Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Variation in Preference for Rhizobium meliloti Within and Between Medicago sativa Cultivars Grown in Soil

Variation in nodulation preferences for Rhizobium strains within and between Medicago sativa cultivars was assessed in the greenhouse with plants grown in Leonard jars and two soils of diverse origin (Lanark and Ottawa), using inocula consisting of effective individual or paired strains of R. meliloti which could be recognized by high-concentration antibiotic resistance. The results indicated considerable variability in host preferences for R. meliloti among plants within cultivars but not between cultivars. The implications of this variation are discussed from the point of view of possible improvement of symbiotic nitrogen fixation. With one exception, the differences in nodulation success between inoculant R. meliloti strains were consistent in Leonard jars and both soils. All introduced strains formed significantly more nodules in Renfrew soil containing few native rhizobia than in Ottawa soil with a large resident R. meliloti population. Plants grown in Lanark soil without inoculation were ineffectively nodulated by native rhizobia and yielded significantly less growth than those receiving inoculation. In contrast, the yield of inoculated plants in Ottawa soil did not significantly differ from those without inoculation due to effective nodulation by native R. meliloti. The data indicated synergistic effects on yield by certain paired strain inocula relative to the same strains inoculated individually in Lanark but not in Ottawa soil or Leonard jars.     

Competition for alfalfa nodulation under metal stress by the metal‐tolerant strain Ochrobactrum cytisi Azn6.2

Legume plants, in association with rhizobia, are gaining increasing interest for heavy metal rhizoremediation. This symbiotic interaction combines the advantages of rhizoremediation and soil nitrogen enrichment. In metal polluted soils, Ochrobactrum cytisi can elicit non‐fixing nodules on legumes, including Medicago sativa. Nodulation kinetics was much slower when M. sativa plants were inoculated with O. cytisi Azn6.2 compared with the natural symbiont Ensifer meliloti 1021 and nodules were ineffective in nitrogen fixation. A competition experiment was performed using alfalfa grown on heavy metals, and co‐inoculated with equal amounts of the metal‐sensitive E. meliloti 1021 and the metal‐resistant O. cytisi Azn6.2. When plants were inoculated in non‐polluted substrates, all nodules were formed by E. meliloti 1021. Nevertheless, under increasing metal concentrations, the number of nodules occupied by O. cytisi grew. At the highest metal concentration, all nodules were elicited by O. cytisi, suggesting that the resistant species can take the place of the natural symbiont. This fact has important ecological and environmental implications when proposing legume–rhizobia symbioses for rhizoremediation and highlights the need of selecting highly resistant rhizobia in order to be competitive in polluted soils.     

The contributions of plant and bacteria genotypes in the growth and nitrogen accumulation of inoculated alfalfa

The symbiotic efficiency of each of 30 alfalfa (16 Medicago sativa and 14 M. varia) cultivars inoculated with 7 Rhizobium meliloti strains was studied in three field experiments. Two-factor analysis of variance of the obtained date demonstrated that the green mass yield and nitrogen accumulation depend on genotypes of both partners. The total contribution of plant and bacterial genotypes to the variation of green mass yield increased from 0–17% in the first year of alfalfa growth to 40–78% in the third year. The effect of the genotypic variability of the symbiotic partners was higher for N accumulation than for the green mass. There was a negative correlation between plant mass and N accumulation in the uninoculated plants with the relative (%) deviations of these parameters in the inoculated plants. In the experiments conducted in the Tashkent region the efficiency of the “alfalfa-R. meliloti” symbiosis was higher than in the experiment conducted in the Tumen region.     

Effect of inoculation and nitrogen application on the elemental composition ofMedicago sativa

Medicato sativa plants were grown with and without inoculation with an effectiveR. meliloti strain on nitrogenfree agar slopes. Half of the uninoculated replications were, furthermore, provided with a sufficient amount of the NH₄NO₃ fertilizer, the fertilizer-treated plants (+N plants) achieved dry matter yield equal to that of inoculated variants. The concentration and uptake of Cl, Mn, Cu, Zn, Br, Sr and Mo were found to be significantly lower in inoculated plants relative to the +N plants indicating that these composition alterations are constitutive to the induced biological nitrogen fixation.     

