Rhizobium Leguminosarum Biovar Trifolii Strain Research Articles

Competitiveness of a native Rhizobium leguminosarum biovar trifolii strain for nodule occupancy is manifested during infection

The stages in the nodulation process that determined the competitiveness of R. leguminosarum bv. trifolii (Rlt) strain 20–15, which proved to be highly competitive for nodulation in Iceland fields tests over several years, is analysed. White clover (Trifolium repens L.) roots were inoculated with inoculum mixtures containing three strains (Rlt 20-15, Rlt 8-9 and Rlt 32-28) in different proportions and cell densities. Competitiveness in root colonization, formation of infection threads and nodule development was assessed for Rlt 20-15 and its weakest competitor, Rlt 32-28. ERIC-polymerase chain reaction (PCR) DNA fingerprinting was used to identify inoculated strains recovered from root surfaces and individual nodules. GFP or DsRed tagged strains were used to determine identity in root hairs and nodules. Both strains colonized the root equally at all inoculum ratios tested. But, Rlt 20-15 initiated significantly more infection threads and formed more nodules than Rlt 32-28. These results show that Rlt 20-15 expresses its nodulation competitiveness during infection, either at infection thread initiation or during successive growth in the infection threads. The data presented support earlier observations that this strain competed well in the field in spite of its inferior ability to survive in the soil.

Symbiotic and saprophytic survival of three unmarked Rhizobium leguminosarum biovar trifolii strains introduced into the field

The symbiotic and saprophytic persistence of three unmarked Rhizobium leguminosarum biovar trifolii (Rlt) strains introduced into a field site in Iceland were followed. This site was free of clover cultivation and initially devoid of clover-nodulating rhizobia as tested by nodulation studies. Nodule occupancy by strains was identified based on their distinct ERIC-polymerase chain reaction (PCR) DNA fingerprint patterns. The survival and persistence of the individual strains in soil were monitored by the quantitative real-time PCR (qRT-PCR) assay, targeting the host-specific nodE gene. The most dominant strain in the nodule population, Rlt 20-15, showed relatively less saprophytic survival ability and maintained high numbers only in the presence of the appropriate host plant. Conversely, the minor nodule occupant, Rlt 32-28, persisted in soil at a relatively higher abundance both in the presence of its host legumes and in the presence of a non-host grass. The qRT-PCR assay was successfully applied to quantify rhizobial strains directly in soil without culturing or nodulation. However, the assay demonstrated less sensitivity compared with the plant infection most-probable-number (MPN) method for estimating the population size of rhizobia in soil. The quantitative detection limit of our qRT-PCR assays was 1 x 10(3) cells per gram of soil, as opposed to the MPN test which has a detection limit of 10 cells per gram of soil.

Complexity of phenotypes and symbiotic behaviour of Rhizobium leguminosarum biovar trifolii exopolysaccharide mutants.

Rhizobium leguminosarum biovar trifolii strain TA1 polysaccharide synthesis (pss) mutants in the pssD, pssP, pssT and pssO genes and altered in exopolysaccharide (EPS) synthesis were investigated. EPS-deficient mutants were also changed in lipopolysaccharide structure. All mutants exhibited varied sensitivities to detergents, ethanol and antibiotics, thus indicating changes in bacterial membrane integrity. Using pss mutants marked with the gusA gene, EPS-deficient mutants were found to have abnormalities in nodule development and to provoke severe plant defence reactions. The pss mutants that produced altered quantities of EPS with a changed degree of polymerisation generally occupied the younger developmental zones of the nodules and elicited moderate plant defence reactions.

Survival and plasmid stability of rhizobia introduced into a contaminated soil

The behaviour of Rhizobium strains introduced separately into soil from a contaminated site with high concentrations of heavy metals (mainly Zn and Hg), and the role of plasmids in the ecology of these rhizobia strains were studied. Six Rhizobium leguminosarum biovar trifolii strains, from different sources and with different plasmid contents, were selected. Two of them were isolated from nodules of subterranean clover plants ( Trifolium subterraneum) grown in the contaminated soil and four were from an uncontaminated soil. After inoculation with approximately 10 7 cells g −1 soil, of each strain, survival and plasmid stability were assessed over a period of 12–18 months. Differences in survival of Rhizobium strains were only detected more than 12 months after inoculation. After 18 months it was clear that survival in contaminated soil was greatest in the two strains originally isolated from that contaminated soil, and also by two of the strains originally isolated from uncontaminated soil. The latter two strains were also the only ones that showed changes in their plasmid profiles. The remaining isolates had the lowest populations, and their plasmid profiles were unchanged and similar to the parent strains.

Erratum to: Two novel chromosomal loci influence cultivar-specific nodulation failure in the interaction between strain ANU794 and subterranean clover cv. Woogenellup.

