Selection for acid tolerance inRhizobium meliloti

In the selection of acid-tolerantRhizobium meliloti, procedures for the collection and isolation of rhizobia, and the assessment of acid tolerance, have not been critically evaluated. Such procedures form the basis of this study. Root nodules were collected fromMedicago spp. found growing on acid soil in Sardinia. Their encumbent bacteria were isolated directly onto media adjusted over a range of pH values, and then assessed for acid tolerance in both the laboratory and field. Strains ofRhizobium meliloti isolated onto low pH media were, in general, more acid-tolerant than sister isolates from high pH media, when tested in both the laboratory and field. Dilution (10 or 100 fold) of the inocula used in the laboratory assessment did not greatly influence the rating derived, although there was some effect of bacterial colony type on growth rating. The link between polysaccharide production and acid tolerance was not strong. There was a poor correlation between the growth ratings derived from the laboratory screening and acid tolerance as expressed in the field.     

Use of an organic buffer for the selection of acid tolerantRhizobium meliloti strains

Summary Nine media used to grow rhizobia were examined for their ability to maintain a stable low pH during the growth ofR. meliloti Large fluctuations in the pH of all media were recorded within 72 h, indicating their unsuitability for use in the selection of acid tolerant rhizobia. Morpholino-ethanesulphonic acid (MES) was assessed for its ability to buffer the pH of the media whilst still permitting rapid growth ofR. meliloti, R. trifolii, andBr. lupini. With 30.7 mM MES, the pH of a defined medium containing galactose, arabinose, and glutamate did not change from the initial value of 5.5 even though rhizobial numbers increased from 10⁴ to 10⁹ cells.ml^–1. Even at a buffer concentration of 15.3 mM, pH only increased from 5.5 to 5.6. There was no effect of the buffer on rhizobial growth.     

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain TA1

Rhizobium leguminosarum bv. trifolii strain TA1 is an aerobic, motile, Gram-negative, non-spore-forming rod that is an effective nitrogen fixing microsymbiont on the perennial clovers originating from Europe and the Mediterranean basin. TA1 however is ineffective with many annual and perennial clovers originating from Africa and America. Here we describe the features of R. leguminosarum bv. trifolii strain TA1, together with genome sequence information and annotation. The 8,618,824 bp high-quality-draft genome is arranged in a 6 scaffold of 32 contigs, contains 8,493 protein-coding genes and 83 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

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 endemic rhizobia on Christmas Island: Implications for agriculture following phosphate mining

Given that phosphate supplies may diminish and become uneconomic to mine after 2020, there is a compelling need to develop alternative industries to support the population on Christmas Island. Former mine sites could be turned into productive agricultural land, however, large-scale commercial agriculture has never been attempted, and, given the uniqueness of the island, the diversity of rhizobia prior to introducing legumes needed evaluation. Therefore, 84 rhizobia isolates were obtained from nine different hosts, both crop and introduced legumes, located at seven sites across the island. Based on 16S rRNA and recA gene sequence analysis, the isolates grouped into 13 clades clustering within the genus Bradyrhizobium, Ensifer, Cupriavidus and Rhizobium. According to the sequences of their symbiosis genes nodC and nifH, the isolates were classified into 12 and 11 clades, respectively, and clustered closest to tropical or crop legume isolates. Moreover, the symbiosis gene phylogeny and Multi Locus Sequence Analysis gene phylogeny suggested vertical transmission in the Alpha-rhizobia but horizontal transmission within the Beta-rhizobia. Furthermore, this study provides evidence of a large diversity of endemic rhizobia associated with both crop and introduced legumes, and highlights the necessity of inoculation for common bean, chickpea and soybean on the Island.     

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N₂) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.     

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.