Dissertations / Theses on the topic 'Grisham'

Harper Lee’s To Kill A Mockingbird is an iconic classic that inspired many street lawyer novels. Examining John Grisham’s A Time To Kill as a low-culture-imprint of Lee’s novel, the thesis analyzes the convergent and divergent points of rhetorical devices that promote colour-blind liberalism across the two texts seeing as they are published 30 years apart. Both pieces of legal fiction act as a reflection and critique of formal legal institutions and through this reflection, the thesis deals with how the texts reinforce, perpetuate and resist the white dominant ideology through the “progressive” race politics of colorblind liberalism.

Orientador: Anete Pereira de Souza
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-18T01:20:36Z (GMT). No. of bitstreams: 1 Schneider_DilaineRoseSilva_D.pdf: 11263694 bytes, checksum: 808a3a965f6f47fb9c2108a4c971b122 (MD5) Previous issue date: 2010
Resumo: A brusone do arroz (rice blast disease) causada pelo ascomiceto fitopatógeno Magnaporthe grisea continua a ter um enorme impacto nas culturas de arroz (Oryza sativa) no Brasil e no mundo. PWL2, uma proteína efetora, é um conhecido produto de um gene AVR (avirulência). O gene PWL2 impede que o fungo infecte weeping lovegrass (Eragrostis curvula). Neste trabalho nós identificamos em uma linhagem de M. grisea um gene que produz uma proteína diferente de PWL2, denominada PWL2D. A seqüência do gene PWL2D tem duas bases que diferem do gene PWL2, as quais produzem alterações nos resíduos de 90 e 142 da proteína. A alteração do resíduo 90 (de D90 para N90) é fundamental para a avirulência. Neste trabalho foram efetuadas a clonagem do gene PWL2D no vetor pET32-Xa/LIC, a expressão em Escherichia coli e a avaliação da estrutura de PWL2D por técnicas espectroscópicas. A proteína PWL2D fusionada à cauda TRX é propensa a agregação, e sua solubilidade é melhorada quando super-expressa sem o seu peptídeo-sinal original. Os resultados estruturais obtidos indicam que a proteína PWL2D possivelmente é intrinsecamente desordenada. Foi elaborado um modelo para a resistência/susceptibilidade do hospedeiro à M. grisea considerando a atuação de PWL2D como uma proteína intrinsecamente desordenada. Os resultados obtidos deverão facilitar a análise estrutural de PWL2D e podem contribuir para a compreensão da função do gene nas interações fungo / planta. Oito diferentes genes de M. grisea, além de PWL2D, foram também estudados neste trabalho. Dentre estes, destacam-se o gene que produz a xilanase XYL5 e seu domínio catalítico, o gene que codifica a chaperona ABC1 e seus dois domínios funcionais, e o gene que codifica a trealase PTH9, sendo todos estes relacionados à patogenicidade do fungo M. grisea. A xilanase XYL5 (EC 3.2.1.8) e seu domínio catalítico conservado (XYL5/DOM) foram fusionados à Maltose Binding Protein (MBP) ou à tiorredoxina (TRX) e expressas em E. coli. A produção de proteína solúvel e ativa foi influenciada pelo tipo de fusão. Os extratos solúveis contendo as proteínas de fusão MBP-XYL5 e MBP-XYL5/DOM apresentaram atividade xilanolítica em relação ao controle. Entretanto, durante o processo de purificação, a atividade foi perdida. Assim, obteve-se pela primeira vez o gene de patogenicidade XYL5 de M. grisea expresso com sucesso em E. coli e sua atividade enzimática xilanolítica foi demonstrada. Não foi possível expressar a chaperona ABC1 na forma solúvel nos sistemas de expressão utilizados, e a sequência gênica referente à trealase PTH9 - por mostrar a presença de introns após o seqüenciamento do gene amplificado na linhagem de M. grisea em estudo, mostrou-se inadequado para a sua expressão protéica no sistema de expressão procariótico utilizado durante a realização deste trabalho
Abstract: The rice blast disease caused by the ascomycete phytopathogen Magnaporthe grisea continues to have a huge impact on crops of rice (Oryza sativa) in Brazil and worldwide. PWL2, an effector protein, is a product of an AVR (avirulence) gene . The gene PWL2 prevents fungus from infecting weeping lovegrass (Eragrostis curvula). In this work we identified in a strain of M. grisea a gene that produces a protein different from PWL2, called PWL2D. The gene sequence PWL2D has two bases that differ from PWL2 gene, which produce changes in residues 90 and 142 of the protein. The change of residue 90 (from D90 to N90) is critical to avirulence. In this work it was realized the cloning of the gene in the vector PWL2D pET32-Xa/LIC, the expression in Escherichia coli and the assessment of PWL2D structure by spectroscopic techniques. The protein fused to the tag PWL2D TRX is prone to aggregation, and its solubility is improved when overexpressed without its original signal peptide. The structural results obtained indicate that possibly the protein PWL2D is intrinsically disordered. A model for the resistance/susceptibility of the host to M. grisea was developed considering the performance of PWL2D as an intrinsically disordered protein. The results should facilitate structural analysis of PWL2D and may contribute to the understanding of gene function in the interactions fungus/plant. Eight different genes of M. grisea, besides PWL2D, were also studied in this work. Among these, stands out the gene that produces xylanase XYL5 and its catalytic domain, the gene that codify the chaperone ABC1 and its two functional domains, and the gene that codify the trehalase PTH9, all them being related to the pathogenicity of the fungus M. grisea. The xylanase XYL5 (EC 3.2.1.8) and its retained catalytic domain (XYL5/DOM) were fused to the solubilizing proteins (MBP) or thioredoxin (TRX) and expressed into E. coli. The production of soluble and active protein was influenced by the type of fusion. The soluble extracts containing the fusion proteins MBP- XYL5 and MBP-XYL5/DOM showed xylanolytic activity compared to the control. However, during the purification process, the activity was lost. Thus, we obtained for the first time the gene pathogenicity XYL5 M. grisea expressed successfully in E. coli and its enzymatic xylanolytic activity was demonstrated. It was not possible to express the chaperone ABC1 in soluble form in the expression systems used, and the gene sequence related to trehalase PTH9 - by showing the presence of introns after the sequencing of the gene amplified in the strain of M. grisea under study, rendered inadequate for its protein expression in the prokaryotic system used during the realization of this work
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular

Mestrado em Engenharia Agronómica - Proteção de Plantas - Instituto Superior de Agronomia
The present work had as objective to determine the duration of the cycle vegetative and to evaluate the susceptibility to rice blast disease of 19 rice advanced lines resulting from the improvement works performed by INIAV,I.P. in collaboration with the COTArroz, in “Salvaterra de Magos” and 12 commercial varieties. The susceptibility was determined under field conditions and greenhouse inoculation studies, having been used five isolated of Magnaporthe grisea of the Portuguese population of the pathogen and one isolated of reference of the Japanese population (JP 56 ). The beginning of tillering of the advanced lines and the varieties occurred between 19 and 29 days after sowing, the booting between 74 and 84 days and heading between 80 and 92 days. The duration of the vegetative cycle varied between 121 days and 140 days, having the majority of the studied advanced lines presented cycle greater than 130 days. In field test 60% of the advanced lines and 58% of commercial varieties were susceptible to leaf or panicle blast, having the advanced lines OP 1227 and OP 1229 and the commercial varieties “Ariete” and “Dardo” been most susceptible to leaf blast. In the study of inoculation 15.8% of the advanced lines presented susceptibility to all isolates, while no commercial variety presented susceptibility to all isolates.

We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

Le cas de l'interaction modèle entre le riz et le champignon pathogène Magnaporthe oryzae a été étudié afin de mieux comprendre les effets d'une pratique culturale, la fertilisation azotée, sur la résistance. Ce travail a permis de mettre au point un système simplifié permettant d'étudier au laboratoire l'augmentation de la sensibilité induite par l'apport azoté, un phénomène dénommé NIS (Nitrogen-Induced Susceptibility), d'analyser les effets de l'azote sur la croissance de M. oryzae et son pouvoir pathogène, d'explorer les effets de l'azote au travers de la diversité du riz et des différents types de résistance (complètes et partielles), et d'identifier par cartographie des zones du génome du riz importantes pour la NIS. L'expression des gènes de défenses ne semble pas altérée en cas de régime riche en azote. Au contraire, le suivi cytologique de l'infection par M. oryzae a mis évidence que la pénétration du champignon n'était pas modifiée par la présence d'azote alors que sa croissance dans la plante est accrue. Le niveau de maladie a pu être augmenté par apport d'acides aminés 24h après le début de l'infection, suggérant qu'une relation trophique est probablement à la base de la NIS. Une analyse de l'effet de l'azote sur la sensibilité à la pyriculariose à travers la diversité du riz nous a permis de mieux caractériser la diversité du phénomène de NIS. La NIS est très polymorphe mais n'est pas corrélée à la différence liée aux sous-groupes indica et japonica du riz. Par ailleurs, un fort apport en azote a réduit la résistance déclenchée par le gène de résistance Pi1, suggérant que la robustesse de ces gènes peut être affectée par la NIS Enfin, un locus qui contrôle la sensibilité du riz à la pyriculariose sous un régime riche en azote a été identifié sur le chromosome 1. Plusieurs éléments suggèrent un lien possible entre l'efficacité de l'utilisation de l'azote (NUE) et la NIS
Nitrogen-Induced Susceptibility (NIS) to plant diseases is a widespread phenomenon. In this work, we set an experimental system in which nitrogen supply strongly affects rice blast susceptibility whereas it is only slightly perturbing plant growth. We show that fungal growth is affected before and after penetration in the plant but that the final penetration rate is not affected; thus a change in penetration is not responsible for increased susceptibility. Differences in total nitrogen amount and defense gene expression before infection are unlikely to be responsible for the observed increase in penetration. After penetration, small changes in plant growth, but not modifications of the transcriptional regulation of defense genes, could be responsible for nitrogen-induced susceptibility. On the other hand, the fungus seems to perceive small differences in nitrogen amount after penetration and this may explain enhanced growth under high nitrogen regime. Indeed, exogenous treatment with some free amino acids after inoculation mimicked Nitrogen-Induced Susceptibility, further arguing that this phenomenon is mostly due to a trophic relation between the plant and the fungus. We also used our experimental system that does not strongly affect plant development to address the question of NIS polymorphism across rice diversity. We show that the capacity of rice to display NIS is highly polymorphic and does not correlate with difference related to indica/japonica sub-groups. We also tested the robustness of three different major resistance genes under high nitrogen. Nitrogen partially breaks down resistance triggered by the Pi1 gene. Cytological examination indicates that penetration rate is not affected by high nitrogen whereas growth of the fungus is increased inside the plant. Using the CSSL mapping population between Nipponbare and Kasalath, we identified a Kasalath locus on chromosome 1, called NIS1, which dominantly increases susceptibility under high nitrogen. We discuss the possible relationships between Nitrogen Use Efficiency (NUE), disease resistance regulation and NIS. This work provides evidences that robust forms of partial resistance exist across diversity and can be genetically mapped. This work also suggests that under certain environmental circumstances, complete resistance may breakdown, irrelevantly of the capacity of the fungus to mutate. These aspects should be considered while breeding for robust forms of resistance to blast disease

