Le système de sécrétion de type III est un des déterminants majeurs de la pathogénicité de Ralstonia pseudosolanacearum qui lui permet d’injecter des effecteurs de type III (les « Rip », « Ralstonia Injected Protein ») directement dans les cellules des plantes hôtes. Les effecteurs RipH1, RipH2 et RipH3 sont des effecteurs de type III conservés dans la plupart des souches séquencées. Au cours de ma thèse, le rôle de ces effecteurs RipH lors de l’infection de différentes plantes a été étudié en prenant comme point d’entrée les protéines de tomates avec lesquelles ces effecteurs interagissent. Un criblage par double hybride dans la levure a permis d’identifier 19 de ces protéines « cibles » de tomate. Des méthodes de génétique inverse ont ensuite été utilisées pour chercher le rôle des orthologues de ces protéines dans différentes plantes modèles lors de l’infection par Ralstonia pseudosolanacearum. Du VIGS chez Nicotiana benthamiana a permis de mettre en évidence l’implication des orthologues de la protéine TOM3 : la multiplication bactérienne est moins importante dans les feuilles lorsque l’expression de ces gènes est diminuée. Dans Arabidopsis thaliana, des mutants d’un gène orthologue de la cible TOM9, décrit comme jouant entre autres un rôle dans la remodélisation de la chromatine, est plus résistant à l’infection par R. pseudosolanacearum. Dans un deuxième chapitre correspondant à un article publié, le rôle de l’effecteur RipAX2 a été étudié dans la résistance des aubergines AG91-25. La présence de cet effecteur dans la souche GMI1000 est nécessaire à l’établissement de la résistance de cette variété dans laquelle le locus de résistance EBWR9 a été mis en évidence. L’ajout de RipAX2 dans la souche PSS4, une souche pathogène de AG91-25 qui ne possède pas cet effecteur naturellement, la rend non pathogène. De plus, le motif protéique putatif « zincbinding » qui est décrit comme nécessaire pour l’induction de réponses de défense chez l’espèce proche de l’aubergine Solanum torvum n’est pas nécessaire pour la résistance de AG91-25. Enfin, la conservation de l’effecteur RipAX2 dans les différentes souches du complexe d’espèces de Ralstonia solanacearum a été étudiée pour évaluer l’efficacité potentielle de cette source de résistance contre différentes souches. / One of the major virulence determinants of plant pathogenic Ralstonia species is the type III secretion system that enables it to inject proteins (also called “Ralstonia Injected Proteins” or Rip) into the host cells. The RipH1,2,3 type III effectors are conserved in different strains of the Ralstonia solanacearum species complex. The role of these effectors during infection has been studied, taking as an entry point the tomato proteins they interact with. Using yeast-two-hybrid screenings we have identified 19 tomato targets of these three RipH. Reverse genetics methods have then been used to study the role of orthologous genes of these targets in other model plants. Virus induced gene silencing in Nicotiana benthamiana showed that the orthologous genes of TOM3 were involved in plant response to Ralstonia pseudosolanacearum, as the bacterial multiplication was diminished in plants silenced for these genes. In Arabidopsis thaliana, mutants of the TOM9 orthologous gene which is described as involved in chromatin remodelisation were more tolerant to infection. In a second chapter corresponding to a published article, the role of RipAX2 has been studied. This effector triggers specific resistance in AG9125 eggplant which carry the major resistance locus EBWR9. This eggplant accession AG9125 is resistant to the wild type R. pseudosolanacearum strain GMI1000, while a ripAX2 defective mutant of this strain can cause wilt. The addition of ripAX2 from GMI1000 to the naturally pathogenic strain PSS4 suppresses its pathogenicity, demonstrating that RipAX2 causes AG9125 resistance. Moreover, a zinc binding motif described as necessary to induce defenses on the eggplant wild relative Solanum torvum upon RipAX2 recognition is not necessary for AG91-25 resistance. The conservation of RipAX2 has been studied in the different strains of the bacteria in order to determine the potential of this resistance source against various strains for breeding

Made available in DSpace on 2015-03-26T13:42:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 559821 bytes, checksum: 5797c4fb6631839d7cef0e75351e4e02 (MD5) Previous issue date: 2011-10-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bacterial wilt caused by Ralstonia solanacearum is currently one of the most important bacterial diseases in eucalyptus. The use of healthy eucalyptus cuttings is the most effective method to prevent the introduction and spread of the pathogen in disease free areas. Infected plants can be identified visually bybacterial cell exudation from the host tissue and confirmed by analysis of PCR. However, in asymptomatic minicuttings with latent infections, in which the xylem shows low concentrations of bacterial cellsit is not possible to visualize wilting symptoms bacterial exudation. Thus, for certification thateucalyptus cuttings are pathogen free, it is necessary to employ a sensitive method able to detect the bacteria, even when present in low concentrations in the host tissue.This study aimed to evaluate the real-time PCR for detection of bacteria in the latent period of infection. Among six primers previously tested, only the oligo224R/224F was specific to R. solanancearum, that amplifies the r16S ribosomal gene region. Subsequently,it was developed a protocol to extract bacterial DNA from infected plant tissue consisting of tissue maceration in liquid nitrogen, addition of saline solution, centrifugation, discard of the supernatant and DNA extraction. Real-time PCR of fourinfected eucalyptus cuttings with R. solanacearum(UFV32)evaluated at 20 days after inoculationshowed 105 to 106 colony forming units (cfu) of R. solanacearum / g of host tissue. The detection limit of real-time PCR was 103cfu / g, copared with 107cfu / mL for the conventional PCR. The method proved to be efficient for detection and quantification of R. solanacearum