The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5'-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

Two novel chromosomal loci influence cultivar-specific nodulation failure in the interaction between strain ANU794 and subterranean clover cv. Woogenellup.

The nodulation failure resulting from the interaction between Rhizobium leguminosarum biovar trifolii strain ANU794 and the Trifolium subterraneum cv. Woogenellup was examined by transposon mutagenesis to resolve whether multiple determinants were involved in cultivar-specificity. Three new transposon-induced mutants of ANU794 (W72, W78 and W710) with significantly enhanced nodulation ability on cv. Woogenellup were identified. The W72 and W78 mutations are chromosomally-located, whereas the W710 mutation isplasmid-located. The ethylene synthesis inhibitor, aminoethoxyvinylglycine, fails to enhance the nodulation ability of ANU794, ANU7943 (csn1::Tn5) and W78 on cv. Woogenellup, but enhances the nodulation ability of W72,W710 and ANU7941 (nodM::Tn5). DNA sequencing of the W78 locus reveals strong homology to an unknown Mycobacterium open reading frame, and to several bacterial non-haem chloroperoxidases. The previously identified csn1 locus showed homology to the 50S ribosomal protein, L9, with the Tn5 insertion being located in the 5'-untranslated region. The results suggest that cultivar-specificity is mediated by at least two independent mechanisms or determinants, and not by a simple gene-for-gene interaction. The role of ethylene in cultivar specificity is discussed. Cultivar-specific interactions may prove useful in identifying pathways involved in efficient nodule formation and plant-microbe interactions.

Competition for Nodule Occupancy Between Introduced Strains of Rhizobium leguminosarum Biovar trifolii and its Influence on Plant Production

Interactions among white clover ( Trifolium repens L.) and selected Rhizobium leguminosarum biovar trifolii strains, and related clover/grass production, were studied in a field experiment during three growing seasons in Iceland. Weather conditions in this country are harsh and rhizobia are rare or absent in the soils. White clover and red fescue ( Festuca rubra L. ‘Leik’) were established in the field in May 1994, using a white clover breeding line (HoKv 9238) which originated from a breeding programme aimed at developing commercial cultivars for cold climates. Rhizobia were initially absent from the field site and clover was inoculated with one of three R. leguminosarum bv. trifolii strains, or a mixture of all three strains. White clover and grass yields were measured during three growing seasons after the establishment year. Nodule occupancy by the three Rhizobium strains was studied by ERIC-PCR DNA fingerprinting. In the mixed inoculum treatment, R. leguminosarum bv. trifolii strain 20-15 occupied 80–90% of the nodules tested, while strain 32-28 was present in up to 3% of the nodules tested. The dominance of strain 20-15 persisted and tended to increase throughout the 4 year experimental period. The introduced rhizobia persisted in high numbers in the soil 1 year after clover was removed and a non-host crop had been grown. ERIC-PCR was an efficient and reliable method by which to identify the Rhizobium strains from cells as well as directly from nodule suspensions. The selected breeding line of white clover persisted well under Icelandic conditions but produced low yield throughout the entire experimental period. Mixed strain inoculation had a positive effect, resulting in both higher white clover yields and higher total plant yields than on plots treated with single strain inoculum.

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii.

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.

Involvement of plasmids in saprophytic performance and sodium chloride tolerance of clover rhizobia W14-2 in vitro

Most information on the role of plasmids in the ecology of rhizobia in soil has been obtained using rhizobial derivatives lacking a single plasmid only. Rhizobium leguminosarum biovar trifolii strain W14-2 contains four plasmids (a, b, c, d). The role of plasmids in saprophytic characteristics and sodium chloride tolerance of W14-2 in vitro was investigated using a library of plasmid-cured W14-2 derivatives that includes derivatives containing only one plasmid and no plasmid, as well as complemented derivatives. Plasmid involvement in certain phenotypes such as swarming (plasmid a), utilization of glycerol (plasmid a), adonitol (plasmid b), rhamnose and sorbitol (plasmid c) was tracked to single plasmids and was not influenced significantly by the other plasmids present in the cell. Presence of plasmid b or c was necessary for the utilization of arabinose and raffinose, whereas presence of both plasmids b and d was required for tolerance to high levels of sodium chloride. The latter phenotype may correspond to tolerance to chloride toxicity or to osmotolerance. Plasmids influenced growth of the strain in liquid HP (peptone-yeast extract-tryptone), a complex medium that was investigated as it contains a wide range of carbon substrates. Positive contributions of plasmids a (shorter duration of the lag phase and increased maximum density of cells), b and c (shorter log-phase generation time) were shown. Plasmid d was detrimental to the strain in HP when it was the only plasmid present in the cell, especially regarding log-phase generation time. Similarly, the derivative with only plasmid d was more sensitive to sodium toxicity than any other derivative, including the plasmidless derivative. Interactions between plasmids influenced growth in HP broth and salt tolerance, and detrimental effects of plasmid d were little expressed if any when the cell contained one plasmid or more besides plasmid d. No plasmid-cured derivative grew better than the wild-type overall, suggesting that the extrachromosomal genome contributes to the saprophytic competence of W14-2 in soil.

