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

L’ingénierie agroécologique vise à produire des savoirs actionnables, pour concevoir des systèmes de cultures économiquement et écologiquement performants, par la valorisation de régulations naturelles. Notre problématique est centrée sur la santé des cultures, et plus particulièrement sur la bactérie phytopathogène Ralstonia solanacearum, agent du flétrissement bactérien alors qu’une souche extrêmement agressive menace la production de tomates en plein champ en Martinique. La nécessité d’explorer et de développer des alternatives aux méthodes conventionnelles de protection des plantes (variétés résistantes, pesticides), actuellement inefficaces, invite à la mise en œuvre d’une démarche de conception innovante. Nos travaux montrent que la mobilisation d’une barrière rhizosphérique est une stratégie de régulation biologique alternative. Différents processus y contribuent, telle que la mycorhization, symbiose entre racines et champignons mycorhiziens à arbuscules, présents dans la plupart des sols. Nous montrons que la mobilisation de réseaux de mycorhizes indigènes à partir d’un sol agricole permet une mycorhization précoce de la tomate. De plus, l’association de plantes aux propriétés mycorhizotrophes et assainissantes en conditions contrôlées montre des effets bioprotecteurs partiels et ouvre de nouvelles perspectives de combinaisons entre processus. Ces combinaisons sont mobilisables par des leviers d’actions multi-scalaires. Nous avons produit une grille d’analyse générique de ces leviers d’action pour la conception, par des trajectoires d’innovation multidirectionnelles, de « systèmes de culture bioprotégés ». Dans le contexte agricole martiniquais, une démarche d’apprentissage permet en effet l’émergence d’une dynamique de co-conception de systèmes de cultures recourant aux plantes mycorhizotrophes. Nos travaux proposent des outils pour une exploration collective de nouvelles stratégies de gestion durable de la santé des cultures. / Agroecological engineering aims to produce actionable knowledge to design economically and environmentally efficient cropping systems, based on the exploitation of natural regulation mechanisms. Our issue is centered on crop health, especially on the plant pathogenic bacterium Ralstonia solanacearum (bacterial wilt agent), of which an extremely aggressive strain threatens field tomato production in Martinique. The need to explore and develop alternatives to conventional methods of plant protection (resistant varieties, pesticides), ineffective in our case, calls for the implementation of an innovative design approach. Our work shows that the protection of the roots via the formation of a self-sustaining rhizospheric barrier may be an alternative biological control strategy. Different processes contribute, such as mycorrhizal symbiosis between roots and arbuscular mycorrhizal fungi, which are present in most soils. We show that the mobilization of indigenous mycorrhizal networks from an agricultural soil allows early mycorrhization of tomatoes. In addition, the association of plants with mycorrhizal and sanitizing properties in controlled conditions showed partial bioprotective effects and opens up new prospects for combinations between processes. These combinations may be exploited in various ways. We produced a generic analysis grid of key levers to design "healthy cropping systems " through multi-directional innovation trajectories. In Martinique's agricultural context, a learning process allows the emergence of a dynamic co-design of cropping systems using mycorrhizal plants. Our work thus provides tools for collective exploration of new sustainable management strategies for crop health.

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.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

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

Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a K_M of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.

