Quantitative comparison of the laboratory and field competitiveness of Rhizobium leguminosarum biovar phaseoli.

Rhizobium leguminosarum bv. phaseoli KIM5s outcompeted strain CE3 in bean (Phaseolus vulgaris L.) root nodulation when plants were grown at any of three field sites, each with a different soil type and indigenous population, or in the laboratory in a sterilized sand, a sterilized peat-vermiculite mixture, or a nonsterile field soil. A mathematical model describing nodulation competitiveness was empirically derived to evaluate the relative competitiveness of the two strains under these conditions. This model relates the proportional representation of the two strains in the inoculum to the proportional representation of nodules occupied by each strain or both strains and provides a measure of competitiveness, which is referred to as the competitiveness index. Statistical comparisons of competitiveness indices showed that the relative competitiveness of KIM5s and CE3 remained constant when the two strains were applied in a constant ratio over a range of inoculum concentrations, from 10(3) to 10(7) cells per seed, and when they were applied in various ratios to six P. vulgaris cultivars. Furthermore, the relative competitiveness of KIM5s and CE3 in the laboratory did not differ significantly from their relative competitiveness at the three field sites studied. Thus, a study of the basis for nodulation competitiveness of KIM5s and CE3 in the laboratory has the potential to provide an understanding of competitiveness both in the laboratory and in the field.     

Quantitative comparison of the laboratory and field competitiveness of Rhizobium leguminosarum biovar phaseoli

Rhizobium leguminosarum bv. phaseoli KIM5s outcompeted strain CE3 in bean (Phaseolus vulgaris L.) root nodulation when plants were grown at any of three field sites, each with a different soil type and indigenous population, or in the laboratory in a sterilized sand, a sterilized peat-vermiculite mixture, or a nonsterile field soil. A mathematical model describing nodulation competitiveness was empirically derived to evaluate the relative competitiveness of the two strains under these conditions. This model relates the proportional representation of the two strains in the inoculum to the proportional representation of nodules occupied by each strain or both strains and provides a measure of competitiveness, which is referred to as the competitiveness index. Statistical comparisons of competitiveness indices showed that the relative competitiveness of KIM5s and CE3 remained constant when the two strains were applied in a constant ratio over a range of inoculum concentrations, from 10(3) to 10(7) cells per seed, and when they were applied in various ratios to six P. vulgaris cultivars. Furthermore, the relative competitiveness of KIM5s and CE3 in the laboratory did not differ significantly from their relative competitiveness at the three field sites studied. Thus, a study of the basis for nodulation competitiveness of KIM5s and CE3 in the laboratory has the potential to provide an understanding of competitiveness both in the laboratory and in the field.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Unaltered Nodulation Competitiveness of a Strain of Bradyrhizobium sp. (Lotus) after a Decade in Soil

A Bradyrhizobium sp. (Lotus) strain that formed a soil population that was highly competitive for nodulation of Lotus pedunculatus 11 years after its introduction into a field soil and a culture of the same strain stored lyophilized were compared with an antibiotic-resistant mutant in respect of their nodulation competitiveness. The mutant was less competitive than the wild-type strain it was isolated from and had to be present at a cell ratio of 5.76:1 in mixed inoculum in sand culture to form 50% of the nodules on L. pedunculatus (50% nodulation value, 5.76). The 50% nodulation values for a soil population of the mutant mixed with soil populations of the lyophilized and field soil strain were, respectively, 6.83 and 5.77, indicating that the field soil strain was not significantly different from the lyophilized strain in nodulation competitiveness. A 50% nodulation value of 11.18 obtained when soil containing a recently established mutant population was mixed with the field soil containing the population established 11 years before, indicating that the plant infection technique underestimated cell numbers of the field soil population by 100%. Nodulation competitiveness was unaffected by the size of the strain populations in the range of 100 to 1,000 cells per g of soil; at 10 cells per g a significant correlation between strain ratios in nodules and in soil was still evident. The results indicated that apparently superior nodulation competitiveness of a well-established soil population relative to that of a subsequently introduced strain may not necessarily reflect the intrinsic competitive abilites of the strain(s) involved. The soil strain did not differ from laboratory-maintained cultures in antigenic properties, effectiveness, or whole cell protein electrophoresis profiles.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

