Rhizobium Meliloti Strain Research Articles

Selecting Rhizobium meliloti for Inoculation of Alfalfa Planted in Acid Soils

AbstractA study was conducted to obtain Rhizobium meliloti strains suitable for use with alfalfa grown in acid soils. Thirteen strains of R. meliloti were examined for their ability to grow in acidified culture medium, and seven of these were characterized for the ability to survive in acid and limed nonsterile soils or grow in the presence of the host legume, Medicago sativa L. The pH values of the most acid, defined medium that permitted growth of the bacteria from a small inoculum ranged from pH 5.3 to 6.0. For R. meliloti 411SE1 and GH1‐1SE1, the minimum pH that allowed for growth, the critical pH, was not a dependable indicator of survival in a more acid medium. Strains of R. meliloti with relatively low critical pH values survived better in a limed soil but not in acid soils than strains with higher critical pH values. Three strains of R. meliloti previously identified as good inoculants for alfalfa in acid soils did not consistently survive better than other strains in a planted or unplanted acid soil of pH 5.3. However, the plants increased the population densities of these three strains more than other strains. These results suggest that R. meliloti strains suitable for inoculation of alfalfa in acid soils may be selected not by simple saprophytic properties but by their stimulation by the host legume in acid soils.

Evaluation of competitive ability of Rhizobium meliloti strains for nodulation in alfalfa

A method was developed for determination of the competitive ability of selected Rhizobium meliloti strains based on recovery from nodules and identification with commercially available antibiotic sensitivity disks. Four strains isolated from soil were used as inocula for alfalfa seedlings under controlled conditions in all possible two-, three-, and four-way combinations and alone as controls. One of the test strains readily formed nodules, but had low expressed nitrogenase activity when used alone. In contrast, another strain with high nitrogenase activity was found to be distinctly noncompetitive with the other strains, based on frequency of recovery from nodules. Nitrogenase activities of groups of plants resulting from growth with some inoculum mixtures showed evidence of linear additivity of the activities based on frequency of recovery of the strains from nodules and the activity of control plants inoculated with single strains. Synergistic responses were found for other inoculum mixtures. Likewise, competitive ability of inoculum strains in the four-way mixtures was not wholly predictable from results obtained in two- and three-way tests, indicating some modification of competitive abilities in multiple-inoculum mixtures. Occupancy of nodules by two strains was found to occur at very low frequency with multiple-strain inocula. The noncompetitive strain was never found in double occupancy with the other strains.

Isolation and identification of β 1 → 2-?-glucans in extracellular polysaccharides of Rhizobium meliloti strain L5-30

Isolation and identification of β 1 → 2-?-glucans in extracellular polysaccharides of Rhizobium meliloti strain L5-30

Localization of symbiotic mutations in Rhizobium meliloti

A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis. Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype. Alternatively, the kanamycin resistance marker of Tn5 was mapped. In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome. It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally. Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes. Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location. With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b. This is in agreement with the genetic mapping data.

Strain-specific serological techniques for the identification of Rhizobium meliloti in commercial alfalfa inoculants

Antisera were prepared against five Rhizobium meliloti strains including three strains adapted to Canadian soil and climate conditions and recently released for use in the production of commercial alfalfa (Medicago spp.) inoculants. The antisera were highly cross-reactive, but specificity was obtained by repeated massive adsorptions of the antisera with cells of the cross-reacting strains. The adsorbed antisera were used in microagglutination tests and the enzyme-linked immunosorbent assay (ELISA) to demonstrate the presence of the Canadian strains in their respective commercial peat-base inoculants at 108–109 viable cells/g. A plant infection technique which was also used to evaluate inoculant quality was specific only at the Rhizobium species level and required up to 4 weeks for completion. Serological techniques made it possible to identify and quantitate inoculant strains of R. meliloti while reducing the time required for testing. The agglutination analysis of inoculants was simple, but required regrowth of each colony isolate to generate sufficient cell numbers. The ELISA technique was used directly with colonies picked from plate counts, and the results were available within 5 days, including the time for colony growth on the plate media.

