Competition between species of Rhizobium for nodulation of Glycine max

Glycine max cv. Malayan is a promiscuously nodulating cultivar which formed nodules with 6 out of 9 strains of Rhizobium spp of diverse origin and all strains of R. japonicum tested. No generalizations can be made as to the probability of strains isolated from a particular host being infective on Malayan as only some isolated from Centrosema pubescens, and Cajanus cajan were able to form nodules. In competition with R. japonicum at 30°C all 20 strains of Rhizobium spp isolated from Malayan grown in Nigeria formed fewer than 50% of the nodules and 14 strains fewer than 25%. Competition was influenced by root temperature. Three strains of Rhizobium spp were poor competitors with R. japonicum between 24° and 33°C but at 36°C they formed more nodules (74–88%) than R. japonicum. Another strain of Rhizobium spp formed the majority of the nodules between 27° and 36°C whereas R. japonicum formed the most at 24°C.     

Interference in culture among Rhizobium strains

Growth interference in culture was shown by 57 strains of Rhizobium belonging to different inoculation groups. The assays were conducted in vitro by spot tests on agar plates and using 1007 paired combinations, 240 cases of interference were observed.A frequency of interference of about 30% was found among strains of R. meliloti and R. meliloti, R. lupini, R. trifolii and R. leguminosarum. Only one case out of 168 showed interference between R. meliloti and R. japonicum.     

Response of Rhizobium strains to acid and aluminium stress

Synchronous-culture, enrichment and isolation trials were done to determine effects of simulated soil acidity stress on growth of Rhizobium sp. (cowpea group), and to test whether tolerance of a strain is stable.In synchronous cultures, acidity and Al reduced the frequency of cell division. Non-dividing cells did not die, but those near division snowed evidence of heightened sensitivity to Al³⁺ and H⁺.Differences in tolerance within single-strain populations were evidently not genetically determined. Prolonged culture under stress (72 generations) failed to enrich a strain in putative tolerant variants. And from six strains of different tolerance, isolates from single colonies that had grown on stress medium were no more tolerant than isolates from colonies on non-stress medium. Tolerance is a consistent and stable strain property.     

Rhizobium inoculation of Calopogonium caeruleum

The shade-tolerant cover legume Calopogonium caeruleum is promiscuous in its nodulating habits. In sand culture, symbiotic effectiveness of the strains tested was variable; 6 strains of rhizobia markedly improved shoot yields and 20 increased shoot N content. In pot experiments using cultivated and non-cultivated soils, inoculation gave no significant increase in shoot yields. When grown under rubber in plantation conditions at four localities, shoot dry matter yields, N content and nodulation also were not different from uninoculated plants when sampled for up to 2 yr after planting. This occurred despite the low numbers (< 10 g^?1 soil) of native rhizobia at some sites and an appreciable establishment (> 70% recovery in nodules) by the inoculant strains.     

Interactions of the microflora from nodulation problem and non-problem soils towards Rhizobium spp on agar culture

Microorganisms (348 fungi, 388 actinomycetes and 319 bacteria) were isolated from a nodulation problem soil, a non-problem virgin soil, a cultivated problem soil and the rhizosphere of clover plants grown in the problem soil. Rhizobium trifolii TA 1 which failed to establish in problem soils was inhibited on laboratory media by a greater number of these soil microorganisms than the better soil colonizing R. trifolii (WU95 and WU290) and R. lupini (WU425). R. lupini was not inhibited or stimulated on agar by many soil or rhizosphere isolates. R. meliloti showed greater stimulation than either R. trifolii or R. lupini and was inhibited by relatively few soil microorganisms so that its poor soil survival was thought to be due to chemical or physical soil conditions rather than to biotic factors. The greatest incidence of rhizobial inhibitors, mainly associated with TA 1, was found among the isolates from the clover rhizosphere. There was a reduction in the relative numbers of rhizobial inhibitors isolated from the cultivated soil compared with the virgin problem soils, a result possibly due to the changed soil environment changing with cultivation, altered vegetation and the addition of superphosphate. Inhibitors of rhizobia were more frequent amongst the bacteria than fungi or actinomycetes. Strong stimulation was more commonly shown by fungi than by actinomycetes or bacteria. The interaction on agar between rhizobia and the soil microflora is related to soil colonization and persistence.     

