Ethiopian soils harbor natural populations of rhizobia that form symbioses with common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

<p>The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.     

Phenotypic and genetic diversity among rhizobia isolated from three Hedysarum species: H. spinosissimum,H. coronarium and H. flexuosum

Cultural, physiological and biochemical properties of 18 strains of rhizobia isolated from root nodules of the forage legume H. spinosissimum were compared with those of rhizobia from the related species H. coronarium (15 strains) and H. flexuosum (four strains). On the basis of 43 characteristics the 37 strains of Hedysarum rhizobia could be divided into two groups by numerical analysis. The H. spinosissimum rhizobia formed the first group and the second group comprised the strains from H. coronarium and H. flexuosum. The reference Rhizobium leguminosarum bv. viceae strain 250A was clustered with the rhizobia from H. coronarium and H. flexuosum. By contrast Bradyrhizobium sp. (Arachis) reference strain 280A was not clustered with any of the strains tested, indicating that the H. spinosissimum rhizobia differ from both Rhizobium and Bradyrhizobium. Serological data also discriminate between H. spinosissimum and H. coronariumrhizobia but not between the latter and H. flexuosum strains. The strains tested exhibit a high degree of specificity for nodulation and nitrogen fixation. We also determined the16SrRNA gene sequence of H. spinosissimum rhizobia (four strains), H. coronarium (two strains) and H. flexuosum (two strains) and found that the four H. spinosissimum isolates share a 98% identity among each other in this region but they showed less than 92% identity to the H. coronarium and H. flexuosum isolates. The H. spinosissimum isolates were closely related to both Mesorhizobium loti and M. ciceri, sharing 97% identity with each species.     

Diversity and effectiveness of rhizobial strains from <Emphasis Type="Italic">Gliricidia sepium</Emphasis> native to Reunion Island,Kenya and New Caledonia

The aim of this study was to use several approaches to assess the diversity occuring in a collection of rhizobial strains that had been isolated from Gliricidia sepium (Jacq.) Walp. originating from Kenya, Reunion Island and New Caledonia. Results showed that G. sepium establishes an effective symbiosis only with fast-growing rhizobia. This suggests that this legume nodulates preferentially with this type of rhizobia, although ineffective nodules were observed on root systems of plants inoculated with slow-growing rhizobia. It was possible to distinguish several groups of strains which have the same behaviour in terms of utilization of amino acids and sources of carbon and intrinsic resistance to antibiotics and salinity. Six molecular groups were distinguished by profiles obtained using PCR/RFLP techniques. No relationship was demonstrated between phenotypic groups and molecular groups. Neither was it possible to find a relationship between the geographic origins of the strains and their distribution in the several phenotypic and/or molecular groups. In an experiment carried out under glasshouse conditions, we observed that all strains from the collection significantly improved the growth of G. sepium cultivated in a substrate totally devoid of nitrogen. The strain GsK5 induced the greatest stimulation of plant growth.     

Characterization of rhizobia fromLeucaena

Seventy-six rhizobia were isolated from the nodules ofLeucaena plants of various genotypes growing in a wide range of soil types and climatic regions. The isolates were fast-growing and acid-producing. In establishing a serological grouping for the isolates, the intrinsic antibiotic resistance (IAR) patterns to low concentrations of eight antibiotics was helpful for selecting the strains for immunization purposes. Eight distinct somatic serogroups ofLeucaena rhizobia were identified by using strain-specific fluorescent antibodies. The results indicated that use of serological markers is a more specific technique than IAR pattern for strain identification. Strains from some different serogroups had the same IAR patterns. The immunofluorescence cross-reactions ofLeucaena rhizobia serogroups among themselves and with other species of fast- and slow-growing rhizobia were very low. Sero-grouping is ideal for use in further ecological studies in field inoculation trials.     

