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.

Made available in DSpace on 2016-12-08T16:44:34Z (GMT). No. of bitstreams: 1 PGPV11MA038.pdf: 1217077 bytes, checksum: 00344fc81180f6d99a71b0ff111924e8 (MD5) Previous issue date: 2011-02-09 / Curtobacterium wilt has become one of the most important emerging diseases in bean plants. Studies aimed at identifying sources of resistance and to know the genetic control of disease are essential for the development of resistant cultivars. The aim of this study was to identify bean genotypes resistant to curtobacterium wilt and determine the inheritance of resistance of this disease on bean, to enable the development of resistant cultivars in a breeding program. Were evaluated 72 genotypes, among which are 67 accessions of the Active Germplasm Bank of beans at the Universidade do Estado de Santa Catarina (UDESC). It was possible to identify Xan 159 as being tolerant to the disease, because it showed the lowest average scores for symptoms of bacterial wilt and is indicated for use in breeding programs to develop resistant cultivars. The study of inheritance of resistance to bacterial wilt was accomplished through a complete diallel of five parents (IAC Carioca Aruã, IAC Carioca Pyatã, IAC Carioca Tybatã, SCS Guará and Pérola) and through study of six generations of people IAC Carioca Aruã x Guara and IAC Carioca Pyatã x Pérola. Diallel analysis showed that although both the additive effects and non-additive are involved, the additive effect is more important in controlling the curtobacterium wilt. The IAC Carioca Pyatã had the highest general combining ability and is recommended for use in breeding programs that aim to develop resistant genotypes. The analysis of the means of generations between genotypes tolerant x susceptible (IAC Carioca Aruã x SCS Guará and IAC Carioca Pyatã x Pérola), demonstrates the importance of the additive effects on the character determination. Analyses of variance of six generations agree with the results found by the analysis of averages, where the additive effect has greater importance for the inheritance of curtobacterium wilt. The narrow sense heritability was around 35%, demonstrating that it is possible to gain with the selection of resistant individuals / A murcha de curtobacterium tem se mostrado uma das doenças emergentes mais importantes na cultura do feijão. Estudos que visem identificar fontes de resistência e conhecer o controle genético da doença são fundamentais para o desenvolvimento de cultivares resistentes. O objetivo deste trabalho foi identificar genótipos de feijão resistentes à murcha de curtobacterium e estudar a herança da resistência desta doença em feijão, para possibilitar o desenvolvimento de cultivares resistentes em programas de melhoramento. Foram avaliados 72 genótipos, dentre os quais estão 67 acessos pertencentes ao Banco Ativo de Germoplasma de Feijão da Universidade do Estado de Santa Catarina (UDESC). Foi possível identificar a linhagem Xan 159 como tolerante à doença, pois apresentou as menores médias para as notas dos sintomas da murcha de curtobacterium, sendo indicado para uso em programas de melhoramento para o desenvolvimento de cultivares resistentes. O estudo da herança da resistência à murcha de curtobacterium foi realizado, através de um dialelo completo com cinco genitores (IAC Carioca Aruã, IAC Carioca Pyatã, IAC Carioca Tybatã, Pérola e SCS Guará) e através do estudo das seis gerações da populações IAC Carioca Aruã x Guará e IAC Carioca Pyatã x Pérola. A análise dialélica mostrou que, embora ambos os efeitos aditivos e não-aditivos estão envolvidos, o efeito aditivo é mais importante no controle da murcha de curtobacterium. O genótipo IAC Carioca Pyatã apresentou a maior capacidade geral de combinação e é recomendado para uso em cruzamentos dirigidos que objetivam o desenvolvimento de genótipos resistentes. A análise das médias das gerações entre genótipos tolerantes x suscetíveis (IAC Carioca Aruã x SCS Guará e IAC Carioca Pyatã x Pérola), demonstra a importância dos efeitos aditivos para a determinação do caráter. As análises das variâncias das seis gerações concordam com os resultados encontrados pela análise das médias, onde o efeito aditivo possui maior importância para a herança da murcha de curtobacterium. A herdabilidade no sentido restrito foi em torno de 35%, demonstrando que há possibilidade de obter ganhos com a seleção de indivíduos resistentes

