Competitive Nodulation Ability Research Articles

Effects of the purL Gene Expression Level on the Competitive Nodulation Ability of Sinorhizobium fredii

Purine pathway in Rhizobium is important during the nodulation processes. The purL gene in Sinorhizobium fredii (S. fredii) has been identified to be required for the whole establishment of a nitrogen-fixing nodule. To get a better understanding of the purL gene's impacts on Rhizobium-plant interaction, the competitive nodulation abilities of S. fredii containing different purL expression plasmids were studied. Several kinds of coinoculations were performed, including using different bacterial concentration ratios, with or without the supplementation of purine source in the plant nutrient solution, and the delayed coinoculation tests. The results indicated that the competitive nodule occupancy of S. fredii was affected significantly by the purL expression level during the early nodulation periods. The mutant strain containing no purL expression could not elicit competitive nodules both in the presence and absence of purine source. A positive linear correlation within certain limits was observed between strain's competitive nodule occupancy and purL gene expression level. All these results suggested that the purL gene played a role in the competitive nodulation of S. fredii.

Screening of Highly Effective Sinorhizobium meliloti Strains for ‘Vector’ Alfalfa and Testing of Its Competitive Nodulation Ability in the Field

Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. meliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA (RAPD) by polymerase chain reaction (PCR) from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a strain suitable for the alfalfa cultivar ‘Vector’ grown in Wuqiao County of Hebei Province was obtained.

Biological characteristics of plasmids of Mesorhizobium huakuii HN3015 from Astragalus sinicus

A Mesorhizobium huakuii strain HN3015 was isolated from Astragalus sinicus in a rice-growing field of Southern China. Strain HN3015 contained three large plasmids. The three indigenous plasmids, named as pMhHN3015a, pMhHN3015b and pMhHN3015c of M. huakuii HN3015, were, respectively, cured by Tn5-sacB insertion. The mutant strain HN3015-1 cured with its largest plasmid pMhHN3015c formed only white null nodules. Mutant HN3015-3 cured with its smallest plasmid pMhHN3015a could form pink effective nodules. However, mutant HN3015-2 cured of the second largest plasmid pMhHN3015b lost nodulation ability. Furthermore, curing of pMhHN3015a had enhanced competitive nodulation ability and symbiotic efficiency of HN3015-3. The results from acidity tolerance assays indicated that the three plasmids in M. huakuii HN3015 had a positive control effect on acidity tolerance of HN3015, and all indigenous plasmids of M. huakuii HN3015 had a negative control effect on the alkali tolerance capacity of HN3015. Surprisingly, all plasmids in M. huakuii HN3015 had also a negative control effect on its growth rate. The results showed an interactive and functional complexity of plasmids in strain HN3015.

Evaluation of the recA-based containment system in Rhizobiummeliloti GR4

Rhizobium meliloti recA strains have been reported as safe hosts for deliberate release experiments due to their reduced survival in soil microcosms. Using R. meliloti integration vectors, two GUS-positive RecA+ and RecA− isogenic derivatives of R. meliloti strain GR4 were constructed. In contrast to previously reported RecA− strains, the RecA− strain GR4KLR showed similar doubling times as its isogenic RecA+ strain GR4KLC. Accordingly, GR4KLR showed normal growth competition capacity when coinoculated with the parental strain in sterile soil microcosms. In addition, the recA mutant showed delayed nodulation on axenically grown alfalfa plants, and its competitive nodulation ability was severely affected. This defect could be corrected by a genetic manipulation based on the use of a multicopy nifA-expressing plasmid. Another novel phenotype associated with the recA mutation in R. meliloti GR4 was an increased rate of plasmid loss. When inoculated in non-sterile soil microcosms, RecA+ and RecA− strains established at high cell densities and no significant differences in their survival were found after 150 days incubation. Our results show that symbiotic performance of a R. meliloti GR4 recA strain may be significantly altered whereas its relative survival in a test soil where it was to be released was not significantly reduced with respect to RecA+ strains.

