Selection of Rhizobium leguminosarum bv. viceae strains for inoculation of Pisum sativum L. cultivars: Analysis of symbiotic efficiency and nodulation competitiveness

From an analysis of 481 Rhizobium leguminosarum bv. viceae strains with 7 pea cultivars in pot and field experiments, we demonstrated that effective strains could be isolated from a rich medium-acid grey forest soil of the Oröl area (Central region of the European part of Russia) but not from a poor acid podzolic soil of the St. Petersburg area (North-West Russia). The proportion of the isolates significantly increasing N accumulation in pea plants (10.2%) is higher than that of strains increasing the shoot dry mass (4.6%) in the pot experiments. The mean values of the increase for N accumulation (33.8%) upon inoculation are also higher than for shoot mass (27.0%) in these experiments. N accumulation in the inoculated pea plants in the pot experiments was significantly correlated with seed yield and seed N accumulation in field experiments, while for shoot dry mass these correlations were either weak or not significant. Two-factor analysis of variance demonstrated that the contribution of plant cultivars to the variation of the major symbiotic efficiency parameters is higher (30.8–31.6%) and contributions of cultivar-strain specificity is lower (5.4–8.8%) than the contributions of strain genotypes (13.4–14.9%). We identified an ineffective R. leguminosarum bv. viceae strain 50 which can be used as a tester for assessing the nodulation competitiveness of the effective strains by an indirect method (analysis of dry mass and N accumulation in pea plants inoculated with the mixture of the tested effective strains and the tester strain). The relative competitive ability (RCA) determined by this method was 75.7–82.8% for strain 52 but only 10.5–13.8% for strain 250a; this difference was confirmed by a direct method (use of the streptomycin-resistant mutants). Results of screening of the diverse collection of 53 effective R. leguminosarum bv. viceae strains by the indirect method permits us to divide them into 3 groups (32 high-competitive, 10 medium-competitive and 11 low-competitive strains) but reveals no correlation between the competitiveness and symbiotic efficiency. N accumulation in the pea shoots is demonstrated to be a much more suitable criterion than the shoot mass for selection either of the highly-effective or of highly-competitive (by the indirect estimation) R. leguminosarum bv. viceae strains in the pot experiments.     

Analysis of N-acyl homoserine-lactone quorum-sensing molecules made by different strains and biovars of Rhizobium leguminosarum containing different symbiotic plasmids

Strains of Rhizobium leguminosarum use a cell density-dependent gene regulatory system to assess their population density. This is achieved by the accumulation of N-acyl-homoserine lactones (AHLs) in the environment during growth of the bacteria and these AHLs stimulate the induction of various bacterial genes that are up-regulated in the late-exponential and stationary phases of growth. A genetically well-characterised strain of R. leguminosarum biovar viciae was found to have four genes, whose products synthesise different AHLs. We have analysed AHL production by four genetically distinct isolates of R. leguminosarum, three of bv. viciae and one of bv. phaseoli. Distinct differences were seen in the pattern of AHLs produced by the bv. viciae strains compared with bv. phaseoli and the increased levels and diversity of AHLs found in bv. viciae strains can be attributed to the rhiI gene, which is located on the symbiotic (Sym) plasmid and is up-regulated when the bacteria are grown in the rhizosphere. Additional complexity to the profile of AHLs is found to be associated with highly transmissible plasmid pRL1JI of R. leguminosarum bv. viciae, but this is not observed with some other strains, including those carrying different transmissible plasmids. In addition to AHLs produced by the products of genes on the symbiotic plasmid, there is clear evidence for the presence of other AHL production loci. Expression levels and patterns of AHLs can change markedly in different growth media. These results indicate that there is a network of quorum-sensing loci in different strains of R. leguminosarum and these loci may play a role in adapting to rhizosphere growth and plasmid transfer.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.     

Host-specific nodulation is encoded on a 14kb DNA fragment in Rhizobium trifolii

Summary The Rhizobium trifolii genes necessary for nodule induction and development have been isolated on a 14.0kb fragment of symbiotic (Sym) plasmid DNA. When cloned into a broad-host-range plasmid vector, these sequences confer a clover nodulation phenotype on a derivative of R. trifolii which has been cured of its endogenous Sym plasmid. Furthermore, these sequences encode both host specificity and nodulation functions since they confer the ability to recognize and nodulate clover plants on Agrobacterium and a fast-growing cowpea Rhizobium. This indicates that the bacterial genes essential for the initial, highly-specific interaction with plants are closely linked.     

repABC-based replication systems of Rhizobium leguminosarum bv. trifolii TA1 plasmids: incompatibility and evolutionary analyses

Soil bacteria of the genus Rhizobium possess complex genomes consisting of a chromosome and in addition, often, multiple extrachromosomal replicons, which are usually equipped with repABC genes that control their replication and partition. The replication regions of four plasmids of Rhizobium leguminosarum bv. trifolii TA1 (RtTA1) were identified and characterized. They all contained a complete set of repABC genes. The structural diversity of the rep regions of RtTA1 plasmids was demonstrated for parS and incα elements, and this was especially apparent in the case of symbiotic plasmid (pSym). Incompatibility assays with recombinant constructs containing parS or incα demonstrated that RtTA1 plasmids belong to different incompatibility groups. Horizontal acquisition was plausibly the main contributor to the origin of RtTA1 plasmids and pSym is probably the newest plasmid of this strain. Phylogenetic and incompatibility analyses of repABC regions of three closely related strains: RtTA1, R. leguminosarum bv. viciae 3841 and Rhizobium etli CFN42, provided data on coexistence of their replicons in a common genomic framework.     

