Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

Intrinsic antibiotic resistance and streptomycin uptake in cowpea rhizobia

Abstract Uptake of [³H]dihydrostreptomycin in sensitive and resistant strains of cowpea rhizobia was examined to explain one of the possible mechanisms of intrinsic antibiotic resistance. The rate of accumulation of labelled streptomycin in resistant strains was drastically reduced when compared to that of sensitive strains. Our results indicated that high levels of intrinsic antibiotic resistance was due to decreased permeability causing failure to accumulate the drug.     

Growth and nutritional characteristics of cowpea rhizobia

Summary Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO₂ and H₂ with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.     

Diverse genomic species and evidences of symbiotic gene lateral transfer detected among the rhizobia associated with Astragalus species grown in the temperate regions of China

Based on the analyses of ribosomal DNA and housekeeping genes, a total of 118 bacterial isolates obtained from 13 Astragalus species grown in the temperate region of China were identified as 19 genomic species of Mesorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium, two of them being putatively new species. Phylogenetic comparison of symbiotic genes (nodC and nifH) and housekeeping genes showed that the symbiotic genes of the Astragalus rhizobia were maintained by both vertical and horizontal transfer. The results demonstrated that the Astragalus species were very promiscuous hosts for rhizobia and that their rhizobia had very diverse genomic and symbiotic gene backgrounds.     

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.     

Diversity of ectomycorrhizal fungi in Britain: a test of the species–area relationship, and the role of host specificity

The host range of ectomycorrhizal (ECM) fungi in Britain was examined by compilation of a data matrix from published literature sources, based primarily on accounts of sporocarp associations with particular host genera. Information was gathered for 577 species of ECM fungi belonging to 51 genera, and 25 genera of host trees, representing the majority of ECM fungal species and host genera recorded in Britain.
Pronounced variation was recorded in the number of ECM fungal species associated with different host genera, with over 200 species recorded with Betula , Fagus , Pinus and Quercus . There was a positive linear relationship ( r ²=0·47, P =0·007) between the number of species of ECM fungi associated with different host genera and the total area occupied by each tree genus in Britain (both values log-transformed). There was also variation in the number of species of ECM fungi which were apparently specific to particular host genera, values ranging from zero (in 15 genera) to >40 in the case of Betula and Fagus . In total, 233 fungal species appeared to be specific to a single host genus (i.e. 40% of those surveyed). Comparison of the ECM mycota associated with different host genera by PCA accounted for 17% of the total variation, with genera belonging to the Fagaceae ( Quercus , Fagus and Castanea ) tending to cluster together, indicating a degree of overlap in their ECM associates. Exotic conifer species, which displayed a lower ECM diversity than would be expected from their distributional areas, were characterized by a high degree of overlap with the ECM associates of Pinus and Betula .
These results indicate that the abundance of different genera of host trees and variation in host specificity could provide a basis for understanding patterns of diversity in ECM fungi within Britain.     

Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

Nodulation and biological nitrogen fixation of 80 soybean cultivars in symbiosis with indigenous rhizobia

Eighty soybean cultivars were assessed for their potential for nodulation and nitrogen fixation with indigenous rhizobia in a Nigerian soil. Seventy-six days after planting (DAP) 87%, 3% and 10% of the soybean cultivars had from 0 to 30, 31 to 60 and over 61 nodules/plant, respectively. Only 8% had a nodule dry weight of 600 to 1100 mg/plant. At 84 DAP the proportion of nitrogen derived from the atmosphere (Ndfa) ranged from 0 to 65% 16% of the cultivars derived 51 to 65% of their N₂ from the atmosphere. The diversity of soybean germplasm and the variation in nodulation and N₂ fixation permitted the selection of the five best cultivars in terms of their compatibility with indigenous rhizobia, % Ndfa and the amount of N₂ which they fixed.     

Rapid preliminary characterisation of host specificity of leaf-beetles (Coleoptera: Chrysomelidae)

Rapid characterisation of host specificity is important both in biological control of weeds and studies in ecology and evolution. A means for doing this was developed and tested on four species of leaf beetles of interest for biological control of the weed Mimosa pigra (Mimosaceae). We identified the most promising for the more detailed tests necessary to obtain release approval. The impact of time-dependent effects and effects of experience were also investigated as part of this study but were not detected. Short-term host specificity tests on adult feeding accurately predicted the results of longer term trials. The long-term trial showed that survival on a plant species depended on feeding on it. Hence short-term feeding trials can predict the longer term survival of adults on other plant species. Different feeding results were obtained in cut foliage versus entire plants but no consistent pattern was shown: of the two insect species tested, one species ate more of the cut material while the opposite was demonstrated for the second species. Species in order of priority for further consideration as biocontrol agents were Syphrea bibiana, Genaphthona sp., Syphrea sp. and Paria sp. The latter three species were probably not sufficiently specific for release. The tests done here allow the identification of the most likely species upon which to conduct the more laborious and difficult larval developmental tests, saving considerable resources.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.     