directly from infected eucalyptus plant tissue. / A murcha bacteriana causada por Ralstonia solanacearum é, atualmente uma das mais importantes doenças bacterianas na eucaliptocultura. O uso de mudas clonais sadias é ométodo mais efetivo para evitar a introdução e disseminação do patógeno em áreas livres da doença. Plantas infectadas podem ser identificadas visualmente ou pela exsudação de pus, porém, em minicepas assintomáticas, mas com infecções latentes, nas quais o xilema apresenta baixas concentrações de células bacterianas, não é possível a visualização de sintomas de murcha ou sinais da doença como a exsudação bacteriana. Assim, para certificação de que as mudas para o plantio estão livres do patógeno, é necessário empregar um método capaz de detectar a bactéria, mesmo quando presente em baixas concentrações.Assim, o presente trabalho objetivou avaliar a PCR em tempo real para detecção da bactéria no período latente de infecção. Primeiramente, dentre seis oligonucleotídeos testados, o oligo 224R/224F, que amplifica a região do gene ribossomal 16S, foi o único específico para R. solanacearum. Subsequentemente desenvolveu-se um protocolo de extração de DNA bacteriano a partir do tecido vegetal infectado que consiste da maceração com nitrogênio líquido, adição de solução salina, centrifugação, descarte do sobrenadante e extração com o kit (Promega) de extração. Para avaliar o método da PCR em tempo real, mudas de eucalipto de quatro clones foram inoculadas com o isolado UFV32 de R. solanacearume o DNA do patógeno foi quantificado aos 20 dias da inoculação. Aproximadamente 105 a 106 unidades formadoras de colônia (ufc)/g de R. solanacearum foram detectadas.. O limite de detecção da PCR em tempo real foi de 103ufc/g contra 107ufc/mL da PCR convencional, ou seja 10.000 vezes maior.. O método mostrou-se eficiente na detecção e quantificação de R. solanacearum diretamente do tecido vegetal de eucalipto.

Made available in DSpace on 2015-03-26T13:37:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1080649 bytes, checksum: 0b734c22ca6fdd3994db4802005b6004 (MD5) Previous issue date: 2012-07-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The Ralstonia wilt of eucalyptus, caused by Ralstonia solanacearum, is potentially one of the major disease of culture. Because of its high genetic and phenotypic variability, currently R. solanacearum is considered a "species complex", subdivided into four taxonomic levels: species, filotipo, sequevar and clone. In order to understand the variability among isolates of R. solanacearum from eucalyptus and develop a protocol for inoculation and screening of resistant clones of eucalyptus to Ralstonia wilt this work was performed. The work was divided into two articles: (i) Molecular characterization of isolates of Ralstonia solanacearum in Eucalyptus spp. and (ii) Method of inoculation and evaluation of resistant clones of Eucalyptus spp. to Ralstonia wilt caused by Ralstonia solanacearum. In Article 1, 19 isolates were analyzed from different regions of eucalyptus in Brazil based on new classification (filotipo and sequevar) and its genetic variability. A product of 372 bp generated by multiplex-PCR amplification using primers Nmult permitted the identification of all isolates analyzed as pertaining to filotipo II. Eighteen isolates were grouped in subclade IIA and just one isolate in IIB. The phylogenetic tree generated from the endoglucanase (egl) gene sequences confirmed the classification of the isolates in filotipo II and separated them into sequevares. The strains AMC22, IBSBF2568 and IBSBF2576 were grouped into a single clade, as well as isolates UFV18 and UFV20, with 89% and 78% posterior probability, respectively, forming two new possible sequevares not defined yet. We also identified isolates belonging to sequevar 41 (100% probability) and 37 (88% probability). However, most of the isolates did not fit in any of previously described sequevar, or formed defined clades. The analysis of results of obtained fragments amplified by ERIC-PCR technique suggested a great genetic diversity among the isolates analyzed in this work. In addition, a high correlation between the geographical origin of isolates and the similarity showed by them was observed. In Article 2, three methods of inoculation and resistant of Eucalyptus spp. clones to Ralstonia wilt was evaluated: (i) soil infestation with R. solanacearum, (ii) dipping of sectioned roots in bacterial suspension, and (iii) injection of a bacterial suspension in the base of the stem. The injection method of a bacterial suspension at the stem base was the most efficient inoculation method of R. solanacearum on eucalyptus. Four, out of 21 clones tested for Ralstonia wilt resistance by this method, were classified as resistant by not showing symptoms of wilt and bacterial exudation up to 30 days after inoculation. / A murcha-de-Ralstonia do eucalipto, causada por Ralstonia solanacearum, constitui potencialmente, uma das principais doenças da cultura. Devido à sua ampla variabilidade, atualmente R. solanacearum é considerada um complexo de espécies , subdividida em quatro níveis taxonômicos: espécie, filotipo, sequevar e clone. Com o intuito de entender a variabilidade entre isolados de R. solanacearum provenientes de eucalipto e desenvolver um protocolo de inoculação e avaliação da resistência de clones de eucalipto à murcha-de-Ralstonia realizou-se este trabalho. Ele foi dividido em dois artigos: (i) Caracterização molecular de isolados de Ralstonia solanacearum de Eucalyptus spp. e (ii) Método de inoculação e avaliação de resistência de clones de Eucalyptus spp. à murcha-de-Ralstonia causada por Ralstonia solanacearum. No artigo 1, foram analisados 19 isolados de R. solanacearum obtidos de eucalipto de diferentes regiões do Brasil quanto à nova classificação (filotipo e sequevar) e quanto à variabilidade genética. Um produto de 372 pb gerado pela amplificação por PCR-multiplex, utilizando primers Nmult, permitiu identificar todos os isolados analisados no filotipo II. Dezoito isolados foram agrupados no subclado IIA e apenas um no IIB. A árvore filogenética