У роботі розглядаються особливості перекладу лексичної категорії заперечення з англійської мови на українську на матеріалі роману J. Grishem "The rainmaker". При цитуванні документа, використовуйте посилання http://essuir.sumdu.edu.ua/handle/123456789/17116

One of the major aims of the study was to characterise spore germination using light microscopy and to investigate the role and relative importance of each of the three conidial cells for successful germination. Analysis of germ tube growth rate showed that there are two modes of germination: a conidium produces either a single germ tube or two slightly slower growing germ tubes. Results from scanning electron microscopy and fluorescence microscopy studies suggest that germ tube emergence is asymmetric and directed towards the substratum. A method was developed by which individual conidial cells could be selectively killed, using localised laser irradiation, a technique which has not been previously used with fungal cells. The data suggest that isolated apical and basal cells are able to germinate and form appressoria. The middle cell never germinated, even when it was the only living cell in a conidium. Furthermore, there was evidence that the apical cell, which germinates most frequently, exerts "apical dominance" over the basal cell. Very little is known about organelle morphology and distribution within living fungal spores, and even less is known abut organellar dynamics during spore germination. Another major aim of this study was to characterise organellar organisation and dynamics in living and potentially pathogenic conidia throughout germination. The analysis used confocal microscopy and vital stains. To obtain biologically meaningful results, an in vitro system which mimicked in vivo conditions as closely as possible was developed and optimised for confocal microscopy. A range of potentially vital dyes were screened as organelle stains in living conidia during germination. Nuclei (stained with SYTO 11), vacuolar compartments (stained with cDFFDA), mitochondria (stained with Rhodamine 123), and the apical vesicle cluster (stained with FM4-64) were identified and characterised during germination. Each cell was shown to contain a single nucleus that changed position within the cell during germination but otherwise did not exhibit extensive movement.

Made available in DSpace on 2015-03-26T13:39:42Z (GMT). No. of bitstreams: 1 texto completo.pdf: 745259 bytes, checksum: d378fcf86e7bec345e93ab6ddc5b11c8 (MD5) Previous issue date: 2011-07-07
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Wheat (Triticum spp.) is a grass grown and used as an energy source worldwide being cultivated in several regions of Brazil. However, some diseases severity and ineffective chemical control have been threatening Brazilian wheat production. Among the diseases, the blast of wheat caused by the fungus Pyricularia grisea, is gaining a prominent role, being able to reduce crop yields by up to 70%. Chemical control of the disease has been unsatisfactory and there is little information on genetic resistance available in the literature. Resistance is the best way to control diseases by both economically and environmentally advantages. Given these facts, the objective of this study was to evaluate the resistance to blast of wheat genotypes for later use in breeding programs. It was obtained 10 different isolates of the cereal producing regions in Brazil. The isolates were transferred to PDA medium (potato dextrose agar) and after development and cleansing of the colonies were transferred to OA medium (oatmeal and agar) and maintained at a temperature of about 25 ° C and light regime of 12 hours for 10 days for sporulation of the fungus to occur. The concentration of the fungus used in the inoculations was adjusted to 1.2 x 105 spores / mL. In the first experiment, plants were inoculated when they had four leaves. The plants were kept under controlled conditions at 25° C and evaluated seven days after inoculation. Plants were classified according to the type of infection and later was calculated the Resistance Spectrum Relative (RSR) (percentage of isolates that the genotype expressed resistance) and the Disease Index (DI) (resistance of a genotype using all range of types of infection). The DI values were considered different (p≤0,05) if their confidence intervals (95%) did not overlap. Genotypes IVI 04033, VI 07443, VI 07505, IVI 04028, VI 07157, VI 04026, VI 98053 and VI 07160 were susceptible to more than 80% of isolates. Five varieties and four lines had a RSR greater than 50% and DIs smaller than 0.6. Among the lines stood out VI 04098 and VI 07094 with RSR greater than 80%, equating to the variety IPR 85. In the second experiment conducted under field conditions, inoculation was done staggered, according to the cycle of the genotypes, when plants reached the stage of 58-60 in Zadoks scale (1974), being applied 1L of suspension of P. grisea at a concentration of 1.2 x 105/mL per plot. Productivity was assessed by harvesting each plot area (3 m2). Disease incidence was assessed by the percentage of infected spikes and severity was assessed by the percentage of infected spikelets in each spike.The yield ranged from 879 to 3983 kg / ha, the incidence of the disease ranged from 0.86 to 84.24% and the severity ranged from 0.48 to 65.29%. Seven genotypes were classified as MR, three genotypes as MS and nine as S. The highlights were the cultivars CD 116, CD 104, IPR 85 and line VI 07094 with yields exceeding 3000 kg / ha and severity lower than 6%. The three variables yield, incidence and severity showed significant correlation with each other.
O trigo (Triticum spp.) é uma gramínea cultivada e utilizada como fonte de energia no mundo todo, sendo cultivado em várias regiões do Brasil. No entanto, a severidade de algumas doenças e o controle químico ineficaz, vêm ameaçando a triticultura brasileira. Entre as doenças, a brusone do trigo causada pelo fungo Pyricularia grisea, vem ganhando um papel de destaque, podendo reduzir a produtividade das lavouras em até 70%. O controle químico da doença tem sido insatisfatório e existem poucas informações sobre resistência genética disponível na literatura. O uso da resistência é a melhor maneira de controle de doenças, tanto pelas vantagens do ponto de vista econômico, quanto ambiental. Diante desses fatos, o objetivo deste trabalho foi avaliar a resistência de genótipos de trigo à brusone para posterior uso em programas de melhoramento genético. Foram obtidos 10 isolados de diferentes regiões produtoras do cereal no Brasil. Os isolados foram repicados para meio BDA (batata, dextrose e Agar) e após desenvolvimento e purificação das colônias foram transferidos para meio AV (aveia e Agar), sendo mantidos sob temperatura de aproximadamente 25ºC e regime de luz de 12 horas, durante 10 dias, para que ocorresse esporulação do fungo. A concentração do fungo empregada nas inoculações foi ajustada para 1,2 x 105 esporos/mL. No primeiro experimento, as plantas foram inoculadas quando apresentavam quatro folhas. As plantas foram mantidas em condições controladas a 25ºC e avaliadas sete dias após a inoculação. As plantas foram classificadas conforme o tipo de infecção e, posteriormente, foram calculados o Espectro de Resistência Relativo (ERR) (porcentagem de isolados que o genótipo expressou resistência) e o Índice de Doença (ID) (resistência de um genótipo usando toda a gama de tipos de infecção). Os valores de ID foram considerados diferentes (p≤0,05) caso seus intervalos de confianças (95%) não se sobrepusessem. Os genótipos IVI 04033, VI 07443, VI 07505, IVI 04028, VI 07157, VI 04026, VI 98053 e VI 07160 apresentaram suscetibilidade a mais de 80% dos isolados. Cinco cultivares e quatro linhagens apresentaram ERR maior que 50% e IDs menores que 0,6. Dentre as linhagens, destacaram-se VI 04098 e VI 07094 com ERR maiores que 80%, se equiparando a variedade IPR 85. No segundo experimento, conduzido em condições de campo, a inoculação foi feita de forma escalonada, de acordo com o ciclo dos genótipos de trigo, quando as plantas atingiram o estádio 58-60 da escala de Zadoks (1974), sendo aplicado 1L de suspensão de conídios de P. grisea na concentração de 1,2 x 105/mL por parcela. A produtividade foi avaliada pela colheita de cada parcela útil (3 m2). A incidência da doença foi avaliada pela porcentagem de espigas infectadas e a severidade foi avaliada pela porcentagem de espiguetas infectadas em cada espiga. A produtividade variou de 879 a 3983 kg/ha; a incidência da doença variou de 0,86 a 84,24% e a severidade variou de 0,48 a 65,29%. Sete genótipos que foram classificados como MR, três genótipos como MS e nove como S. Destacaram-se as cultivares CD 116, CD 104, IPR 85 e a linhagem VI 07094 com produtividades superiores a 3000 kg/ha e severidades menores que 6%. As três variáveis: incidência, produtividade e severidade, apresentaram correlação significativa entre si.