Diversity in the nutritional requirements of strains of various Rhizobium species

An examination of 85 strains of bacteria from five species of rhizobia (Rhizobium sp., R. japonicum, R. lupini, R. meliloti and R. trifolii), using a new semi-quantitative assay procedure, disclosed wide diversity among the strains in their requirement for, and response to, vitamins, carbon sources, and nitrogen sources. Approximately half of the strains in the first four species grew as well without vitamins as they did when supplied with a vitamin mixture or with yeast extract, but the other strains showed considerable variation in their requirements. Some strains were inhibited by yeast extract, or showed best growth in basic media supplemented with only one vitamin. The strains within the species differed widely in their utilization of gluconate, mannitol and arabinose as C-sources; there was less diversity in their use of glutamine, histidine, NH₄⁺-N and NO₃^?-N as N-sources. The significance of these observations in the culture of rhizobia in the laboratory, in their ecological adaptation to particular environments, and in their ability to form an effective symbiosis with particular host legumes, is discussed.     

Influence oF Pseudomonas putida on nodulation of Phaseolus vulgaris

The effect of Pseudomonas putida (isolate M17) on Rhizobium phaseoli nodulation of the common bean, Phaseolus vulgaris, was investigated under field and greenhouse conditions. The results indicated that P. putida markedly increased nodulation compared to R. phaseoli controls. Furthermore, 2-ketogluconic acid, a phosphate-solubiliring compound, was detected in P. putida M17. This could imply an increased P supply to roots of P. vutgaris, which may function to increase nodules. Bean yields and shoot fresh weight were not significantly altered by the addition of P. putida M17.     

Field inoculation of Medicago with V-A mycorrhiza and Rhizobium in phosphate-fixing agricultural soil

Field inoculation of Medicago sativa with its symbiotic partners Rhizobium meliloti and the endomycorrhizal fungus Glomus mosseae was assayed under standard agricultural conditions in untreated arable phosphate-fixing soil. Glomus mosseae was successfully introduced and efficiently stimulated plant growth, N incorporation and P uptake. In contrast with a previous filed experiment, Rhizobium meliloti was also effective when inoculated alone. The dual inoculation of Rhizobium + Glomus more than doubled yield compared to an uninoculated control.     

Abundance and characterization of cowpea miscellany Rhizobium from sudanese soils

Sudan is the fourth largest exporter of groundnuts in the world, yet little is known concerning the plant-rhizobial symbiosis. A study was made on the abundance of groundnut-nodulating rhizobia in the soils of Sudan as related to soil properties and the duration since groundnuts were last planted. Also, physiological, serological and nitrogen-fixing characteristics of Sudanese rhizobia are reported. All but one of 32 sites contained more than 300 rhizobia g^?1 soil capable of forming nodules on siratro (Macroptilium atropurpureum). Several of these soils had never been planted to groundnut. A correlation matrix indicated no relationship was present between soil rhizobial populations and any of the measured soil properties, or between soil rhizobial populations and the time since groundnuts were last planted in the rotation. Individual isolates of Rhizobium from six legumes: groundnut (Arachis hypogaea), mung bean (Vigna radiata), lubia (Dolichos lablab), cowpea (Vigna unguiculata), pigeonpea (Cajanus cajan) and bambara groundnut (Voandzeia subterranea) were obtained from four locations in Sudan. All isolates were able to nodulate each of the six legumes when grown in sterile vermiculite. The isolates grew in 0.1% NaCl-amended media, but growth was variable in 2.0% amended media. Most isolates grew after exposure to moist heat for 15 min at 50°C. Optimum pH for growth was, in general, between pH 6 and 8. Agglutination reactions indicated isolates from groundnuts, as well as isolates from other legumes, belonged to several serological groupings. Some isolates formed a large number of nodules on a Sudanese groundnut cultivar, whereas other isolates formed only few nodules.     