Rhizobial Populations in Soils from Natural Acacia senegal and Acacia nilotica Forests in Mauritania and the Senegal River Valley

Eighty-two strains of rhizobia were isolated from soils taken from several sites in Mauritania and Senegal. These soil samples were collected from natural stands of Acacia nilotica and Acacia senegal. The soils from Mauritania were less rich in native rhizobia than the soils from Senegal. The strains were characterized using polymerase chain reaction–restriction fragment length polymorphism and by sequencing the rDNA 16S–23S intergenic spacer region (IGS). They were sorted into seven IGS groups. These groups were not associated with the geographical origin of the strains or with the host-plant species at the site where the soils were collected. Most of the strains were in three of the IGS groups (I, IV, and V). One representative strain from each IGS group was sequenced and showed that the strains were from the genus Mesorhizobium. IGS groups I, IV, and VI were close to the species M. plurifarium (AF34563), IGS groups IIand III were close to the species Mesorhizobium sp. (AF510360), IGS group V was close to the species Mesorhizobium sp. (AF510366), and IGS group VII was close to Mesorhizobium sp. (AF510346).     

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

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

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.     

Selection of Portuguese Rhizobium leguminosarum bv. trifolii strains for production of legume inoculants

From several native clover species, growing in six different soil types, 170 Rhizobium leguminosarum biovar trifolii strains were isolated, covering the central and southern regions of Portugal. The effectiveness of the strains varied from ineffective to highly effective on T. subterraneum cv. Clare and on T. fragiferum cv. Palestine, with a predominance of medium and high effectiveness on both host plants. The effectiveness was not influenced by provenence (soil or plant), except for the strains from the rankers soils and for the strains isolated from T. pratense, that were ineffective or medium effective on T. subterraneum.Selected strains were evaluated for effectiveness on T. subterraneum cv. Clare, using the commercial strain TA1 as reference. Several of the isolated strains were more effective than TA1, indicating that local strains may be used to produce better inoculants.     

Genetic diversity of indigenous tropical fast-growing rhizobia isolated from soybean nodules

This study characterized genetically 30 fast-growing rhizobial strains isolated from nodules of Asian and modern soybean genotypes that had been inoculated with soils from disparate regions of Brazil. Analyses by rep-PCR (ERIC and REP) and RAPD indicated a high level of genetic diversity among the strains. The RFLP-PCR and sequencing analysis of the 16S rRNA genes indicated that none of the strains was related to Sinorhizobium (Ensifer) fredii, whereas most were related to Rhizobium tropici (although they were unable to nodulate Phaseolus vulgaris) and to Rhizobium genomic species Q. One strain was related to Rhizobium sp. OR 191, while two others were closely related to Agrobacterium (Rhizobium) spp.; furthermore, symbiotic effectiveness with soybean was maintained in those strains. Five strains were related to Bradyrhizobium japonicum and B. elkanii, with four of them being similar to strains carried in Brazilian inoculants, therefore modifications in physiological properties, as a shorter doubling time might have resulted from adaptation to local conditions. Phospholipid fatty acid analysis (PFLA) was less precise in delineating taxonomic relationships. The strains fit into eight Nod-factor profiles that were related to rhizobial species, but not to N₂-fixation capacity or competitiveness. The data obtained highlight the diversity and promiscuity of rhizobia in the tropics, being capable of nodulating exotic legumes and might reflect ecological strategies to survive in N-poor soils; in addition, the diversity could also represent an important source of efficient and competitive rhizobial strains for the tropics. Putative new rhizobial species were detected only in undisturbed soils. Three species (R. tropici, B. japonicum and B. elkanii) were found under the more sustainable management system known as no-till, while the only species isolated from soils under conventional till was R. tropici. Those results emphasize that from the moment that agriculture was introduced into undisturbed soils rhizobial diversity has changed, being drastically reduced when a less sustainable soil management system was adopted.     

Molecular diversity,effectiveness and competitiveness of indigenous rhizobial population infecting mungbean <Emphasis Type="Italic">Vigna radiata</Emphasis> (L. Wilczek) under semi-arid conditions

