Le modèle étudié est l’agent phytopathogène vasculaire Ralstonia solanacearum, en portant une attention particulière aux souches de phylotype II. Cette bactérie d’origine tellurique est très diversifiée, tant au plan génétique que phénotypique. Sa classification en constante évolution témoigne d’une volonté de clarifier cette biodiversité inhabituellement forte, tout en cherchant à reconnaître les écotypes structurant ce complexe d’espèces, i.e., des groupes de souches partageant à la fois des traits génotypiques et biologiques spécifiques. Dans le cadre de ce pathosystème modèle, nous nous sommes attachés dans un premier temps à revisiter de façon précise les pathotypes au sein d’écotypes bien décrits dans la littérature, ou à en faire la description (phylotype III africain). Nous avons observé une forte convergence phénotypique entre les souches de phylotype III des hauts plateaux africains et les souches Brown rot de phylotype IIB-1, capables de flétrir la pomme de terre et d’autres Solanacées à température froide. L’adaptation de souches aussi diverses pour la tolérance au froid nous a conduits à dresser un bilan de la situation R. solanacearum en Europe et in extenso dans le bassin méditerranéen. Cette approche a permis d’apprécier les degrés de divergence significative dans le pouvoir pathogène (virulence et agressivité) sur Solanaceae au sein de souches quasi clonales unifiant l’écotype Brown rot, qui s’établissent aussi sous forme d’infections latentes dans les tissus vasculaires de bananiers (Musacées). Dans le même temps, le phénotype de souches pathogènes du bananier, unifiant l’écotype Moko, a aussi été revisité sur Solanaceae qu’elles parviennent à flétrir, y compris des ressources génétiques résistantes au flétrissement bactérien. L’ensemble de ces données expérimentales a permis de dégager les critères de sélection pour le choix de trois nouvelles souches du complexe d’espèces R. solanacearum, dont nous avons obtenu les séquences génomiques. Notre approche en génomique comparative a permis de décrire le premier pangénome chez cet agent pathogène : l’ensemble les gènes repérés de l’espèce. Ces données ont été exploitées par différentes approches bio-informatiques et permettent de concevoir une refonte pertinente du complexe d’espèces R. solanacearum en trois nouvelles espèces génomiques, regroupant les souches de phylotypes I (Asie) et III (Afrique) d’une part, puis les souches de phylotype II (Amérique), et enfin les souches de phylotype IV (Indonésie) d’autre part. Ce pangénome a ensuite été exploité en concevant et développant une puce à ADN, un outil permettant l’exploration à haut débit d’une grande quantité de souches. La densité des données expérimentales accumulées permet une démarche vers l’écologie moléculaire et de reconstituer certains pans du passé évolutif des souches de phylotype II chez R. solanacearum. Par ailleurs, l’analyse approfondie de ces données de génomique, associant phylogéographie et structuration des populations de l’écotype Brown rot, montre une double situation épidémiologique en Europe, recoupant des influences andines et africaines. De la même façon, l’écotype Moko présente trois structures génétiques distinctes. Ces données ont été analysées de manière à retracer les principaux flux de gènes dans les états ancestraux des phylotypes et de dégager la forte contribution de la partie mobile du génome, des gènes relatifs à l’adaptation environnementale et à la pathogénie, comme moteurs dans l’évolution de cet important organisme phytopathogène. / The studied model is the vascular plant pathogen Ralstonia solanacearum, with a particular focus on phylotype II strains. This telluric bacterium has a wide diversity, both on genotypic and phenotypic levels. Its evolving classification reflects the need to clarify its unusual biodiversity and seek to identify ecotype structure in this species complex, i.e., groups of strains with both genotypic and specific biological traits. Within the framework of this model pathosystem, we initially focused on deeply revisiting pathotypes among ecotypes, although well described in the literature, or describing new ecotypes (African phylotype III). We observed high phenotypic convergence between strains from phylotype III from the African highlands and Brown rot strains from phylotype IIB-1, both able to trigger wilt symptoms on potato and other Solanaceae at cold temperatures. Adaptation of diverse strains for cold tolerance led us to investigate the R. solanacearum situation in Europe and more specifically in the Mediterranean regions. This strategy allowed us to appreciate the