Sabe-se que a grande maioria dos fitopatógenos depende quase que exclusivamente de vetores para disseminarem-se para novos hospedeiros, porém pouco foi estudado no que se refere aos efeitos dos micro-organismos sobre seus insetos vetores. Sendo Bemisia tabaci uma praga de elevada importância e vetora de inúmeros vírus para plantas cultiváveis, é de extrema relevância estudar os efeitos provocados pelos vírus sobre seu desempenho biológico. Assim, esta pesquisa objetivou: a) avaliar os efeitos direto e indireto dos begomovírus Tomato severe rugose vírus (ToSRV) e Tomato yellow leaf curl virus (TYLCV) sobre parâmetros biológicos de B. tabaci biótipo B e Q respectivamente, dos quais: duração e viabilidade do período ninfal; razão sexual; fecundidade; fertilidade e longevidade. Os resultados encontrados variam de positivos, neutros a negativos, dependendo do parâmetro, efeito e espécie do vírus estudado. Sendo assim, observou-se que há efeito direto de ambos os vírus na duração do período ninfal, sendo menor em insetos virulíferos que em sadios. Ademais, há um incremento no número médio de ovos depositados por insetos virulíferos com ToSRV (225,2 ovos/fêmea) quando comparado com insetos sadios (180,1 ovos/fêmea). Já TYLCV afetou diretamente a longevidade de machos os quais tiveram a duração da fase adulta incrementada quando virulíferos (30 dias) e 24 dias quando sadios. Há um efeito indireto negativo de ToSRV sobre a viabilidade ninfal de seu vetor, sendo de 52% quando estes são mantidos em plantas infectadas e 86% em plantas sadias de tomate. A razão sexual também foi afetada por este vírus, favorecendo as fêmeas, sendo de 2:1 a proporção entre fêmeas e machos em plantas infectadas. Ademais, a longevidade de machos foi reduzida drasticamente quando em plantas infectadas com ToSRV. Também foi detectado um efeito indireto positivo do TYLCV sobre a fecundidade de fêmeas de B. tabaci biótipo Q, as quais depositaram em média maior quantidade de ovos em plantas infectadas que em plantas sadias de tomate, sendo 52,8 e 33,2 ovos respectivamente. Tais resultados permitem concluir que, nas condições em que os ensaios foram realizados, ToSRV afeta diretamente de forma positiva seu vetor, enquanto possui efeitos indiretos principalmente negativos sobre parâmetros biológicos de B. tabaci biótipo B. Igualmente TYLCV possui efeitos diretos positivos sobre o biótipo Q da espécie de mosca-branca. Já indiretamente este vírus, diferentemente de ToSRV, afeta positivamente a biologia de seu vetor B. tabaci biótipo Q, favorecendo a fecundidade dos indivíduos que se desenvolveram em plantas infectadas de tomate. / It is known that the vast majority of pathogens relies almost exclusively vector for spreading to new hosts, but little has been studied regarding the effects of micro-organisms on its insect vectors. Bemisia tabaci is a pest of high importance vector of numerous virus to cultivated plants, it is extremely important to study the effects caused by viruses on its biological performance. Thus, this study aimed to: a) evaluate the direct and indirect effects of the begomovirus Tomato severe rugose virus (ToSRV) and Tomato yellow leaf curl virus (TYLCV) on biological parameters of B. tabaci biotype B and Q respectively, of which duration and viability of nymphal development; sex ratio; fecundity; fertility and longevity. The results range from positive, neutral to negative, depending on the parameter, effect and species of the virus studied. Thus, it was observed that there is a direct effect of the both virus in the duration of nymphal development, being less than viruliferous insects in healthy individuals. Moreover, there is an increase in the average number of eggs laid by viruliferous insects with ToSRV (225,2 eggs / female) when compared with healthy insects (180,1 eggs / female). Already TYLCV directly affected the longevity of males which lasted adulthood increased when viruliferous (30 days and 24 when healthy). There is an indirect negative effect on the viability of ToSRV nymphal of its vector, and 52% when they are kept in infected plants and 86% in healthy tomato plants. The sex ratio was also affected by this virus, favoring females, with a ratio of 2:1 between females and males in infected plants. Furthermore, the longevity of males was reduced dramatically when plants infected with ToSRV. We also detected a positive indirect effect of TYLCV on fertility of female B. tabaci biotype Q, which placed greater average number of eggs in infected plants than on healthy plants of tomato, 52,8 and 33,2 eggs respectively. These results indicate that, under conditions in which the tests were performed, ToSRV directly affects positively its vector, while indirect effects has mostly negative on biological parameters of B. tabaci biotype B. TYLCV also has positive direct effects on the Q biotype of the whitefly species. Already indirectly this virus, unlike ToSRV, positively affects the biology of its vector B. tabaci biotype Q, favoring the fecundity of individuals that developed in infected tomato plants.