O fungo Humicola grisea var. thermoidea foi submetido a diferentes condições de cultivo com o intuito de determinarem-se as melhores condições para produção de conídios, crescimento e produção de metabólitos secundários com atividade biológica. Para tal foram desenvolvidos experimentos avaliando dois parâmetros: meio fermentativo e tempo de incubação. Os meios fermentativos líquidos utilizados foram Czapek, Jackson e Vogel em condições de agitação (120 rpm), e os tempos avaliados foram 72, 96, 120, 144, 168 e 192 horas. Também foi desenvolvida cultura por fermentação em substrato sólido, meio de arroz enriquecido, em diferentes tempos de incubação, sendo escolhido o período de 60 e 90 dias como os mais promissores. Todos os cultivos foram realizados em temperatura de 40ºC. Dos cultivos em meio líquido foram obtidos extratos acetoetílicos e butanólicos dos filtrados das culturas e metanólicos dos micélios. Dos cultivos a partir do meio sólido foram obtidos extratos acetoetílicos. Os extratos foram avaliados pela técnica de bioautografia na concentração de 30 mg/mL (10 L), utilizando-se como indicadores biológicos as cepas Kocuria rhizophila (ATCC 9341), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) e Pseudomonas aeruginosa (ATCC 14885). Os extratos acetoetílicos e metanólicos obtidos a partir dos cultivos em Czapek e Vogel, nos tempos 144, 168 e 192 horas se mostraram ativos contra as cepas K. rhizophila e S. aureus. Os extratos obtidos do meio sólido e o extrato acetoetílico (192 horas) obtido do meio de cultivo de Czapek foram avaliados pelo método de microdiluição em microplaca com o objetivo de determinarem-se os valores de CIM, utilizando como microrganismos indicadores K. rhizophila e S. aureus. O extrato mais promissor foi o extrato acetoetílico do cultivo em meio de arroz enriquecido, durante 90 dias, sendo os valores de concentração inibitória mínima de 350 g/mL e de 250 g/mL, contra S. aureus e K. rhizophila, respectivamente. Os extratos foram avaliados por cromatografia líquida de alta eficiência, sendo a princípio considerados promissores os extratos em acetato de etila de Czapek e arroz enriquecido. O fungo foi cultivado em arroz (60 e 90 dias), devido ao maior rendimento, manejo mais simples, menores valores de concentração inibitória mínima quando comparados aos cultivos desenvolvidos nos meios fermentativos líquidos avaliados. Deste modo, foi promovida ampliação da escala de cultivo em meio sólido e os extratos obtidos foram submetidos a processos cromatográficos visando o isolamento de substâncias ativas. Foram isoladas seis substâncias, e elucidadas três: dimetil tereftalato, ácido p-hidroxifenilacético e uma lactama, a qual foi isolada pela primeira vez de fungos e havia sido isolada apenas da esponja marinha (Halichondria melanodocia) infestada por alga. Os resultados dos ensaios de microdiluição em microplaca de tais substâncias se mostraram bastante discretos. Entretanto, outros ensaios biológicos, dentre eles, antitumorais e antiparasitários serão realizados para as substâncias isoladas.
The fungos Humicola grisea var. thermoidea was submmited to different growth conditions, aiming to determine the best condition for the conidia production, mycelia growth and production of secondary metabolites bearing biological activities, as well. For that, it was evaluated both deferent fermentative media (Czapek, Jackson and Vogel) and incubation time (72, 96, 120, 144, 168 e 192 hours), under 120 RPM. The fungus was also cultivated in solid medium using enriched rice in different times of incubation in two batches of 60 and 90 days of fermentation. All fermentations were undertaken at 40ºC. From the fermetation in liquid media it was obtained the extracts in ethyl acetate and n- butanol, from the broth, respectively, and in methanol from the mycelium. From the solid media (rice) it was obtained the ethyl acetate extract. The obtained extracts were submitted to bioautography assay by applying 10 L of a solution containing 30 mg/mL of each extract against the following bacteria strains: Kocuria rhizophila (ATCC 9341), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 14885). The extracts em ethyl acetate and methanol obtained from from Czapek and Vogel media at 144, 168 e 192 hours displayed activity against K. rhizophila and S. aureus. The extracts obtained from the solid medium and the ethyl acetate extract from the Czapek nediun at 192 hours were evaluated using the Minimum Inhibitory Concentration (MIC) in microplates, aiming to determine the MIC values for these extracts against both K. rhizophila and S. aureus. The most active extract (ethyl acetate) was the one obtained from solid media cultivated for 90 days, furnishing the MIC values of 350 g/mL and 250 g/mL against S. aureus and K. rhizophila, respectively. All the obtained extracts were submitted to HPLC chromatography and the best chromatographic profiles were displayed by both ethyl acetate extracts from Czapek and enriched rice media. The cultivation in rice media was simple and produced the highest yield of extract, as well as displayed the lowest CIM in comparison with the extracts obtained from the broth of liquid media. Therefore, the fungus was cultivated in a larger scale in rice aiming to obtain enough amount of ethyl acetate extract to allow the isolation of the major active compounds by chromatographic means. Thus, six compounds were isolated, from which three were identified so far: dimethyl tereftalate, p-hidroxyphenylacetic acid and the one lactam. The lactam was isolated for the firs time from a soil fungus, and it was previous isolated only from a marine sponge (Halichondria melanodocia) infested by algae. However, the obtained MIC values for these compounds were very high. Even though, other biological assays, such as: anticancer and antiparasitic, among other will be undertaken to evaluate the potential of the isolated compounds.

Magnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.

'The ascomycetal fungus Magnaporthe grisea causes the most damaging fungal disease of rice, blast. Resistant rice cultivars are typically dependent on the presence of one, or a few, major genes that are effective only toward particular M. grisea isolates. These isolates have avirulence genes that correspond to specific rice resistance genes, in a gene-for-gene relationship. Results presented here show the genetic identification of AVR2-MARA, a M. grisea a gene that confers avirulence toward the rice cultivar Maratelli. Two techniques were used for determining the chromosomal location of AVR2-MARA, restriction fragment length polymorphism analysis (RFLP), and bulked segregant analysis. Through RFLP analysis AVR2-MARA was mapped to Chromosome 7, between markers cos196 and cos209. Bacterial Artificial Chromosome (BAC) clones, as well as cosmids, were utilized in chromosome walking to the locus. Walks were initiated from markers on both sides of the locus, allowing the identification of sequences that were inseparable from AVR2-MARA. However, I was unable to clone the complete locus. In contrast, avr2-MARA , the virulent locus, was isolated from the virulent parental strain O-135. Sequence analysis of markers inseparable from the AVR2-MARA locus showed a higher AT content than typically observed in the M. grisea genome. Sequence analysis of a fragment inseparable from the AVR2-MARA locus also revealed a putative open-reading-frame (ORF) with significant homology to AVR-Pita. During this study I found that the segregation of avirulence on Maratelli corresponded with the segregation of avirulence towards the rice cultivars M-103 and M-201. AVR-M201, conferring avirulence toward the rice cultivar M-201, was identified several years ago (Valent et al. 1991), but never mapped. In our mapping cross, consisting of 65 progeny, AVR2-MARA and AVR-M201 are inseparable. Furthermore, we isolated two virulent mutants of AVR2-MARA via UV mutagenesis and both had also gained virulence on cultivars M-103 and M-201. This suggests that AVR2-MARA, AVR-M201 (and AVR-M103) are the same avirulence gene. Because AVR2-MARA originated in a finger millet isolate, and confers avirulence toward several different rice cultivars, it may represent a host-species-specificity factor. Such rice cultivars may be guarded from infection by finger millet isolates due to recognition of the AVR2-MARA gene product.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T18:33:23Z No. of bitstreams: 1 texto completo.pdf: 20005358 bytes, checksum: 1121af69167ca6262a2059e1892c3134 (MD5)
Made available in DSpace on 2017-04-20T18:33:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 20005358 bytes, checksum: 1121af69167ca6262a2059e1892c3134 (MD5) Previous issue date: 2003-12-12
Conselho Nacional de Desenvolvimento Científico e Tecnológico
Em Magnaporthe grisea, agente causal da brusone, mutagênese insercional mediada por transformação tem constituído estratégia para a identificação de genes essenciais para a patogenicidade em arroz. A técnica REMI, integração mediada por enzima de restrição, merece destaque em virtude da eficiência de transformação e da predominância de integrações simples. Visando a implantação de programa de mutagênese insercional em M. grisea, os objetivos deste trabalho incluíram: (1) adequar as condições para a obtenção e regeneração de protoplastos do ascomiceto, (2) estabelecer sistema de transformação REMI em M. grisea, avaliando o potencial dos protoplastos e do vetor pAN7-1 e (3) selecionar e caracterizar mutantes com patogenicidade alterada em plantas de arroz. Produção eficiente de protoplastos foi alcançada com o uso simultâneo de 10 mg de Lysing Enzymes e 10 mg de Cellulase Onozuka R10 em 3 mL de MgSO 4 a 1,2 M / NaH 2 PO 4 a 0,01 M (pH = 5,8). Protoplastos de M. grisea I-22 liberados com 3 horas de hidrólise enzimática apresentaram maior capacidade de regeneração da parede celular. Quando expostos ao vetor pAN7-1, os protoplastos foram prontamente transformados para a resistência à higromicina. Quando pAN7-1- HindIII foi usado para transformar I-22 na presença de HindIII, a freqüência de transformantes foi 1,1 a 8,1 vezes superior ao tratamento sem a adição da endonuclease de restrição. No geral, a melhor concentração de HindIII foi 5 unidades/reação de transformação. A partir de testes de patogenicidade envolvendo 125 transformantes, principalmente gerados por REMI, foi possível selecionar cinco mutantes com alterações consistentes na patogênese. Dois desses mutantes, T108 e T93, causaram poucas lesões em folhas de arroz, enquanto o mutante T251 não foi patogênico. A alteração na patogenicidade de T108 foi acompanhada pela menor capacidade de desenvolvimento in vitro. Quando inoculado em plantas de arroz, o mutante T41 apresentou agressividade reduzida, caracterizada por lesões arredondadas de tamanho limitado. Por sua vez, o período de incubação para o mutante T72 foi mais longo do que o do isolado selvagem. Além disso, atrasos consideráveis na germinação de conídios e na formação de apressórios foram detectados em T72. Os mutantes T93 e T251 apresentaram fenótipos semelhantes quando em cultura, caracterizados pela pigmentação marrom. Análises Southern blots de quatro mutantes indicaram que em 50 % dos casos, T108 e T251, apenas uma cópia de pAN7-1 se integrou em um único sítio no genoma. No mutante T251 ocorreu evento REMI propriamente dito. Os mutantes T41 e T93 apresentaram padrões de integração mais complexos.
Transformation mediated-insertional mutagenesis of phytopathogenic fungi is an important tool to identify genes involved in pathogenicity. An improved version of this method is the restriction enzyme mediated integration, or REMI. We chose REMI to begin an insertional mutagenesis project in M. grisea. In this work, were reported the: (1) protoplasts production and regeneration of M. grisea, (2) transformation of protoplasts with pAN7-1 mediated by restriction enzyme and (3) identification and characterization of five transformants with pathogenicity defects in rice, at the phenotypic and molecular levels. The highest protoplasts production was obtained with Lysing Enzymes plus Cellulase Onozuka R-10 and the osmotic buffer MgSO 4 at 1.2 M / NaH 2 PO 4 at 0.01 M (pH = 5.8). The highest regeneration frequency was obtained with protoplasts produced after 3 hours of incubation. The I-22 protoplasts were readily transformed for hygromycin resistance. When pAN7-1-HindIII was used to transform fungal protoplasts in the presence of the HindIII, the transformation efficiency was increased 1.1 to 8.1-fold. The optimal HindIII concentration for enhanced transformation corresponded to 5 unit/transformation mix. Out of 125 transformants screened for the ability to infect rice plants, five showed changes in pathogenicity. The T108 and T93 mutants caused few lesions in rice leaves, while the T251 mutant was non-pathogenic. The alteration in pathogenicity of T108 was accompanied by reduced development in culture. The T41 mutant caused small and limited round lesions. The incubation period of the T72 mutant was longer than of wild type. Furthermore, late germination and appresorium formation was detected in the T72 mutant. The T93 and T251 mutants had similar phenotypes, characterized by a brown-pigmented colony. Four mutants (T41, T93, T108 and T251) were examined by Southern blots. The T108 and T251 mutants contained one copy of the vector integrated at a single site in the genome. REMI event occurred in the T251 mutant. More complex integration events were observed in the T41 and T93 mutants.
Tese importada do Alexandria