La marchitez bacteriana de la papa (MB) causada por Ralstonia solanacearum (Smith, 1896) (Yabuuchi et al., 1995), es una de las más devastadoras enfermedades que afectan el cultivo de la papa en los países en desarrollo. En la actualidad, no existe un control químico efectivo. De todas las medidas de control, la utilización de variedades genéticamente resistentes es la mejor estrategia de manejo de la enfermedad y la que ofrece mejores perspectivas. El objetivo principal de este estudio fue identificar genotipos de especies silvestres de papa que presenten altos niveles de resistencia a la MB con énfasis en la resistencia a la infección latente en tubérculos. Para ello, se evaluaron 4461 genotipos diferentes de papa silvestre del Banco de Germoplasma del Centro Internacional de la Papa (CIP). 10 plantas por genotipo fueron inoculadas con una suspensión de la cepa CIP204 de R. solanacearum a una concentración de 1 x 108 ufc/g de suelo. Para asegurar la durabilidad de la resistencia, los genotipos resistentes fueron evaluados nuevamente contra 7 cepas de R. solanacearum con alta agresividad. Los genotipos resistentes a por lo menos 5 cepas fueron sometidos a una tercera prueba donde se evaluó además la resistencia a la infección en tubérculos en condiciones menos severas. Se encontraron 178 genotipos resistentes a la cepa CIP204 (4% de los tamizados). De 162 genotipos resistentes analizados, la resistencia pudo ser confirmada en sólo 52 genotipos en tamizados consecutivos con varias cepas del patógeno, de los cuales 9 genotipos también son resistentes al tizón tardío de la papa. De 26 genotipos analizados para la infección en tubérculos, 7 genotipos (6 de Solanum acaule y 1 de Solanum chacoense) fueron resistentes a la marchitez en planta y no mostraron infección latente ni en tallos ni en tubérculos. S. acaule y S. chacoense son las especies más promisorias para los futuros programas de mejoramiento. Esta es la primera evidencia de que altos niveles de resistencia a la marchitez bacteriana existe en la naturaleza. Palabras clave: MB, papa, resistencia, silvestres, infección latente. / --- The Bacterial Wilt of the Potato(BW) caused by Ralstonia solanacearum (Smith, 1896) (Yabuuchi et al., 1995), is one of the most devastating illnesses that affect the potato’s cultivation in the developing countries. At the present time an effective chemical control does not exist. Of all the control measures of BW, the use of genetically resistant varieties is the best handling strategy of the illness and the one that offer better perspectives. The main objective of this study was to identify genotypes of wild potato species that show high levels of resistance to bacterial wilt with emphasis in the resistance to the latent infection in tubers. For that reason, 4461 different genotypes from the Germoplasm Bank at the International Potato Center (CIP) Lima-Perú,were evaluated. Ten plants per genotype were inoculated with a suspension of the strain CIP204 of R. solanacearum to a concentration of 108 ufc/g soil. To ensure the durability of resistance, resistant genotypes were tested against seven strains of R. solanacearum with various levels of aggressiveness. The genotypes resistant to at least 5 strains of R. solanacearum were re-exposed to the pathogen in less severe conditions to assess the presence of latent infection in tubers. 178 genotypes (4%) of all screened were found to be resistant to CIP204 strain. From162 resistant genotypes analyzed, resistance could be confirmed in only 52 in subsequent screenings with several strains of the pathogen. From these, 9 genotypes are also resistant lo Late Blight. From 28 genotypes tested for tuber infection, seven (6 of S. acaule and 1 of S. chacoense) were found resistant to plant wilt and did not harbour any stem or tuber latent infection. S. acaule and S. chacoense are the most promising species for future breeding programs.This is the first evidence that high resistance levels of resistance to Bacterial Wilt exist in the nature. Keywords: BW, potato, resistance, wild, latent infection

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-08-25T16:15:33Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_karine_laste_macagnan.pdf: 989328 bytes, checksum: a2029037b34e4625051db4296d4fad83 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:59:34Z (GMT) No. of bitstreams: 2 dissertacao_karine_laste_macagnan.pdf: 989328 bytes, checksum: a2029037b34e4625051db4296d4fad83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T20:59:41Z (GMT) No. of bitstreams: 2 dissertacao_karine_laste_macagnan.pdf: 989328 bytes, checksum: a2029037b34e4625051db4296d4fad83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-28T20:59:48Z (GMT). No. of bitstreams: 2 dissertacao_karine_laste_macagnan.pdf: 989328 bytes, checksum: a2029037b34e4625051db4296d4fad83 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-12-04 / Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGS / O poli(3-hidroxibutirato) [P(3HB)] é o biopolímero mais estudado e caracterizado dentre a família dos polihidroxialcanoatos (PHAs), termoplásticos que têm como principais características a rápida biodegradabilidade e a biocompatibilidade. O processo de