Parasitism of cowpea by Striga gesnerioides: variation in virulence and discovery of a new source of host resistance

In glasshouse experiments in the United Kingdom, Striga gesnerioides-resistant cowpea varieties, Suvita-2 and 58–57, were assessed alongside a susceptible variety for their responses to seventeen samples of S. gesnerioides from four West African countries. The susceptible variety was attacked by all samples, while Suvita-2 was resistant to samples from Burkina Faso and Mali, and 58–57 was resistant only to samples from Burkina Faso.
At least three physiological groups of the parasite can be distinguished: samples from Burkina Faso attacking the smallest range of cowpea varieties; those from Mali being intermediate; those from Niger and Nigeria attacking all the cowpea varieties. A sample from Cameroon behaved slightly differently and may represent a fourth type.
A cowpea line from Botswana, B.301, selected for its field resistance to Alectra vogelii proved resistant to all Striga populations to which it was exposed, including those from Niger and Cameroon.     

Gluconate catabolism in cowpea rhizobia: evidence for a ketogluconate pathway

Enzymatic and radiorespirometric analysis of several strains of cowpea rhizobia revealed the presence of key enzymes of the Entner-Doudoroff (ED) pathway with the operation of the hexose cycle for the dissimilation of gluconate. These bacteria lack the oxidative pentose phosphate (PP) pathway when grown on gluconate. Gluconate-grown cells possessed an operational tricarboxylic acid (TAC) cycle. Enzymes of an ancillary pathway, the ketogluconate (KG) pathway for gluconate catabolism were detected. The presence of this pathway was confirmed by techniques of thin-layer chromatography and radiorespirometry.Abbreviations ED Entner Doudoroff - PP pentose phosphate - EMP Embden-Meyerhof-Parnas - KG ketogluconate - TCA tricarboxylic acid - DKG diketogluconate - PFK phosphofructokinase     

Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates

The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by 'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N(2). Hence, host sanctions by a diverse set of legumes play a role in maintaining N(2) fixation.     

Silicon promotes nodule formation and nodule function in symbiotic cowpea (Vigna unguiculata)

Applying silicon in the form of metasilicic acid (H₄SiO₃) or silicic acid (H₄SiO₃) to Bradyrhizobium -infected, hydroponically grown cowpea seedlings resulted in a significant ( P 0.05) increase in the number of nodules, nodule dry matter, and nitrogen fixed on a per plant basis. Total dry matter of plants increased with silicon supply, and the differences were significant ( P 0.05) at the higher silicon concentrations. Cowpea plants cultured in sand were also assessed for their response to silicic acid. Nodule number and nodule mass increased with silicon supply to sand cultured plants, though nitrogen fixation was unaltered. Although silicon is not essential for growth of cowpea, it is important for nodule formation and nodule functioning in hydroponically grown plants. Consequently, data collected and conclusions drawn from earlier glasshouse experiments, which have excluded silicon from nutrient solutions, may be flawed. Future studies on nodulation and nitrogen fixation using legumes in liquid culture must therefore include silicon as a nutrient element.     

Body size and feeding specificity: macrolepidoptera in Britain

Within a geographic assemblage, large-bodied species of macrolepidopteran moths tend, on average, to be less host-specific than small-bodied. Five possible explanations for this pattern are identified, based respectively on (i) phylogenetic relationships between species, (ii) latitudinal gradients in body size and feeding specificity, (iii) the relationship between range size and body size, (iv) larger body size as a buffer from environmental variation, and (v) the relationship between endophagous host associations and small body size. These mechanisms are tested using data for British macrolepidoptera and also evaluated using evidence from the literature at large. Although some of their assumptions are found to be justified, there is no significant support for any single mechanism. This lack of evidence for previously proposed mechanisms is discussed in the light of a recently proposed alternative explanation which combines theories of host quality and host defence mechanisms.     

Specificity in the symbiotic association of Lotus corniculatus and Rhizobium loti from natural populations

To test whether Rhizobium loti are coadapted to nodulate local plant genotypes, we competed R. loti strains in a common environment with clonally propagated Lotus corniculatus. Both the plants and bacterial strains were originally collected from natural populations in three localities and the R. loti strains used were distinguishable by enzyme electrophoretic markers and differed in geographical origin relative to host plant origin. The proportions of nodules occupied by symbiont strains varied widely and depended on both host plant and symbiont genotype. Nonrandom nodulation patterns resulted primarily from preferential nodulation of host genotypes by the symbiont strain that had been associated with the host in the natural environment. Symbionts nodulating their original hosts were preferentially found in nodules on adventitious tap roots as opposed to the younger, lateral roots (for one host-symbiont pair) or in large nodules, independent of location on the root system (for a second host-symbiont pair). The proportion of nodules occupied by a symbiont on novel host genotypes varied, ranging from nearly random expectation to a significant reduction in the proportion of nodules occupied. The analysis of the bacteria recovered from 994 nodules by multilocus enzyme electrophoresis revealed that 952 (95.8%) nodules were occupied by one of the four inoculant strains and 11 (1.1%) were co-occupied by two inoculant strains. A total of 31 (3.1%) nodules were occupied by strains that did not match the electrophoretic profiles of the original inoculant strains. Based on the comparison of multilocus profiles for 23 enzyme loci, we concluded that these bacteria were foreign strains and not recombinants of the original inoculant strains. Our findings indicate a strong host genotype by strain interaction underlying the outcome of rhizobial competition for nodulation sites and suggest there are distinct mechanisms leading to differential recognition of compatible host and symbiont genotypes.     

Nodule morphology,symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis s.l.

Background and Aims

The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules.

Methods

Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N₂ fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy.

Key Results

Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen.

Conclusions

Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.