An Ecological Study of Marked Rhizobium trifolii Strains on the Host Plant Trifolium repens var. Huai in an Acidic Peat and a Neutral Mineral Soil

An ecological study of the nodulation of Trifolium repens var. grassland Huai by genetically marked Rhizobium trifolii was carried out in two Irish soils, a neutral mineral and an acidic peat. An indigenous population of 2 x 10⁴ R. trifolii /g was found in the mineral soil. In the peat soil, 4 x 10¹ R. trifolii /g was found in the uninoculated peat. This number increased to 4.5 x 10⁵ R. trifolii /g, however, eight weeks after the peat soil was neutralized, supplemented with nutrients and sown with uninoculated clover seed. Indigenous R. trifolii strains from the mineral soil were effective whereas strains from the peat soil were ineffective on the host plant T. repens under plant room conditions. The introduced strains were inoculated on to clover seed at the rate of 1 x 10⁵ R. trifolii /seed. In the mineral soil, the introduced inoculum failed to establish at any period during the growing season. In the peat soil, the percentage establishment of the introduced inoculum varied from 40-50% of nodules selected eight weeks after sowing to 70-90% of nodules selected at the end of the growing season.     

Toxicity of Sodium and Chloride Ions to Rhizobium spp. in Broth and Peat Culture

The growth of a strain of Rhizobium trifolii and of R. meliloti was studied in broth and peat cultures to determine the relative toxicity of Na⁺ and Cl^-. The following salts were added in a range of concentrations: Na₂HPO₄ as a source of Na⁺, CaCl₂.2H₂O as a source of Cl^-, and NaCl. Disodium hydrogen orthophosphate affected the growth rate of both strains in broth culture but not in peat culture. Unexpectedly, calcium chloride was more toxic than NaCl in broth and peat culture. The toxicity of NaCl can be ascribed to the Cl^-. Rhizobium meliloti strains grew on 3·5% NaCl after adaptation during a long period. Rhizobia for soya bean and cowpea grew at 0·5% NaCl and those for clover and pea, at 1·0% NaCl.     

Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera.

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.     

A Survey of Rhizobia in Farm Soils at Wye College, Kent

S ummary : Numbers of Rhizobium meliloti, R. trifolii, R. leguminosarum and R. lupini in different fields near Wye, Kent, were determined by the 'plant dilution'method. R. trifolii was most abundant, followed by R. leguminosarum , with R. meliloti and R. lupini less abundant and more restricted in their distribution. Isolates were made from nodules taken from the highest dilutions of soil that produced nodules and were tested for effectiveness in fixing nitrogen in agar tube culture. In general, isolates were effective or fairly effective. Highly effective isolates of R. trifolii were found in a very calcareous soil which had grown barley for 9 years. A permanent pasture which received higher doses of N fertilizer than other fields, contained strains of R. trifolii with a wider range of effectiveness, some giving only 50% of the dry matter production of the standard strain. The survival of R. meliloti 2001 in fields in which lucerne had not been grown for 9—14 years was studied, using a serological technique. No isolate was identified with certainty as strain 2001, but 55 of the 110 isolates tested showed some common features with this strain.     

Production of Antimicrobial and Bacteriocin-Like Substances by Rhizobium trifolii

Rhizobium trifolii strains IARI and Rel-1 produced substances with broad and narrow activity spectra, respectively. Reproducible inhibitory zones of various sizes produced by R. trifolii IARI (2 to 14 mm) and R. trifolii Rel-1 (2 to 6 mm) were detected, depending upon the indicator organism used. The maximum production of these substances by both strains of R. trifolii was observed on l-arabinose agar. A preliminary characterization of the antimicrobial substance produced by strain IARI showed resistance to heat (75 to 80 degrees C for 45 min), trypsin, lysozyme, DNase I, and RNase A. On the other hand, the substance produced by strain Rel-1 showed sensitivity to heat (75 to 80 degrees C for 45 min) and trypsin, but resistance to lysozyme, RNase A, and DNase I.     

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin.     

Examining growth and survival of cowpea rhizobia in Jamaican peat

We have examined the survival of four cowpea rhizobia strains in Jamaican peat to determine its suitability as inoculant carrier. All strains survived well since more than 10⁷ cells of rhizobia per gram of peat were recovered from the inoculant after storage for 6 months at 30C. Survival of cowpea rhizobia was better when inoculants were stored at 4 than 30C. The native strains JRC29 and JRW3 (isolated in Jamaica) survived much better than the introduced strains MI-50A and IRC291 (isolated in West Africa). Survival of cowpea rhizobia was not significantly increased when peat was mixed with 1% sucrose. Our results suggest that Jamaican peat may be used as a carrier for inoculant production.     

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.     

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.     

