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1.
BMC Microbiol ; 21(1): 118, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33874906

RESUMO

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex is an important soil-borne disease worldwide that affects more than 450 plant species, including peanut, leading to great yield and quality losses. However, there are no effective measures to control bacterial wilt. The reason is the lack of research on the pathogenic mechanism of bacterial wilt. RESULTS: Here, we report the complete genome of a toxic Ralstonia solanacearum species complex strain, Rs-P.362200, a peanut pathogen, with a total genome size of 5.86 Mb, encoding 5056 genes and the average G + C content of 67%. Among the coding genes, 75 type III effector proteins and 12 pseudogenes were predicted. Phylogenetic analysis of 41 strains including Rs-P.362200 shows that genetic distance mainly depended on geographic origins then phylotypes and host species, which associated with the complexity of the strain. The distribution and numbers of effectors and other virulence factors changed among different strains. Comparative genomic analysis showed that 29 families of 113 genes were unique to this strain compared with the other four pathogenic strains. Through the analysis of specific genes, two homologous genes (gene ID: 2_657 and 3_83), encoding virulence protein (such as RipP1) may be associated with the host range of the Rs-P.362200 strain. It was found that the bacteria contained 30 pathogenicity islands and 6 prophages containing 378 genes, 7 effectors and 363 genes, 8 effectors, respectively, which may be related to the mechanism of horizontal gene transfer and pathogenicity evaluation. Although the hosts of HA4-1 and Rs-P.362200 strains are the same, they have specific genes to their own genomes. The number of genomic islands and prophages in HA4-1 genome is more than that in Rs-P.36220, indicating a rapid change of the bacterial wilt pathogens. CONCLUSION: The complete genome sequence analysis of peanut bacterial wilt pathogen enhanced the information of R. solanacearum genome. This research lays a theoretical foundation for future research on the interaction between Ralstonia solanacearum and peanut.


Assuntos
Genoma Bacteriano/genética , Ralstonia solanacearum/genética , Arachis/microbiologia , Composição de Bases/genética , Ilhas Genômicas/genética , Filogenia , Ralstonia solanacearum/química , Ralstonia solanacearum/classificação
2.
Phytopathology ; 109(11): 1922-1931, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31272278

RESUMO

Bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is a serious threat to potato production in Uganda. However, little is known about the extent of the disease and the type of the pathogen strains involved. A nationwide survey was conducted to study BW prevalence and incidence in potato, and potato tuber and stem samples of potential alternative hosts were collected for pathogen isolation. DNA was extracted from pure cultures for genetic diversity studies. The pathogen was phylotyped by multiplex PCR; then, a subset of isolates was typed at sequevar level. Isolates of the same sequevar were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. BW prevalence and incidence in potato farms were 81.4 and 1.7%, respectively. Three RSSC phylotypes were identified, with the majority of the strains belonging to Phylotype II (80%) followed by Phylotype I (18.5%) and III (1.5%). Phylotype I strains belonged to Sequevar 31, and Phylotype II strains belonged to Sequevar 1. Potato-associated Phylotype II Sequevar 1 strains were more diverse (27 TRST haplotypes) than nonpotato Phylotype I (5 TRST haplotypes). Mapping of TRST haplotypes revealed that three TRST haplotypes of Phylotype II Sequevar 1 strains play an important epidemiological role in BW of potato in Uganda being disseminated via latently infected seed.[Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Epidemiologia Molecular , Ralstonia solanacearum , Solanum tuberosum , Tipagem Molecular , Filogenia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Solanum tuberosum/microbiologia , Uganda
3.
Plant Dis ; 102(11): 2258-2267, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30192708

RESUMO

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.


Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Variação Genética , Doenças das Plantas/microbiologia , Ralstonia solanacearum/classificação , Rosa/microbiologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Países Baixos , Filogenia , Ralstonia solanacearum/genética , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/patogenicidade , Solanum melongena/microbiologia
4.
Genes Genomics ; 40(6): 657-668, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29892946