Nodules from mungbean crop raised for the first time at Ram Dhan Singh (RDS) farm of Chaudhary Charan Singh (CCS) Haryana Agricultural University, Hisar were collected from 17 different locations. Twenty-five mungbean rhizobia were isolated and authenticated by plant infection test. DNA of all these rhizobia was extracted purified and amplified using enterobacterial repetitive intergenic consensus (ERIC) primers. All the mungbean rhizobial isolates were clustered into 4 groups at 65% of similarity and were further divided into 17 subclusters at 80% of similarity. All the 4 types of rhizobia were not present at any of the location and group 2 or 4 rhizobia were invariably present. Efficacy of these rhizobia in terms of nodulation, nitrogen uptake and chlorophyll a fluorescence was determined under pot culture conditions. Strain MB 307 showed maximum nitrogen uptake of 31.9 mg N plant⁻¹ followed by strain MB 1205, MB 1206(2), MB 308, MB 1524 and strain MB 1521 was found to be the least efficient in terms of N 2 fixation. Nodule occupancy by different rhizobia ranged from 5.5 to 40.3%. Most of the strains belonging to the 2nd group which clustered maximum number of strains were comparatively better competitors and formed 19.5–40.3% of the nodules and were also effective. Isolate MB 307, the most efficient strain, was found to have nodule occupancy of 31.5%. Such type of predominant, efficient and better competitor strains should be selected for enhancing nodule competitiveness.     

Indigenous plasmids and cultural characteristics of rhizobia nodulating chickpeas (Cicer arietinum L.)

We examined 27 strains of chickpea rhizobia from different geographic origins for indigenous plasmids, location and organization of nitrogen fixation (nif) genes, and cultural properties currently used to separate fast- and slow-growing groups of rhizobia. By using an in-well lysis and electrophoresis procedure one to three plasmids of molecular weights ranging from 35 to higher than 380 Mdal were demonstrated in each of 19 strains, whereas no plasmids were detected in the eight remaining strains. Nitrogenase structural genes homologous to Rhizobium meliloti nifHD, were not detected in plasmids of 26 out of the 27 strains tested. Hybridization of EcoRI digested total DNA from these 26 strains to the nif probe from R. meliloti indicated that the organization of nifHD genes was highly conserved in chickpea rhizobia. The only exception was strain IC-72 M which harboured a plasmid of 140 Mdal with homology to the R. meliloti nif DNA and exhibited also a unique organization of nifHD genes. The chickpea rhizobia strains showed a wide variation of growth rates (generation times ranged from 4.0 to 14.5 h) in yeast extract-mannitol medium but appear to be relatively homogeneous in terms of acid production in this medium and acid reaction in litmus milk. Although strains with fast and slow growth rates were identified, DNA/DNA hybridization experiments using a nifHD-specific probe, and the cultural properties examined so far do not support the separation of chickpea rhizobia into two distinct groups of the classical fast- and slow-growing types of rhizobia.     

Genetic relationships between clinical and non-clinical strains of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> biovar 1A as revealed by multilocus enzyme electrophoresis and multilocus restriction typing

Background  

Genetic relationships among 81 strains of Y. enterocolitica biovar 1A isolated from clinical and non-clinical sources were discerned by multilocus enzyme electrophoresis (MLEE) and multilocus restriction typing (MLRT) using six loci each. Such studies may reveal associations between the genotypes of the strains and their sources of isolation.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Molecular methods of analysing bacterial diversity: the case of Rhizobia

The distribution of rhizobia, bacteria which nodulate the roots of leguminous plants, was surveyed for 91 species of the familyLeguminosae. A total of 117 strains of rhizobia were isolated, and 28 strains were obtained from culture collections. The sample total of 145 strains was discriminated by rapid ribosomal RNA (rRNA) sequencing. The partial sequences (157 bases from position (inEscherichia coli) 1220 to 1377 from 5 end) of 16S rRNA revealed the presence of 16 groups in these rhizobia. Further, DNA-DNA homology studies suggested that the differences of the 16 groups were enough to justify establishing at least 16 species.     

A numerical taxonomic study of some pigmented bacteria isolated from Organic Lake,an antarctic hypersaline lake

A study was made of a group of moderately halophilic, heterotrophic, pigmented strains isolated from Organic Lake, Antarctica. These strains were Gram-negative, non-motile, had an aerobic metabolism and a mol% G+C content of their DNA in the range 35–41, indicating that they may be members of the Flavobacterium-Cytophaga group. A numerical taxonomic study involving 134 characteristics compared the antarctic strains with reference strains from Flavobacterium, Cytophaga and Flectobacillus. The antarctic strains formed two clusters that did not contain any reference strains suggesting that they may represent two new species of the genus Flavobacterium.     

