Rhizobium Leguminosarum Research Articles

Structure–function characterization of an aldo–keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Deoxynivalenol (DON) is a mycotoxin, produced by filamentous fungi such as Fusarium graminearum, that causes significant yield losses of cereal grain crops worldwide. One of the most promising methods to detoxify this mycotoxin involves its enzymatic epimerization to 3-epi-DON. DepB plays a critical role in this process by reducing 3-keto-DON, an intermediate in the epimerization process, to 3-epi-DON. DepBRleg from Rhizobium leguminosarum is a member of the new aldo–keto reductase family, AKR18, and it has the unusual ability to utilize both NADH and NADPH as coenzymes, albeit with a 40-fold higher catalytic efficiency with NADPH compared to NADH. Structural analysis of DepBRleg revealed the putative roles of Lys-217, Arg-290, and Gln-294 in NADPH specificity. Replacement of these residues by site-specific mutagenesis to negatively charged amino acids compromised NADPH binding with minimal effects on NADH binding. The substrate-binding site of DepBRleg is larger than its closest structural homolog, AKR6A2, likely contributing to its ability to utilize a wide range of aldehydes and ketones, including the mycotoxin, patulin, as substrates. The structure of DepBRleg also suggests that 3-keto-DON can adopt two binding modes to facilitate 4-pro-R hydride transfer to either the re- or si-face of the C3 ketone providing a possible explanation for the enzyme’s ability to convert 3-keto-DON to 3-epi-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively.

Residual Effect of Finely-Ground Biochar Inoculated with Bio-Fertilization Impact on Productivity in a Lentil–Maize Cropping System

Biochar fertilization improves soil fertility and carbon sequestration, implying agricultural and environmental advantages. The effect of different sized previously applied biochar and biofertilizer agents on succeeding crops remains poorly known for legume–cereal cropping cycles. This study compared different particle-sized biochar and biofertilizer strains applied to lentils for their residual impact on subsequent maize growth, nutrition, and soil fertility without further polluting the environment. Three particle sizes (<2, 2–5, 5–10 mm) of Babul tree (Acacia arabica) wood biochar was obtained through grinding and sieving and applied prior to the lentil (first) crop at a rate of 500 g m−2. The commercial Rhizobium leguminosarum products Biozote-N and Rhizogold were inoculated to lentil seeds before sowing. The effect of biochar and biofertilizer agents on the succeeding maize (second) crops was evaluated for soil and crop performance. Findings revealed that particle sizes of <2 mm biochar and Biozote-N inoculation enhanced plant height, leaf area and leaf area index, biological yield, and thousand grain weight of the subsequent maize crop. Maize grain yield was enhanced by 2.5%, tissue N uptake by 15%, nitrogen uptake efficiency by 17%, grain protein content by 15%, extractable P by 17%, and soil bulk density by 3% with a residual biochar particle size of <2 mm and Biozote-N inoculation. It was concluded that the finely grounded (<2 mm) biochar particle combined with inoculation of Biozote-N was superior to larger particle sizes for enhancing crop growth and improving soil fertility status at the residual level, benefiting the subsequent crop in a legume–cereal rotation system.

Legume plant defenses and nutrients mediate indirect interactions between soil rhizobia and chewing herbivores

Soil bacteria that associate with plant roots promote host vigor. Legume plants form mutualisms with rhizobial bacteria, and legumes grown with rhizobia have more nutrients and defenses than those grown without rhizobia. However, few studies have tested how stressors such as herbivores affect soil rhizobia, and the mechanisms mediating these interactions. Here we tested reciprocal interactions between a chewing herbivore, Sitona lineatus (pea leaf weevil), and Pisum sativum (pea) plants grown with or without rhizobia (Rhizobium leguminosarum biovar. viciae), and the plant-defense and nutritional mechanisms mediating these interactions. We hypothesized that plants grown with rhizobia would have less feeding from S. lineatus due to greater expression of phytohormones or physical defenses. We also predicted that herbivory might impede the mutualism between P. sativum and rhizobia. Our experiments showed that leaf defoliation by S. lineatus was indeed lowest on plants with rhizobia. Plants grown with rhizobia had increased gene transcript expression associated with hormone-related defense (jasmonic acid, ethylene, abscisic acid) as well as physical and antioxidant-related defense, which may explain reduced feeding by S. lineatus. Conversely, S. lineatus feeding reduced the number of root nodules and nodule fresh weight, suggesting a disruption of the symbiosis between plants and rhizobia. Our study shows that aboveground herbivores can engage in mutually antagonistic interactions with soil microbes that are mediated through multiple plant-mediated pathways.

