Rhizobium Leguminosarum Biovar Research Articles

Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar. phaseoli

Rhizobium leguminosarum bv. phaseoli strains P31 and R1, Serratia sp. strain 22b, Pseudomonas sp. strain 24 and Rhizopus sp. strain 68 were examined for their plant growth-promoting potential on lettuce and forage maize. All these phosphate solubilizing microorganisms (PSM) were isolated from Quebec soils. The plants were grown in field conditions in three sites having high to low amounts of available P. In site 1 (very fertile soil), strains R1 and 22b tended to increase the dry matter yield of lettuce shoots (p≤0.10). Lettuce inoculated with rhizobia R1 had a 6% higher P concentration (p≤0.10) than the uninoculated control. In site 2 (poorly fertile soil), the dry matter of lettuce shoots was significantly increased (p≤0.05) by inoculation with strain P31 and 24 plus 35 kg ha-1 P-superphosphate, or with strain 68 plus 70 kg ha-1 P-superphosphate. In site 3 (moderately fertile soil), the dry matter of maize shoots was significantly increased (p≤0.05) by inoculation with strain 24 plus 17.5 kg ha-1 P-superphosphate, or with strain P31 plus 35 kg ha-1 P-superphosphate. Inoculation with PSM did not affect lettuce P uptake in the less fertile soil in site 2. In site 3 with the moderately fertile soil, maize plants inoculated with strain R1 had 8% higher P concentration than the uninoculated control (p≤0.01), and 6% with strains P31 and 68 (p≤0.05). The results clearly demonstrate that rhizobia specifically selected for P solubilization function as plant growth promoting rhizobacteria with the nonlegumes lettuce and maize. The P solubilization effect seems to be the most important mechanism of plant growth promotion in moderately fertile and very fertile soils when P uptake was increased with rhizobia and other PSM.

Population dynamics of indigenous and genetically modified rhizobia in the field

SUMMARYMany factors have been shown to affect rhizobial populations in soil. These include: soil fertility; physical properties such as pH and clay content; biotic factors such as distribution of the host plant and the prevalence of predators; and climatic effects including temperature and rainfall. Extremes of soil pH, temperature and moisture are not favourable to rhizobia. In certain circumstances, some rhizobial species seem to survive as part of the saprophytic soil microflora, whereas others cannot be detected unless their leguminous plant host is present. There have been numerous reports over the past century on factors which influence rhizobial survival, reflecting the importance of rhizobial inoculants in agriculture. Many of these results appear to be contradictory, presumably because of the complexity of the interactions between different influences, which this review of the literature attempts to clarify. Results from monitoring population dynamics of different rhizobial species and biovars under various crops at Rothamsted demonstrate that populations of Rhizobium leguminosarum biovars survive in the absence of their host plants at c. 104–105 modulating cells g‐1 soil, and increase only about threefold following cultivation of the host. In contrast, Sinorhizobium meliloti could not be detected before its host was grown, but increased to 106 nodulating cells g‐1 soil when lucerne was cultivated. The behaviour of a genetically modified R. leguminosarum bv. viciae strain, RSM2004, following field release as an inoculant, was similar to that of the indigenous population, numbers remaining stable in the absence of the host following an initial decline, with a small but significant numerical advantage being conferred by the presence of the host in the subsequent years. A second genetically modified R. leguminosarum bv. viciae inoculant, CT0370, was found to survive in numbers similar to those of the indigenous population.

Growth and symbiotic performance of faba bean inoculated withRhizobium leguminosarumbiovar.viciaestrains tolerant to salt

Abstract Strain GRA19 of Rhizobium leguminosarum biovar. viciae was found to be tolerant to low levels of salt (50 mM) by comparing growth under stress conditions to that in the absence of stress. Growth and symbiotic N2 fixation (acetylene reduction activity) under saline conditions of the faba bean (Vicia faba L.) cultivar Alameda inoculated with GRA19 were reduced. Salinity significantly decreased shoot and root dry weight, nodule dry weight, and average individual nodule dry weight. Nitrogen fixation was more severely affected by salinity than plant growth and nodulation. Evidence presented here suggests the need for selecting faba bean cultivars that are tolerant to salt stress for breeding programs in order to improve the symbiotic performance of R. leguminosarum biovar. viciae.

Growth and nitrogen assimilation in nodules in response to nitrate levels inVicia fabaunder salt stress

This study analyses the effects of salt on the effective symbiosis of faba bean (Vicia faba L. var. minor cv. Alborea) and salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA19 grown with two KNO 3 levels (2 and 8 mM). The addition of 8 mM KNO 3 to the growth medium increases plant tolerance to salinity even with a concentration of 100 mM NaCl. This KNO 3 level in control plants reduced the N 2 fixation. For 2 and 8 mM KNO 3 the plants treated with NaCl reduced N 2 fixation to identical values. The activity of the enzymes mediating ammonium assimilation in nodules (GS, NADH-GOGAT and NADH-GDH) was decreased by high KNO 3 levels. The results show that NADH-GOGAT activity was more markedly inhibited than was GS activity by salinity, therefore NADH-GOGAT limits the ammonium assimilation by nodules in V. faba under salt stress. The total proline content in the nodule was not related to salt tolerance and thus does not serve as a salttolerance index for V. faba.