Os psilídeos (Hemiptera: Psylloidea) são insetos sugadores de floema que se destacam como vetores de bactérias associadas a doenças emergentes em várias culturas agrícolas, como \'Candidatus Liberibacter solanacearum\' (CaLsol), que afeta batata, cenoura e tomate na América do Norte e Central, Europa, África e Nova Zelandia. Estudos recentes mostram associações de CaLsol com diversas espécies de olerícolas e psilídeos vetores, sugerindo a existência de mais interações do que as atualmente conhecidas. No Brasil, ainda não há registros de CaLsol nem dos psilídeos já conhecidos como vetores. Embora Psylloidea seja um grupo diversificado, apenas 89 espécies encontram-se identificadas no Brasil, sendo a maioria delas em áreas de floresta, havendo poucas informações sobre psilídeos em olerícolas. Assim, os objetivos do presente trabalho foram: 1) Avaliar a diversidade de espécies de Psylloidea associadas a cultivos de olerícolas na região Centro-Sul do Brasil, buscando-se identificar a ocorrência de espécies vetoras ou potenciais vetoras de CaLsol; 2) Avaliar a preferência hospedeira, sobrevivência e desenvolvimento de duas espécies de psilídeos associados a olerícolas, Russeliana capsici Burckhardt e R. solanicola Tuthill, em diferentes espécies vegetais que poderiam atuar como hospedeiras de CaLsol ou dos vetores; 3) Caracterizar o comportamento alimentar de adultos e imaturos de R. solanicola em plantas com diferentes níveis de adequação para o seu desenvolvimento, usando a técnica Electrical Penetration Graph (EPG). No levantamento realizado com métodos diretos (rede entomológica, batida de planta e inspeção visual) e indiretos (bandeja amarela com água) em várias localidades de seis estados brasileiros, foram encontrados 2813 exemplares de psilídeos, pertencentes a 7 famílias e 20 gêneros, com maior frequência de R. solanicola (95,1%), R. capsici (91,6%) e Isogonoceraia divergipennis (85,4%) em Daucus carota, Capsicum annuum (pimenta) e Solanum tuberosum, respectivamente. Observaram-se imaturos de R. capsici em C. annuum, e de R. solanicola em D. carota e na planta espontânea Parthenium hysterophorus. Em ensaios de escolha, houve preferência para pouso e oviposição de R. solanicola em D. carota e P. hysterophorus, e de R. capsici em C. annuum, Nicandra physalodes e S. americanum. Observou-se desenvolvimento completo (ovo-adulto) de R. solanicola em D. carota, S. tuberosum, N. physalodes, P. hysterophorus, e Bidens pilosa, enquanto que R. capsici mostrou especificidade hospedeira, desenvolvendo-se apenas em C. annum (pimentão e pimenta). O estudo de EPG mostrou que ninfas e adultos de R. solanicola atingem o floema tanto em plantas hospedeiras (D. carota e P. hysterophorus) como em não hospedeiras (C. annuum e S. lycopersicum), mas com variações significativas em parâmetros de atividade estiletar que permitem diferenciar o comportamento alimentar nas diferentes espécies vegetais. Os estudos demonstram uma íntima associação de R. solanicola e R. capsici com cultivos de olerícolas e seu potencial como vetores de CaLsol, em caso de introdução deste patógeno no Brasil. / Psyllids (Hemiptera: Psylloidea) are phloem-feeding insects with increased importance as vectors of phloem-limited bacteria associated with emerging diseases in agricultural crops, e.g. \'Candidatus Liberibacter solanacearum\' (CaLsol), which causes severe losses in potatoes, carrots and tomatoes in North and Central America, Europe, Africa and New Zealand. Recent studies show associations of CaLsol with diverse species of vegetables and psyllid vectors, suggesting that more interactions than those currently known are likely. In Brazil, there are no records of CaLson and psyllid species already known as vectors. Although Psylloidea is a diversified group, only 73 species were identified in Brazil and most of them on forest trees, with scarce information about psyllids on vegetable crops. Therefore, the objectives of this work were: 1) To survey the diversity of psyllid species vectors associated with vegetable crops in the Central-South region of Brazil, aiming to identify vector or potential vector species of CaLsol; 2) To evaluate host plant preference, survival and egg-adult development of two psyllid species associated with vegetable crops, Russeliana capsici and R. solanicola, on various plant species that could serve as hosts of the pathogen or vectors; 3) To characterize the feeding behavior of adults and nymphs of R. solanicola on plants with variable levels of suitability as developing hosts, by using the Electrical Penetration Graph (EPG) technique. In the survey carried out with direct (sweep net, stem-tap, visual inspection) and indirect (yellow pan trap) methods in various localities of six Brazilian states, 2,813 psyllid speciments of 7 families and 20 genera were collected, with higher frequencies of R. solanicola (95,1%), R. capsici (91,6%) and Isogonoceraia divergipennis (85,4%) on Daucus carota, Capsicum annuum (pepper) and Solanum tuberosum, respectively. Immatures of R. capsici were observed on C. annuum, and those of R. solanicola were observed on D. carota and on the weed Parthenium hysterophorus. Choice tests in the laboratory revealed settling and oviposition preferences of R. solanicola on D. carota and P. hysterophorus, and of R. capsici on C. annuum, Nicandra physalodes and S. americanum. Complete egg-adult development was observed for R. solanicola on D. carota, S. tuberosum, Nicandra physalodes, P. hysterophorus and Bidens pilosa, whereas R. capsici showed host specificity by developing only on C. annum (green pepper and capsicum). The EPG study showed that R. solanicola nymphs and adults reach the phloem both on host (D. carota e P. hysterophorus) and non-host (C. annuum e S. lycopersicum) plants, but with significant differences on stylect activity parameters, allowing distinction of the feeding behavior among the different plant species. Overall, the studies indicate a close association of R. solanicola and R. capsici with vegetable crops, as well as their potential as vectors of CaLsol in case of introduction of this pathogen in Brazil.