Rhizobium leguminosarum symbiovar viciae strains are natural wheat endophytes that can stimulate root development.

Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.

Carbohydrate utilization by belowground organs defines the capacity of faba bean to exploit organic phosphorus sources

AbstractBackgroundLimited phosphate (Pi) reserves and the economic and adverse environmental impacts of excessive application of mineral fertilizers in soils are alarming. Therefore, the use of organic phosphorus (Porg) is suggested to reduce reliance on chemical fertilizers.AimsThere is currently little information regarding the accessibility of Porg to N2 fixation plants. Therefore, the present study aims to address the question of to what extent Porg substrates can serve as reliable sources of phosphorus (P) for faba bean in its nodulated phase. Specifically, we intended to explore whether N2‐fixing plants employ intrinsic mechanisms to facilitate Porg availability.MethodsA pot experiment was conducted to assess the inherent capacity of two faba bean varieties (Tiffany vs. Mallory) to utilize Porg supplied as phytic acid and Ca‐glycerophosphate. Plants were inoculated with Rhizobium leguminosarum bv. viciae 3841 strain and cultivated on sterilized loamy sand under greenhouse conditions.ResultsPhytic acid was relatively less efficient than Ca‐glycerophosphate in sustaining plant growth. Compared to Mallory, the variety Tiffany displayed a higher capability to use Porg, as evidenced by the relatively higher biomass production, nodulation, phosphorus use efficiency, and P‐ and N‐accumulations in plant tissues. Porg exploitation was intimately linked to the efficient utilization of the host's carbohydrate budget into nutrient‐acquiring structures (roots and nodules). In addition, the preferential allocation of Pi to prolific nodules is essential for plants to thrive best under these circumstances.ConclusionsTogether with N profitability, these findings suggested that N2‐fixing plants (e.g., faba bean) can promote the sustainable exploitation of Porg, thereby diminishing the sizeable environmental footprint.

Enhancing systemic resistance in faba bean (Vicia faba L.) to Bean yellow mosaic virus via soil application and foliar spray of nitrogen-fixing Rhizobium leguminosarum bv. viciae strain 33504-Alex1.

Rhizobium spp. manifests strong nitrogen fixation ability in legumes. However, their significance as biocontrol agents and antivirals has rarely been investigated. Under greenhouse conditions, the molecularly identified nitrogen-fixing plant growth-promoting rhizobacteria (PGPR), Rhizobium leguminosarum bv. viciae strain 33504-Alex1, isolated from the root nodules of faba bean plants, was tested as a soil inoculum or a foliar application to trigger faba bean plants’ resistance against Bean yellow mosaic virus (BYMV) infection. Compared to the non-treated faba bean plants, the applications of 33504-Alex1 in either soil or foliar application significantly promoted growth and improved total chlorophyll content, resulting in a considerable reduction in disease incidence and severity and the inhibition index of BYMV in the treated faba bean plants. Furthermore, the protective activities of 33504-Alex1 were associated with significant reductions in non-enzymatic oxidative stress markers [hydrogen peroxide (H2O2) and malondialdehyde (MDA)] and remarkably increased DPPH free radical scavenging activity and total phenolic content compared to the BYMV treatment at 20 days post-inoculation. Additionally, an increase in reactive oxygen species scavenging enzymes [superoxide dismutase (SOD) and polyphenol oxidase (PPO)] and induced transcriptional levels of pathogenesis-related (PR) proteins (PR-1, PR-3, and PR-5) were observed. Of the 19 polyphenolic compounds detected in faba bean leaves by high-performance liquid chromatography (HPLC) analysis, gallic and vanillic acids were completely shut down in BYMV treatment. Interestingly, the 33504-Alex1 treatments were associated with the induction and accumulation of the most detected polyphenolic compounds. Gas chromatography-mass spectrometry (GC-MS) analysis showed hexadecanoic acid 2,3-dihydroxypropyl ester, tetraneurin-A-Diol, oleic acid, and isochiapin B are the major compounds in the ethyl acetate extract of 33504-Alex1 culture filtrate (CF), suggesting it acts as an elicitor for the induction of systemic acquired resistance (SAR) in faba bean plants. Consequently, the capacity of R. leguminosarum bv. viciae strain 33504-Alex1 to enhance plant growth and induce systemic resistance to BYMV infection will support the incorporation of 33504-Alex1 as a fertilizer and biocontrol agent and offer a new strategy for crop protection, sustainability, and environmental safety in agriculture production.

