Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Identification of the host-range DNA which allows Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas

Summary The special ability of Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas had previously been shown to be determined by the symbiotic plasmid, pRL5JI, of this strain. A region of pRL5JI, 2.0 kb in size, was found to confer the ability to nodulate cv. Afghanistan peas when transferred to strains of R. leguminosarum which normally fail to nodulate this host. This region of pRL5JI, responsible for the extension of host-range, was closely linked to, but did not include, the genes required for root hair curling. Although extensive homology has been found between the R. leguminosarum nod genes on pRL5JI and those on the normal symbiotic plasmid pRL1JI, a fragment from the 2.0 kb region involved in nodulation of cv. Afghanistan has been identified, which was not homologous to DNA in strains which do not nodulate cv. Afghanistan.     

The Rhizobium leguminosarum bv. trifolii pssB gene product is an inositol monophosphatase that influences exopolysaccharide synthesis

The pssB gene of Rhizobium leguminosarum bv. trifolii encodes a protein of 284 amino acids with sequence similarity to eukaryotic inositol monophosphatases. The gene was cloned and overexpressed in Escherichia coli. The purified gene product of pssB showed inositol monophosphatase activity with a Km of 0.23 mM, and a Vmax of 3.27 mumol Pi min-1 (mg protein)-1. Its substrate specificity, Mg+2 requirement, Li+ inhibition, and subunit association (dimerization) were studied and compared to those of other inositol monophosphatases. Western immunoblotting with anti-PssB antibodies showed the presence of PssB in R. leguminosarum bv. trifolii strain TA1 and lack of this protein in the pssB mutant strain Rt12A. The presence of PssB protein in R. leguminosarum bv. trifolii TA1 was correlated with phosphatase activity with myo-inositol 1-phosphate as a substrate. Evidence for a regulatory function of PssB protein in exopolysaccharide (EPS) synthesis is presented. The mutation in pssB caused EPS overproduction, and introduction of pssB into the wild-type TA1 strain reduced EPS synthesis. The changes in the level of EPS production were correlated with a non-nitrogen-fixing phenotype of rhizobia.     

Increased metabolic potential of Rhizobium spp. is associated with bacterial competitiveness

Of 105 rhizobial isolates obtained from nodules of commonly cultivated legumes, we selected 19 strains on the basis of a high rate of symbiotic plant growth promotion. Individual strains within the species Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and Rhizobium etli displayed variation not only in plasmid sizes and numbers but also in the chromosomal 16S-23S internal transcribed spacer. The strains were tagged with gusA gene and their competitiveness was examined in relation to an indigenous population of rhizobia under greenhouse conditions. A group of 9 strains was thus isolated that were competitive in relation to native rhizobia in pot experiments. Nineteen selected competitive and uncompetitive strains were examined with respect to their ability to utilize various carbon and energy sources by means of commercial Biolog GN2 microplate test. The ability of the selected strains to metabolize a wide range of nutrients differed markedly and the competitive strains were able to utilize more carbon and energy sources than uncompetitive ones. A major difference concerned the utilization of amino and organic acids, which were metabolized by most of the competitive and only a few uncompetitive strains, whereas sugars and their derivatives were commonly utilized by both groups of strains. A statistically significant correlation between the ability to metabolize a broad range of substrates and nodulation competitiveness was found, indicating that metabolic properties may be an essential trait in determining the competitiveness of rhizobia.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Exopolysaccharide depolymerases induced by Rhizobium bacteriophages.

Enzymes induced by two Rhizobium trifolii bacteriophages caused depolymerization of exopolysaccharides from most R. trifolii and R. leguminosarum strains tested, but did not, in general, attack the exopolysaccharides of R. meliloti, the slow-growing rhizobia, or Agrobacterium. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains.     

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2- O -methylfucose residue at the reducing-terminal N -acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro ( Macroptilium atropurpureum ). Here we show that transfer of nodZ to Rhizobium leguminosarum biovar (bv) viciae , which produces LCOs that are not modified at the reducing-terminal N -acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N -acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum Glycine soja Vigna unguiculata and Leucaena leucocephala . Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.     

Effect of the fungicide captafol on the survival and symbiotic properties of Rhizobium trifolii

The fungicide captafol is toxic to Rhizobium trifolii at concentrations greater than 75 μg/ml, and at lower concentrations it affects growth adversely. Captafol-resistant mutants were isolated and all were found to have lost the same plasmid and the ability to nodulate clovers. Nodulation plasmids transferred from a R. leguminosarum or R. trifolii donor to the resistant mutants conferred the ability to nodulate peas and clover plants, respectively. The rhizobia remained resistant to captafol indicating that the genetic alteration leading to captafol resistance was not necessarily detrimental to the ability of the bacteria to form nitrogen fixing nodules. These results indicate that captafol may act as a plasmid-curing agent in R. trifolii.     