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-19T14:23:07Z No. of bitstreams: 2 Dissertação - Julio Miguel Grández Rios - 2015.pdf: 691183 bytes, checksum: 9355a2c6be0d96cd9448a56e587ba28c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-04-19T14:25:56Z (GMT) No. of bitstreams: 2 Dissertação - Julio Miguel Grández Rios - 2015.pdf: 691183 bytes, checksum: 9355a2c6be0d96cd9448a56e587ba28c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-04-19T14:25:56Z (GMT). No. of bitstreams: 2 Dissertação - Julio Miguel Grández Rios - 2015.pdf: 691183 bytes, checksum: 9355a2c6be0d96cd9448a56e587ba28c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-20 / The replacement of native plants by introduced or invasive exotic plants may have significant impacts on the fauna of herbivorous insects. These impacts can occur both directly, through changes in reproductive success and population size herbivorous insects that consume the exotic plants, and indirectly through changes in the availability of native host plants. This study is divided into two parts. In the first chapter, I evaluated the effect of phylogenetic isolation on the richness and composition of species of herbivorous insects on exotic plants. In addition, I also examined possible differences in response to phylogenetic isolation of exotic plants between herbivore assemblages made up exclusively of endophagous and exophagous insects.In the second chapter, I evaluated the effect of phylogenetic isolation of host plants on the mean specialization of herbivorous insects fauna associated with host plants. In addition,I also analyzed the effect of phylogenetic isolation of host plants differs between native and exotic plants. Taken together, the results of the two chapters exemplify how the richness, composition and specialization of herbivores respond to phylogenetic isolation of their host plants. / A substituição de plantas nativas por plantas exóticas invasoras ou introduzidas pode ter impactos significativos sobre a fauna de insetos herbívoros.Este impacto pode ocorrer tanto diretamente, por meio de mudanças no sucesso reprodutivo e no tamanho populacional de insetos herbívoros que consomem as plantas exóticas, quanto indiretamente, através da alteraçãona disponibilidade de plantas hospedeiras nativas. O presente trabalho está dividido em duas partes. No primeiro capítulo, avaliei o efeito do isolamento filogenético sobre a riquezae composição de espécies de insetos herbívoros em plantas exóticas. Além disso, examinei possíveis diferenças na resposta ao isolamento filogenético de plantas exóticas entre assembleias de herbívoros compostas exclusivamente por insetos endófagos einsetos exófagos. No segundo capítulo, avaliei o efeito do isolamento filogenético das plantas hospedeiras sobre a especialização média das faunas de insetos herbívoros associadas às plantas hospedeiras. Além disso, também analisei se o efeito do isolamento filogenético das plantas hospedeiras difere entre as plantas nativas e exóticas. Tomados em conjunto, os resultados dos dois capítulos exemplificam como a riqueza, a composição e a especialização dos insetos herbívoros respondem ao isolamento filogenético de suas plantas hospedeiras.

Fungos associados às plantas desempenham diversas funções biológicas importantes e constituem imensos reservatórios de novos compostos químicos, atividades biológicas e processos biotecnológicos ainda subexplorados. A diversidade estimada para esses microrganismos é enorme, porém menos de 7 % das espécies é conhecida. Em cana-de-açúcar, uma das principais culturas agrícolas do Brasil, estudos de interação e diversidade são recentes e ainda incipientes, principalmente com relação à comunidade fúngica e sua interação com plantas transgênicas. Uma das espécies presentes na comunidade fúngica associada à cana-de-açúcar é o fungo Trichoderma virens, o qual apresenta grande potencial de exploração como agente de controle biológico, promotor de crescimento vegetal e produtor de enzimas e metabólitos secundários. Nesse sentido, o presente trabalho buscou primeiramente determinar a estrutura e diversidade da comunidade de fungos (endófitos de raiz e rizosfera) associados a duas variedades de cana-de-açúcar, uma convencional (SP80-1842) e outra transgênica (IMI-1, expressando resistência ao herbicida imazapir), e avaliar possíveis efeitos da transgênese, do estágio de crescimento e do manejo agrícola. Além disso, uma linhagem de T. virens, isolada como endófito de raiz dessa planta, foi selecionada para estudos de interação fungo-planta e determinação dos mecanismos envolvidos na sua atividade de controle biológico. Na primeira etapa do trabalho, que compreendeu isolamento, caracterização e avaliação de alterações na comunidade fúngica, os resultados obtidos mostraram que a comunidade fúngica associada à cana-de-açúcar é formada por pelo menos 35 gêneros diferentes, a maioria do filo Ascomicota, sendo que a sua estrutura incluiu um grande número de gêneros pouco frequentes e um pequeno número de gêneros altamente frequentes (Penicillium, Fusarium, Aspergillus, Trichoderma e Epicoccum), dos quais alguns apresentam especificidade ao local de isolamento (raiz ou rizosfera). A avaliação de possíveis efeitos sobre a comunidade fúngica mostrou que a idade da planta foi o único fator que influenciou de forma significativa as características dessa comunidade, sendo que os efeitos da transgênese, se existentes, devem ser secundários quando comparados às fontes naturais de variação. Na segunda etapa, a linhagem de T. virens T.v.223 foi transformada pelo sistema Agrobacterium tumefaciens e o transformante gerado (expressando resistência à higromicina B e a proteína GFP) foi utilizado em ensaios de interação via reisolamento e microscopia. Os resultados revelaram que esse fungo não promoveu alterações fenotípicas visíveis na planta hospedeira, colonizou predominantemente as raízes, onde formou camadas densas de micélio ao seu redor, antes de penetrar o espaço intercelular das primeiras camadas de célula da epiderme do tecido radicular. Por último, mutantes de T. virens deficientes para produção de quitinases foram gerados por deleção gênica e silenciamento gênico via RNAi e avaliados quanto à capacidade de controle biológico de diferentes fitopatógenos em ensaios em casa de vegetação. Curiosamente, os resultados mostraram que as enzimas quitinolíticas podem ser essenciais à atividade de controle biológico efetuada por T. virens, mas que a importância e a participação dessas enzimas no processo dependem do tipo de planta e de patógeno envolvidos na interação, já que mais de um mecanismo deve contribuir para o controle biológico por T. virens, como por exemplo, a indução de resistência da planta. / Plant-associated fungi perform several important biological functions and are considered a vast source of novel chemical compounds, biological activities and biotechnological processes, whose potential is underexplored. The estimated diversity for these microorganisms is massive, but less than 7% of the species are already known. Sugarcane, one of the most important crops in Brazil, has only recently