Phage sensitivity and host range of Rhizobium strains isolated from root nodules of temperate legumes

Twenty-five Rhizobium strains were isolated from root nodules of Astragalus spp. (10), Hedysarum alpinum (7), Glycyrrhiza pallidiflora (3) and Ononis arvensis (5). The sensitivity of these strains to bacteriophages of Rhizobium loti, R. meliloti, R. galegae and R. leguminosarum was studied. Phages specific to R. loti strains were shown to induce the phage lysis of several Astragalus, Hedysarum and Ononis rhizobia. Ten R. loti strains tested for nodulation abilities on the plant hosts under investigation were able to develop nitrogen-fixing nodules on the Ononis arvensis roots. On the other hand, rhizobia from Ononis and Glycyrrhiza could form an effective symbiosis with Lotus corniculatus plants, so these bacteria are considered to belong to the Rhizobium loti taxon. Bacterial strains isolated from Astragalus and Hedysarum were observed to cross-nodulate their plant hosts as well as Oxytropis campestris, Glycyrrhiza uralensis and Ononis arvensis plants, whereas they could not nodulate Lotus plants. It is concluded that these Rhizobium strains comprise a cross-inoculation group related to Rhizobium loti. ei]{gnR O D}{fnDixon}     

Response of common bean lines to inoculation: comparison between the <Emphasis Type="Italic">Rhizobium tropici</Emphasis> CIAT899 and the native <Emphasis Type="Italic">Rhizobium etli</Emphasis> 12a3 and their persistence in Tunisian soils

Since Phaseolus vulgaris (L) is poorly nodulated in all regions of Tunisia where this crop is grown, the response of common-bean lines CocoT and Flamingo to inoculation with reference Rhizobium tropici CIAT 899 or native rhizobia, namely Sinorhizobium fredii 1a6, Rhizobium etli 12a3, and Rhizobium gallicum 8a3, was studied in a field station. Since R. etli 12a3 was found to be the most effective native rhizobium, it was subsequently compared with R. tropici CIAT 899 in a broader study in two stations over 3 years. A significant interaction between bean and rhizobia was observed for nodule number, shoot dry weight, grain yield, and contents of nitrogen and chlorophyll. The native rhizobia was more efficient than CIAT899 for Flamingo, though not for CocoT. The Enzyme-linked immunosorbent assay technique was used with polyclonal antibody to assess the occupancy in nodule and persistence in soil of the inoculated rhizobia. For both stations the nodule occupancy was 100% during the first year for each rhizobium, but during the next 2 years, between 7 and 15% of nodules were formed by the rhizobia inoculated in the neighboring plot. It is concluded that the first-year inoculation is sufficient to maintain an adequate rate of nodulation during three growth cycles, and that the native R etli can be recommended for the common-bean inoculation in similar soils of Tunisia.     

Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.     

Thermophilic amylase-producing bacteria from Vietnamese soils

Of 25 bacterial isolates from Vietnamese soils, two were identified asBacillus stearothermophilus and one asThermoactinomyces thalpophilus, both thermophilic, amylase-producing bacteria. Amylase activity was highest in the presence of cassava starch as carbon source and (NH₄)₂HPO₄ as nitrogen source. The strains exhibit a high amylase productivity within the first 5 to 7 h of cultivation at 55°C. The crude enzyme had optima of pH 6.5 and 70°C.     

Symbiotic effectiveness of strains of Rhizobium leguminosarum biovar phaseoli isolated from soils of Rwanda

We have isolated 48 strains of Rhizobium leguminosarum biovar phaseoli from nodules of Phaseolus vulgaris L. cultivated on 32 different soils at 22 various locations in Rwanda, Central Africa. The symbiotic effectiveness of the strains was appraised in the greenhouse by measuring shoots dry matter and total plant nitrogen content after six weeks of growth. Of the strains tested 19%, 58% and 23% were rated very effective, effective and ineffective, respectively. A very significant correlation (r=0.96, P<0.01) was observed between shoots dry matter and total N content. By using the total nitrogen balance method, it was estimated that in the presence of a very effective strain, up to 86% of the N present in the shoots comes from N₂ fixation. No significant correlations were observed between the symbiotic effectiveness of the strains and the pH of the soils from which they originated, the tolerance of the strains to acidity or their ability to produce organic acids. The nine very effective strains selected were highly competitive against two ineffective strains with the two P. vulgaris cultivars Rubona-5 and Kiryumukwe.Contribution no 367, Station de recherches, Agriculture Canada.Contribution no 367, Station de recherches, Agriculture Canada.