Genetic diversity of microsymbionts nodulating Trifolium pratense in subpolar and temperate climate regions

Rhizobia are soil-borne bacteria forming symbiotic associations with legumes and fixing atmospheric dinitrogen. The nitrogen-fixation potential depends on the type of host plants and microsymbionts as well as environmental factors that affect the distribution of rhizobia. In this study, we compared genetic diversity of bacteria isolated from root nodules of Trifolium pratense grown in two geographical regions (Tromsø, Norway and Lublin, Poland) located in distinct climatic (subpolar and temperate) zones. To characterize these isolates genetically, three PCR-based techniques (ERIC, BOX, and RFLP of the 16S-23S rRNA intergenic spacer), 16S rRNA sequencing, and multi-locus sequence analysis of chromosomal house-keeping genes (atpD, recA, rpoB, gyrB, and glnII) were done. Our results indicate that a great majority of the isolates are T. pratense microsymbionts belonging to Rhizobium leguminosarum sv. trifolii. A high diversity among these strains was detected. However, a lower diversity within the population derived from the subpolar region in comparison to that of the temperate region was found. Multi-locus sequence analysis showed that a majority of the strains formed distinct clusters characteristic for the individual climatic regions. The subpolar strains belonged to two (A and B) and the temperate strains to three R. leguminosarum genospecies (B, E, and K), respectively.

Synergistic Interaction between Symbiotic N2 Fixing Bacteria and Bacillus strains to Improve Growth, Physiological Parameters, Antioxidant Enzymes and Ni Accumulation in Faba Bean Plants (Vicia faba) under Nickel Stress.

Several activities in the agriculture sector lead to the accumulation of Nickel (Ni) in soil. Therefore, effective and economical ways to reduce soil bioavailability of Ni must be identified. Five isolates of Rhizobium leguminosarum biovar Viceae (ICARDA 441, ICARDA 36, ICARDA 39, TAL–1148, and ARC–207) and three bacterial strains (Bacillus subtilis, B. circulance, and B. coagulans) were evaluated for tolerance and biosorption of different levels of Ni (0, 20, 40, 60, and 80 mg L−1). Pot experiments were conducted during the 2019/2020 and 2020/2021 seasons using four inoculation treatments (inoculation with the most tolerant Rhizobium (TAL–1148), inoculation with the most tolerant Rhizobium (TAL–1148) + B. subtilis, inoculation with the most tolerant Rhizobium (TAL–1148) + B. circulance, and inoculation with the most tolerant Rhizobium (TAL–1148) + B. coagulans) under different levels of Ni (0, 200, 400, and 600 mg kg−1), and their effects on growth, physiological characteristics, antioxidant enzymes, and Ni accumulation in faba bean plants (Vicia faba C.V. Nobaria 1) were determined. The results showed that Rhizobium (TAL–1148) and B. subtilis were the most tolerant of Ni. In pot trials, inoculation with the most tolerant Rhizobium TAL–1148 + B. subtilis treatment was shown to be more effective in terms of growth parameters (dry weight of plant, plant height, number of nodules, and N2 content), and this was reflected in physiological characteristics and antioxidant enzymes under 600 mg kg−1 Ni compared to the other treatments in the 2019/2020 season. In the second season, 2020/2021, a similar pattern was observed. Additionally, lower concentrations of Ni were found in faba bean plants (roots and shoots). Therefore, a combination of the most tolerant Rhizobium (TAL–1148) + B. subtilis treatment might be used to reduce Ni toxicity.