La culture de l’aubergine est confrontée au flétrissement bactérien, maladie causée par le complexe d’espèces Ralstonia solanacearum. La résistance variétale est la méthode la plus efficace pour contrôler cette maladie. Un QTL majeur (ERs1) a précédemment été cartographié dans une population de lignées recombinantes (RIL) issue du croisement aubergine sensible (S) MM738 × aubergine résistante (R) AG91-25. Initialement, ERs1 a été détecté avec 3 souches du phylotype I, alors qu’il est contourné par la souche PSS4 de ce même phylotype. Les objectifs de cette thèse étaient (i) de préciser la position d’ERs1 et de définir son spectre d’action, (ii) d’identifier d’autres QTLs contrôlant les souches virulentes sur AG91-25, et (iii) d’introgresser certains de ces QTLs dans des cultivars S. Pour cela, 2 populations d'haploïdes doublés (HD), MM152 (R) × MM738 (S) et EG203 (R) × MM738 (S), ont été créées. La population RIL a été phénotypée avec 4 souches supplémentaires appartenant aux phylotypes I, IIA, IIB et III, tandis que les populations HD l’ont été avec les souches virulentes PSS4 et R3598. Les analyses de cartographie génétique ont confirmé l’existence d’ERs1 (renommé EBWR9), défini sa position sur le chromosome (chr) 9 et validé son contrôle spécifique de 3 souches du phylotype I. EBWR2 et EBWR14, 2 autres QTLs à large spectre, ont été détectés sur les chr 2 et 5. Les analyses QTL ont mis en évidence un système de résistance de type polygénique chez EG203. Le transfert de la résistance dans 2 cultivars locaux a été initié et a permis l’introgression d’EBWR9 et d’EBWR2. Ces résultats ouvrent des perspectives quant à la création de variétés à large spectre de résistance. / Eggplant cultivation is confronted by the bacterial wilt disease caused by the Ralstonia solanacearum species complex. Breeding resistant cultivars is the most effective strategy to control the disease but is limited by the pathogen’s extensive genetic diversity. A major QTL (ERs1) was previously mapped in a recombinant inbred lines (RIL) population from the cross of susceptible (S) MM738 × resistant (R) AG91-25 lines. ERs1 was originally found to control 3 strains from phylotype I, while being ineffective against the strain PSS4 from the same phylotype. The objectives of this thesis was to (i) clarify the position of ERs1 and define its spectrum of action, (ii) found other QTLs, promptly to control virulent strains on AG91-25 and (iii) introgress some of the QTLs into two S cultivars. For this purpose, the new doubled haploid (DH) populations MM152 (R) × MM738 (S) and EG203 (R) × MM738 (S) were created. The RIL population was phenotyped with 4 additional RSSC strains belonging to phylotypes I, IIA, IIB and III and the DH populations were phenotyped with virulent strains PSS4 and R3598. QTL mapping confirmed the existence of ERs1 (renamed EBWR9), defined its position on chromosome (chr) 9 and validated its specific control of 3 phylotype I strains. EBWR2 and EBWR14, 2 broad-spectrum resistance QTLs, were detected on chr 2 and 5. QTL analysis reveals a polygenic system of resistance in EG203. The transfer of resistance into 2 local cultivars was initiated and allowed the introgression of EBWR9 and EBWR2 QTLs through a backcross scheme. These results offer perspectives to breed broad-spectrum R cultivars.

