Combined effects of intermittent radio frequency heating with cinnamon oil vapor on microbial control and quality changes of alfalfa seeds.

Alfalfa sprouts have high nutritional values when consumed raw, but are easily contaminated by food pathogens. This study aimed to evaluate effects of essential oil (EO) vapors, RF heating and their combinations on microbial inactivation, germination rate and generated sprouts' quality of alfalfa seeds at moisture contents of 7.52%, 9.53% and 11.45% wet basis (w.b.). Results showed that cinnamon oil and oregano oil vapors both had antimicrobial effects against Salmonella on alfalfa seeds and cinnamon oil vapor had a little better activity. Weibull model well fitted the Salmonella inactivation curves under RF heating. The rate of Salmonella inactivation increased but germination rate decreased with increasing temperature and moisture content of seeds. The intermittent RF treatments improved the heating uniformity and germination rate of the whole batch of seeds as compared to the continuous RF heating. The combination of intermittent RF heating and cinnamon oil vapor exhibited additive antimicrobial effects, up to 3.99-4.12 log CFU/g Salmonella reductions, and maintained the germination rate above 90%. However, for natural microbial decontamination, combined treatments only caused 0.56-0.82 log CFU/g reductions of total bacterial counts. The fresh weight, length, flavor, the content of phenolics and ascorbic acid, and antioxidant capacity of generated sprouts were not significantly impacted. The study provided an effective method for microbial control on sprouting seeds.

Sustainable effect of a symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti on nodulation and photosynthetic traits of four leguminous plants under low moisture stress environment.

Sustainable effect of a nitrogen-fixing bacterium Sinorhizobium meliloti on nodulation and photosynthetic traits (phenomenological fluxes) in four leguminous plants species under low moisture stress (20-25% soil moisture content) environment was studied. Sinorhizobium meliloti was isolated from fenugreek (Trigonella foenum-graecum) root nodules, and later, it was cultured and purified. Nodulation and photosynthetic ability in the presence of S. meliloti were tested in four leguminous plant species, that is, kidney bean (cv. lobia-2000), black bean (cv. NM-97), mung bean (cv. NM-2006) and chickpea (cv. Pb-2008). Plants of each species were grown in sterilized soil that was previously treated with 25 ml suspension containing S. meliloti at 41 × 106  CFU ml-1  kg-1 pot. One-month-old plants were subjected to low soil moisture stress conditions for 15 days, and soil moisture contents were maintained to 20-25% throughout the experimental period. The ability to fix nitrogen, nodule formation, and their subsequent effect on phenomenological fluxes in low moisture treated legumes were studied.

Biochemical mechanisms of rhizospheric Bacillus subtilis-facilitated phytoextraction by alfalfa under cadmium stress - Microbial diversity and metabolomics analyses.

The effects of Bacillus subtilis inoculation on the growth and Cd uptake of alfalfa were evaluated in this research using pot experiments, and the relevant biochemical mechanisms were first investigated by combined microbial diversity and nontarget metabolomics analyses. The results indicated that inoculation with alfalfa significantly decreased the amount of plant malondialdehyde (MDA) and improved the activities of plant antioxidant enzymes and soil nutrient cycling-involved enzymes, thereby promoting biomass by 29.4%. Inoculation also increased Cd bioavailability in rhizosphere soil by 12.0% and Cd removal efficiency by 139.3%. The biochemical mechanisms included enhanced bacterial diversity, transformed microbial community composition, regulated amounts of amino acids, fatty acids, carbohydrates, flavonoids and phenols in rhizosphere soil metabolites, and modulations of the corresponding Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These responses were beneficial to microbial activity, nutrient cycling, and Cd mobilization, detoxification, and decontamination by alfalfa in soil. This study, especially the newly identified differential metabolites and metabolic pathways, provides new insights into mechanism revelation and strategy development in microbe-assisted phytomanagement of heavy metal-contaminated soils.

Arbuscular mycorrhizal fungus alleviates alfalfa leaf spots caused by Phoma medicaginis revealed by RNA-seq analysis.

