O fungo Magnaporthe oryzae, agente causal da brusone em trigo e em várias outras gramíneas, desde o seu primeiro relato no Brasil tem sido alvo de inúmeras pesquisas. O entendimento da morfologia, fisiologia e parâmetros bioquímicos deste ascomiceto é o primeiro passo para a adoção de medidas eficientes de controle da doença. Os objetivos deste trabalho foram avaliar os aspectos morfológicos, fisiológicos e bioquímicos relativos à patogenicidade do fungo M. oryzae em trigo, além de determinar in vitro as condições ótimas de temperatura e fotoperíodo para o crescimento e esporulação dos isolados testados, quantificar a pressão de turgor exercida pelo apressório no momento da penetração do substrato, verificar a presença de enzimas extracelulares produzidas pelo patógeno e o papel de cada uma delas na degradação da parede celular e, também, demonstrar a possível produção e ação de metabólitos tóxicos do fungo em plântulas de trigo. Dois isolados do fungo, Py5003 e Py6017, foram repicados para os meios de cultivo cenoura, milho-cenoura, batata-dextrose-ágar (BDA), aveia e V8, incubados, de maneira independente, sob luz contínua, escuro contínuo e fotoperíodo de 12h, por 20 dias, sendo avaliados periodicamente quanto ao crescimento micelial total e esporulação do patógeno. Em seguida, conídios germinados e com apressórios formados foram submetidos a diferentes concentrações de PEG-8000 para a mensuração da pressão de turgor apressorial. Além disso, o teor de proteínas totais foi mensurado e a atividade celulolítica determinada, por espectrofotometria indireta para os isolados cultivados em meio Merlin-Norkrans Modificado (MNM), a partir das enzimas exo e endo-?-1,4-glucanase e ?-glicosidase. A produção de metabólitos tóxicos foi determinada por condutivimetria, após o cultivo dos isolados por 5 e 10 dias em batata-dextrose (BD) e obtenção dos filtrados após esse período, sendo que, a ação destes, foi observada em plântulas de trigo com 25 dias de idade. Os resultados evidenciaram melhor crescimento micelial e esporulação dos isolados em meio de cultivo composto de aveia, incubado a 25 °C e fotoperíodo de 12 h. A pressão de turgor apressorial, tanto para Py5003 quanto para Py6017, concentrouse na faixa dos 7,5 MPa. Não houve diferença estatística entre os teores de proteínas totais apresentados pelo isolados. Os dois isolados exibiram alta atividade celulolítica, com diminuição da atividade observada para endo-?-1,4-glucanase e ?- glicosidase na presença de glicose e celobiose, respectivamente. A exposição de plântulas de trigo aos filtrados dos isolados do fungo M. oryzae provocou alta liberação de eletrólitos, sendo, em termos absolutos, Py5003 maior do que Py6017. Plântulas expostas ao filtrado, autoclavado ou não, e ao extrato bruto obtido a partir da separação dos metabólitos tóxicos em acetato de etila, apresentaram-se cloróticas e/ou necróticas. Os resultados sugerem um mecanismo de parasitismo desenvolvido por M. oryzae no desenvolvimento da brusone em trigo, indo desde a fase de pré-penetração, passando pelo estabelecimento das relações parasitárias estáveis e o aparecimento de sintomas e sinais na planta hospedeira. / The fungus Magnaporthe oryzae, causal agent of blast in wheat and several other grasses, since its first occurrence in Brazil has been the subject of extensive research. The understanding of the morphology, physiology and biochemical parameters of this ascomycete is the first step for the adoption of efficient measures to control the disease. The objectives of this study were to evaluate the morphological, physiological and biochemical aspects related to the pathogenicity of the fungus M. oryzae on wheat. Thus, it were determined in vitro the optimal conditions of temperature and photoperiod for growth and sporulation of the isolates tested, quantified the pressure exerted by appressorium at the time of substrate penetration, verified the presence of extracellular enzymes produced by the pathogen and their role in the degradation of cell wall and also demonstrated the possible production and action of toxic metabolites of the fungus in wheat seedlings. Two isolates of the fungus, Py5003 and Py6017, were grown on carrot, maize-carrot, PDA, oat and V8 media and measure periodically for 20 days and after that period, being evaluated the total mycelia growth and sporulation of the pathogen. Then, germinated conidia and appressoria were subjected to different concentrations of PEG-8000 for the measurement of appressorial turgor pressure. In addition, the concentration of total proteins was measured and enzyme activity (exo and endo-?- 1,4-glucanase, ?-glucosidase) was determined by indirect spectrophotometry, for the isolates grown in Modified Melin-Nokrans (MMN) medium. The production of toxic metabolites was determined after growing the isolates for 5 and 10 days in potatodextrose medium (PD) and obtaining the filtrate. The toxic action was evaluated in wheat seedlings (25 days old) by measuring electrolyte leakage. The results showed better mycelial growth and sporulation of the isolates in oat medium incubated at 25 °C and under a photoperiod of 12 h. Appressorial turgor pressure for both isolates were at the range of 7.5 MPa.There were no statistical differences between the levels of total protein presented by the isolates. The two isolates showed high cellulolytic activity, with decreased activity observed for endo-?-1,4-glucanase and ?- glucosidase in the presence of glucose and celobiose, respectively. The exposure of wheat seedlings to the filtrates of the fungus M. oryzae caused high electrolyte release, being, in absolute terms, Py5003 greater than Py6017. Seedlings exposed to the filtrate, autoclaved or not, and the crude extract obtained from the separation of the toxic metabolites in ethyl acetate, exibited clorotic and/or necrotic. These results suggest a mechanism of parasitism developed by M. oryzae on wheat, ranging from pre-penetration through the establishment of stable parasitic relationships and the appearance of symptoms and signals in the host plant.

