Made available in DSpace on 2016-12-08T16:44:37Z (GMT). No. of bitstreams: 1 PGPV12MA064.pdf: 1012596 bytes, checksum: c4f66f0fa2a1b45c1ad39219f45c6b2d (MD5) Previous issue date: 2012-07-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) has showed one of the most important diseases in the bean crop. Its symptoms occur from wilts at leaves to death of the infected plant. The control is based on the use of healthy seeds, crop rotation and especially resistant cultivars. The goal of this study was to estimate the efficiency of selection in segregating populations of beans for bacterial wilt resistance and indicate the best time of the cycle to make the selection of resistant plants and also to test in field the effectiveness selection at greenhouse for resistance to this disease. To achieve these objectives were used converging crosses between Aruã x Guará, Pyatã x Guará and Pyatã x Pérola, originating F2, F2:3, F3:4 which were subsequently inoculated with Curtobacterium flaccumfaciens pv. flaccumfaciens cff 2634 and evaluated according to a scale related to the common symptoms of the disease at 20, 40 and 60 days after inoculation. The field test of the effectiveness selection at greenhouse was used the same crosses, however, the populations used were in the generations F2:3, F3:4 and F4:5, subsequently measured the agronomic traits such as plant height, first pod, stem diameter, number of pods per plant, number of grains per plant, grain weight in grams per line and resistance to bacterial wilt. Through the data can be established that the populations of Aruã x Guará, Pyatã x Guará and Pyatã x Pérola have similar behavior in relation to resistance to the bacterial wilt symptoms. The selection was effected for the coming populations of Aruã x Guará and Pyatã x Pérola. The favorable time for distinguishing and selecting reliably segregating plants is between 40 and 60 days after inoculation. It was likewise observed that there is genetic diversity and superiority of selected plants on not select plants and their parents, indicating in the field that the greenhouse selection for resistance to bacterial wilt was effective / A murcha de curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) tem se mostrado uma das doenças emergentes mais importantes na cultura do feijão. Seus sintomas ocorrem desde murchas nas folhas até à morte da planta infectada. O controle está baseado em uso de sementes sadias, rotação de cultura e principalmente cultivares resistentes. Assim, o objetivo deste trabalho foi estimar a eficiência da seleção em populações segregantes de feijão para resistentes à murcha de curtobacterium e indicar o melhor período do ciclo da cultura para proceder a seleção das plantas resistentes e também testar a campo a efetividade da seleção em casa de vegetação. Para atingir estes objetivos foram usados cruzamentos convergentes entre Aruã x Guará, Pyatã x Guará e Pyatã x Pérola, dando origem a populações segregantes que nas das gerações F2, F2:3 e F3:4 foram inoculadas com Curtobacterium flaccumfaciens pv. flaccumfaciens cff 2634 e avaliadas segundo uma escala de notas referentes aos sintomas comuns da doença aos 20, 40 e 60 dias após a inoculação. O teste a campo da efetividade da seleção em casa de vegetação usou os mesmos cruzamentos, entretanto as populações avaliadas são relativas às gerações F2:3, F3:4 e F4:5 sendo posteriormente mensurados os caracteres agronômicos como altura de planta, inserção do primeiro legume, diâmetro do caule, número de legumes por planta, número de grãos por planta, peso em gramas de grãos por linha e resistência à murcha de curtobacterium. Através dos dados pôde se estabelecer que as populações segregantes de Aruã x Guará, Pyatã x Guará e Pyatã x Pérola possuem comportamento semelhante em relação à resistência aos sintomas da murcha de curtobacterium. A seleção foi efetiva para as populações oriundas de Aruã x Guará e Pyatã x Pérola e o período propício para distinguir e selecionar de maneira confiável as plantas segregantes ocorre entre 40 e 60 dias após a inoculação. Foi da mesma forma observado que existe divergência genética e superioridade de plantas selecionadas sobre plantas não selecionadas e genitores, indicando a campo que a seleção em casa de vegetação visando resistência à murcha de curtobacterium foi efetiva