Orientador: Antonio Carlos Maringoni / Banca: Marli Teixeira de Almeida Minhoni / Banca: Renate Krause Sakate / Banca: Luís Otavio Saggion Beriam / Banca: Ivan Paulo Bedendo / Resumo: A murcha-de-curtobacterium, causada por Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), é uma das principais doenças bacterianas da cultura do feijoeiro, acarretando grandes perdas na produção dessa cultura. Até o momento existem poucas informações sobre os diferentes nichos de sobrevivência desta bactéria e de sua gama de hospedeiras. Em vista disso, este trabalho teve por objetivos principais verificar a capacidade de sobrevivência saprofítica de Cff em restos de cultura de feijoeiro mantidos na superfície do solo e enterrados à 20 cm de profundidade; a influência da temperatura, umidade e do tipo de solo no período de sobrevivência da bactéria em solo; determinar a gama de hospedeiras de Cff inoculadas artificialmente, tanto por ferimento no caule, como por aspersão de suspensão bacteriana na parte aérea das plantas; a capacidade de colonização de Cff do rizoplano de plantas de aveia e trigo; e o efeito do pré-plantio de aveia e trigo na ocorrência da murcha-de-curtobacterium. Quanto à capacidade de sobrevivência de Cff em restos de cultura de feijoeiro, foi demonstrado que a bactéria possui menor capacidade de sobrevivência quando os restos vegetais são incorporados ao solo e também em épocas com maiores índices de precipitação e temperaturas mais altas. O período de sobrevivência do patógeno nos restos culturais de feijoeiro mantidos na superfície do solo variou entre 165 e 240 dias e nos restos vegetais enterrados a 20 cm de profundidade, o período de sobrevivência foi inferior a 30 dias. Quanto à sobrevivência de Cff na forma de células livres no solo, foi verificado que a temperatura, a umidade e o tipo do solo têm influencia na capacidade de sobrevivência da bactéria. O tempo de sobrevivência de Cff variou entre dois e quinze dias. Das 30 espécies botânicas inoculadas artificialmente com Cff, a bactéria causou lesões na parte... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) is one of the main bacterial diseases affecting bean culture, leading to great losses in its production. So far there is scarce information about the different survival niches and host range of this bacterium. Thus, the present study had as major aims to verify Cff capability of saprophytically surviving in bean debris kept on the soil surface and buried at 20 cm depth; to assess the influence of temperature, humidity and soil type on the survival period of this bacterium in soil; to determine the host range for artificially inoculated Cff, either through stem injury or through bacterial sprinkling onto the shoot of plants; to verify Cff capability of colonizing the rhizoplane of oat and wheat plants; and to assess the effect of oat and wheat pre-planting on the occurrence of bean bacterial wilt. Cff had decreased capability of surviving in bean debris when the latter were incorporated into the soil and during periods of higher rainfall rates and temperatures. The pathogen survival period in bean culture remnants kept on the soil surface ranged from 165 and 240 days, while in plant debris buried at 20 cm depth the survival period was inferior to 45 days. The survival capability of Cff as free cells in soil was influenced by temperature, humidity and soil type. Cff survival time varied between two and fifteen days. Of 30 plant species artificially inoculated with Cff, bean and soy shoot had lesions caused by the bacterium which endophytically colonized wheat leaves and soy and wheat stem and leaves. Cff was also shown to have no capability of colonizing oat and wheat rhizoplane while the pre-planting of these grass plants, before bean culture establishment, had no effect on the occurrence of bean bacterial wilt / Doutor

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-06-29T19:03:41Z No. of bitstreams: 1 2015 - Francine Yuriko Otsuka Rocha.pdf: 2503949 bytes, checksum: b9cea1d05fae4647a2e2459b6401ecc0 (MD5) / Made available in DSpace on 2017-06-29T19:03:41Z (GMT). No. of bitstreams: 1 2015 - Francine Yuriko Otsuka Rocha.pdf: 2503949 bytes, checksum: b9cea1d05fae4647a2e2459b6401ecc0 (MD5) Previous issue date: 2015-09-09 / CAPES / The fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici is a disease of economic importance to tomato crop (Lycopersicon esculentum Mill.) because of the losses caused in the production. This study aimed to select bacteria belonging to the genus Bacillus which have antagonistic activity to fungi present in tomato producing areas. It was obtained 193 bacterial isolates from tomato roots and stems. We performed the complete sequencing 16S rRNA subunit of 32 bacteria from the work collection using the 