Comparative Analyses in Lotus : The Cytogenetic Map of Lotus uliginosus Schkuhr

A comparative cytogenetic map was built for Lotusuliginosus (2n = 12), expanding previous analyses that revealed intra- and interspecific chromosomal rearrangements in the model legume L. japonicus, L. filicaulis, and L. burttii. This species is positioned in a sister clade of the previously-mapped species and is proposed as one of the progenitors of L. corniculatus, the main forage crop of the genus. The cytogenetic map allowed the location of 12 genomic regions to be compared between these species. A high macrosynteny was revealed, but it was interrupted by a translocation involving chromosomes 3 and 5, a new rearrangement for the genus. Also, a transposition on chromosome 2 was found in L. japonicus 'Miyakojima'. Furthermore, changes in the number, size, and position of rDNA sites were observed, as well as an intraspecific size heteromorphism of the 5S rDNA site on L. uliginosus chromosome 6. The karyotype differences observed are proportional to the phylogenetic distance among these species.     

Burkholderia species are ancient symbionts of legumes

Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia . Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia . The symbiosis-related genes formed deep Burkholderia -specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.     

Phylogenetic analyses of Bradyrhizobium symbionts associated with invasive Crotalaria zanzibarica and its coexisting legumes in Taiwan

In this study, the genetic diversity and identification of Bradyrhizobium symbionts of Crotalaria zanzibarica, the most widely-distributed invasive legume in Taiwan, and other sympatric legume species growing along riverbanks of Taiwan were evaluated for the first time. In total, 59 and 54 Bradyrhizobium isolates were obtained from C. zanzibarica and its coexisting legume species, respectively. Based on the multilocus sequence analysis (MLSA) of concatenated four housekeeping genes (dnaK-glnII-recA-rpoB gene sequences, 1901 bp), the 113 isolates displayed 53 unique haplotypes and grouped into 21 clades. Of these clades, 11 were found to be congruent to already defined Bradyrhizobium species, while the other 10 clades were found to not be congruent to any defined species. In particular, the C. zanzibarica isolates belong to 14 MLSA clades, six of which overlapped with the isolates of coexisting legumes. According to the nodA gene sequences (555 bp) obtained from the 105 isolates, these isolates were classified into three known nodA clades, III.2, III.3 and VII and were further clustered into 10 groups. Furthermore, the C. zanzibarica isolates were clustered into 8 nodA groups, five of which overlapped with the isolates from coexisting legumes. Additionally, the nodA genes of the isolates from native species were dominated by Asian origin, while those from C. zanzibarica were dominated by American origin. In conclusion, C. zanzibarica is a promiscuous host capable of recruiting diverse Bradyrhizobium symbionts, some of which are phylogenetically similar to the symbionts of coexisting legumes in Taiwan.     

Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

Diversity and relationships of bradyrhizobia from legumes native to eastern North America

DNA sequencing and polymerase chain reaction (PCR) assays with lineage-specific primers were used to analyze the diversity of 276 isolates of Bradyrhizobium sp. nodule bacteria associated with 13 native legumes species in the northeastern United States, representing eight genera in six legume tribes. A PCR screen with two primer pairs in the rRNA region indicated that seven of the legume species were exclusively associated with strains having markers resembling Bradyrhizobium elkanii, while the remaining six host species harbored strains related to both B. elkanii and Bradyrhizobium japonicum. Sequence analysis of 22 isolates for portions of 16S rRNA and 23S rRNA yielded congruent phylogenetic trees and showed that isolates from different legume genera often shared similar or identical sequences. However, trees inferred from portions of two other genes (alpha-ketoglutarate dioxygenase gene (tfdA), the alpha-subunit of nitrogenase (nifD)) differed significantly from the rRNA phylogeny. Thus, for Bradyrhizobium populations in this region, lateral gene transfer events appear to have altered genealogical relationships of different portions of the genome. These results extend the number of likely cases of gene transfer between divergent taxa of Bradyrhizobium (from members of the B. elkanii lineage to the B. japonicum group) and suggest that transfers have also occurred among separate subgroups of the B. elkanii lineage.     

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.     

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

Aims

In the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.

Methods

Glasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.

Results

Complex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.

Conclusions

Effective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.     

Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden

Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.     

