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Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia.

Common beans (Phaseolus vulgaris L.) have centers of origin in both Mesoamerica and Andean South America, and have been domesticated in each region for perhaps 5000 years. A third major gene pool may exist in Ecuador and Northern Peru. The diversity of the rhizobia associated with beans has also been studied, but to date with an emphasis on the Mesoamerican center of origin. In this study we compared bean rhizobia from Mexico and Andean South America using both phenotypic and phylogenetic approaches. When differences between the rhizobia of these two regions were shown, we then examined the influence of bean cultivar on the most probable number (MPN) count and biodiversity of rhizobia recovered from different soils. Three clusters of bean rhizobia were distinguished using phenotypic analysis and principal-component analysis of Box AIR-PCR banding patterns. They corresponded principally to isolates from Mexico, and the northern and southern Andean regions, with isolates from southern Ecuador exhibiting significant genetic diversity. Rhizobia from Dalea spp., which are infective and effective on beans, may have contributed to the apparent diversity of rhizobia recovered from the Mesoamerican region, while the rhizobia of wild Phaseolus aborigineus from Argentina showed only limited similarity to the other bean rhizobia tested. Use of P. vulgaris cultivars from the Mesoamerican and Andean Phaseolus gene pools as trap hosts did not significantly affect MPN counts of bean rhizobia from the soils of each region, but did influence the diversity of the rhizobia recovered. Such differences in compatibility of host and Rhizobium could be a factor in the poor reputation for nodulation and N2 fixation in this crop.

Colony Count, Microbial↗

Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.

Amino Acid Sequence↗

A new peroxidase cDNA from white clover: its characterization and expression in root tissue challenged with homologous rhizobia, heterologous rhizobia, or Pseudomonas syringae.

Temporal reverse transcription-polymerase chain reaction (RT-PCR) expression analyses were performed on Trprx2, a new white clover peroxidase, with roots challenged with homologous rhizobia, heterologous rhizobia, and a pathogen, Pseudomonas syringae. Low levels of Trprx2 expression were evident in all rhizobial treatments but in P.syringae-treated clover background expression was dramatically reduced within 1 h and was undetectable in treatments inoculated for more than 3 h. Spraying 4 mM salicylic acid onto seedlings increased Trprx2 expression. These data suggest a defensive role for Trprx2 in white clover and indicate active defense suppression by the pathogen.

Amino Acid Sequence↗

Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes.

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 x 10 g of soil for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P </= 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N(2)-fixing crops indicates a potential for improving inoculation technology, the N(2) fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.

Journal Article↗

Ecology and genetics of tropical rhizobia species.

Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation. So far most information on Rhizobium concerns fast-growing rhizobia and their host legume. Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils. The majority of the developing countries are in tropical and sub-tropical regions. Except for R. japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species. In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia. By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions. In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested. This involves close cooperation between molecular biologists and microbial ecologists.

Journal Article↗

Certain environmental factors affecting rhizobia and symbiotic systems.

The interrelation between rhizobia and certain fungi, bacteria, actinomycetes, nematodes, and seed-coat diffusates of Phaseolus vulgaris were investigated. The effect of pesticides, i.e. fungicides, herbicides, and nematocides on growth of rhizobia, and the symbiotic systems between rhizobia and their respective host is reported. Degradation of certain herbicides and insecticides is shown. The movement of rhizobia in soil as affected by water tension, tolerance of salts, and soil temperatures are discussed. Environmental factors may affect the successful establishment of an effective symbiosis between rhizobia and their hosts at any or all the three stages. They may 1) affect occurrence, growth, and survival of root nodule bacteria, 2) modify nodule formation, or 3) affect the function of the formed nodules (VINCENT 1962). The environmental aspect considered here include the antagonistic factors against rhizobia, the pesticides, and some ecological aspects of rhizobia in soil, e.g., the movement and salts and heat tolerance. These aspects were investigated by Egyptian workers over the period 1948-1972. Comprehensive reviews on the effect of environmental factors on rhizobia were reported by VINCENT (1962) and NUTMAN (1972).

Actinomycetales↗

Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology.

Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N2-fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N2-fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.

Biotechnology↗

Enzymes of the glyoxylate cycle in rhizobia and nodules of legumes.