27F and Amp2 primers. These bacteria were randomly selected, and 20 bacteria were obtained from healthy tomato plants. The results showed a high similarity to species of the genus Bacillus: B. thuringiensis, B. megaterium, B. cereus, B. toyonensis, B. stratosphericus and Paenibacillus barcinonensis. Evaluation of the functional characteristics of the bacterial strains showed that 72% possess proteolytic activity, 78% have cellulolytic activity, 48% are able to solubilize inorganic phosphate, 42% solubilize zinc oxide and 40.8% are able to produce indols. Antagonism tests carried out in vitro showed that some bacterial strains reduced the mycelial growth of the three races of the fungus when subjected to direct method (parallel streaks). An experiment was conducted using the genotypes Debora Plus, Santa Clara Miss Brazil, Perinha and Carolina in order to select a sensitive tomato genotype to the three fungal races. The Perinha variety was the most susceptible for races 1, 2 and 3 and presented the highest percentage of fungal colonization. Thus, this variety was used in a greenhouse experiments to evaluate the antagonistic role of the Bacillus strains against three fungal races. The antagonistic activity of bacteria was assessed 15 days after transplanting of the tomato seedlings to contaminated substrate. The results showed that the B. stratosphericus strain contributed to control at least 93.25% of the damage caused by race 1, strains from species B. megaterium and B. thuringiensis controlled 100% the damage caused by race 2 and at least 67.35% of the disease caused by the race 3 of the fungus. The results suggest that some strains have the potential to control race 1, 2 and 3 of the F. oxysporum f. sp. lycopersici and have functional activities that may contribute to improving the growth of tomato plants. However, field studies are needed to confirm the role of these Bacillus species during the interaction with tomato plants grown in soil contaminated with the race 1, 2 and 3 of the fusarium wilt. / A murcha-de-fus?rio causada por Fusarium oxysporum f. sp. lycopersici ? uma doen?a de grande import?ncia econ?mica para a cultura do tomateiro (Lycopersicon esculentum Mill.) devido as perdas que ocasiona na produ??o. O presente estudo teve como objetivo selecionar bact?rias pertencentes ao g?nero Bacillus que apresentam atividade antag?nica para aplica??o em ?reas produtoras de tomate infestadas pelo fungo. Foram obtidos 193 isolados de bact?rias de ra?zes e hastes de tomateiro. Foi realizado o sequenciamento completo da subunidade 16S RNAr de 32 isolados da cole??o de trabalho, utilizando os iniciadores 27F e Amp2. Essas bact?rias foram selecionadas aleatoriamente, sendo que 20 bact?rias foram obtidas de tomateiro sadio. Os resultados mostraram uma alta similaridade das estirpes com as esp?cies do g?nero Bacillus: B. thuringiensis, B. megaterium, B. cereus, B. toyonensis, B. stratosphericus, al?m da esp?cie Paenibacillus barcinonensis. Testes para avaliar as caracter?sticas funcionais das estirpes bacterianas in vitro mostraram que 72% das estirpes possuem atividade proteol?tica, 78% possuem atividade celulol?tica, 48% solubilizam fosfato inorg?nico, 42% solubilizam zinco e 40,8% s?o capazes de produzir indol. Testes de antagonismo realizados in vitro mostraram que algumas estirpes bacterianas retardaram ou reduziram o crescimento micelial das tr?s ra?as do fungo quando submetidos ao m?todo de a??o direta (estrias paralelas). Com o objetivo de selecionar um gen?tipo de tomateiro sens?vel as tr?s ra?as do fungo, foi instalado um experimento utilizando os gen?tipos D?bora Plus, Santa Clara Miss Brasil, Perinha e Carolina. A variedade Perinha foi a mais suscet?vel as ra?as 1, 2 e 3 j? que apresentou um maior percentual de coloniza??o do fungo. A atividade antag?nica das bact?rias foi avaliada 15 dias ap?s o transplantio das mudas de tomate da variedade Perinha para o substrato contaminado. Os resultados mostraram que a estirpe da esp?cie B. stratosphericus controlou cerca de 93,25% das plantas crescidas na presen?a da ra?a 1 do fungo. J? as esp?cies B. megaterium e B. thuringiensis controlaram 100% da doen?a das plantas de tomateiro cultivadas em substrato com a ra?a 2 e de 67,35% das plantas cultivadas em substrato com a ra?a 3 do fungo. Os resultados obtidos sugerem que algumas estirpes possuem potencial para controle de F. oxysporum f. sp. lycopersici ra?a 1, 2 e 3 e possuem atividades funcionais que podem contribuir para a melhoria do crescimento das plantas de tomateiro cultivadas em ?reas infestadas com o fungo. Entretanto, estudos em campo s?o necess?rios para confirmar o papel dessas esp?cies de Bacillus na intera??o com plantas de tomateiro cultivadas na presen?a das ra?as 1, 2 e 3 da murcha de fus?rio.