AIMS: One of the major limitations to the production of alfalfa (Medicago sativa) is the fungus Phoma medicaginis, which infects alfalfa and causes leaf spots. This study aims to understand alfalfa's response to P. medicaginis infection, the colonization of arbuscular mycorrhizal fungus (AMF) and the effect of AMF on plant-pathogen interactions. METHODS AND RESULTS: Transcriptome analysis (RNA-seq) was used to identify differentially expressed genes (DEGs) in alfalfa infected by P. medicaginis and colonized by AMF Rhizophagus intraradices. AMF ameliorated the effects of P. medicaginis infection on alfalfa by reducing leaf spot incidence and disease index by 39·48 and 56·18% respectively. Inoculation with pathogen and AMF induced the activity of defence pathways, including peroxidase (POD), polyphenol oxidase activities and jasmonic acid (JA), salicylic acid concentration. Plants showed differential expression of P. medicaginis resistance-related genes, including genes belonging to pathogenesis-related (PR) proteins, chitinase activity, flavonoid biosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, phenylalanine metabolism and photosynthesis. Inoculation with AMF led to changes in the expression of genes involved in PR proteins, chitinase activity, phenylalanine metabolism and photosynthesis. CONCLUSION: The physiological and transcriptional changes caused by P. medicaginis infection in non-mycorrhizal and mycorrhizal alfalfa provides crucial information for understanding AMF's association with pathogenic systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that AMF alleviated alfalfa leaf spots demonstrating that AMF can serve as a biocontrol strategy for alfalfa disease management.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level.

BACKGROUND: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness. RESULTS: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants. CONCLUSIONS: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

Influences of arbuscular mycorrhizae, phosphorus fertiliser and biochar on alfalfa growth, nutrient status and cadmium uptake.

The objective of the study was to explore the influences of arbuscular mycorrhizae (AM), phosphorus (P) fertiliser, biochar application (BC) and their interactions on Medicago sativa growth, nutrient, Cd content and AM fungi-plant symbioses. Applications of both P fertiliser and BC significantly increased total biomass and P and potassium (K) uptake, regardless of AM. When no P fertiliser or BC was used, the shoot biomass and nitrogen (N), P, and K contents in the +AM treatments were 1.39, 1.54, 4.53 and 2.06 times higher than those in the -AM treatments, respectively. AM fungi only elevated the total P uptake by 44.03% when P fertiliser was applied at a rate of 30 mg P kg-1 in the absence of BC addition. With BC application or high-P fertiliser input (100 mg P kg-1), the soil available P was significantly higher than that in the other treatments, and AM fungi significantly reduced the shoot biomass. The minimum Cd concentration occurred in the shoots of alfalfas treated with BC and high-P fertiliser inputs; this concentration was lower than the maximum permitted concentration in China. Although the BC and high-P inputs could eliminate the positive mycorrhizal response, the results suggested that BC application in combination with high-P fertiliser input could not only increase forage yields but also lower Cd concentrations to meet the forage safety standards by the dilution effect.

Arbuscular Mycorrhizal Symbiosis Mitigates Iron (Fe)-Deficiency Retardation in Alfalfa (Medicago sativa L.) Through the Enhancement of Fe Accumulation and Sulfur-Assisted Antioxidant Defense.

Iron (Fe)-deficiency is one of the major constraints affecting growth, yield and nutritional quality in plants. This study was performed to elucidate how arbuscular mycorrhizal fungi (AMF) alleviate Fe-deficiency retardation in alfalfa (Medicago sativa L.). AMF supplementation improved plant biomass, chlorophyll score, Fv/Fm (quantum efficiency of photosystem II), and Pi_ABS (photosynthesis performance index), and reduced cell death, electrolyte leakage, and hydrogen peroxide accumulation in alfalfa. Moreover, AMF enhanced ferric chelate reductase activity as well as Fe, Zn, S and P in alfalfa under Fe-deficiency. Although Fe-transporters (MsIRT1 and MsNramp1) did not induce in root but MsFRO1 significantly induced by AMF under Fe deficiency in roots, suggesting that AMF-mediated Fe enhancement is related to the bioavailability of Fe at rhizosphere/root apoplast rather than the upregulation of Fe transporters under Fe deficiency in alfalfa. Several S-transporters (MsSULTR1;1, MsSULTR1;2, MsSULTR1;3, and MsSULTR3;1) markedly increased following AMF supplementation with or without Fe-deficiency alfalfa. Our study further suggests that Fe uptake system is independently influenced by AMF regardless of the S status in alfalfa. However, the increase of S in alfalfa is correlated with the elevation of GR and S-metabolites (glutathione and cysteine) associated with antioxidant defense under Fe deficiency.

Phenotypically and Genotypically Heterogeneous Strains of Pseudomonas syringae Associated With Alfalfa Leaf Spot Disease in Iran.

