Selection of Rhizobium strains on their competitive ability for nodulation

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

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.     

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.     

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.     

Lime pelleting inoculated serradella (Ornithopus spp.) increases nodulation and yield

Lime pelleting of the inoculated seed is recommended for most pasture legume species to improve survival of the rhizobia on the seed and to counter deleterious effects of soil or fertiliser acidity on rhizobial numbers. Except for New South Wales, lime pelleting is specifically not recommended for serradella (Ornithopus spp.). Our objectives were to evaluate effects of lime pelleting on bradyrhizobial numbers on seed, and nodulation and growth of the serradella plants. Three experiments are reported at two acid-soil sites in northern New South Wales involving four cultivars of yellow serradella (Ornithopus compressus) and Bradyrhizobium sp. (Lupinus) strains WSM471 (current inoculant strain) and WU425 and WSM480. Lime pelleting increased bradyrhizobial numbers on seed, 24 h after inoculation, by an average of 90%. Similarly, lime pelleting increased nodulation and shoot dry matter of the inoculated plants by an average of 57 and 28%, respectively. The three strains were similar in effects on plant growth. Relative values for shoot dry weight, averaged over sites, were 100 for WSM471 and 98 for both WU425 and WSM480. Our results confirmed previous research that lime pelleting inoculated serradella seed was not deleterious to survival of the bradyrhizobial inoculum, and showed that it could result in enhanced symbiotic activity of the inoculum in some instances. We recommend lime pelleting of serradella and that WSM471 remain the inoculant strain.     

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.     

Interactions between Sclerotium rolfsii and Trichoderma spp: Relationship between antagonism and disease control

Ten isolates of Trichoderma spp were examined for their ability to antagonize growth and to parasitize mycelium of Sclerotium rolfsii (Sr-1) on agar media, to inhibit germination of sclerotia of S. rolfsii on natural soil plates and to sporulate on the sclerotia, and to protect bean seedlings against the pathogen in the greenhouse. A high negative correlation (r = ?0.844) was observed between plant stand in the greenhouse and sclerotial germination on soil plates but not with antagonism on agar plates. Three isolates of T. harzianum (Th-7, Th-20, WT-6) and one of T. hamatum (TRI-4) were especially effective in reducing sclerotial germination and controlling disease in the greenhouse. Three isolates of Trichoderma spp (WT-6, TMP, and TRI-4), effective in reducing sclerotial germination of isolate Sr-1, also prevented sclerotial germination in four out of five additional S. rolfsii isolates studied.     

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.     

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.     

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.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

Isolation and characterization of Thiobacillus from alkali soils

Alkali soils are poor in sulphur-oxidizing bacteria. By appropriate enrichment, two bacterial strains resembling Thiobacillus thiooxidans and T. novellus have been isolated from an alkali soil. The strain of T. novellus possesses characteristics which make it suitable in reclamation of alkali soils by sulphur amendment.     

Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biolog™ system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species.     

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.     

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.     

Changes in Mycobacterium spp. population structure and pyrene mineralization in polycyclic aromatic hydrocarbon-amended soils

The effect of pyrene and phenanthrene contamination on soil Mycobacterium spp. community structure was examined using PCR-amplification of 16S rRNA genes with primers specific for the fast-growing group of Mycobacterium spp. and separation of phylotypes by temperature gradient gel electrophoresis (TGGE). The degradative potential of the soil microbial community was measured over time by mineralization of ¹⁴C-pyrene added to the contaminated soils. PCR-TGGE profiles, in combination with band sequencing and phylogenetic analysis of the prominent phylotypes, indicated shifts in the Mycobacterium spp. community during incubation. Reductions in species diversity and enrichments of specific populations were observed in all pyrene- and phenanthrene-treated soils, in contrast to the relatively stable control soil profiles. Mineralization studies indicated the shortest acclimation periods and the highest initial rates of pyrene degradation occurred in soils pre-exposed to phenanthrene, or a mixture of phenanthrene and pyrene, for 14 weeks. Pre-exposure of soil microorganisms to a single dose of pyrene for the same length of time also decreased the acclimation period for the degradation of pyrene. Monthly application of either pyrene or phenanthrene to soils, however, resulted in an increase in pyrene degradative potential 6 weeks after the first pre-exposure, but a decrease in degradative potential 14 weeks after the first pre-exposure. Similar PCR-TGGE profiles were obtained from soils with comparable pyrene mineralization curves or degradative potentials.     

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.     

Germination and hyphal growth of Glomus caledonicum on water agar containing benomyl

Concentrations of benomyl greater than 10 ng ml^?1 inhibited germination and hyphal growth of the vesicular-arbuscular mycorrhizal fungus Glomus caledonicum on water agar. This effect was attributable to only the activity of methyl-2-benzimidazole carbamate, one of the two hydrolysis products of benomyl. Hyphal growth but not germination was affected by the pH of water agar buffered with 10 mm 2-(N-morpholino)ethane sulphonic acid.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.