recuperação do bioplástico consiste em uma etapa importante no processo de produção. O desenvolvimento de metodologias mais seguras, econômicas e ambientalmente corretas, e que permitam um alto rendimento desse biopolímero, torna-se necessário para uma produção de P(3HB) economicamente e ecologicamente atrativa. Portanto, o objetivo do trabalho foi otimizar a metodologia clássica de recuperação de poli(3-hidroxibutirato) utilizando clorofórmio como solvente. O bioprocesso foi realizado em duas etapas, utilizando linhagem de Ralstonia solanacearum. O inoculo foi produzido em Erlenmeyers aletados de 500 mL, contendo 160 mL de meio YM e 40 mL de suspensão bacteriana, mantidos em incubador agitador orbital por 24 h, 150 rpm e 28 °C. A segunda etapa, produção de P(3HB), foi realizada utilizando Erlenmeyers aletados de 500 mL, contendo 160 mL de meio F4 e 20 % de inóculo, mantidos em 28 °C, 200 rpm e 72 h em incubador agitador orbital. Após a fermentação, as células foram separadas por centrifugação a 10.000 x g, lavadas com solução salina 0,89 % e secas a 56 °C. O acúmulo de P(3HB) na célula foi quantificado por cromatografia gasosa. A metodologia de extração foi otimizada em relação aos parâmetros: tempo de extração (2 h a 15 min), separação da biomassa/solução extrativa (papel filtro ou funil de separação), estado de célula (seca ou fresca) e proporção de solvente (10:1, 20:1 e 40:1 v/m). Por evaporação da solução extrativa foram obtidos filmes poliméricos. Os filmes recuperados foram analisados física e quimicamente por Espectroscopia de Infravermelho por Transformada de Fourier (FTIR), Calorimetria Diferencial de Varredura (DSC), Análise Termogravimétrica (TGA) e Cromatografia de Permeação em Gel (GPC). O acúmulo de P(3HB) foi de 51,15 %. Os maiores rendimentos de filme foram obtidos após 30 min de aquecimento, utilizando funil de decantação para separar a solução extrativa da biomassa, célula seca, e proporção de solvente 40:1 v/m, alcançando-se recuperação de 98 % do polímero acumulado. A análise dos filmes através de FTIR resultou em bandas características de P(3HB). Os filmes oriundos de células secas tiveram temperaturas inicial e final de degradação e grau de cristalinidade superiores aos filmes de célula fresca. Todavia, os últimos apresentaram massa molar maior do que os primeiros. A metodologia clássica de extração química de poli(3-hidroxibutirato) por clorofórmio com aquecimento pode ser otimizada, resultando em redução de 75 % do tempo de aquecimento e separação da biomassa/solução extrativa mais rápida e econômica. Porém, não foi possível substituir a utilização de massa celular seca por fresca nem reduzir a proporção inicial de solvente de 40:1 (v/m). / Poly (3-hydroxybutyrate) [P(3HB)] is the most important biopolymer from the polyhydroalcanoates (PHAs) families, and they are thermoplastics with rapid biodegradability and biocompatibility. The recovery process of bioplastics is an important stage in the production process. Development of safe, economic and environmentally friendly methodologies, that result in high yield biopolymer, is necessary for the attractive P(3HB) production. Therefore, the objective was to optimize the classical methodology for poly (3-hydroxybutyrate) recovery using chloroform as solvent. The bioprocess was performed in two phases, with Ralstonia solanacearum strain. In the first phase, inoculum production, was performed in Erlenmeyer 500 mL flasks, containing 160 mL medium YM and 40 mL bacterial suspension. The flasks were incubated in shaker for 24 h, 150 rpm and 28 °C. The second phase, P(3HB) production, was performed using Erlenmeyer 500 mL flask, containing 160 mL medium F4 and 20 % (v/v) inoculum, maintained in 28 °C, 200 rpm and 72 h. After fermentation cells were separated by centrifugation at 10,000 x g, washed with saline (0,89 % w/v) and dried at 56 °C. P(3HB), accumulation was quantified by gas chromatography. The extraction methodology was optimized according to: extraction time (2 h - 15 min), extractive solution/biomass separation (paper filter or separation funnel), state of cell (dry or fresh), and solvent ratio (10:1, 20:1 and 40:1 v/m). Polymeric films were obtained by evaporation from extractive solutions and they were analysed by Fourier Transform Infrared (FTIR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Gel Permeation Chromatography (GPC). The P(3HB) accumulation was 51,15 %. Highest yields of film were obtained after 30 min heating, by funnel for extractive solution recovery, dry cell and solvent ratio 40:1 v/m; and highest recovery was 98 % of cumulated polymer in the cell. Characteristic bands of P(3HB) were obtained to produced films by FTIR analysis. Films from dried cells showed initial and final degradation temperature and crystallinity degree higher than and films these from fresh cell, however, they showed higher molar weight than the first ones. The classical chemical extraction of P(3HB) by chloroform with heating can be optimized, resulting in a 75 % reduction as well heating time extraction solution most fastest and economical. However, wasn’t possible replace the dry cell mass use by fresh cell mass or reduce the initial solvent proportion [40:1 (v/m)].