Movement by rhizobium and nodulation of legumes

Several Rhizobium spp were tested for chemotaxis using a soft agar assay. Bacteria migrated in chemotactic bands in response to substances present in the agar medium. R. japonicum S-110 formed chemotactic bands in response to l-arabinose, l-canavanine, and yeast extract but not to several other common compounds, including several amino acids and sugars. These results suggested that simple compounds present in legume root exudates may attract these bacteria and aid in their rhizosphere accumulation and, thus, in the nodulation process. A slow-migrating mutant of R. japonicum S-110 was isolated. Microscopic examination showed that mutant populations had a much lower proportion of motile cells than did wild-type populations. Greenhouse nodulation tests, in which mutant and wild-type cells were mixed and used as inoculum, indicated that the mutant had less nodulation potential.     

Indole acetic acid production by Rhizobium: Effect of 2-ketoglutaric acid

I ndole acetic acid (IAA) production from tryptophan by cell suspensions of Rhizobium trifolii, R. leguminosarum, R. phaseoli and R. lupini was studied in the presence or in the absence of 2-ketoglutaric acid. In R. lupini, production of IAA was strongly enhanced by the ketoacid, but in fast growing rhizobia it was less enhanced or unaffected. On the other hand, glutamic acid inhibits IAA production by R. meliloti, but stimulates IAA production in both R. leguminosarum and R. phaseoli. A hypothesis is proposed to explain these results.     

Survival of fast- and slow-growing Rhizobium spp under conditions of relatively mild desiccation

When subjected to desiccation with Ca(NO₃)₂ at 27°C, strains of the fast growing Rhizobium meliloti and R. trifolii, survived better than slow-growing strains of R. japonicum and of the “cowpea miscellany”. At lower vapour pressures in a forced-draught oven, the pattern of survival changed and strains of slow-glowing Rhizobium withstood desiccation better than those of fast-growing species. The results are considered to be consistent with the interpretation that a lower internal water-retaining ability at any relative vapour pressure, renders strains of slow-growing Rhizobium more resistant to severe desiccation than strains of fast-growing species. It is suggested that under conditions of milder desiccation, this property is disadvantageous to the slow-growing Rhizobium because insufficient moisture is available to allow for the functioning of vital enzymes.     

Salinity tolerance of Rhizobium mutants: Growth and relative efficiency of symbiotic nitrogen fixation

A salinity-tolerant strains of Rhizohium able to grow and fix nitrogen in symbiosis with lentil (Lens esculenta) in saline soil was derived frorn effective Rhizobium strain RL 5. A forced mutation with the mutagen nitrosoguanidine resulted in the isolation of five different mutant strains. The salinity tolerance, streptomycin resistance, growth, nodulation behaviour and relative efficiency of symbiotic N₂-fixation of these strains were studied. Among the five mutants and parent, LM 4 and LM 1 successfully tolerated 200 μ g ml^?1 streptomycin and 1.5%NaCl. These two mutants also significantly increased number and dry weight of nodules per plant, dry matter yield of the crop and N₂-fixation. Between the two, LM 4 seemed generally the better.     

Soil temperature,mycorrhizal infection and nodulation of Medicago truncatula and Trifolium subterraneum

Establishment of vesicular-arbuscular mycorrhizal fungi in plant roots involves a pre-infection phase of propagule germination, hyphal growth and appressorium formation, followed by growth of the fungus within the root. The effect of soil temperature on the pre-infection stage was examined by counting the numbers of fungal “entry-points” on the main roots of Medicago truncatula and Trifolium subterraneum, grown at soil temperatures of 12°, 16°, 20° and 25°C for periods up to 12 days. Increased root temperature was positively associated with increased numbers of “entry-points”. This effect was more marked between 12° and 16°C than at higher temperatures, as shown by comparing plants at the same stage of development (emergence of spade leaf) and by calculating the results as entry points per cm root.The first root nodules appeared sooner at higher temperatures (20° and 25°), but subsequent development of nodules (measured as nodule number and aggregate volume of nodules per plant, up to 21 days) was best at 16°C for both host Rhizobium combinations in non-sterile and autoclaved soil. There was no evidence that competition between mycorrhizal fungi and Rhizobium for infection sites occurred.A method of obtaining numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi in soil is described.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Differences between strains of rhizobium trifolii in ability to colonize soil and plant roots in the absence of their specific host plants

Evidence is presented of strain differences within Rhizobium trifolii in ability to colonize soil surrounding common pasture species, both legume and non-legume, in problem sandy soils in Western Australia. R. lupini and R. trifolii are shown to spread better than R. meliloti in these soils.     