La pyriculariose provoquée par le champignon Magnaporthe oryzae constitue l’une des principales maladies du riz, avec des épidémies récurrentes et très destructrices qui touchent toutes les régions rizicoles. Outre les enjeux économiques et humanitaires liés à cette maladie, le statut de céréale modèle du riz et de champignon parasite modèle de M. oryzae font de l’interaction entre ces deux partenaires un modèle privilégié pour l’étude du contrôle génétique et moléculaire des relations hôte-pathogène. Les protéines effectrices, Avr, de M. oryzae sont sécrétées au cours de l’infection de la plante par ce champignon. Chez certaines variétés de riz ces effecteurs agissent comme des protéines d’avirulence. Elles sont reconnues par les protéines de résistance, R, et induisent une forte réponse de défense dont une mort cellulaire programmée appelée HR (hypersensitive response) à l’origine de la résistance de la plante. L’objectif de cette thèse est de déterminer les structures des protéines Avr, et R et celles des complexes Avr-R. L'analyse structure-fonction permettra d’identifier les domaines et les acides aminés importants pour l'activité des effecteurs et des protéines de résistance. Pour la réalisation de cette thèse, une large gamme de techniques de biologie moléculaire (PCR, clonage moléculaire), de biophysique (Fluorescence) et de biologie structurale (RMN, cristallographie aux RX) disponibles au laboratoire seront utilisées. Du point de vue fonctionnel, ces études devraient apporter de nouveaux éléments sur les fonctions végétales qui sont modulées au cours de l’infection et permettent ainsi à la maladie de s'établir en créant des conditions favorables à la mise en place du processus infectieux. / MAGNAPORTHE oryzae causes a devastating disease of rice known as rice blast, affecting all regions cultivating rice. In addition to the economic and humanitarian issues associated with the disease, rice and M. oryzae are widely accepted as models of cereal and fungus for studying genetic control and host-pathogen molecular interactions. The effector proteins, Avr of M. oryzae, are secreted during plant infection by the fungus. In some of the rice cultivation these effectors act as avirulence proteins. They are recognized by resistance proteins R, and induce a strong host response, including programmed cell death known as HR (hypersensitive response) leading to plant resistance. The purpose of the thesis is to elucidate the atomic structures of the proteins Avr, R and Avr-R complexes. Subsequent structure-function analysis will identify domains and important amino acids needed for the activity of the effector and resistance proteins. To carry out this thesis, a wide range of molecular biology techniques (pcr, molecular cloning), biophysics (fluorescence) and structural biology (NMR, X-ray crystallography) will be used. From the functional point of view, these studies will provide insight into mechanisms affecting host functions that are modulated during infection, thus creating favorable conditions for the infection process.

La résistance partielle aux agents pathogènes représente une source importante pour l'amélioration des plantes. Cependant les mécanismes moléculaires sous-jacents à ce type de résistance sont encore mal connus. L'interaction entre le riz et le champignon Magnaporthe oryzae est un modèle de choix pour ce type d'analyse, de nombreuses ressources génétiques et outils d'analyse fonctionnelle étant disponibles. Chez le riz, hormis le gène Pi21 qui contrôle la résistance partielle, aucune information biologique et fonctionnelle ne permet d'expliquer cette forme de résistance. En amont de ce travail de thèse, le phénomène de défense préformée a récemment été identifié ; il est défini par la corrélation entre l'expression des gènes liés à la défense avant infection et la résistance partielle à M. oryzae. L'identification de régulateurs de la résistance partielle et des défenses préformées a été l'objectif de cette thèse. Deux stratégies ont été adoptées. Une étude du transcriptome visant à sélectionner et caractériser des gènes candidats sur la base de leur profil d'expression constitutive a été réalisée. Une méthode de sélection par « guilt-by-association » s'est avérée efficace pour identifier des gènes impliqués dans la résistance de la plante. Les gènes AGO18, Z-BED, HSF23 et CaMBP ont été identifiés comme des régulateurs positifs des défenses de la plante. Les gènes HSF23 et CaMBP contrôlent l'expression constitutive des gènes liés à la défense mais leur sur-expression modifie la croissance des plantes. La sur-expression des gènes Z-BED et AGO18 n'a entraîné aucune modification de la croissance de la plante mais une augmentation de la résistance à M. oryzae, sans modification apparente de l'expression des gènes de défense testés. Le gène Z-BED code pour un facteur de transcription putatif dont on peut faire l'hypothèse qu'il contrôle un ensemble encore inconnu de l'arsenal de défense. Le gène AGO18 code pour une protéine argonaute potentiellement impliquée dans l'extinction de gène via la méthylation de la chromatine. Enfin le gène OB-fold est un régulateur négatif des défenses de la plante dont les cibles, potentiellement des ARN, restent à identifier.La deuxième approche a consisté en une détection de loci contrôlant la densité de lésions causées par M. oryzae. Une zone du génome, PRM1, contrôle ce phénotype, confère une résistance à un spectre de souches relativement large, semble contrôler l'expression de gènes de défense avant et au cours de l'infection, et enfin semble ralentir la croissance du champignon avant pénétration. Cette approche sans a priori renforce l'hypothèse que l'expression des gènes de défense avant infection est associée à la résistance partielle du riz à M. oryzae.De plus amples investigations seront nécessaires pour relier les phénotypes de résistance partielle tels que l'inhibition de la croissance pré-pénétration et la densité de lésions entre eux d'une part et d'autre part à l'expression des gènes de défenses avant infection / Partial resistance to pathogens is a major source for plant breeding. However, the molecular mechanisms underlying this type of resistance are still poorly understood. The interaction between rice and the fungus Magnaporthe oryzae is a model of choice for this type of analysis, many genetic and functional analysis tools being available. In rice, except for the Pi21 gene that controls partial resistance, no biological and functional information can explain this form of resistance. Prior to this thesis, the phenomenon of preformed defense has recently been identified; it is defined by the correlation between the expression of genes related to defense before infection and partial resistance to M. oryzae. Identification of partial resistance and preformed defense regulators has been the objective of this thesis. Two strategies were adopted.A transcriptome analysis to select and characterize candidate genes based on their constitutive expression pattern was performed. A method of selection by "guilt-by-association" has been effective in identifying genes involved in plant resistance. The genes AGO18, Z-BED, HSF23 and CaMBP were identified as positive regulators of plant defenses. The genes HSF23 and CaMBP control the constitutive expression of defense related genes, but their over-expression modifies plant growth. Over-expression of Z-BED and AGO18 genes does not affect plant growth but increases the resistance to M. oryzae, without apparent change in the expression of the defense genes tested. The Z-BED gene encodes for a putative transcription factor that likely controls an unknown set of the defense arsenal. The AGO18 gene encodes an Argonaute protein potentially involved in gene silencing via chromatin methylation. Finally the OB-fold gene is a negative plant defense regulator, and its hypothetical RNA targets remain to be identified.The second approach consisted of detection of loci controlling the lesions density caused by M. oryzae. A region of the genome, PRM1, controls this phenotype, confers resistance to a relatively wide range of isolates, appears to control the expression of defense genes before and during the infection, and finally seems to inhibit the growth of the fungus before penetration. This approach without a priori supports the hypothesis that the expression of defense genes before infection is associated with partial resistance of rice to M. oryzae.Further investigations are needed to link the resistance phenotypes such as partial inhibition of fungal growth pre-penetration and density of these lesions on the one hand, and the defense gene expression before infection on the other hand.

