Strains Of Rhizobium Trifolii Research Articles

The structure of the O-antigenic chain of the lipopolysaccharide of Rhizobium trifolii 4s

The structure of the O-antigen chain of the lipopolysaccharide (LPS) of Rhizobium trifolii 4s has been determined by a combination of chemical and spectroscopic methods. The glycosyl components were found to be l-rhamnose, N-acetyl- d-glucosamine, and N-acetyl- d-mannosamine in 3:1:1 molar proportion, as determined by gas chromatography and gas chromatography-mass spectrometry of alditol acetate and persilylated ( R)-2-hydroxybutyl glycoside derivatives. The linkage positions and configurations of the glycosyl residues were obtained by 1D and 2D NMR spectroscopy. The polymer has a pentasaccharide repeating-unit containing rhammose and N-acetylglucosamine in the main chain and N-acetylmannosamine as the sole-side chain component. This latter residue is linked to a main-chain rhamnose residue. This result was suggested by NMR spectroscopy and confirmed by periodate oxidation. The sequence was deduced by 1D and 2D NMR NOE experiments and by partial hydrolysis studies. The repeating unit of the polysaccharide is shown. This constitutes the first complete structure of an O-antigenic chain of the lipopolysaccharide of any strain of Rhizobium trifolii. ▪

The 2,4-D-induced wheat para-nodules are modified lateral roots with structure enhanced by rhizobial inoculation

Nodular outgrowths (para-nodules or p-nodules) on the roots of wheat (Triticum aestivum L.) cv. Miskle seedlings were induced by treatment with 0.3 and 0.6 mg L-1, 2,4-dichlorophenoxyacetate (2,4-D). When co-inoculated with Rhizobium trifolii strain ATCC 14480, more p-nodules were formed at these levels and p-nodulation occured at 0.1 mg L-1 indicating that inoculation enhances 2,4-D-induced p-nodulation. Similar to lateral roots, the p-nodules arose from the pericycle opposite the phloem tissues and were free from the cortical cells of the parental root at all stages of development. Structurally, the p-nodules exhibited tissue differentiation. They possessed a highly organized central vascular cylinder connected to that of the parent root, an endodermis, a cap, and an apical and lateral meristems. P-nodules formed by 2,4-D treatment alone were irregularly lobed due to uncoordinated activity of the apical meristem, while those in the combined 2,4-D and inoculation treatment were more globose. The results of the present study indicate that the 2,4-D-induced wheat p-nodules are modified lateral roots, the structure of which is enhanced by rhizobial inoculation.

Yield and nitrogen fixation of berseem clover as a potential winter forage crop under semiarid conditions

Abstract Field inoculation studies were carried out on loamy clay, brown steppe soil to test the potential and yield of berseem clover (Trifolium alexandrinum) planted separately and together with ryegrass at 50:50 ratio. The experimental site was located on the border of the desert zone of Israel, with an annual average rainfall of about 350 mm. Four cuttings were carried out during the growth period: 88, 121, 158, and 183 days after planting. Clover dry matter and N uptake obtained at the first cutting from the plots inoculated by an effective Rhizobium trifolii strain outyielded those in the uninoculated plots by 39 and 36%, respectively. In four cuts inoculated clover:ryegrass mixture produced 11 880 kg ha‐1 dry matter containing 2349 kg of crude protein, which was 11% more than inoculated clover grown alone. These figures were not significantly different from the yields of ryegrass fertilized with 500 kg N ha‐1. The N2 fixation rates of clover plants grown in mixture with ryegrass were higher than in...

Genetic analysis and cellular localization of the Rhizobium host specificity-determining NodE protein.