PERFORMANCE OF Rhizobium meliloti STRAINS SELECTED FOR LOW-pH TOLERANCE

Twelve strains of Rhizobium meliloti were examined for their ability to nodulate alfalfa (Medicago sativa) and fix nitrogen under acid soil conditions. The strains were separated into a low-pH-sensitive group and a low-pH-tolerant group on the basis of plant dry matter yields in greenhouse experiments. The dry matter yields of alfalfa inoculated with the low-pH-tolerant strains were 5.9, 10.0 and 1.3 times greater than the yields with the low-pH-sensitive strains at soil pH 5.0, 5.5 and 6.0, respectively. Yields of alfalfa at soil pH 6.7 were the same for both groups. Four strains were selected from the low-pH-tolerant group for further evaluation. Plant yield, nitrogen fixation, specific nitrogenase activity, nodule number and nodule weight were measured for each strain in relation to soil pH, inoculum level and alfalfa cultivar. Soil pH, inoculum level and cultivar had significant effects on most of the characteristics measured. There were no significant differences in yield and nitrogen fixation within the four strains, and only minor differences in nodule number, nodule weight and specific nitrogenase activity. The low-pH-tolerant strains were competitive with indigenous strains and effective on a range of alfalfa cultivars. Inoculation rates at 105 rhizobia per seed were required to obtain maximum effect from the applied inoculum. R. meliloti strains selected for low-pH tolerance provide the potential for substantially increasing alfalfa yields on moderately acid soils.

Succinate dehydrogenase mutant of Rhizobium meliloti.

A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis. It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates. The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective. A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.

Root Surface Association in Relation to Nodulation of Medicago sativa

Nine strains of Rhizobium meliloti, ranging in competitive ability on Medicago sativa from excellent to poor in autoclaved soils, were paired in 29 combinations and used to inoculate M. sativa in a liquid rooting medium. A positive correlation (r = 0.545) between strain ratios in nodules after 28 days and root surface cell ratios after 7 days was determined. Two cell fractions from the root surface, representing loosely and firmly adhering cells, were investigated. Infectivity was linked to the more firmly adhering cells. A significant relationship was established between the cell ratios of competing strains in the two fractions. In another experiment, adherence of cells of both infective and noninfective Rhizobium strains to roots of M. sativa and Trifolium repens was demonstrated; the ratios of loosely to firmly adhering cells on the root surface were significantly narrower with the infective combinations than with noninfective strain-legume associations.

Absence of accumulation of ppGpp and RNA during amino acid starvation in Rhizobium meliloti.

Lack of three different amino acids or treatment with the analogue DL-serine hydroxamate does not induce the accumulation of ppGpp and pppGpp, the 3'-pyrophosphates of GDP and GTP, respectively, in Rhizobium meliloti strain 41. Surprisingly, RNA accumulation is controlled under the above mentioned conditions stringently. Moreover, no significant RNA accumulation was found during chloramphenicol, tetracycline, and streptomycin treatment, suggesting that R. meliloti, unlike any other bacteria in investigated so far, is not able to accumulate RNA without ongoing protein synthesis. On the other hand, lack of carbon source and ammonium starvation result in a significant ppGpp accumulation.

Host Plant and Rhizobium Effects on Acetylene Reduction in Alfalfa during Regrowth 1

The effects of host plant and Rhizobium strain variation on apparent N2 fixation (C2H2 reduction) and total nonstructural carbohydrate (TNC) of the root system during regrowth of alfalfa (Medicago sativa L.) were studied to determine the prevalence of the known decline in C2H2-reduction activity following shoot harvest. Harvest at 10% bloom in ramets of a ‘Dohfari’ clone inoculated with Rhizobium meliloti strain 102F28 decreased shoot carbon exchange rate (CER) 99% immediately, but C2H2 reduction declined only 75% in the following 3 days. Thereafter, CER and C2H2 reduction increased in parallel during shoot regeneration. Those data provided circumstantial evidence that carbohydrates stored in roots or root nodules were used to maintain low rates of C2H2 reduction during the intial period of regrowth. In experiments with seedlings of 10 alfalfa cultivars inoculated with R. meliloti 102F28 and in trials with Dohfari and ‘Vernal’ clones inoculated with one of 10 different R. meliloti strains, shoot harvest produced a significant (p ≤0.001) decline in C2H2 reduction within 3 days to values ranging from 4-30% of the original rate. Root system TNC generally declined over the same period, but in only two of the 30 host-symbiont combinations examined was that change significant (p ≤0.05). No significant correlation was observed between C2H2-reduction rate and root system TNC in the same plants at harvest (day 0) or on day 3 in any host-symbiont combination. Results from this study suggest that although nonstructural carbohydrates are consumed in alfalfa roots during defoliation, the), are not used to maintain maximum (day 0) rates of N2 fixation during the early stages of regrowth in widely different host-symbiont combinations. This evidence is consistent with the concept that the recovery of N2 fixation following shoot harvest depends upon the regrowth of new shoots.