Dimorphandra mollis Benth. (Fabaceae - Caesalpinioideae), popularmente conhecida como faveiro, fava d\'anta ou faveleiro, é uma espécie nativa do Cerrado brasileiro, que apresenta registros de ocorrência por todo o Brasil Central. Sua principal importância econômica está associada às elevadas concentrações de rutina, um flavonóide alvo de muitos estudos farmacológicos e de largo emprego na indústria farmacêutica, sendo esta a segunda espécie mais utilizada como fonte de rutina no mercado mundial. Apesar do amplo uso econômico, sua exploração não apresenta características agrícolas, sendo realizada por vias extrativistas. Além disso, há carência de informações sobre sua biologia, o que torna a exploração um fator de risco à conservação. Há poucos estudos relacionados à sua composição química de seus órgãos e tecidos, além disso, estudos de germinação in vitro para obtenção de calos, suspensões celulares e clones não apresentaram sucesso. Este trabalho teve por objetivo desenvolver uma detalhada investigação da composição flavonoídica de folhas, flores, frutos, madeira, cascas, plântulas, embriões, exsudados radiculares de plântulas, e calos originados da desdiferenciação de diversos explantes pelo emprego da técnica de cromatografia de alta eficiência com detector de arranjo de diodos acoplado ao espectrômetro de massas, com o intuito de contribuir para uma maior compreensão da composição química de seus órgãos e tecidos, e assim, subsidiar novos estudos sobre a biossíntese de flavonoides, assim como, contribuir para a própria exploração racional dos recursos econômicos inerentes a essa espécie. Os resultados obtidos levam à conclusão de que nos diversos órgãos e tecidos de D. mollis investigados, predominam estereoisômeros de astilbina, rutina e quercitrina. Os calos apresentaram perfil flavonoídico muito simples, contendo apenas pequenas quantidades de apigenina, não sendo viáveis à obtenção de flavonoides de interesse por meio de seu cultivo. Dimorphandra mollis Benth. (Fabaceae - Caesalpinioideae), popularmente conhecida como faveiro, fava d\'anta ou faveleiro, é uma espécie nativa do Cerrado brasileiro, que apresenta registros de ocorrência por todo o Brasil Central. Sua principal importância econômica está associada às elevadas concentrações de rutina, um flavonóide alvo de muitos estudos farmacológicos e de largo emprego na indústria farmacêutica, sendo esta a segunda espécie mais utilizada como fonte de rutina no mercado mundial. Alguns artigos acadêmicos reportam a detecção de propriedades tóxicas de extratos metanólicos de D. mollis sobre operárias de Apis mellifera, e atribuem a toxicidade ao flavonóide astilbina (3-β-Ο/-rhamnosideo de 5,7,3’,4’-tetraidroxi-2,3-diidroflavonol) isolado dos pedúnculos e flores. Seus resultados indicam que a astilbina reduziu a sobrevivência média das abelhas tratadas. Também é atribuída a astilbina a atividade inibidora sobre o desenvolvimento de larvas de Anticarsia gemmatalis Hübner (Lepdoptera: Noctuidae) e Spodoptera frugiperda Smith (Lepdoptara: Noctuidae), relatando redução do peso corporal durante a fase larval e, prolongamento, tanto da fase larval quanto da fase pupal, para as duas espécies de lepidópteros, consideram a possibilidade de uso deste flavonóide como um biopesticida. Neste estudo, foram testadas as interferências de extratos flavonoídicos de diversos órgãos de D. mollis sobre o forrageio e o ciclo de vida de lagartas de Spodoptera frugiperda (Lepidoptera: Noctuidae) e de adultos de Sitophilus oryzae (Coleoptera: Curculionidae). Nenhum resultado negativo estatisticamente significativo foi observado no forrageio das duas espécies em questão. Tal resultado pode ser devido a uma tolerância desses animais aos flavonoides majoritários encontrados nos extratos, astilbina, rutina e quercitrina, ou ainda a mecanismos de detoxificação. / Dimorphandra mollis Benth. (Fabaceae - Caesalpinioideae), popularly known as faveiro, fava d\'anta or faveleiro, is a native species of the Brazilian Cerrado, which has records of occurrence throughout the Central Brazil. Its main economic importance is associated with high concentrations of rutin, a flavonoid target of many pharmacological studies and widespread use in the pharmaceutical industry. D. mollis is the second most commonly used plant species as a source of rutin in the global market. Despite their broad economic use, their exploitation occurs in a very primitive way. In addition, there is no subsidiary information about their biology, which makes exploring this species a risk factor for its conservation. There are few studies that deals with the chemical composition of D. mollis organs and tissues, in addition, in vitro germination studies to obtain calluses, cell suspensions and clones showed no success. This work aimed to develop a detailed research flavonoid composition of leaves, flowers, fruits, wood, bark, seedlings, embryos, seedlings root exudates, callus using high performance liquid chromatography with diode array detector coupled to a mass spectrometer in order to contribute to a greater understanding of the chemical composition of their organs and tissues, and thus support new studies on flavonoid biosynthesis, as well as contribute to the very rational exploitation the financial resources inherent in this species. The principal constituents found were