A murcha bacteriana, causada por Ralstonia solanacearum, afeta principalmente as solanáceas, destacando-se as culturas da batata, berinjela, jiló, pimentão e tomate. No presente trabalho foi conduzida a caracterização molecular de isolados de R. solanacearum e sua possível relação com características relacionadas à morfologia, bioquímica, patogenicidade, agressividade e distribuição geográfica. Foram utilizados 51 isolados pertencentes à biovar 2, sendo 9 provenientes de berinjela e 42 de batata, coletados em diversas regiões brasileiras. A análise molecular permitiu separar os isolados em quatro grupos distintos de padrões de bandas para os iniciadores BOX e ERIC, e em cinco para o iniciador REP. Não foi encontrada relação dos grupos de isolados caracterizados molecularmente com tamanho de colônias, ocorrência de mutantes, produção de melanina, capacidade de colonização do sistema radicular e resistência a antibióticos/fungicidas. A identificação de isolados de batata, como biovar 2-A, e de berinjela, como biovar 2-T, com base em teste bioquímico do uso de trealose, foi confirmadas pela análise molecular. Não houve variação de agressividade entre os isolados inoculados em batata e berinjela, exceção feita ao isolado avirulento CNPH-65. Portanto, isolados das biovares 2-A e 2-T podem infectar estas duas hospedeiras com a mesma intensidade sob altas temperaturas. Para todos os isolados, o desenvolvimento da população bacteriana foi significativamente maior no sistema radicular de plantas das cultivares suscetíveis, tanto para batata como para berinjela. No entanto, dentro de cada cultivar, os isolados se comportaram de maneira semelhante, não sendo possível fazer distinção entre os mesmos. A tentativa de se associar grupos de isolados caracterizados molecularmente com os locais de origem revelou alguns aspectos interessantes. O grupo I agregou somente isolados do Paraná. No grupo II ficaram isolados da Bahia, Distrito Federal e do Paraná. No Grupo III, foram reunidos todos os isolados de berinjela e um único de batata, sendo todos procedentes do Distrito Federal. O grupo IV, de forma semelhante ao grupo II, reuniu isolados de locais diversos como Paraná, Goiás, Rio Grande do Sul e Distrito Federal. Portanto, nos grupos I e III parece haver uma tendência de relação entre grupamento molecular e local de origem, enquanto que para os grupos II e IV, isolados de características genéticas similares são provenientes de locais distintos, apontando considerável diversidade genética do patógeno. / The bacterial wilt disease caused by Ralstonia solonacearum affects mainly the solanaceous species, specially potato, eggplant, peppers, tomato and brazilian gilo (Solanum gilo). This work reports the molecular characterization of R. solanacearum biovar 2 isolates and the possible relationship of this molecular data with other characteristics related to morphology, biochemistry, pathogenicity, aggressiveness and geographical distribution. Fifty-one biovar 2 isolates were studied, 9 isolated from eggplant and 42 from potato, all of them collected from different regions of Brazil. According to the molecular analysis, the isolates were clustered in four different groups, with distinct band patterns to the primers BOX and ERIC, and five groups to the primers REP. There was no relationship between the groups clustered through molecular analyses and phenotypic characteristics, such as colony size, presence of mutants, melanin presence, capability of root system colonization and antibiotic/fungicide resistance. The identification of potato isolates as the biovar 2-A, and the eggplant isolates as biovar 2-T, based on biochemical tests using trealose were confirmed with the molecular analyses. There was no variation of aggressiveness in the isolates inoculated on potato an eggplant, except the avirulent isolate CNPH-65. Consequently, isolates of biovars 2-A and 2-T are able to infect both hosts with the same aggressiveness under high temperatures. The population of all isolates developed in significant levels at the root system of susceptible cultivars of both hosts, potato and eggplant. However, considering each cultivar tested, there was no difference between isolates. Interesting results were observed when the isolates clustered based on molecular data were associated with the geographical region of their collection. The group I clustered only the isolates collected in Paraná. The group II clustered the isolates collected in Bahia, Federal District and some in Paraná. The group III clustered all isolates from eggplant and only one of potato, all of them collected in the Federal District. The group IV, as the group II, clustered isolates from different regions, like Paraná, Goiás, Rio Grande do Sul and Federal District. These results suggest a relationship between the isolates clustered through molecular analysis in the groups II and III and their geographical region of collection. The isolates clustered in the same way, with similar genetic background in the groups II and IV, were however collected in different regions, showing the great genetic variation of this pathogen.

