Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil

Phaseolus vulgaris L. (common bean) is nodulated by rhizobia present in the fields around the Seibersdorf laboratory despite the fact that common bean has not been grown for a long time. Using PCR analysis with repetitive primers, plasmid profiles, nifH profiles, PCR-RFLP analysis of the 16S rRNA gene and of the 16S rRNA-23S rRNA intergenic spacer and the nodulation phenotype, two well-differentiating groups could be distinguished. One group showed high similarity to Rhizobium sp. R602sp, isolated from common bean in France, while the other showed the same characteristics as R. etli . We detected little variation in the symbiotic regions but found higher diversity when using approaches targeting the whole genome. Many isolates obtained in this study might have diverged from a limited number of strains, therefore the Austrian isolates showed high saprophytic and nodulation competence in that particular soil.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 M Al at pH 5.5. Three strains were tolerant of 20 M Al at pH 5.5 and 10 M Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 M Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 M Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

The accumulation of trehalose in nodules of several cultivars of common bean (Phaseolus vulgaris) and its correlation with resistance to drought stress

Nine cultivars of common bean were grown in the presence of a natural microflora without exogenous rhizobial inoculation. Nodules were harvested 30 days post planting (early flowering stage) and the presence of trehalose determined. Amounts present varied according to cultivar and were between 0.20 and 1.63 mg g⁻¹ nodule dry weight. Rhizobial strains were isolated from the nodules of three selected cultivars (Canario 101, Flor de Mayo Bajio and Flor de Mayo 38). Trehalose levels in nodules produced after either mixed strain reinfection, or after axenic homologous reinfection or after axenic cross‐reinfection could be manipulated by applying drought stress. Mixed reinfection nodules from stressed plants accumulated between two and six times the trehalose concentration found in non‐stressed control plants. After axenic cross‐reinfection up to 48‐fold increases in nodule trehalose content were recorded during drought stress. Those cultivars exhibiting high nodule trehalose levels and/or a high degree of trehalose stimulation in response to drought stress also exhibited a high leaf relative water content and were also the most drought resistant. During drought stress nodule trehalase levels rose only slightly.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Feedback limitation of photosynthesis of Phaseolus vulgaris L grown in elevated CO2

The capacity for photosynthesis is often affected when plants are grown in air with elevated CO₂ partial pressure. We grew Phaseolus vulgaris L. in 35 and 65 Pa CO₂ and measured photosynthetic parameters. When assayed at the growth CO₂ level, photosynthesis was equal in the two CO₂ treatments. The maximum rate of ribulose-1,5-bisphosphate (RuBP) consumption was lower in plants grown at 65 Pa, but the CO₂ partial pressure at which the maximum occurred was higher in the high-CO₂-grown plants, indicating acclimation to high CO₂. The acclimation of RuBP consumption to CO₂ involved a reduction of the activity of RuBP carboxylase which resulted from reduced carbamylation, not a loss of protein. The rate of RuBP consumption declined with CO₂ when the CO₂ partial pressure was above 50Pa in plants grown under both CO₂ levels. This was caused by feedback inhibition as judged by a lack of response to removing O₂ from the air stream. The rate of photosynthesis at high CO₂ was lower in the high-CO₂-grown plants and this was correlated with reduced activity of sucrose-phosphate synthase. This is only the second report of O₂-insensitive photosynthesis under growth conditions for plants grown in high CO₂.     

Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress

Common bean plants inoculated with salt-tolerant Rhizobium tropici wild-type strain CIAT899 formed a more active symbiosis than did its decreased salt-tolerance (DST) mutant derivatives (HB8, HB10, HB12 and HB13). The mutants formed partially effective (HB10, HB12) or almost ineffective (HB8, HB13) nodules (Fix(d)) under non-saline conditions. The DST mutant formed nodules that accumulated more proline than did the wild-type nodules, while soluble sugars were accumulated mainly in ineffective nodules. Under salt stress, plant growth, nitrogen fixation, and the activities of the antioxidant defense enzymes of nodules were affected in all symbioses tested. Overall, mutant nodules showed lower antioxidant enzyme activities than wild-type nodules. Levels of nodule catalase appeared to correlate with symbiotic nitrogen-fixing efficiency. Superoxide dismutase and dehydroascorbate reductase seem to function in the molecular mechanisms underlying the tolerance of nodules to salinity.     