The relatively high level of fatty acids in soybean nodules and rhizobia from soybean nodules suggested that the glyoxylate cycle might have a role in nodule metabolism. Several species of rhizobia in pure culture were found to have malate synthetase activity when grown on a number of different carbon sources. Significant isocitrate lyase activity was induced when oleate, which presumably may act as an acetyl CoA precursor, was utilized as the principle carbon source. Malate synthetase was active in extracts of rhizobia from nodules of bush bean (Phaseolus vulgaris L.), cowpea (Vigna sinensis L.), lupine (Lupinus angustifolius L.) and soybean (Glycine max L. Merr.). Activity of malate synthetase was, however, barely detectable in rhizobia from alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.) and pea (Pisum sativum L.) nodules. Appreciable isocitrate lyase activity was not detected in rhizobia from nodules nor was it induced by depletion of endogenous substrates by incubation of excised bush bean nodules. Although rhizobia has the potential for the formation of the key enzymes of the glyoxylate cycle, the absence of isocitrate lyase activity in bacteria isolated from nodules indicated that the glyoxylate cycle does not operate in the symbiotic growth of rhizobia and that the observed high content of fatty acids in nodules and nodule bacteria probably is related to a structural role.

Journal Article↗

Survival and efficiency of cowpea rhizobia on pelleted and non-pelleted peanut seeds, treated with fungicides.

Peanut seeds were either normally inoculated with the legume inoculant Okadin, containing cowpea rhizobia, or pelleted and treated with each of the fungicides Brassical, Thiram, Orthocide 75, Falisan, Vitavax 75, and Agrosan. The seeds were then incubated at room (+/- 25 degrees C) or refrigeration temperatures (+/- 5 degrees C). Survival tests were made after 2 and 10 days. Treated seeds were also planted in pots containing Nile silt for testing the efficiency of rhizobia as affected by the fungicide and the pelleting treatments. Pelleting of peanut seeds enhanced the survival of rhizobia whether seeds were incubated at room or refrigeration temperature. Protection was more pronounced when seeds were kept at low temperature. This was true of the fungicides Brassical, Orthocide 75, Vitavax 75, Thiram, and Agrosan. Falisan, however, did not help the rhizobia to survive. All the fungicides tested reduced the number of rhizobia to nil within 10 days when the seeds were normally inoculated and then treated and incubated at room temperature. The numbers of rhizobia were appreciably reduced when incubated at refrigeration temperature. Pelleting tended to prevent the harmful effect of the fungicides. This was clearly demonstrated with a tendency of an increase in the total nitrogen of the plants. On the contrary, normally inoculated and treated seeds grew into plants with reduced amounts of total nitrogen fixed.

Arachis↗

In vitro studies on the effects of herbicides on the growth of rhizobia.

AIMS: To study the possible adverse effect of herbicides on nodulation and nitrogen fixation in legumes by affecting the nitrogen-fixing rhizobia. METHODS AND RESULTS: Experiments were conducted to study the effect of four herbicides (terbutryn/terbuthylazine, trietazine/simazine, prometryn and bentazone) on the growth of nitrogen-fixing pea rhizobia (Rhizobium leguminosarum) in vitro by measuring optical density. Terbutryn/terbuthylazine, trietazine/simazine and prometryn had an adverse effect on the growth of rhizobia whereas bentazone was safe to rhizobia. CONCLUSIONS: The above herbicides could be used in pea at the recommended rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The adverse effects of herbicides on rhizobia were observed at concentrations not normally expected to occur under field conditions. Further, previously observed adverse effects of these herbicides on nodulation and nitrogen fixation of peas were, possibly, not due to their effects in rhizobia but to their adverse effects on the plant growth itself.

Culture Media↗

Interaction of lectins from soybean and peanut with rhizobia that nodulate soybean, peanut, or both plants.

Four of 14 strains of Rhizobium japonicum from soybean nodulated peanut (Arachis hypogaea L. cultivar Jumbo Virginia), and 3 of 8 Rhizobium sp. strains from peanut nodulate soybean (Glycine max (L.) Merr. cultivar Harosoy 63). Cells of three peanut rhizobia bound fluorescent- and radioisotope-labeled soybean lectin. Two of these strains failed to nodulate soybean, and conversely, two peanut strains that nodulated soybean did not bind to soybean lectin. Both culture medium and age had pronounced effects of the number of peanut rhizobia cells that bound fluorescent-labeled soybean lectin. Harosoy 63 soybean root exudates stimulated the growth of peanut rhizobia, but had no consistent influence on the number of cells that bound soybean lectin. Although extracellular soybean lectin receptors were present in culture fluids from each of the peanut rhizobia whose cells bound the lectin, the titer of receptors was greatest for strains 3G4b5. The affinity constants for the adherence of soybean lectin to Rhizobium sp. 3G4b5 cells from cultures of various ages ranged from 4.2 X 10(6) to 4.9 X 10(6) M-1, and the number of lectin binding sites per cell decreased as cells aged. Cells of the soybean and peanut rhizobia did not bind fluorescent- or radioisotope-labeled peanut lectin. The results indicate that there is no relationship between the ability of peanut and soybean rhizobia to nodulate the reciprocal host plant and their ability to bind to the lectin of that plant.