The nucleotide sequence of the nodE gene of Rhizobium trifolii strain ANU843 was determined. Like the nodE gene of R. leguminosarum strain 248 it encodes a protein with a predicted mol. wt of 42.0 kd. The predicted NodE proteins of R.trifolii and R.leguminosarum have a homology of 78%. Using antibodies raised against the NodE protein of R.trifolii it was shown that the NodE products of R.leguminosarum and R.trifolii are localized in the cytoplasmic membrane. Furthermore, these NodE proteins are predicted to contain at least two non-polar transbilayer alpha-helices. The nodE genes of R.trifolii and R.leguminosarum were separated from the nodF genes that precede them in the respective nodFE operons. Using the resulting clones, in which NodE was produced under control of the flavonoid-inducible nodABCIJ promoter of R.leguminosarum, it was shown that the NodE product is the main factor that distinguishes the host range of nodulation of R.trifolii and R.leguminosarum. Hybrid nodE genes, which consist of a 5' part of the R.leguminosarum nodE gene and a 3' part of the R.trifolii gene, were constructed. From the properties of these hybrid genes it was concluded that a central region of 185 amino acids at the most, containing only 44 non-identical amino acids, determines the difference in the host-specific characteristics of the two NodE proteins.

Siderophore production and utilization byRhizobium trifolii

Several strains ofRhizobium trifolii were tested for their ability to synthesize and utilize phenolate or hydroxamate types of siderophores. None of the nodulating strains ofR. trifolii was able to produce detectable amounts of siderophores. Only the non-nodulating strainR. trifolii AR6 formed a phenolate siderophore, which stimulated the growth of the siderophore-negative mutant AR65. Other strains ofR. trifolii could not utilize iron from exogenously supplied Desferal, pseudobactin or citrate. The siderophore fromR. trifolii AR6 and 2,3-dihydroxybenzoic acid slightly stimulated the growth of someR. trifolii strains.

The influence of side-chain modifications on the solution behavior of the capsular polysaccharide from Rhizobium trifolii strain TA-1

The effect of chemical modification of the side chain on the solution properties of the capsular polysaccharide from Rhizobium trifolii strain TA-1 has been investigated. The gel stability of the unmodified polymer has been measured by the sigmoidal change of optical activity as a function of temperature. The transition temperature decreases with increasing side-chain modification by periodate oxidation and further borohydride reduction. A series of carboxylic derivatives has been obtained, in addition to the non-ionic ones, by chlorite oxidation after the periodate splitting. Polyelectrolytic ( i.e., apparent pK) and optical dissymmetric properties ( i.e. circular dichroism, c.d.) have been studied on the ionic derivatives as a function of pH. The change of c.d. bands as a function of the degree of oxidation is related to the fractions of the oxidized sites by assuming that periodate reaction occurs according to the magnitude of the rate constants found for simple sugars.

Acid-tolerance and symbiotic effectiveness of Rhizobium trifolii associated with a Trifolium subterraneum L.-based pasture growing in an acid soil

The critical range of pH, below which growth in solid media of several strains of Rhizobium trifolii was inhibited, was between pH 4.55 and 4.85. This was consistent with growth of the strains over a range of pH in liquid culture. Concentrations of Al in excess of 150 μM were necessary to inhibit growth of R. trifolii in agar-culture at pH 5.10. Sensitivity of growth to Al was also influenced by the concentration of P in the medium. Acid-tolerance of R. trifolii isolates collected from a very acid soil was considered to be poor and was similar to that observed for R. trifolii strains TA1 and WU95. Only 96 of 481 isolates were capable of growth in solid media at pH 4.70, whereas all isolates made growth at pH 5.10. The proportion of isolates capable of growth at pH 4.70 was not related to the pH of the soil from which they were isolated, but was influenced by the method of isolation. A higher proportion of more acid-tolerant isolates were obtained from clover nodules formed in dilute soil suspensions than from single nodules removed from plants in the field. The symbiotic eflectiveness of 127 isolates of R. trifolii from the acid soil, relative to the effectiveness of R. trifolii strain TA1 in association with Trifolium subterraneum, ranged between 27 and 112% with a mean of 74% over all isolates. Significant negative correlations were found between the symbiotic eflectiveness and the growth of individual isolates in solid media at low pH.