In this study, we provide a polyphasic characterization of 18 Pseudomonas spp. strains associated with alfalfa leaf spot symptoms in Iran. All of the strains were pathogenic on alfalfa, although the aggressiveness and symptomology varied among the strains. All strains but one were pathogenic on broad bean, cucumber, honeydew, and zucchini, whereas only a fraction of the strains were pathogenic on sugar beet, tomato, and wheat. Syringomycin biosynthesis genes (syrB1 and syrP) were detected using the corresponding PCR primers in all of the strains isolated from alfalfa. Phylogenetic analyses using the sequences of four housekeeping genes (gapA, gltA, gyrB, and rpoD) revealed that all of the strains except one (Als34) belong to phylogroup 2b of P. syringae sensu lato, whereas strain Als34 placed within phylogroup 1 close to the type strain of P. syringae pv. apii. Among the phylogroup 2b strains, nine strains were phylogenetically close to the P. syringae pv. aptata clade, whereas the remainder were scattered among P. syringae pv. atrofaciens and P. syringae pv. syringae strains. Pathogenicity and host range assays of the bacterial strains evaluated in this study on a set of taxonomically diverse plant species did not allow us to assign a "pathovar" status to the alfalfa strains. However, these results provide novel insight into the host range and phylogenetic position of the alfalfa-pathogenic members of P. syringae sensu lato, and they reveal that phenotypically and genotypically heterogeneous strains of the pathogen cause bacterial leaf spot of alfalfa.

Rhizophagus irregularis modulates cadmium uptake, metal transporter, and chelator gene expression in Medicago sativa.

Arbuscular mycorrhizal fungi (AMF) are considered a potential biotechnological tool for mitigating heavy metal (HM) toxicity. A greenhouse experiment was conducted to evaluate the impacts of the AM fungus Rhizophagus irregularis on cadmium (Cd) uptake, mycorrhizal colonization, and some plant growth parameters of Medicago sativa (alfalfa) in Cd-polluted soils. In addition, expression of two metal chelators (MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)) and two metal transporter genes (MsIRT1 and MsNramp1) was analyzed using quantitative real-time PCR (qRT-PCR). Cd addition had a significant negative effect on mycorrhizal colonization. However, AMF symbiosis promoted the accumulation of biomass under both stressed and unstressed conditions compared with non-mycorrhizal (NM) plants. Results also showed that inoculation with R. irregularis significantly reduced shoot Cd concentration in polluted soils. Transcripts abundance of MsPCS1, MsMT2, MsIRT1, and MsNRAMP1 genes were downregulated compared with NM plants indicating that metal sequestration within hyphal fungi probably made Cd concentration insufficient in root cells for induction of these genes. These results suggest that reduction of shoot Cd concentration in M. sativa colonized by R. irregularis could be a promising strategy for safe production of this plant in Cd-polluted soils.

Effects of arbuscular mycorrhizal fungi, biochar and cadmium on the yield and element uptake of Medicago sativa.

The synergistic effects of arbuscular mycorrhizal fungi (AMF) inoculation and biochar application on plant growth and heavy metal uptake remain unclear. A pot experiment was carried out to investigate the influence of AMF inoculation, biochar and cadmium (Cd) addition on the growth, nutrient and cadmium uptake of Medicago sativa, as well as soil biological and chemical characteristics. In comparison to the non-Cd pollution treatment, Cd addition significantly decreased mycorrhizal colonization, biomass, and N, P, Ca and Mg contents of shoots and roots in the absence of biochar. Biochar amendment did not increase mycorrhizal colonization at either Cd levels. Regardless of the biochar amendment, AMF inoculation significantly promoted contents of N and P in plant shoots grown in the Cd-contaminated soils. Nevertheless, in the presence of Cd pollution, biochar dramatically elevated the biomass and N, P, K and Ca contents of plant tissues in both AMF inoculation treatments. Biochar addition significantly reduced soil DTPA-extracted Cd. The treatments with AMF inoculation and biochar amendment showed the lowest shoot Cd concentrations and contents, highest plant tissue N and P contents in the Cd addition group. These results suggested that combined use of AMF inoculation and biochar amendment had significant synergistic effects not only on nutrient uptake but also on the reduction in cadmium uptake of alfalfa grown in Cd-polluted soil.

Insight into Bacterial Community Diversity and Monthly Fluctuations of Medicago sativa Rhizosphere Soil in Response to Hydrogen Gas Using Illumina High-Throughput Sequencing.