RapD Is a Multimeric Calcium-Binding Protein That Interacts With the Rhizobium leguminosarum Biofilm Exopolysaccharide, Influencing the Polymer Lengths

Rhizobium leguminosarum synthesizes an acidic polysaccharide mostly secreted to the extracellular medium, known as exopolysaccharide (EPS) and partially retained on the bacterial surface as a capsular polysaccharide (CPS). Rap proteins, extracellular protein substrates of the PrsDE type I secretion system (TISS), share at least one Ra/CHDL (cadherin-like) domain and are involved in biofilm matrix development either through cleaving the polysaccharide by Ply glycanases or by altering the bacterial adhesive properties. It was shown that the absence or excess of extracellular RapA2 (a monomeric CPS calcium-binding lectin) alters the biofilm matrix’s properties. Here, we show evidence of the role of a new Rap protein, RapD, which comprises an N-terminal Ra/CHDL domain and a C-terminal region of unknown function. RapD was completely released to the extracellular medium and co-secreted with the other Rap proteins in a PrsDE-dependent manner. Furthermore, high levels of RapD secretion were found in biofilms under conditions that favor EPS production. Interestingly, size exclusion chromatography of the EPS produced by the ΔrapA2ΔrapD double mutant showed a profile of EPS molecules of smaller sizes than those of the single mutants and the wild type strain, suggesting that both RapA2 and RapD proteins influence EPS processing on the cell surface. Biophysical studies showed that calcium triggers proper folding and multimerization of recombinant RapD. Besides, further conformational changes were observed in the presence of EPS. Enzyme-Linked ImmunoSorbent Assay (ELISA) and Binding Inhibition Assays (BIA) indicated that RapD specifically binds the EPS and that galactose residues would be involved in this interaction. Taken together, these observations indicate that RapD is a biofilm matrix-associated multimeric protein that influences the properties of the EPS, the main structural component of the rhizobial biofilm.

Effect of Rhizobium Leguminosarum Inoculum, Rice Hulls, and Urea Fertilizer on the Availability of Phosphorous and Potassium for the Growth and Yield of Broad Bean (Vicia faba L.)

Aiming to investigate the effect of Rhizobium Leguminosarum inoculum, rice hulls, and urea fertilizer on the availability of phosphorous and potassium regarding the growth and yield traits of broad bean plant (Vicia faba L.), a field trial was carried out during the autumn agricultural season 2021 in Al-Daghara district, 17 km north of the center of Al-Diwaniyah Governorate-Iraq. Two levels of Rhizobium Leguminosarum (R0) the control, and (R1) bacterial inoculation, three levels of organic fertilizer (rice hulls) S0, S3, and S5 t/ha-1, and three levels of urea fertilizer (U0, U50, and U75) kg/ha-1 and their overlap, were used and studied. The results of the interaction treatment (bacterial inoculum + rice hulls (5 t/ha-1) + urea fertilizer (75 t/ha-1)) yielded the highest significant increase in the traits of root-knot number, root-knot weight, potassium concentration, phosphorous concentration) compared to the control, 23,851 knots/ plant, 0.550 / 122.6 kg.soil-1 and 23.812 kg/soil-1, respectively.

ISOLATION AND DIAGNOSIS OF CADMIUM-RESISTANT BACTERIA AND ITS POTENTIAL PHYTOREMEDIATION WITH THE BROAD BEAN PLANT

Results of the study proved that phytoremediation can be a promising technique to treat cadmium (Cd)-contaminated soil. Four bacteria types were isolated from the soil; two are autotrophic and others are heterotrophic. Autotrophic bacteria were dominant in soils with 42 mg Cd Kg-1. The total count and diversity of both bacteria types decreased with the increase of Cd in media and reached their minimum limit of tolerance at 60 mg Cd L-1 in terms of the heterotrophic bacteria, while the minimum limit of tolerance in the case of autotrophic bacteria was at 110 mg Cd L-1. The four isolates can form biofilms that ranged in thickness between 2.8–4.3 mm. The tolerant isolates belong to Rhizobium leguminosarum, Pseudomonas fluorescens, Actinobacteria, and Corynebacterium. Shoot and dry weight significantly varied according to the changes in Cd concentrations and isolate types. The level in either shoot or root exceeded critical levels, however, its concentration was higher in the root compared with the shoot. The effect of Cd on broad bean plants began at 80 and 100 mg Cd L-1. The broad bean plant was resistant to growing in the contaminated area by Cd even at 120 mg Cd Kg-1 DW. The presence of heterotrophic bacteria was noticeably useful for autotrophic bacteria, as well as, for enhancing Cd resistance. The study showed that cooperative phytoremediation could be a safe and active technique to apply in the field soil contaminated with heavy metals.

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. “salignae” is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. “cyanophyllae” is proposed. Isolates formed effective nodules on A. saligna.