Characterization of Aberrant Infection Events Induced on Trifolium subterraneum by Rhizobium trifolii Region II Mutants

Summary The early infection events of subterranean clover by the wild-type Rhizobium trifolii strain ANU843 was compared to the infective ability of two nodulation-defective mutants derived from this strain (261 and 262). Strain ANU843 readily infected the newly emerging root hair cells which were present behind the root tip at the time of inoculation although the zone of maximum susceptibility occurred behind the root tip in an area where no root hairs had yet emerged. Both mutant strains were capable of infecting the most susceptible root hair cells at the time of inoculation and inducing nodules but did so with a>50 % reduction in efficiency and nodules were slow to appear and develop. After direct «spot inoculation» of root cells in the most susceptible zone, the progress of the infections initiated by ANU843 and mutant 261 was assessed at 24, 48, 72 h and several days post inoculation using microscopic observation. The rate of the infection process initiated by strain ANU843 on subterranean clovers was similar to those described for the infection of other legumes: marked root hair curling and the initial penetration of the root hair was already apparent after 24 h; extensive cortical cell division ahead of the infection thread was prominent at 72 h and by this time, the infection thread had penetrated to the root cortex. In contrast, infection threads initiated by mutant 261 were still confined to the root hair cell even after 72 h and the infection thread induced rarely penetrated to the root cortex. Microscopic analysis indicated that the development of the infection thread was slow. Cortical cell division was initiated by the mutant strain but was not as extensive as that induced by the parent strain. The poor infective and nodulation ability of mutant strain 261 suggests that the gene(s) affected are required by the bacterium to initiate normal infection thread development in the plant host.

Variation in speed of infection of “no root hair zone” of white clover and nodulating competitiveness among strains of Rhizobium trifolii

Using a marking technique to determine the location of infectible regions on the roots of white clover, nodulation frequency profiles for three strains of Rhizobium trifolii were obtained every 2 days for 10 days at pH 6.7 and 5.0 in N-free rooting solution. For all strains, the first nodules were formed in the region of the primary root which bore no root hairs (NRH zone) at the time of inoculation. Strains differed significantly in the speed with which they nodulated this zone. There were also similar significant differences in the percentage of plants nodulated by each strain in the NRH zone 10 days after inoculation. At both solution pH values, there was a positive correlation between the speed with which a strain nodulated the NRH zone in single culture and its nodulating competitiveness in mixed culture.

Acidity, aluminium and multiplication of Rhizobium tripolii: Possible mechanisms of aluminium toxicity

In the absence of Al the lower limit for multiplication by Rhizobium trifolii strain HP3 was pH 4.8–5.0 and for BELI192 was pH 4.4–4.6. 50 μm Al caused inhibition of multiplication of both strains in the range pH 4.6–5.6 despite precipitation of Al at pH values greater than 4.6. An increase in phosphate concentration from 10 to 100 μ m for cells which had been exposed to Al for 14 days at pH 4.5 and 5.5 did not remove the inhibitory effect of Al. Transfer of these cells to fresh Al medium caused a further decrease in numbers although transfer to non-stress medium allowed multiplication to occur. Equilibration of Al medium for 18 months or centrifugation of the medium before inoculation did not reduce the inhibitory effect of Al. Data from these experiments indicate that Rhizobium cells exposed to Al suffer an initial irreversible toxic effect and cells which survive this stage, perhaps due to environmental variation, then suffer a bacteriostatic effect. Calculation of ion activity products pAL + 3 p OH and pAl + pPO 4 and comparison with solubility products for AI(OH) 3 and AIPO 4 successfully predicted the precipitation of Al in the medium, but not the conditions under which Al was toxic to Rhizobium. Therefore, both the toxic form of Al and the mechanism(s) of toxicity remain unclear.