Persistence, population dynamics and competitiveness for nodulation of marker gene-tagged Rhizobium galegae strains in field lysimeters in the boreal climatic zone

A non-indigenous wild-type strain Rhizobium galegae HAMBI 540, which specifically nodulates perennial goat’s rue ( Galega orientalis), and its marker gene-tagged derivatives R. galegae HAMBI 2363( luc), R. galegae HAMBI 2368( gusA21) and R. galegae HAMBI 2364( gusA30) were used to evaluate the persistence, population dynamics and competitiveness for nodulation of rhizobia under field conditions in Finland. The lysimeters were filled with clean or diesel oil-polluted (3000 μg g −1) agricultural soil. During the first 2 years of the field release luc- and gusA21-tagged strains could be effectively detected by cultivation, reinforced with colony polymerase chain reaction. The population densities remained relatively stable from 10 4 to 10 5 cfu g −1 dry soil from spring until late autumn. Replicate limiting dilution polymerase chain reaction analysis gave comparable results with cultivation with strain HAMBI 2363 until 49 weeks after inoculation. GUS activity of strain HAMBI 2368 could be stably detected in nodules and soil. On the other hand, luc activity weakened clearly in cold conditions along with decreased metabolic activity of rhizobia. The competitive ability for nodulation of the gusA30-tagged strain decreased slowly with time compared to the wild-type strain. Moderate soil pollution did not have significant effects on target bacteria or plant growth. Limited vertical movement of target bacteria outside the rhizosphere was detected from percolated water.

In situ analysis of introduced Frankia populations in root nodules of Alnus glutinosa grown under different water availability

The competitive ability for nodulation of Alnus glutinosa (L.) Gaertn. plants by Frankia strains inoculated into soil with indigenous Frankia populations was studied at two matric potentials representing "dry" (-0.016 MPa) and "wet" (-0.001 MPa) conditions. In pots kept at a matric potential of -0.001 MPa, nitrate concentrations decreased within 3 weeks more than 10-fold to an average of approx. 200 µmol·(g soil dry wt.)-1. After 4 months, nitrate concentrations in these pots were 16 ± 16 and 277 ± 328 µmol·(g soil dry wt.)-1 (mean ± SD) for non-inoculated and inoculated soils, respectively. At a matric potential of -0.016 MPa, nitrate concentrations for non-inoculated and inoculated soils were 687 ± 491 and 1796 ± 1746 µmol·(g soil dry wt.)-1, respectively. Inoculated plants always grew better than their non-inoculated counterparts. The largest plants were found on inoculated soil at a matric potential of -0.001 MPa, whereas the smallest plants were found on non-inoculated soil at the same matric potential. At a matric potential of -0.016 MPa, plants grown on non-inoculated soil were not as tall as those grown on inoculated soil and were slightly chlorotic, indicating that the high level of nitrate in the soil was not providing optimal plant growth conditions. The number of nodule lobes formed on plants was not significantly different among treatments, though size and weight of lobes differed. Nodules from plants grown on inoculated soils always harbored vesicle-producing Frankia populations, while nodules from plants grown on non-inoculated soils harbored only Frankia with distorted vesicles or no Frankia at all. All strains in nodules from plants grown on non-inoculated soil were of Alnus host infection group IIIa. Nodules from plants grown on soil inoculated with strains ArI3 (group IIIa), Ag45/Mut15 (group IV), and AgB1.9 (group I) were also infected by Frankia strain Ag45/Mut15. These results indicate that by inoculation, Frankia populations could be established under conditions that did not favour vesicle formation in root nodules formed by the indigenous Frankia population. Inoculation even in soils with high nitrogen content might therefore be an appropriate strategy to enhance plant growth.Key words: competition, fluorescent oligonucleotide probes, inoculation, in situ hybridization, matric potential, nitrate, rRNA.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn 5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Evaluation of the recA-based containment system in Rhizobiummeliloti GR4