stereoisomers of astilbin, rutin and quercitrin. The callus culture presented a very simple flavonoid profile containing only small amounts of apigenin. Dimorphandra mollis Benth. (Fabaceae - Caesalpinioideae), popularly known as faveiro, fava d\'anta or faveleiro, is a native species of the Brazilian Cerrado, which has occurrence records throughout the Central Brazil. Its main economic importance is associated with high concentrations of rutin, a flavonoid target of many pharmacological studies and widespread use in the pharmaceutical industry, which is the second species most commonly used as a source of rutin in the world market. Some scientific papers report toxic activities of methanol extracts of D. mollis on Apis mellifera of workers, and attribute the toxicity astilbin flavonoid (3-β-Ο-rhamnoside of 5,7,3’,4’-tetraidroxi- 2,3-diidroflavonol) isolated from the stems and flowers of this species. These results suggest that astilbin reduced the mean survival of the treated bees. Also is attributed to astilbin the inhibitory activity against the development of Anticarsia gemmatalis Hübner (Lepdoptera: Noctuidae) larvae and Spodoptera frugiperda Smith (Lepdoptara: Noctuidae), reporting body weight loss during the larval stage and extending both larval and pupal stage for the two Lepidopterans. These results suggests the use of astilbin as a biopesticide. In the present study, was tested the interference of the flavonoid extracts from several D. mollis parts on the foraging and life cycle of both Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae and adults of Sitophilus oryzae (Coleoptera: Curculionidae). No statistically significant negative result was observed in foraging of both species concerned. This result may be due to a tolerance of these species to flavonoids majorly found in D. mollis extracts, astilbin, rutin and quercitrin, or due detoxification mechanisms.

Xylanase was purified from the crude culture of Aspergillus oryzae DSM1863 by sephadex G200 and DEAE – cellulose ion exchange chromatography. The molecular mass of the purified xylanase determined by SDS–PAGE was 21 kDa with a specific activity of 6768 U/mg towards 1% (w/v) of birch wood xylan. The optimum temperature was observed at 60°C. The enzyme was thermostable in the temperature range of 37-50°C with a high residual activity of 62-74% (650.6- 775.9 U/mg protein). / Enzyme xylanase được tinh sạch từ dịch lên men của chủng Aspergillus oryzae DSM1863 sau khi qua cột sắc ký lọc gel sephadex G200 và sắc ký trao đổi ion DEAE – cellulose. Khối lượng phân tử của enzyme xylanase tinh sạch được xác định bằng điên di đồ SDS- PAGE. Xylanase tinh sạch có kích thước là 21 kDa với hoạt tính đặc hiệu đạt 6768 U/mg sau khi được xác định với nồng độ cơ chất là 1% birch wood xylan. Nhiệt độ tối ưu để enzyme hoạt động mạnh nhất là 60°C. Enzyme xylanase khá bền nhiệt. Hoạt tính của enzyme vẫn còn duy trì 62-74% (hoạt tính đặc hiệu đạt 650.6-775.9 U/mg protein) sau khi 8 giờ ủ ở 37-50°C.

La bactériose vasculaire du riz (BLB) et bactériose non-vasculaire du riz (BLS), causées respectivement par Xanthomonas oryzae pv. oryzae (Xoo) et X. oryzae pv. oryzicola (Xoc), sont les deux plus importantes maladies bactériennes du riz. Ces maladies limitent le rendement de la production de riz dans les zones rizicoles en Asie et dans certaines régions d'Afrique. L'infection et la multiplication bactérienne dans les tissus de l'hôte dépendent souvent des facteurs de virulence de ces bactéries dont le type le système de sécrétion de type III (T3SS) et ses substrats. Dans cette thèse, nous avons identifié neuf effecteurs non-TAL (transcription Transcription activator-like) sécrétés par des effecteurs de type III de la souche chinoise 13751 de Xoo en utilisant le domaine d'induction HR de la protéine avirulente AvrBs1 comme gène reporter. Parmi eux, XopAE13751 a été expérimentalement confirmé pour la première fois comme étant un effecteur non-TAL. Ensuite, par l'analyse mutationnelle de ces gènes effecteurs identifiés dans Xoo, nous avons constaté que l'effecteur non-TAL XopR13751 était nécessaire pour la virulence de la souche chinoise de Xoo sur le riz hybride Teyou63. En parallèle, nous avons démontré que le gène rsmA (repressor of secondary metabolism) - comme le gène rsmAXoo de l'espèce chinoise Xoo 13751- régule positivement l'expression des gènes associés aux facteurs de virulence, tels que le système de sécrétion de type III, les enzymes extracellulaires et le DSF (diffusible signal factor). De plus, le gène effecteur non-TAL xopO s'est avéré être peu répandu chez les Xanthomonas puisqu'il est présent uniquement chez X. euvesicatoria (Xe) et Xoc mais est absent chez Xoo. En considérant les deux pathovars de X. oryzae, avec deux modes d'infection différents, xopO a été examiné comme un facteur de la spécificité du tissu par l'inactivation mutationnelle du gène dans Xoc et par l'expression du gène dans Xoo. Les résultats ont montré que xopO n'est pas la cause déterminante de la spécificité de tissu chez Xoc. Enfin, nous avons étudié les VNTRs (Variable Number of Tandem Repeats) comme outil de typage moléculaire rapide, fiable et rentable, pour améliorer le contrôle des épidémies et pour évaluer la structure de population des souches de Xoc. 28 loci candidats VNTR ont été prédits par le criblage de trois génomes de Xoc (souche