Cloning and mutagenesis of nodulation genes from Rhizobium leguminosarum TOM, a strain with extended host range

Summary Some primitive pea lines, e.g. cultivar Afghanistan, are resistant to nodulation by most strains of Rhizobium leguminosarum. However the Turkish strain TOM can nodulate cv. Afghanistan in addition to commercial pea varieties, and this extended host range is a property of its symbiotic plasmid, pRL5JI. A gene bank was constructed using DNA from a strain of R. leguminosarum containing pRL5JI. Following transfer to a strain of R. leguminosarum that had been cured of its symbiotic plasmid, two derivatives were isolated that contained cloned nodulation determinants, and were able to nodulate both cv. Afghanistan and a commercial pea variety. In addition, these clones conferred the ability to nodulate peas to a strain of R. phaseoli that had been previously cured of its symbiotic plasmid. One of these clones was subjected to mutagenesis with transposon Tn5, and 11 mutants were identified that were affected in nodulation ability. The sites of Tn5 insertions were mapped using restriction endonucleases and all were found to be within a region of 5 kb. The mutants fell into three classes on the basis of their map positions and their phenotypes on the two different pea lines tested. One class of mutants was affected in gene functions that were common to the nodulation of both pea hosts; a second class was impaired specifically in the nodulation of the commercial pea variety; a third class of mutant failed to confer on a normal strain of R. leguminosarum the supplementary ability to nodulate cv. Afghanistan.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

Lectin-enhanced accumulation of manganese-limited Rhizobium leguminosarum cells on pea root hair tips.

The ability of Rhizobium leguminosarum 248 to attach to developing Pisum sativum root hairs was investigated during various phases of bacterial growth in yeast extract-mannitol medium. Direct cell counting revealed that growth of the rhizobia transiently stopped three successive times during batch culture in yeast extract-mannitol medium. These interruptions of growth, as well as the simultaneous autoagglutination of the bacteria, appeared to be caused by manganese limitation. Rhizobia harvested during the transient phases of growth inhibition appeared to have a better attachment ability than did exponentially growing rhizobia. The attachment characteristics of these manganese-limited rhizobia were compared with those of carbon-limited rhizobia (G. Smit, J. W. Kijne, and B. J. J. Lugtenberg, J. Bacteriol. 168:821-827, 1986, and J. Bacteriol. 169:4294-4301, 1987). In contrast to the attachment of carbon-limited cells, accumulation of manganese-limited rhizobia (cap formation) was already in full progress after 10 min of incubation; significantly delayed by 3-O-methyl-D-glucose, a pea lectin haptenic monosaccharide; partially resistant to sodium chloride; and partially resistant to pretreatment of the bacteria with cellulase. Binding of single bacteria to the root hair tips was not inhibited by 3-O-methyl-D-glucose. Whereas attachment of single R. leguminosarum cells to the surface of pea root hair tips seemed to be similar for both carbon- and manganese-limited cells, the subsequent accumulation of manganese-limited rhizobia at the root hair tips is apparently accelerated by pea lectin molecules. Moreover, spot inoculation tests with rhizobia grown under various culture conditions indicated that differences in attachment between manganese- and carbon-limited R. leguminosarum cells are correlated with a significant difference in infectivity in that manganese-limited rhizobia, in contrast to carbon-limited rhizobia, are infective. This growth-medium-dependent behavior offers and explanation for the seemingly conflicting data on the involvement of host plant lectins in attachment of rhizobia to root hairs of leguminous plants. Sym plasmid-borne genes do not play a role in manganese-limitation-induced attachment of R. leguminosarum.     

Performance of phaseolus bean rhizobia in soils from the major production sites in the Nile Delta

The symbiotic and competitive performances of two highly effective rhizobia nodulating French bean P. vulgaris were studied in silty loam and clayey soils. The experiments were carried out to address the performance of two rhizobia strains (CE3 and Ph. 163] and the mixture thereof with the two major cultivated bean cultivars in two soil types from major growing French bean areas in Egypt. Clay and silty loam soils from Menoufia and Ismailia respectively were planted with Bronco and Giza 6 phaseolus bean cultivars. The data obtained from this study indicated that rhizobial inoculation of Giza 6 cultivar in clayey soil showed a positive response to inoculation in terms of nodule numbers and dry weight. This response was also positive in dry matter and biomass accumulation by the plants. The inoculant of strain CE3 enhanced plant growth and N-uptake relative to Ph. 163. However, the mixed inoculant strains were not always as good as single strain inoculants. The competition for nodulation was assessed using two techniques namely fluorescent antibody testing (FA) and REP-PCR fingerprinting. The nodule occupancy by inoculant strain Ph. 163 in both soils occupied 30-40% and 38-50 of nodules of cultivar Bronco. The mixed inocula resulted in higher proportions of nodules containing CE3 in silty loam soil and Ph. 163 in clayey soil. The native rhizobia occupied at least 50% of the nodules on the Bronco cultivar. For cultivar Giza 6, the native rhizobia were more competitive with the inoculant strains. Therefore, we suggest using the studied strains as commercial inocula for phaseolus bean.     