Host genes inPisum sativum L. conferring resistance to EuropeanRhizobium leguminosarum strains

Summary Using primitive and wild pea plants from Afghanistan, Iran and Turkey, three host genes were detected, which confer resistance to nodulation by Rhizobium strains of cultivated peas from Europe. A dominant gene Sym 1 controls temperature-sensitive nodulation in pea cv. Iran. Another gene Sym 2 confers general resistance to a large number of European Rhizobium strains at all temperatures used. The degree of dominance of the latter gene is dependent on the Rhizobium strain used. A third gene Sym 4 is responsible for specific resistance to a single Rhizobium strain.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

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.Selected strains were evaluated for effectiveness on T. subterraneum cv. Clare, using the commercial strain TA1 as reference. Several of the isolated strains were more effective than TA1, indicating that local strains may be used to produce better inoculants.     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Modification of symbiotic interaction of pea (Pisum sativum L.) andRhizobium leguminosarum by induced mutations

Summary In pea (Pisum sativum L.), mutants could be induced, modified in the symbiotic interaction withRhizobium leguminosarum. Among 250 M₂-families, two nodulation resistant mutants (K₅ and K₉) were obtained. In mutant K₅ the nodulation resistance was monogenic recessive and not Rhizobium strain specific. Out of 220 M₂-families one mutant nod₃ was found which could form nodules at high nitrate concentrations (15 mM KNO₃). This mutant nodulated abundantly with severalRhizobium strains, both in the absence and presence of nitrate. Probably as the result of a pleiotropic effect, its root morphology was also changed. Among 1800 M₂-families, five nitrate reductase deficient mutants were obtained and one of them (mutant E₁) was used to study the inhibitory effect of nitrate on nodulation and nitrogen fixation.The results of the present investigation show that pea mutants which are modified in their symbiosis withRhizobium leguminosarum, can readily be obtained. The significance of such mutants for fundamental studies of the legume-Rhizobium symbiosis and for applications in plant breeding is discussed.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Characterization of two novel Rhizobium leguminosarum bacteriophages from a field release site of genetically-modified rhizobia

Two Rhizobium leguminosarum biovar viceae bacteriophages with contrasting properties were isolated from a field site in which the survival of genetically modified R. leguminosarum inoculants had been monitored for several years. Inoculant strain RSM2004 was used as the indicator for phage isolation and propagation. One phage, RL1RES, was temperate and could not replicate in any of the 42 indigenous R. leguminosarum field isolates tested although nested PCR indicated that phage sequences were present in six of the isolates. The second phage, RL2RES, was virulent, capable of generalised transduction, contained DNA with modified cytosine residues, and was capable of infecting all field isolates tested although the GM inoculant strain CT0370 was resistant. Sequence with homology to RL2RES was detected by nested PCR in six of the 42 field-isolates. These were not the same isolates that showed homology to RL1RES. The implication of these findings for the survival of rhizobial inoculants, and the ecology of phages and their host bacteria, are discussed.     

Location of nif genes on large plasmids in Rhizobium strains isolated from legume tree root nodules

Abstract The presence of plasmids in Rhizobium strains isolated from legume tree root nodules has been studied. Bacteria isolated from Acacia melanoxylon, A. cyanophylla, Prosopis chilensis and Sophora microphylla harbour 2 to 5 plasmids of an M _r higher than 115 · 10⁶. Hybridization experiments have shown DNA homology between plasmid pID1 ( R. meliloti 41 nifH and D genes) and one of the plasmids in each of the bacteria studied.     

Sugar-binding activity of pea (Pisum sativum) lectin is essential for heterologous infection of transgenic white clover hairy roots by Rhizobium leguminosarum biovar viciae

Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium. Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R. leguminosarum biovar viciae (R.l. viciae). Legume lectins contain a specific sugar-binding site. Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l. viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V). For every construct tested, nodulation by the homologous symbiont R.l. trifolii was normal. These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l. viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.     

Characterization of Rhizobium loti strains from the Salado River Basin

Thirty indigenous rhizobia strains, isolated from Lotus tenuis in the area of Chascomús and other regions of the Salado River Basin (Argentina), were characterized based on generation time, acid production, carbon utilization, protein profile, and molecular characterization by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by the polymerase chain reaction (PCR). The results indicated that native rhizobia isolates from the Chascomús area are predominantly fast and intermediate-growers. The unclassified rhizobia examined by PCR-RFLP were found to be closely related to the reference strains of validly described Rhizobium species.     

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394–5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using ¹H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.