Differential stimulation and inhibition of growth of Rhizobium trifolii strain T1 and other Rhizobium species by various carbon sources

The physiological properties of Rhizobium trifolii strain T1 were studied in detail, since this strain has many useful characteristics and appears ideal for development as a reference strain for R. trifolii. Some tricarboxylic acid cycle intermediates and related compounds were found to stimulate growth in the presence of sucrose and arabinose, while others inhibited growth partially or completely. Other R. trifolii strains behaved likewise. Moreover, similar responses were also observed with other Rhizobium species, both fast-growing and slow-growing. On the basis of these growth responses, the various species of fast-growing and slow-growing rhizobia could be differentiated. Of the fast-growers tested, R. trifolii and R. leguminosarum are much more closely related to each other than either is to R. meliloti. Similarly, the slow-growing cowpea rhizobia are more closely related to R. japonicum than either group is to R. lupini. It is proposed that strain T1 should be developed as the reference strain for Rhizobium trifolii.     

Use of plasmid R68.45 for constructing a circular linkage map of the Rhizobium trifolii chromosome.

Plasmid R68.45 was used to promote conjugal transfer of chromosomal markers in Rhizobium trifolii RS55. Analysis of two-factor and three-factor crosses among R. trifolii strains enabled construction of a circular linkage map of the R. trifolii chromosome, containing 17 nutritional and resistance markers.     

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

AUTOMATED 35S-LABELLED PROTEIN ELECTROPHORESIS FOR IDENTIFICATION OF STRAINS OF RHIZOBIUM LEGUMINOSARUM bv. TRIFOLII

A technique for strain-level identification within a Rhizobium biovar is described, based on automated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of ³⁵S-labelled proteins, offering substantial improvements on existing SDS-PAGE methods particularly in the areas of standardization of electrophoresis conditions and rapidity of positive identification. Gels were analysed with a β-scanner, the beta particle emission data being directly relayed to an IBM PC/AT computer for subsequent manipulation. Analysis of the total protein profiles obtained by this method revealed regions of variability between strains of R. leguminosarum bv. trifolii. Automated comparison of these regions enabled identification of strains. The method was successfully used for identifying root nodule isolates obtained from competition studies between known pairs of R. leguminosarum bv. trifolii strains inoculated onto Trifolium repens.     

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.     

Trifolin: a Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.     

Expression of an agrocin-encoding plasmid of Agrobacterium tumefaciens in Rhizobium meliloti

Plasmid RP4 was used to mobilize the agrocin 84-encoding plasmid, pAg396, from Agrobacterium tumefaciens strain 396 to A. tumefaciens C58 and C58CI as well as Rhizobium meliloti. It was transferred to, but not stably maintained in, R. leguminosarum. It could not be transferred to R. lupini, R. japonicum or R. trifolii. Plasmid pAg396 did not segregate in R. meliloti and produced levels of agrocin comparable to the parental strain A. tumefaciens 396. The potential of agrocin producing R. meliloti in biological control of crown gall is being investigated.     

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.     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Rhizobium lipopolysaccharide modulates infection thread development in white clover root hairs.

The interaction between Rhizobium lipopolysaccharide (LPS) and white clover roots was examined. The Limulus lysate assay indicated that Rhizobium leguminosarum bv. trifolii (hereafter called R. trifolii) released LPS into the external root environment of slide cultures. Immunofluorescence and immunoelectron microscopy showed that purified LPS from R. trifolii 0403 bound rapidly to root hair tips and infiltrated across the root hair wall. Infection thread formation in root hairs was promoted by preinoculation treatment of roots with R. trifolii LPS at a low dose (up to 5 micrograms per plant) but inhibited at a higher dose. This biological activity of LPS was restricted to the region of the root present at the time of exposure to LPS, higher with LPS from cells in the early stationary phase than in the mid-exponential phase, incubation time dependent, incapable of reversing inhibition of infection by NO3- or NH4+, and conserved among serologically distinct LPSs from several wild-type R. trifolii strains (0403, 2S-2, and ANU843). In contrast, infections were not increased by preinoculation treatment of roots with LPSs from R. leguminosarum bv. viciae strain 300, R. meliloti 102F28, or members of the family Enterobacteriaceae. Most infection threads developed successfully in root hairs pretreated with R. trifolii LPS, whereas many infections aborted near their origins and accumulated brown deposits if pretreated with LPS from R. meliloti 102F28. LPS from R. leguminosarum 300 also caused most infection threads to abort. Other specific responses of root hairs to infection-stimulating LPS from R. trifolii included acceleration of cytoplasmic streaming and production of novel proteins. Combined gas chromatography-mass spectroscopy and proton nuclear magnetic resonance analyses indicated that biologically active LPS from R. trifolii 0403 in the early stationary phase had less fucose but more 2-O-methylfucose, quinovosamine, 3,6-dideoxy-3-(methylamino)galactose, and noncarbohydrate substituents (O-methyl, N-methyl, and acetyl groups) on glycosyl components than did inactive LPS in the mid-exponential phase. We conclude that LPS-root hair interactions trigger metabolic events that have a significant impact on successful development of infection threads in this Rhizobium-legume symbiosis.     

Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region.

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.