Influence of host genotypes on growth, symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N2 fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.

Biosynthesis of cyclic ?-(1,2)-glucans in Rhizobium leguminosarum biovars viciae, phaseoli and trifolii

Inner membranes of Rhizobium leguminosarum biovars viciae, phaseoli, and trifolii synthesized a heterogenous family of neutral cyclic β-(1,2)-glucans in a reaction system that used oligosaccharide intermediates covalently bound to a large protein. This glucoprotein showed a slightly lower mobility on SDS-polyacrylamide gels (apparent mol. mass of 320 kDa) than the β-(1,2)-glucan intermediate protein of Rhizobium meliloti. In vivo pulse-label experiments with growing cells of R. leguminosarum biovar trifolii RS800 using radioactive glucose showed that few species of cyclic β-(1,2)-glucans were synthesized and up to 30% were substituted with charged non-glycosidic residues, probably sn-1-phosphoglycerol.

Biosynthesis of cyclic ?-(1,2)-glucans in Rhizobium leguminosarum biovars viciae, phaseoli and trifolii

Biosynthesis of cyclic ?-(1,2)-glucans in Rhizobium leguminosarum biovars viciae, phaseoli and trifolii

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of polymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C18:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viciae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the C18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

AbstractA collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.

Rhizobiumhost-range extension by introduction ofRhizobium trifolii nodGenes

Abstract Although the symbiosis between legume and root nodule bacteria is generally considered to be highly specific, the degree of specificity varies widely among rhizobia (Gabriel and Rolfe 1990; Young and Johnston 1989). Some strains show a very narrow host-range, for example, Rhizobium leguminosarum biovar trifolii nodulates only Trifolium. In contrast, Rhizobium sp. NGR 234 nodulates 35 legume genera, in addition to the non-legume Parasponia (Lewin et al. 1987).

Improved growth and yield of Faba beans (Vicia faba cv. Fiord) by inoculation with strains of Rhizobium leguminosarum biovar. viciae in acid soils in south west Victoria

The response of faba beans (Vicia faba L. cv. Fiord) to seed inoculation with eight strains of Rhizobium leguminosarum biovar. viciae was examined in field experiments at six sites on acid soils in south-west Victoria. At two of the sites, two additional strains were examined, and in 1988, 14 strains were examined at one site. Very low natural populations of R. leguminosarum bv. viciae were found at the experimental sites. Most strains resulted in improved early nodulation and increased grain yield at all sites, when compared to inoculation with the commercial strain of rhizobia (SU391). Plant dry matter production and nitrogen accumulation in the plant shoot tissue was also increased at one site during the flowering period by some strains. Large visual differences between plots inoculated with SU391 and other strains were evident at most sites. Most uninoculated treatments were not nodulated and yielded very poorly. Treatments inoculated with the strain SU391 performed similarly to the uninoculated treatments.

Number of Rhizobia and delayed inoculation influence nodulation of clovers

The relationship between numbers of rhizobia and nodulation response of legumes is of considerable practical importance. Experiments were done under controlled conditions to determine the influence of numbers of Rhizobium leguminosarum biovar. trifolii on nodulation of arrowleaf clover (Trifolium vesiculosum Savi.) and crimson clover (T. incarnatum L.). Numbers of rhizobia in excess of 1000 per seed did not substantially increase earliness of nodulation or total number of nodules formed on the taproot. Nodules, however, were formed nearer the top of the taproot as numbers of rhizobia increased to 100,000 per seed. Delayed inoculation experiments indicated that nodulation sites for these clovers only remained susceptible to infection for less than 1 day. Delaying inoculation for 4 days resulted in only a 1 to 2 day delay in nodulation for arrowleaf and crimson clovers respectively and no delay for subterranean clover (T. subterraneum L.). Apparently, larger seedlings nodulated faster.

The survival of bacteria exposed to desiccation on surfaces associated with farm buildings.