Resumo: O presente trabalho teve como objetivo avaliar a agressividade de linhagens de Ralstonia solanacearum provenientes de solanáceas, plantas ornamentais e eucalipto, em plantas de batata, tomate e fumo, bem como caracterizar as linhagens por meio de técnicas moleculares. Vinte e duas linhagens foram utilizadas nos ensaios de avaliação da agressividade, em experimentos conduzidos em casa-de-vegetação evidenciaram alta severidade da doença pelas linhagens de R. solanacearum quando inoculadas em plantas de tomate e batata, sendo a batata mais afetada nas inoculações. Todas as linhagens mostraram-se agressivas, sendo que o fumo mostrou baixa suscetibilidade ao ataque das bactérias. As linhagens mais agressivas em plantas de tomate foram IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 e IBSBF 2000, pertencentes às biovares I, II e III. As linhagens mais agressivas às plantas de fumo foram IBSBF 309, IBSBF 2131 e IBSBF 292T, pertencentes à biovar I. Foi efetuado também ensaio de microbiolização in vitro em sementes de eucalipto, a fim de se identificar possíveis linhagens patogênicas a esta espécie vegetal e concluiu-se que todas as linhagens utilizadas infectaram plantas de eucalipto ou afetaram seu crescimento. A caracterização molecular de 41 linhagens de Ralstonia solanacearum, provenientes de diversas plantas hospedeiras, incluindo solanáceas, bananeira, helicônia, plantas ornamentais e eucalipto, foi efetuada empregando-se ERIC e BOX-PCR e os resultados mostraram grande diversidade genética entre as linhagens. A análise de PCR-RFLP da região espaçadora 16S-23S DNAr permitiu distinguir os isolados pertencentes à biovar III das demais biovares (I, II, IIA e IIT), quando digeridos com as enzimas Taq I e Hin6 I. A análise de sequenciamento de parte dos genes Endoglucanase (Egl) e MutS possibilitou a classificação em filotipos e os resultados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study aimed to evaluate the aggressiveness of strains of Ralstonia solanacearum from solanaceus, ornamental and eucalyptus plants, on potato, tomato and tobacco, and to characterize the strains through molecular techniques. Twenty-two strains were used in this study to evaluate the aggressiveness and, the experiments conducted in a greenhouse revealed the high susceptibility of tomato and potato plants, with the potato being the most affected on through the inoculations. All isolates proved to be aggressive and higher tolerance to the attack of bacteria was verified on tobacco plants. Strains more aggressive on tomato were IBSBF 309, IBSBF 1712, IBSBF 1839, IBSBF 1882, IBSBF 1883 and IBSBF 2000, belonging to biovars I, II and III. The more aggressive strains on the tobacco plants were IBSBF 309, IBSBF 292T and IBSBF 2131 belonging to biovar I. Tests in vitro of microbiolization of eucalyptus seeds were also performed in order to identify possible pathogenic strains to this species and the results showed that all strains used cause infection on emerging plants or affected their growth. To molecular characterization of 41 strains of Ralstonia solanacearum from several host plants including solanaceous, banana, heliconia, ornamentals and eucalyptus were employed to ERIC and BOX-PCR, and the results showed high genetic diversity among strains. The analysis of PCR-RFLP of 16S-23S spacer region rDNA allowed us to distinguish the isolates belonging to biovar III from the others (biovars I, II, IIA and IIT) when digested with enzymes Taq I and Hin6 I. The sequence analysis of the partial of Endoglucanase (Egl) and MutS genes allowed the classification in phylotypes and the results revealed a predominance of the phylotype II in Brazil, and four isolates were classified in the phylotype I, all belonging to biovar III / Orientador: Antonio Carlos Maringoni / Coorientador: Suzete Aparecida Lanza Destéfano / Banca: Valdemar Atilio Malavolta Junior / Banca: Ivan Paulo Bedendo / Mestre

Le flétrissement bactérien causé par Ralstonia solanacearum limite la production mondiale de nombreuses cultures. La diversité génétique étendue de la bactérie a permis au cours des dernières années de concevoir le concept de complexe d'espèces de R. solanacearum (RSSC). Le séquençage génomique de plus de 30 souches représentatives de chaque groupe phylogénétique a élargi notre connaissance de l'évolution et de la spéciation du RSSC. Cela a permis d'identifier de nouvelles fonctions associées à la virulence. En outre, un grand nombre d'études ont été réalisées sur l'interaction plantes hôtes-R. solanacearum et éclairent la génétique, la biologie moléculaire et le développement de la maladie. Ces études ont documenté les stratégies d'infection qu'utilise R. solanacearum pour faire face aux défenses immunitaires des plantes. Bien