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-21T13:35:42Z No. of bitstreams: 1 Christtianno de Lima Rollemberg.pdf: 2445206 bytes, checksum: 4da0aaed00880d13a23b17cdb6cae239 (MD5) / Made available in DSpace on 2017-02-21T13:35:42Z (GMT). No. of bitstreams: 1 Christtianno de Lima Rollemberg.pdf: 2445206 bytes, checksum: 4da0aaed00880d13a23b17cdb6cae239 (MD5) Previous issue date: 2013-04-30 / This study evaluated the use of silicon (Si) in micropropagation of banana 'Silk' and 'Pacovan Ken' aiming to reduce the severity of fusarium wilt caused by Fusarium oxysporum f. sp. cubense and moko disease caused by Ralstonia solanacearum race 2. The banana plantlets were produced in vitro by adding calcium silicate and potassium silicate (0, 0.25, 0.5, 0.75 and 1 g L-1) to MS medium in the phases of multiplication and rooting. After in vitro culture, the plants were transferred to plastic tubes containing substrate plus the same sources of Si, and maintained in a greenhouse for 45 days, when they were inoculated with the pathogens. With respect to fusarium wilt in cultivars Silk and Pacovan Ken, the elevation of Si increased the incubation period (IP) and reduced the disease index (DI) and area under the disease progress curve (AUDPC). In cultivar Silk but not in Pacovan Ken calcium silicate was significantly more effective than potassium silicate. In shoots and roots of both cultivars in both sources, before and after acclimatization Si concentration was greater at a dose of 1.0 g L-1 compared to the control without Si. Before acclimatization, calcium silicate provided higher Si concentration in the shoots than potassium silicate. The opposite happened with the Si concentration in the roots. After acclimatization, there was no difference between the calcium silicate and potassium silicate, for both cultivars. In general, for both cultivars and sources of Si there were positive correlations with the concentration of Si and IP, and negative correlations with DI and AUDPC. Before and after acclimatization, the anatomical variable of roots: thickness of the root epidermis, cortex, endodermis and central cylinder of banana 'Silk' and 'Ken Pacovan' were influenced by Si sources. Calcium silicate was more efficient in increasing the thickness of the root epidermis, cortex and central cylinder, while potassium silicate was more efficient in thickening of the endodermis. In general, there were positive correlations among anatomical variable of roots with PI and negative correlations with DI and AUDPC, except for potassium silicate in cultivar Silk. The research conducted with moko disease showed that increase of Si in Silk and Pacovan Ken cultivars caused increase in IP and decreases the DI and AUDPC. At the dosage of 1.0 g L-1 AUCPD was reduced by 27.3%. In cultivar Silk, calcium silicate was more effective than potassium silicate (P≤0.05), while in „Pacovan Ken‟ there was no difference. In both cultivars, plants treated with Si showed, in general, concentrations of chlorophylls a, b and total higher than plants Si- up to six days after inoculation, which may have influenced the disease IP. In general, both the enzymes related to oxidative stress (CAT, SOD and APX), as the plant defense (POX, PPO, CHI and GLU), had increased its activities in plants treated with Si, especially those with calcium silicate, indicating a possible role in reducing the severity of the disease. The supply of Si in micropropagation of banana 'Silk' and 'Pacovan Ken' promoted reduction of Fusarium wilt and moko disease, and therefore can be used as a new technology in the management of these diseases. / Este estudo avaliou o uso do silício (Si) na micropropagação de bananeira „Maçã‟ e „Pacovan Ken‟ visando a redução da severidade da murcha-de-fusário, causada pelo Fusarium oxysporum f. sp. cubense e do moko da bananeira causado por Ralstonia solanacearum raça 2. As mudas de bananeira foram produzidas in vitro com adição de silicato de cálcio e silicato de potássio (0; 0,25; 0,5; 0,75 e 1 g L-1) ao meio de cultivo MS nas fases de multiplicação e enraizamento. Após o cultivo in vitro, as plantas foram transferidas para tubetes contendo substrato acrescido das mesmas fontes de Si, e mantidas em casa de vegetação por 45 dias, quando foram inoculadas com os patógenos. Com relação à murcha-de-fusário, nas cultivares Maçã e Pacovan Ken, a elevação das doses de Si aumentou o período de incubação (PI) e reduziu o índice de doença (IDO) e a área abaixo da curva de progresso da doença (AACPD). Em „Maçã‟ o silicato de cálcio foi significativamente mais eficiente que o silicato de potássio, o que