Acidity, aluminium and multiplication of Rhizobium trifolii: Effects of initial inoculum density and growth phase

Factors influencing the response of Rhizobium strains to acidity and aluminium were studied in an arabinose-galactose-glutamate liquid medium. Rhizobium trifolii strains HP3 and BEL 1192 multiplied at the same rate at pH S.S. BEL1192 multiplied at a slower rate at pH 4.5, and all produced visible turbidity from a low initial cell density. HP3 multiplied at pH 4.5 but produced no turbidity. The pH value of the medium increased when cell densities reached 10 6-10 7 cfu ml −1. Diauxic growth responses occurred, only at pH 4.5, and HP3 demonstrated a population effect at pH 4.5 with visible turbidity produced from high initial cell densities. 50 μm Al caused a decrease in numbers of BEL 1192 at pH4.5 and HP3 at pH 5.5 irrespective of initial cell densities. 50 μ m Al was more toxic to HP3 cells in log phase than in stationary phase.

Alteration of surface properties in a Tn5 mutant strain of Rhizobium trifolii 0403.

A symbiotically defective mutant strain of Rhizobium trifolii, UR251, was obtained by transposon Tn5 mutagenesis of R. trifolii 0403 rif and recognized by its partially ineffective (Fix +/-) phenotype on white clover plants. UR251 had a single Tn5 insertion in plasmid DNA, a wild-type plasmid pattern, and no detectable Mu DNA sequences originally present in the vector used for Tn5 mutagenesis. Agglutination by the clover lectin trifoliin A and attachment to clover root hairs was higher with UR251 than with the wild-type strain. The capsular polysaccharide (CPS) of UR251 was altered, as shown by a slower rate of CPS depolymerization with a CPS beta-lyase, PD-I; more pyruvate and less acetate and 3-hydroxybutanoate noncarbohydrate substitutions as quantitated by 1H nuclear magnetic resonance; and a higher pyruvyl transferase activity (enzymatic pyruvylation of lipid-bound saccharides). The site of increased pyruvylation in the CPS of UR251 was on the terminal galactose of the branch of the repeating oligosaccharide unit. These results show that the level of noncarbohydrate substitutions of the CPS as well as pyruvyl transferase activity are altered in R. trifolii UR251 and that trifoliin A-binding ability and clover root hair attachment are improved in this mutant strain of R. trifolii 0403 rif.

Comparative growth responses of N2-fixing and N-fertilized subterranean clover on acid soils

Effects of soil chemical properties on biological N2-fixation (BNF) byTrifolium subterraneum L. ‘Mt. Barker’ —Rhizobium trifolii strain 162×95 were studied on thirteen soils in a greenhouse experiment. Dry matter production of inoculated/+N plants indicated three groups of soils. Mean soil solution activities of Al3+ were 29.4, 9.8 and 0.1 μM for groups of soils with no BNF, limited BNF, and unlimited BNF, respectively. Using easily measurable soil chemical properties, the following equation related relative dry matter production to pH and soil Al saturation: Inoculated/+N×100=−140+46.83 (soil solution pH) −0.31 (Al concentration ×100/CEC) (r2=0.66).

Precautions in the use of yeast extract mannitol medium for evaluation of legume seed toxicity to Rhizobium

Toxicity of clover seeds to rhizobia has been reported. Yeast extract medium and a defined medium were used to test whether the growth medium was involved in the toxic response. Complete inhibition of four Rhizobium trifolii strains by subterranean clover ( Trifolium subterraneum L.), arrowleaf clover ( T. risiculosum Savi.), and red clover ( T. pratense L.) seeds consistently occurred on yeast extract amended agar but not on a defined medium. The toxic response probably involved a reaction between tannic acid from seeds and iron in the medium. Addition of 200 μM FeEDTA to yeast extract medium alleviated the growth inhibition response of rhizobia to all seeds tested.

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.