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

Summary A field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha^–1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha^–1 of¹⁵N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N₂ fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N₂ fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha^–1 respectively. These two cultivars produced 991 and 883 kg ha^–1 of grain, respectively, when inoculated and 663 and 620 kg ha^–1 with the addition of 100 kg N ha^–1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.     

Propyl gallate,a free radical scavenger,counteracts the benefits of exogenously applied salicylic acid and aggravates the deleterious effects of the southern bean mosaic virus in Rhizobium-nodulated Phaseolus vulgaris plants

Infection of Rhizobium-nodulated Phaseolus vulgaris by the southern bean mosaic virus (SBMV) markedly decreased the growth and nodulation of plants. Exogenous applications of salicylic acid (SA) at concentrations ≥10 μM further decreased growth and nodulation of virus-infected (V) plants. Only SA concentration of 5 μM in the solution improved the growth, nodulation, chlorophyll concentration and the catabolism of ureide in leaves of V plants. The spray of leaves with 2 mM propyl gallate (+Pg) decreased growth, nodulation as well as the chlorophyll and leaf ureide concentrations in V plants, regardless of the concentration of SA at which plants were grown. Ultrastructural damages in leaf cells caused by SBMV were also enhanced in V+Pg plants. The massive proliferation of virus particles and the presence of virus crystalline arrays within symbiosomes of nodules in V+Pg plants were attributed to proliferation of branched plasmodesmata in leaf vascular-parenchyma cell walls that facilitated virus movement. Virus particles were never observed in leaf and nodule tissues of V plants not sprayed with Pg. Exogenous applications of SA hindered while Pg increased the symbiotic performance of H plants, pointing out the complexity to be addressed in breeding for virus resistance in Rhizobium-nodulated beans.     

Polymorphism of a complex resistance gene candidate family in wild populations of common bean (Phaseolus vulgaris) in Argentina: comparison with phenotypic resistance polymorphism

Fifteen populations of wild bean (Phaseolus vulgaris), located in three provinces in Argentina, were analysed for their polymorphism for a complex resistance gene candidate (RGC) family clustered on the genome and for resistance phenotypes to strains of Colletotrichum lindemuthianum. Results indicate that RGC polymorphism is high. Population structure obtained for markers related to resistance was compared to population structure obtained for RAPD markers in order to infer the evolutionary forces driving polymorphism for resistance in wild populations at both molecular and phenotypic levels. Hierarchical analysis of differentiation showed that, within provinces, populations were differentiated for RAPD as well as for molecular and phenotypic markers of resistance. In contrast, provinces were differentiated only for RAPD and RGC markers and not for resistance phenotypes. The discrepancies found between diversity structures for molecular markers (RAPD and RGCs) and for resistance phenotypes suggest an effect of selection and indicate that diversity for resistance may not be driven by the same selective forces at the molecular and phenotypic levels. We further discuss whether specific selective forces are exerted on RGC markers and underline the importance of spatial scale of analysis for demonstrating an impact of selection.     

Use of the gusA gene marker in a competition study of the Rhizobium strains nodulating the common bean (Phaseolus vulgaris) in Senegal soils

Indigenous rhizobial population is among the factors which influence increased crop yield through inoculation with elite strains. In this study, we compared in greenhouse conditions the competitiveness of Rhizobium strain ISRA 355 for nodulation of the common bean (Phaseolus vulgaris) cultivated in different unsterile Senegal soils in terms of pH, N and C contents. The strain ISRA 355 produced a stable GUS+ transconjugant which was used for competition with indigenous soil rhizobia in six localities. At Bayakh, the transconjugant ISRA 355gusA was less competitive than the indigenous rhizobial strains, whereas in the other localities, it was more competitive since it occupied more than 90% of the nodules. Thus the Rhizobium strain ISRA 355 should be used for successfully inoculating the common bean in Senegal soils.     