The microbial diversity and the monthly fluctuations in Medicago sativa field soil in response to hydrogen gas were investigated. Illumina high-throughput sequencing was used to analyze the bacteria in raw and hydrogen-treated rhizosphere soil. Among the 18 soil samples, the abundance change of the predominant phyla Proteobacteria and Actinobacteria showed opposite trends. The diversity index analysis of the nine leguminous soil samples showed the highest diversity of the microbial community in July. In the other nine soil samples treated with hydrogen, the microbial diversity decreased and the diversity of soil microorganisms in September was higher than that in July, but not significantly so. The heat map analysis revealed that the microbial community composition of the soil samples was different before and after the hydrogen treatment. After the soil samples were treated with hydrogen, the dominant genera were Nocardioide, Pseudomonas, Janibacter, Microbacterium, Microvirga, Streptomyces, and Phenylobacterium in May; Bradyrhizobium, Haliangium, Sphingomonas, Blastocatella, Lysobacter, and Sphingopyxis in July; and Aeromicrobium, Pseudonocardia, Lentzea, and Skermanella in September. This study indicates that time and hydrogen gas have significant effects on the diversity of microbes in M. sativa rhizospheric soil.

Rhizobium inoculation enhances copper tolerance by affecting copper uptake and regulating the ascorbate-glutathione cycle and phytochelatin biosynthesis-related gene expression in Medicago sativa seedlings.

Despite numerous reports that legume-rhizobium symbiosis alleviates Cu stress in plants, the possible roles of legume-rhizobium symbiosis and the regulatory mechanisms in counteracting Cu toxicity remain unclear. Here, Sinorhizobium meliloti CCNWSX0020 was used for analyzing the effects of rhizobium inoculation on plant growth in Medicago sativa seedlings under Cu stress. Our results showed that rhizobium inoculation alleviated Cu-induced growth inhibition, and increased nitrogen concentration in M. sativa seedlings. Moreover, the total amount of Cu uptake in inoculated plants was significantly increased compared with non-inoculated plants, and the increase in the roots was much higher than that in the shoots, thus decreasing the transfer coefficient and promoting Cu phytostabilization. Cu stress induced lipid peroxidation and reactive oxygen species production, but rhizobium inoculation reduced these components' accumulation through altering antioxidant enzyme activities and regulating ascorbate-glutathione cycles. Furthermore, legume-rhizobium symbiosis regulated the gene expression involved in antioxidant responses, phytochelatin (PC) biosynthesis, and metallothionein biosynthesis in M. sativa seedlings under Cu stress. Our results demonstrate that rhizobium inoculation enhanced Cu tolerance by affecting Cu uptake, regulating antioxidant enzyme activities and the ascorbate-glutathione cycle, and influencing PC biosynthesis-related gene expression in M. sativa. The results provide an efficient strategy for phytoremediation of Cu-contaminated soils.

The LsrB Protein Is Required for Agrobacterium tumefaciens Interaction with Host Plants.

Agrobacterium tumefaciens infects and causes crown galls in dicot plants by transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion system. This process requires appropriate environmental conditions, certain plant secretions, and bacterial regulators. In our previous work, a member of the LysR family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to modulate its symbiotic interactions with the host plant alfalfa. However, the function of its homolog in A. tumefaciens remains unclear. In this study, we show that the LsrB protein of A. tumefaciens is required for efficient transformation of host plants. A lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in succinoglycan production, attachment, and resistance to oxidative stress and iron limitation. RNA-sequencing analysis indicated that 465 genes were significantly differentially expressed (upregulation of 162 genes and downregulation of 303 genes) in the mutant, compared with the wild-type strain, including those involved in succinoglycan production, iron transporter, and detoxification enzymes for oxidative stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus, or A. tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB deletion mutant. Our findings suggest that a conserved mechanism of LsrB function exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.

Sinorhizobium meliloti Glutathione Reductase Is Required for both Redox Homeostasis and Symbiosis.

Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti, is required for the development of alfalfa (Medicago sativa) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. melilotigor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H2O2 and sodium nitroprusside. Interestingly, the expression of gshA, gshB1, katA, and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa.IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti, the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis.

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium.