L'intégrité de paroi cellulaire est cruciale pour la survie du champignon pendant son cycle de développement ou en réaction à des conditions de stress. Chez la levure, les voies de signalisation de MAP kinase, Slt2, et calcineurin, régulent la réparation de paroi cellulaire. MPS1, l'orthologue de SLT2, chez Magnaporthe grisea est essentiel pour la réparation de la paroi cellulaire et pour la pénétration de l'appressorium (Xu, et al., 1998). Chez la levure, Slt2 active les facteurs de transcription Rlm1, Swi4 et Swi6, alors que le calcineurin active Crz1. Par ailleurs, PaIdc1 a un rôle dans la localisation nucléaire de PaMpk1 (orthologue de Slt2 et Mps1). (Corinne Jamet-Vierny, et al., 2007). MgIDC1, Le gène orthologue de PaIDC1 a été, de même, identifié chez M. grisea. ScAGS1 (α-glucan synthase), est un composant principal de la paroi principal des champignons. CaCAS5, est un régulateur transcriptionel, régule plusieurs gènes de la paroi cellulaire et ScKnr4 est impliqué dans la régulation de l'activité de 1,3--glucan synthase et la formation de la paroi cellulaire Ces gènes ont été identifiés chez M. grisea et des mutants de délétion ont été construits par le remplacement de gène chez M. grisea. Les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δidc1 montrent une forte réduction de mycélium aérien. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δswi4 ont une très forte réduction de sporulation. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δcrz1 ont une forte réduction de pouvoir pathogène. De plus, les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δswi4 sont inhibé, de 100% en présence d'un mélange de l'Aculéacine et Nikkomycine à faible dose (DI20). Les résultats montrent que chez M. grisea, il existe deux voies impliquées dans l'intégrité de la paroi cellulaire. La voie MAPK MPS1, qui régule les facteurs de transcription Rlm1, Swi4, Swi6 et la voie de Calcineurine, qui régule Crz1. D'après les analyses, SWI4 est impliquée dans la régulation des gènes de glucan synthases et chitine synthase, quand RLM1 n'est impliqué que dans la régulation des gènes de chitine synthase. Dans la voie de Calcineurine, CRZ1 contrôle les gènes de chitine synthase aussi. Ces études suggèrent que les facteurs de transcription qui sont régulés par Mps1 et Calcineurine sont spécialisés dans la régulation des gènes de cible spécifiques à l'intégrité et la réparation de la paroi cellulaire.

Les isolats de Magnaporthe grisea possédant le gène d'avirulence ACE1 sont spécifiquement reconnus par les variétés de riz possédant le gène de résistance Pi33. ACE1 code pour une enzyme du métabolisme secondaire (un hybride polycétide synthase/peptide synthétase). L'activité enzymatique d'Ace1 étant nécessaire à l'avirulence, le signal reconnu par les plantes résistantes possédant Pi33 pourrait être le métabolite secondaire dont la biosynthèse dépend d'Ace1. ACE1 est exclusivement exprimé dans les appressoria pendant l'étape de pénétration, aussi bien sur plante que sur certaines membranes artificielles. Aucune expression n'a été détectée dans les appressoria du mutant déficient en mélanine bufl, incapable de développer une pression appressoriale. L'ajout de solutions osmotiques sur des appressoria de bufl rétablit l'expression d'ACE1. Nos résultats suggèrent que l'expression d'ACE1 est dépendante d'un stade de développement atteint juste avant la pénétration et de la pression appressoriale. Des délétions du promoteur d'ACE1 ont révélé une région de 200 bp nécessaire à la transcription dans l' appressorium. L'analyse de l'allèle ACE1 dans le descendant virulent 2/0/3 a révélé l'insertion d'un nouveau retroposon (MINE) dans la POL d'ACE1. La majorité des isolats récoltés dans le monde sont avirulents pour Pi33 et possèdent tous le même allèle ACE1 (ACE1-GUY11. 1). Les isolats virulents ont été majoritairement trouvés en Asie et Amérique du Sud, son génétiquement liés et peuvent être classés en trois groupes en fonction de leur génotype ACE1. Un groupe possède l'allèle virulent (ACE1-GUY11. 2) qui présente 99 % d'identité avec ACEl-GUY11. 1. Le deuxième groupe possède l'allèle virulent (ACE1-CM28) qui présente 88 % d'identité avec ACE1-GUY11. 1. Le troisième groupe possède les allèles virulents ACEl-GUYll. 2 et ACEl-CM28. Ces allèles sont localisés sur des chromosomes différents, indiquant que ces isolats normalement haploi͏̈des sont partiellement diploi͏̈des au locus ACEl
Isolates of the rice blast fungus Magnaporthe grisea that carry the avirulence gene ACE1 are specifically recognized by rice cultivars carrying the resistance gene Pi33. ACE1 encodes an enzyme of the secondary metabolism (a hybrid polyketide synthase/non-ribosomal peptide synthase). Since Acel enzymatic activity is required for avirulence, the signal recognized by rice cultivars carrying Pi33 should be a secondary metabolite. ACE1 is expressed exclusively in appressoria during the penetration process, either on plant or artificial surfaces. ACE1 was not expressed in appressoria differentiated on Mylar or in appressoria from the melanin-deficient mutant bufl, unable to build up appresorial turgor. Addition of hyper-osmotic solutions to bufl appressoria restored ACE1 expression. Our results suggest that ACE1 expression requires an appressorial developmental stage reached before penetration and turgor. Deletion analysis of ACE1 promoter revealed a 200-bp region required for appressorium specific transcription. Characterization of ACE1 structure in the virulent progeny 2/0/3 revealed an insertion of a new retroposon (MINE) into ACE1 ORF. Most worldwide M grisea isolates were avirulent towards Pi33 and carried the same ACE1 avirulent allele (ACE1-GY11. 1). Isolates virulent towards Pi33 were mostly detected in Asia and South America and classified into three groups according to their ACE1 genotypes. The first group has a virulent allele (ACE1-GY11. 2) that is 99% identical to ACEl-GY11. 1. The second group has a virulent allele (ACE1-CM28) that is 88% identical to ACE1-GY11. 1. The third group has two virulent ACE1 alleles (ACE1-GY11. 1 and ACE1-CM28). These two alleles are localized on different chromosomes, indicating that these normally haploid isolates are partially diploid for ACE1. Typing of isolates from these groups using neutral markers (micro-satellites, SNIPS) revealed that they are genetically related, suggesting that they derive from a single complex event