gerada a partir das sequências do gene da endoglucanase (egl) confirmou a classificação dos isolados no filotipo II e os separou em sequevares. Os isolados AMC22, IBSBF2568 e IBSBF2576 agruparam-se em um único clado, assim como os isolados UFV18 e UFV20, com 89% e 78% de probabilidade posteriori, respectivamente, compondo dois novos possíveis sequevares, ainda não definidos. Foram identificados também isolados pertencentes ao sequevar 41 (100% de probabilidade) e 37 (88% de probabilidade). Entretanto, a maioria dos isolados não se enquadrou em nenhum sequevar descrito, nem formaram clados definidos. O resultado da análise de fragmentos amplificados pela técnica de ERIC-PCR indicou grande diversidade genética entre os isolados avaliados neste trabalho, havendo, em geral, uma alta correlação entre a origem geográfica dos isolados e a similaridade entre eles. No artigo 2, avaliaram-se três métodos de inoculação e a resistência de clones de Eucalyptus spp. à murcha-de-Ralstonia: (i) infestação do solo com R. solanacearum; (ii) imersão de raízes seccionadas em suspensão bacteriana; e (iii) injeção de suspensão bacteriana na base do caule. O método de inoculação de injeção de suspensão bacteriana na base do caule destacou-se como o mais eficiente para inoculações de R. solanacearum em eucalipto. De 21 clones avaliados quanto à resistência à murcha-de-Ralstonia por esse método, apenas quatro foram classificados como resistentes por não apresentarem sintomas de murcha e exsudação bacteriana até 30 dias após a inoculação.

Made available in DSpace on 2015-04-11T13:55:36Z (GMT). No. of bitstreams: 1 Maria do Socorro Monteiro da Silva.pdf: 2303257 bytes, checksum: 2f6b50384f8da9d8bbccbe4568a506b1 (MD5) Previous issue date: 2011-04-06 / Fundação de Amparo à Pesquisa do Estado do Amazonas / The tomato, it belongs to the Solanaceae family, is one of the most consumed vegetables in Amazonas State, and the most marketed fruits come from the southern and southeast states, where weather conditions are more pleasant. Bacterial wilt, caused by the bacterium Ralstonia solanacearum, was established as one of the most important diseases for the tomato crop. Due to soil and climatic requirements, the tomato production is practiced with more intensity in the coldest and the driest regions in Brazil (Southeast, South and Midwest). In the humid tropical regions, the climatic conditions, permanently hot and humid, favors the survival of this bacterium that is the main reason that discourages the production of this crop in this region due to low productivity. With the purpose of indicating the technological innovations that enable the production of this vegetable in large scale, this study aims to evaluate the potential production and trade of two tomato cultivars and management techniques to decrease the incidence of bacterial wilt in protected cultivation systems, in the region of Iranduba - Amazon. The tomato cultivars used were C-38 and Santa Barbara, both developed by Embrapa Eastern Amazon, Belém/PA with a history of tolerance bacterial wilt, they were planted succeeding maize, in two treatments and in other, in successive crops. The data evidences the importance of combining techniques with resistant plant material from crops, because in the management systems that the tomato was planted in succession to maize, mortality was lesser than conventional management system and the productivity (approximately 22,65 t ha-¹) was higher than the average of the Amazonas State (approximately 14,60 t ha-¹). Comparated to the Santa Barbara cultivars, the C-38 cultivar showed higher productivity and characteristics of shape, weight and number of fruit with greater acceptance by the interal market / O tomate, pertencente à família das solanáceas, representa uma das hortaliças mais consumidas no Estado do Amazonas, sendo que a maioria dos frutos comercializados é proveniente dos estados do sul, sudeste e centro-oeste, onde as condições climáticas são mais amenas. A Murcha Bacteriana, causada pela bactéria Ralstonia solanacearum, constitui-se como uma das doenças mais importante para a cultura. Devido às exigências edafoclimáticas do tomateiro, sua produção é praticada com mais intensidade nas regiões mais frias e secas do Brasil. Já nas regiões do trópico úmido, as condições de clima, permanentemente quente e úmido, favorecendo a sobrevivência desta bactéria é o principal motivo que desestimula a produção da cultura nesta região devido a baixa produtividade. Com a finalidade de indicação de inovações tecnológicas que possibilitem a produção desta hortaliça em grande escala, este trabalho objetiva avaliar o potencial produtivo e comercial de duas cultivares de tomate e técnicas de manejo visando diminuir a incidência de murcha bacteriana, em sistema de cultivo protegido, na região de Iranduba AM. Os cultivares utilizados, C-38 e Santa Bárbara, ambos desenvolvidos pela Embrapa Amazônia Oriental, Belém/PA com histórico de tolerância à murcha bacteriana, foram plantados sucedendo o milho, em dois tratamentos e em outro, em cultivos sucessivos. Os dados obtidos evidenciaram a importância da combinação de material vegetal resistente com técnicas de cultivos, pois nos sistemas de manejo em que o tomate foi plantado em sucessão ao milho, apresentou mortalidade inferior ao sistema de manejo convencional, sendo que a produtividade obtida, na média do experimento, de 22,65 t ha-1foisuperior à média do estado do Amazonas(14,60 t ha-1). Comparativamente ao cultivar Santa Bárbara, o cultivar C-38 apresentou maior produtividade e características de formato e número de frutos com maior aceitação pelo mercado interno