RESUMO

Ralstonia solanacearum is a soil-borne phytopathogen associated with bacterial wilt disease of sesame. R. solanacearum is the predominant agent causing damping-off from tropical to temperate regions. Because bacterial wilt has decreased the sesame industry yield, we sequenced the SEPPX05 genome using PacBio and Illumina HiSeq 2500 systems and revealed that R. solanacearum strain SEPPX05 carries a bipartite genome consisting of a 3,930,849 bp chromosome and a 2,066,085 bp megaplasmid with 66.84% G+C content that harbors 5,427 coding sequences. Based on the whole genome, phylogenetic analysis showed that strain SEPPX05 is grouped with two phylotype I strains (EP1 and GMI1000). Pan-genomic analysis shows that R. solanacearum is a complex species with high biological diversity and was able to colonize various environments during evolution. Despite deletions, insertions, and inversions, most genes of strain SEPPX05 have relatively high levels of synteny compared with strain GMI1000. We identified 104 genes involved in virulence-related factors in the SEPPX05 genome and eight absent genes encoding T3Es of GMI1000. Comparing SEPPX05 with other species, we found highly conserved secretion systems central to modulating interactions of host bacteria. These data may provide important clues for understanding underlying pathogenic mechanisms of R. solanacearum and help in the control of sesame bacterial wilt.


Assuntos
Ralstonia solanacearum/genética , Proteínas de Bactérias/genética , Composição de Bases/genética , Sequência de Bases/genética , Genoma Bacteriano/genética , Genômica/métodos , Filogenia , Doenças das Plantas/microbiologia , Ralstonia/genética , Ralstonia solanacearum/classificação , Análise de Sequência de DNA/métodos , Sesamum/microbiologia , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodos
5.
Lett Appl Microbiol ; 66(5): 384-393, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29446102

RESUMO

Bacterial wilt of Curcuma alismatifolia (Patumma) caused by Ralstonia solanacearum is a major disease affecting the quality of rhizome exports. Traditionally, R. solanacearum is classified into five races based on differences in host range and six biovars based on biochemical properties. Recently a classification scheme based on phylotypes and sequevars was presented by the scientific community as a tool for determining phylogenetic relationships within R. solanacearum. This study used traditional and molecular methods to identify R. solanacearum strains from Patumma. All the strains were identified as biovar 4. A phylotype-specific multiplex PCR-based phylotyping of all the isolates detected the phylotype I-specific amplicon of 144 bp and the R. solanacearum-specific 281 bp amplicon. Phylogenetic analyses of endoglucanase (egl) sequences clustered all three strains of Patumma into phylotype I, sequevar 48 with reference strains M2 and M6. The study determined that the R. solanacearum strains from Patumma belong to biovar 4, phylotype I that originated from Asia, and sequevar 48. SIGNIFICANCE AND IMPACT OF THE STUDY: Phylotype and sequevar of Ralstonia solanacearum were associated with geographic region and geographic distribution. This is the first study to identify phylotype and sequevar of R. solanacearum from Patumma in Chiang Mai, Thailand. This will be useful for study of disease epidemiology and could help management for control of bacterial wilt diseases in this host.


Assuntos
Curcuma/microbiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Proteínas de Bactérias/genética , Celulase/genética , Proteínas de Ligação a DNA/genética , Tipagem Molecular/métodos , Filogenia , Ralstonia solanacearum/isolamento & purificação , Tailândia , Fatores de Transcrição/genética
6.
Braz. j. microbiol ; 48(2): 193-195, April.-June 2017. tab
Artigo em Inglês | LILACS | ID: biblio-839391

RESUMO

Abstract Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.


Assuntos
Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , DNA Bacteriano/química , Genoma Bacteriano , Análise de Sequência de DNA , Ralstonia solanacearum/genética , Genótipo , DNA Bacteriano/genética , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/classificação , Índia
7.
Nucleic Acids Res ; 45(W1): W265-W269, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28482090

RESUMO

Peptidome similarity analysis enables researchers to gain insights into differential peptide profiles, providing a robust tool to discriminate strain-specific peptides, true intra-species differences among biological replicates or even microorganism-phenotype variations. However, no in silico peptide fingerprinting software existed to facilitate such phylogeny inference. Hence, we developed the Peptidomes for Phylogenies (P4P) web tool, which enables the survey of similarities between microbial proteomes and simplifies the process of obtaining new biological insights into their phylogeny. P4P can be used to analyze different peptide datasets, i.e. bacteria, viruses, eukaryotic species or even metaproteomes. Also, it is able to work with whole proteome datasets and experimental mass-to-charge lists originated from mass spectrometers. The ultimate aim is to generate a valid and manageable list of peptides that have phylogenetic signal and are potentially sample-specific. Sample-to-sample comparison is based on a consensus peak set matrix, which can be further submitted to phylogenetic analysis. P4P holds great potential for improving phylogenetic analyses in challenging taxonomic groups, biomarker identification or epidemiologic studies. Notably, P4P can be of interest for applications handling large proteomic datasets, which it is able to reduce to small matrices while maintaining high phylogenetic resolution. The web server is available at http://sing-group.org/p4p.