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, 2009.
Submitted by Larissa Ferreira dos Angelos (ferreirangelos@gmail.com) on 2010-03-29T18:28:40Z No. of bitstreams: 1 2009_LeandroNogueiraRamos.pdf: 1947338 bytes, checksum: b0d1d970d899c30989e722fb6a263700 (MD5)
Approved for entry into archive by Daniel Ribeiro(daniel@bce.unb.br) on 2010-04-06T19:43:46Z (GMT) No. of bitstreams: 1 2009_LeandroNogueiraRamos.pdf: 1947338 bytes, checksum: b0d1d970d899c30989e722fb6a263700 (MD5)
Made available in DSpace on 2010-04-06T19:43:46Z (GMT). No. of bitstreams: 1 2009_LeandroNogueiraRamos.pdf: 1947338 bytes, checksum: b0d1d970d899c30989e722fb6a263700 (MD5) Previous issue date: 2009
O arroz (Oryza sativa L.) é um dos principais cereais cultivados no Brasil, desempenhando um papel social e econômico de grande importância para as mais diversas regiões do país. Esse cereal é conduzido no Brasil sob dois principais sistemas de cultivo, o irrigado, tipicamente utilizado nos estados da região Sul do país e o de sequeiro, tradicional na abertura de novas áreas e nas regiões Centro, Norte e Nordeste do Brasil. Para cada sistema de cultivo são utilizados grupos de cultivares adaptadas a condições específicas, principalmente quanto ao fornecimento de água. (sem parágrafo) A brusone, causada pelo fungo Pyricularia grisea (Cooke) Sacc. cuja fase teleomórfica corresponde a Magnaporthe grisea (Barr), é um dos principais fatores limitantes para a cultura do arroz no Brasil e no mundo. O controle é feito, principalmente, pelo uso de cultivares resistentes e fungidas. No entanto, o controle químico através de fungicidas específicos, além de elevar o custo de produção do cereal, causa vários danos ao meio ambiente, e deve ser utilizado integrado a outras técnicas de manejo. Programas de melhoramento genético de arroz são conduzidos no país por diferentes instituições, gerando, a cada ano, novas cultivares de arroz com resistência vertical a brusone. A estreita base genética dos programas de melhoramento tem contribuído para a quebra de resistência das novas cultivares lançadas por estes programas em apenas 1 ou 2 anos após o lançamento, especialmente no Estado do Tocantins. Quebras de resistência em período tão curto apontam para a necessidade busca de fontes de resistência duráveis à brusone, especialmente nas regiões tropicais de cultivo de arroz, onde a variabilidade genética do patógeno é alta. Para tanto, é de suma importância o conhecimento sobre as populações do patógeno e de suas interações com as plantas hospedeiras. O conhecimento mais aprofundado das características das populações de Magnaporthe grisea da região Centro-Norte do Brasil é condição fundamental na orientação dos programas de melhoramento do hospedeiro. O patossistema arroz/brusone é estudado há muito tempo em várias partes do mundo. Porém, no Brasil ainda foi pouco explorado, havendo necessidade de pesquisas em regiões com alto potencial produtivo, como os Estados do Pará, Rondônia, Acre, Tocantins e Maranhão, especialmente em virtude da expansão da fronteira agrícola do arroz de sequeiro. Objetivou-se, a partir do presente trabalho, identificar a estabilidade da cultivar Oryzica Llanos 5 (modelo de cultivar com resistência durável, oriunda da Colômbia) quando inoculada com isolados de M. grisea coletados na região Centro-Norte do Brasil, mediante avaliações de parâmetros epidemiológicos como período de incubação, latência, severidade e agressividade da doença. Além disso, objetivou-se detectar, a partir de estudos moleculares, a variabilidade genética e a estrutura populacional de isolados coletados no Tocantins, Goiás e Pará. No capítulo 1 desta dissertação descreve-se a ocorrência de isolados de M. grisea, coletados nos estados do Centro-Norte do Brasil, capazes de quebrar a resistência genética na cultivar Oryzica Llanos 5, padrão de resistência à doença, e examinam-se possíveis interações entre a virulência a esta cultivar e alguns parâmetros monocíclicos das epidemias de brusone. Para tanto, foram avaliados 35 isolados monospóricos de M. grisea obtidos nos municípios de Lagoa da Confusão, Dueré, Formoso do Araguaia (Tocantins), Luiz Alves do Araguaia (Goiás) e Paragominas (Pará). Objetivando-se a quantificação do desenvolvimento micelial dos isolados in vitro, calculou-se, a cada 3 dias, a área da colônia, a partir da qual obteve-se a área sob a curva de crescimento micelial (ASCCM) para cada um dos isolados. Os parâmetros epidemiológicos (período de incubação, período de latência, severidade da doença e área sob a curva de progresso da doença ASCPD), foram determinados em casa de vegetação, para todos os 35 isolados em três variedades de arroz (Caloro e Fanny suscetíveis; Oryzica Llanos 5 resistente). As plantas foram avaliadas a cada três dias através de análise visual baseada em uma escala de notas de sete classes (0, 1, 3, 4, 5, 7 e 9). No estudo in vitro, houve diferença significativa (p0,05) da ASCCM entre os isolados, independente do local de coleta. Nos experimentos de casa de vegetação, o período de incubação nas cultivares suscetíveis variou entre 1,00 dia (isolado F-2) e 5,75 dias (isolado LA-2) em Caloro; e entre 1,00 dia (isolado F- 2) e 6,25 dias (isolado LA-2) em Fanny. Quando os isolados monospóricos foram inoculados na cultivar resistente Oryzica Llanos 5, observou-se que 34% dos isolados estudados apresentaram quebra de resistência (F-1, F-2, F-5, F-9, F-10, LC-1, LC-2, LC-4, D-2, P-2 e P- 6). Os isolados que quebraram resistência apresentaram os menores períodos de incubação (ex. isolado D-1, 2,0 dias), enquanto que os isolados que não quebraram resistência apresentaram os maiores períodos de incubação (ex. isolado LA-2, 6,25 dias). Isolados que quebraram resistência apresentaram também o menor período de latência. Observou-se forte correlação positiva entre a severidade da doença aos 6, 9 e 12 DAI e a ASCPD, e uma significativa correlação negativa entre período de incubação ou de latência e a ASCPD. As ASCPDs nas cultivares suscetíveis foram maiores que no padrão de resistência. É importante ressaltar a quebra de resistência genética da cultivar Oryzica Llanos 5, até o momento considerada como padrão de resistência, por isolados de M. grisea coletados no Centro-Norte do Brasil. O resultado desses estudos demonstra claramente a alta diversidade genética do patógeno nesta região. No capítulo 2, objetivou-se identificar a diversidade genética e a estrutura de população de isolados monospóricos coletados em lavouras comerciais de arroz nos Estados de Tocantins, Goiás e Pará. Para tanto, conduziram-se ensaios na Embrapa-CENARGEN, onde foram avaliados 140 isolados monospóricos de M. grisea obtidos nos municípios de Lagoa da Confusão, Dueré e Formoso do Araguaia (Tocantins), Luiz Alves do Araguaia (Goiás) e Paragominas (Pará). Neste estudo, 34 marcadores microssatélites marcados com fluorocromo foram utilizados para a genotipagem automática em sequenciador de DNA. Quatorze marcadores microssatélites mostraram-se altamente eficientes para estimar a diversidade genética e detectar estruturação em população de isolados monospóricos de M. grisea. Alguns deles apresentaram um conteúdo informativo elevado, facilitando a genotipagem em escala e propiciando alta eficiência na análise de polimorfismo de DNA. A média do número de alelos por loco foi 6,35, variando de 2 alelos para os marcadores ms 109 - 110, ms 115 - 116, ms 61 - 62 a 16 alelos para o marcador PG 27. Os índices de diversidade genética (DG), conteúdo polimórfico informativo (PIC) e probabilidade de identidade (PI), confirmaram a eficiência destes marcadores. A população de isolados de M. grisea do Centro Norte do Brasil apresentou substruturação em três sub-populações (K=3). Os isolados de Goiás (Luiz Alves) em sua maioria foram observados no Grupo 1, os isolados de Tocantins (Formoso, Dueré e Lagoa da Confusão) em sua maioria foram observados no Grupo 2 e os isolados do Pará (Paragominas) foram observados no Grupo 3. Os isolados do Pará são os mais distantes geneticamente em relação aos isolados de Goiás e Tocantins. A alta diversidade genética observada na metapopulação de M. grisea do Centro-Norte brasileiro, assim como a evidência de estruturação, sugerem a necessidade de monitoramento específico e emprego de estratégias adequadas pelos programas de melhoramento genético para as sub-populações detectadas. _______________________________________________________________________________ ABSTRACT
Rice (Oryza sativa L.) is one of the main cereals cultivated in Brazil, carrying an important social and economical role in several geographical regions. Rice is cultivated in Brazil in two main cropping systems, irrigated, typical of the Southern region and dry land (sequeiro), tradicionally used for opening new areas in the Center, Northern and Northeastern parts of Brazil. For each cultivation system, a group of cultivars, especially adapted to each condition, are used. Rice blast, caused by the fungus Pyricularia grisea (Cooke) Sacc. teleomorph Magnaporthe grisea (Barr), is one of the main limiting factors to rice production in Brazil and elsewhere. Control is mainly by a combination of resistant cultivars and fungicides. However, the use of fungicides increases production costs and may be damaging to the environment, and should be used integrated with other disease management techniques. Several rice breeding programs are conducted in Brazil by a number of different research institutions, every year offering new rice cultivars with vertical resistance to blast to growers. Unfortunately, the narrow genetic basis of the breeding programs has contributed to resistance breakdown of these new rice cultivars just 1 or 2 years after release, as has been observed in the State of Tocantins. Resistance breakdown in such a short period of time points towards the need to find and select durable sources of resistance, especially in the tropical regions, where genetic variability of the pathogen is seemingly very high. However, before beginning the search of sources of host resistance, knowledge of the pathogen population variability is very important, as well as of its interactions with its plant host. Deeper knowledge of the populations of Magnaporthe grisea from Central-North Brazil is a fundamental condition for the orientation of rice breeding programs. The rice blast pathosystem has been studied for some time in other geographic regions, but is still hardly explored in Brazil. Therefore, there is a pressing need to conduct prospective diversity research of the pathogen in regions with high yielding potential, such as the States of Pará, Rondônia, Acre, Tocantins and Maranhão, especially taking into account the expansion of dry land rice. This study aimed to describe the stability of rice cultivar Oryzica Llanos 5 (standard of durable resistance, originated in Colômbia) when inoculated with M. grisea isolates of collected in the Center-Northern Brazil, by estimations of epidemiological parameters, such as period of incubation, latent period, severity and pathogen aggressiveness. In addition, the genetic variability and the populational structure of M. grisea isolates collected in the States of Tocantins, Goiás and Pará were examined by molecular studies. Chapter 1 describes the discovery of isolates of M. grisea from the States of Tocantins (TO), Goiás (GO) and Pará (PA), capable of breaking the resistance of cv. Oryzica Llanos 5, the standard of genetic resistance to rice blast, and examines possible interactions between virulence and some monocyclic components of rice blast epidemics. Towards that end, thirtyfive monosporic isolates of M. grisea from the counties of Lagoa da Confusão, Dueré, Formoso do Araguaia (TO), Luiz Alves do Araguaia (GO) and Paragominas (PA) were studied. Radial mycelial growth was determined in vitro, every third day, and the area under the curve of mycelial growth (AUCMG) of each isolate was compared. Epidemiological parameters (period of incubation, latent period, disease severity and the area under disease progress curve AUDPC) were estimated in the greenhouse, for each of the thirty-four isolates in three rice cultivars (Caloro and Fanny susceptible; Oryzica Llanos 5 resistant). Plants were evaluated visually every third day, with the aid of a diagrammatic scale of seven classes (0, 1, 3, 4, 5, 7 and 9). In the in vitro study, the AUCMG was significantly different among isolates (p0,05), irrespective of geographical origin. Period of incubation in susceptible cultivars varied from 1.00 day (isolate F-2) to 5.75 days (isolate LA-2) on Caloro; and between 1.00 day (isolate F-2) to 6.25 days (isolate LA-2) on Fanny. When the monosporic isolates were inoculated on resistant cv. Oryzica Llanos 5, 34% broke its resistance (F-1, F-2, F-5, F-9, F-10, LC-1, LC-2, LC-4, D-2, P-2 e P-6). The isolates that broke Oryzica Llanos 5 resistance also had the shortest incubation periods (e.g. isolate D-1, 2.0 days), while the isolates that did not show resistance breakdown, generally had the longest incubation periods (e.g. isolate LA-2, 6.25 days). Isolates virulent on Oryzica Llanos also had the shortest latent periods. A significant and positive correlation was observed between disease severity at 6, 9 and 12 days after inoculation and the AUDPC, and a significant and negative correlation was generally observed between period of incubation or latent period and the AUDPC. The AUDPCs of susceptible cultivars were larger than in the resistant cultivar. The resistance breakdown of cv. Oryzica Llanos 5, so far the resistant standard, by isolates of M. grisea from the Brazilian Central-North region is reported here for the first time and is a clear indication of the high genetic diversity of the pathogen in this region, which therefore poses a great threat to rice production in Brazil. In chapter 2, monosporic isolates collected at commercial rice farming at the states of Tocantins, Goiás and Pará were examined for genetic diversity and population structure. In order to examine the diversity, essays were conducted at Embrapa-CENARGEN, where 140 M. grisea monosporic isolates obtained from the counties of Lagoa da Confusão, Dueré and Formoso do Araguaia (Tocantins), Luiz Alves do Araguaia (Goiás) and Paragominas (Pará) were evaluated. For this study, 34 microsatelite markers marked with fluorochrome were used for automatic genotyping in DNA sequencing. Fourteen microsatelites markers appeared highly efficient to estimate genetic diversity and to detect structuration in the M. grisea metapopulation from Central-North Brazil. Some of them had high informative content, facilitating genotyping in scale and DNA polymorphism analysis. The medium number of alleles per loco was 6.35, varying from 2 alleles by markers ms 109 110, ms 115 - 116, ms 61 62 to 16 alleles by marker PG 27. Genetic diversity index (GD), informative polymorphic content (IPC) and identity portability (IP), confirmed these markers efficiency. M. grisea metapopulation from Central-North Brazil was substructured in three sub-populations (K=3). Goiás (Luiz Alves) isolates were mostly observed in Group 1, Tocantins (Formoso, Dueré and Lagoa da Confusão) isolates were mainly observed in Group 2 and Pará (Paragominas) isolates were all observed in Group 3. Group 3 isolates (Pará) were the most genetically distant in relation to isolates of Goiás and Tocantins. The high genetic diversity observed in the M. grisea metapopulation, as well as the evidence of populational sub-structures, suggest the need of specific monitoring and the use of adequate strategies by genetic improvement programs to each of the sub-populations identified.