que ces données qualitatives soient fondamentales pour comprendre l'infection par R. solanacearum, elles sont insuffisantes pour savoir si une plante sera infectées ou non. Cette question fondamentale nécessiteune compréhension quantitative des processus responsables de l'infection et colonisation de la plante par la populations de R. solanacearum. Ce travail a permis une étude quantitative permettant de répondre à la question suivante:"Combien d'individus de R. solanacearum entrent de la racine pour établir la base de l'infection donnant lieu a la maladie bactérienne dans la plante?" Cela nous a permis de déterminer quels facteurs contrôlent cette dynamique d'infection de R. solanacearum au sein de la plante hôte. Cette question scientifique nécessite un réexamen du cycle de vie de R. solanacearum et la caractérisation précise de l'ensemble du processus d'infection dans la plante. Sept paramètres dynamiques ont été affinés à partir de cinq étapes d'infection de R. solanacearum. Ensuite, nous avons établi un modèle mathématique de la dynamique dans l'hôte de la bactérie par l'intégration de ces paramètres. Le modèle suggère que toute la dynamique de la population influe sur la taille de la population de R. solanacearum. L'évaluation in vivo des paramètres et de leurs interactions prédit une petite taille de la population fondatrice, autour de quatre centaines de celluels bactériennes, ce qui a été confirmé par des mesures expérimentales avec une approche probabiliste. Pour comprendre les mécanismes qui restreignent la population de R. solanacearum, nous avons étudié les impacts de la virulence bactérienne, des barrières des plantes et du facteur environnemental sur la population fondatrice de l'infection. Nous avons montré que le goulot d'étranglement des infections est principalement modulé par l'agent pathogène (arsenal de virulence), l'hôte (caractéristiques physiques et immunitaires) et les conditions environnementales (température) en accord avec des études qualitatives. / Bacterial wilt caused by Ralstonia solanacearum limits the global production of many crops. The extensive genetic diversity of the bacterium has in recent years led to the concept of a R. solanacearum Species Complex (RSSC). Genome sequencing of over 30 representative strains of the each phylogenetic groups has broadened our knowledge of the evolution and speciation of RSSC. This enabled the identification of novel virulence-associated functions. Furthermore, a large number of studies have been carried on plants-R. solanacearum interactions and shed light on the genetics, molecular biology, and disease development. These studies have documented the infection strategies of R. solanacearum employed to cope with plant immune defenses. Although these qualitative investigations are a basis to understand the pathogenesis of R. solanacearum, they are insufficient to know whether or not plants will be diseased. These fundamental questions require a quantitative understanding of the processes responsible for the rise, dissemination and fall of the infection populations of R. solanacearum. This work pioneered the quantitative study in plant bacterial pathogens by addressing the following question: "How many R. solanacearum individuals enter from the root to establish the bacterial wilt disease in plant?" It allowed us to determine what factors control the infection dynamics of R. solanacearum within the host plant. This scientific question requires a re-examination on the life cycle of R. solanacearum and the precise characterization of the whole infection process in plant. Seven dynamical parameters were refined from five subsequent infection steps of R. solanacearum in host plant. Then, we established a mathematical model of within-host dynamics of the bacterium by the integration of these parameters. The model suggests that the whole population dynamics influences the founding population/bottleneck size of R. solanacearum. The in vivo assessment of parameters and their interactions predicted a small founding population size, around four hundred bacterial cells, which was confirmed by experimental measurements with a probabilistic approach. To understand mechanisms restricting R. solanacearum population, we further investigated impacts of bacterial virulence, plant barriers and environmental factor on the infection founding population. We showed that infection bottleneck is mainly modulated by the pathogen (virulence arsenal), the host (physical and immunity traits) and the environmental conditions (temperature).