been studied regarding interactions and diversity, but these studies are still incipient, mainly concerning fungal community and its interaction with transgenic plants. One of the species found on sugarcane fungal community is the fungus Trichoderma virens, whose potential on biological control, growth promotion, and enzymes and secondary metabolites production is huge. Taking this into consideration, the current work aimed to determine the structure and diversity of the fungal community (root endophytes and rhizosphere) associated to two varieties of sugarcane, the conventional (SP80-1842) and the transgenic counterpart (IMI-1, expressing imazapyr herbicide resistance), and assess possible effects from transgenese, growth stage and management. In addition, a strain of T. virens, isolated as a sugarcane endophyte, was selected to perform the fungal-plant interaction assays and to determine its biological control mechanisms. The results of the first part of this work, including the isolation, characterization and evaluation of fungal community changes, showed that the sugarcane fungal community is made up by at least 35 different genera, most of them belonging to Ascomycota phylum, and its structure included many genera observed in very low frequencies, and a few genera highly frequent (Penicillium, Fusarium, Aspergillus, Trichoderma and Epicoccum), from which some have specificity to the place of isolation (root or rhizosphere). Assessing possible effects upon the fungal community showed that the growth stage was the only factor significantly influencing the communitys features, besides, if transgenese effects are present, they may be minor compared to other natural sources of variation. The second part of this work included the Agrobacterium tumefaciens-mediated transformation of T. virens strain T.v.223 and utilization of the generated transformant (hygromycin B resistant and GFPexpressing) to perform interaction studies by re-isolation and microscopy. The results revealed that this fungus did not promote any phenotypic change in the host plant, it was found mostly in roots, formed a dense mycelia cover over the roots and was able to penetrate intercellular spaces of root epidermis first layers. Finally, T. virens chitinase-deficient mutants were generated by gene deletion and RNAi gene silencing, and tested for biological control activity against different phytopathogens in greenhouse assays. Curiously, the results showed that chitinolytic activity may be essential to the biocontrol activity of T. virens, but its significance and the input of each chitinase depends on the plant and pathogen playing the interaction, since more than one mechanism may account for T. virens biological control, for instance, the induction of plant resistance.

Une invasion biologique réussie se décompose en trois étapes : l'introduction d'une population allochtone sur une nouvelle aire de répartition géographique, l'établissement de populations viables et leur prolifération exponentielle dans l'aire d'introduction. En 2002, Shea et Chesson ont énoncé l'hypothèse d'Opportunité de Niche dans le but d'expliquer la réussite de l'établissement et de la prolifération des espèces envahissantes. Selon ces auteurs, les espèces exogènes introduites sur une nouvelle aire géographique peuvent y profiter d'une part de la diminution de la pression exercée par leurs ennemis naturels et d'autre part d'une augmentation de la disponibilité de leurs ressources. Drosophila suzukii est une de ces espèces dites envahissantes. Originaire d'Asie, sa présence est aujourd'hui largement recensée en Amérique du Nord et dans tout l'ouest de l'Europe. Depuis son introduction en 2008, en Europe notamment, l'invasion de D. suzukii est particulièrement rapide et les dégâts engendrés dans les cultures par cette espèce qui se développe sur les fruits en cours de maturité sont très préoccupants. Au cours de nos travaux, nous avons mis en évidence certains caractères chez cette espèce comme étant des éléments facilitant le succès de son invasion. D. suzukii présente tout d'abord une grande résistance face à ses principaux ennemis naturels, les parasitoïdes. Nous avons ensuite montré l'existence d'un lien entre sa capacité à mettre en échec les parasitoïdes larvaires et son importante charge hémocytaire. Par ailleurs, grâce à un échantillonnage très large, nous avons illustré la grande polyphagie de cette espèce. Ce caractère lui assure potentiellement une disponibilité en ressources tout au long de l'année. Enfin, à l'aide d'une étude portant sur les relations tritrophiques, nous avons montré que la qualité nutritive des plantes hôtes utilisées par D. suzukii peut avoir un impact sur le niveau de sa résistance aux entomophages. D. suzukii parvient en effet à augmenter sa résistance à plusieurs espèces de parasitoïdes lorsqu'elle réalise son développement sur un substrat riche en alcaloïde. Nos derniers résultats suggèrent qu'elle serait même capable de réaliser une forme d'automédication. Les différentes études réalisées au cours de cette thèse semblent confirmer que D. suzukii profite d'une opportunité de niche en Europe tempérée / Successful biological invasion is divided into three stages: the introduction of a non-native population on a new geographical area, the establishment of viable populations and their exponential growth in the area of introduction. In 2002, Shea and Chesson proposed the Opportunity Niche Hypothesis in order to explain the success of the establishment and the spread of invasive species. According to these authors, alien species introduced into a new range can jointly benefit from a decrease of the pressure applied by natural enemies and from an increase in the availability of resources. Drosophila suzukii has been reported to be an invasive species. Native of Asia, it has been now widely observed in North America and throughout Western Europe. Since its introduction in 2008, especially in Europe, the invasion of D. suzukii is particularly rapid and, as it grows on ripe fruits, a lot of concerns have been raised on the damage it causes on crops. In our work, we have highlighted some characters of this species as elements that facilitate the success of its invasion. D. suzukii is highly resistant against parasitoids, known as its major natural enemies. We have then showed the existence of a link between the ability to resist to larval parasitoids and a high hemocyte load. Furthermore, thanks to a very large sample, we have illustrated the great polyphagia of this species. This property provides available resources throughout the year. Finally, with a study of tritrophic interactions, we have shown that the nutritional quality of host plants used by D. suzukii can have an impact on the level of resistance to entomophages. Indeed, D. suzukii increases its resistance to several species of parasitoids when it growths on an alkaloid-rich substrate. Our recent results suggest that it would even be able to achieve a form of self-medication. The different studies performed in this thesis seem to confirm that D. suzukii makes the most of niche opportunity to efficiently invade temperate Europe