Nodulating Competitiveness of a Nonmotile Tn7 Mutant of Bradyrhizobium japonicum in Nonsterile Soil

A nonmotile mutant of Bradyrhizobium japonicum serogroup 127 was generated by Tn7 mutagenesis and matched with the wild type against a common competitor in studies of soybean nodulation in nonsterile soil. The Tn7 mutant was very similar to the wild type in growth rate in culture, soybean lectin-binding ability, flagellar morphology, and nodulating capability, but it had a longer lag phase. Competing strains were distributed uniformly in soil in various ratios and at different population densities prior to planting. Mutant and wild type were equally prevalent in the seedling rhizosphere at about the time of nodule initiation, suggesting that motility conferred no advantage in rhizosphere colonization. Nodulation success of the Tn7 mutant was lower than that of the wild type under all test conditions. Differences were greatest at low soil populations of competitors and much less pronounced at initial populations of 10⁷ g⁻¹. The longer lag phase of the Tn7 mutant may have contributed to its decreased competitiveness, especially at the higher inoculation levels. The antibiotic and motility markers were stable, and the rifampin resistance derived from the parent did not affect adversely the competitiveness of the Tn7 mutant. We found motility to be of limited importance to the competitiveness of a strain in normal nonsterile soil, where the significance, if any, of this ability may be in migration at the immediate root surface in soils sparsely populated with rhizobial symbionts.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa.

A transposon Tn5-induced mutant of Rhizobium meliloti Rm2011, designated Rm6963, showed a rough colony morphology on rich and minimal media and an altered lipopolysaccharide (LPS). Major differences from the wild-type LPS were observed in (i) hexose and 2-keto-3-deoxyoctonate elution profiles of crude phenol extracts chromatographed in Sepharose CL-4B, (ii) silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis patterns of crude and purified LPS fractions, and (iii) immunoreactivities otherwise present in purified LPS of the parental strain Rm2011. In addition, Rm6963 lost the ability to grow in Luria-Bertani medium containing the hydrophobic compounds sodium deoxycholate or SDS and showed a decrease in survival in TY medium supplemented with high calcium concentrations. The mutant also had altered symbiotic properties. Rm6963 formed nodules that fixed nitrogen but showed a delayed or even reduced ability to nodulate the primary root of alfalfa without showing changes in the position of nodule distribution profiles along the roots. Furthermore, 2 to 3 weeks after inoculation, plants nodulated by Rm6963 were smaller than control plants inoculated with wild-type bacteria in correlation with a transient decrease in nitrogen fixation. In most experiments, the plants recovered later by expressing a full nitrogen-fixing phenotype and developing an abnormally high number of small nodules in lateral roots after 1 month. Rm6963 was also deficient in the ability to compete for nodulation. In coinoculation experiments with equal bacterial numbers of both mutant and wild-type rhizobia, only the parent was recovered from the uppermost root nodules. A strain ratio of approximately 100 to 1 favoring the mutant was necessary to obtain an equal ratio (1:1) of nodule occupancy. These results show that alterations in Rm6963 which include LPS changes lead to an altered symbiotic phenotype during the association with alfalfa that affects the timing of nodule emergence, the progress of nitrogen fixation, and the strain competitiveness for nodulation.     