Rhizobium meliloti inoculation of alfalfa selected for tolerance to acid,aluminum-rich soils

Alfalfa (Medicago sativa L.) growth and nodulation in acid soil is reduced because the plant and its bacterial symbiontRhizobium meliloti cannot tolerate acid, aluminum-rich soil. A study was conducted to determine if a relatively acid-tolerant alfalfa germplasm combined with a relatively acid-tolerantR. meliloti strain could overcome these limitations. In a light room study, an acid-tolerant alfalfa germplasm inoculated with a more acid-tolerantR. meliloti strain produced greater top growth, nodule number and weight, and acetylene reduction values in an unlimed soil (pH 4.6) than the same germplasm inoculated with a relatively acid-sensitiveR. meliloti strain or an acid-sensitive germplasm inoculated with either a relatively acid-tolerant or acid-sensitiveR. meliloti strain.     

Nodulation of annual medicago by strains ofR. Meliloti in a commercial inoculant as influenced by soil phosphorus and pH

Summary Four species of annual medics showed poor growth and little or no nodulation in an inoculated, acid soil low in available phosphorus. Nodulation was significantly increased by adding phosphorus to the soil. The number of nodules found on the roots of each species was approximately the same for soil at pH 5.5 or 6.8. A commercial inoculant ofRhizobium meliloti was adequate for nodulation of all four species and two strains of rhizobia predominated in the nodules which were assayed by immunodiffusion.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Influence of Location, Host Cultivar, and Inoculation on the Composition of Naturalized Populations of Rhizobium meliloti in Medicago sativa Nodules

A phage typing system was used to evaluate the composition of indigenous populations of Rhizobium meliloti inhabiting nodules of Medicago sativa cultivars grown with and without inoculation at two field sites during 1983 and 1984. Soil at both locations contained established populations of R. meliloti at planting. Analysis of 1,920 nodule isolates revealed 55 unique phage types of indigenous R. meliloti at one site and 65 indigenous types at the other location. The distributions of phage types differed markedly between locations. At one site, the nodule population was dominated by two phage types; seven others occurred consistently but at lower frequency, and the remainder were encountered infrequently. No indigenous types predominated at the other location, although nine occurred more frequently than the remaining types. Indigenous R. meliloti predominated in nodules from inoculated plots at both sites, with inoculant recovery varying between 10 and 38% in each of two years. The frequency of occurrence of particular phage types at one location was significantly influenced by both M. sativa cultivar and inoculation. At this location, the interaction of cultivar and inoculation on the incidence of phage types suggests that the presence of an inoculant strain differentially affected nodule occupancy of M. sativa cultivars by members of the indigenous R. meliloti population. At both sites, the frequency of specific phage types differed between years. The data emphasize the importance of understanding the ecology and characteristics of indigenous Rhizobium populations as a prerequisite for elucidating problems of inoculant establishment and persistence in competitive situations.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Mixture of endophytic Agrobacterium and Sinorhizobium meliloti strains could induce nonspecific nodulation on some woody legumes

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.     

Derivatives of Rhizobium meliloti strains carrying a plasmid of Rhizobium leguminosarum specifying hydrogen uptake and pea-specific symbiotic functions

pIJ1008, a Rhizobium leguminosarum plasmid which determines hydrogen uptake ability and symbiotic functions in pea was transferable to three of seven natural isolates of R. meliloti tested. In these three strains, pIJ1008 was maintained stably with the respective sym megaplasmid indigenous to each R. meliloti strain. These strains carrying both plasmids nodulated alfalfa but not pea. By reisolation and examination of the strains from alfalfa nodule tissue, it was shown that pIJ1008 continued to be maintained but that pea-nodulation ability was suppressed.In one strain of R. meliloti which carries a 200 kb cryptic plasmid (in addition to a megaplasmid), the transfer and selection for pIJ1008 resulted in the loss of the cryptic plasmid.In three separate plant growth experiments, alfalfa nodules induced by each of the R. meliloti strain carrying both sym plasmids were assayed for hydrogen uptake activity. The average activity was 40-, 3.5-and 2-fold higher than with the respective pIJ1008-free strains. However, this higher activity was not accompanied by an increase in plant biomass or nitrogen content of shoots.C.B.R.I. Contribution Number: 1478     

Symbiotic effectiveness of Rhizobium meliloti at low root temperature

Laboratory, growth chamber and field experiments were conducted to select among 226 isolates of Rhizobium meliloti for the ability to grow, nodulate alfalfa (Medicago sativa L.) and support N₂-dependent plant growth between 9° and 12°C. There was wide variation in the abilities of R. meliloti isolates to grow and form nodules at 10°C. Culture doubling times (t_d) varied from 1 to 155h, and the number of nodules formed on alfalfa in growth pouches in 2 weeks varied from 0 to 3.8 nodules per plant. Nodulation occurred at 9°C, but there was no significant N₂-dependent plant growth at this temperature. However, several isolates of R. meliloti had the ability to nodulate alfalfa and produce N₂-dependent growth at root temperatures between 10° and 12°C root temperature than did 14 other isolates tested. In field experiments, inoculation with strain NRG-34 resulted in greater nodule numbers, nodule weight, proportion of nodules occupied by the inoculant strain and plant weight than did inoculation with a commercial strain (NRG-185). These results permitted selection of a strain with better low-temperature competitive abilities than the currently available commercial strains.     