Nodule formation by clover-Rhizobium carrying chromosomal nod genes.

Plasmid pCINod1 carrying attP of a lysogenic rhizobiophage and the nod genes was constructed. Inoculation of a conjugation mixture of Escherichia coli harbouring pCINod1 and a nodulation-defective Rhizobium leguminosarum biovar trifolii strain resulted in formation of root nodules on white clover. Rhizobium transconjugant strain CNH1 isolated from these nodules had integrated pCINod1 into its chromosome. The light and electron microscopic observation of the nodulation process revealed that the nod genes on the chromosome of strain CNH1 were completely functional.

The construction, detection and use of bioluminescent Rhizobium leguminosarum biovar trifolii strains

A. CRESSWELL, L. SKOT AND A.R. COOKSON. 1994. The gene encoding the firefly luciferase enzyme (luc) was introduced to Rhizobium leguminosarum biovar trifolii strains with a view to using the resulting bioluminescent strains to study the survival of genetically-engineered rhizobia in soil microcosms. The genetically-engineered micro-organisms (GEMs) behaved similarly to their parent strains with respect to growth rate in laboratory media and in their symbiotic performance with their host plants. No gene transfer could be detected in laboratory mating experiments. When inoculated onto a non-sterile soil the population of the GEM declined sharply from an initial cell density of 2 times 1077 g-1 soil to approach a stable cell density of approximately 3 times 102 g-1 after 150 d. Direct photography of bioluminescent rhizobia enabled the detection of colonies as small as 0.1 mm in diameter without the need for transferring colonies onto filter paper. When a Rhizobium strain carrying the luc marker on a plasmid was used as inoculant it was possible to visualize differences in colonization of the rhizosphere of white clover and ryegrass by contact print and colour transparency films. The photographic detection methods described here demonstrate the possibilities of using bioluminescent rhizobia for assessing their survival in soil, and for looking at rhizosphere populations which may be an important site for potential gene transfer.

Oxygen sensitivity and metal ion-dependent transcriptional activation by NIFA protein from Rhizobium leguminosarum biovar trifolii.

The NIFA protein from Rhizobium leguminosarum biovar trifolii (R. trifolii) strain ANU843 lacks an N-terminal domain present in homologous NIFA proteins from other diazotrophs. The R. trifolii nifA gene product is unstable when expressed in Escherichia coli under both aerobic and microaerobic conditions. Stability is increased by fusion of additional amino acids to the N-terminus of the protein or by expression of nifA in sno mutant (presumed protease deficient) strains of E. coli. Transcriptional activation in vivo by R. trifolii NIFA decreases under aerobic growth conditions, or when cultures are depleted of metal ions. In sno mutant strains this decrease in activity reflects a loss of specific activity rather than proteolytic degradation, implying that R. trifolii NIFA requires metal ions for activity and is oxygen sensitive. The addition of 30 amino acids to the amino-terminus of R. trifoli NIFA results in an oxygen-tolerant protein, with metal ion-dependent activity. Metal ions are therefore not only required for oxygen sensing by R. trifolii NIFA but may play an additional role in determining NIFA structure or activity.

The nodDABC Genes of Rhizobium leguminosarum biovar trifolii Confer Root-Hair Curling Ability to a Diverse Range of Soil Bacteria and the Ability to Induce Novel Root Swellings on Beans

Cloned DNA fragments coding for the nodDABC genes of Rhizobium leguminosarum biovar trifolii strain ANU843 were introduced into Rhizobium strains possessing Sym plasmid deletions. These strains were able to: (a) synthesise four butanol-soluble Nod metabolites; (b) affect the normal growth pattern of plant root hairs of a wide range of host and non-host legumes; and (c) induce many root outgrowths on Phaseolus plants. The four Nod metabolites produced by these strains were labelled by supplying cultures with 14C-acetate in the presence of a flavonoid inducer of nod gene expression. In contrast, more than ten Nod metabolites were synthesised by wild-type strains or constructed strains containing the full complement of R. leguminosarum biovar. trifolii nodulation and host specific nodulation genes. Strain ANU845 containing nodDABC did not induce infection threads or nodule initiation sites but distorted and curled cells in plant root hairs. However strain ANU845 induced root outgrowths on beans (Phaseolus vulgaris) that appeared to result from a proliferation of the epidermal tissue. Transfer of plasmids bearing nodDABC to various Gram-negative bacteria, Agrobacterium tumefaciens, Pseudomonas aeruginosa, Lignobacter sp., Azospirillum brasilense and Escherichia coli, and different non-nodulating mutant rhizobia conferred on these strains the ability to cause root-hair curling and distortions. Several strains induced root-hair curling on clover and a range of other non-host legumes. We suggest that the expression of nodDABC in a range of soil bacteria may extend or alter the effects of these soil bacteria on the roots of host plants.

Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants

From several native clover species, growing in six different soil types, 170 Rhizobium leguminosarum biovar trifolii strains were isolated, covering the central and southern regions of Portugal. The effectiveness of the strains varied from ineffective to highly effective on T. subterraneum cv. Clare and on T. fragiferum cv. Palestine, with a predominance of medium and high effectiveness on both host plants. The effectiveness was not influenced by provenence (soil or plant), except for the strains from the rankers soils and for the strains isolated from T. pratense, that were ineffective or medium effective on T. subterraneum.

NodT, a positively-acting cultivar specificity determinant controlling nodulation of Trifolium subterraneum by Rhizobium leguminosarum biovar trifolii

Rhizobium leguminosarum biovar trifolii strain TA1 nodulates a range of Trifolium plants including red, white and subterranean clovers. Nitrogen-fixing nodules are promptly initiated on the tap roots of these plants at the site of inoculation. In contrast to these associations, strain TA1 has a 'Nod-' phenotype on a particular cultivar of subterranean clover called Woogenellup (A.H. Gibson, Aust J Agric Sci 19: (1968) 907-918) where it induces rare, poorly developed, slow-to-appear and ineffective lateral root nodules. By comparing the nodulation gene region of strain TA1 with that of another R. leguminosarum bv. trifolii strain ANU843, which is capable of efficiently nodulating cv. Woogenellup, we have shown that the nodT gene (B.P. Surin et al., Mol Microbiol 4: (1990) 245-252) is essential for nodulation on cv. Woogenellup. The nodT gene is naturally absent in strain TA1. A cosmid clone spanning the entire nodulation gene region of strain TA1 was capable of conferring nodulation ability to R.l. bv. trifolii strains deleted for nodulation genes, but only on cultivars of subterranean clovers nodulated by strain TA1. This shows that cultivar recognition events are, in part, determined by genes in the nodulation region of strain TA1. Complementation studies also indicated that strain TA1 contains negatively-acting genes located on the Sym plasmid and elsewhere, which specifically block nodulation of cv. Woogenellup.

Production and epitope analysis of monoclonal antibodies against a Rhizobium leguminosarum biovar trifolii strain

Heat-treated cells of Rhizobium leguminosarum biovar trifolii strain 162X95 were used to produce monoclonal antibodies (MAbs). The fusion produced three cross-reactive MAbs and eight MAbs specific for the immunizing strain and a group of five other R. trifolii strains from the same geographic region where 162X95 was isolated (California). Seven MAbs were analyzed by competitive enzyme-linked immunosorbent assay to determine the number of different epitopes detectable on strain 162X95. The results indicated that six MAbs reacted with the same or overlapping epitopes, and the seventh MAb gave inconclusive results.

Specificity of association between Bacillus isolates and genotypes of Lolium perenne and Trifolium repens from a grass – legume pasture

Specificity between plants and associated rhizosphere bacteria was investigated using species and genotypic mixtures of Lolium perenne L. (perennial ryegrass) and Trifolium repens L. (white clover) inoculated with Bacillus strains isolated from rhizosphere soil of the pasture plants. The genotypic identity of plants was controlled by using stolon tips (Trifolium) and tillers (Lolium) of three genotypes of each species collected from a 45-year-old permanent pasture. Inoculation of plants in the greenhouse with Bacillus isolates that had coexisted in the field with the Lolium component of a Lolium–Trifolium mixture increased Lolium root and shoot weight. Root and nodule weight of Trifolium in mixture regardless of its genotype was also higher when coexistent Bacillus–Lolium combinations were present. The presence of other coexistent Bacillus–plant or plant–plant combinations did not enhance performance of either pasture species. The identity of the Rhizobium leguminosarum biovar trifolii strains that formed Trifolium root nodules was unaffected by inoculation with Bacillus. Key words: Trifolium repens, Lolium perenne, Bacillus, specificity, rhizosphere, yield.

Rhizobium population genetics: Effect of clover variety and inoculum dilution on the genetic diversity sampled from natural populations

Strains ofRhizobium leguminosarum biovartrifolii were isolated from nodules formed on white clover plants (Trifolium repens) following inoculation with soil suspensions, and examined for allozyme variation. A similar spectrum of genetic variants was recovered from each of four different clover cultivars, and over a 104-fold range of soil dilutions. The frequency of allozyme variants was not significantly affected by the choice of cultivar or dilution.