Phylogenetic supermatrix analysis of GenBank sequences from 2228 papilionoid legumes

A comprehensive phylogeny of papilionoid legumes was inferred from sequences of 2228 taxa in GenBank release 147. A semiautomated analysis pipeline was constructed to download, parse, assemble, align, combine, and build trees from a pool of 11,881 sequences. Initial steps included all-against-all BLAST similarity searches coupled with assembly, using a novel strategy for building length-homogeneous primary sequence clusters. This was followed by a combination of global and local alignment protocols to build larger secondary clusters of locally aligned sequences, thus taking into account the dramatic differences in length of the heterogeneous coding and noncoding sequence data present in GenBank. Next, clusters were checked for the presence of duplicate genes and other potentially misleading sequences and examined for combinability with other clusters on the basis of taxon overlap. Finally, two supermatrices were constructed: a "sparse" matrix based on the primary clusters alone (1794 taxa x 53,977 characters), and a somewhat more "dense" matrix based on the secondary clusters (2228 taxa x 33,168 characters). Both matrices were very sparse, with 95% of their cells containing gaps or question marks. These were subjected to extensive heuristic parsimony analyses using deterministic and stochastic heuristics, including bootstrap analyses. A "reduced consensus" bootstrap analysis was also performed to detect cryptic signal in a subtree of the data set corresponding to a "backbone" phylogeny proposed in previous studies. Overall, the dense supermatrix appeared to provide much more satisfying results, indicated by better resolution of the bootstrap tree, excellent agreement with the backbone papilionoid tree in the reduced bootstrap consensus analysis, few problematic large polytomies in the strict consensus, and less fragmentation of conventionally recognized genera. Nevertheless, at lower taxonomic levels several problems were identified and diagnosed. A large number of methodological issues in supermatrix construction at this scale are discussed, including detection of annotation errors in GenBank sequences; the shortage of effective algorithms and software for local multiple sequence alignment; the difficulty of overcoming effects of fragmentation of data into nearly disjoint blocks in sparse supermatrices; and the lack of informative tools to assess confidence limits in very large trees.     

Comparative phylogeography of Amphicarpaea legumes and their root-nodule symbionts in Japan and North America

Aim Relationships of eastern Asian and eastern North American populations of legumes in the genus Amphicarpaea Elliot ex. Nuttall (Phaseoleae–Glycininae) and their root nodule bacteria (Bradyrhizobium Jordan) were analysed to test whether both organisms share an identical biogeographic history. Location Japan and eastern North America (New York and Illinois). Methods Sequences of three plant genes (chloroplast trnL region, nuclear ribosomal ITS, and histone H3‐D) and a segment of the bacterial ribosomal region (partial 16S rRNA and 23S rRNA genes, and the 16S rRNA–23S rRNA ITS) were used to analyse phylogenetic relationships. Results For plants, Japanese populations formed a sister group to a well‐supported clade of all North American genotypes. For nodule bacteria associated with Amphicarpaea, isolates from North America did not form a single clade relative to Asian genotypes. Japanese Bradyrhizobium isolates were closely related to particular sub‐groups of North American bacteria (lineages ‘B’ and ‘C’), with other American bacteria branching earlier. Main conclusions Plants and bacteria showed clear deviations from a pattern of parallel cladogenesis. The most basal Amphicarpaea lineage was associated with a recently‐diverged bacterial group, while one recently‐diverged plant lineage had symbionts that branched in a basal position relative to the other Amphicarpaea bacteria. When analysed with data on symbiotic compatibility from inoculation experiments, the molecular phylogenies suggested that for plants, at least one transition has occurred toward more promiscuous nodulation behaviour. Among bacteria, strains with narrow host range on Amphicarpaea appear to be ancestral to symbiotic generalists.     

Seasonality of Rhizome and Shoot Production and Nitrogen Fixation in Lotus uliginosus under Upland Conditions in South-west Scotland

Development, growth and nitrogen fixation were monitored over3 years in marsh birdsfoot trefoil (Lotus uliginosus Schkuhr),at an upland site in south-west Scotland Plants exhibited markedseasonality of growth, with production of both aerial shootsand rhizome reaching a peak in September and October Renewedaerial shoot growth in spring was initiated mainly from nodeson rhizomes formed in the previous autumn Nitrogen fixationwas measured in soil cores using the acetylene reduction techniqueFixation commenced in early June, coinciding with increasingsoil temperature, and thereafter rose to a peak in mid-summerThe subsequent decline in fixation was associated with increasingrainfall, decreasing soil temperature, flowering and the onsetof rhizome production Lotus uliginosus Schkuhr, marsh birdsfoot trefoil, seasonality, rhizome production, nitrogen fixation, acetylene reduction     

Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici

Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.     

Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean)

Los Tuxtlas is the northernmost rain forest in North America and is rich in Bradyrhizobium with an unprecedented number of novel lineages. ITS sequence analysis of legumes in polycultures from Los Tuxtlas led to the identification of Phaseolus lunatus and Vigna unguiculata in addition to Phaseolus vulgaris as legumes associated with maize in crops. Bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus and V. unguiculata was revealed using ERIC-PCR and PCR-RFLP of rpoB genes, and sequencing of recA, nodZ and nifH genes. P. lunatus and V. unguiculata nodule bacteria corresponded to bradyrhizobia closely related to certain native bradyrhizobia from the Los Tuxtlas forest and novel groups were found. This is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication.     