Os microrganismos endofíticos são aqueles, cultiváveis ou não, que habitam o interior da planta hospedeira sem causar danos aparentes ou estruturas externas visíveis. Essa interação planta-microrganismo é intrínseca a determinadas espécies de plantas e/ou bactérias. Nas últimas décadas os estudos de microrganismos endofíticos têm sido realizados em diversas plantas hospedeiras, sendo esses estudos direcionados, principalmente, para a diversidade e características benéficas induzidas, como o controle biológico de doenças. As doenças com sintomas de murcha e cancro são consideradas emergentes no setor florestal. O Brasil está entre os maiores produtores mundiais de Eucalyptus e a expansão do setor, juntamente com o cultivo clonal, tem acarretado o aumento da incidência de patógenos. O surgimento de novas doenças exige estudos relacionados tanto a interação do agente patogênico com hospedeiro quanto de todos os componentes do patossistema. Neste contexto, os microrganismos endofíticos e do rizoplano têm sido descritos como potenciais controladores biológicos de doenças. Dessa forma, o presente trabalho teve por objetivos avaliar a interação da comunidade bacteriana associada a Eucalyptus sp. com fungos causadores de murcha e cancro. Foi também estudada a diversidade e colonização desses endófitos em sementes e o efeito da germinação nessa interação. Adicionalmente, foi realizada uma busca por bactérias associadas a Eucalypus sp. com capacidade de controlar fungos causadores de murcha e cancro. Foi observado que a densidade bacteriana endofítica não difere entre as espécies de Eucalyptus citriodora, E. urophylla e E. globulus x E. grandis avaliadas e que esta comunidade cultivada é composta por bactérias pertencentes às classes Actinobacteria, Proteobacteria e Firmicutes. Com uso de diferentes ferramentas de estudo foi observado que a quantidade de bactérias endofíticas nas sementes aumenta proporcionalmente ao tempo de germinação. Foi observado também que os sintomas de murcha e cancro causados, principalmente, por Ceratocystis fimbriata podem ser causados por outros fungos patogênicos. Foram identificados 41 isolados bacterianos endofíticos e do rizoplano com capacidade de controlar fungos patogênicos causadores de murcha e cancro em Eucalyptus sp. Adicionalmente ao potencial para uso no controle biológico do cancro e da murcha de Eucalyptus, as bactérias de sementes aqui relatadas, podem ter papel importante de proteção das plantas no campo. Futuros estudos devem ser direcionados para identificação de genes associados a essa inibição, bem com análise dos fatores responsáveis por este controle biológico no campo. / The endophytic microorganisms are those, cultivated or not, that inhabit the interior of the plant host without causing apparent damages or visible external structures. This interaction plant-microorganism is specific to certain species of plants and/or bacteria. In the last few years studies of endophytic microorganisms have been carried out for several plant hosts, which are focused mainly to diversity and biotechnological potential, such as biological control of disease. Brazil is one of the largest world Eucalyptus producers and the increasing of the Eucalyptus production associated to clonally reproduction has allowed an increasing incidence of some pathogens. These new disease demands on studies related to causal agent and biotic and abiotic factors associated to the diseases. The canker of Eucalyptus is considered an emerging disease by several reforestation companies. In that way, the rhizoplane and endophytic microorganisms has been described as potential diseases biological control agents. In this context, the aims of the present work were to assess the bacterial community associated to Eucalyptus sp. plants and seeds, besides the interaction with the wilt and canker pathogenic fungi. We also assessed the diversity and colonization effect of seed germination on bacterial community. Additionally, was carried out a screening of the Eucalypus sp. associated bacteria against pathogenic fungi that cause wilt and canker. We observed that endophytic bacterial density doesn\'t differ among the species of Eucalyptus citriodora, E. urophylla and E. globulus x E. grandis and that this cultivated community is composed by follow bacterial classes Actinobacteria, Proteobacteria and Firmicutes. Using different study approaches was observed that the amount of endophytic bacteria in the seeds increases proportionally at the germination time. We also observed that the symptoms of wilt and canker caused, mainly, by Ceratocystis fimbriata may be caused by other pathogenic fungi too. Additionally, were identified 41 endophytic and rhizoplane bacterial isolates able to control pathogenic fungi that cause Eucalyptus wilt and canker. Besides to the wilt and canker biological control potential of that bacteria, the seed endophyte identified in the present work can have important function on plant protection in the field. New studies should be addressed for identification of genes related to antagonism, besides of factors responsible for biological control occurrence in the field.