não ocorreu na „Pacovan Ken‟. A concentração de Si na parte aérea e raízes das cultivares, em ambas as fontes, antes e após a aclimatização foi maior na dose de 1,0 g L-1 em relação à testemunha sem Si. Antes da aclimatização, o silicato de cálcio proporcionou maior concentração de Si na parte aérea que o silicato de potássio. O contrário aconteceu com a concentração de Si nas raízes. Após aclimatização, não houve diferença entre o silicato de cálcio e o silicato de potássio, para as duas cultivares. Em geral, para ambas as cultivares e fontes de Si foram observadas correlações positivas da concentração de Si com PI e correlações negativas com IDO e AACPD. Antes e após a aclimatização das plantas, as espessuras da epiderme radicular, córtex, endoderme e cilindro central das bananeiras „Maçã‟ e „Pacovan Ken‟ foram influenciadas pelas fontes de Si. O silicato de cálcio foi mais eficiente no aumento da espessura da epiderme radicular, córtex e cilindro central, enquanto o silicato de potássio foi mais eficiente no aumento da espessura da endoderme. Em geral, foram observadas correlações positivas das variáveis anatômicas das raízes com PI e correlações negativas com IDO e AACPD, exceto para silicato de potássio em bananeira „Maçã‟. Na pesquisa desenvolvida com o moko da bananeira, a elevação das doses de Si nas cultivares Maçã e Pacovan Ken causou aumento no PI e reduções do IDO e AACPD. Na dosagem de 1,0 g L-1, a AACPD foi reduzida em até 27,3%. Em bananeira „Maçã‟ o silicato de cálcio foi mais eficiente que o silicato de potássio (P≤0,05), enquanto na „Pacovan Ken‟ não houve diferença. Nas duas cultivares, plantas tratadas com Si apresentaram, de maneira geral, concentrações de clorofilas a, b e total maiores que as plantas Si- até os seis dias após inoculação, o que pode ter influenciado o PI da doença. Em geral, tanto as enzimas relacionadas ao estresse oxidativo (CAT, SOD e APX), quanto as de defesa da planta (POX, PFO, GLU e QUI), tiveram suas atividades aumentadas nos tratamentos com silício, especialmente naqueles com silicato de cálcio, indicando uma possível participação na redução da severidade da doença. O fornecimento de Si na micropropagação de bananeiras „Maçã‟ e „Pacovan Ken‟ promoveu redução da murcha-de-fusário e moko da bananeira, podendo ser utilizado como uma nova tecnologia no manejo dessas doenças.

Photosystem I (PSI) from plants, algae, and cyanobacteria can mediate H2 evolution in vivo and in vitro. A simple, self-platinization procedure that permits stable PSI-mediated H2 evolution in vitro has been developed. The H2 evolution capabilities of PSI from Thermosynechococcus elongatus have been characterized. This organism utilizes cytochrome c6 (cyt c6) as the e- donor to P700. Using a solution-based, self-organized platinization of the PSI nanoparticles, this study demonstrates a sodium ascorbate-cyt-PSI-Pt-H2 electron transport and proton reduction system that yields light-dependent H2. The system was thermostable with H2 evolution increasing up to 55°C. In addition, stability studies have shown the H2 evolution to be very stable, with no significant decrease over the 80 days investigated. Through simple optimization a H2 production rate of ~5.5 mol H2/h/mg Chl [micro-mole H2 per hour per milligram chlorophyll] was attained. To further optimize the H2 production Asc-cyt-PSI-Pt-H2 system, response surface methodology (RSM) was employed. The process parameter studied included temperature, light intensity and platinum salt concentration. The results showed that experimental data had a good fit to the proposed model (R2=0.99 and p < 0.001). Platinum salt concentration, temperature and the interaction between platinum salt concentration and temperature showed significant effects on the total H2 yield. Light intensity had minimal effect of the total H2 yield within the region studied. The optimum parameters for H2 photoproduction were light intensity of 240 μE/m2/s, [micro-eistien per square meter per second], platinum salt concentration of 636 μM [micro-mol/liter] and temperature of 310C. Finally, studies that will improve the H2 yield by increasing the kinetics of electron transfer were done. A hybrid protein was formed by engineering a gene to express a fusion of the membrane-bound [Ni-Fe] hydrogenase from Ralstonia eutropha H16 and the stromal-exposed subunits PsaE and PsaD of PSI from T. elongatus. A PsaE-free mutant of PSI was simultaneously formed by genetically disrupting the expression of the PsaE subunit of a native PSI; that will allow in vitro reconstitution of the desired PsaE-hydrogenase fusion protein with PsaE-free PSI.