Selection of alfalfa genotypes for resistance to the foliar pathogen Curvularia geniculata

ABSTRACT Foliar diseases impose severe restrictions on the persistence and productivity of Medicago sativa, both of which may be increased by developing disease resistant and more competitive genotypes that can improve pasture quality. We found Curvularia geniculata as the principal alfalfa foliar pathogen in the Brazilian state of Rio Grande do Sul (RS). Growth chamber experiments evaluated the resistance of alfalfa genotypes 'E1C4', 'CPPSul', 'ABT 805' and 'CUF-101' to C. geniculata as compared the control 'Crioula' genotype. These genotypes were also evaluated in field trials at a sea level site in Eldorado do Sul in central RS and at two sites £200 m above sea level, one in Bagé municipality in south west RS and the other at a farm near the town of Alto Feliz in north east RS. Plants were spray-inoculated with 1.6 x 106 ml-1 of C. geniculata spores and visually evaluated for leaf damage 14 days later. The C. geniculata infection rates varied from zero to 100%. Alfalfa persistence and forage mean dry mass (DM) production at the Eldorado site were measured during different seasons from November 2013 to January 2015 by calculating the incidence of invasive plants and morphologically separating leaves from stems and calculating both leaf and stem DM. Data were analysed using mixed statistical models. The best results for persistence and forage DM were shown by the 'CPPSul' genotypes (DM = 16,600 kg ha-1) and 'Crioula' (DM = 15,750 kg ha-1). These two genotypes will be used for subsequent investigations and selection cycles.

Genotyping-by-sequencing-based genome-wide association studies on Verticillium wilt resistance in autotetraploid alfalfa (Medicago sativa L.).

Verticillium wilt (VW) is a fungal disease that causes severe yield losses in alfalfa. The most effective method to control the disease is through the development and use of resistant varieties. The identification of marker loci linked to VW resistance can facilitate breeding for disease-resistant alfalfa. In the present investigation, we applied an integrated framework of genome-wide association with genotyping-by-sequencing (GBS) to identify VW resistance loci in a panel of elite alfalfa breeding lines. Phenotyping was performed by manual inoculation of the pathogen to healthy seedlings, and scoring for disease resistance was carried out according to the standard test of the North America Alfalfa Improvement Conference (NAAIC). Marker-trait association by linkage disequilibrium identified 10 single nucleotide polymorphism (SNP) markers significantly associated with VW resistance. Alignment of the SNP marker sequences to the M. truncatula genome revealed multiple quantitative trait loci (QTLs). Three, two, one and five markers were located on chromosomes 5, 6, 7 and 8, respectively. Resistance loci found on chromosomes 7 and 8 in the present study co-localized with the QTLs reported previously. A pairwise alignment (blastn) using the flanking sequences of the resistance loci against the M. truncatula genome identified potential candidate genes with putative disease resistance function. With further investigation, these markers may be implemented into breeding programmes using marker-assisted selection, ultimately leading to improved VW resistance in alfalfa.

Sinorhizobium meliloti chemotaxis to quaternary ammonium compounds is mediated by the chemoreceptor McpX.

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpXPR and McpX34-306 ) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpXPR with dissociation constants (Kd ) in the nanomolar range for choline and glycine betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant-derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding.

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa.

UNLABELLED: The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCE: The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system.

[Root Nodule Bacteria Sinorhizobium meliloti: Tolerance to Salinity and Bacterial Genetic Determinants].

The theoretical and experimental data on salt tolerance of root nodule bacteria Sinorhizobium meliloti (Ensifer meliloti), an alfalfa symbiont, and on genetic determination of this feature are reviewed. Extensive data on the genes affecting adaptation of proteobacteria are provided, as well as on the groups of genes with activity depending on the osmolarity of the medium. Structural and functional polymorphism of the bet genes involved in betaine synthesis and transport in S. meliloti is discussed. The phenotypic and. genotypic polymorphism in 282 environmental rhizobial strains isolated from the centers of alfalfa diversity affected by aridity and salinity is discussed. The isolates from the Aral Sea area and northern Caucasus were shown to possess the betC gene represented by two types of alleles: the dominant A-type allele found in Rm 1021 and the less common divergent E-type allele, which was revealed in regions at the frequencies at the frequencies of 0.35 and 0.48, respectively. In the isolates with the salt-tolerant phenotype, which were isolated from root nodules and subsequently formed less effective symbioses with alfalfa, the frequency of E-type alleles was 2.5 times higher. Analysis of the nucleotide and amino acid sequences of the E-type allele of the betC gene revealed that establishment of this allele in the population was a result of positive selection. It is concluded that diversification of the functionally diverse bet genes occurring in S. meliloti affects the salt tolerance and symbiotic effectivity of rhizobia.