Suitability of oven-dried root nodules for Rhizobium strain identification by immunofluorescence and agglutination

Legume root-nodules, dried at oven temperature (70°C for 48 h) were suitable for Rhizobium strain identification by immunofluorescence and agglutination. The fluorescence of bacteroids of R. japonicum, R. leguminosarum, R. meliloti, R. phaseoli , and Rhizobium spp. from oven-dried nodules was the same as those from frozen, desiccated, or nodules dried at room temperature (28°C). Oven-dried nodules did not require further steaming for agglutination. Bacteroid agglutinations gave 2–16 fold lower titres than those of the cultured cells. Fresh and oven-dried soybean rhizobia from a mixed inoculation gave exactly the same results when identified by immunofluorescence or agglutination.     

Early Interactions of Rhizobium leguminosarum bv. phaseoli and Bean Roots: Specificity in the Process of Adsorption and Its Requirement of Ca(sup2+) and Mg(sup2+) Ions

Roots of Phaseolus vulgaris L. were incubated with dilute suspensions (1 x 10(sup3) to 3 x 10(sup3) bacteria ml(sup-1)) of an antibiotic-resistant indicator strain of Rhizobium leguminosarum bv. phaseoli in mineral medium and washed four times by a standardized procedure prior to quantitation of adsorption (G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:371-376, 1986). The population of rhizobia remaining adsorbed on roots after washing was homogeneous, as indicated by the first-order course of its desorption by hydrodynamic shear. Rhizobia were maximally active for adsorption in the early stationary phase of growth. The process leading to adsorption was rapid, without an initial lag, and slowed down after 1 h. Adsorption of the indicator strain at 10(sup3) bacteria ml(sup-1) was inhibited to different extents in the presence of 10(sup3) to 10(sup8) antibiotic-sensitive competitor rhizobia ml(sup-1). After a steep rise above 10(sup4) bacteria ml(sup-1), inhibition by heterologous competitors in the concentration range of 10(sup5) to 10(sup7) bacteria ml(sup-1) was markedly less than by homologous strains, while at 10(sup8) bacteria ml(sup-1) it approached the high level of inhibition by the latter. At 10(sup7) bacteria ml(sup-1), all of the heterologous strains tested were consistently less inhibitory than homologous competitors (P < 0.001). These differences in competitive behavior indicate that in the process of adsorption of R. leguminosarum bv. phaseoli to its host bean roots, different modes of adsorption occur and that some of these modes are specific for the microsymbiont (as previously reported for the alfalfa system [G. Caetano-Anolles and G. Favelukes, Appl. Environ. Microbiol. 52:377-381, 1986]). Moreover, whereas the nonspecific process occurred either in the absence or in the presence of Ca(sup2+) and Mg(sup2+) ions, expression of specificity was totally dependent on the presence of those cations. R. leguminosarum bv. phaseoli bacteria adsorbed in the presence of Ca(sup2+) and Mg(sup2+) were more resistant to desorption by shear forces than were rhizobia adsorbed in their absence. These results indicate that (i) symbiotic specificity in the P. vulgaris-R. leguminosarum bv. phaseoli system is expressed already during the early process of rhizobial adsorption to roots, (ii) Ca(sup2+) and Mg(sup2+) ions are required by R. leguminosarum bv. phaseoli for that specificity, and (iii) those cations cause tighter binding of rhizobia to roots.     

Identification of the rhizobium strains in pea root nodules using genetic markers.

Pea plants were inoculated jointly with pairs of genetically marked strains of Rhizobium leguminosarum. Out of 297 modules examined 56 contained both inoculant strains. The ratios of the strains in the inoculum did not affect the frequencies of mixed nodules. Generally one of the strains consistently occupied the majority of the nodules and ithe mixed nodules comprised the majority of bacteria. Transfer of the P-group R factor, RP4, between certain strains of Rhizobium within mixed nodules was detected. In some cases the non-parental progeny comprised 10% of the rhizobia isolated from such nodules.