Ralstonia solanacearum, une béta-proteobactérie d'origine tellurique, est l'une des phytobactérioses les plus nuisibles au niveau mondial. Cette bactérie est capable d'infecter plus de 250 espèces différentes dont certaines présentent un intérêt économique majeur (tomate, pomme de terre, tabac). R. solanacearum est divisée en 4 phylotypes distincts présentant des origines géographiques différentes : I (asiatique), IIA et IIB (américain), III (africain), IV (indonésien). Parmi ces phylotypes, le phylotype I est en expansion démographique, hautement recombinogène, réparti mondialement et possède une large gamme d'hôtes. Il possède donc un fort potentiel évolutif (sensu McDonald et Linde, 2002). Afin de contrôler cette bactérie, la lutte génétique reste la méthode la plus prometteuse : elle consiste à déployer des cultivars possédant différents sources de résistance (i.e., des gènes de résistance). La variété d'aubergine AG91-25 (E6) possède un gène majeur de résistance (ERs1) lui permettant de contrôler certaines souches de R. solanacearum de phylotype I. Cependant, la gestion de cette résistance requiert d'étudier au préalable sa durabilité afin d'en éviter le contournement. Cette durabilité peut être estimée en étudiant le potentiel évolutif d'un agent pathogène face à cette source de résistance, ainsi qu'en décryptant les mécanismes moléculaires de l'interaction entre l'hôte (gène R) et le pathogène (effecteur de types trois). Afin d'étudier la dynamique évolutive de R. solanacearum sous une pression de sélection exercée par la variété résistante E6, nous avons mis en place un essai d'évolution expérimentale au champ. Cet essai est composé de trois couples de microparcelles d'aubergines résistantes E6 et d'aubergines sensibles E8, implantées deux fois par an, pendant trois ans (soit 5 cycles). Un schéma MLVA (« Multi-Locus VNTR Analysis ») composé de 8 loci minisatellites a été développé afin de caractériser les souches extraites de ces cycles de cultures. Ces VNTR sont spécifiques aux souches de R. solanacearum de phylotype I, hautement polymorphes et discriminants à toutes les échelles : mondiale, régionale et locale. Nos résultats démontrent une absence de contournement de la résistance d'E6 par les populations parcellaires de R. solanacearum, confirmant le caractère durable de cette résistance. Cette variété aurait fortement réduit les populations bactériennes du sol, ne leur permettant plus d'infecter l'hôte résistant. Parallèlement, 100% des plants d'E8 sont morts à partir du cycle 2. La maladie au sein des microparcelles semble progresser selon une dynamique de « plante-à-plante ». Une baisse de la diversité génétique a aussi été observée au cours des cycles de culture répétés d'E8, associée à l'augmentation en fréquence de deux haplotypes. Cependant, aucune structuration génétique claire n'a été observée à l'échelle de la parcelle entière ou de la microparcelle. En revanche, les données d'isolement par la distance semblent indiquer qu'une structure spatiale semble être en cours d'établissement. L'ensemble de nos résultats suggère une structure épidémique clonale de nos populations parcellaires. Nous nous sommes aussi intéressés à l'implication de 10 ET3 dans l'interaction R. solanacearum vs aubergine résistante (E6). La distribution des 10 ET3 candidats est variable au sein d'une collection de souches phylogénétiquement diverses (91 souches) : ripAJ et ripE1 sont les ET3 les plus partagés alors que ripP1 et ripP2 sont les moins fréquemment. Certains ET3 présentent peu (ripAJ) voire pas (ripE1 et ripP2) de polymorphisme de taille, alors que d'autres (ripAU) sont extrêmement polymorphes. Cependant la composition en effecteurs d'une souche ne semble pas être corrélée à un phénotype sur aubergine E6. Nous avons identifié le gène d'effecteur ripAX2 comme ayant une fonction d'avirulence sur aubergine résistante E6. Sa reconnaissance par E6 semble s'opérer au niveau de la zone hypocotylaire. / Ralstonia