A bacteriophage typing system for Rhizobium meliloti

Fifteen specific bacteriophages, each active on particular strains of Rhizobium meliloti, have been selected from those isolated by enrichment of local soils. Variables affecting phage–host interaction have been examined and standardized. The differential susceptibility of individual R. meliloti strains to each phage produces a distinctive pattern of response which allows the segregation of each strain into one of the 80 different groups identified to date. Discrimination between strains with this typing system is reproducible with no change in phage type following extended subculture or plant infection and reisolation of strains from root nodules of alfalfa. The large number of different strains recognized by the system should make it very useful in experimentally controlled tests of selected strains in the nodulation of alfalfa plants.

Strain identification inRhizobium meliloti using the antibiotic disk susceptibility test

The antibiotic disk susceptibility test was used to measure the variation in the intrinsic resistance of 49 strains ofRhizobium meliloti to 9 antibiotics. Several strains had unique patterns of resistance. However, during cluster analysis, when a minimum Euclidean distance equal to 4 was used as a discriminating tool, the strains were grouped in 12 groups. The largest group contained 74% of the strains but 9 strains (2 very effective, 4 effective and 3 ineffective) showed very unique patterns of resistance and formed 9 distinct groups.R. meliloti strains in general showed high intrinsic resistance to the 9 antibiotics tested.

Rhizobial Strain Preference of Alfalfa Populations Selected for Characteristics Associated with N 2 Fixation 1

Preference for strains of Rhizobium meliloti by alfalfa (Medicago sativa L.) genotypes was studied in a series of experiments to determine whether selection for physiological and morphological traits associated with N2 fixation capability modified the preference of alfalfa for rhizobial strains. In the first two experiments, an unselected alfalfa population and eight subpopulations were inoculated with antibiotic-resistant mutants of Rhizobium to study the effect of plant selection on preference for strains of Rhizobium. A series of indigenous isolates of Rhizobium was obtained from nodules of alfalfa subpopulations grown in nonsterile field soil and tested for effectiveness. A second set of experiments was similarly conducted with five populations: two unselected alfalfa populations, a population derived from each unselected population by three cycles of selection for enhanced nitrogenase activity, and the F1 between the two third-cycle populations. Plant selection for high and low levels of N2 fixation potential modified the preference of alfalfa for antibiotic resistant mutant strains of R.meliloti. Selected alfalfa subpopulations showed increased nodule occupancy by indigenous and by effective mutant strains compared with the unselected original population. In other experiments, nodule occupancy by an effective mutant increased on selected alfalfa populations compared with that of the unselected populations, while nodule occupancy by indigenous Rhizobium decreased. Generally, the indigenous isolates from the alfalfa entries selected for high levels of N2 fixation potential were more effective than were the isolates of Rhizobium from unselected populations and subpopulations selected for low levels of N2 fixation potential. It was concluded that plant selectlon programs should be conducted with the most effective and competitive strains of Rhizobium available. This should increase the likelihood that selected alfalfa populations will show preference for very effective strains of indigenous Rhizobium.

Chemotaxis by Rhizobium meliloti

Chemotaxis by Rhizobium meliloti strain Ve 26 has been studied and conditions required for chemotaxis have been defined, using the Adler capillary assay technique. Several sugars and amino-acids were shown to be attractants with varying effectiveness for this organism: sugars are weak attractants (except gluconate) and amino-acids are good attractants (except unpolar amino-acids).

Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti.

Large plasmids of molecular weight varying from 90 to around 200 x 10(6) have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation - phenol extraction procedure. With a less destructive method (Eckardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300 x 10(6) (= megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids. All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nifDH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nifDH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.