Distribution and diversity of rhizobia nodulating agroforestry legumes in soils from three continents in the tropics

The natural rhizobial populations of Calliandra calothyrsus, Gliricidia sepium, Leucaena leucocephala and Sesbania sesban were assessed in soils from nine sites across tropical areas of three continents. The rhizobial population size varied from undetectable numbers to 1.8 x 104 cells/g of soil depending on the trap host and the soil. Calliandra calothyrsus was the most promiscuous legume, nodulating in eight soils, while S. sesban nodulated in only one of the soils. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analyses of the 16S rRNA gene and the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes were used to assess the diversity and relative abundance of rhizobia trapped from seven of the soils by C. calothyrsus, G. sepium and L. leucocephala. Representatives of the 16S rRNA RFLP groups were also subjected to sequence analysis of the first 950 base pairs of the 16S rRNA gene. Eighty ITS groups were obtained, with none of the ITS types being sampled in more than one soil. RFLP analysis of the 16S rRNA yielded 23 'species' groups distributed among the Rhizobium, Mesorhizobium, Sinorhizobium and Agrobacterium branches of the rhizobial phylogenetic tree. The phylogeny of the isolates was independent of the site or host of isolation, with different rhizobial groups associated with each host across the soils from widely separated geographical regions. Although rhizobial populations in soils sampled from the centre of diversity of the host legumes were the most genetically diverse, soil acidity was highly correlated with the diversity of ITS types. Our results support the hypothesis that the success of these tree legumes in soils throughout the tropics is the result of their relative promiscuity (permissiveness) allowing nodulation with diverse indigenous rhizobial types.     

Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia

The taxonomic diversity of thirty-seven Rhizobium strains, isolated from nodules of leguminous trees and herbs growing in Ethiopia, was studied using multilocus sequence analyses (MLSA) of six core and two symbiosis-related genes. Phylogenetic analysis based on the 16S rRNA gene grouped them into five clusters related to nine Rhizobium reference species (99–100% sequence similarity). In addition, two test strains occupied their own independent branches on the phylogenetic tree (AC86a2 along with R. tibeticum; 99.1% similarity and AC100b along with R. multihospitium; 99.5% similarity). One strain from Milletia ferruginea was closely related (>99%) to the genus Shinella, further corroborating earlier findings that nitrogen-fixing bacteria are distributed among phylogenetically unrelated taxa. Sequence analyses of five housekeeping genes also separated the strains into five well-supported clusters, three of which grouped with previously studied Ethiopian common bean rhizobia. Three of the five clusters could potentially be described into new species. Based on the nifH genes, most of the test strains from crop legumes were closely related to several strains of Ethiopian common bean rhizobia and other symbionts of bean plants (R. etli and R. gallicum sv. phaseoli). The grouping of the test strains based on the symbiosis-related genes was not in agreement with the housekeeping genes, signifying differences in their evolutionary history. Our earlier studies revealing a large diversity of Mesorhizobium and Ensifer microsymbionts isolated from Ethiopian legumes, together with the results from the present analysis of Rhizobium strains, suggest that this region might be a potential hotspot for rhizobial biodiversity.     

Phylogenetic and symbiotic characterization of rhizobial bacteria nodulating Argyrolobium uniflorum in Tunisian arid soils

Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tunisia were characterized by phenotypic features, RFLP, and sequencing of PCR-amplified 16S rRNA genes. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at temperatures up to 40 degrees C. Phylogenetically, the new isolates were grouped in the genera Sinorhizobium (27), Rhizobium (13), and Agrobacterium (2). Except for the 2 Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among them. Sinorhizobium sp. strains STM 4034, STM 4036, and STM 4039, forming the most effective symbiosis, are potential candidates for inoculants in revegetalisation programs.     

Crystal types and their distribution in the bark of African genistoid legumes (Fabaceae tribes Sophoreae,Podalyrieae, Crotalarieae and Genisteae)

The occurrence and distribution of seven crystal types in 114 bark samples from 25 genera and 91 species, representing all four tribes of African genistoid legumes, are reported. The seven types are prismatic, druse (including irregular crystal clusters), navicular (including truncated navicular, here reported for the first time), spherical crystal cluster, styloid, crystal sand and acicular crystals in sheaf‐like aggregates. Unlike most studies, the elemental composition of the crystals was examined using X‐ray microanalyses. With the exception of acicular crystals, all crystals showed the typical peaks of calcium (sometimes accompanied by small amounts of magnesium). Acicular crystals in sheaf‐like aggregates were composed only of carbon and oxygen, indicating that they are organic and precipitate during drying or after fixation in alcohol. These crystals are found only in the two early‐diverging lineages of Podalyrieae (Cyclopia and Virgilia+Calpurnia clades), consistent with the phylogenetic pattern in the tribe (indicating a secondary loss). Navicular crystals are restricted to Podalyrieae and Crotalarieae. Prismatic crystals in bark are proposed to be the ancestral condition, with multiple losses (or reversals) in Podalyrieae, Crotalarieae and Genisteae. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 620–632.