Arachis↗

Characteristics of the rhizobia associated with Dalea spp. in the Ordway, Kellogg-Weaver Dunes, and Hayden prairies.

Habitat fragmentation affects the biodiversity and function of aboveground organisms in natural ecosystems but has not been studied for effects on belowground species. In this paper, we consider the diversity of the rhizobia associated with the indigenous legume Dalea purpurea in 3 residual prairie areas in Minnesota and Iowa. Using Dalea purpurea as a trap host, 218 rhizobia were recovered from these soils then characterized using BOXA1R PCR. Three major and 13 minor groups were distinguished based on a similarity of greater than 75% in fingerprint patterns. Each major group consisted almost exclusively of rhizobia from a single prairie, with the diversity of Dalea rhizobia recovered from the Hayden Prairie less than that obtained with rhizobia from the other prairies. Based on 16S rRNA gene sequence analysis, isolates from the Hayden, Ordway, and Kellogg-Weaver Dunes prairies were most similar to Rhizobium etli and Rhizobium leguminosarum, Rhizobium gallicum, and Mesorhizobium amorphae and Mesorhizobium huakuii, respectively. This variation in the dominant microsymbiont species across the 3 prairies studied was unexpected but could have been influenced by the limited number of samples that we were allowed to take, by unanticipated cross-nodulation between native legumes, and by variation in the range of legume species present in each residual prairie area. While some of the rhizobia from Dalea nodulated Phaseolus vulgaris, Macroptilium atropurpureum, Leucaena leucocephala, and Onobrychis viciifolia in addition to the Dalea species tested, others nodulated Astragalus canadensis or Amorpha canescens.

DNA, Bacterial↗

[Diversity and resistance of rhizobia isolated from Caragana intermedia in Maowusu sandland].

Fifteen rhizobia strains were isolated from wild shrubby legume Caragana intermedia in Maowusu sandland. A dendrogram was constructed based on esterase profiles, showing a rich diversity of these rhizobia. Many biochemical characteristics were detected, including acid or alkali production, catalase activity, utilization of sole carbon sources, and resistance to salt, acid-alkali and temperature variation. The results indicated that all the rhizobia strains isolated from Caragana intermedia could excrete H+ on YMA agar and produce catalase. 73.3% strains could tolerate NaCl stress at 3% concentration, and 80% strains could grow at 50 degrees C. Except the difference in lactose and starch utilization, rhizobia strains had no bias on the rest carbon sources. However, the difference in resistance to stress existed among strains, which might be related to the adaptation of rhizobia to diverse landscapes in Maowusu sandland. It was revealed that rhizobia nodulating Caragana intermedia could be used as a new germplasm to fix nitrogen under severe environment.

Caragana↗

[Isolation and biological characteristics of rhizobia strains from Abrus cantoniensis].

OBJECTIVE: The research aimed at studying the biological characteristics of rhizobia isolated from Abrus cantoniensis. METHOD: The rhizobia strains, isolated from different environments in Guangxi, were studied for their growing characters and the generation time. They were also compared for survival capabilities under stresses caused by NaCl, pH, temperature, and different kinds and concentration of antibiotics. RESULT: The strains obtained from A. cantoniensis in subtropical zone produced alkali in YMA medium, the average generation time was 14.8 hours, and thus they belong to slow-growing rhizobia. Rhizobia strains differed greatly in respect to tolerance of high temperature, adaptability of acidic environment and sensitivity to four antibiotics, but they had the same abilities of using different carbon and nitrogen sources. After 70 days from inoculated strains, the seedling formed nodules on the root (85.0%), and the dry matter of vine was increased by 51.1%. CONCLUSION: The rhizobia strains isolated from different ecological environments are good germplasm resources of tolerances to high temperature and acidic environment. The research will greatly help utilize the rhizobia resources and enhance the quality of crude drugs of medicinal leguminosae.

Abrus↗

Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt.

About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.

Bacterial Proteins↗