philippine BLS256, souche chinoise GX01 et souche malienne MAI10). Des paires d'amorces pour l'amplification de PCR de chacun des 28 loci ont été conçues et testées à un pannel de 20 souches de Xoc provenant de l'Asie et de l'Afrique. Le séquençage des amplicons de PCR a confirmé 25 loci VNTR robustes et polymorphes communs entre les souches Xoc asiatiques et africaines. Un dendrogramme, construit à partir de la combinaison des 25 loci de VNTR (MLVA-25), a montré que la plupart des souches asiatiques sont clairement distinguables des souches africaines. Cependant, en accord avec de précédents rapports, une souche Malienne se distingue et semble être liée aux souches asiatiques, suggérant une introduction possible de souches sur le continent africain. Ce nouvel outil de typage basé sur les VNTR sera utile pour l'étude de structures de populations et pour la surveillance épidémiologique de Xoc. / Bacterial leaf blight (BLB) and Bacterial leaf streak (BLS), caused respectively by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), are two most important bacterial diseases on rice, constraining severely the rice yield in the rice-growing areas in Asia and in parts of Africa. Successful infection and bacterial multiplication in host tissue often depend on virulence factors from these bacteria including type Ⅲ secretion system (T3SS) and its substrates. In this thesis, we identified nine type Ⅲ secreted non-TAL (Transcription activator-like) effectors of Xoo Chinese strain 13751 using HR-inducing domain of avirulence protein AvrBs1 as the reporter, among them, XopAE13751 was first found experimentally to be non-TAL effector. Subsequently, through mutational analysis of these identified effector genes in Xoo, we showed non-TAL effector XopR13751 was found to be required for full virulence of Xoo Chinese strain in hybrid rice Teyou63. In parallel, we demonstrated that rsmA (repressor of secondary metabolism)-like gene rsmAXoo of Xoo Chinese strain 13751 positively regulated the expression of genes associated with virulence factors such as type Ⅲ secretion system, extracellular enzymes and diffusible signal factor (DSF). Furthermore, non-TAL effector gene xopO was found to be narrowly distributed in Xanthomonas, which was only present in X. euvesicatoria (Xe) and Xoc, but not in Xoo. Based on the consideration of two X. oryzae pathovars carrying two different infection ways, xopO was tested in host and tissue specificity by analysis of mutational analysis of the gene in Xoc and expression of the gene in Xoo. The results showed that xopO of Xoc did not function as a determinant in host and tissue specificity. Finally, we explored Variable Number of Tandem Repeats (VNTRs) as a fast, reliable and cost-effective molecular typing tool, to better monitoring epidemics and assess the population structure of Xoc strains. 28 candidate VNTR loci were predicted by screening of three Xoc genome sequences (Philippine strain BLS256, Chinese strain GX01 and Malian strain MAI10). Primer pairs for PCR amplification of all 28 loci were designed and applied to a panel of 20 Xoc strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci which are shared among Asian and African strains of Xoc. A dendrogram was constructed from 25 VNTR loci-combinating data (MLVA-25), indicating that most Asian strains were clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali appeared to be related to Asian strains, pointing to a possible introduction of strains to the African continent. A detailed analysis of the evolutionary relationships among a larger set of Xoc strains from China will be presented, considering different spatial scales. In conclusion, a new VNTR-based tool useful for studies of population structures and epidemiological monitoring of Xoc was successfully established.

Os micro-organismos que habitam o interior das plantas (endofíticos ou endófitos) tornaram-se foco de interesse por estarem envolvidos na produção de compostos químicos como enzimas, alcalóides, antibióticos, anticancerígenas e diferentes metabólitos. Os ecossistemas de regiões tropicais tem sido alvo de busca de compostos naturais por causa da riqueza de espécies e nichos ecológicos presentes nestas comunidades. O objetivo deste trabalho o isolamento, identificação e a bioprospecção de fungos endofíticos obtidos de Combretum leprosum e a detecção nos extratos de planta e micro-organismos da presença do composto combretastatin (CA4). Folhas, galhos, frutos e raízes de C. leprosum foram coletados de cinco estados dentro da zona de semiárido brasileiro: Bahia, Piauí, Ceará, Paraíba, Rio Grande do Norte. Partes das amostras foram triturados e submetidos à maceração primeiramente em diclorometano, seguidos de tetrahidrofurano e acetona de acordo com Pettit et al.