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No. of bitstreams: 1 Liane Cristine Rebouças Demosthenes - Tese.pdf: 1077147 bytes, checksum: b3915a604b50db5ab23367233a0eb874 (MD5) Previous issue date: 2013-12-17 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases of solanaceous. Present in all national territory, causes rapid wilting of plants and affects several crops of agronomic interest. In Amazonas, the high temperature and humidity combined with the genetic diversity of this pathogen diversity of hosts favor the development and aggressiveness of the pathogen and complicate the management of this disease. The aim of this study was to evaluate the genetic diversity of R. solanacearum using AFLP markers and evaluate defense responses submitted by Capsicum spp. natives of the Amazon region, verifying the expression of pathogenesis-related proteins, resulting from infection by the pathogen. Samples were collected in four counties vegetable growers in the metropolitan area of Manaus to establish a collection of 30 isolates of the pathogen. We performed classical biochemical characterization of the isolates and characterization of genetic diversity using AFLP molecular markers. The 24 primer combinations were tested and selected the six best informative combinations . The six combinations of primers generated a 432 bands , ranging from 47 to 103 loci in the combination E + AT / M + C. The Jaccard similarity coefficient estimated between 30 isolates ranged from 0.01 to 0.98. From the similarity matrix a dendrogram was generated by UPGMA method being possible separate the isolates into six groups with cophenetic correlation coefficient of r = 0.98. The defense response submitted by Capsicum spp. were also evaluated by biochemical methods for the determination of total protein, phenylalanine ammonia lyase activity and peroxidase activity phenoloxidases. The enzymatic activity varied according to the level of resistance of the evaluated accessions, being higher in the first hours after inoculation and decreased after 72 hours. It was possible to identify two resistant accessions ( BC 05 and NT ) showed that grade point average of 1.17 xi and 0.94 . The BC had access resistance of accessions was associated with increased activity of PPO and FAL, indicating that these enzymes make up the defense mechanism in plants of Capsicum. The POX activity was lower when compared with the activity of PPO and FAL / A murcha bacteriana causada por Ralstonia solanacearum é uma das doenças mais importantes das solanáceas. Presente em todo o território nacional, causa murcha rápida das plantas e afeta várias culturas de interesse agronômico. No Amazonas, as condições de altas temperatura e umidade do ar aliados à ampla diversidade genética deste patógeno, diversidade de hospedeiros e agressividade favorecem o desenvolvimento do patógeno e dificultam o manejo desta doença. O objetivo deste trabalho foi avaliar a variabilidade genética de R. solanacearum utilizando marcadores AFLP e avaliar as respostas bioquímicas de defesa apresentadas por acessos de Capsicum spp. nativos da região Amazônica, verificando a expressão de proteínas relacionadas com a patogênese, decorrentes da infecção pelo patógeno. Foram realizadas coletas em quatro municípios produtores de hortaliças da área metropolitana de Manaus para estabelecer uma coleção com 30 isolados do patógeno. Foi realizada a caracterização bioquímica clássica dos isolados, testes de patogenicidade e caracterização da diversidade genética utilizando marcadores moleculares AFLP. Foram testadas 24 combinações de primers e seleciondas as seis combinações mais informativas. As seis combinações de primers utilizadas apresentaram um total de 432 