A la différence des mammifères, les plantes ne possèdent pas de cellules spécialisées dans la défense face aux pathogènes. Chaque cellule végétale peut déclencher une réponse immunitaire. Pour interagir avec succès avec la plante, les pathogènes doivent alors supprimer ou contourner les défenses de l’hôte. Afin d’y parvenir, les bactéries pathogènes disposent des effecteurs, des protéines qui peuvent être injectées dans la cellule végétale. De nombreux effecteurs sont connus pour cibler les organites lors de l’infection, confirmant l’importance du chloroplaste et de la mitochondrie dans les mécanismes de défense des cellules végétales. Dans ces conditions, il demeure vital pour la plante de garder la main sur l’expression des gènes des organites afin d’assurer une réponse proportionnée au risque encouru sans pénaliser la croissance de façon disproportionnée. A la différence de l’expression des gènes nucléaires, la régulation de l’expression des gènes des organites se fait principalement lors d’étapes très complexes de maturation post-transcriptionnelle. Parmi les protéines impliquées dans ces étapes de maturation, on trouve les protéines pentatricopeptide repeat (PPR). Les protéines PPR sont impliquées dans de nombreuses étapes de maturation des ARN des organites, comme l’édition C vers U ou l’épissage. Elles sont également présentes chez d’autres eucaryotes, mais n’ont jamais été étudiées chez les bactéries. L’hypothèse testée dans le cadre de la thèse est que ces protéines PPR, qu’elles soient d’origine exogène ou endogène, sont impliquées dans des modifications de l’expression des gènes des organites en condition de stress biotique. Afin de tester notre hypothèse, nous nous sommes intéressés à PGN (Pentatricopeptide repeat protein for Germination on NaCl) chez la plante modèle A. thaliana. Caractérisée par Laluk et al. (2011), le mutant KO montre une sensibilité accrue au nécrotrophe Botrytis cinerea, et l’expression du gène codant pour cette protéine est induite après infection. Nous avons mis en évidence des défauts d’édition dans la séquence non codante en amont de nad6 et dans cox2, deux gènes mitochondriaux. Leur édition ne varie cependant pas en condition d’infection par Botrytis cinerea. Dans la même optique, à la suite d’un crible bio-informatique, nous nous sommes intéressés à deux protéines PPR bactériennes que nous avons trouvées chez les phytopathogènes Erwinia amylovora et Ralstonia solanacearum. Probablement obtenues par les bactéries par transfert horizontal de gènes, il s’agit de la première caractérisation de PPR bactériennes à notre connaissance. Ces protéines possèdent des caractéristiques d’effecteurs, c’est—dire des protéines injectées par la bactérie dans la plante durant l’infection. Si nous n’avons pas vu de modification du transcriptome des organites de la plante provoqué par la surexpression de ces protéines PPR exogènes, nous avons cependant mis en évidence une baisse significative du taux d’incidence de la maladie provoquée par E. amylovora en l’absence d’un gène fonctionnel codant pour sa PPR chez la plante hôte Malus domestica « Golden delicious ». Pour la PPR d’E. amylovora comme celle de R. solanacearum, nous avons également trouvés plusieurs interactants en double hybride levure chez A. thaliana, représentant de nombreuses cibles putatives à étudier. Afin de réaliser ces expérimentations et d’obtenir ces résultats, nous avons eu besoins d’outils particuliers. Nous avons donc développé un pipeline spécifique d’analyse de données de séquençage d’ARN ainsi qu’une méthode améliorée de prédiction des zones de fixation des protéines PPR, ouvrant la voie à une caractérisation simplifiée de nombreuses protéines. / Compared to mammals, plants do not have highly specialized cells involved in defense against pathogens. Each plant cell is able to start an immune response. To interact successfully with plants, pathogens have to block or bypass host defenses. To do so, phytopathogenic bacteria can use effectors, which are basically bacterial proteins injected in the plant cell during infection. Several effectors are known to target organelles during infection, supporting the idea that chloroplasts and mitochondria are key players in plant cell defense. As a reason, it remains necessary for the plant to keep organellar gene expression under control in order to ensure a response in proportion to the risk, without penalizing growth. Unlike nuclear gene expression, organellar gene expression regulation goes through highly complex post-transcriptional maturation steps. Among proteins involved in these events, PPR proteins (for pentatricopeptide repeat) are known to be very important. PPR proteins are involved in several RNA maturation steps in organelles, like C to U editing or splicing. They are studied in several eukaryotes, but not in bacteria. During my PhD studies, the hypothesis is exogenous or endogenous PPR proteins are involved in organellar gene expression modifications during biotic stress. To test our hypothesis, we work on PGN (Pentatricopeptide repeat protein for Germination on NaCl) in plant model A. thaliana. Characterized by Laluk et al. (2011), the KO mutant displays an enhanced sensitivity to the necrotrophic pathogen Botrytis cinerea, and PGN gene expression is induced after infection. We find two editing defects for the KO mutant, in nad6 5’ non coding sequence and in cox2 coding sequence. However, editing at these two sites does not vary in wild type plants during Botrytis cinerea infection.Using a bioinformatic screen, we find several bacterial PPR proteins. Two of them are encoded by bacterial plant pathogens: Erwinia amylovora and Ralstonia solanacearum. To our knowledge, these proteins, putatively obtained through horizontal gene transfer, are the first bacterial PPR proteins to be characterized. They also share similarities with bacterial effectors. If overexpression of these bacterial PPR proteins in A. thaliana does not unveil organellar transcriptome modifications, we show a decrease of the incidence rate of the disease caused by E. amylovora in the host plant Malus domestica “Golden delicious” without a functional gene coding for the PPR protein. For both Erwinia and Ralstonia PPR, we find several interactants in A. thaliana using Yeast Two Hybrid, each of them representing a potential target that could be studied. In order to perform these experiments and obtain these results, we needed very specific tools. During the PhD studies, we develop an RNAseq analysis pipeline and an enhanced method to predict PPR binding sites, opening the way to an easier characterization of several PPR proteins.

Cupriavidus metallidurans CH34 est une béta-protéobactérie, résistante aux métaux lourds, isolée des sédiments d'une usine de métallurgie non-ferreuse en Belgique. <p>Le génome de cette bactérie contient un chromosome (3.6 Mb), un mégaplasmide (2.6 Mb) et deux plasmides pMOL28 (171 kb) et pMOL30 (234 kb) déjà connus pour porter des gènes de résistance aux métaux lourds. <p>Nous avons d'abord fait le catalogue des gènes impliqués dans la résistance aux métaux lourds et, ensuite, cherché à mesurer leur expression par deux approches transcriptomiques :RT-PCR et puces à ADN.<p> L'analyse du génome montre au moins 170 gènes relatifs à la résistance aux ions métalliques localisés sur les 4 réplicons, principalement sur les deux plasmides. Ces gènes codent essentiellement pour des systèmes d'efflux tel que les HME-RND (transport chimioosmotique avec flux de protons à contresens), les ATPases de type P ou encore pour le système de résistance aux ions Cu(II). Dans le génome de C. metallidurans, nous avons identifié 13 opérons qui codent pour des systèmes HME-RND, seuls trois, localisés sur les plasmides, sont surexprimés en présence de métaux lourds. Huit gènes codent pour des ATPases de type P, dont deux appartiennent à une classe dont les substrats ne sont pas métalliques. Deux ATPases appartiennent à une famille spécialisée pour l'efflux du Cu(II) et les quatre autres à une autre grande famille impliquée dans l'efflux des ions Cd(II), Pb(II) et Zn(II). Les analyses transcriptomiques montrent la surexpression des deux premières classes d'ATPases P en présence des métaux lourds. La mutagenèse du gène zntA (mégaplasmide), codant pour l'une des ATPases, provoque une diminution de la viabilité en présence de Zn(II), Cd(II) et dans une moindre mesure de Pb(II), Tl(I) et Bi(III). <p>Sur pMOL30, la résistance au cuivre implique un groupe de 19 gènes cop codant pour la résistance au cuivre au niveau du périplasme et du cytoplasme, et vraisemblablement pour une forme de stockage du cuivre essentiel. Ces 19 gènes sont surexprimés en présence de cuivre, mais une quinzaine de gènes proches semblent aussi requis pour une expression optimale de la résistance au cuivre. <p>L'annotation des plasmides a mis en évidence la parenté du plasmide pMOL28 avec le plasmide pHG1 (hydrogénotrophie, fixation du CO2) de C. eutrophus H16 et le plasmide pSym (fixation de l'azote) de C. taiwanensis, et chez pMOL30, la présence de deux îlots génomiques concentrant la plupart des résistances aux métaux lourds. Les puces montrent la surexpression de 83 sur 164 gènes dans pMOL28, et de 143 sur 250 gènes dans pMOL30. Elles montrent aussi que les gènes présents sur les deux plasmides sont davantage surexprimés que ceux localisés sur les deux mégaréplicons. Parmi les gènes surexprimés les plus intéressants du plasmide pMOL30, il faut mentionner des transposases tronquées et des gènes impliqués dans la synthèse des membranes (glycosyltransférases). L'analyse de l'expression des gènes plasmidiens de résistance aux métaux lourds montre la surexpression en présence de plusieurs ions métalliques ajoutés indépendamment et pas seulement par les substrats métalliques de ces opérons, ce qui suggère l'intervention de deux types de régulation dont les gènes correspondants sont aussi localisés sur le chromosome et le mégaplasmide.<p>Ce travail met en évidence la spécialisation de la bactérie dans la réponse à un grand spectre de concentrations de métaux lourds, jusqu'à la limite majeure de la toxicité observée pour les bactéries mésophiles hétérotrophes. Cette spécialisation correspond bien aux biotopes industriels de divers continents dans lesquels on l'a trouvée. <p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Nous avons exploré la diversité génétique du complexe d'espèces Ralstonia solanacearum (ceRs) pour caractériser et comprendre les graves épidémies de flétrissement bactérien qui sévissent à Madagascar. Une large collection de souches (n=1224; 75 sites) est constituée. Les souches sont assignées aux phylotypes I, III, et la grande majorité associée à l'épidémie appartiennent au groupe IIB-1 (‘Brown rot’ des anglo-saxons) signalé pour la première fois à Madagascar. L'approche MLVA (RS2-MLVA9) a révélé leur apparenté aux souches IIB-1 distribuées mondialement, suggérant ainsi une introduction malheureuse à Madagascar. Trois populations clonales se propagent par des tubercules infectées. Le génotypage des souches du phylotype III, avec le schéma hautement résolutif RS3-MLVA16 que nous avons développé, a révélé une forte diversité génétique structurée géographiquement en onze populations clonales bien différenciées. Cette structure reflète un caractère endémique des populations suggérant l'absence de transmission par tubercules. Les souches malgaches sont différentes de celles d'Afrique continentale. Les souches IIB-1 et III présentent deux modèles épidémiologiques différents. Les variétés de pomme de terre cultivées à Madagascar n'ont montré aucune résistance génétique vis-à-vis du panel de souches malgaches. Cependant, dans nos conditions expérimentales les accessions 720118 et 800934 sont résistantes aux souches I-31 non détectées pour l'instant à Madagascar, mais prévalentes dans l'océan Indien. Nous disposons ainsi d'un outil robuste pouvant être appliqué à l'étude du phylotype III, d'une vue globale de la structure des populations et d'épidémiologie du ceRs. / This thesis is exploring genetic diversity, population structure and molecular epidemiology of the Ralstonia solanacearum species complex (Rssc) causing potato bacterial wilt outbreaks in Madagascar. We characterized a large collection of strains (n=1224; 75 sites) collected from potato production areas. Surprisingly, the large outbreaks were associated with IIB-1 strains (Brown rot) while a few were associated with phylotypes I and III. This is the first report of phylotype IIB-1 in Madagascar. The IIB-1 strains were genotyped based on MLVA (RS2-MLVA9). And Malagasy phylotype IIB-1 clustered with worldwide distributed strains. Fine scale genetic investigation suggested three clonal populations that were introduced and spread through latently infected tuber-seeds. Phylotype III strains were genotyped with the highly discriminatory RS3-MLVA16 scheme we developed. Genetic population analyses revealed a high genetic diversity within phylotype III strains that geographically structured into 11 clonal populations. This support the endemic character of the phylotype III population in Madagascar and suggests no transmission with potato tubers. Malagasy strains were distinct from continental African strains. A clear-cut epidemiological profile is shown between IIB-1 and III strains. Genetically, no bacterial wilt resistance properties were shown for the most popular Malagasy potato cultivars, except two cultivars: 720118 and 800934 that showed strong resistance to phylotype I-31 strain that are predominantly distributed in the Indian Ocean. This study offered tool to genotype phylotype III strains and gives an insights into population structure and epidemiology of the Rssc.

Dans les îles du sud-ouest de l'océan Indien (SOOI) (Comores, Maurice, Mayotte, Réunion, Rodrigues et Seychelles), le flétrissement bactérien causé par le complexe d'espèces Ralstonia solanacearum (ceRs) est une phytobactériose considérée comme l'une des plus nuisibles pour les productions vivrières ou d'exportation. Les travaux de thèse présentés dans ce manuscrit avaient pour principal objectif l'exploration du niveau et de la distribution de la diversité génétique du ce Rs et de la structure génétique de ses populations dans le SOOI. Nous avons mené de vastes campagnes d'échantillonnage qui ont permis de constituer une large collection de 1704 isolats, principalement à partir de Solanacées (tomate, pomme de terre, piment, aubergine, poivron) et de géranium rosat. L'assignation phylogénétique des isolats a montré une très forte prévalence du phylotype I (88 %), qui est distribué dans chaque île du SOOI, tandis que les phylotypes II (9 %) et III (3 %) ne sont trouvés qu'à La Réunion. Deux souches de phylotype IV ont par ailleurs été signalées à l'île Maurice, représentant le premier rapport de ce groupe phylogénétique dans le SOOI. Une approche phylogénétique et de génotypage (MLSA/MLST) basée sur l'analyse de séquences de 6 gènes de ménage et 1 gène associé à la virulence (egl) a permis de révéler les relations génétiques entre 145 souches représentatives (diversité géographique + hôte d'isolement) du SOOI et 90 souches mondiales de référence. Le développement et l'application d'un schéma MLVA basé sur 17 séquences répétées en tandem (VNTR) sur près de 1300 souches a permis de révéler que les populations de phylotype I sont organisées en complexes clonaux dans le SOOI et que le niveau de diversité génétique est très contrasté selon les îles, Maurice présentant la plus forte diversité génétique. Un résultat majeur de cette thèse est la mise en évidence du déploiement d’une lignée génétique (sequevar I-31 ; STI-13 ; MT-035), surreprésentée dans les îles du SOOI, qui pourrait avoir été introduite via du matériel végétal contaminé depuis l'Afrique de Sud ou l’Afrique de l'Ouest. Nos études préliminaires montrent que l'haplotype majoritaire MT-035 (i) est le probable haplotype fondateur du complexe clonal le plus prévalent dans le SOOI, (ii) présente un pouvoir pathogène élevé (large gamme d'hôtes comprenant des plantes cultivées et des adventices, et forte agressivité sur Solanacées) et (iii) possède une forte aptitude à la compétition dans l'environnement via la production de bactériocines. Ces travaux permettront in fine de renforcer l'épidémiosurveillance et orienter les stratégies de lutte vis-à-vis de cet agent phytopathogène, notamment via le déploiement de cultivars résistants. / In the southwest Indian Ocean (SWIO) islands (Comoros, Mauritius, Mayotte, Réunion, Rodrigues and Seychelles), bacterial wilt caused by the Ralstonia solanacearum species complex (Rssc) is considered one of the most harmful plant disease for food crops or export. The main objective of this work presented in this manuscript was to explore the level and the distribution of the genetic diversity of Rssc and the genetic structure of its populations in SWIO. We conducted extensive sampling campaigns that resulted in a large collection of 1704 isolates, mainly from Solanaceae (tomato, potato, chilli, eggplant, pepper) and geranium rosat. The phylogenetic assignment of the isolates showed a very high prevalence of phylotype I (88 %), which is distributed in each island of the SWIO, while phylotypes II (9 %) and III (3 %) are found only in Réunion. Two phylotype IV strains have also been reported in Mauritius, representing the first report of this phylogenetic group in SWIO. A phylogenetic and genotyping approach (MLSA/MLST) based on sequence analysis of 6 housekeeping genes and 1 gene associated with virulence (egl) revealed the genetic relationships between 145 representative SWIO strains (geographic diversity + host) and 90 global reference strains. The development and application of MLVA scheme based on 17 variable number of tandem repeat sequences (VNTR) on nearly 1300 strains revealed that phylotype I populations are organized into clonal complexes in SWIO and that the level of genetic diversity is highly contrasted according to the islands, with Mauritius having the highest genetic diversity. This work highlights the deployment of a genetic lineage (Sequevar I-31, STI-13, MT-035), overrepresented in SWIO islands, which could have been introduced via contaminated plant material from South Africa or West Africa. Our preliminary studies show that the main haplotype MT-035 (i) is the probable founding haplotype of the most prevalent clonal complex in SWIO, (ii) has high pathogenicity (wide range of hosts including cultivated plants and weeds, and high aggressiveness on Solanaceae) and (iii) has a strong ability to compete in the environment via the production of bacteriocins. This work will ultimately strengthen epidemiosurveillance and guide control strategies of this plant pathogen, including the deployment of resistant cultivars.

Made available in DSpace on 2015-03-26T13:37:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 275601 bytes, checksum: 8dc4b9237b3474a70a7b5c112c54fb7d (MD5) Previous issue date: 2009-02-13 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The occurrence of bacterial wilt is one of the main restrictions to the tomato production in tropical areas, mainly due to the lack of effective methods of control. The research had the objective to evaluate the efficiency of the essential oil of mustard in the treatment of the soil for the suppression of the population of Ralstonia solanacearum. Initially, different selective mediums were evaluated for the detection and quantification of the bacterial population in the soil. Among the appraised mediums, only two mediums showed appropriate sensibility for the detection of the bacteria, and the South Africa Selective Medium - Elphinstone (SMSA-E) showed the highest recovery tax of the bacteria in the soil, the lowest repression index to the R. solanacearum and highest suppression index of background microorganisms. In the evaluation of the EOM gaseous effect against R. solanacearum, this was capable to inhibit it in vitro development completely. The bacterial colonies that were exposited to the EOM gaseous increased the linkage of cellular metabolites. Anaerobic and gram-positive bacterias were less sensitivity to the product than R. solanacearum. The recovery of the bacteria was not observed with the selective medium SMSA-E, after substratum fumigation for seven days with EOM in concentrations over 100µL of ITCA/L of soil. The tomato plants cultivated in the substratum submitted to this same treatment had not present symptoms of bacterial wilt after 45 days of the transplant. / A ocorrência de murcha bacteriana é um dos principais fatores limitantes ao cultivo de tomate em regiões tropicais, principalmente devido à falta de métodos efetivos de controle. O presente trabalho teve como objetivo avaliar a eficiência do óleo essencial de mostarda no tratamento do solo para a supressão da população de Ralstonia solanacearum. Inicialmente, avaliaram-se diferentes meios seletivos para a detecção e monitoramento da população da bactéria no solo. Dos meios avaliados, apenas dois apresentaram sensibilidade adequada para a detecção da bactéria, sendo que, o meio South Africa Selective Medium Elphinstone (SMSA-E) possibilitou a maior taxa de recuperação da bactéria no solo, com baixo índice de repressão ao patógeno alvo e alto índice de supressão à microrganismos contaminantes. Quanto ao efeito dos vapores do EOM sobre R. solanacearum, estes foram capazes de inibir completamente o seu desenvolvimento in vitro. A exposição das colônias bacterianas aos vapores do EOM ocasionou aumento do extravasamento de metabólitos celulares. Bactérias anaeróbicas facultativas e/ou gram-positivas foram menos sensíveis ao produto que R. solanacearum. Após fumigação por sete dias do substrato com EOM em concentrações a partir de 100µL de ITCA/L de solo, não se observou a recuperação da bactéria com o meio seletivo SMSA-E. As plantas de tomate cultivadas no substrato submetido a este mesmo tratamento não apresentaram sintomas de murcha bacteriana após 45 dias do transplante.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-09-19T14:09:02Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) / Made available in DSpace on 2016-09-19T14:09:02Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Doutorado_Carolina_Barbosa_Malafaia_2016_PPGCB_UFPE.pdf: 5843362 bytes, checksum: c164be8fbab90a37d6db77986071cad9 (MD5) Previous issue date: 2016-01-25 / CAPES / A formação de biofilme é uma característica importante para as bactérias, por ser uma formação natural e altamente influenciada pelo ambiente circundante, confere aos microrganismos alta tolerância às adversidades e torna-se importante na virulência para patógenos. Sendo assim, apresentamos nesta tese uma investigação da adesão bacteriana e desenvolvimento de biofilme das fitobactérias Ralstonia solanacearum (Rsol) e Acidovorax citrulli (Acc), agronomicamente importantes, sobre superfícies hidrofóbicas, foi investigado também o emprego de extratos vegetais de plantas oriundas da Caatinga, na inibição da adesão bacteriana e sua capacidade bactericida contra R. solanacearum, e foi determinado o método mais eficiente na preparação amostras proteicas para R. solanacearum, A. citrulli e Pectobacterium carotovorum