significant divergence towards pathogenicity (virulence and aggressiveness) on Solanaceae within clonal-like structure of strains in the Brown rot ecotype, which also established latent interactions in the banana vascular system. In the mean time, phenotypes of banana pathogenic strains unifying the Moko ecotype, was also revisited on Solanaceae, and was able to trigger symptoms on both susceptible and resistant genetic resources to bacterial wilt. All these experimental data yielded selection criteria for choosing three new candidate strains in the R. solanacearum species complex for complete genome sequencing. Our genomic comparative approach allowed us to describe the first pangenome of this pathogen: all targeted identified genes of this species complex. These data were analyzed by various bioinformatic approaches and allowed us to design a complete reshaping of R. solanacearum species complex into three distinct genomic species, firstly clustering strains from phylotype I (Asia) with strains from phylotype III (Africa); strains from phylotype II (America); and lastly, strains from phylotype IV (Indonesia). This pangenome was then used for designing a DNA microarray, a high resolution tool that allowed us to explore a wide set of genomes. The density of accumulated data allowed for a molecular ecological approach to retrieve a certain amount of the evolutionary past of R. solanacearum phylotype II strains. Furthermore, a deeper analysis of these genomic data, combining phylogeography with population structure analysis of the Brown rot ecotype, revealed a dual epidemic situation in Europe, both across Andean and African influences. Similarly, the Moko ecotype presents three distinct genetic structures. These data were analyzed within the purpose of tracking the main gene flows in the ancestral states of phylotypes and to unravel the strong contribution of the mobile elements, genes related to environmental adaptation, and pathogenicity as a major driving force into the evolution of this successful plant pathogen.

O tomateiro (Solanum lycopersicum L.) é uma das plantas cultivadas de maior importância no mundo, sendo uma das principais hortaliças em importância econômica no Brasil. A murcha-bacteriana é uma doença dentre as mais limitantes para a cultura do tomateiro e o uso de cultivares resistentes à Ralstonia spp. é uma das únicas alternativas para evitar as perdas causadas por esse patógeno. Contudo, existem raríssimos estudos sobre a base genética de genótipos resistentes aos principais isolados de Ralstonia spp. Objetivou-se nesse trabalho estudar parâmetros genéticos da resistência à murcha-bacteriana em linhagens e híbridos usados como porta-enxertos de tomateiro, por meio de abordagens complementares que envolvem desde o ajuste das metodologias para indução artificial da doença, até estimativas das capacidades de combinação e a heterose entre linhagens, visando identificar cruzamentos promissores para o estabelecimento de um programa de melhoramento genético para resistência à murcha-bacteriana em tomateiro para porta-enxertos. Comparando-se quatro métodos de indução da resistência verificou-se que o método da lesão das raízes do torrão com objeto cortante (bisturi) e pulverização de 5 mL da suspensão bacteriana no torrão, comparativamente aos outros métodos avaliados, apresenta fácil execução, requer quantidades reduzidas de suspensão bacteriana e proporciona boas quantidades de plantas de tomateiro com sintomas de murcha-bacteriana, sendo o mais adequado entre os métodos testados. Foi estimada a heterose e capacidade de combinação entre linhagens de tomateiro para um isolado do biovar 2 de Ralstonia solanacearum em estágio de plântulas, em ambiente protegido, e em um solo naturalmente infestado por Ralstonia spp. Entre as linhagens avaliadas, apenas o genótipo Hawaii 7996 apresentou níveis aceitáveis de resistência, sendo necessário encontrar novas fontes de resistência para o início de um programa de melhoramento. Verificou-se que nenhuma das progênies derivadas dos cruzamentos desse estudo apresentaram resistência superior ao parental superior Além disso, foi verificada a importância dos efeitos aditivos e, consequentemente, os cruzamentos de genitores com alta CGC para o caráter e a seleção recorrente seriam as melhores alternativas no melhoramento para resistência à murcha-bacteriana. Finalmente, foram estudados os genitores e seus híbridos, utilizados como porta-enxerto em condições de campo e em ambiente protegido visando o aumento da resistência à murcha-bacteriana causada por Ralstonia spp., e incremento dos parâmetros produtivos