A rápida perda da resistência das cultivares de arroz à brusone, causada por Magnaporthe grisea (Herb.) Barr, é geralmente atribuída à variabilidade genética do patógeno ou ainda à ocorrência de escape durante a seleção de novas cultivares. Com o objetivo de caracterizar a estrutura genética de um grupo de isolados de M. grisea em Santa Catarina (SC), plantas com sintomas de brusone foram coletadas em 12 municípios e na Estação Experimental da Epagri em Itajaí (EEI), SC. Os isolados foram analisados através de rep-PCR baseado no transposon Pot-2. Para a identificação das raças, um subgrupo de isolados foi inoculado na série internacional de cultivares diferenciais de arroz. As raças identificadas tiveram seu espectro de virulência avaliado em um grupo de cultivares com genes de resistência conhecidos (isolinhas). Isolados que apresentaram características genéticas distintas, foram avaliados quanto a capacidade de recombinação parassexual. Noventa e dois isolados monoconidiais obtidos foram agrupados através de rep-PCR em 13 haplótipos e 2 linhagens. Nove haplótipos encontrados na amostragem dos municípios não foram encontrados na EEI, indicando a ocorrência de escape. Seis raças de M. grisea foram identificadas a partir da inoculação de 28 isolados, representando 12 haplótipos na série diferencial, sendo que, 5 destas, foram avirulentas à isolinha que possui os genes de resistência Pi-1 e Pi-11. Por outro lado, o isolado 25, agrupado na raça IA-65, superou 4 das 5 isolinhas testadas. A análise da capacidade de recombinação parassexual mostra a existência de compatibilidade vegetativa associado à ocorrência de anastomoses entre três haplótipos, sendo identificados três possíveis isolados recombinantes.

L’obbiettivo del nostro studio è stato quello di comprendere il meccanismo che controlla la regolazione del flusso sanguigno cerebrale nel corpo calloso (CC) umano con il fine di spiegare l’effetto BOLD, precedentemente scoperto da Fabri e collaboratori nel 2011. Le analisi per determinare la presenza, il numero, la distribuzione e la morfologia delle cellule immunopositive all’enzima ossido nitrico sintetasi neuronale (nNOS) sono eseguite sul corpo calloso e sul’indusium griseum (IG). L’Ossido nitrico (NO) è un neurotrasmettitore gassoso largamente diffuso nel cervello umano ed ha, tra le altre funzioni, un potente effetto vasodilatante e perciò contribuisce a regolare il flusso sanguigno cerebrale. Sezioni seriali sagittali ottenute da blocchetti in paraffina e da taglio del tessuto in congelato sono state utilizzate per l’analisi immunoistochimica sul CC e sul IG. L’intensa marcatura che si è ottenuta ha dimostrato la presenza di cellule immunopositive al nNOS, e grazie alla microscopia a fluorescenza ne è stata confermata la loro natura neuronale e astrocitaria. Cellule neuronali immunopositive all’nNOS sono state osservate sia nel CC che nell’IG mentre gli astrociti che lo esprimono erano solo presenti nel CC. I neuroni nNOS positivi hanno mostrato diverse morfologie e risultavano essere piu abbondanti a 1mm a partire dalla linea mediana, per l’IG e a 4mm per il CC con un picco di massima abbondanza nel corpo del CC. In alcuni casi questi nueroni erano localizzati principalmente nell’interfaccia tra IG e CC e piu specificatamente in prossimita delle arterie piali, arteriole che originano dall’arteria sopracallosale che poi si affosano nel corpo calloso. La presenza di neuroni nNOS positivi in prossimità di vasi sanguigni, in questi due tessuti suggeriscono un loro plausibile ruolo nella regolazione neurovascolare del CC che potrebbero quindi ipoteticamente spiegare l’effetto BOLD osservato in risonanza. l’IG inoltre potrebbe giocare un ruolo attivo nel controllo della vascolarizzazione del CC, ed è quindi probabile e che non sia solo un tessuto di rimanenza embrionale come precedentemente si riteneva ma un tessuto con un sua funzione specifica. Infine è stata osservata per la prima volta la presenza di astrociti nNOS positivi nel corpo calloso umano. Queste cellule variavano nella presenza, nel numero e nelle loro distribuzione in base a diverse condizioni di ossigenazione sistemica che avvenivano nel momento del decesso. Infatti nei soggetti deceduti dopo una breve ipossia gli astrociti nNOS positivi erano assenti, mentre risultavano essere molto abbondanti nei soggetti deceduti con un ipossia prolungata. L’immunopositività degli astrociti sembrerebbe quindi essere correlata alla durata dell’ipossia cerebrale al momento della morte.
The aim of the present study is to investigate the possible mechanism for the control of cerebral blood flow in the corpus callosum (CC), that could explain the BOLD effect previously found (Fabri et al., 2011). The presence, number, distribution and morphology of neuronal Nitric Oxyde Sinthase (nNOS) positive cells was investigated in the corpus callosum (CC) and indusium griseum (IG). Nitric Oxyde (NO) is a gaseous neurotransmitter largely diffused in the brain, whichexerts a powerful vasodilatory effect, and therefore it can contribute to regulate the cerebral blood flow. Sagittal serial sections from paraffin or frozen autoptic specimens of human adult CC and overlying IG were processed for immunohistochemistry and immunofluorescence analysis, using an antibody against the neuronal form of the enzyme Nitric Oxyde Synthase (nNOS). The stainings revealed the presence of many nNOS immunopositive cells. By double labeling technique with immunofluorescence at confocal microscopy, it has been shown that in the CC both neurons and astrocytes positive to nNOS antibody were present, and their number varied in different conditions, as detailed below. In the IG, only neurons nNOS positive were found. Neurons showed different morphologies, were more numerous 1 mm apart from the medial line in IG and 4 mm in CC, with a peak over the body of the CC. In some cases, they were located at the boundary between IG and CC, more densely packed in proximity to the pial arteries penetrating into the CC. The significant presence of nNOS immunopositive neurons in these two structures suggests that they might have a role in the neurovascular regulation of CC, moreover the IG could plays a functional role in the adult brain. The presence of nNOS positive astrocytes in the human CC has been here demostrated for the first time. As previously mentioned, their number and distribution varied in different conditions: nNOS positive astrocytes were absent in samples from subjects deceased after a short hypoxia; their number and labeling intensity increased with the hypoxia prolongation. Neuronal NOS immunopositivity of CC astrocytes seems thus related to the hypoxia duration and the consequent brain damage.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-06T12:00:41Z No. of bitstreams: 3 Dissertação - Lorraine Andrade Malaspina - 2014 - (1).pdf: 19847965 bytes, checksum: 80a80b846a7ec7bf574c1a4bbeb45756 (MD5) Dissertação - Lorraine Andrade Malaspina - 2014 - (2).pdf: 481787 bytes, checksum: 75b49000c4d8d2964d52487b43437104 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)
Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-03-06T12:06:22Z (GMT) No. of bitstreams: 3 Dissertação - Lorraine Andrade Malaspina - 2014 - (1).pdf: 19847965 bytes, checksum: 80a80b846a7ec7bf574c1a4bbeb45756 (MD5) Dissertação - Lorraine Andrade Malaspina - 2014 - (2).pdf: 481787 bytes, checksum: 75b49000c4d8d2964d52487b43437104 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5)
Made available in DSpace on 2015-03-06T12:06:22Z (GMT). No. of bitstreams: 3 Dissertação - Lorraine Andrade Malaspina - 2014 - (1).pdf: 19847965 bytes, checksum: 80a80b846a7ec7bf574c1a4bbeb45756 (MD5) Dissertação - Lorraine Andrade Malaspina - 2014 - (2).pdf: 481787 bytes, checksum: 75b49000c4d8d2964d52487b43437104 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-12-08
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Obtaining 3-dimensional structure of an enzyme can be used as an initial step in projects of genetic engineering and enzymatic engineering when aiming for optimization of catalytic activity and/or production of target enzymes on an industrial scale. Currently, crystallography is the most widely used method for the determination of three-dimensional structures of macromolecules. The plant biomass in the form of cellulose, hemicellulose and lignin, have great potential for biotechnological applications. With the growing demand for renewable energy sources, its been proposed it's use to obtain energy, called biofuels. For such purpose, the main approach is the search of the degradation of biomass via enzymatic hydrolysis. In this context, the study of microorganisms capable of carrying out the degradation of biomass and the study of the enzymes involved in this process play a key role. Particularly, the thermophilic fungus Humicola grisea var. thermoidea presents signi cant production of active lignocellulolytic enzymes at high temperature and has been considered a strong candidate for industrial applications. However, the scienti c literature still lacks of structural information on fungal enzymes involved in the hydrolysis of lignocellulose. In previous work, the cellulolytic enzyme cellobiohydrolase (CBH1.2) from Humicola grisea has been identi ed and cloned into Pichia pastoris as well as one of the endoxylanases (HXYN2) from this same organism. In this master's project, the enzymes CBH1.2 and HXYN2 were expressed using heterologous expression system obtaining satisfactory yield for in vitro assays. Puri - cation protocols were established via precipitation by ammonium sulfate and initial experiments of enzyme activity were performed via the reducing sugars method for both enzymes. XX Crystallization conditions were found for the enzyme CBH1.2r, where small needleshaped crystals were obtained in crystallization trials. In addition to this, the cloning of the enzyme CBH1.2 from Humicola grisea with the pHIL-D2 expression vector (for extracellular expression) was performed. This vector was used to transform GS115 and SMD1168 strains of the yeast P. pastoris both with the genotype his4-. Transformants that were able to secrete active protein were detected in both strains.
A obten c~ao da estrutura tridimensional de uma enzima pode ser utilizada como passo inicial em projetos de engenharia gen etica e engenharia enzim atica com intuito de optimiza c~ao da atividade catal tica e/ou a produ c~ao em escala industrial de enzimas alvo. Atualmente, a cristalogra a e o m etodo mais empregado para a determina c~ao de estruturas tridimensionais de macromol eculas. A biomassa vegetal, na forma de celulose, hemicelulose e lignina, apresenta grande potencial para aplica c~oes biotecnol ogicas. Com a crescente demanda por fontes renov aveis de energia, tem-se proposto sua utiliza c~ao para a obten c~ao de energia, os ditos biocombust veis. Para esse m, a principal abordagem que se tem procurado e a degrada c~ao da biomassa via hidr olise enzim atica. Neste contexto, o estudo de micro-organismos capazes de realizar a degrada c~ao da biomassa e o estudo das enzimas envolvidas no processo apresentam papel chave. Particularmente, o fungo term o lo Humicola grisea var. thermoidea apresenta produ c~ao signi cativa de enzimas lignocelulol ticas ativas a alta temperatura e tem sido considerado um forte candidato para aplica c~oes industriais. Contudo, a literatura cient ca ainda carece de informa c~oes estruturais sobre as enzimas do fungo envolvidas na hidr olise da lignocelulose. Em trabalhos anteriores, a enzima celulol tica celobiohidrolase (CBH1.2) de H. grisea foi identi cada e clonada em Pichia pastoris bem como uma das endoxilanases (HXYN2) deste mesmo organismo. Neste projeto de mestrado, foram expressadas as enzimas CBH1.2 e HXYN2 utilizando sistema heter ologo de express~ao, obtendo rendimento satisfat orio para ensaios in vitro. Foram estabelecidos protocolos de puri ca c~ao via precipita c~ao por sulfato de am^onio e realizados experimentos iniciais de atividade enzim atica via o m etodo dos a c ucares redutores para as duas enzimas. XVIII Foi encontrada condi c~ao de cristaliza c~ao para a enzima CBH1.2r onde foram obtidos pequenos cristais em forma de agulha nos ensaios de cristaliza c~ao. Al em deste, tamb em foi realizada a clonagem da enzima CBH1.2 de Humicola grisea no vetor de express~ao pHIL-D2 (para express~ao extracelular). Este vetor foi utilizado para transformar as linhagens SMD1168 e GS115 da levedura P. pastoris ambas com o gen otipo his4-. Foram detectados transformantes capazes de secretar a prote na ativa em ambas as linhagens.