bandas, variando de 47 loci à 103 na combinação E+AT/M+C. O coeficiente de similaridade de Jaccard estimado entre os 30 isolados avaliados variou de 0,01 à 0,98. A partir da matriz de similaridade foi gerado o dendrograma pelo método UPGMA sendo possível separar os isolados em seis grupos com coeficiente de correlação cofenética no valor de r = 0,98. Também foram avaliadas as resposta de defesa apresentadas por acessos de Capsicum spp. através de métodos bioquímicos de determinação de proteínas totais, atividade da fenilalanina amônia liase, atividade de fenoloxidases e peroxidases. A atividade enzimática variou conforme o nível de resistência ix dos acessos avaliados, sendo maior nas primeiras horas após a inoculação e decrescendo após 72 horas. Foi possível identificar dois acessos resistentes (BC 05 e NT) que apresentaram média de notas de 1,17 e 0,94. O acesso BC apresentou A resistência dos acessos foi associada com a maior atividade da PPO e FAL, indicando que estas enzimas compõem o mecanismo de defesa em plantas de Capsicum. A atividade da POX foi menor quando comparada com a atividade da PPO e FAL.

Please read the abstract in the section 07back of this document / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Ralstonia solanacearum, the causal agent of bacterial wilt disease, has been found to affect numerous economically important plants. Understanding the molecular basis of resistance, tolerance and susceptibility of plants to pathogens such as R. solanacearum is a major goal of molecular plant pathologists. Prior to this study it was thought that Arabidopsis accession Kil-0 shows gene-for-gene “resistance” to an African Eucalyptus isolate of R. solanacearum, BCCF402. However, a subsequent preliminary study indicated that Kil-0 may exhibit “tolerance” which is defined as the plant’s ability to support high pathogen numbers without displaying disease symptoms or a reduction in host fitness. The aim of this study was to determine if Kil-0 was tolerant to R. solanacearum BCCF402. The bacterial load of R. solanacearum was quantified in accessions Kil-0 and Be-0 using dilution plating and quantitative PCR methods. The cytC gene region was used to quantify R. solanacearum in Arabidopsis plants and the amount of bacterial DNA was normalized to “alien” DNA that was spiked into each sample. High bacterial concentrations of BCCF402 were found in Kil-0 but plants exhibited no wilting symptoms. Additionally, Kil-0 plants inoculated with BCCF402 showed no significant reduction in fitness compared to control Kil-0 plants. In contrast, high bacterial numbers and severe disease symptoms were observed in the susceptible Be-0 plants, whereas Nd1 plants contained a low number of bacteria and no disease symptoms indicative of a resistance response. These results illustrated that Kil-0 is tolerant to R. solanacearum isolate BCCF402. A tool for the visualization of R. solanacearum in Arabidopsis plants was designed. R. solanacearum isolate BCCF402 was tagged with two mCherry-containing plasmids under the constitutive expression of the tac promoter. The expression levels of mCherry were suitable for successful visualization in planta. BCCF402 cells transformed with the mCherry-containing plasmids were not affected in terms of virulence or disease progression compared to wildtype BCCF402 cells. A plasmid loss of 30-35% was observed in mCherry-tagged BCCF402 cells at later stages of Arabidopsis infection. mCherry-tagged BCCF402 was successfully visualized in Kil-0 leaves at early infection stages. / Dissertation (MSc)--University of Pretoria, 2014. / gm2014 / Plant Science / unrestricted

Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / Unrestricted

No description available.

