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.     

Methods To Alter the Recovery and Nodule Location of Bradyrhizobium japonicum Inoculant Strains on Field-Grown Soybeans

Three strains of Bradyrhizobium japonicum, I17, 110, and 61A76, were evaluated for their ability to form nodules on field-grown soybeans in soil with a highly competitive indigenous B. japonicum population. The predominant indigenous strain, 0336, in the field site used was unlike the more common isolates from Midwestern soils which belong to the 123 or 138 serogroups. This strain persisted in the soil for at least 30 years without any soybean crops. The three inoculant strains differed in their ability to compete with indigenous strains for nodule formation. Four different inoculation treatments were tested in three adjacent fields. When the amount of inoculum was increased, a higher proportion of nodules contained the inoculant strain. The most competitive inoculant strain was I17, a recent field isolate. Strain 61A76 was better than 110. There was no difference in recovery of the inoculant strains on the Hodgson or Corsoy soybean cultivars, nor was there a difference in recovery of the inoculant strains during the growing season. The vertical distribution of nodules containing the inoculant strains was affected by the method of adding the inoculant to the soil. Inoculant added to the seed furrow produced nodules mainly in the top region of the soybean root. Inoculant tilled into the soil produced nodules primarily in the bottom part of the root. The nodules that were produced in the bottom part of the root are younger and may contribute significant amounts of fixed nitrogen to the soybean during seed formation.     

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.     

Effectiveness of Rhizobium Strains Used in Inoculants after Their Introduction into Soil

Rhizobium strains used in inoculants for Trifolium spp., Medicago spp., Glycine max, and Lotus pedunculatus were isolated from nodules of these legumes grown in soils into which the rhizobia had been introduced 4 to 8 years before. Isolations were made from a total of 420 nodules. Nodule occupancy by the inoculant strains varied from 17.7% for a soybean strain to 100% in the case of L. pedunculatus whose specific rhizobia did not occur in the soils studied. In general, inoculant strains isolated from nodules did not differ in effectiveness from cultures of the same strains concurrently maintained in lyophilized form. The average effectiveness of all of the isolates (identified and unidentified) from a legume was 7.1 to 73.3% higher than that of the unidentified isolates alone, demonstrating the prolonged effect that a single-seed inoculation has on the rhizobial population in a soil which had not been planted with legumes before. Relatively weak recovery of a Rhizobium japonicum strain introduced into soil 4 years after soybean seed inoculated with a different strain had been planted in the same soil confirmed the advantage of a resident population over an introduced inoculant strain.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.     

Growth of Indigenous Rhizobium leguminosarum and Rhizobium meliloti in Soils Amended with Organic Nutrients

The ability of indigenous Rhizobium leguminosarum and Rhizobium meliloti to use organic nutrients as growth substrates in soil was assessed by indirect bacteriophage analysis. A total of 17 organic compounds, including 9 carbohydrates, 3 organic acids, and 5 amino acids, were tested (1,000 μg g⁻¹) in three soils with different cropping histories. Four additional soils were screened with a glucose amendment. Nutrient amendments stimulated growth of indigenous rhizobia, allowing subsequent replication of indigenous bacteriophages. Phage populations were enumerated by plating soil extracts on 19 R. leguminosarum and 9 R. meliloti indicator strains, including root nodule isolates from the soils assayed. On the basis of indirect phage analysis, all soils contained native rhizobia similar to one or more of the indicator strains, although not all indicator strains were detected in soil. All organic compounds stimulated growth of indigenous rhizobia, but the growth response varied for each rhizobial strain depending on the nutrient, the nutrient concentration, and the soil. Indigenous rhizobia readily utilized most organic compounds except phenylalanine, glycine, and aspartic acid. The ability of indigenous rhizobia to utilize a wide range of organic compounds as growth substrates in situ indicates their ability to successfully compete with other soil bacteria for nutrients in these soils.     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

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.     

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.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Competition Among Rhizobium spp. for Nodulation of Leucaena leucocephala in Two Tropical Soils

The successful nodulation of legumes by a Rhizobium strain is determined by the competitive ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena spp. (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia in both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules.     

Survival of Rhizobium in Acid Soils

A Rhizobium strain nodulating cowpeas did not decline in abundance after it was added to sterile soils at pH 6.9 and 4.4, and the numbers fell slowly in nonsterile soils at pH 5.5 and 4.1. A strain of R. phaseoli grew when added to sterile soils at pH 6.7 and 6.9; it maintained large, stable populations in soils of pH 4.4, 5.5, and 6.0, but the numbers fell markedly and then reached a stable population size in sterile soils at pH 4.3 and 4.4. The abundance of R. phaseoli added to nonsterile soils with pH values of 4.3 to 6.7 decreased similarly with time regardless of soil acidity, and the final numbers were less than in the comparable sterile soils. The minimum pH values for the growth of strains of R. meliloti in liquid media ranged from 5.3 to 5.9. Two R. meliloti strains, which differed in acid tolerance for growth in culture, did not differ in numbers or decline when added to sterile soils at pH 4.8, 5.2, and 6.3. The population size of these two strains was reduced after they were introduced into nonsterile soils at pH 4.8, 5.4, and 6.4, and the number of survivors was related to the soil pH. The R. meliloti strain that was more acid sensitive in culture declined more readily in sterile soil at pH 4.6 than did the less sensitive strain, and only the former strain was eliminated from nonsterile soil at pH 4.8; however, the less sensitive strain also survived better in limed soil. The cell density of the two R. meliloti strains was increased in pH 6.4 soil in the presence of growing alfalfa. The decline and elimination of the tolerant, but not the sensitive, strain was delayed in soil at pH 4.6 by roots of growing alfalfa.     

Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti

The development and function of the Rhizobium meliloti-Medicago sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable. The intracellular pH of R. meliloti was measured using either radioactively labeled weak acids (5,5-dimethyloxazolidine-2,4-dione and butyric acid) or pH-sensitive fluorescent compounds; both methods gave similar values. Six acid-tolerant strains (WSM419, WSM533, WSM539, WSM540, WSM852, and WSM870) maintained an alkaline intracellular pH when the external pH was between 5.6 and 7.2. In contrast, two Australian commercial inoculant strains (CC169 and U45) and four acid-sensitive strains from alkaline soils in Iraq (WSM244, WSM301, WSM365, and WSM367) maintained an alkaline intracellular pH when the external pH was ≥6.5, but had intracellular pH values of ≤6.8 when the external pH was ≤6.0. Four transposon Tn5-induced mutants of acid-tolerant strain WSM419, impaired in their ability to grow at pH 5.6, showed limited control over the intracellular pH. The ability to generate a large pH gradient under acid conditions may be a better indicator of acid tolerance in R. meliloti under field conditions than is growth on acidic agar plates.     

Assessing Genotypic Diversity and Symbiotic Efficiency of Five Rhizobial Legume Interactions Under Cadium Stress for Soil Phytoremediation

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima<M. truncatula<M. marina<L. sativus<L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

Method for Testing Degree of Infectivity of Rhizobium meliloti Strains

The infectiveness of different strains of Rhizobium meliloti was tested with a technique that uses the addition of tetracycline to the root medium. To stop the infection, the antibiotic was added some time after the inoculation of Medicago sativa plants. A coefficient of infectivity for each strain was calculated according to the number of nodules that appeared with and without the addition of the antibiotic. This method seems useful in infectivity studies and is simpler and easier to perform than the test of competence between strains.     

Genetic diversity of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> associated with alfalfa in Chilean volcanic soils and their symbiotic effectiveness under acidic conditions

A study was conducted with the aim of evaluating the genetic diversity of alfalfa rhizobia isolated from volcanic soils in southern Chile and their ability to establish an effective symbiosis with alfalfa. Rhizobial strains isolated from nodules were identified and selected based on PCR analyses and acid tolerance. Symbiotic effectiveness (nodulation and shoot dry weight) of acid-tolerant rhizobia was evaluated in glasshouse experiments under acidic conditions. The results revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules with a high genetic diversity at strain level grouped in three major clusters. There was a close relationship (r ² = 0.895, P ≤ 0.001, n = 40) between soil pH and the size of rhizobial populations. Representative isolates from major cluster groups showed wide variation in acid tolerance expressed on buffered agar plates (pH 4.5–7.0) and symbiotic effectiveness with alfalfa. One isolate (NS11) appears to be suitable as an inoculant for alfalfa according to its acid tolerance and symbiotic effectiveness at low pH (5.5). The isolation and selection of naturalized S. meliloti strains with high symbiotic effectiveness under acidic conditions is an alternative approach to improving the productivity of alfalfa and for reducing the application of synthetic fertilizers in Chile.     

Survival ofRhizobium leguminosarum biovarviceae subjected to heat,drought and salinity in soil

Two strains (RCR 1001 and 1044) and a commercial inoculant (Okadin) ofRhizobium leguminosarum biovarviceae were tested for their ability to survive in autoclaved clay soil for up to four months under heat, salinity and drought stress. Resistance to heat was tested by incubating rhizobia in soil at 27, 37 and 42 °C. Tolerance of rhizobia to salinity was investigated by growing rhizobia in soil salinized with 1 and 2 % NaCl (m/m). Drought resistance was tested by subjecting bacteria to soil moisture contents of 20, 10 and 5%. Strain RCR 1001 was more resistant to heat and nodulated faba bean better than other tested strains. A commercial inoculant Okadin survived more (plate count method) and nodulated faba bean (plant infectivity, most probable number, MPN) at moisture content of 5% and 2% NaCl. Although, strains RCR 1001 and 1044 resisted these stress conditions (plate count) they lost their abilities to nodulate faba bean (MPN-test). There is a possibility for selection of effective rhizobia which are more tolerant to harsh conditions.     

Estimation of host-strain compatibility for symbiotic N-fixation between Rhizobium meliloti,several annual species of Medicago and Medicago sativa

Standard methods for estimating host-strain compatibility for N-fixation in vitro proved inadequate for examination of the symbiosis between Rhizobium meliloti and Medicago sativa, M. littoralis, M. tornataas well as for hybrids between M. littoralisand M. truncatula. When screening procedures in soil free of background R. melilotiwere adopted, nitrogen fixation appeared to be unrestricted by the screening environment. During the experiments it became clear that the single commercial inoculant strain available in Australia (CC169) was poorly effective at N-fixation with many of the lucerne and annual medic cultivars examined. Interspecific hybridisation between M. truncatulaand M. littoralis also produced genotypes that were particularly difficult to satisfy in a symbiotic sense. Despite the existence of substantial host-strain genotype interactions between the symbiotic partners, several alternative strains of R. meliloti proved to be broadly effective at N-fixation. One of these, WSM826, has since become commercially available in Australia.     

Motility and Chemotaxis of Rhizobium meliloti in Soil

The spreading of Rhizobium meliloti strains in various autoclaved soils was measured by using simple modifications of established techniques. Behavioral mutants were used to demonstrate that efficient spreading requires active motility and chemotaxis. The rate of spreading was affected by physical or chemical differences among soils and by changes in water content.