La céréaliculture occupe une place importante dans l’agriculture mondiale et contribue à la sécurité alimentaire des populations. Pour assurer la production des céréales (orge, blé, avoine), il est nécessaire de lutter contre ses ravageurs, essentiellement les pucerons qui sont capables de transmettre plusieurs virus. L’analyse des génomes des pucerons tels que Rhopalosiphum padi, R. maidis, Sitobion avenae, Schizaphis graminum, de leur évolution et de leur relation avec les plantes hôtes (céréales) pourrait contribuer à la mise en place de moyens de lutte pour contrôler les populations de ces ravageurs. Dans ce contexte, cette étude s’est focalisée sur la recherche des éléments transposables des deux superfamilles Tc1-mariner-IS630 et des piggyBac. Les ETs, considérés comme des moteurs de la plasticité génomique et de l’évolution des espèces, sont utilisés en biotechnologie pour développer des outils de transfert de gènes. Dans un premier temps, nous avons recherché des éléments de la famille mariner, ou apparentés à cette famille, dans les génomes séquencés de trois espèces de pucerons : Acyrthosiphon pisum, Myzus persicae et Diuraphis noxia. Sur la base de similitude de séquences, nous avons pu caractériser 183 éléments répartis en trois clades. Le premier, commun aux trois espèces, correspond au clade de la sous-famille irritans DD34D. Il est subdivisé en trois tribus Macrosiphinimar, Batmar-like elements et Dnomar-like elements. Le deuxième comprend l’élément rosa DD41D qui appartient à une famille phylogénétiquement proche de mariner. Le troisième comprend des séquences avec de longues répétitions terminales inversées et inclut deux tribus DD40-41D. Ces deux derniers clades, plus répandus chez A. pisum, dérivent vraisemblablement d’un ancêtre commun et formeraient une nouvelle famille.Dans un deuxième temps, nous avons exploité les résultats de la recherche in silico pour identifier in vitro des éléments de la sous-famille irritans chez les pucerons des céréales et chez leur plante hôte. Deux types d’éléments délétées (MITEs) ont été identifiés chez les pucerons, l’un commun à toutes les espèces avec un pourcentage d’identité supérieur à 98% (Aphidmar) et l’autre spécifique à S. avenae (Samar2). Par ailleurs, les génomes des céréales (orge, blé, brachypodium, égilope) ont été analysés en utilisant comme requêtes des séquences d’éléments de la sous-famille irritans trouvés chez les aphides. Un seul contig de l’orge cultivar barke comprend un élément tronqué de 320 pb, flanqué par de l’ADN génomique de pucerons. La vérification in vitro de la présence de cette séquence chez plusieurs cultivars d’orge révèle deux types de séquences. Le premier est similaire à celui trouvé in silico chez l’orge, le second correspond à l’élément Samar2 délété de 7 nucléotides au niveau du point de cassure de la délétion initiale. Ceci suggère l’existence d’un transfert horizontal entre pucerons des céréales et l’orge. Enfin, l’abondance de données génomiques et la rareté des travaux approfondis portant sur les membres de la superfamille piggyBac, nous ont amenés à analyser in silico leurs caractéristiques, leur distribution et leur évolution. Un total de 117 séquences protéiques de PBLE (éléments autonomes) et de PGBD (éléments domestiqués), ont été utilisées comme requêtes. Quatre groupes structuraux de PBLE ont été définis en fonction de la présence ou absence de répétitions sub-terminales (directes/inversées). Toutefois, il n'existe aucune relation entre ces quatre groupes et la phylogénie des PBLE. Les PGBD soumis à une forte sélection purifiante, sont clairement structurés en neuf groupes dont un correspondant à un nouvel ensemble d’éléments domestiqués trouvé chez les Néopterygiens. L’analyse fine des PGBD révèle que le domaine catalytique de la transposase ancestrale n'est pas toujours conservé. La phylogénie générale des PBLE et des PGBD suggère des événements multiples de domestication des PGBD à partir de différents ancêtres PBLE. / Cereal farming plays an important role in world agriculture and contributes to the food security of the populations. To improve the production of cereals (barley, wheat, oats ...), it is necessary to fight against their pests, especially aphids, able to transmit several viruses. The analysis of aphid’s genomes such as Rhopalosiphum padi, R. maidis, Sitobions avenae and Schizaphis graminum, their evolution and their relationships with their host plants could contribute to define strategies against pest populations. In this context, this work focused on the analysis of transposable elements belonging to Tc1-mariner-IS630 and piggyBac superfamilies. Indeed, TEs are involved in genomic plasticity and evolution of species, and are also used in biotechnology to develop gene transfer tools. In the first chapter, we investigate three available genomes of aphids, namely Acyrthosiphon pisum, Myzus persicae and Diuraphis noxia, to search for elements of the mariner family or close to it. Based on sequence similarities, we were able to characterize 183 elements distributed in three clades. The first one, common to the three species, corresponds to the clade of irritans subfamily DD34D, and is subdivided into three tribes Macrosiphinimar, Batmar-like elements and Dnomar-like elements. The second one includes the rosa element DD41D belonging to a group close to the mariner family. The third one includes sequences with long Terminal Inverted Repeats and is subdivided into two DD40-41D tribes. These two latter clades, more common in A. pisum, likely derive from a common ancestor and would form a new family. In the second chapter, the results of in silico research were exploited, to identify in vitro, elements of the irritans subfamily in cereal aphids and in their host plants as well. Two types of deleted elements (MITEs) have been identified in aphids, one common to all species with a percentage of identity higher than 98% (Aphidmar) and the other one specific to S. avenae (Samar2). In addition, the genomes of cereals (barley, wheat, brachypodium, aegilops) were investigated using as queries