Solanacearum is a soilborn beta-proteobacterium responsible of bacterial wilt on Solanaceaous crops. This bacterium is considered as one of the most harmful plant disease worldwide. This bacterium possesses the ability to infect more than 250 different species, including crops with major economic importance (tomato, potato, tobacco, eucalyptus…). R. solanacearum is divided into four phylotypes originated from different areas: I (Asian), IIA and IIB (American), III (African), IV (Indonesian). Among these phylotype, phylotype I is currently in demographic expansion, is highly recombinogenic and has a wide hosts range. Thus, altogether, these characteristics demonstrated that this phylotype has a high evolutionary potential (sensu McDonald and Linde, 2002). In order to control this bacterium, genetic plant resistance seems to be the most promising method. This method consists in using cultivars with different source of resistance such as resistance genes and/or resistant QTLs. The AG91-25 (E6), an eggplant cultivar possessing a major resistance gene (ERs1), is capable to control some of phylotype I strains of R. solanacearum. However, in order to optimize the management of this resistance and to avoid its fast breakdown, we need to deeply investigate the durability of this resistant gene. Durability can be estimated by studying the evolutionary potential of our pathogen faced to E6 source of resistance and by understanding the molecular mechanisms underlying the interaction between the host (R gene) and its pathogene (Type III Effector – T3E). In order to study R. solanacearum evolutionary dynamics under selective pressure from E6 resistant cultivar, we set up an experimental evolution trial in the field. This trial consisted of three couples of resistant (E6) and susceptible eggplants (E8) microplots, implanted twice a year during three years, hence consisting of 5 cycles. A Multi-Locus VNTR Analysis (MLVA) scheme, consisting of 8 minisatellite loci, was developed in order to characterize the strains extracted from these crop cycles. These VNTRs were specific to R. solanacearum phylotype I strains, they were highly polymorphic and discriminatory at different scale: globally, regionally and locally.Our results showed no breakdown of E6 resistance by R. solanacearum populations, which confirms that this resistance is durable. It seemed that this cultivar reduced the soil bacterial population, preventing bacterial population to infest the resistant host. At the same time, 100% of the E8 plants have died, starting at cycle 2. Bacterial wilt seemed to spread with a “plant-to-plant” dynamics within each microplot. Genetic diversity reduction was also observed during the successive cycle of susceptible eggplant, associated with the increase of frequency of two main haplotypes. However, we failed to identify a clear genetic structuration, neither at the plot scale nor at the microplot scale. Nevertheless, isolation-by-distance data seemed to show that a spatial structure is currently establishing. Altogether, our results suggested that our plot populations appeared to have a clonal epidemic structure.We also looked into 10 T3Es' involvement in the interaction between R. solanacearum and the resistant eggplant (E6). Their distribution was completely different within a collection of phylogenetically diverse strains (91 strains): ripAJ and ripE1 are the most shared T3Es whereas ripP1 and ripP2 were the less common T3E whithin our collection of strains. Some T3Es showed few (ripAJ) or no length polymorphism at all (ripE1 and ripP2) whereas some other (ripAU) are extremely polymorphic. Nevertheless, the T3E effector repertoire did not seemed to be correlated to a specific phenotype on E6 eggplant. Its recognition by E6 seemed to occur in the hypocotyle region rather than in the mesophyll, highlighting a possible organ-specificity of the interaction between ERs1 and ripAX2.

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