subsp. carotovorum (Pcc) para aplicação em estudos futuros de investigação molecular da formação de biofilmes fitopatogênicos. A formação de biofilme por diferentes isolados bacterianos após 24h de incubação em diferentes meios de cultura foi quantificado pelo método de cristal violeta e suas estruturas observadas por microscopia eletrônica de varredura e microscopia confocal. Foram avaliados também 22 extratos aquosos de 16 plantas coletadas na Caatinga quanto a capacidade de inibição da formação de biofilme de Rsol. Quanto à eficiência na obtenção de proteínas, foram testados os métodos de Trizol, Fenol, Centrifugação e Lise e avaliados através de eletroforese uni e bidimensional. Quanto a formação de biofilme os resultados obtidos indicam que, nas condições testadas, isolados de Rsol se mostrou diferente entre os isolados tanto quantitativa quando morfologicamente onde os isolados B5-5 CGH26 CGH8 e SCN 21 foram moderados ou fortes produtores de biofilme. Já os isolados de Acc não foram bons produtores de biofilme, apresentando apenas os isolados Acc1.43 e Acc 1.73 como fortes formadores de biofilme com quantidade e morfologia semelhantes. No screening de atividade antibiofilme, dentre os extratos testados apenas ramos de Harpochilus neesianus e folhas de Myroxylon peruiferum apresentaram atividade antibiofilme superior a 83% e 50%, respectivamente, e Jacaranda rugosa apresentou atividade antimicrobiana contra todos os isolados de Rsol testados. Quanto à extração de proteínas de alta qualidade o método de Lise foi o mais eficiente para Rsol e Pcc, apresentando respectivamente 369 ± 4 e 212 ± 3 diferentes spots de proteínas, contudo para Acc o método de centrifugação foi o mais indicado com 224 ± 8 spots. De acordo com os resultados deste estudo conclui-se que a formação de biofilme pode ser quantitativa e estruturalmente distinta entre isolados da mesma espécie. O screening das propriedades antimicrobianas das plantas fornece base de dados para o desenvolvimento de novos agentes antibacterianos naturais contra fitopatógenos seguros para o meio ambiente e para o desenvolvimento de estudos moleculares da formação de biofilme faz-se necessária uma prévia determinação de métodos para obtenção das macromoléculas a serem analisadas, sendo assim a seleção de métodos de extração é um ponto crucial para obtenção de amostras de qualidade para analises confiáveis. / Biofilm formation is an important feature for bacteria due to its naturally occurring and highly influence by the surrounding environment, giving to the microorganisms high tolerance to adversity and becoming essential in virulence for pathogens. Thus, we present in this thesis an investigation about bacterial adhesion and biofilm development of the phytobacteria Ralstonia solanacearum (Rsol) and Acidovorax citrulli (Acc), agronomically important, on hydrophobic surfaces; it was also investigated the use of plant extracts from the Caatinga region through the inhibition of the bacterial adhesion and its bactericidal activity against R. solanacearum. The most efficient method to prepare protein samples for R. solanacearum, A. citrulli and Pectobacterium carotovorum subsp. carotovorum (Pcc) was determined to be applied in future studies of molecular investigation of the formation of pathogenic biofilms. The biofilm formation by different bacterial strains after 24 h incubation in distinct culture media was quantified by the crystal violet method and its structures were observed by scanning electron microscopy and confocal microscopy. There were also evaluated 22 aqueous extracts from 16 plants collected in the Caatinga as its potential of inhibition of Rsol biofilm formation. In what concerns the efficiency in obtaining proteins, Trizol, Phenol, centrifugation, and Lyse were the methods evaluated by one- and two-dimensional electrophoresis. The results for biofilm formation demonstrate that, under the tested conditions, Rsol strains were different, both quantitatively and morphologically, and the strains namely B5-5, CGH26, CGH8, and SCN 21 were moderate or strong biofilm producers. Regarding the results for Acc strains, it is possible to note that they were not good biofilm producers, unless the strains Acc1.43 and Acc1.73 that were considered strong biofilm producers with similar quantity and morphology patterns. In relation to the screening of antibiofilm activity, only branches of Harpochilus neesianus and leaves of Myroxylon peruiferum presented antibiofilm activity with values higher than 83% and 50%, respectively, and Jacaranda rugosa showed activity antimicrobial against all the tested Rsol strains. The extraction of high quality proteins was performed most efficiently by the Lysis method for Rsol and Pcc, respectively with 369 ± 4 and 212 ± 3 different spots of proteins, however the centrifuge method was better for Acc with 224 ± 8 spots. According to the results of this study it is possible to conclude that biofilm formation can be quantitatively and structurally distinct from strains of the same species. The screening of the antimicrobial properties of the plants provides data as a basis for the development of new natural antibacterial agents against safe phytopathogens for the environment; in addition, for the development of molecular studies about the biofilm formation it is necessary a preliminary determination of the methods suitable for obtaining the macromolecules to be analyzed, so the selection of extraction methods is a crucial point for obtaining quality samples for reliable analysis.