Abundance and Diversity of Soybean-Nodulating Rhizobia in Black Soil Are Impacted by Land Use and Crop Management

To investigate the effects of land use and crop management on soybean rhizobial communities, 280 nodule isolates were trapped from 7 fields with different land use and culture histories. Besides the known Bradyrhizobium japonicum, three novel genospecies were isolated from these fields. Grassland (GL) maintained a higher diversity of soybean bradyrhizobia than the other cultivation systems. Two genospecies (Bradyrhizobium spp. I and III) were distributed widely in all treatments, while Bradyrhizobium sp. II was found only in GL treatment. Cultivation with soybeans increased the rhizobial abundance and diversity, except for the soybean monoculture (S-S) treatment. In monoculture systems, soybeans favored Bradyrhizobium sp. I, while maize and wheat favored Bradyrhizobium sp. III. Fertilization decreased the rhizobial diversity indexes but did not change the species composition. The organic carbon (OC) and available phosphorus (AP) contents and pH were the main soil parameters positively correlated with the distribution of Bradyrhizobium spp. I and II and Bradyrhizobium japonicum and negatively correlated with Bradyrhizobium sp. III. These results revealed that different land uses and crop management could not only alter the diversity and abundance of soybean rhizobia, but also change interactions between rhizobia and legume or nonlegume plants, which offered novel information about the biogeography of rhizobia.     

Genetic diversity and biogeography of rhizobia associated with Caragana species in three ecological regions of China

Twenty-two genospecies belonging mainly to Mesorhizobium, and occasionally to Rhizobium and Bradyrhizobium, were defined among the 174 rhizobia strains isolated from Caragana species. Highly similar nodC genes were found in the sole Bradyrhizobium strain and among all the detected Mesorhizobium strains. A clear correlation between rhizobial genospecies and the eco-regions where they were isolated was found using homogeneity analysis. All these results demonstrated that Caragana species had stringently selected the rhizobia symbiotic genotype, but not the genomic background; lateral transfer of symbiotic genes from Mesorhizobium to Bradyrhizobium and among the Mesorhizobium species has happened in the Caragana rhizobia; and biogeography of Caragana rhizobia exists. Furthermore, a combined cluster analysis, based upon the patterns obtained from amplified 16S rRNA gene and 16S–23S intergenic spacer restriction analyses, BOX PCR and SDS-PAGE of proteins, was reported to be an efficient method to define the genospecies.     

Changes in soil microbial community structure with planting years and cultivars of tree peony (Paeonia suffruticosa)

To understand the effects of planting tree peony (Paeonia suffruticosa) on soil microbial community structure, soil samples were collected from the tree peony gardens with three peony cultivars and three planting years, and adjacent wasteland at Luoyang, Henan Province of China. Soil microbial communities were analyzed by the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) of partial 16S rDNA and sequencing methods. With the succeeding development of tree peony garden ecosystems, soil pH, organic C, total P, and available P increased. Soil total N, the cell numbers of bacteria, fungi, and actinomycetes, the Shannon’s diversity index (H), richness (S), and Evenness (E _H ) first showed an increasing trend after wasteland was reclaimed and then a decreasing trend became apparent after 5 years of planting. Principal component analysis based on DGGE banding patterns showed that the microbial community structures were influenced by tree peony cultivars and planting years, and the influences of planting years were greater than those of tree peony cultivars. Sequence analysis of the DGGE bands revealed that the dominant bacteria in tree peony garden soils belonged to Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, Planctomycetes, Euryarchaeota, and Unclassified-bacteria. Moreover, some bacteria closely related to Bradyrhizobium, Sphingopyxis, Novosphingobium, and Sphingomonas, which have been associated with nitrogen fixation and recalcitrant compounds degradation, disappeared with the increasing planting years of tree peony. The bacteria had similarity of 100 % compared with Pseudomonas mandelii which was a denitrifying bacteria, and increased gradually with increasing planting years of tree peony.     

Centrosema is a promiscuous legume nodulated by several new putative species and symbiovars of Bradyrhizobium in various American countries

Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidic and degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a poor acid savanna soil from Venezuela was characterized, and the members of the collection were compared to other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulating Centrosema in American countries were closely related to different species of the genus Bradyrhizobium. However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that they formed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineages that were divergent among themselves and with respect to those formed by Centrosema strains isolated in other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed for the first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhizobium. The nodC genes of the Centrosema strains were divergent among themselves and with respect to the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume. According to these results, the currently known Centrosema-nodulating strains represent several new putative species and symbiovars of the genus Bradyrhizobium.     

Heterogeneity among Mycobacterium ulcerans from French Guiana Revealed by Multilocus Variable Number Tandem Repeat Analysis (MLVA)

Buruli ulcer is an emerging and neglected tropical disease caused by Mycobacterium ulcerans. Few cases have been reported so far in the Americas. With 250 cases reported since 1969, French Guiana is the only Buruli ulcer endemic area in the continent. Thus far, no genetic diversity studies of strains of M. ulcerans from French Guiana have been reported. Our goal in the present study was to examine the genetic diversity of M. ulcerans strains in this region by using the Multilocus Variable Number Tandem Repeat Analysis (MLVA) approach. A total of 23 DNA samples were purified from ulcer biopsies or derived from pure cultures. MVLA was used in the study of six previously-described Variable Number of Tandem Repeat (VNTR) markers. A total of three allelic combinations were characterized in our study: genotype I which has been described previously, genotype III which is very similar to genotype I, and genotype II which has distinctly different characteristics in comparison with the other two genotypes. This high degree of genetic diversity appears to be uncommon for M. ulcerans. Further research based on complete genome sequencing of strains belonging to genotypes I and II is in progress and should lead soon to a better understanding of genetic specificities of M. ulcerans strains from French Guiana.     

PCR-Ribotyping of Xenorhabdus and Photorhabdus Isolates from the Caribbean Region in Relation to the Taxonomy and Geographic Distribution of Their Nematode Hosts

The genetic diversity of symbiotic Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to Xenorhabdus and Photorhabdus spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus Xenorhabdus from the genus Photorhabdus. The genus Xenorhabdus appears more diverse than the genus Photorhabdus, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.     

Diversity of Bacteria Carried by Pinewood Nematode in USA and Phylogenetic Comparison with Isolates from Other Countries

Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Lately, mutualistic relationship has been suggested between the pinewood nematode (PWN), Bursaphelenchus xylophilus the causal nematode agent of PWD, and bacteria. In countries where PWN occurs, nematodes from diseased trees were reported to carry bacteria from several genera. However no data exists for the United States. The objective of this study was to evaluate the diversity of the bacterial community carried by B. xylophilus, isolated from different Pinus spp. with PWD in Nebraska, United States. The bacteria carried by PWN belonged to Gammaproteobacteria (79.9%), Betaproteobacteria (11.7%), Bacilli (5.0%), Alphaproteobacteria (1.7%) and Flavobacteriia (1.7%). Strains from the genera Chryseobacterium and Pigmentiphaga were found associated with the nematode for the first time. These results were compared to results from similar studies conducted from other countries of three continents in order to assess the diversity of bacteria with associated with PWN. The isolates from the United States, Portugal and China belonged to 25 different genera and only strains from the genus Pseudomonas were found in nematodes from all countries. The strains from China were closely related to P. fluorescens and the strains isolated from Portugal and USA were phylogenetically related to P. mohnii and P. lutea. Nematodes from the different countries are associated with bacteria of different species, not supporting a relationship between PWN with a particular bacterial species. Moreover, the diversity of the bacteria carried by the pinewood nematode seems to be related to the geographic area and the Pinus species. The roles these bacteria play within the pine trees or when associated with the nematodes, might be independent of the presence of the nematode in the tree and only related on the bacteria''s relationship with the tree.     

Differential Susceptibilities of Aedes aegypti and Aedes albopictus from the Americas to Zika Virus

BackgroundSince the major outbreak in 2007 in the Yap Island, Zika virus (ZIKV) causing dengue-like syndromes has affected multiple islands of the South Pacific region. In May 2015, the virus was detected in Brazil and then spread through South and Central America. In December 2015, ZIKV was detected in French Guiana and Martinique. The aim of the study was to evaluate the vector competence of the mosquito spp. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), South America (Brazil, French Guiana) for the currently circulating Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia.Conclusions/SignificanceThis study suggests that although susceptible to infection, Ae. aegypti and Ae. albopictus were unexpectedly low competent vectors for ZIKV. This may suggest that other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak.     

Detection of the type III secretion system and its phylogenetic and symbiotic characterization in peanut bradyrhizobia isolated from Guangdong Province,China

The distribution of rhcRST and rhcJ-C1 fragments located in different loci of the type III secretion system (T3SS) gene cluster in the peanut-nodulating bradyrhizobia isolated from Guangdong Province, China was investigated by PCR-based sequencing. T3SS was detected in approximately one-third of the peanut bradyrhizobial strains and the T3SS-harboring strains belonging to different Bradyrhizobium genomic species. Diverse T3SS groups corresponding to different symbiotic gene types were defined among the 23 T3SS-harboring strains. The same or similar T3SS genes were detected in different genospecies, indicating that interspecies horizontal transfer of symbiotic genes had occurred in the Bradyrhizobium genus.     

Genetic divergence among Bradyrhizobium strains nodulating wild and cultivated Kummerowia spp. in China

Distribution of rhizobial species is affected by geographical isolation and selected by leguminous hosts, however, little is known about the molecular evolution of rhizobia nodulating the same legume in different eco-environments. In present study, the microevolution of Bradyrhizobium associated with the leguminous grass Kummerowia grown in exurban areas and cultivated in urban areas in China was investigated. Total 14 genospecies, including seven new groups, were identified based on a concatenated sequence analysis of taxonomic markers (SMc00019, truA and thrA) for 94 representative strains. Results demonstrated that lower levels of nucleotide diversity were found in the strains isolated from urban areas compared with those isolated from exurban areas, based on the evolutional analyses of three housekeeping genes (atpD, glnII and recA), two symbiosis-related genes (nodC and nifH), and the taxonomic markers. Moreover, compared with urban areas, gene exchange and recombination occurred more frequently among the genospecies isolated from exurban areas, regardless of the geographical distribution. Finally, the evolutionary lineage of Bradyrhizobium strains isolated from urban areas was independent of that of the strains isolated from exurban areas. In summary, the evolutionary history of Kummerowia bradyrhizobia may have been gradually segregated to different evolutionary lineages, irrespective of distinct biogeography.     

Geographic Separation of Domestic and Wild Strains of Toxoplasma gondii in French Guiana Correlates with a Monomorphic Version of Chromosome1a

Background

Previous studies have stressed the genetic divergence and high pathogenicity of strains of T. gondii from French Guiana. Although strains from coastal, human adapted environments (so called anthropized) resemble those found in other regions of the Caribbean, strains collected from inland jungle environment are genetically quite diverse. To better understand the composition of these distinct strain types, we undertook a more in depth analysis of T. gondii strains from French Guiana including profiling of chromosome 1a (Chr1a), which is often shared as a single monomorphic haplotype among lineages that are otherwise genetically distinct.

Methodology/Principal Findings

Comparison of intron sequences from selectively neutral genes indicated that anthropized strains were most closely related to clonal type III strains from North America, although wider RFLP analysis revealed that they are natural hybrids. In contrast, strains isolated from the jungle were genetically very diverse. Remarkably, nearly all anthropized strains contained the monomorphic version of Chr1a while wild stains were extremely divergent. The presence of the monomorphic Chr1a strongly correlated with greater transmission in domestic cats, although there were several exceptions, indicating that other factors also contribute. Anthropized strains also varied in their virulence in laboratory mice, and this pattern could not be explained by the simple combination of previously identified virulence factors, indicating that other genetic determinants influence pathogenicity.

Conclusions/Significance

Our studies underscore the marked genetic separation of anthropized and wild strains of T. gondii in French Guiana and provide additional evidence that the presence of Chr1a is associated with successful expansion of widely different lineages within diverse geographic areas. The predominance of Chr1a among strains in the anthropized environment suggests that it may confer an advantage for transmission in this environment, and thus potentially contribute to the spread of pathogenecity determinants.     

Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece

Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48^T and B. liaoningense USDA 3622^T and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar.     

Root nodules of Genista germanica harbor Bradyrhizobium and Rhizobium bacteria exchanging nodC and nodZ genes

A collection of 18 previously unstudied strains isolated from root nodules of Genista germanica (German greenweed) grown in southeast Poland was evaluated for the level of genetic diversity using the BOX-PCR technique and the phylogenetic relationship based on both core (16S rRNA, dnaK, ftsA, glnII, gyrB, recA, rpoB) and nodulation (nodC and nodZ) gene sequences. Each of the 18 G. germanica root nodule isolates displayed unique BOX-PCR patterns, indicating their high level of genomic heterogeneity. Based on the comparative 16S rDNA sequence analysis, 12 isolates were affiliated to the Bradyrhizobium genus and the other strains were most similar to Rhizobium species. Phylogenetic analysis of the core gene sequences indicated that the studied Bradyrhizobium bacteria were most closely related to Bradyrhizobium japonicum, whereas Rhizobium isolates were most closely related to Rhizobium lusitanum and R. leguminosarum. The phylogenies of nodC and nodZ for the Rhizobium strains were incongruent with each other and with the phylogenies inferred from the core gene sequences. All Rhizobium nodZ gene sequences acquired in this study were grouped with the sequences of Bradyrhizobium strains. Some of the studied Rhizobium isolates were placed in the nodC phylogenetic tree together with reference Rhizobium species, while the others were closely related to Bradyrhizobium bacteria. The results provided evidence for horizontal transfer of nodulation genes between Bradyrhizobium and Rhizobium. However, the horizontal transfer of nod genes was not sufficient for Rhizobium strains to form nodules on G. germanica roots, suggesting that symbiotic genes have to be adapted to the bacterial genome.     

Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt

The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense)

Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolor-nodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Isolation and characterization of microsatellite markers in Pterocarpus officinalis Jacq.

The leguminous Pterocarpus officinalis Jacq. is one of the dominant freshwater wetland tree species in the Caribbean basin. Anthromorphic factors threaten to reduce its population. In order to investigate the genetic diversity and structure of this species, we developed eight pairs of primers for nuclear microsatellites. One hundred ninety‐one individuals were analysed within nine Caribbean and continental populations. These loci were polymorphic in all the populations, with four to 20 alleles per locus. Significant Hardy–Weinberg deviation was detected and was interpreted as a result of Wahlund effect. These loci constitute a powerful tool to investigate the genetic patterns within populations of the swamp species P. officinalis.     

Bradyrhizobium canariense and Bradyrhizobium japonicum are the two dominant rhizobium species in root nodules of lupin and serradella plants growing in Europe

Forty three Bradyrhizobium strains isolated in Poland from root nodules of lupin species (Lupinus albus, L. angustifolius and L. luteus), and pink serradella (Ornithopus sativus) were examined based on phylogenetic analyses of three housekeeping (atpD, glnII and recA) and nodulation (nodA) gene sequences. Additionally, seven strains originating from root-nodules of yellow serradella (O. compressus) from Asinara Island (Italy) were included in this study. Phylogenetic trees revealed that 15 serradella strains, including all yellow serradella isolates, and six lupin strains grouped in Bradyrhizobium canariense (BC) clade, whereas eight strains from pink serradella and 15 lupin strains were assigned to Bradyrhizobium japonicum (BJ1). Apparently, these species are the two dominant groups in soils of central Europe, in the nodules of lupin and serradella plants. Only three strains belonged to other chromosomal lineages: one formed a cluster that was sister to B. canariense, one strain grouped outside the branch formed by B. japonicum super-group, and one strain occupied a distant position in the genus Bradyrhizobium, clustering with strains of the Rhodopseudomonas genus. All strains in nodulation nodA gene tree grouped in a cluster referred to as Clade II, which is in line with earlier data on this clade dominance among Bradyrhizobium strains in Europe. The nodA tree revealed four well-supported subgroups within Clade II (II.1-II.4). Interestingly, all B. canariense strains clustered in subgroup II.1 whereas B. japonicum strains dominated subgroups II.2-II.4.     

Diversity and Evolution of Hydrogenase Systems in Rhizobia

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.