Assuntos
Bactérias/classificação , Mapeamento de Peptídeos , Filogenia , Proteômica , Software , Bacillus cereus/classificação , Bacillus cereus/genética , Bactérias/genética , Bifidobacterium animalis/classificação , Bifidobacterium animalis/genética , Internet , Peptídeos/análise , Peptídeos/química , Proteoma , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética
8.
Braz J Microbiol ; 48(2): 193-195, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28041840

RESUMO

Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen.


Assuntos
DNA Bacteriano/química , Genoma Bacteriano , Genótipo , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Análise de Sequência de DNA , Solanum tuberosum/microbiologia , DNA Bacteriano/genética , Índia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/isolamento & purificação
9.
Appl Environ Microbiol ; 83(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28003195

RESUMO

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations.IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies.


Assuntos
Evolução Molecular , Genótipo , Repetições Minissatélites/genética , Filogenia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Adaptação Biológica/genética , DNA Bacteriano , Monitoramento Epidemiológico , Marcadores Genéticos , Variação Genética/genética , Epidemiologia Molecular , Tipagem Molecular/métodos , Família Multigênica , Doenças das Plantas/microbiologia , Caules de Planta/microbiologia , Polimorfismo Genético , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/patogenicidade , Rizosfera , Análise de Sequência de DNA , Microbiologia do Solo , Especificidade da Espécie , Virulência
10.
Curr Microbiol ; 73(4): 542-9, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27402488

RESUMO

Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Solanum tuberosum/microbiologia , Técnicas de Tipagem Bacteriana/instrumentação , Primers do DNA/genética , Ralstonia solanacearum/classificação , Reação em Cadeia da Polimerase em Tempo Real/instrumentação
11.
BMC Genomics ; 17: 90, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26830494

RESUMO

BACKGROUND: The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. RESULTS: We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species. CONCLUSIONS: Together, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species.


Assuntos
Genoma Bacteriano , Genômica , Proteômica , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Variação Genética , Genômica/métodos , Filogenia , Doenças das Plantas/microbiologia , Proteômica/métodos , Ralstonia solanacearum/classificação
12.
J Microbiol Methods ; 123: 101-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26876453

RESUMO

The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase (egl) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need of bacterial isolation. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our collection with no prior sequevar information, as well as 4 strains in infected plant samples, using their partial egl sequences amplified and sequenced with primers designed in this study. The Ralstonia solanacearum typing program does not require expertise or specific knowledge to use, gives results in seconds, and provides data interpretation for the user. The program and primers can help expert or non-expert users to quickly type an unknown R. solanacearum species-complex strain and determine whether it is a highly regulated R. solanacearum strain. The program can also serve as a confirmation method, since it is the only method that can easily and directly determine whether the strain in question is a sequevar 1 or 2 strain of R. solanacearum.


Assuntos
Proteínas de Bactérias/genética , Celulase/genética , Ralstonia solanacearum/enzimologia , Análise de Sequência de DNA/métodos , Proteínas de Bactérias/química , Sequência de Bases , Celulase/química , Genótipo , Filogenia , Ralstonia solanacearum/química , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Alinhamento de Sequência , Análise de Sequência de DNA/instrumentação , Software
13.
J Biol Chem ; 291(13): 6813-30, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26823466

RESUMO

The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits γ-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ∼30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other γ-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ralstonia solanacearum/genética , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/genética , gama-Glutamilciclotransferase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes , Glutationa/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Filogenia , Plantas/microbiologia , Estrutura Terciária de Proteína , Ralstonia solanacearum/classificação , Ralstonia solanacearum/enzimologia , Ralstonia solanacearum/patogenicidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , gama-Glutamilciclotransferase/química , gama-Glutamilciclotransferase/metabolismo
14.
Microb Pathog ; 90: 84-92, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26606869

RESUMO

Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Doenças das Plantas/microbiologia , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/patogenicidade , Técnicas de Cultura de Células , Ralstonia solanacearum/classificação , Ralstonia solanacearum/citologia , Virulência
15.
Antonie Van Leeuwenhoek ; 109(1): 71-82, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26494208