Rhizobium leguminosarum bv. trifolii exopolysaccharide and sunflower husk biochar as factors affecting immobilization of both tetracycline and Cd2+ ions on soil solid phase

Rhizobium leguminosarum bv. trifolii exopolysaccharide and sunflower husk biochar as factors affecting immobilization of both tetracycline and Cd2+ ions on soil solid phase

Fruit-peel-degrading Potential of Some Legumes-associated Bacteria

This research aimed at determining the digestive ability of nine legumes-associated bacteria on the peels of some fruits (orange, watermelon, plantain, banana, pineapple and pawpaw). The bacteria were cultivated separately on each peel for 18 hours at 28oC; the amount of glucose released was quantified using Dinitrosalicylic acid reagent method. The results obtained showed that the bacteria degraded all the peels with the highest (0.297 mg/mL) and lowest (0.087 mg/mL) glucose concentrations produced by Rhizobium leguminosarum FUBO001 and Bonitrorhizobium winogradskyi FUBO004 in banana and pawpaw peels, respectively. The Bo. winogradskyi FUBO004 synthesized 0.101 mg/mL as minimum sugar amount in the former peel while Bradyrhizobium nigeriasis FUBO005 produced the highest glucose quantity (0.167 mg/mL) in the latter peel. The lowest amounts of glucose in orange, watermelon, pineapple and plantain peels produced by Bradyrhizobium nigeriasis FUBO003, Rhizobium nigeriasis, R. nigeriasis and Br. nigeriasis FUBO003 were 0.095, 0.132, 0.09 and 0.248 mg/mL respectively. In these peels, the highest amount of the reducing sugar made was 0.131 mg/mL by Br. nigeriasis FUBO005, 0.211 mg/mL by Br. nigeriasis FUBO005, 0.156 mg/mL by Bo. winogradskyi FUBO004 and 0.291 mg/mL by R. nigeriasis. These results suggest that the bacteria catabolized the fruit peels, reflecting their high potential in the conversion of the fruit peels to useful products.

Extracting Information from Gene Coexpression Networks of Rhizobium leguminosarum.

Nitrogen uptake in legumes is facilitated by bacteria such as Rhizobium leguminosarum. For this bacterium, gene expression data are available, but functional gene annotation is less well developed than for other model organisms. More annotations could lead to a better understanding of the pathways for growth, plant colonization, and nitrogen fixation in R. leguminosarum. In this study, we present a pipeline that combines novel scores from gene coexpression network analysis in a principled way to identify the genes that are associated with certain growth conditions or highly coexpressed with a predefined set of genes of interest. This association may lead to putative functional annotation or to a prioritized list of genes for further study.

Biosynthesis of eriodictyol from tyrosine by Corynebacterium glutamicum

BackgroundEriodictyol is a bioactive flavonoid compound that shows potential applications in medicine development and food processing. Microbial synthesis of eriodictyol has been attracting increasing attention due to several benefits. In this study, we employed a GRAS strain Corynebacterium glutamicum as the host to produce eriodictyol directly from tyrosine.ResultsWe firstly optimized the biosynthetic module of naringenin, the upstream intermediate for eriodictyol production, through screening of different gene orthologues. Next, to improve the level of the precursor malonyl-CoA necessary for naringenin production, we introduced matB and matC from Rhizobium trifolii into C. glutamicum to convert extracellular malonate to intracellular malonyl-CoA. This combinatorial engineering resulted in around 35-fold increase in naringenin production from tyrosine compared to the initial recombinant C. glutamicum. Subsequently, the hpaBC genes from E. coli encoding 4-hydroxyphenylacetate 3-hydroxylase were expressed in C. glutamicum to synthesize eriodictyol from naringenin. Further optimization of the biotransformation process parameters led to the production of 14.10 mg/L eriodictyol.ConclusionsThe biosynthesis of the ortho-hydroxylated flavonoid eriodictyol in C. glutamicum was achieved for the first time via functional expression of E. coli hpaBC, providing a baseline strain for biosynthesis of other complex flavonoids. Our study demonstrates the potential application of C. glutamicum as a host microbe for the biosynthesis of value-added natural compounds from tyrosine.

An Alliance of Trifolium repens-Rhizobium leguminosarum bv. trifolii-Mycorrhizal Fungi From an Old Zn-Pb-Cd Rich Waste Heap as a Promising Tripartite System for Phytostabilization of Metal Polluted Soils.

