RAPD and rep PCR‐based fingerprinting of vascular bacterial pathogens of <i>Musa</i> spp.

Thwaites, Richard; Mansfield, J.; Eden-Green, S. J.; Seal, Susan

doi:10.1046/j.1365-3059.1999.00321.x

Cited by 27 publications

(18 citation statements)

References 23 publications

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“…R. solanacearum is no exception; many studies revisited its diversity at the genus level (8,9,40), the species complex level (14,26,41), or the ecological level (10,37,39). Hence, as a high-throughput alternative to the full sequencing of entire bacterial genomes, the pangenomic comparative genomic hybridization (CGH) microarray developed in this study was designed for fine investigation of the phylogenetic diversity of R. solanacearum.…”

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Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

Cellier

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Chiroleu

et al. 2012

Appl Environ Microbiol

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The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.

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Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

Cellier

Remenant

Chiroleu

et al. 2012

Appl Environ Microbiol

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“…Whilst this method can be very useful for typing of some xanthomonads, it does not provide the necessary resolution or reproducibility to be useful for all species. More recently, a major and comprehensive analysis of the genus was completed (Rademaker et al, 2005) that compared amplicon profiles produced by using PCR primers designed to repeated sequences dispersed in bacterial genomes (Martin et al, 1992;Louws et al, 1994;Thwaites et al, 1999). This study utilized all three commonly used repetitive PCR methods (REP, BOX and ERIC) to produce a high-resolution identification scheme, and encompassed many of the pathovars of X. campestris that could not be resolved in the earlier study by Vauterin…”

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“…The conserved primers were designed from accrued gyrB alignments from this study and can be used for gyrB amplification from all species. The PCR cycling conditions were 2.5 min at 94 uC, then 30 s at 94 uC, 45 s at 50 uC and 1 min at 68 uC for 34 cycles, then 7 min at 68 uC to end.Purity and yield of PCR product for sequencing were checked by running 5 ml reaction mixture on a 1.5 % agarose gel and poststaining using ethidium bromide, according to standard protocols (Thwaites et al, 1999). The remaining mixture was purified by using a commercial kit to remove residual primers and other reaction components (Wizard PCR Clean-up kit; Promega).…”

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Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences

Parkinson¹,

Aritua

Heeney³

et al. 2007

International Journal of Systematic and Evolutionary Microbiology

141

102

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The genus Xanthomonas currently comprises 27 species with validly published names that are important crop and horticultural pathogens. We have constructed a phylogram from alignment of gyrase B (gyrB) sequences for all xanthomonad species, both to indicate inter-species relatedness and as an aid for rapid and accurate species-level identification. The phylogeny indicated a monophyletic group, with X. albilineans and X. sacchari as the most ancestral species. Three species, X. hyacinthi, X. translucens and X. theicola, formed an early-branching group. Three clades were supported by high bootstrap values: group 1 comprised X. cucurbitae, X. cassavae and X. codiaei; group 2 comprised X. arboricola, X. campestris, X. populi, X. hortorum, X. gardneri and X. cynarae; group 3 contained the remaining species, within which two further clades, supported by a 100 % bootstrap value, were identified. Group 3A comprised X. axonopodis, X. euvesicatoria, X. perforans and X. melonis, together with X. alfalfae, X. citri and X. fuscans, whose names were recently validly published. Group 3B contained the monocot pathogens X. vasicola and X. oryzae. Two recently identified species, X. cynarae and X. gardneri, were poorly discriminated and were related closely to X. hortorum. Three species, X. perforans, X. euvesicatoria and X. alfalfae, had identical gyrB sequences. Partial sequencing of a further five genes from these species found only minor sequence differences that confirmed their close relatedness. Although branch lengths between species varied, indicating different degrees of genetic distinctiveness, the majority (n521) were well-differentiated, indicating the utility of the method as an identification tool, and we now use this method for routine diagnosis of xanthomonad species. INTRODUCTIONClassification of species within the genus Xanthomonas, which was hitherto based on biochemical characteristics (Van den Mooter & Swings, 1990), underwent major revision after a comprehensive spectrophotometric DNA-DNA hybridization study of the genus (Vauterin et al., 1995), which resulted in the recognition of 20 constituent species. Following this study, names of seven further species have been validly published: X. cynarae (Trébaol et al., 2000), X. euvesicatoria, X. perforans, X. gardneri (Jones et al., 2004; Euzéby, 2006), X. citri, X. fuscans and X. alfalfae (Gabriel et al., 1989; Euzéby, 2007;Schaad et al., 2005Schaad et al., , 2006Schaad et al., , 2007.The complexity of the genus and resource requirements for comprehensive pairwise analyses required for DNA-DNA hybridization analysis make this technique unsuitable for routine identification in most diagnostic laboratories. A rapid characterization method based on analysing cell-wall fatty acids using GC was developed and applied to the identification of plant pathogens (Stead, 1989(Stead, , 1992. Whilst this method can be very useful for typing of some xanthomonads, it does not provide the necessary resolution or reproducibility to be useful for all species. More recently, a majo...