Management of Subterranean Clover in Pine Forested Range

Management of Subterranean Clover in Pine Forested Range

The response of white clover to different strains ofRhizobium trifolii in hill land reseeding: A second trial

S. 184 white clover was surface seeded into natural molinia pasture on wet stagnogley soil containing no indigenousRhizobium trifolii. Seedlings were ‘spray inoculated’ after emergence with each of three strains ofR. trifolii. The best of these treatments produced an eight fold improvement in dry matter in the seeding year, followed by a 28% improvement in the following year. The results confirm the potential benefits which may be achieved by inoculating clover with suitable strains of rhizobia. The data are compared with a previously reported trial on an adjacent site where benefits were much greater in the first year. The difference is attributed to the overall advantages conferred in the present trial by much higher seedling populations and less severe competition from native species in the establishment phase.

Selective synthesis of polysaccharides byRhizobium trifolii, strain TA-1

Rhizobium trifolii strain TA-1, produces one of each of the exocellular polysaccharides EPS, CPS and β-1,2-glucans as a major product during cultivation in glutamic acid-mannitol-salts (GMS) medium at 25°. In batch culture, the major exocellular polysaccharide product was acidic exopolysaccharide (EPS) under conditions of air saturation; capsular polysaccharide (CPS) under conditions of N-limitation and moderate oxygen supply; and cyclic β-1,2-glucans at high cell density and severe oxygen limitation.

The discernible, structural features of the acidic polysaccharides secreted by different Rhizobium species are the same

Octasaccharide repeating-units have been isolated from the acidic polysaccharides secreted by Rhizobium trifolii strain NA30, R. trifolii strain LPR5, R. leguminosarum strain LPR1, and R. phaseoli strain LPR49. ( R. trifolii is the symbiont of clover, R. leguminosarum, of peas, and R. phaseoli, of beans). The repeating units were formed by treating the polysaccharides with an enzyme produced by a bacteriophage. The glycosyl sequence and the structures and locations of the non-glycosyl substituents were shown to be identical for repeating units derived from all of these polysaccharides, except for that derived from the polysaccharide produced by R. trifolii NA30. Therefore, the discernible structural features of the acidic polysaccharides secreted by Rhizobium species cannot be the determinant of host specificity. In support of this conclusion is the observation that R. trifolii LPR5045, produced by curing R. trifolii LPR5 of its Sym plasmid (the Sym plasmid is required for symbiosis and host specificity), secreted a polysaccharide having the same structure (including identities and locations of nonglycosyl substituents) as that of the polysaccharide secreted by its plasmid-containing parent. Thus, the structural genes that encode for synthesis of the acidic polysaccharide secreted by R. trifolii LPR5045 are not located on the Sym plasmid, and neither are the genes that encode for synthesis and attachment of non-glycosyl substituents of the polysaccharide. The possibility remains that a quantitatively minor component of the acidic polysaccharide could be a host-specific determinant.

Effect of biosynthesis conditions on the chemical composition of the water-soluble polysaccharides of fast-growing rhizobia.

Water-soluble polysaccharides of fast-growing Rhizobia strains were produced in three conditions of biosynthesis: cells growing in a yeast extract medium, cells growing in a mineral medium and cells resting. Under these three conditions, three strains of Rhizobium leguminosarum (L 1S, L2S, L3S), three strains of Rhizobium phaseoli (P2S, P7S, P8S), and two strains of Rhizobium trifolii (T7S, T15S) produced polysaccharides which were precipitated with one volume of ethanol and purified. These polysaccharides contained (in % w/w): glucose (51.7-64.8), galactose (8.9-14.7), glucuronic acid (14-23), and some pyruvic and acetic acids which varied with the conditions of polysaccharide production. The polysaccharides produced in the conditions cited above, by four strains of Rhizobium meliloti (M5N1, M11S, M1-5, L5-30) differed from those of R. leguminosarum, R. phaseoli, and R. trifolii. The polysaccharides of R. meliloti contained (in% w/w): glucose (63.7-74), galactose (8.2-11), no glucuronic acid (except for R. meliloti M11S: 1%) and some pyruvic, acetic and succinic acids which varied with the conditions of polysaccharide biosynthesis.