The Bolesław waste heap in South Poland, with total soil Zn concentrations higher than 50,000 mg kg–1, 5,000 mg Pb kg–1, and 500 mg Cd kg–1, is a unique habitat for metallicolous plants, such as Trifolium repens L. The purpose of this study was to characterize the association between T. repens and its microbial symbionts, i.e., Rhizobium leguminosarum bv. trifolii and mycorrhizal fungi and to evaluate its applicability for phytostabilization of metal-polluted soils. Rhizobia originating from the nutrient-poor waste heap area showed to be efficient in plant nodulation and nitrogen fixation. They demonstrated not only potential plant growth promotion traits in vitro, but they also improved the growth of T. repens plants to a similar extent as strains from a non-polluted reference area. Our results revealed that the adaptations of T. repens to high Zn-Pb-Cd concentrations are related to the storage of metals predominantly in the roots (excluder strategy) due to nodule apoplast modifications (i.e., thickening and suberization of cell walls, vacuolar storage), and symbiosis with arbuscular mycorrhizal fungi of a substantial genetic diversity. As a result, the rhizobia-mycorrhizal fungi-T. repens association appears to be a promising tool for phytostabilization of Zn-Pb-Cd-polluted soils.

Inability to Catabolize Rhamnose by Sinorhizobium meliloti Rm1021 Affects Competition for Nodule Occupancy.

Rhizobium leguminosarum strains unable to grow on rhamnose as a sole carbon source are less competitive for nodule occupancy. To determine if the ability to use rhamnose as a sole carbon source affects competition for nodule occupancy in Sinorhizobium meliloti, Tn5 mutants unable to use rhamnose as a sole carbon source were isolated. S. meliloti mutations affecting rhamnose utilization were found in two operons syntenous to those of R. leguminosarum. Although the S. meliloti Tn5 mutants were complemented using an R. leguminosarum cosmid that contains the entire wild-type rhamnose catabolic locus, complementation did not occur if the cosmids carried Tn5 insertions within the locus. Through a series of heterologous complementation experiments, enzyme assays, gene fusion, and transport experiments, we show that the S. meliloti regulator, RhaR, is dominant to its R. leguminosarum counterpart. In addition, the data support the hypothesis that the R. leguminosarum kinase is capable of directly phosphorylating rhamnose and rhamnulose, whereas the S. meliloti kinase does not possess rhamnose kinase activity. In nodule competition assays, S. meliloti mutants incapable of rhamnose transport were shown to be less competitive than the wild-type and had a decreased ability to bind plant roots in the presence of rhamnose. The data suggests that rhamnose catabolism is a general determinant in competition for nodule occupancy that spans across rhizobial species.

Draft Genome Sequence of Rhizobium ruizarguesonis (Rhizobium leguminosarum) Strain 1TK341.

ABSTRACTRhizobium ruizarguesonis (Rhizobium leguminosarum) strain 1TK341 was isolated from pink nodules of fixation-negative mutant line P61 of pea (Pisum sativum L.) grown in soil. Here, we report the draft genome sequence of the strain.

Rhizobium croatiense sp. nov. and Rhizobium redzepovicii sp. nov., two new species isolated from nodules of Phaseolus vulgaris in Croatia

Phaseolus vulgaris is a legume indigenous to America which is nodulated by strains of genus Rhizobium in Croatia. Four of these strains, 13TT, 9T, 18TT and 8Z are phylogenetically close to the species from the Rhizobium leguminosarum phylogenetic complex in the 16S rRNA gene analysis. The results of both the analyses of the concatenated recA and atpD genes and whole genomes revealed that the strains 13TT and 9T clustered with Rhizobium sophoriradicis CCBAU 03470T and the strains 18TT and 8Z with Rhizobium ecuadorense CNPSO 671T. Whole genome average nucleotide identity blast (ANIb) and dDDH values between the strains 13TT and the type strain of R. sophoriradicis and between the strains 18TT and the type strain of R. ecuadorense were lower than 95% and 70%, respectively, which are the threshold values recommended for bacterial species differentiation. These results combined with those of chemotaxonomic and phenotypic analyses support the affiliation of these strains to two novel species within the genus Rhizobium for which we propose the names Rhizobium croatiense sp. nov. 13TT (=LMG 32397T, = HAMBI 3740T) as type strain and Rhizobium redzepovicii sp. nov. 18TT (=LMG 32398T, = HAMBI 3741T) as type strain.