RESUMO

The genus Ralstonia contains species that are devastating plant pathogens, opportunistic human pathogens, and/or important degraders of xenobiotic and recalcitrant compounds. However, significant nomenclature problems exist, especially for the Ralstonia solanacearum species complex which consists of four phylotypes. Phylogenomics of the Ralstonia genus was investigated via a comprehensive analysis of 39 Ralstonia genomes as well as four genomes of Cupriavidus necator (more commonly known by its previous name Ralstonia eutropha). These data revealed 686 single-copy orthologs that could be extracted from the Ralstonia core-genome and used to reconstruct the phylogeny of the genus Ralstonia. The generated tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome. Our data confirmed that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of the genus Ralstonia, e.g. strains of Ralstonia solanacearum phylotype IIA and IIB may represent two subspecies of R. solanacearum, and strains of R. solanacearum phylotype I and III may be classified into two subspecies of Ralstonia pseudosolanacearum. Recently, strains of R. solanacearum phylotype IV were proposed to be reclassified into different subspecies of Ralstonia syzygii; our study, however, showed that phylotype IV strains had high isDDH values (83.8-96.1 %), indicating it may be not appropriate to classify these closely related strains into different subspecies. We also evaluated the performance of six chromosomal housekeeping genes (gdhA, mutS, adk, leuS, rplB and gyrB) used in Ralstonia phylogenetic inference. The multilocus sequence analysis of these six marker genes was able to reliably infer the phylogenetic relationships of the genus Ralstonia.


Assuntos
Genoma Bacteriano , Filogenia , Ralstonia/genética , Hibridização Genômica Comparativa , DNA Bacteriano/genética , Dosagem de Genes , Genes Bacterianos , Genes Essenciais , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , Ralstonia/classificação , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Alinhamento de Sequência , Análise de Sequência de DNA
16.
PLoS One ; 10(10): e0139637, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26426354

RESUMO

Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.


Assuntos
Bioensaio , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças das Plantas/genética , Ralstonia solanacearum/genética , Solanum lycopersicum/genética , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/classificação
17.
PLoS One ; 10(3): e0122182, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25811378

RESUMO

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.


Assuntos
Musa/microbiologia , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Biodiversidade , Brasil , Musa/virologia , Filogenia , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Appl Environ Microbiol ; 81(10): 3542-51, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25769835

RESUMO

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.


Assuntos
Doenças das Plantas/microbiologia , Ralstonia solanacearum/fisiologia , Solanum lycopersicum/microbiologia , Adaptação Fisiológica , Ralstonia solanacearum/classificação , Ralstonia solanacearum/genética , Ralstonia solanacearum/isolamento & purificação , Temperatura , Clima Tropical
19.
Int J Syst Evol Microbiol ; 64(Pt 9): 3087-3103, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24944341

RESUMO

The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T) = LMG 27706(T) = DSM 27477(T)) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (type strain UQRS 461(T) = LMG 9673(T) = NCPPB 1029(T)) for the strains of R. solanacearum phylotypes I and III.


Assuntos
Filogenia , Ralstonia solanacearum/classificação , Ralstonia/classificação , Composição de Bases , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Ácidos Graxos/química , Indonésia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Doenças das Plantas/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA
20.
Phytopathology ; 104(6): 586-96, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24283538

RESUMO

Although bacterial wilt remains a major plant disease throughout South America and the Caribbean, the diversity of prevalent Ralstonia solanacearum populations is largely unknown. The genetic and phenotypic diversity of R. solanacearum strains in French Guiana was assessed using diagnostic polymerase chain reactions and sequence-based (egl and mutS) genotyping on a 239-strain collection sampled on the families Solanaceae and Cucurbitaceae, revealing an unexpectedly high diversity. Strains were distributed within phylotypes I (46.9%), IIA (26.8%), and IIB (26.3%), with one new endoglucanase sequence type (egl ST) found within each group. Phylotype IIB strains consisted mostly (97%) of strains with the emerging ecotype (IIB/sequevar 4NPB). Host range of IIB/4NPB strains from French Guiana matched the original emerging reference strain from Martinique. They were virulent on cucumber; virulent and highly aggressive on tomato, including the resistant reference Hawaii 7996; and only controlled by eggplant SM6 and Surya accessions. The emerging ecotype IIB/4NPB is fully established in French Guiana in both cultivated fields and uncultivated forest, rendering the hypothesis of introduction via ornamental or banana cuttings unlikely. Thus, this ecotype may have originated from the Amazonian region and spread throughout the Caribbean region.


Assuntos
Cucurbitaceae/microbiologia , Variação Genética , Genoma Bacteriano/genética , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Solanaceae/microbiologia , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Ecótipo , Guiana Francesa , Genótipo , Geografia , Especificidade de Hospedeiro , Tipagem Molecular , Fenótipo , Filogenia , Reação em Cadeia da Polimerase , Ralstonia solanacearum/classificação , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/patogenicidade , Análise de Sequência de DNA , Virulência
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