de um híbrido comercial utilizado como copa. Verificou-se influência dos diferentes porta-enxertos em caracteres de produção e massa dos frutos e no desenvimento de copa. Caracteres relacionados ao sistema radicular dos porta-enxertos apresentaram heterose em relação aos seus genitores, com efeitos aditivos e não aditivos importantes, mostrando o desempenho das linhagens e sua contribuição para a produção de híbridos superiores. Por fim, este estudo possibilitou o melhor entendimento sobre a base genética da resistência em genótipos de tomateiro que possuem uso potencial como porta-enxertos resistentes à murcha-bacteriana. / The tomato (Solanum lycopersicum L.) is one of the most important cultivated plants in the world, being one of the main vegetables of economic importance in Brazil. The bacterial wilt is one of the most limiting diseases for the tomato crop and the use of resistant cultivars to Ralstonia spp. is one of the only alternatives to avoid losses caused by this pathogen. However, there are very few studies on the genetic basis of genotypes resistant to the main isolates of Ralstonia spp. The objective of this work was to study the genetic parameters of resistance to bacterial wilt in lines and hybrids used as tomato rootstocks, through complementary approaches that range from the adjustment of the methodologies for artificial induction of the disease, to estimates of the combining abilities and heterosis among lines, aiming to identify promising crosses for the establishment of a rootstock genetic breeding program for resistance to bacterial wilt in tomatoes. Comparing four resistance induction methods, it was verified that the method of injuring roots with cutting object (scalpel) and spraying 5 mL of the bacterial suspension in the clod compared to the other\'s methods evaluated, is easiest to perform, requires less amounts of bacterial suspension and provides good amounts of tomato plants with wilt symptoms, being the most suitable among the methods tested. The heterosis and combining ability of tomato lines for a strain of biovar 2 of Ralstonia solanacearum in greenhouse at seedling stage and in soil naturally infested by Ralstonia spp were estimated. Among the lines evaluated, only the Hawaii 7996 genotype showed acceptable levels of resistance, and it is necessary to find new sources of resistance for the beginning of a breeding program. It was verified that none of the progenies derived from the crosses of this study presented superior resistance to the superior parental. In addition, the importance of the additive effects was verified and, consequently, the crossings of parents with high CGC for the character and the recurrent selection would be the best alternatives in the improvement for resistance to bacterial wilt. Finally, we studied the parents and their hybrids, used as rootstocks under field conditions and in a protected environment aiming the increasing resistance to bacterial wilt caused by Ralstonia spp., and increasing of the yield parameters of a commercial hybrid used as a scion. The influence of the different rootstocks was verified in characters of yield and mass of the fruits and in the development of the scion. Characteristics related to the root system of rootstocks showed heterosis in relation to their parents, with important additive and non-additive effects, showing the performance of the lines and their contribution to the production of superior hybrids. Finally, this study allowed a better understanding of the genetic basis of resistance in tomato genotypes that have potential use as bacterial wilt resistant rootstocks.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-12-04T18:41:29Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_final_Adenilson.pdf: 1632453 bytes, checksum: ca3bab925a332585e719109a07462e72 (MD5) / Made available in DSpace on 2018-12-04T18:41:29Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação_final_Adenilson.pdf: 1632453 bytes, checksum: ca3bab925a332585e719109a07462e72 (MD5) Previous issue date: 2018-09-27 / Os objetivos deste trabalho foram a obtenção de um mapa genético de alta densidade utilizando marcadores SNP (Single Nucleotide Polymorphism) obtidos através de genotipagem por sequenciamento (GBS) e identificar as regiões genômicas ligadas à tolerância à murcha bacteriana (Ralstonia solanacearum Smith) em uma população de duplo-haploides (DH) de tabaco. As linhagens endogâmicas NC95 (tolerante) e NC2326 (suscetível) foram cruzadas entre si gerando a população F1, anteras foram coletadas destas plantas para produção de haploides e posterior duplicação cromossômica