Rhizobium meliloti recA strains have been reported as safe hosts for deliberate release experiments due to their reduced survival in soil microcosms. Using R. meliloti integration vectors, two GUS-positive RecA + and RecA − isogenic derivatives of R. meliloti strain GR4 were constructed. In contrast to previously reported RecA − strains, the RecA − strain GR4KLR showed similar doubling times as its isogenic RecA + strain GR4KLC. Accordingly, GR4KLR showed normal growth competition capacity when coinoculated with the parental strain in sterile soil microcosms. In addition, the recA mutant showed delayed nodulation on axenically grown alfalfa plants, and its competitive nodulation ability was severely affected. This defect could be corrected by a genetic manipulation based on the use of a multicopy nifA-expressing plasmid. Another novel phenotype associated with the recA mutation in R. meliloti GR4 was an increased rate of plasmid loss. When inoculated in non-sterile soil microcosms, RecA + and RecA − strains established at high cell densities and no significant differences in their survival were found after 150 days incubation. Our results show that symbiotic performance of a R. meliloti GR4 recA strain may be significantly altered whereas its relative survival in a test soil where it was to be released was not significantly reduced with respect to RecA + strains.

Increase in the Competitive Nodulation Ability of Bradyrhizobium japonicum Strains Grown in Purified Water.

Increase in the Competitive Nodulation Ability of Bradyrhizobium japonicum Strains Grown in Purified Water.

Isolation of transposon Tn5-induced hydrophobic mutants of aBradyrhizohium japonicumstrain with improved competitive nodulation abilities

Abstract Hydrophobic mutants of the Bradyrhizohium japonicum strain 138NR were obtained by transposon Tn5 mutagenesis followed by replica-plating on polystyrene plates. Fifteen mutants were isolated at a frequency of 106. Gel-filtration analysis of the exopolysaccharides revealed that the hydrophobic mutants produced a significantly smaller amount of low-molecular-weight polysaccharides than the parent. Four of the isolated mutants formed a larger number of nodules on soybean (Glycine max L. Merr. cv. Tamahomare) than the parent, two were superior in symbiotic nitrogen fixation, and two were symbiotically defective on soybean. Competitive nodulation abilities of the mutants were examined by inoculating them to soybean with B. japonicum strain 123ET as a competitor and determining the nodule occupancy based on the antibiotic resistance. All the mutants tested except for the symbiotically defective ones were superior in their competitive nodulation ability to the parent strain. When inoculated 24 h before t...

Correlation between competitive nodulation ability and cell surface hydrophobicity of Bradyrhizobium japonicum.

Cell surface hydrophobicity of Bradyrhizobium japonicum strains 123ET and 138NR was studied as a factor in competition for the attachment to root and the nodulation of soybean. Hydrophobicity of the strains, which was measured by a biphasic water-hydrocarbon system, significantly changed with culture age having a maximum value in mid-log phase. Three days-old soybean (Glycine max cv. Tamahomare) were inoculated with the mixture of two strains, and grown for 3 weeks in sterilized vermiculite tubes. Bradyrhizobia attached to the seedling roots were released at 90 min after the inoculation by sonication and enumerated by plate counting. Two strains were distinguished by the difference in antibiotic resistance. Nodule occupancies by the strains were determined by the ratio of the strains on the seedling root surface, not in the inocula. Logarithm of the ratio of the strains on the root was highly correlated with the difference in hydrophobicity of the strains.

Competitive modulation ability of Bradyrhizobium japonicum strains incubated in soil

Six doubly-marked antibiotic-resistant strains of BradyRhizobium japonicum were introduced separately into soil, incubated, and examined for competitive nodulation ability. Mixtures of these strains in soil suspension and strains cultured in yeast extract mannitol broth were used to inoculate soybean ( Glycine max) seedlings. Nodule occupancy was determined by the antibiotic resistance of bacteria in each nodule. N50 (the inoculum ratio yielding a 50% nodule occupancy rate) was used to measure the competitive nodulation ability of each strain. Three weeks of soil incubation increased the competitive ability of strains 31Nal fRif R and 123 Ery RTet Rand decreased that of strains 29Ery RTet R and 138Nal RRif R. Soil incubation had no significant effect on the competitive nodulation abilities of strains 122Ery'Tet' and 143Nal'RiP. These results show that the soil environment of B. japonicum strains can affect their competitiveness for nodulation.