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Summary The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod⁺ and Fix⁺ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.     

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.     

Plant defence and delayed infection of alfalfa pseudonodules induced by an exopolysaccharide (EPS I)-deficient Rhizobium meliloti mutant

Mutants of the symbiotic soil bacterium Rhizobium meliloti that fail to synthesize the acidic exopolysaccharide EPS I were unable to induce infected root nodules on Medicago sativa L. (alfalfa). These strains, however, elicited pseudonodules that contained no infection threads or bacteroids. The cortical cell walls of the pseudonodules were abnormally thick and incrusted with an autofluorescent material. Parts of these cell walls and wall appositions contained callose. Biochemical analysis of nodules induced by the EPS I-deficient R. meliloti mutant revealed an increase of phenolic compounds bound to the nodule cell walls when compared with the wild-type strain. These microscopic and biochemical data indicated that a general plant defence response against the EPS I-deficient mutant of R. meliloti was induced in alfalfa pseudonodules. Following prolonged incubation with the EPS I-deficient R. meliloti mutant, the defence system of the alfalfa plant could be overcome by the rhizobium mutant. In the case of the delayed infections, the mutants colonized lobes of the pseudonodules, but the infection threads in these nodules had an abnormal morphology. They were greatly enlarged and did not contain the typical gum-like matrix inside. The bacteria were tightly packed. Based on the mechanism of phytopathogenic interactions, we propose that EPS I or a related compound may act as a suppressor of the alfalfa plant defence system, enabling R. meliloti to infect the plant.     

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V _max and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.     

Microscopic studies of cell divisions induced in alfalfa roots by Rhizobium meliloti

We have used spot-inoculation and new cytological procedures to observe the earliest events stimulated in alfalfa (Medicago sativa L.) roots by Rhizobium meliloti. Roots were inoculated with 1–10 nl of concentrated bacteria, fixed in paraformaldehyde, and after embedding and sectioning stained with a combination of acridine orange and DAPI (4-6-diamidino-2-phenylindole hydrochloride). Normal R. meliloti provoke cell dedifferentiation and mitosis in the inner cortex of the root within 21–24 h after inoculation. This activation of root cells spreads progressively, leading to nodule formation. In contrast, the R. meliloti nodA and nodC mutants do not stimulate any activation or mitosis. Thus the primary and earliest effect of Rhizobium nod gene action is plant cellular activation. A rapid, whole-mount visualization by lactic acid shows that the pattern of nodule form varies widely. Some R. meliloti strains were found to be capable of stimulating on alfalfa roots both normal nodules and a hybrid structure intermediate between a nodule and a lateral root.     

Some physiological characteristics ofRhizobium meliloti andR. trifolii in relation to efficiency of symbiosis with lucerne and Egyptian clover

Summary Several strains ofRhizobium meliloti andR. trifolii were screened for efficiency of symbiosis with their respective hosts, lucerne (Medicago sativa L.) and Egyptian clover or berseem (Trifolium alexandrinum L.) under bacteriologically controlled conditions on agar slopes. Based on the dry weight of plants which was taken as the parameter of symbiotic efficiency, the strains of the two species of rhizobia were grouped into different categories with the aim of correlating some physiological properties of bacteria with their symbiotic efficiency. The physiological properties taken into account were the abilities of bacteria to produce total Salkowski positive compounds (SPC) in terms of indole acetic acid (IAA) equivalents on a tryptophane containing substrate, to catabolize added IAA, to reduce nitrate, to utilize nitrite and to reduce triphenyl tetrazolium chloride (TTC).No positive and significant correlations could be established between symbiotic efficiency and the physiological properties of bacteria. Nevertheless, significant inverse relationships between formation of SPC from tryptophane and IAA destruction (in bothR. meliloti andR. trifolii), between nitrite formation and nitrite utilization (only inR. trifolii) and between symbiotic efficiency and nitrite utilization (only inR. meliloti) were apparent.     

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti

Background  

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H₂O₂ and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria.