| 1. |
Parker MA,
Lafay B,
Burdon JJ,
van Berkum P,
( 2002 ) Conflicting phylogeographic patterns in rRNA and nifD indicate regionally restricted gene transfer in Bradyrhizobium. PMID : 12177349 : DOI : 10.1099/00221287-148-8-2557 Abstract >>
Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.
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2. |
Yasuta T,
Okazaki S,
Mitsui H,
Yuhashi K,
Ezura H,
Minamisawa K,
( 2001 ) DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii. PMID : 11679318 : DOI : 10.1128/AEM.67.11.4999-5009.2001 PMC : PMC93263 Abstract >>
We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii. The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA. A large-deletion mutant of B. elkanii, USDA94 Delta rtx::Omega 1, which lacks rtxA, ORF1 (rtxC), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94 Delta rtx::Omega 1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.
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3. |
Gu CT,
Wang ET,
Sui XH,
Chen WF,
Chen WX,
( 2007 ) Diversity and geographical distribution of rhizobia associated with Lespedeza spp. in temperate and subtropical regions of China. PMID : 17530227 : DOI : 10.1007/s00203-007-0256-3 Abstract >>
Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins.
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4. |
Parker MA,
Kennedy DA,
( 2006 ) Diversity and relationships of bradyrhizobia from legumes native to eastern North America. PMID : 17473884 : DOI : 10.1139/w06-076 Abstract >>
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.
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5. |
Stepkowski T,
Hughes CE,
Law IJ,
Markiewicz ?,
Gurda D,
Chlebicka A,
Moulin L,
( 2007 ) Diversification of lupine Bradyrhizobium strains: evidence from nodulation gene trees. PMID : 17400786 : DOI : 10.1128/AEM.02125-06 PMC : PMC1907101 Abstract >>
Bradyrhizobium strains isolated in Europe from Genisteae and serradella legumes form a distinct lineage, designated clade II, on nodulation gene trees. Clade II bradyrhizobia appear to prevail also in the soils of Western Australia and South Africa following probably accidental introduction with seeds of their lupine and serradella hosts. Given this potential for dispersal, we investigated Bradyrhizobium isolates originating from a range of native New World lupines, based on phylogenetic analyses of nodulation (nodA, nodZ, noeI) and housekeeping (atpD, dnaK, glnII, recA) genes. The housekeeping gene trees revealed considerable diversity among lupine bradyrhizobia, with most isolates placed in the Bradyrhizobium japonicum lineage, while some European strains were closely related to Bradyrhizobium canariense. The nodA gene tree resolved seven strongly supported groups (clades I to VII) that correlated with strain geographical origins and to some extent with major Lupinus clades. All European strains were placed in clade II, whereas only a minority of New World strains was placed in this clade. This work, as well as our previous studies, suggests that clade II diversified predominately in the Old World, possibly in the Mediterranean. Most New World isolates formed subclade III.2, nested in a large "pantropical" clade III, which appears to be New World in origin, although it also includes strains originating from nonlupine legumes. Trees generated using nodZ and noeI gene sequences accorded well with the nodA tree, but evidence is presented that the noeI gene may not be required for nodulation of lupine and that loss of this gene is occurring.
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6. |
Martens M,
Delaere M,
Coopman R,
De Vos P,
Gillis M,
Willems A,
( 2007 ) Multilocus sequence analysis of Ensifer and related taxa. PMID : 17329774 : DOI : 10.1099/ijs.0.64344-0 Abstract >>
Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.
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7. |
Batista JS,
Hungria M,
Barcellos FG,
Ferreira MC,
Mendes IC,
( 2007 ) Variability in Bradyrhizobium japonicum and B. elkanii seven years after introduction of both the exotic microsymbiont and the soybean host in a cerrados soil. PMID : 17265000 : DOI : 10.1007/s00248-006-9149-2 Abstract >>
The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.
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8. |
Barcellos FG,
Menna P,
da Silva Batista JS,
Hungria M,
( 2007 ) Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah soil. PMID : 17308185 : DOI : 10.1128/AEM.01823-06 PMC : PMC1855619 Abstract >>
The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N(2)-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.
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9. |
Vinuesa P,
Silva C,
Werner D,
Martínez-Romero E,
( 2005 ) Population genetics and phylogenetic inference in bacterial molecular systematics: the roles of migration and recombination in Bradyrhizobium species cohesion and delineation. PMID : 15579380 : DOI : 10.1016/j.ympev.2004.08.020 Abstract >>
A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.
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10. |
Weir BS,
Turner SJ,
Silvester WB,
Park DC,
Young JM,
( 2004 ) Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species. PMID : 15466541 : DOI : 10.1128/AEM.70.10.5980-5987.2004 PMC : PMC522066 Abstract >>
The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.
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11. |
Itoh K,
Tashiro Y,
Uobe K,
Kamagata Y,
Suyama K,
Yamamoto H,
( 2004 ) Root nodule Bradyrhizobium spp. harbor tfdAalpha and cadA, homologous with genes encoding 2,4-dichlorophenoxyacetic acid-degrading proteins. PMID : 15066803 : DOI : 10.1128/aem.70.4.2110-2118.2004 PMC : PMC383140 Abstract >>
The distribution of tfdAalpha and cadA, genes encoding 2,4-dichlorophenoxyacetate (2,4-D)-degrading proteins which are characteristic of the 2,4-D-degrading Bradyrhizobium sp. isolated from pristine environments, was examined by PCR and Southern hybridization in several Bradyrhizobium strains including type strains of Bradyrhizobium japonicum USDA110 and Bradyrhizobium elkanii USDA94, in phylogenetically closely related Agromonas oligotrophica and Rhodopseudomonas palustris, and in 2,4-D-degrading Sphingomonas strains. All strains showed positive signals for tfdAalpha, and its phylogenetic tree was congruent with that of 16S rRNA genes in alpha-Proteobacteria, indicating evolution of tfdAalpha without horizontal gene transfer. The nucleotide sequence identities between tfdAalpha and canonical tfdA in beta- and gamma-Proteobacteria were 46 to 57%, and the deduced amino acid sequence of TfdAalpha revealed conserved residues characteristic of the active site of alpha-ketoglutarate-dependent dioxygenases. On the other hand, cadA showed limited distribution in 2,4-D-degrading Bradyrhizobium sp. and Sphingomonas sp. and some strains of non-2,4-D-degrading B. elkanii. The cadA genes were phylogenetically separated between 2,4-D-degrading and nondegrading strains, and the cadA genes of 2,4-D degrading strains were further separated between Bradyrhizobium sp. and Sphingomonas sp., indicating the incongruency of cadA with 16S rRNA genes. The nucleotide sequence identities between cadA and tftA of 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were 46 to 53%. Although all root nodule Bradyrhizobium strains were unable to degrade 2,4-D, three strains carrying cadA homologs degraded 4-chlorophenoxyacetate with the accumulation of 4-chlorophenol as an intermediate, suggesting the involvement of cadA homologs in the cleavage of the aryl ether linkage. Based on codon usage patterns and GC content, it was suggested that the cadA genes of 2,4-D-degrading and nondegrading Bradyrhizobium spp. have different origins and that the genes would be obtained in the former through horizontal gene transfer.
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12. |
Parker MA,
( 2004 ) RRNA and dnaK relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legume trees in Costa Rica. PMID : 15214639 : DOI : 10.1078/0723-2020-00266 Abstract >>
Enzyme electrophoresis and sequencing of rRNA and dnaK genes revealed high genetic diversity among root nodule bacteria from the Costa Rican trees Andira inermis, Dalbergia retusa, Platymiscium pinnatum (Papilionoideae tribe Dalbergieae) and Lonchocarpus atropurpureus (Papilionoideae tribe Millettieae). A total of 21 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 36 isolates analyzed, and no ETs were shared in common by isolates from different legume hosts. However, three of the ETs from D. retusa were identical to Bradyrhizobium sp. isolates detected in prior studies of several other legume genera in both Costa Rica and Panama. Nearly full-length 16S rRNA sequences and partial 23S rRNA sequences confirmed that two isolates from D. retusa were highly similar or identical to Bradyrhizobium strains isolated from the legumes Erythrina and Clitoria (Papilionoideae tribe Phaseoleae) in Panama. rRNA sequences for five isolates from L. atropurpureus, P. pinnatum and A. inermis were not closely related to any currently known strains from Central America or elsewhere, but had affinities to the reference strains Bradyrhizobium japonicum USDA 110 (three isolates) or to B. elkanii USDA 76 (two isolates). A phylogenetic tree for 21 Bradyrhizobium strains based on 603 bp of the dnaK gene showed several significant conflicts with the rRNA tree, suggesting that genealogical relationships may have been altered by lateral gene transfer events.
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13. |
Okazaki S,
Sugawara M,
Minamisawa K,
( 2004 ) Bradyrhizobium elkanii rtxC gene is required for expression of symbiotic phenotypes in the final step of rhizobitoxine biosynthesis. PMID : 14711685 : DOI : 10.1128/aem.70.1.535-541.2004 PMC : PMC321310 Abstract >>
We disrupted the rtxC gene on the chromosome of Bradyrhizobium elkanii USDA94 by insertion of a nonpolar aph cartridge. The rtxC mutant, designated DeltartxC, produced serinol and dihydrorhizobitoxine but no rhizobitoxine, both in culture and in planta. The introduction of cosmids harboring the rtxC gene into the DeltartxC mutant complemented rhizobitoxine production, suggesting that rtxC is involved in the final step of rhizobitoxine biosynthesis in B. elkanii USDA94. Glycine max cv. Lee inoculated with DeltartxC or with a null mutant, Deltartx::Omega1, showed no foliar chlorosis, whereas the wild-type strain USDA94 caused severe foliar chlorosis. The two mutants showed significantly less nodulation competitiveness than the wild-type strain on Macroptilium atropurpureum. These results indicate that dihydrorhizobitoxine, the immediate precursor of the oxidative form of rhizobitoxine, has no distinct effect on nodulation phenotype in these legumes. Thus, desaturation of dihydrorhizobitoxine by rtxC-encoded protein is essential for the bacterium to show rhizobitoxine phenotypes in planta. In addition, complementation analysis of rtxC by cosmids differing in rtxC transcription levels suggested that rhizobitoxine production correlates with the amount of rtxC transcript.
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14. |
Stepkowski T,
Czapli?ska M,
Miedzinska K,
Moulin L,
( 2003 ) The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria. PMID : 14666974 : DOI : 10.1078/072320203770865765 Abstract >>
DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins. Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies. In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria. A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus. Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical. In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA. The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny. The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R. radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium. The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus. Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B. japonicum, B. elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants. Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).
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15. |
Chang YL,
Wang ET,
Sui XH,
Zhang XX,
Chen WX,
( 2011 ) Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China. PMID : 21498018 : DOI : 10.1016/j.syapm.2010.12.004 Abstract >>
As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.
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16. |
Sánchez-Cañizares C,
Rey L,
Durán D,
Temprano F,
Sánchez-Jiménez P,
Navarro A,
Polajnar M,
Imperial J,
Ruiz-Argüeso T,
( 2011 ) Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus. PMID : 21420266 : DOI : 10.1016/j.syapm.2010.11.020 Abstract >>
Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.
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17. |
Menna P,
Hungria M,
( 2011 ) Phylogeny of nodulation and nitrogen-fixation genes in Bradyrhizobium: supporting evidence for the theory of monophyletic origin, and spread and maintenance by both horizontal and vertical transfer. PMID : 21357454 : DOI : 10.1099/ijs.0.028803-0 Abstract >>
Bacteria belonging to the genus Bradyrhizobium are capable of establishing symbiotic relationships with a broad range of plants belonging to the three subfamilies of the family Leguminosae (=Fabaceae), with the formation of specialized structures on the roots called nodules, where fixation of atmospheric nitrogen takes place. Symbiosis is under the control of finely tuned expression of common and host-specific nodulation genes and also of genes related to the assembly and activity of the nitrogenase, which, in Bradyrhizobium strains investigated so far, are clustered in a symbiotic island. Information about the diversity of these genes is essential to improve our current poor understanding of their origin, spread and maintenance and, in this study, we provide information on 40 Bradyrhizobium strains, mostly of tropical origin. For the nodulation trait, common (nodA), Bradyrhizobium-specific (nodY/K) and host-specific (nodZ) nodulation genes were studied, whereas for fixation ability, the diversity of nifH was investigated. In general, clustering of strains in all nod and nifH trees was similar and the Bradyrhizobium group could be clearly separated from other rhizobial genera. However, the congruence of nod and nif genes with ribosomal and housekeeping genes was low. nodA and nodY/K were not detected in three strains by amplification or hybridization with probes using Bradyrhizobium japonicum and Bradyrhizobium elkanii type strains, indicating the high diversity of these genes or that strains other than photosynthetic Bradyrhizobium must have alternative mechanisms to initiate the process of nodulation. For a large group of strains, the high diversity of nod genes (with an emphasis on nodZ), the low relationship between nod genes and the host legume, and some evidence of horizontal gene transfer might indicate strategies to increase host range. On the other hand, in a group of five symbionts of Acacia mearnsii, the high congruence between nod and ribosomal/housekeeping genes, in addition to shorter nodY/K sequences and the absence of nodZ, highlights a co-evolution process. Additionally, in a group of B. japonicum strains that were symbionts of soybean, vertical transfer seemed to represent the main genetic event. In conclusion, clustering of nodA and nifH gives additional support to the theory of monophyletic origin of the symbiotic genes in Bradyrhizobium and, in addition to the analysis of nodY/K and nodZ, indicates spread and maintenance of nod and nif genes through both vertical and horizontal transmission, apparently with the dominance of one or other of these events in some groups of strains.
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18. |
Zhang YM,
Li Y,
Chen WF,
Wang ET,
Tian CF,
Li QQ,
Zhang YZ,
Sui XH,
Chen WX,
( 2011 ) Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain. PMID : 21784912 : DOI : 10.1128/AEM.00542-11 PMC : PMC3187167 Abstract >>
As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.
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19. |
Wu LJ,
Wang HQ,
Wang ET,
Chen WX,
Tian CF,
( 2011 ) Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. PMID : 21303397 : DOI : 10.1111/j.1574-6941.2011.01064.x Abstract >>
A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.
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20. |
Li QQ,
Wang ET,
Zhang YZ,
Zhang YM,
Tian CF,
Sui XH,
Chen WF,
Chen WX,
( 2011 ) Diversity and biogeography of rhizobia isolated from root nodules of Glycine max grown in Hebei Province, China. PMID : 21340735 : DOI : 10.1007/s00248-011-9820-0 Abstract >>
A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S-23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline-saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.
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21. |
Risal CP,
Yokoyama T,
Ohkama-Ohtsu N,
Djedidi S,
Sekimoto H,
( 2010 ) Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal. PMID : 20851547 : DOI : 10.1016/j.syapm.2010.06.008 Abstract >>
Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.
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22. |
Menna P,
Barcellos FG,
Hungria M,
( 2009 ) Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes. PMID : 19628593 : DOI : 10.1099/ijs.0.009779-0 Abstract >>
The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species.
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23. |
Zhang YF,
Sui XH,
Chen WF,
Chen WX,
Han LL,
Wang ET,
Lu YL,
( 2009 ) Bradyrhizobium spp. and Sinorhizobium fredii are predominant in root nodules of Vigna angularis, a native legume crop in the subtropical region of China. PMID : 19557346 : DOI : 10.1007/s12275-009-0001-5 Abstract >>
Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters.
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24. |
Ramírez-Bahena MH,
Peix A,
Rivas R,
Camacho M,
Rodríguez-Navarro DN,
Mateos PF,
Martínez-Molina E,
Willems A,
Velázquez E,
( 2009 ) Bradyrhizobium pachyrhizi sp. nov. and Bradyrhizobium jicamae sp. nov., isolated from effective nodules of Pachyrhizus erosus. PMID : 19567584 : DOI : 10.1099/ijs.0.006320-0 Abstract >>
Several strains isolated from the legume Pachyrhizus erosus were characterized on the basis of diverse genetic, phenotypic and symbiotic approaches. These novel strains formed two groups closely related to Bradyrhizobium elkanii according to their 16S rRNA gene sequences. Strains PAC48T and PAC68T, designated as the type strains of these two groups, presented 99.8 and 99.1% similarity, respectively, in their 16S rRNA gene sequences with respect to B. elkanii USDA 76T. In spite of these high similarity values, the analysis of additional phylogenetic markers such as atpD and glnII genes and the 16S-23S intergenic spacer (ITS) showed that strains PAC48T and PAC68T represented two separate novel species of the genus Bradyrhizobium with B. elkanii as their closest relative. Phenotypic differences among the novel strains isolated from Pachyrhizus and B. elkanii were found regarding the assimilation of carbon sources and antibiotic resistance. All these differences were congruent with DNA-DNA hybridization analysis which revealed 21% genetic relatedness between strains PAC48T and PAC68T and 46% and 25%, respectively, between these strains and B. elkanii LMG 6134T. The nodD and nifH genes of strains PAC48T and PAC68T were phylogenetically divergent from those of bradyrhizobia species that nodulate soybean. Soybean was not nodulated by the novel Pachyrhizus isolates. Based on the genotypic and phenotypic data obtained in this study, the new strains represent two novel species for which the names Bradyrhizobium pachyrhizi sp. nov. (type strain PAC48T=LMG 24246T=CECT 7396T) and Bradyrhizobium jicamae sp. nov. (type strain PAC68T=LMG 24556T=CECT 7395T) are proposed.
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25. |
Zhao CT,
Wang ET,
Chen WF,
Chen WX,
( 2008 ) Diverse genomic species and evidences of symbiotic gene lateral transfer detected among the rhizobia associated with Astragalus species grown in the temperate regions of China. PMID : 18657113 : DOI : 10.1111/j.1574-6968.2008.01282.x Abstract >>
Based on the analyses of ribosomal DNA and housekeeping genes, a total of 118 bacterial isolates obtained from 13 Astragalus species grown in the temperate region of China were identified as 19 genomic species of Mesorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium, two of them being putatively new species. Phylogenetic comparison of symbiotic genes (nodC and nifH) and housekeeping genes showed that the symbiotic genes of the Astragalus rhizobia were maintained by both vertical and horizontal transfer. The results demonstrated that the Astragalus species were very promiscuous hosts for rhizobia and that their rhizobia had very diverse genomic and symbiotic gene backgrounds.
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26. |
Steenkamp ET,
Stepkowski T,
Przymusiak A,
Botha WJ,
Law IJ,
( 2008 ) Cowpea and peanut in southern Africa are nodulated by diverse Bradyrhizobium strains harboring nodulation genes that belong to the large pantropical clade common in Africa. PMID : 18539053 : DOI : 10.1016/j.ympev.2008.04.032 Abstract >>
Cowpea (Vigna unguiculata) and peanut (Arachis hypogaea) in southern Africa are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the identity of these bacteria, a collection of 22 isolates originating from the root nodules of both hosts in Botswana and South Africa was investigated using the combined sequences for the core genome genes rrs, recA, and glnII. These data separated the majority of the isolates into one of three unique lineages that most likely represent novel Bradyrhizobium species. Some isolates were also conspecific with B. yuanmingense and with B. elkanii, although none grouped with B. japonicum, B. canariense or B. liaoningense. To study the evolution of nodulation genes in these bacteria, the common nodulation gene, nodA, and host-specific nodulation genes, nodZ, noeE, and noeI, were analyzed. The nodA phylogeny showed that the cowpea and peanut Bradyrhizobium isolates represent various locally adapted groups or ecotypes that form part of Clade III of the seven known BradyrhizobiumnodA clades. This large and highly diverse clade comprises all strains from sub-Saharan Africa, as well as some originating from the Americas, Australia, Indonesia, China and Japan. Some similar groupings were supported by the other nodulation genes, although the overall phylogenies for the nodulation genes were incongruent with that inferred from the core genome genes, suggesting that horizontal gene transfer significantly influences the evolution of cowpea and peanut root-nodule bacteria. Furthermore, identification of the nodZ, noeI, and noeE genes in the isolates tested indicates that African Bradyrhizobium species may produce highly decorated nodulation factors, which potentially represent an important adaptation enabling nodulation of a great variety of legumes inhabiting the African continent.
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27. |
Vinuesa P,
Rojas-Jiménez K,
Contreras-Moreira B,
Mahna SK,
Prasad BN,
Moe H,
Selvaraju SB,
Thierfelder H,
Werner D,
( 2008 ) Multilocus sequence analysis for assessment of the biogeography and evolutionary genetics of four Bradyrhizobium species that nodulate soybeans on the asiatic continent. PMID : 18791003 : DOI : 10.1128/AEM.00875-08 PMC : PMC2583495 Abstract >>
A highly supported maximum-likelihood species phylogeny for the genus Bradyrhizobium was inferred from a supermatrix obtained from the concatenation of partial atpD, recA, glnII, and rpoB sequences corresponding to 33 reference strains and 76 bradyrhizobia isolated from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam. The power of the multigene approach using multiple strains per species was evaluated in terms of overall tree resolution and phylogenetic congruence, representing a practical and portable option for bacterial molecular systematics. Potential pitfalls of the approach are highlighted. Seventy-five of the isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense, B. yuanmingense, or B. elkanii, whereas one represented a novel Bradyrhizobium lineage. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence against the monophyly of the of B. japonicum I and Ia lineages was found. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage are misclassified as B. japonicum. The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones and had broad geographic and environmental distribution ranges, as revealed by mapping climate types and geographic origins of the isolates on the species tree.
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28. |
Yokoyama T,
( 2008 ) Flavonoid-responsive nodY-lacZ expression in three phylogenetically different Bradyrhizobium groups. PMID : 18449225 : DOI : 10.1139/w08-021 Abstract >>
Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4',5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not.
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29. |
Moulin L,
Jaffré T,
Fardoux J,
Giraud E,
Dreyfus B,
Bailly X,
Chaintreuil C,
Rigault F,
( 2007 ) Nickel resistance determinants in bradyrhizobium strains from nodules of the endemic New Caledonia legume Serianthes calycina. PMID : 17951443 : DOI : 10.1128/AEM.01431-07 PMC : PMC2168143 Abstract >>
Bradyrhizobium strains, isolated in New Caledonia from nodules of the endemic legume Serianthes calycina growing in nickel-rich soils, were able to grow in the presence of 15 mM NiCl2. The genomes of these strains harbored two Ni resistance determinants, the cnr and nre operons. By constructing a cnrA mutant, we demonstrated that the cnr operon determines the high nickel resistance in Bradyrhizobium strains.
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30. |
Fossou RK,
Ziegler D,
Zézé A,
Barja F,
Perret X,
( 2016 ) Two Major Clades of Bradyrhizobia Dominate Symbiotic Interactions with Pigeonpea in Fields of C?te d'Ivoire. PMID : 27891120 : DOI : 10.3389/fmicb.2016.01793 PMC : PMC5104742 Abstract >>
In smallholder farms of C?te d'Ivoire, particularly in the northeast of the country, Cajanus cajan (pigeonpea) has become an important crop because of its multiple beneficial facets. Pigeonpea seeds provide food to make ends meet, are sold on local markets, and aerial parts serve as forage for animals. Since it fixes atmospheric nitrogen in symbiosis with soil bacteria collectively known as rhizobia, C. cajan also improves soil fertility and reduces fallow time. Yet, seed yields remain low mostly because farmers cannot afford chemical fertilizers. To identify local rhizobial strains susceptible to be used as bio-inoculants to foster pigeonpea growth, root nodules were collected in six fields of three geographically distant regions of C?te d'Ivoire. Nodule bacteria were isolated and characterized using various molecular techniques including matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) and DNA sequencing. These molecular analyses showed that 63 out of 85 nodule isolates belonged to two major clades of bradyrhizobia, one of which is known as the Bradyrhizobium elkanii super clade. Phylogenies of housekeeping (16S-ITS-23S, rpoB) and symbiotic (nifH) genes were not always congruent suggesting that lateral transfer of nitrogen fixation genes also contributed to define the genome of these bradyrhizobial isolates. Interestingly, no field-, plant-, or cultivar-specific effect was found to shape the profiles of symbiotic strains. In addition, nodule isolates CI-1B, CI-36E, and CI-41A that belong to distinct species, showed similar symbiotic efficiencies suggesting that any of these strains might serve as a proficient inoculant for C. cajan.
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31. |
Manszewski T,
Singh K,
Imiolczyk B,
Jaskolski M,
( 2015 ) An enzyme captured in two conformational states: crystal structure of S-adenosyl-L-homocysteine hydrolase from Bradyrhizobium elkanii. PMID : 26627650 : DOI : 10.1107/S1399004715018659 Abstract >>
S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 ? resolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.
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32. |
Faruque OM,
Miwa H,
Yasuda M,
Fujii Y,
Kaneko T,
Sato S,
Okazaki S,
( 2015 ) Identification of Bradyrhizobium elkanii Genes Involved in Incompatibility with Soybean Plants Carrying the Rj4 Allele. PMID : 26187957 : DOI : 10.1128/AEM.01942-15 PMC : PMC4561682 Abstract >>
Symbioses between leguminous plants and soil bacteria known as rhizobia are of great importance to agricultural production and nitrogen cycling. While these mutualistic symbioses can involve a wide range of rhizobia, some legumes exhibit incompatibility with specific strains, resulting in ineffective nodulation. The formation of nodules in soybean plants (Glycine max) is controlled by several host genes, which are referred to as Rj genes. The soybean cultivar BARC2 carries the Rj4 gene, which restricts nodulation by specific strains, including Bradyrhizobium elkanii USDA61. Here we employed transposon mutagenesis to identify the genetic locus in USDA61 that determines incompatibility with soybean varieties carrying the Rj4 allele. Introduction of the Tn5 transposon into USDA61 resulted in the formation of nitrogen fixation nodules on the roots of soybean cultivar BARC2 (Rj4 Rj4). Sequencing analysis of the sequence flanking the Tn5 insertion revealed that six genes encoding a putative histidine kinase, transcriptional regulator, DNA-binding transcriptional activator, helix-turn-helix-type transcriptional regulator, phage shock protein, and cysteine protease were disrupted. The cysteine protease mutant had a high degree of similarity with the type 3 effector protein XopD of Xanthomonas campestris. Our findings shed light on the diverse and complicated mechanisms that underlie these highly host-specific interactions and indicate the involvement of a type 3 effector in Rj4 nodulation restriction, suggesting that Rj4 incompatibility is partly mediated by effector-triggered immunity.
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33. |
Parker MA,
Rousteau A,
( 2014 ) Mosaic origins of Bradyrhizobium legume symbionts on the Caribbean island of Guadeloupe. PMID : 24747127 : DOI : 10.1016/j.ympev.2014.04.011 Abstract >>
To analyze geographic affinities of Bradyrhizobium sp. symbionts associated with the diverse legume flora on the Caribbean island of Guadeloupe, 39 isolates from 18 legume genera were compared to a reference set of 269 Bradyrhizobium strains from North America, Central America, Puerto Rico and the Philippines. A multilocus sequence analysis (4192 bp) showed that nucleotide diversity in Guadeloupe equaled or exceeded that found in all other regional Bradyrhizobium populations examined. Bayesian phylogenetic tree analysis grouped the Guadeloupe Bradyrhizobium strains into clades with at least 20 distinct sets of non-Guadeloupe relatives, implying that the island was colonized numerous times from multiple source regions. However, for 18% of the Guadeloupe isolates, inferred geographic affinities for the nifD locus, in the symbiosis island region of the Bradyrhizobium chromosome, conflicted with the source region deduced from a tree based on six concatenated housekeeping genes. Geographic mosaic ancestry was therefore evident among Guadeloupe bradyrhizobia. Horizontal gene transfer subsequent to island colonization appears to have generated strains that carry combinations of genes from disparate source regions.
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34. |
Chaloupkova R,
Prudnikova T,
Rezacova P,
Prokop Z,
Koudelakova T,
Daniel L,
Brezovsky J,
Ikeda-Ohtsubo W,
Sato Y,
Kuty M,
Nagata Y,
Kuta Smatanova I,
Damborsky J,
( 2014 ) Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites. PMID : 25004965 : DOI : 10.1107/S1399004714009018 Abstract >>
The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
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35. |
Delamuta JR,
Ribeiro RA,
Menna P,
Bangel EV,
Hungria M,
( 2012 ) Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria. PMID : 24031882 : DOI : 10.1590/S1517-83822012000200035 PMC : PMC3768805 Abstract >>
Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.
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36. |
Wang YC,
Wang F,
Hou BC,
Wang ET,
Chen WF,
Sui XH,
Chen WX,
Li Y,
Zhang YB,
( 2013 ) Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov. PMID : 23759600 : DOI : 10.1016/j.syapm.2013.05.001 Abstract >>
In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA-DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732(T)=LMG 26835(T)=HAMBI 3286(T)) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729(T)=LMG 26833(T)=HAMBI 3287(T)). They had a DNA G+C mol% (Tm) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.
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37. |
Parker MA,
( 2012 ) Legumes select symbiosis island sequence variants in Bradyrhizobium. PMID : 22369247 : DOI : 10.1111/j.1365-294X.2012.05497.x Abstract >>
Bradyrhizobium strains sampled from 14 legume genera native to eastern North America showed substantial host-related phylogenetic clustering at three loci in the symbiotic island (SI) region (nodC, nifD, nifH), indicating selection of distinct suites of SI lineages by different legumes. Bacteria assorted consistently with particular legumes across two regions separated by 800 km, implying recurrent assembly of the same symbiotic combinations. High genetic polymorphism of all three SI loci relative to four nonsymbiotic loci supported the inference that a form of multiple-niche balancing selection has acted on the SI region, arising from differential symbiont utilization by different legume taxa. Extensive discordance between the tree for SI variants and a phylogenetic tree inferred for four housekeeping loci implied that lateral transfer of the symbiosis island region has been common (at least 26 transfer events among 85 Bradyrhizobium strains analysed). Patterns of linkage disequilibrium also supported the conclusion that recombination has impacted symbiotic and nonsymbiotic regions unequally. The high prevalence of lateral transfer suggests that acquisition of a novel SI variant may often confer a strong selective advantage for recipient cells.
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38. |
van Berkum P,
Elia P,
Song Q,
Eardly BD,
( 2012 ) Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr). PMID : 22074348 : DOI : 10.1094/MPMI-09-11-0241 Abstract >>
A multilocus sequence typing (MLST) method based on allelic variation of seven chromosomal loci was developed for characterizing genotypes (GT) within the genus Bradyrhizobium. With the method, 29 distinct multilocus GT were identified among 190 culture collection soybean strains. The occupancy of 347 nodules taken from uninoculated field-grown soybean plants also was determined. The bacteroid GT were either the same as or were closely related to GT identified among strains in the culture collection. Double-nodule occupancy estimates of 2.9% were much lower than values published based on serology. Of the 347 nodules examined, 337 and 10 were occupied by Bradyrhizobium japonicum and B. elkanii, respectively. The collection strains within the species B. japonicum and B. elkaniialso were compared with Bradyrhizobium cultures from other legumes. In many cases, the observed GT varied more according to their geographic origin than by their trap hosts of isolation. In other cases, there were no apparent relationships with either the legume or geographic source. The MLST method that was developed should be a useful tool in determining the influence of geographic location, temperature, season, soil type, and host plant cultivar on the distribution of GT of Bradyrhizobium spp.
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39. |
Geurts R,
Squartini A,
Roelofsen W,
Op den Camp RH,
( 2012 ) Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness. PMID : 22668002 : DOI : 10.1094/MPMI-11-11-0304 Abstract >>
The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.
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40. |
Mishra RP,
Tisseyre P,
Melkonian R,
Chaintreuil C,
Miché L,
Klonowska A,
Gonzalez S,
Bena G,
Laguerre G,
Moulin L,
( 2012 ) Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia. PMID : 22093060 : DOI : 10.1111/j.1574-6941.2011.01235.x Abstract >>
The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.
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41. |
Ormeño-Orrillo E,
Rogel-Hernández MA,
Lloret L,
López-López A,
Martínez J,
Barois I,
Martínez-Romero E,
( 2012 ) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). PMID : 22109095 : DOI : 10.1007/s00248-011-9974-9 Abstract >>
Nitrogen-fixing bacteria of the Bradyrhizobium genus are major symbionts of legume plants in American tropical forests, but little is known about the effects of deforestation and change in land use on their diversity and community structure. Forest clearing is followed by cropping of bean (Phaseolus vulgaris) and maize as intercropped plants in Los Tuxtlas tropical forest of Mexico. The identity of bean-nodulating rhizobia in this area is not known. Using promiscuous trap plants, bradyrhizobia were isolated from soil samples collected in Los Tuxtlas undisturbed forest, and in areas where forest was cleared and land was used as crop fields or as pastures, or where secondary forests were established. Rhizobia were also trapped by using bean plants. Bradyrhizobium strains were classified into genospecies by dnaK sequence analysis supported by recA, glnII and 16S-23S rDNA IGS loci analyses. A total of 29 genospecies were identified, 24 of which did not correspond to any described taxa. A reduction in Bradyrhizobium diversity was observed when forest was turned to crop fields or pastures. Diversity seemed to recover to primary forest levels in secondary forests that derived from abandoned crop fields or pastures. The shifts in diversity were not related to soil characteristics but seemingly to the density of nodulating legumes present at each land use system (LUS). Bradyrhizobium community composition in soils was dependent on land use; however, similarities were observed between crop fields and pastures but not among forest and secondary forest. Most Bradyrhizobium genospecies present in forest were not recovered or become rare in the other LUS. Rhizobium etli was found as the dominant bean-nodulating rhizobia present in crop fields and pastures, and evidence was found that this species was introduced in Los Tuxtlas forest.
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42. |
( N/A ) DNA sequence of the common nodulation genes of Bradyrhizobium elkanii and their phylogenetic relationship to those of other nodulating bacteria. PMID : 7949325 : Abstract >>
A 6.6-kb BamHI fragment containing the common nodulation genes of Bradyrhizobium elkanii USDA94 was identified by southern hybridization using the common nod genes of B. japonicum as a probe. This fragment was cloned and sequenced. Analysis of the sequence showed open reading frames highly homologous to nolA, nodD2, nodD1, and nodKABC from other bradyrhizobial sources. The sequence showed higher homology to the common nod genes of Bradyrhizobium sp. (Parasponia) than to those from B. japonicum. The open reading frame identified between nodD1 and nodA in the B. elkanii sequence was far more similar to nodK from Bradyrhizobium sp. (Parasponia) than to nodY from B. japonicum. The molecular phylogeny of nodD and nodAB from many sources was analyzed. The genetic distance between the nod genes is far greater than the distance between the 16S rRNA and nifH genes. The differences between the nod genes among the species of Rhizobium is as great as that between Bradyrhizobium and Rhizobium. The host range of the microsymbiont was found to be a better predictor of the similarities of the common nod genes than the 16S rRNA or nifH genes. We propose two groups of nod genes among the rhizobia and bradyrhizobia, based on molecular phylogenetic analysis: those which nodulate legumes of temperate origin in the tribes Vicieae or Trifolieae and those which nodulate legumes of tropical origin in the tribe Phaseoleae.
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43. |
Ueda T,
Suga Y,
Yahiro N,
Matsuguchi T,
( 1995 ) Phylogeny of Sym plasmids of rhizobia by PCR-based sequencing of a nodC segment. PMID : 7814339 : DOI : 10.1128/jb.177.2.468-472.1995 PMC : PMC176613 Abstract >>
To understand the host specificity of rhizobia and the relationship between the evolution of Sym plasmids and that of host plants, we determined partial nodC sequences of 10 representative rhizobium strains and then constructed an evolutionary tree for the deduced amino acid sequences with four published sequences. These coding sequences yield a phylogenetic tree similar to that for leghemoglobin of host plants, suggesting that the evolution of common nodulation genes may be linked to host legume evolution and speciation.
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44. |
Kalita M,
Ma?ek W,
( 2019 ) The ftsA gene as a molecular marker for phylogenetic studies in Bradyrhizobium and identification of Bradyrhizobium japonicum. PMID : 30417315 : DOI : 10.1007/s13353-018-0479-9 PMC : PMC6373400 Abstract >>
The use of ftsA gene sequences for taxonomic studies of the genus Bradyrhizobium bacteria was assessed. The ftsA gene codes for an actin-like protein involved in prokaryotic cell division. Up to now, this gene has not been used as a phylogenetic marker for analysis of bacteria establishing root nodule symbiosis with Fabaceae plants. In this study, the ftsA gene sequences obtained for bradyrhizobia forming N2 fixing symbiosis with four Genisteae tribe plants growing in Poland and most of the type strains of the genus Bradyrhizobium species were analyzed and evaluated as molecular markers for phylogenetic studies of these bacteria for the first time. The ftsA gene sequences of all bradyrhizobial strains with completely or partially sequenced genomes, available in the GenBank database, were also included into the analysis. The phylogeny of the ftsA gene was compared to the phylogenies of other chromosomal genes commonly used in the studies of Bradyrhizobium bacteria. The results showed that the phylogenies of ftsA and the core genes recA and glnII were congruent, making the ftsA gene useful as a phylogenetic marker. Analysis of the ftsA gene sequences revealed a single-nucleotide polymorphism unique to Bradyrhizobium japonicum strains, and the potential use of this SNP for identification of this species was discussed.
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45. |
Yang SH,
Chen WH,
Wang ET,
Chen WF,
Yan J,
Han XZ,
Tian CF,
Sui XH,
Singh RP,
Jiang GM,
Chen WX,
( 2018 ) Rhizobial biogeography and inoculation application to soybean in four regions across China. PMID : 29719942 : DOI : 10.1111/jam.13897 Abstract >>
The aim of the study was to survey rhizobial biogeography and to inoculate soybean with selected rhizobia in China to enhance symbiotic nitrogen fixation (SNF). Biogeography, genetic diversity and phylogeny of soybean rhizobia were surveyed. Inocula were prepared and applied to soybean. Results showed that Bradyrhizobium elkanii and Ensifer fredii were widely distributed in acid and alkaline soils respectively. Available iron was detected as the first determinant for distribution of the two rhizobia and the soybean varieties did not greatly affect the rhizobial compatibility. Geographical latitude and precipitation in June were the main geographical and climatic factors affecting the rhizobial distribution. Inoculation with selected rhizobia increased the nodule number, fresh weight, occupation ratio, seed protein content and soybean yields. Selection and application of effective soybean rhizobia across China according to biogeography were clarified to promote the SNF, thereby improving soybean yield. Rhizobial diversity and biogeography were evaluated systematically in six sites across China. Available iron and soil pH are found to be the most important determinants for the distribution of soybean rhizobia. Inoculation to soybean enhances SNF, positively correlating to the increase in soybean yield and seed protein content.
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46. |
Nguyen HP,
Miwa H,
Kaneko T,
Sato S,
Okazaki S,
( 2017 ) Identification of Bradyrhizobium elkanii Genes Involved in Incompatibility with Vigna radiata. PMID : 29292795 : DOI : 10.3390/genes8120374 PMC : PMC5748692 Abstract >>
The establishment of a root nodule symbiosis between a leguminous plant and a rhizobium requires complex molecular interactions between the two partners. Compatible interactions lead to the formation of nitrogen-fixing nodules, however, some legumes exhibit incompatibility with specific rhizobial strains and restrict nodulation by the strains. Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean cultivars carrying the Rj4 allele. Here, we explored genetic loci in USDA61 that determine incompatibility with V. radiata KPS1. We identified five novel B. elkanii genes that contribute to this incompatibility. Four of these genes also control incompatibility with soybean cultivars carrying the Rj4 allele, suggesting that a common mechanism underlies nodulation restriction in both legumes. The fifth gene encodes a hypothetical protein that contains a tts box in its promoter region. The tts box is conserved in genes encoding the type III secretion system (T3SS), which is known for its delivery of virulence effectors by pathogenic bacteria. These findings revealed both common and unique genes that are involved in the incompatibility of B. elkanii with mung bean and soybean. Of particular interest is the novel T3SS-related gene, which causes incompatibility specifically with mung bean cv. KPS1.
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47. |
Chibeba AM,
Kyei-Boahen S,
Guimarães MF,
Nogueira MA,
Hungria M,
( 2017 ) Isolation, characterization and selection of indigenous Bradyrhizobium strains with outstanding symbiotic performance to increase soybean yields in Mozambique. PMID : 28775390 : DOI : 10.1016/j.agee.2017.06.017 PMC : PMC5521954 Abstract >>
Soybean inoculation with effective rhizobial strains makes unnecessary the use of N-fertilizers in the tropics. A frequently reported problem is the failure of the inoculant strains to overcome the competition imposed by indigenous rhizobial populations. The screening of indigenous rhizobia, already adapted to local conditions, searching for highly effective strains for use as inoculants represents a promising strategy in overcoming inoculation failure. The objective of this study was to isolate and characterize indigenous rhizobia and to identify strains that hold potential to be included in inoculant formulations for soybean production, with both promiscuous and non-promiscuous soybean cultivars, in Mozambican agro-climatic conditions. A total of 105 isolates obtained from nodules of promiscuous soybean grown at 15 sites were screened for N2-fixation effectiveness in the greenhouse along with five commercial strains. Eighty-seven isolates confirmed the ability to form effective nodules on soybean and were used for genetic characterization by rep-PCR (BOX) and sequencing of the 16S rRNA gene, and also for symbiotic effectiveness. BOX-PCR fingerprinting revealed remarkable genetic diversity, with 41 clusters formed, considering a similarity level of 65%. The 16S rRNA analysis assigned the isolates to the genera Bradyrhizobium (75%) and Agrobacterium/Rhizobium (25%). Great variability in symbiotic effectiveness was detected among the indigenous rhizobia from Mozambique, with ten isolates performing better than the commercial strain B. diazoefficiens USDA 110, the best reference strain, and 51 isolates with lower performance than all reference strains. Thirteen of the best isolates from the first greenhouse trial were evaluated, along with the five commercial strains, in two promiscuous (TGx 1963-3F and TGx 1835-10E) and one non-promiscuous (BRS 284) soybean cultivars in a second greenhouse trial. In general the promiscous soybeans responded better to inoculation. The 13 isolates were also characterized for tolerance to acidity and alkalinity (pH 3.5 and 9.0, respectively), salinity (0.1, 0.3 and 0.5 mol L-1 of NaCl) and high temperatures (35, 40 and 45 �XC) in vitro. Five isolates, three (Moz 4, Moz 19 and Moz 22) belonging to the superclade B. elkanii and two (Moz 27 and Moz 61) assigned to the superclade B. japonicum, consistently showed high symbiotic effectiveness, suggesting that the inoculation with indigenous rhizobia adapted to local conditions represents a possible strategy for increasing soybean yields in Mozambique. Phylogenetic position of the five elite isolates was confirmed by the MLSA with four protein-coding housekeeping genes, dnaK, glnII, gyrB and recA.
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48. |
( 2013 ) Phylogenetic evidence of the transfer of nodZ and nolL genes from Bradyrhizobium to other rhizobia. PMID : 23499616 : DOI : 10.1016/j.ympev.2013.03.003 Abstract >>
Nod factor modifications mediated by nodZ and nolL gene products (fucosylation and acetylation of fucose residues, respectively) were probably later acquisitions in the nodulation process. Novel phylogenetic analyses suggest that nodZ and nolL genes were transferred from Bradyrhizobium to other nodule bacteria. These bradyrhizobial genes are highly diverse while rhizobial, sinorhizobial and mesorhizobial nodZ and nolL genes are represented by few branches among those from bradyrhizobia. These genes in novel rhizobial backgrounds may have favored efficient nodulation in legume hosts commonly associated with Bradyrhizobium strains.
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49. |
Delamuta JRM,
Menna P,
Ribeiro RA,
Hungria M,
( 2017 ) Phylogenies of symbiotic genes of Bradyrhizobium symbionts of legumes of economic and environmental importance in Brazil support the definition of the new symbiovars pachyrhizi and sojae. PMID : 28647304 : DOI : 10.1016/j.syapm.2017.04.005 Abstract >>
Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil (Arachis hypogaea, Acacia auriculiformis, Glycine max, Lespedeza striata, Lupinus albus, Stylosanthes sp. and Vigna unguiculata) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades-B. japonicum and B. elkanii-with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII-recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi, the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium.
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50. |
( 2013 ) Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean). PMID : 23280323 : DOI : 10.1016/j.syapm.2012.10.006 Abstract >>
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.
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51. |
( 2012 ) Robust markers reflecting phylogeny and taxonomy of rhizobia. PMID : 23028691 : DOI : 10.1371/journal.pone.0044936 PMC : PMC3444505 Abstract >>
Genomic ANI (Average Nucleotide Identity) has been found to be able to replace DNA-DNA hybridization in prokaryote taxonomy. The ANI of each of the core genes that has a phylogeny congruent with the reference species tree of rhizobia was compared to the genomic ANI. This allowed us to identify three housekeeping genes (SMc00019-truA-thrA) whose ANI reflected the intraspecies and interspecies genomic ANI among rhizobial strains, revealing an ANI gap (?2%) between the inter- and intra-species comparisons. The intraspecies (96%) and interspecies (94%) ANI boundaries calculated from three genes (SMc00019-truA-thrA) provided a criterion for bacterial species definition and confirmed 621/629 of known interspecies relationships within Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium. Some widely studied strains should be renamed. The SMc00019-truA-thrA ANI also correlates well with the genomic ANI of strains in Agrobacterium, Methylobacterium, Ralstonia, Rhodopseudomonas, Cupriavidus and Burkholderia, suggesting their wide applicability in other bacteria.
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