através da cultura de anteras, gerando 180 famílias duplo-haploides que foram avaliadas em ambiente controlado quanto à tolerância à murcha bacteriana, após inoculação com R. solanacearum, através de uma escala de notas com amplitude de 0 a 4. As famílias DH foram genotipadas utilizando a metodologia de GBS e os dados resultantes desta genotipagem foram alinhados com o genoma de referência do tabaco para posterior obtenção dos marcadores SNP utilizados na construção do mapa de ligação. O mapa de ligação juntamente com os dados de fenotipagem foram utilizados para realizar o mapeamento de QTLs através do mapeamento por intervalo composto. Foram identificados 6.842 SNPs, utilizados para construção de um mapa de ligação com 70.583 cM, sendo este o maior mapa de ligação utilizando marcadores SNP disponível para tabaco e com o maior número de marcadores. Utilizando este mapa de ligação foram mapeados 13 QTLs para tolerância à murcha bacteriana em oito grupos de ligação, dos quais oito QTLs ainda não tinham sido identificados na literatura especializada. Os locos presentes nos grupos de ligação 3, 17 e 22 apresentaram os maiores efeitos na variação fenotípica. O elevado número de QTLs mapeados nesta população confirma o padrão de herança quantitativa da tolerância de tabaco à murcha bacteriana causada por R. solanacearum. / The objectives of this work were to obtain a high-density genetic map using SNP (Single Nucleotide Polymorphism) markers obtained through Genotyping-by-Sequencing (GBS) and to identify the genomic regions linked to bacterial wilt tolerance (Ralstonia solanacearum Smith) in a tobacco double-haploid (DH) population. The inbred lines NC95 (tolerant) and NC2326 (susceptible) were crossed generating the F1 population, anthers were collected from these plants for haploid production and subsequent chromosomic duplication using anthers culture, generating 180 double-haploid families that were evaluated in a controlled environment for tolerance to bacterial wilt after inoculation with R. solanacearum, using an assessment scale from 0 to 4. The DH families were genotyped using the GBS methodology and the resulting data from this genotyping were aligned with the reference genome and then to obtain the SNP markers used to construct the genetic linkage map. The linkage map jointly with the phenotyping data were used to QTL mapping through the composite interval mapping method. A total of 6,842 SNPs was identified and used to construct a linkage map with 70,583 cM, being the largest SNP-based genetic linkage map available for tobacco and presenting the highest number of markers. Using this linkage map, 13 QTLs were mapped for bacterial wilt tolerance in eight linkage groups, from those eight QTLs had not yet been identified in the specialized literature. The loci present in linkage groups 3, 17 and 22 had the highest effects on phenotypic variation. The high number of QTLs mapped in this population confirms the quantitative genetic control of tobacco tolerance to bacterial wilt caused by R. solanacearum.

O tomateiro (Solanum lycopersicum L.) é uma das plantas cultivadas de maior importância no mundo, sendo uma das principais hortaliças em importância econômica no Brasil. A murcha-bacteriana é uma doença dentre as mais limitantes para a cultura do tomateiro e o uso de cultivares resistentes à Ralstonia spp. é uma das únicas alternativas para evitar as perdas causadas por esse patógeno. Contudo, existem raríssimos estudos sobre a base genética de genótipos resistentes aos principais isolados de Ralstonia spp. Objetivou-se nesse trabalho estudar parâmetros genéticos da resistência à murcha-bacteriana em linhagens e híbridos usados como porta-enxertos de tomateiro, por meio de abordagens complementares que envolvem desde o ajuste das metodologias para indução artificial da doença, até estimativas das capacidades de combinação e a heterose entre linhagens, visando identificar cruzamentos promissores para o estabelecimento de um programa de melhoramento genético para resistência à murcha-bacteriana em tomateiro para porta-enxertos. Comparando-se quatro métodos de indução da resistência verificou-se que o método da lesão das raízes do torrão com objeto cortante (bisturi) e pulverização de 5 mL da suspensão bacteriana no torrão, comparativamente aos outros métodos avaliados, apresenta fácil execução, requer quantidades reduzidas de suspensão bacteriana e proporciona boas quantidades de plantas de tomateiro com sintomas de murcha-bacteriana, sendo o mais adequado entre os métodos testados. Foi estimada a heterose e capacidade de combinação entre linhagens de tomateiro para um isolado do biovar 2 de Ralstonia solanacearum em estágio de plântulas, em ambiente protegido, e em um solo naturalmente infestado por Ralstonia spp. Entre as linhagens avaliadas, apenas o genótipo Hawaii 7996 apresentou níveis aceitáveis de resistência, sendo necessário encontrar novas fontes de resistência para o início de um programa de melhoramento. Verificou-se que nenhuma das progênies derivadas dos cruzamentos desse estudo apresentaram resistência superior ao parental superior Além disso, foi verificada a importância dos efeitos aditivos e, consequentemente, os cruzamentos de genitores com alta CGC para o caráter e a seleção recorrente seriam as melhores alternativas no melhoramento para resistência à murcha-bacteriana. Finalmente, foram estudados os genitores e seus híbridos, utilizados como porta-enxerto em condições de campo e em ambiente protegido visando o aumento da resistência à murcha-bacteriana causada por Ralstonia spp., e incremento dos parâmetros produtivos de um híbrido comercial utilizado como copa. Verificou-se influência dos diferentes porta-enxertos em caracteres de produção e massa dos frutos e no desenvimento de copa. Caracteres relacionados ao sistema radicular dos porta-enxertos apresentaram heterose em relação aos seus genitores, com efeitos aditivos e não aditivos importantes, mostrando o desempenho das linhagens e sua contribuição para a produção de híbridos superiores. Por fim, este estudo possibilitou o melhor entendimento sobre a base genética da resistência em genótipos de tomateiro que possuem uso potencial como porta-enxertos resistentes à murcha-bacteriana. / The tomato (Solanum lycopersicum L.) is one of the most important cultivated plants in the world, being one of the main vegetables of economic importance in Brazil. The bacterial wilt is one of the most limiting diseases for the tomato crop and the use of resistant cultivars to Ralstonia spp. is one of the only alternatives to avoid losses caused by this pathogen. However, there are very few studies on the genetic basis of genotypes resistant to the main isolates of Ralstonia spp. The objective of this work was to study the genetic parameters of resistance to bacterial wilt in lines and hybrids used as tomato rootstocks, through complementary approaches that range from the adjustment of the methodologies for artificial induction of the disease, to estimates of the combining abilities and heterosis among lines, aiming to identify promising crosses for the establishment of a rootstock genetic breeding program for resistance to bacterial wilt in tomatoes. Comparing four resistance induction methods, it was verified that the method of injuring roots with cutting object (scalpel) and spraying 5 mL of the bacterial suspension in the clod compared to the other\'s methods evaluated, is easiest to perform, requires less amounts of bacterial suspension and provides good amounts of tomato plants with wilt symptoms, being the most suitable among the methods tested. The heterosis and combining ability of tomato lines for a strain of biovar 2 of Ralstonia solanacearum in greenhouse at seedling stage and in soil naturally infested by Ralstonia spp were estimated. Among the lines evaluated, only the Hawaii 7996 genotype showed acceptable levels of resistance, and it is necessary to find new sources of resistance for the beginning of a breeding program. It was verified that none of the progenies derived from the crosses of this study presented superior resistance to the superior parental. In addition, the importance of the additive effects was verified and, consequently, the crossings of parents with high CGC for the character and the recurrent selection would be the best alternatives in the improvement for resistance to bacterial wilt. Finally, we studied the parents and their hybrids, used as rootstocks under field conditions and in a protected environment aiming the increasing resistance to bacterial wilt caused by Ralstonia spp., and increasing of the yield parameters of a commercial hybrid used as a scion. The influence of the different rootstocks was verified in characters of yield and mass of the fruits and in the development of the scion. Characteristics related to the root system of rootstocks showed heterosis in relation to their parents, with important additive and non-additive effects, showing the performance of the lines and their contribution to the production of superior hybrids. Finally, this study allowed a better understanding of the genetic basis of resistance in tomato genotypes that have potential use as bacterial wilt resistant rootstocks.

Made available in DSpace on 2016-04-28T14:57:35Z (GMT). No. of bitstreams: 1 Ana Rosa de Figueiredo Fernandes.pdf: 5291957 bytes, checksum: e9ba6c76361eb4a2e901846a5bb5c2dd (MD5) Previous issue date: 2009-10-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / ?The pathogenicity of Pratylenchus brachyurus on tobacco (Nicotiana tabacum) cultivar K326 was evaluated in experiments testing different initial population densities of the nematode (Pi = 0; 1,000; 3,000; 9,000 and 27,000 eggs and larvae per plant), in eight replications. The isolate was monoxenically multiplied on carrot discs. At the end of the experiment (70 days after inoculation), the above ground part of each plant was weighed and measured and, the respective root system was processed. The reproduction rate of Pratylenchus brachyurus was estimated by extracting the nematodes of the roots and of the soil. Necroses on the roots of the tobacco plants were observed. Pratylenchus brachyurus caused well-delimited root lesions on tobacco root. None of the analyzed plants died; however plant growth was reduced. Also, it was observed a decrease of the fresh mass of roots and of the dry top weight, mainly under the highest population density (27,000 nematodes per plant). The reproduction rate was low. Besides the interaction between Pratylenchus brachyurus and Ralstonia solanacearum in two tobacco varieties, Virg?nia K326 and Burley By21 was evaluated. Test plants were inoculated with the nematode in the initial population density of 3,000 individuals (eggs and larvae) by plant and, 15 days later, the plants were inoculated with a sample of soil infested by the bacterium, in estimated population density of 106 CFU.g-1 of soil. Besides the mixed inoculation with Pratylenchus brachyurus and Ralstonia solanacearum, tobacco plants were single inoculated. The plants were monitored daily. Dates of the beginning of symptom expression were registered, as well as symptom evolution. When the plants were inoculated with Pratylenchus brachyurus and Ralstonia solanacearum, symptom of wilt that is caused by the bacterium Ralstonia solanacearum began earlier than in single inoculated plants, and they also evolved quicker, inclusive in the variety Virg?nia K326 that was considered more tolerant. In this case, the presence of Pratylenchus brachyurus, in the root system of these plants, probably favored the infection by Ralstonia solanacearum, even before the symptoms caused by the nematodes could be detected. This study was done in greenhouse and under laboratory conditions. / ?A patogenicidade de Pratylenchus brachyurus em fumo Virg?nia K326 foi avaliada em experimento no qual foram testadas diferentes densidades populacionais iniciais do nemat?ide (Pi = 0; 1.000; 3.000; 9.000 e 27.000 ovos e larvas por planta), em oito repeti??es. O isolado foi multiplicado axenicamente em discos de cenoura. Ao final do experimento (85 dias), as plantas tiveram sua parte a?rea pesada e medida e foi processado o sistema radicular de cada uma. O fator de reprodu??o foi estimado por extra??o dos nemat?ides das ra?zes e do solo. Necroses nas ra?zes das plantas foram observadas. Pratylenchus brachyurus causou les?es radiculares delimitadas nas ra?zes de fumo. N?o houve morte de nenhuma planta analisada, por?m a altura das plantas foi afetada e tamb?m a massa seca da parte a?rea, principalmente sob a densidade populacional mais alta, de 27.000 nemat?ides por planta. O fator de reprodu??o foi baixo. Al?m disto, avaliou-se a intera??o entre Pratylenchus brachyurus e Ralstonia solanacearum em fumo Virg?nia variedade K326 e fumo Burley variedade BY21. As plantas foram inicialmente inoculadas com o nemat?ide na densidade populacional de 3.000 ovos e larvas por planta e 15 dias ap?s, foram inoculadas com uma amostra de solo infestado pela bact?ria, na densidade populacional estimada de 106 UFC.g-1 de solo. Al?m da inocula??o mista com Pratylenchus brachyurus e Ralstonia solanacearum, testemunhas foram inoculadas isoladamente. As plantas foram monitoradas diariamente, anotando-se a data de in?cio e da evolu??o dos sintomas. Quando as plantas foram inoculadas com os Pratylenchus brachyurus e Ralstonia solanacearum, os sintomas de murcha, que s?o causados pela bact?ria se manifestaram mais cedo e tamb?m evolu?ram mais r?pido, inclusive na variedade K326 que foi considerada mais tolerante. Neste caso, provavelmente a presen?a de Pratylenchus brachyurus, no sistema radicular dessas plantas, favoreceu a infec??o por Ralstonia solanacearum, antes mesmo que os sintomas causados pelo nemat?ide pudessem ser observados. Este trabalho foi realizado em casa de vegeta??o e em laborat?rio.

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.

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.