment greatly reduced SCN reproduction and significantly promoted plant growth and yield production in both greenhouse and field trials, suggesting that SN101 is a promising seed-coating agent that may be used as an alternative bio-nematicide for controlling SCN in soybean fields. Our findings also demonstrate that combination of multiple rhizobacterial strains needs to be considered in the seed coating for better management of plant nematodes.

Specific Host-Responsive Associations Between Medicago truncatula Accessions and Sinorhizobium Strains.

Legume plants interact with rhizobia to form nitrogen-fixing root nodules. Legume-rhizobium interactions are specific and only compatible rhizobia and plant species will lead to nodule formation. Even within compatible interactions, the genotype of both the plant and the bacterial symbiont will impact on the efficiency of nodule functioning and nitrogen-fixation activity. The model legume Medicago truncatula forms nodules with several species of the Sinorhizobium genus. However, the efficiency of these bacterial strains is highly variable. In this study, we compared the symbiotic efficiency of Sinorhizobium meliloti strains Sm1021, 102F34, and FSM-MA, and Sinorhizobium medicae strain WSM419 on the two widely used M. truncatula accessions A17 and R108. The efficiency of the interactions was determined by multiple parameters. We found a high effectiveness of the FSM-MA strain with both M. truncatula accessions. In contrast, specific highly efficient interactions were obtained for the A17-WSM419 and R108-102F34 combinations. Remarkably, the widely used Sm1021 strain performed weakly on both hosts. We showed that Sm1021 efficiently induced nodule organogenesis but cannot fully activate the differentiation of the symbiotic nodule cells, explaining its weaker performance. These results will be informative for the selection of appropriate rhizobium strains in functional studies on symbiosis using these M. truncatula accessions, particularly for research focusing on late stages of the nodulation process.

Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression.

The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.