Quantitative Study of Nodulation Competitiveness in Rhizobium Strains

We compared the nodulation competitiveness of three strains of Rhizobium leguminosarum by counting the number of nodules formed on faba bean plants after the application at sowing time of different concentrations of the strains to soils already containing Rhizobium strains of the same species. A relationship of type y = axⁿ was found to exist between the ratio of the nodules formed by the applied inoculum strain to the nodules formed by the soil strains and the ratio of Rhizobium cells in the inoculum to the cells in the soil. This relationship was also confirmed in another competition experiment in which two R. meliloti strains of identical competitiveness were mixed in various proportions. The relationship can also be applied to the majority of results reported in the literature. Should it prove to be more widely applicable, it could be used to estimate the relative competitiveness of Rhizobium strains and thus predict the performance of an inoculum in a given soil.     

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Competition among<Emphasis Type="Italic">Bradyrhizobium</Emphasis> strains for nodulation of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis>): Use of dark-nodule strain

The competitiveness of dual-strain inoculum of Bradyrhizobium strains S24 and GR4 was demonstrated for nodulation of green gram (Vigna radiata). Strain S24 formed pink nodules, GR4 produced visually distinguishable dark-brown nodules. When a mixture of these Bradyrhizobium strains was applied as inoculum, nodules of both pink and dark-brown types were formed on the same root. The strain GR4, which was less competitive than strain S24, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine to obtain pigment-diverse mutants and six selected mutants were screened for symbiotic parameters. One mutant produced pink nodules and appreciably increased plant dry mass. The competitive ability of this mutant lacking brown pigment was compared with that of strain S24 by using antibiotic resistance markers; it showed increased nodulation competitiveness than its parent strain GR4. The dark-brown nodule-phenotype could be useful in evaluating nodulation competitiveness of "cowpea miscellany" bradyrhizobia in soil where dark-brown nodule-forming strains are not indigenous.     

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

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.     

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.     

2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant

When Rhizobium etli CE3 was grown in the presence of Phaseolus vulgaris seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-O-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen. R. etli strain CE395 was isolated after Tn5 mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-O-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with P. vulgaris. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-O-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than lps⁺ strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.     

Autoregulatory response of Phaseolus vulgaris L. to symbiotic mutants of Rhizobium leguminosarum bv. phaseoli.

In Rhizobium-legume symbiosis, the plant host controls and optimizes the nodulation process by autoregulation. Tn5 mutants of Rhizobium leguminosarum bv. phaseoli TAL 182 which are impaired at various stages of symbiotic development, were used to examine autoregulation in the common bean (Phaseolus vulgaris L.). Class I mutants were nonnodulating, class II mutants induced small, distinct swellings on the roots, and a class III mutant formed pink, bacterium-containing, but ineffective nodules. A purine mutant (Ade-) was nonnodulating, while a pyrimidine mutant (Ura-) formed small swellings on the roots. Amino acid mutants (Leu-, Phe-, and Cys-) formed mostly empty white nodules. Each of the mutants was used as a primary inoculant on one side of a split-root system to assess its ability to suppress secondary nodulation by the wild type on the other side. All mutants with defects in nodulation ability, regardless of the particular stage of blockage, failed to induce a suppression response from the host. Only the nodulation-competent, bacterium-containing, but ineffective class III mutant induced a suppression response similar to that induced by the wild type. Suppression was correlated with the ability of the microsymbiont to proliferate inside the nodules but not with the ability to initiate nodule formation or the ability to fix nitrogen. Thus, the presence of bacteria inside the nodules may be required for the induction of nodulation suppression in the common bean.     

Successful inoculation of Vigna radiata (L) Wilczek grown in rice fallow soil containing indigenous cowpea rhizobia

Field experiments in rice fallow soil with two Rhizobium strains, VUW.2 and VRS.6, showed that both were capable of competing with native rhizobia in infecting the roots of the mung bean and occupying the nodules to an extent of 35–75%. Simultaneous infection with both strains was most effective in increasing growth and nodulation efficiency. It was also observed that the inoculated rhizobial strains increased the grain yield 18.7–23.6% above control. The maximum increase of 23.6% in grain yield was obtained from seeds inoculated with both strains simultaneously.     

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.