(1987) para possível extração da CA4. Além disso, para avaliação in vitro da atividade citotóxica e antimicrobiana foram realizadas extrações em acetato de etila, clorofórmio e metanol. Foram detectados a possível presença da CA4 em todos os órgãos das plantas extraídos com tetrahidrofurano e as maiores concentrações foram observadas nas folhas. A atividade antitumoral dos extratos vegetais apresentaram as maiores inibições contra carcinoma (ovário IC50 10µg/mL-1, rim IC50 8,7µg/mL-1 e mama IC50 14,1µg/mL-1) e glioma.IC50 13,5µg/mL-1. A outra parte das amostras (folhas, caules e raízes) foram desinfetadas, fragmentadas e colocadas em meios de cultivo (Martin, BDA, Agar água) por 60 dias, 28°C. Foram isolados 405 fungos endofíticos e 159 apresentaram atividade contra fitopatogênicos, 72% para Rhizoctonia solani e 28% para Pythium aphanidermatum. As vinte e três linhagens que apresentaram as melhores atividades antifitopatogênicas foram submetidas a crescimento em Czapec em cultura estacionaria, por 30 dias, a 28°C, os respectivos metabólitos foram obtidos em múltiplo (3.0 e 11.0) e avaliados a atividade antimicrobiana contra bactérias patogênicas e fungos. Quatro linhagens foram selecionadas, identificadas pelo sequenciamento da região 18S, CFE177 como Fusarium oxysporum, CFE03 como Hypocrea koningii, linhagem CFE108 como Aspesgillus oryzae e CFE391 como Fusarium solani e avaliadas in vitro pelos testes biológicos: atividade antitumoral, antioxidante e antimicobactéria. Os compostos produzidos por A. oryzae CFE108 apresentaram potencial para bioprospecção, e de acordo com as atividade citotóxicas as maiores ações foram contra as linhagens linfoma histiocística (J744), mieloma murino (B16F10) e baixa citotóxidade para carcinoma de bexiga (ECV304) e leucemia eritroblástica humana (k562) na concentração de 1mg/mL-1. Foram isolados dois compostos: SS-XL-32-01 identificado como bis-(2-etilhexil) ftalato (DEHP) e SS-XL-20-1 identificado como fenol, 2.2 metilenobis[6-(1,1-dimetiletil)-4- etil], ambos com atividade anticâncer para células HeLa com percentual de ate 98% e 71%, de morte, respectivamente. Alem disso, a modificação através da reação de metilação do composto SS-XL-32-1 resultou na quebra do anel aromático, formação de 4 subprodutos e perda da atividade, sendo um indicativo do sitio ativo da molécula responsável pela atividade observada. Portanto, fungos endofíticos de 18 plantas do semiárido brasileiro podem ser considerados fonte de bioprospecção para novas moléculas bioativas com atividade antitumoral. / The micro-organisms that reside in the aerial tissues and roots of plants (endophytic or endophyte) became the focus of interest for being involved in the chemical production such as enzymes, alkaloids, antibiotics, anticancer and different metabolites. The ecosystems of tropical region have been targeted search of natural compounds because of the richness of species and ecological niches present in these communities. The aim of this work was the isolation, identification and bioprospection for endophytic fungi from Combretum leprosum and detection in extracts of the plant and micro-organisms for the presence of the combretastatin (CA4). Leaves, stems, fruits and roots of C. leprosum were collected from five states within the semi-arid zone of Brazil: Bahia, Piaui, Ceara, Paraiba, Rio Grande do Norte. Part of the samples were crushed and subjected to maceration in dichloromethane, followed by tetrahydrofuran and acetone according to Pettit et al. (1987) for extracting the possible CA4. Moreover, for in vitro evaluation of the cytotoxic and antimicrobial activity extractions were carried out in ethyl acetate, chloroform and methanol. Were detected the possible presence of CA4 all plant organs extracted with tetrahydrofuran and the highest concentrations were observed on the leaves. The antitumor activity of plant extracts showed the highest inhibition against carcinoma (ovary IC50 10µg/mL-1, kidney IC50 8.7 µg/mL-1 and breast IC50 14.1 µg/mL-1) glioma IC50 and 13.5 mg-/mL-1. The other part of the samples (leaves, stems and roots) were disinfected, fragmented and placed in culture media (Martin, PDA, water agar) for 60 days, 28°C. 405 Endophytic fungi were isolated and 159 showed activity against phytopathogenic, 72% for Rhizoctonia solani and 28% for Pythium aphanidermatum. Twenty-three strains that showed good activities antiphytopathogenic, were grow on medium Czapec in static culture, for 30 days at 28°C, the respective metabolites were obtained in multiples pH (3.0 and 11.0) and evaluated the antimicrobial activity against pathogenic bacteria and fungi. Four strains were selected, identified by sequencing the 18S region, CFE177 as Fusarium oxysporum, CFE03 as Hypocrea koningii, strain CFE108 as Aspesgillus oryzae and CFE391 Fusarium solani, and evaluated by in vitro biological tests: antitumor, antioxidant and antimicobactérium activity. The compounds produced by A. oryzae CFE108 had biological potential and in accordance with the cytotoxic activity, showed the highest activities against lymphoma lines (J744), murine myeloma (B16F10) and low cytotoxicity for carcinoma of the bladder (ECV304) and leukemia erythroblastic human (K562) in 1mg/mL-1 concentration. Two compounds were isolated: SS-XL-32- 01 identified as bis-(2-ethylhexyl)phthalate (DEHP), and SS-XL-20-1 as phenol 2.2methylenobis [6-(1,1-dimethylethyl) - 4-ethyl], both with anticancer activity for HeLa cells with a percentage of up to 98% and 71%, of death, respectively. In addition, modified by methylation reaction of the compound SS-XL-32-1 resulted in the breaking of the aromatic ring and result in formation of four product and loss of activity being indicative of the active site of the molecule can be the aromatic ring. Therefore, endophytic fungi in semiarid Brazil plant can be considered a source of bioprospection for new bioactive molecules with anticancer activity.

The blast is the main disease of flooded rice. The integrated management of this disease is based on the adoption of cultural, genetic and chemical strategies of control. The objective of this work was to evaluate the effect of eight chemical control programs of Pyricularia oryzae applied on IRGA 424, IRGA 428, GURI INTA CL, PUITA INTA CL, QM 10 10 CL and INOV CL rice varieties, and 10/30/2013 and 11/26/2013 sowing times. This work was conducted in the 2013/2014-crop season, under field conditions, in lowland area in Sao Sepé, RS, Brazil. The treatments were arranged in a randomized experimental blocks design, in a split plot and five replications. The variables analyzed were AUCPD on the flag leaf and entire plant, panicle blast, yield and the whole grains percentage. The delay in sowing date increased significantly the incidence and severity of the blast in flooded rice. IRGA 424, IRGA 428 and QM 10 10 CL showed resistance to Pyricularia oryzae, while the GURI INTA CL, PUITA INTA CL and INOV CL were susceptible. The addition of Tricyclazole fungicide to Kresoxim-methyl + Epoxiconazole fungicides is critical to the efficient control of Pyricularia oryzae. The applications of fungicides in the R1 + R2 + R4 and R2 + R4 stages are the most effective in the chemical control of rice blast. The integration among cultural, genetic and chemical methods is critical to the efficient management of blast in flooded rice. / A brusone é a principal doença da cultura do arroz irrigado. O manejo integrado desta doença é baseado na adoção de um conjunto de métodos de controle, como o controle cultural, controle genético e o controle químico. O objetivo deste trabalho foi avaliar o efeito de oito programas de controle químico de Pyricularia oryzae, nas cultivares de arroz irrigado IRGA 424, IRGA 428, GURI INTA CL, PUITÁ INTA CL, QM 10 10 CL e INOV CL, nas datas de semeadura de 30/10/2013 e 26/11/2013. O experimento foi conduzido em São Sepé/RS, na safra 2013/14, obedecendo o delineamento experimental de blocos ao acaso, em parcelas subsubdivididas e cinco repetições. As variáveis consideradas foram AACPB na folha bandeira e na planta inteira da cultura do arroz, o percentual de incidência de brusone na base da panícula, a produtividade e o percentual de grãos inteiros. O atraso na época de semeadura aumentou significativamente a incidência e a severidade da brusone na cultura do arroz irrigado. IRGA 424, IRGA 428 e QM 10 10 CL apresentaram resistência à Pyricularia oryzae, enquanto que GURI INTA CL, PUITÀ INTA CL e INOV CL foram suscetíveis. A adição do fungicida Triciclazol aos fungicidas Cresoxim-metilico + Epoxiconazol foi eficiente para controle de Pyricularia oryzae. As aplicações de fungicidas para controle químico da brusone foram mais eficazes quando realizadas nos estádios R1 + R2 + R4 e R2 + R4. A integração entre os métodos cultural, genético e químico foi eficiente para o manejo da brusone na cultura do arroz irrigado.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diseases are limiting factors for the expression of the high yield potential of rice cultivars in the state of Rio Grande do Sul. Blast (Pyricularia oryzae) is considered the main disease, causing damage to yield and milling grain quality. Losses vary depending on the cultivar, management practices and climatic conditions. The integrated management practices for disease control has been used in order to minimize fungicide applications. In the season 2014/15 were set up two experiments in order to evaluate the influence of nitrogen, silicates and fungicides on the occurrence of rice blast (Chapter I) and the behavior and the quality of rice grains of cultivar Guri INTA CL (Chapter II ). The experiment was conducted in a lowland area at Department of Crop Sciences of the Federal University of Santa Maria (UFSM), with the experimental design of randomized blocks in a factorial (4x4x2) and four replications. In the experiment I, the treatments consisted of nitrogen rates (N) applied preflood pre and post flood (0, 60, 120 and 180 kg ha-1), and magnesium (Mg) and calcium (Ca) silicate applied to the soil (0, 3000, 6000 and 9000 kg ha-1) and whether or not fungicide the mixture tricyclazole, azoxystrobin and difenoconazole. The treatments experiment II were: nitrogen rates applied pre-flood pre and post flood (0, 60, 120 and 180 kg ha-1), application of potassium silicate (K) to the leaves (0; 2,9; 5,8 and 8,7 L ha-1) and whether or not fungicide. It was observed that rates above 60 kg ha-1 N provide plants to greater severity of rice blast, requiring the use of fungicides for effective disease control and keep grain yield. In the absence of conditions of chemical control, the occurrence of blast reduces the industrial quality of rice grains, increasing the percentage of chalky grains, white belly and chalk area. The silicate effects of both Ca and Mg and K have higher responses in the absence of fungicides cultivar of rice Guri INTA CL. / As doenças são fatores limitantes para a expressão do alto potencial produtivo das cultivares de arroz irrigado no estado do Rio Grande do Sul. A brusone (Pyricularia oryzae) é considerada sua principal doença e, causa danos à produtividade e qualidade dos grãos. As perdas variam em função da cultivar utilizada, das práticas de manejo adotadas na lavoura e das condições climáticas. O manejo integrado de práticas para o controle da doença vem sendo utilizado com o intuito de minimizar as aplicações de fungicidas. Na safra agrícola 2014/15 foram instalados dois experimentos com o objetivo de avaliar a influência do nitrogênio, silicatos e fungicidas na ocorrência de brusone (Capítulo I) e o comportamento quanto a qualidade industrial dos grãos de arroz irrigado da cultivar Guri INTA CL (Capítulo II). Os experimentos foram conduzidos a campo, na área de várzea do Departamento de Fitotecnia da Universidade Federal de Santa Maria (UFSM), com o delineamento experimental de blocos ao acaso, em esquema fatorial (4x4x2) com quatro repetições. No experimento I, os tratamentos foram constituídos de doses de nitrogênio (N) aplicados em cobertura (0, 60, 120 e 180 kg ha-1), doses de silicato de cálcio (Ca) e magnésio (Mg) aplicado via solo (0, 3000, 6000 e 9000 kg ha-1) e aplicação ou não de fungicidas da mistura triciclazol, azoxistrobina e difenoconazol. Os tratamentos do experimento II foram: doses de nitrogênio aplicados em cobertura (0, 60, 120 e 180 kg ha-1), aplicação de silicato de potássio (K) via foliar (0; 2,9; 5,8 e 8,7 L ha-1) e aplicação ou não de fungicidas. Observou-se que doses acima de 60 kg ha-1 de N predispõem as plantas à maior severidade de brusone, sendo necessário a utilização de fungicidas para o controle eficiente da doença e manutenção do rendimento de grãos. Em condições de ausência do controle químico, a ocorrência de brusone reduz a qualidade industrial de grãos de arroz, aumentando os percentuais de grãos gessados, barriga branca e área gessada. Os efeitos de silicato tanto de Ca e Mg como de K apresentam maiores respostas na ausência de fungicidas na cultivar de arroz irrigado Guri INTA CL.

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No. of bitstreams: 2 Dissertação - Rafaela Araújo Guimarães - 2014.pdf: 1551849 bytes, checksum: b39011c17618c122fad17d142b0288ec (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Sarocladium oryzae, the causal agent of rice sheath rot disease is described as antagonistic to rice pathogens. Rice blast is a major rice disease and is responsible for losses up to 100% in productivity. The disease control is done by integrated management, where the main practices are use of resistant cultivars and chemical control. The biocontrol agents or their metabolites may to be more practical to be including them as components in the management. The in objectives of this study a consist to evaluate of S. oryzae isolates for the morphological variability, genetics, biochemistry and antagonistic activity to rice pathogens; evaluate effect of S. oryzae filtrade on conidial germination and appressorium formation of M. oryzae; evaluate potential of conidia and filtrate S. oryzae in the suppression of leaf blast severity and quantify activity of enzymes involved in interaction M. oryzae x rice plant x S. oryzae. Isolates were characterized for color, texture, colony diameter, conidia size and hyphae thickness. In genetic studies, we used RAPD marker primers, and cerulenin production was quantified by HPLC. Antagonism in vitro was assessed by dual culture method. The effect of S. oryzae on conidial germination and appressorium formation of M. oryzae was evaluated using hydrophobic surface. Rice cultivar BRS Primavera, M. oryzae isolate (Py 10.900) and S. oryzae, isolate So 03, were utilized to study plant-pathogen-antagonist relationship. Plants were sprayed, with conidial suspension (CS), 3x105 conídios.mL-1 and culture filtrate (CF) 100% of S. oryzae, two days before inoculation with M. oryzae. S. oryzae isolates showed morphological variability, polymorphism in DNA. A majority of S. oryzae isolates (60%) ware able to produce cerulenin and over 55% were antagonistic to C. miyabeans, M. oryzae, M. albescens and T. cucumeris. The isolate So 29 showed largest inhibition zone. Filtrate of isolates So 03 and So 29 delayed conidia germination by 89.5% and inhibited appressoria formation of M. oryzae by 85%. CS reduced of leaf blast severity in 68.8% and CF in 75.5%. The enzymes β-1,3-glucanase and peroxidase exhibited maximum activity in plants sprayed with CF, when as the activity was high for SA and lipoxygenase in relation to CS treatment, compared to their respective controles, in the absence of M. oryzae. After inoculation with M. oryzae, the lipoxygenase and phenylalanine ammonia-lyase activity in both treatments CF and CS, showed differences compared to controls (plants inoculated with M. oryzae and water only). S. oryzae presented variability to characteristics evaluated and showed potential antagonism against to rice pathogens. Changes in enzyme activity indicate their role in induction resistance in plants in M. oryzae x rice x S. oryzae interaction. / Sarocladium oryzae, agente causal da podridão da bainha do arroz, é descrito como antagonista a patógenos de arroz. A brusone, principal doença do arroz é responsável por perdas de até 100% na produtividade. O controle desta doença é feito pelo uso do manejo integrado, onde as principais práticas são o uso de cultivares resistentes e o controle químico. O uso de agentes de biocontrole ou seus metabólitos pode ser mais uma prática a ser inserida ao manejo. Os objetivos deste estudo foram: avaliar isolados de S. oryzae quanto à variabilidade morfológica, genética, bioquímica e quanto à atividade antagônica aos patógenos de arroz; avaliar o efeito do filtrado de S. oryzae na germinação de conídios e na formação de apressórios de M. oryzae; avaliar o potencial da suspensão de conídios e do filtrado de S. oryzae na supressão da brusone foliar e quantificar a atividade das enzimas relacionadas à patogênese e do fitohormônio ácido salicílico (AS) envolvidos na interação M. oryzae x arroz x S. oryzae. Os isolados foram caracterizados quanto à cor, textura, diâmetro da colônia, tamanho dos conídios e espessura das hifas. No estudo genético, utilizou-se marcador RAPD (Random Amplified Polymorphic DNA) e a produção de cerulenina foi quantificada em HPLC. O antagonismo in vitro foi avaliado pelo método da cultura pareada. A ação de S. oryzae sobre a germinação de conídios e a formação de apressórios de M. oryzae foi avaliada em superfície hidrofóbica. Plantas de arroz da cultivar BRS Primavera, um isolado virulento de M. oryzae (Py 10.900) e o isolado So 03 de S. oryzae, foram avaliados no estabelecimento das relações planta-patógeno-antagonista. As plantas foram pulverizadas com S. oryzae, na forma de suspensão de conídios (SC) - 3x105 conídios.mL-1 e filtrado (FI) 100% concentrado, dois dias antes da inoculação com M. oryzae. Os isolados de S. oryzae apresentaram variabilidade morfológica e polimorfismo no DNA. A maioria dos isolados de S. oryzae (60%) foi capaz de produzir cerulenina e mais de 55% foram antagônicos a C. miyabeans, M. oryzae, M. albescens e T. cucumeris. O isolado So 29 apresentou o maior halo de inibição no pareamento. Os filtrados dos isolados So 03 e So 29 retardaram a germinação dos conídios em 89,5% e inibiram a formação dos apressórios de M. oryzae em 85%. A SC reduziu a severidade da brusone foliar em 68,8% e o FI em 75,5%. Os maiores valores de atividade enzimática específica em relação ao controle antes da presença de M. oryzae foram para β-1,3-glucanase e peroxidase no tratamento com FI, enquanto que, na SC foram lipoxigenase e AS. Depois da presença de M. oryzae a lipoxigenase e a fenilalanina-amônia liase apresentaram atividade tanto com FI quanto na SC, diferindo dos controles (plantas inoculadas com água e com M. oryzae, somente). S. oryzae apresentou variabilidade para as características avaliadas e potencial antagônico aos patógenos do arroz. As alterações na atividade enzimática indicam a indução de resistência em plantas na interação M. oryzae x arroz x S. oryzae.