sequences of irritans subfamily found in aphids. A single contig identified in Hordeum vulgare (cultivar barke) contains a 320 bp truncated element flanked by genomic DNA of aphids. The presence of this sequence was checked in several barley cultivars by an in vitro approach. Two types of sequences were found. The first one similar to that found in barley from the in silico approach, the second one corresponding to Samar2 element, lacking seven nucleotides at the breaking points of the initial deletion. This suggests a possible horizontal transfer between cereal aphids and barley. In the last chapter, the abundance of genomic data and the scarcity of in-depth research covering all members of the piggyBac superfamily led us to determine in silico their characteristic, their distribution and their evolution. A total of 117 proteic sequences of the PBLE (autonomous elements) and PGBD (domesticated elements) have been used as queries. Four structural groups of PBLE have been identified depending on the presence or absence of sub-terminal repeats (direct / inverted). However, there is no relationship between the structural groups and the phylogeny of these PBLE elements. PGBD are clearly structured into nine main groups including a new group of domesticated elements found in Neopterygii. The catalytic domain of the ancestral transposase is not always preserved, but all these domesticated elements are subjected to a strong purifying selection. The general phylogeny of PBLEs and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors.

No description available.

Mealybugs (Order Hemiptera: Superfamily Coccoidea) represent a persistent problem in grape-growing regions globally, with a notable increase in infestations in recent times. The current study investigates the species composition and seasonal dynamics of mealybugs in vineyards. Pseudococcus maritimus and Ferissia gilli remain the dominant species, while Pseudococcus viburni was also spotted during two separate instances and Planococcus ficus remained absent. Commercially available pheromone delta traps and adhesive bands were deployed to capture the male mealybugs, while numerically higher efficacy was observed in the adhesive band trap. Notably, the placement of adhesive band traps on the cordon and trunk revealed differential male mealybug captures, probably indicating their movement within the vines. The research also divulges into the life cycle of mealybugs and their population densities during the sampling season. The main highlight has been the lower population density of earlier nymphal stages of the first generation of mealybugs followed by the colonization of grapevines by the second-generation crawlers. Adults of second generations give rise to the overwintering stages of mealybugs. Traditionally the research also explores the species and seasonal population dynamics of ants along with mealybugs and their management in Virginia vineyards. The activity of fifteen genera of ants in the vineyard was recorded, with some of the dominant ant genera like Tetramorium, Crematogaster, and Lasius were recorded in the vineyard actively tending and defending the mealybugs and moving them around. Fruit cluster infestation was also higher in the control treatment in comparison to sugar dispenser and ant bait dispenser treatments. This comprehensive assessment of ant diversity further deepens our understanding of the intricate ecosystem within vineyards. A spray trial using three different insecticides with distinct modes of action (Buprofezin, Bifenthrin, and Spirotetramat) in two of the commercial vineyards. Preliminary results indicate that on some days after treatment, insecticides effectively controlled mealybug populations, offering a glimmer of hope to vineyard owners grappling with infestations. Mealybugs pose a significant threat to grape cultivation, and this research provides valuable insights that can help vineyard owners and grape growers develop more targeted and effective control strategies. Species identification, understanding their behavior, and exploring potential allies in the fight against mealybugs are all crucial steps toward maintaining the health and productivity of vineyards in Virginia. While challenges remain in the battle against mealybugs, this research marks a significant step forward in safeguarding grape cultivation in Virginia and potentially reshaping strategies for controlling these persistent pests in vineyards. Growers and researchers alike eagerly await further developments and the practical application of these findings to protect the flourishing vineyards of the region. / Doctor of Philosophy / Mealybugs are common grapevine pests, recently causing a nuisance in vineyards across the United States and Canada. Some of the previously documented species of mealybugs common in the eastern part of the States include grape mealybug, obscure mealybug, Gill's mealybug, and long-tailed mealybugs. The research has mainly addressed the mealybugs and their tending ant species and their management in vineyards in Virginia. The study identifies grape mealybug and Gill's mealybug as the dominant species with occasional sightings of obscure mealybug and a lack of vine mealybug throughout the sampled vineyard sites. The male mealybugs were monitored using commercially available pheromone delta traps and adhesive band traps. Placement of adhesive band traps in the cordon and trunk of grapevines revealed different male mealybug capture data suggesting their movement within the grapevines. Two generations of mealybugs including the first generation with lower population density emerging out from the overwintering life-stage and the subsequent second generation responsible for colonizing different parts of the grapevines and producing overwintering life-stages were observed. Fifteen genera of ants were sampled from the vineyard via pitfall trap with some ants like pavement ants, acrobat ants, and medium garden ants actively tending and protecting the mealybugs. The use of sugar dispensers in the vineyards also led to lower fruit cluster infestations with mealybugs in comparison to the area without dispensers. The insecticidal spray trial using three different insecticides was also effective in controlling populations of mealybugs on some of the dates after treatment. The research offers valuable insights for grape growers, aiding in the development of effective control strategies for mealybug control. It emphasizes the importance of identifying the species present, understanding their behavior, and exploring potential allies and insecticide options in pest management.