Mediation of sterol-induced calmodulin synthesis in Phaseolus vulgaris roots by Ca2+ and its possible relationship to plant growth regulators

Vitamin D₃ and stigmasterol have been previously shown to stimulate growth, Ca²⁺ fluxes and calmodulin synthesis in Phaseolus vulgaris roots. In this study, these sterols (10⁻⁹ M ) were shown to accelerate the incorporation of [³H]-thymidine into DNA in Phaseolus vulgaris (L. cv. Contraancha) root apices, similarly to a mixture of the mitogenic plant growth factors 2,4-dichlorophenoxyacetic acid and kinetin (4.6 μ M each). The effects of stigmasterol were blocked by flufenazine, a calmodulin antagonist. Analogously to stigmasterol, the plant hormones stimulated calmodulin synthesis as shown by double labeling of root proteins with [¹⁴C]-leucine and [³H]-leucine, respectively, followed by their separation on sodium dodecyl sulfate-po-lyacrylamide gels and a calmodulin affinity column, immunoblot analysis and cyclic AMP phosphodiesterase activation assays. The stimulation of root calmodulin formation by stigmasterol was abolished in the absence of Ca²⁺ in the incubation medium and was mimicked by the Ca²⁺ ionophore A–23187. The results suggest that the sterols, like plant mitogenic hormones, promote DNA synthesis, and that these compounds stimulate calmodulin synthesis as a consequence of their mitogenic activity. ^Ca2+ appears to mediate the action of the sterols.     

Salt-tolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2-fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti

Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR–RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR–RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N₂-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. II. Linkage relations and utility of a dominant gene for lethal systemic necrosis to soybean mosaic virus

Summary A single dominant factor, Hss, that conditions a rapid lethal necrotic response to soybean mosaic virus (SMV) has been identified in Phaseolus vulgaris L. cv. Black Turtle Soup, line BT-1. Inoculated plants carrying this factor developed pinpoint necrotic lesions on inoculated tissue followed by systemic vascular necrosis and plant death within about 7 days, regardless of ambient temperature. BT-1 also carries dominant resistance to potyviruses attributed to the tightly linked or identical factors, I, Bcm, Cam, and Hsw, so linkage with Hss was evaluated. No recombinants were identified among 381 F₃ families segregating for potyvirus susceptibility, thus if Hss is a distinct factor, it is tightly linked to I, Bcm, Cam, and Hsw. BT-1 was also crossed reciprocally with the line Great Northern 1140 (GN 1140) in which the dominant gene, Smv, for systemic resistance to SMV was first identified. Smv and Hss segregated independently and are co-dominant. The (GN 1140 x BT-1) F₁ populations showed a seasonal shift of the codominant phenotype. Evaluation of the (GN 1140 x BT-1) F₂ population under conditions where Smv is partially dominant allowed additional phenotypic classes to be distinguished. Pathotype specificity has not been demonstrated for either Smv or Hss. Genotypes that are homozygous for both dominant alleles are systemically resistant to the virus and in addition show undetectable local viral replication or and no seed transmission. This work demonstrates that a gene which conditions a systemic lethal response to a pathogen may be combined with additional gene(s) to create an improved resistant phenotype.     

Selection of symbiotically energy efficient strains of Rhizobium japonicum by their ability to induce a H2-uptake hydrogenase in the free-living state

The capacity of inducing a H₂-uptake hydrogenase in free-living cultures was examined in 21 strains of Rhizobium japonicum. Four strains were found to take up H₂ at rapid rates after 3 days of growth on agar slants inside sealed vials provided with an atmosphere of 5% H₂ in air. Soybean nodules from these strains lost little or no H₂ in air and their bacteroids oxidized H₂ at rates that were similar to those observed in free-living cultures. In contrast, three randomly chosen strains of R. japonicum that showed no H₂-uptake capacity in free-living state produced nodules which lost large amounts of H₂ and the corresponding bacteroids had no hydrogenase activity. A screening procedure is described for the selection of Rhizobium strains producing high energy-efficient nodules based on a test of their ability to induce a H₂-uptake hydrogenase in asymbiotic conditions.