Made available in DSpace on 2014-06-11T19:35:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-11-25Bitstream added on 2014-06-13T19:05:03Z : No. of bitstreams: 1 theodoro_gf_dr_botfca.pdf: 486962 bytes, checksum: 133c964b71961b171a0d897af6f2f21e (MD5) / Universidade Estadual Paulista (UNESP) / Este trabalho visou avaliar a ocorrência da bactéria Curtobacterium flaccumfaciens pv. flaccumfaciens em lavouras de feijoeiro localizadas em alguns municípios do Estado de Santa Catarina nas safras 2002/03 e 2003/04; a reação de genótipos de feijoeiro à murcha-de-curtobacterium; e o efeito de doses de nitrogênio (N) e potássio (K) sobre a severidade da doença, peso da matéria seca e o conteúdo de diversos nutrientes na parte aérea das cultivares IAC Carioca Pyatã, IPR 88 - Uirapuru e SCS 202 - Guará. Foram coletadas plantas com sintoma de murcha em lavouras de feijoeiro em 12 municípios. Posteriormente, procedeu-se ao isolamento, à purificação e à caracterização da bactéria e aos testes de patogenicidade. Aos 10 dias após a semeadura, plântulas de 24 genótipos de feijoeiro, conduzidas em casa-de-vegetação, foram inoculadas com o isolado FJ 36. A severidade da murcha-de-curtobacterium foi avaliada, a cada cinco dias, até aos 25 dias após a inoculação. Tanto no experimento que avaliou o efeito do N (uréia) quanto do K (cloreto de potássio) na severidade da doença, empregou-se a dose recomendada pela análise de solo e variações de 25 e 50 % abaixo e acima da mesma. Foi coletada a parte aérea das plantas antes e após as adubações, aos 25 DAS, para se aferir o peso da matéria seca e o conteúdo de N, fósforo (P), K, cálcio (Ca) e magnésio (Mg). Constatou-se que C. f. pv. flaccumfaciens esteve presente em plantas cultivadas nos municípios de Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada e Tigrinhos. Dos genótipos avaliados, somente as cultivares IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considerados como padrões de resistência à doença, apresentaram-se com as menores notas de severidade média e valores de área abaixo da curva do progresso da murcha-de-curtobacterium (AACPMC)... . / This work aimed evaluate the occurrence of the bacteria Curtobacterium flaccumfaciens pv. flaccumfaciens in common bean fields of Santa Catarina State, during the harvest of 2002/03 and 2003/04; the reaction of bean genotypes to the bacterial wilt; and the effect of nitrogen (N) and potassium (K) levels on the severity of the disease, the weight of the dry mass and the content of nutrients I nthe aerial part of the cultivars IAC Carioca Pyatã, IPR 88 - Uirapuru and SCS 202 - Guará cultivars. Plants with symptoms of wilt were collected in bean fields of 12 cities. It was made the isolation, purification and cultural characterization of the bacteria and the procedures to fulfill the Kochþs postulates. The inoculation with the strain FJ 36 was done in the 10th day after the sow of 24 common bean genotypes, under greenhouse conditions. The bacterial wilt severity was evaluated, in each five days, until the 25th day after inoculation. It was adopted as treatments the recommended level of N (urea) and K (potassium chloride), by the soil analysis, as well levels 25% and 50% under and below it. The aerial part of the plants was collected before and after the fertilizations, to determine the weight of the dry mass and the content of N, phosphorus (P), K, calcium (Ca) and magnesium (Mg). Curtobacterium flaccumfaciens pv. flaccumfaciens was detected in plants cultivated in Campos Novos, Faxinal dos Guedes, Guatambu, Ipuaçu, Ponte Serrada and Tigrinhos. Regarding the evaluated genotypes, only the cultivars IAC Carioca Akytã, IAC Carioca Aruã e IAC Carioca Pyatã, considered as patterns of resistance, had the lower values of average severity and area under the bacterial wilt progress curve (AUBWPC). However, although to have been considered susceptible, the cultivars SCS 202 - Guará and IPR Graúna showed relatively low values of AUBWPC... (Complete abstract, click electronic address below).

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.