Antibiotic sensitivity in Rhizobium and Agrobacterium

Summary The resistance of 81 Rhizobium strains and 11 Agrobacterium strains to 16 antibiotics was investigated. Most strains were resistant even to high concentrations of lincomycin. All the strains of the species Rhizobium meliloti, Rhizobium phaseoli, Rhizobium japonicum and the genus Agrobacterium reacted relatively homogeneously to all the antibiotics, unlike Rhizobium trifolii, Rhizobium leguminosarum and Rhizobium spp. The majority of the fast-growing species of Rhizobium tested was found to be resistant to > 32 μg/ml penicillin and to Rhizobium were susceptible to penicillin concentrations 1 μg/ml. The majority of the Agrobacterium strains tested were resistant to streptomycin concentrations > 32 μg/ml, allowing them to be distinguished from strains of R. meliloti which were rarely resistant to such high concentrations.

Structural elucidation, using h.p.l.c.-m.s. and g.l.c.-m.s., of the acidic polysaccharide secreted by rhizobium meliloti strain 1021

An improved, glycosyl-sequencing method has been used for elucidating the structure of the acidic polysaccharide secreted by Rhizobium meliloti strain 1021. The polysaccharide was methylated, the ether partially hydrolysed, the products were reduced, and the alditols ethylated; the resulting mixture of peralkylated oligosaccharide-alditols was analyzed by h.p.l.c.-m.s. Chemical ionization (c.i.) mass spectra were obtained at 2-s intervals, as the various, peralkylated oligosaccharide-alditols were eluted from the h.p.l.c. column. Peralkylated mono-, di-, tri-, and tetra-saccharide-alditols could be readily detected, and at least partially identified, by their M + I ions, in conjunction with other characteristic ions present in their c.i.-mass spectra. The peralkylated di- and tri-saccharide-alditols were further analyzed by g.l.c.-m.s. in the electron-impact mode. The structure of the acidic polysaccharide secreted by R. meliloti strain 1021 is the same as that of the previously characterized polysaccharide secreted by R. meliloti strain U-27.

Genetic Variation for Acetylene Reduction Rate and Other Characters in Alfalfa

The objective of this study was to determine the general (GCA) and specific (SCA) combining abilities of alfalfa (Medicago sativa L.) for rate of acetylene reduction, plant dry weight, N content (%), total plant N and nodule score. The parental clones were selected from the commercial cultivars ‘Beaver,’ ‘Roamer,’ ‘Saranac,’ and ‘Rambler.’ Five strains of Rhizobium meliloti Dang., including four single strains and one commercial inoculant ‘Balsac’ were used. The most effective strain of Rhizobium was NRG61, while RM110 was the poorest. Diallel analysis of the six‐parent half diallel cross revealed that mean squares of both GCA and SCA were highly significant for all characters except the SCA mean square for nodule score. The highly significant interactions of GCA and SCA with Rhizobium strains suggested differential expressions of gene effects for alfalfa genotypes with different strains of Rhizobium. The GCA variance components were higher than SCA for total plant dry weight, total plant N and nodule score. This suggested that the major portion of the total genetic variation was additive. Recurrent selection for these characters should be possible. However, the additive and non‐additive gene effects were equally important for acetylene reduction rate with Rhizobium strains NRG43, NRG61, and NRG118. Reciprocal recurrent selection method might be necessary to improve this character with these single Rhizobium strains. Combining ability analysis for each of the parental clones indicated that Clone 4 had consistently high and positive GCA effects over all Rhizobium treatments. Clone 2 was on the other extreme with negative GCA effects in all cases. None of the other parental clones produced consistently positive GCA effects across all the Rhizobium strains.

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.

EFFET DU SOUFRE ET DE L’AZOTE SUR LA FIXATION SYMBIOTIQUE D’AZOTE CHEZ LES PLANTULES DE LUZERNE (Medicago Sativa L.)

The effect of the addition of sulphur and nitrogen on the aerial mass, root mass, nodulation and nitrogenase activity was studied on alfalfa cv. Saranac seedlings inoculated with a very efficient strain of Rhizobium meliloti. The aerial mass, root mass and a nodulation index increase with the addition of increasing concentrations of nitrogen and sulphur. The rate of increase of aerial mass and the nodulation index with sulphur are not constant over the range of the concentrations studied. Sulphur and nitrogen appeared to have a very important multiplicative effect on aerial mass and root mass. In the absence of sulphur, nitrogen did not affect nitrogenase activity. The aerial mass and the nodulation index are strongly correlated to the root mass and nitrogenase activity. However, nitrogenase activity and the root mass were not significantly correlated.