Huanglongbing é a mais destrutiva doença da citricultura mundial. Seu recente relato no Brasil, em 2004, e a detecção de duas espécies bacterianas associadas à doença -Candidatus Liberibacter asiaticus e Candidatus Liberibacter americanus resultaram em poucos estudos do comportamento do Huanglongbing nas condições dos pomares brasileiros. Os objetivos deste trabalho foram (i) comparar a eficiência de infecção por Candidatus Liberibacter asiaticus e Candidatus Liberibacter americanus em plantas cítricas inoculadas por meio de enxertia com as duas espécies bacterianas, separadamente, e mantidas sob diferentes temperaturas, e (ii) comparar o progresso temporal e espacial do Huanglongbing causado por ambas as espécies, a partir da infecção natural por Diaphorina citri, tendo plantas de citros ou Murraya paniculata (murta) como fonte primária de inóculo. Para alcançar o primeiro objetivo, dois experimentos foram conduzidos em câmaras de crescimento com temperatura e fotofase controladas. A incidência da doença foi determinada por Real-Time PCR de amostras foliares. As temperaturas mais amenas foram favoráveis à infecção por Candidatus Liberibacter americanus, enquanto que altas temperaturas não afetaram a infecção de Candidatus Liberibacter asiaticus. A eficiência de infecção para esta última espécie foi favorecida nas temperaturas de 17/22°C e 22/27°C (noturna/diurna, respectivamente). Para cumprir com o segundo objetivo, um experimento foi conduzido com diferentes fontes de inóculo, que corresponderam aos seguintes tratamentos: (i) plantas de laranjeira doce da variedade Natal infectadas por Candidatus Liberibacter asiaticus ou Candidatus Liberibacter americanus, (ii) unicamente plantas de Murraya paniculata infectadas por Candidatus Liberibacter americanus e (iii) unicamente plantas de laranjeira doce da variedade Natal infectadas por Candidatus Liberibacter americanus. Cada tratamento foi aplicado em um compartimento isolado de uma casade- vegetação. O primeiro tratamento foi repetido. Em todos os compartimentos, 156 a 158 plantas de laranjeira doce da variedade Valência foram dispostas ao redor das plantas fonte de inóculo. Psilídeos livres das bactérias foram periodicamente confinados em plantas fonte de inóculo e, posteriormente foram liberados na casa-de-vegetação para livre movimentação e multiplicação. Quando as duas espécies bacterianas estavam presentes nas diferentes plantas cítricas utilizadas como fonte de inóculo, houve maior detecção de Candidatus Liberibacter asiaticus e maior eficiência de aquisição desta espécie bacteriana por psilídeos. Quando somente Candidatus Liberibacter americanus estava presente nas plantas fonte de inóculo, a eficiência de transmissão foi maior quando Murraya paniculata foi utilizada como fonte de inóculo, mostrando que Diaphorina citri pode adquirir naturalmente Candidatus Liberibacter 12 americanus de Murraya paniculata e transmiti-la para plantas cítricas. A eficiência de aquisição da espécie Candidatus Liberibacter americanus também foi maior tendo Murraya paniculata como planta fonte de inóculo e não citros. / Huanglongbing is the most destructive disease of citrus worldwide. There are few studies on the behavior of Huanglongbing in the Brazilian orchards conditions due to the recent report in Brazil, in 2004. Two bacterial species were associated with the disease in Brazil: Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus. The aims of this work were (i) to compare the efficiency of infection by Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus in citrus graft-inoculated plants with both bacterial species, separately, and kept under different temperatures, and (ii) to compare the spatio-temporal progress of Huanglongbing caused by both species, naturally transmitted by Diaphorina citri, having infected citrus plants or Murraya paniculata (jasmine orange) as primary inoculum sources. To achieve the first objective, two experiments were carried out in plant growth chambers with controlled temperature and light. Disease incidence in the inoculated plants was determined by Real-Time PCR of the leaf samples. The milder temperatures were favorable to Candidatus Liberibacter americanus infection, while the higher temperatures did not affect the infection by Candidatus Liberibacter asiaticus. The infection by this latter species was favored by temperatures of 17/22ºC and 22/27°C (night/day, respectively). To achieve the second objective, an experiment was carried out with different sources of inoculum, corresponding to the following treatments: (i) Natal sweet orange plants infected by Candidatus Liberibacter asiaticus or Candidatus Liberibacter americanus, (ii) only Murraya paniculata plants infected by Candidatus Liberibacter americanus and (iii) only Natal sweet orange plants infected by Candidatus Liberibacter americanus. Each treatment was applied in an isolated compartment of a screenhouse. The first treatment was repeated. In all of the compartments, from 156 to 158 Valencia sweet orange plants were located around the source of inoculum plants. Psyllids free of the bacteria were periodically confined in source of inoculum plants and, after this period, they were set free, and allowed to the free movement and multiplication inside the screenhouse. When both bacterial species were present in the different citrus plants as source of inoculum, there was higher detection of Candidatus Liberibacter asiaticus than Candidatus Liberibacter americanus and higher acquisition efficiency of the former bacterial species than the later by psyllids. When only Candidatus Liberibacter americanus was present in the source of inoculum plants, the higher transmission efficiency was reached when Murraya paniculata served as source of inoculum, showing that Diaphorina citri can naturally acquire Candidatus Liberibacter americanus from Murraya paniculata and transmit it to citrus plants. The acquisition efficiency of the 14 Candidatus Liberibacter americanus species was higher with Murraya paniculata as source of inoculum than with citrus plants was the source of inoculum.