Influence of a vesicular-arbuscular mycorrhizal fungus on the competitive ability of Bradyrhizobium spp. for nodulation of cowpea Vigna unguiculata (L.) Walp in non-sterilized soil

Summary We examined the influence of a vesicular-arbuscular (VAM) fungus (Glomus pallidum Hall) on the competitive ability of introduced and native Bradyrhizobium strains to nodulate cowpeas [Vigna unguiculata (L) Walp]. Our experiments in non-sterilized soil revealed that in the presence of VAM fungus, introduced Bradyrhizobium spp. strains become more competitive than native rhizobia. For example, strain JRC29 occupied 59.2% of the total nodules when inoculated alone, but this figure increased to 71.2% when JRC29 was used in dual inoculations with VAM fungus. A similar pattern of enhanced competitiveness for nodule formation was observed with the two other strains in the presence of the VAM fungus. Our results suggest that the competitiveness of rhizobia can be enhanced by co-inoculating with a selected strain of a VAM fungus.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

Manganese modulates the responses of nitrogen-supplied and Rhizobium-nodulated Phaseolus vulgaris L. to inoculation with arbuscular mycorrhizal fungi

In Venezuela, low yields of black bean crops are attributed, in part, to the low manganese (Mn) and phosphorus (P) contents in the Quartzipsamment soils where this crop is usually sown. To test this hypothesis, black bean plants were grown in sterilized sand to simulate soil physical properties, were fertilized with increasing Mn concentrations (0.1-20 μM) and inoculated with a commercial mixture of Rhizobium leguminosarum bv phaseoli strains 127K44, 127K89, 127K105 (+Rh), in combination with arbuscular mycorrhizal fungi Scutellospora heterogama and Entrophospora colombiana (+AMF). Non-inoculated plants fertilized with 6 mM NO₃ and 2 mM P served as controls. Plants were harvested at 18, 25, 33, and 40 days after emergence. At all harvests, the greatest growth and highest P and iron (Fe) leaf concentrations occurred in control plants grown in 5 μM Mn. The growth of +AMF plants was promoted at 0.1 μM Mn and inhibited at higher than 1 μM Mn. Whereas, concentrations of 5-10 μM Mn enhanced the growth and the Mn concentrations in leaves of +Rh plants 40 days after emergence. The tripartite symbiosis (+Rh+AMF) decreased growth, nodulation and leaf ureide and chlorophyll concentrations in plants grown in less than 20 μM Mn, imputed to severe ultrastructural alterations in the leaf and nodule tissues. Only +Rh+AMF plants grown in 20 μM Mn were effectively nodulated, AMF colonized and reached the flowering stage, although with diminished growth and low chlorophyll concentrations. Results confirm the high Mn requirement of +Rh plants for growth and nodulation and question the implementation of the tripartite symbiosis to improve yields in early flowering black bean varieties planted in soils deficient in Mn and P.     

Effect of low root temperature on symbiotic nitrogen fixation and competitive nodulation of Onobrychis viciifolia (sainfoin) by strains of arctic and temperate rhizobia

Summary Symbiotic effectiveness and competitive nodulation of the temperate forage legume sainfoin (Onobrychis viciifolia cv. Melrose) by two strains of arctic rhizobia (from Astragalus or Oxytropis sp.) and two strains of temperate rhizobia (from sainfoin) were evaluated at temperatures of 9, 12, or 15°C for roots and 20°C (day) and 15°C (night) for shoots. The inocula consisted of effective individual or paired arctic and temperate strains which were identified on the basis of differential growth. At 9°C, the arctic strains were generally more competitive than the temperate strains, whereas at the highest temperature tested, the converse was apparent. Symbiotic effectiveness (shoot dry weight, nitrogenase activity, and number of nodules) was similarly affected by root temperature, except that at 15°C the arctic strains were generally as effective as the temperate strains. The marked interaction between temperature and strain on competitiveness or on effectiveness indicates symbiotic adaptation of the arctic rhizobia to low temperatures. On the basis of these data we suggest that the use of selected, cold-adapted rhizobia in sainfoin inoculants may be beneficial in temperate regions where low soil temperatures occur early in the growing season.Contribution no. 413 and 1334 of the Agriculture Canada Sainte-Foy Research Station and Plant Research Centre, respectively