Competitive Nodulation Ability of Root-Nodule Bacteria in Soil

Competitive Nodulation Ability of Root-Nodule Bacteria in Soil

Evaluation of Alfalfa and Rhizobium meliloti for Compatibility in Nodulation and Nodule Effectiveness1

The purpose of this investigation was to develop a procedure for testing strains of Rhizobium meliloti and cultivars of alfalfa (Medicago sativa L.) for symbiotic compatibility. Antiblotic‐resistant mutants were developed from four strains of R. meliloti that differed for plant dry matter yield, days to initial nodulation and nodule numbers when inoculated onto one cultivar of alfalfa. The mutant strains were not significantly different from their corresponding parental strains in either days to initial nodulation or host dry matter production on N‐free medium. The preference of alfalfa for different strains of Rhizobium was not altered by 0 to 300 ppm N and 33 to 300 ppm potassium phosphate.The competitive ability for nodulation of effective and ineffective strains of R. meliloti was investigated using ‘Saranac’ and MnNC 4, an experimental population of alfalfa. Antibiotic‐resistant mutants were used to verify the relative proportions of effective and ineffective nodules on each plant. A significant positive correlation was observed between the percentage of nodules produced by the effective strains and the dry matter production of plants grown at zero N. Acetylene reduction assays indicated that differences in competitive ability of the effective strains against the ineffective strains measured by the antibiotic‐resistant mutant technique and seedling growth were associated with nitrogenase activity.A procedure of inoculating alfalfa plants with a mixture of effective and ineffective strains at zero N and measuring shoot dry weight might be used to identify strains of Rhizobium of high competitive ability and plants with increased preference for effective strains of Rhizobium. This approach should expedite attempts to select Rhizobium‐alfalfa combinations with enhanced N2‐fixation capacity.

Selection of Rhizobium strains on their competitive ability for nodulation

The shoot dry weight of alfalfa inoculated with an effective strain of Rhizobium meliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leguminosarum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia faba planted in a soil containing indigenous R. leguminosarum. The percentage of recovery of the inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitiveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.

Studies on double strain occupancy of nodules and the competitive ability of Rhizobium trifolii on red and white clover grown in soil and agar

SUMMARYAn investigation concerning double strain occupancy of nodules and the competitive ability for nodulation of Rhizobium trifolii strains 7A strr and 32 spcr was carried out using red and white clover varieties grown in agar and soil.Only two nodules of the 498 typed from plants grown in soil contained both inoculated strains and none of those analysed by the replica plating technique were formed by both 32 spcr and an indigenous strain. In contrast, plants grown on agar produced between 17% and 25% doubly infected nodules. Strain 32 spcr was more competitive for nodulation than 7A strr both in soil and agar. Indigenous rhizobia in soil were highly competitive for nodulation with the introduced strains; increasing the density of the inoculum resulted in an increase in the proportion of nodules occupied by the inoculated strains. The strains used as mixed inocula in soil were less successful in nodulation than the same strains used as pure inocula. The results also indicate that there was a host effect on the nodulation success of 7A strr.

The competitive ability and symbiotic effectiveness of doubly labelled antibiotic resistant mutants of Rhizobium trifolii

SUMMARYDoubly labelled mutants of Rhizobium trifolii, resistant to streptomycin and spectinomycin, were studied in respect of nodulating competitiveness and symbiotic effectiveness relative to the ‘wild‐type’ parent strains using Trifolium repens cv. S184 as the host plant.A combination of antibiotic resistance, differential absorption of congo‐red and the fluorescent antibody technique permitted the rapid differentiation of all Rhizobium strains used, either from mixed inocula or from nodules. The doubly labelled antibiotic resistant mutants were inferior in terms of competitive ability for nodulation with an ineffective strain compared with the ‘wild‐type’ parent strains. A rapid method for evaluating effective antibiotic resistant strains for nodulating competitiveness is suggested. All the mutants examined were also found to be less symbiotically effective than the respective ‘wild‐type’ strains although these differences generally did not reach statistical significance. The reduced symbiotic effectiveness of the antibiotic resistant mutants was associated with an increase in magnitude of the variances for shoot dry weights, relative to that shown by the parent strains. A possible explanation for this phenomenon is presented.