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“…Nevertheless, in such a case, a RAPD fingerprint is produced from the bacterial DNA, but this pattern is usually nonreproducible, since the selected genomic fragments to be amplified may change from one experiment to another, resulting in different RAPD fingerprints (29). Therefore, by using a set of eight different bacteria (four X. cynarae, two xanthomonad-like bacteria isolated from artichoke but nonpathogenic to this plant, and two other Xanthomonas species), we selected 40 RAPD primers (out of 340 tested) for their ability to produce reproducible fingerprints as in other studies (7,18,21,31,32). Moreover, we took into account only the intensive bands that are highly reproducible (6,9,13,21) when analyzing RAPD fingerprints.…”

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confidence: 99%

Assessment of the Genetic Diversity among Strains of Xanthomonas cynarae by Randomly Amplified Polymorphic DNA Analysis and Development of Specific Characterized Amplified Regions for the Rapid Identification of X. cynarae

Trébaol¹,

Manceau

Tirilly³

et al. 2001

Appl Environ Microbiol

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The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.Water-soaked and dark green spots on capitulum bracts of artichoke (Cynara scolymus L.) were observed for the first time in 1954 in Brittany and near Angers (France). Warm and humid periods are favorable for development of this disease, which has been observed in numerous artichoke crops in Brittany and has resulted in substantial economic losses for the last decade. The causal agent of this disease was first isolated by Ridé (25) from such bract spots and was identified as a phytopathogenic bacterium belonging to the genus Xanthomonas (25). It was recently classified at the species level as Xanthomonas cynarae (33). An identification test of X. cynarae was described based on biochemical, physiological, and pathogenicity tests (33). Colonies of X. cynarae are yellow and surrounded with a white halo when grown on a Tween medium (17) and produce the typical symptoms of this disease when inoculated to detached scarified bracts of artichoke. A pathogenicity test is required, since other Xanthomonas species look like X. cynarae when grown on Tween medium. This identification procedure, including pathogenicity tests, was used to monitor bacterial populations in artichoke fields and showed that X. cynarae is present on the leaf surface of artichoke before the capitulum development.A rapid and specific identification test would be very useful to monitor the contamination of artichoke plants in order to develop strategies to control the disease in fields. Since the diagnostic test described above is time consuming and requires, for the pathogenicity t...

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RAPD and rep PCR‐based fingerprinting of vascular bacterial pathogens of Musa spp.

Cited by 27 publications

References 23 publications

Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

Phylogenetic analysis of Xanthomonas species by comparison of partial gyrase B gene sequences

Assessment of the Genetic Diversity among Strains of Xanthomonas cynarae by Randomly Amplified Polymorphic DNA Analysis and Development of Specific Characterized Amplified Regions for the Rapid Identification of X. cynarae

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