The survival of 11 species of Gram-negative and Gram-positive bacteria was examined on different surfaces exposed to desiccation. There were large variations between species; Pseudomonas spp. and Rhizobium leguminosarum biovars survived for less than 2 d, whilst Enterococcus spp. survived for more than 11 weeks. The type of surface on to which the bacteria were deposited affected survival, but with different effects between species. In addition the survival of spontaneous nalidixic acid-resistant (Nal-r) mutants of a natural Escherichia coli isolate were compared. Overall the differences were slight, but of seven resistant mutants, five survived better than the parent whilst one survived less well. Nine transposon insertion derivatives of one of the Nal-r mutants (ECO80) which survived better than the parent were compared; all survived similarly to the parent except ECO883 which survived less well. The growth characteristics of ECO883 and ECO80 were compared; at high osmotic pressures (> 0.4 mol 1-1 NaCl) ECO883 grew more slowly and showed a longer lag time than the parent. Of the osmoregulatory functions studied, ECO883 appeared to be altered with respect to K+ transport or accumulation, although the transposon insertion had occurred in a gene distant from known K+ transport genes.

A Rhizobium tropici DNA region carrying the amino‐terminal half of a nodD gene and a nod‐box‐like sequence confers host‐range extension

Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculenta and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.

Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov.

A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

Evaluation of a Strategy for Identifying Nodulation Competitiveness Genes in Rhizobium Leguminosarum Biovar Phaseoli

Rhizobium leguminosarum biovar phaseoli strain KIM5s is consistently much more competitive than strain CE3 in nodulation of beans (Phaseolus vulgaris L.) in the laboratory and in the field. To identify genes that contribute to the competitiveness of KIM5s, we transferred a cosmid library containing KIM5s DNA into CE3 and applied the transconjugants to bean plants to allow the plants to enrich for those with enhanced nodulation competitiveness. The nodule isolates were then applied to plants for further enrichment. Of 75 isolates from nodules sampled after the two enrichments, 9 were more competitive than CE3. For example, when outnumbered in the inocula 40-fold by a reference strain, these nine strains typically occupied 15-40% of the nodules compared with 0-3% for CE3. However, when these strains were cured of the cosmids, they remained highly competitive, demonstrating that the enhanced competitiveness of the strains was not associated with the cosmids. We found no evidence for cosmid insertion into the chromosome or for cosmid-induced genetic changes in these cured strains. We found some evidence suggesting that their altered competitiveness was due to spontaneous genetic changes that did not involve the cosmids. Although these highly competitive variants remain genetically uncharacterized, they may provide insight into bacterial traits that contribute to, or detract from, successful nodulation competitiveness.

Enumeration of indigenous Rhizobium leguminosarum biovar Trifolii in soils previously treated with metal-contaminated sewage sludge

The effects of heavy metals from metal-contaminated sewage sludge on the indigenous population of Rhizobium leguminosarum biovar trifolii in the soils of two well controlled field experiments at Braunschweig in northeast Germany are reported. Both experiments were in the same field, but one was on an old arable soil and the other on an ex-woodland soil. The following treatments were applied to both experiments: inorganic fertilizer at 180 kg N ha −1 yr −1 (control soils); uncontaminated sludge at 100m 3 ha −1 yr −1 or 300m 3 ha −1 yr −1; or metal-contaminated sludge at the same two rates. In the ex-woodland experimental soil, rhizobial numbers decreased by an order of magnitude in plots treated with 100 m 3 yr −1 contaminated sludge compared to the control plots and plots receiving the same amount of uncontaminated sludge. The total Zn and Cd concentrations in these plots were close to the German limits, but were well below the U.K. and E.C. upper limits. Even with 300m 3 yr −1 uncontaminated sludge rhizobial numbers decreased by several orders of magnitude compared to the control soils, in both field experiments: at both sites one of the four replicate plots had no rhizobia, whereas the other three plots had decreased numbers. Metal concentrations in these plots were well below the U.K. and E.C. upper limits, except Zn which was close to the German limit, and ranged from (mg kg −1): Zn, 200–250; Cu, 46–62; Ni, 16–23 and Cd, 0.9–1.6. In plots receiving 300m 3 yr −1 contaminated sludge, in both field experiments, rhizobial numbers were further decreased compared to plots receiving uncontaminated sludge at the same rate and were several orders of magnitude smaller than in the control plots. For example, in the old arable site, three out of four plots had no rhizobia, with one plot containing 20 cell g −1soil. In the ex-woodland site, two plots out of four had no rhizobia, whereas, the other two had <4 and 9 cells g −1 soil. Metal concentrations in these plots were above the U.K. and E.C. upper limits for Zn, but were still below the corresponding limits for Cd. Copper concentrations in one old arable plot and three ex-woodland plots were slightly above the U.K. limits for soils of pH 5–6. The German limits for Zn, Cu, and Cd, but not Ni, were considerably exceeded in these plots. The major factor influencing rhizobial numbers, in both field experiments, was not the soil pH, nor the organic carbon content, but rather the metal concentrations in the soil. The smaller N contents, chlorosis and stunting of clover plants grown in soils from plots containing no, or very few, rhizobia was not due to phytotoxicity, but was shown by the addition of nitrogen fertilizer to be due to lack of N 2-fixation. Our results indicate that although several metals were accumulated simultaneously in both field experiments there was a strong Zn effect on the numbers of rhizobia in these soils. Significant reductions in rhizobial numbers occurred even at metal concentrations well below the current U.K. and E.C. upper limits for all metals, but close to the much more strict German limits for Zn and Cd. These results are discussed in relation to the setting of safe limits for the long-term protection of soils.