Methylobacterium mesophilicum SR1.6/6 é uma bactéria endofítica isolada de ramo de citros previamente esterilizado superficialmente. Esta bactéria possui a capacidade de associar-se com uma ampla variedade de espécies e tecidos de plantas, principalmente nas raízes, mediado pela formação de biofilme e superfícies do hipocótilo de plântulas in vitro. Este trabalho teve como objetivo o desenvolvimento e a aplicação de um modelo para o estudo in vitro da associação entre esta bactéria e plântulas de soja. Metodologias de genômica e transcriptômica foram aplicadas para a obtenção do draft genômico desta bactéria e de um amplo perfil de sequências expressas, obtidas em dois tratamentos distintos; i) células de biofilme células bacterianas aderidas às raízes das plântulas removidas por sonicação, e ii) células planctônicas células bacterianas em suspensão (i.e. interagindo somente com exsudados radiculares). Os dados genômicos obtidos por 454-pirosequenciamento resultaram em uma cobertura de 37 vezes o tamanho do genoma e geraram 242 contigs. Entre estes, 187 contigs grandes representaram 96% do genoma (tamanho estimado de 6.8 Mb), com um conteúdo GC de 69.5%. Considerando a análise da expressão gênica, um procedimento para monitorar a aderência das células bacterianas às raízes das plântulas foi realizado por meio de microscopia eletrônica de varredura de amostras de raízes coletadas durante o experimento (i.e. 24, 48 e 72h após a inoculação). As células bacterianas obtidas em cada tratamento foram inicialmente submetidas à extração de RNA total, seguida de um processo de enriquecimento de mRNA e sequenciamento por meio da tecnologia de 454-pirosequenciamento (RNA-Seq). O amplo perfil de expressão gênica obtido foi mapeado no draft genômico, resultando em um total de 1.930 clusters gênicos. Posteriormente, estes clusters foram filtrados de acordo com sua abundância e ocorrência diferencial em cada tratamento, resultando em 280 genes diferencialmente expressos. Funções relacionadas ao metabolismo de etanol/metanol, divisão celular, resposta ao estresse oxidativo, produção de sideróforos, biossíntese de peptidoglicanos e hopanóides foram induzidas nas células bacterianas aderidas às raízes das plântulas, enquanto que genes relacionados ao metabolismo essencial das células foram observados principalmente nos tratamentos controle e planctônico. Estes dados fornecem uma base para estudos relacionados a mecanismos moduladores da interação bactériaplanta, distinguindo significativamente os tratamentos biofilme e planctônico, mostrando assim que o contato físico é essencial para o sucesso da interação em estudo. Por fim, estas análises permitiram uma ampla visualização de perfis de expressão gênica desta bactéria, utilizando o draft genômico primeiramente obtido como base para o estudo desta interação com a planta hospedeira. Estudos futuros podem ser desenvolvidos visando caracterizar os mecanismos adaptativos desta bactéria, como seu metabolismo metilotrófico e outros metabolismos específicos, os quais podem dar suporte ao comportamento endofítico deste organismo. / Methylobacterium mesophilicum strain SR1.6/6 is an endophytic bacterium, which has originally been isolated from surface-sterilized healthy citrus branch. This bacterium is able to associate with a range of plant species, rather in the roots, mediated by a biofilm structure, and in hypocotyl surfaces of in vitro seedlings. The aim of the present study was the development and application of a model to study in vitro the association between this bacterium and soybean seedlings. Genomic and transcriptomic approaches were applied resulting a draft of this bacterium genome and a broad profile of mapped mRNA sequences obtained in two different treatments; i) biofilm cells root adhered bacterial cells were removed by sonication, and ii) planktonic cells bacteria cells in suspension (i.e. interacting only with root exudates). Genomic data, obtained by 454-pyrosequencing have had an average depth of 37-fold coverage of the genome and yielded 242 contigs. Among these, 187 large contigs represented 96% of the genome sequence (estimate size of 6.8 Mb) with a GC content of 69.5%. Concerning the gene expression survey, the process to monitor the adherence of bacteria cells to the roots was performed by scanning electron microscopy of roots collected along the experiment (i.e. 24, 48 and 72 hours after inoculation). Bacterial cells obtained in each treatment were firstly submitted to RNA extraction, followed by mRNA enrichment and RNA-Seq using 454-pyrosequencing technology. The broad gene expression profile obtained was mapped into the drafted genome, resulting in a total of 1.930 gene clusters. After that, these clusters were filtered according to their abundance and differential occurrence in each treatment resulting in 280 differential expressed genes. Functions related to methanol/etanol metabolism, cell division, oxidative stress response, siderophore production, peptidoglycan and hopanoid biosynthesis were induced in bacterial cells adhered to plant roots, while genes related to essential cell metabolism were observed mostly in control and planktonic treatment. Also, these data provide insights into the mechanisms modulating plant microbe-interaction, significantly distinguishing biofilm and planktonic treatment, showing that the physical contact is a crucial step on plant-microbe interactions. In conclusion, results allowed a strongly supported analysis of gene expression, based on the genome draft of an endophytic bacterium interacting with the host plant. Further studies should focus on the adaptive mechanisms present in this bacterium, like the methylotrophic lifestyle and other specific metabolisms which might support its behavior as an endophytic bacterium.