Construction of gene replacement vectors for Gram? bacteria using a genetically modified sacRB gene as a positve selection marker

Based on the wide-host-range suicide vector pSUP102 (Simon et al. 1983), gene replacement vectors (pWS232/pWS233) have been developed that facilitate the identification by a positive selection procedure of double recombination events in Gram− bacteria. The vectors contain the tetracycline and gentamicin resistance gene as selectable markers, as well as a modified sacRB gene mediating sucrose sensitivity. In order to increase the versatility of the sacRB gene as a positive selection marker and, hence, of vectors that carry this gene, an EcoRI as well as a HindIII site located within the coding region of the sacRB gene were removed by in-vitro mutagenesis. To test the suitability of the vectors, a Tn5-carrying EcoRI fragment of Rhizobium leguminosarum biovar. viciae VF39 was homogenotized into the wild-type strain, resulting in double recombinants with a Lac− phenotype. Although the Tn5 insertion was flanked on one side by only approximately 100 bp of VF39 homologous DNA, this was sufficient for homologous recombination to occur, and double recombinants could readily be isolated.

The effect of heavy metals on dinitrogen fixation by Rhizobium-white colover in a range of long-term sewage sludge amended and metal-contaminated soils

An investigation was conducted to determine whether effective strains of Rhizobium leguminosarum biovar. trifolii capable of symbiotic N 2 fixation with white clover ( Trifolium repens) were present in a range of metal-contaminated soils. A number of historically sewage-amended sites (including experimental, pasture grassland and arable sites) were selected and compared with highly contaminated samples from abandoned heavy metal mines. Many sites had metal concentrations above the limits established by the UK Government, based on those developed by the European Commission (EC) for sludge-amended soils. Acetylene reduction activity (ARA) was used to screen the samples for effective N 2 fixation. When the host plant was indigenous to the sward, rhizobia were found in the nodules and in the soil rhizosphere at all the sites tested. They were shown to be capable of effective symbiosis and N 2 fixation, even though metal concentrations greatly exceeded the soil metal limits in some cases. However, nodulation failed to occur in some cases where T. repens was not indigenous to metal-contaminated soils. This indicated either that an ineffective rhizobial population was present, or that effective cells were absent from the soil. The influence of individual metals on ARA could not be determined conclusively because of the confounding effects of soil physicochemical variability and the presence of different metals at high concentrations together in the soil. However, Cd concentrations appeared to be particularly important in determining the presence of effective ARA in soils with no indigenous clover. In contrast to previous studies, the results presented here suggest that heavy metals may have had a quantitative effect on the free-living population of rhizobia, rather than a genetic effect.

Transfer of symbiotically fixed nitrogen from berseem (Trifolium alexandrinum L.) to maize via vesicular-arbuscular mycorrhizal hyphae.

In order to examine whether hyphae of VA mycorrhizal fungi may aid the transfer of symbiotically fixed nitrogen from a legume to a non-legume, the roots of berseem plants inoculated with the VAM fungus Glomus intraradices and Rhizobium leguminosarum biovar. trifolii (strain RCR 5) were separated by a 2 cm root-free zone from the roots of maize plants. A 40 um pore size nylon net allowed VAM hyphae to pass through the root-free zone but prevented root penetration. Replacement of this net by a 0.45 μm pore size membrane prevented penetration of VAM hyphae to the root-free compartment. When the berseem plants were 63 d old, the plant chambers were sealed in plastic bags to allow the roots of the plants to be exposed to a l0 N2 -enriched atmosphere for 5 d. Berseem plants that were exposed to a 15 N2 -enriched atmosphere fixed more 15 N than did berseem plants maintained under air at natural 15 N abundance. The total 15 N excess content of berseem was similar whether or not the mycorrhiza had access to the maize roots. Plant mass (dry weight) of maize was not affected by the mycorrhizal fungus, but total nitrogen content tended to be higher in VAM-colonized maize than in non-colonized maize. Furthermore, both atom %15 N excess and 15 N excess content of mycorrhizal maize were significantly increased as compared with non-mycorrhizal maize or with maize controls without berseem. The amounts of 15 N transferred were small, accounting for less than 4% of the 15 N in the N2 -fixing plant. In spite of the 2 cm root-free separating zone, the presence of the mycorrhizal fungus in the non-legume increased the transfer of symbiotically fixed nitrogen from berseem to maize plants that were physiologically younger. The implications of these results for nitrogen transfer from a legume to an accompanying non-legume via VAM hyphae are discussed.