Dissertação (mestrado)—Universidade de Brasília, Departamento de Fitopatologia, do Instituto de Ciências Biológicas, 2011.
Submitted by Alaíde Gonçalves dos Santos (alaide@unb.br) on 2012-09-11T12:36:07Z No. of bitstreams: 1 2011_AnaPauladaSilvaPagani.pdf: 2923412 bytes, checksum: b63fe23aa025c53e6ccfa31879633f54 (MD5)
Rejected by Jaqueline Ferreira de Souza(jaquefs.braz@gmail.com), reason: on 2012-09-12T12:25:57Z (GMT)
Submitted by Alaíde Gonçalves dos Santos (alaide@unb.br) on 2012-09-12T12:40:20Z No. of bitstreams: 1 2011_AnaPauladaSilvaPagani_Parcial.pdf: 2764145 bytes, checksum: c938fbb8af2dd6254bd1650d6f471d08 (MD5)
Approved for entry into archive by Jaqueline Ferreira de Souza(jaquefs.braz@gmail.com) on 2012-09-12T12:42:32Z (GMT) No. of bitstreams: 1 2011_AnaPauladaSilvaPagani_Parcial.pdf: 2764145 bytes, checksum: c938fbb8af2dd6254bd1650d6f471d08 (MD5)
Made available in DSpace on 2012-09-12T12:42:32Z (GMT). No. of bitstreams: 1 2011_AnaPauladaSilvaPagani_Parcial.pdf: 2764145 bytes, checksum: c938fbb8af2dd6254bd1650d6f471d08 (MD5)
A brusone do trigo, causada pelo fungo Magnaporthe grisea, embora de ocorrência esporádica devido às exigências climáticas específicas, tais como alta umidade e temperatura é uma doença muito severa, acarretando em elevadas perdas de produtividade. O Capítulo 1 desta dissertação descreve a reação de 147 genótipos de trigo comum e sintético à doença em condições de campo, visando resistência e tolerância à brusone. Tolerância foi definida como resiliência quanto à produção, mesmo com reação suscetível. A intensidade da doença variou entre os plantios, sendo baixa em 2010 e elevada incidência em 2011. A grande maioria dos materiais avaliados foi suscetível à doença. Apenas 3,4% dos genótipos apresentaram reação de resistência (incidência nas espigas menor que 5 %), enquanto outros 16 % dos genótipos foram moderadamente resistentes. Genótipos resistentes incluem Melchior, Safira, Jesuita, CASW94Y00116S (ciclo médio) e Trintecinco (ciclo longo). Os materiais de ciclo curto foram os que apresentaram os maiores percentuais de doença nas espigas. Com relação à tolerância, destacaram-se os materiais BH1146 e PF909 (ciclo curto) e África 43 (ciclo médio).O Capítulo 2 teve como objetivo verificar a eficiência de fungicidas sintéticos e de métodos alternativos de controle da brusone do trigo, em quatro experimentos de campo. Dois experimentos, análogos e repetidos em 2010 e em 2011, examinaram o efeito de aplicações de silicato de Ca e Mg em duas cultivares de trigo com diferentes níveis de resistência de campo à brusone, BRS-264 (Altamente Suscetível) e BR-18 Terena (Moderadamente Resistente). Foram determinadas a incidência e a severidade da brusone (utilizando uma escala de notas). Dois outros experimentos, análogos e instalados nas mesmas épocas, examinaram o efeito de aplicação de fosfito de K e de fungicidas sintéticos no controle da doença na cv. BR- 264. No ano de 2010 a intensidade média da doença foi bastante inferior que a observada em 2011, mas a cv. BR-18 apresentou consistentemente menor incidência e severidade em 2010 e 2011, respectivamente. Em 2010 foi encontrada interação significativa (p ≤ 0,05) entre os genótipos e a aplicação de Si quanto à incidência de brusone e apenas BRS-264 apresentou significativa redução da incidência com aplicações de Si, via sulco ou via foliar. Quanto à severidade em 2010, não houve interação genótipo e Si, e menores severidades também foram observadas com Si via sulco e via foliar. No ano de 2011 não houve interação significativa (p > 0,05) entre genótipos e Si, e a aplicação de Si via foliar resultou em menores severidades da doença. Com relação ao uso de fosfito e de fungicidas, em 2010, todos os tratamentos apresentaram controle superior à testemunha (p ≤ 0,05) tanto para incidência quanto para severidade. Em 2011, a aplicação de fosfito resultou em resposta intermediária, não se distinguindo dos tratamentos com fungicidas sintéticos nem da testemunha (p > 0,05). Em média, os fungicidas apresentaram apenas 34 % de controle da brusone. No Capítulo 3, estimou-se a diversidade genética de 40 isolados monospóricos de Magnaporthe de trigo e de arroz, coletados na região central do país. Foram empregados 25 marcadores microssatélites com fluorescência para a genotipagem. A análise filogenética separou dois grandes grupos, um constituído apenas por isolados oriundos de cultivares de trigo e outro de apenas de cultivares de arroz. A similaridade genética entre os isolados de M. grisea provenientes de plantas de arroz e de trigo foi de apenas 3 %. Em conclusão, este estudo indica que fontes de resistência ou tolerância a brusone são raras no germoplasma de trigo, embora alguns materiais potencialmente úteis tenham sido identificados. Além disso, constata-se que o controle atual da brusone do trigo no Cerrado necessita da integração de várias ferramentas de manejo, uma vez que as opções químicas, genéticas e alternativas apresentaram apenas efeitos parciais em condições ambientais favoráveis. Por fim, este trabalho indica diferenças fundamentais entre os isolados de trigo e de arroz, o que sugere significativo isolamento genético e possível origem filogenética distinta entre essas populações. _______________________________________________________________________________________ ABSTRACT
DISSERTATION ABSTRACT Wheat blast, caused by Magnaporthe grisea, is a severe disease, causing significant yield losses. The disease occurs sporadically, due to specific climatic conditions, such as high temperature and humidity. Dissertation Chapter 1 describes the field reaction of 147 wheat lines and commercial cultivars to blast, aiming to identify disease resistance and tolerance (i.e., yield resilience in susceptible genotypes). Disease intensity varied among years, was low in 2010 and very high in 2011. The great majority of genotypes was susceptible to the disease. Only 3.4 % were resistant (ear incidence < 5 %), while another 16 % were classified as moderately resistant. Resistant genotypes included Melchior, Safira, Jesuita, CASW94Y00116S (intermediate cycle) and Trintecinco (late maturing cycle). Early maturing genotypes had the highest disease incidence in the ears. Genotypes BH1146 e PF909 (early) and África 43 (intermediate) were distinguished as tolerant. Chapter 2 aimed to estimate the efficiency of synthetic fungicides and alternative methods of wheat blast control, by means of four field experiments. Two similar experiments, replicated in 2010 and 2011, examined the effect of Ca and MG silicates in two wheat cultivars with different levels of field resistance to blast, BRS-264 (Highly Resistant) and BR-18 Terena (Moderately Resistant). Blast incidence and severity (with a disease scale) were determined. Two other separate experiments, carried out concunently in 2010 and 2011, investigated the effect of K phosphite and fungicide applications on disease control in cv. BRS-264. Overall disease intensity was much lower in 2010 than in 2011, but cultivar BR-18 consistently displayed lower incidence and severity values in 2010 and 2011, respectively. In 2010, a significant (p ≤ 0.05) interaction was found between genotypes and Si for incidence, and only BRS-264 showed a significant incidence reduction by Si applications via planting furrow or via leaf applications. For severity in 2010, there was no genotype x Si interaction and lower severities were again observed by Si applications in the planting furrow or by foliar applications. In 2011, no significant (p > 0.05) genotype x Si interaction was found, and foliar Si applications resulted in lower disease severity. In the fungicide-phosphite experiment, in 2010 all treatments reduced disease component to the non-treated control plots (p ≤ 0.05), both in incidence and severity. In 2011, the K phosphite treatment was intermediate, not significantly different from synthetic fungicides, nor from the untreated control. (p > 0.05). Overall, synthetic fungicides were not rather inefficient for wheat blast control, reducing disease levels by only 34 %. Finally, Chapter 3 estimated the genetic diversity among 40 monosporic wheat and rice isolates of Magnaporthe grisea, collected in commercial fields in the Brazilian Mid-West. Twenty-five fluorescent microsatellite markers were employed for genotyping and the filogenetic analysis separated two great groups, one comprising only isolates from wheat and another with only rice isolates. Genetic similarity among Magnaporthe grisea isolates collected on rice or wheat plants was only 3 %. In conclusion, this study indicates that sources of resistance or tolerance to blast are relatively rare in the wheat germoplasm, even if some potentially promising materials have been identified. Furthermore, these data shows that present control of wheat blast in the Brazilian Mid-West necessitates the integration of several complementary methods, once no single chemical, genetic or alternative option is sufficient for disease control in climate conditions conducive to the disease. Finally, this work indicates fundamental differences among wheat and rice isolates, suggesting genetic isolation and a probable distinct phylogenetic origin of these two populations.

Le beta-1,3-d-glucane est un constituant essentiel de la paroi cellulaire des champignons, synthetise chez les vegetaux principalement dans la fonction cicatricielle (callose). L'enzyme qui le synthetise, joue un role important dans la croissance mycelienne et peut etre une cible interessante pour des fongicides. La beta-1,3-d-glucane synthase (ec 2. 4. 1. 34) de pyricularia oryzae, champignon responsable de la fletrissure du riz, a ete caracterisee. Nous avons dans un premier temps mis au point un protocole d'extraction des membranes par des centrifugations fractionnees. L'enzyme a ensuite ete solubilisee a l'aide du detergent polyoxyethylene ether w1. Le produit synthetise a ete identifie par digestion a l'aide d'hydrolases specifiques, puis par chromatographie. En conditions optimales, l'activite specifique de l'extrait enzymatique partiellement purifie peut atteindre 10 nmoles de glucose incorporees / mg de proteines / minute. Les etudes biochimiques ont montre les caracteristiques de regulation du systeme enzymatique: l'activation par les ions calcium et magnesium, l'activation par les nucleotides triphosphates a faible concentration (gtp 10#-#8m, atp 10#-#6m) et l'absence de forme zymogene. L'importance de la partie lipophile des molecules inhibitrices a ete montree en etudiant l'inhibition par les detergents de la famille de l'octylglucoside. La fraction enzymatique solubilisee, puis purifiee par centrifugation en gradient de densite, a ete analysee par electrophorese en condition denaturante. Les peptides de 32, 46 et 54 kda sont correles a l'activite et pourraient correspondre a des sous-unites du complexe enzymatique. Une banque d'adn genomique en phage de p. Oryzae a ete construite. Un fragment du gene etg1 de s. Cerevisiae, codant pour une proteine membranaire impliquee dans la synthese de beta-1,3-glucane, a ete amplifie par pcr et utilise comme sonde. Des experiences d'hybridation ont revele la presence de sequences homologues chez p. Oryzae. Le criblage de la banque genomique a permis d'isoler un clone, en cours de sous-clonage pour le sequencage

Magnaporthe grisea is a filamentous ascomycete fungus that causes blast disease on rice and other grasses. Blast is a serious deterrent to rice production and negatively affects production of other cereals, forage crops and economically important grasses. The primary means of blast disease management involves the development and implementation of genetically resistant plants. Understanding the molecular basis of plant resistance is the foundation for the development of unique and durable plant protection. The results presented here focus on genes in the rice blast fungus called avirulence genes that encode molecules acting as effectors of host resistance. Until recently, two avirulence loci had been shown to induce resistance in rice cultivar Maratelli. This study gives an update on the current status of one, the AVR1-MARA locus, and describes a new Maratelli avirulence locus that is not allelic to AVR1-MARA or AVR2-MARA. Additionally, evidence is given that indicates a genome rearrangement is responsible for generation of the newly described avirulence locus. Genetic data, hybridization results and DNA sequence analysis demonstrate the translocation of a large AT-rich fragment from one chromosomal location to another. Molecular detection of the translocation is demonstrated by hybridization of certain AVR1-MARA markers that only follow the avirulent phenotype in strains after the rearrangement. The rearrangement is detectable genetically, as the avirulent phenotype controlled at this locus segregates independently from progenitor strains that also contain a single Maratelli-specific avirulence gene. CHEF electrophoretic separation of chromosome-sized DNA shows that the AT rich sequences are located on one of the larger M. grisea chromosomes both before and after cross 4134. Hybridization of CHEF blots indicates that two chromosomes may have been involved in a translocation, however a reorganization of chromosome 2 cannot be ruled out. A homing enzyme strategy for determining the size of the translocated fragment is described. These results demonstrate an example of genomic plasticity leading to a translocation and creation of a new avirulence locus in the rice blast fungus M. grisea.

The Faint Infrared Grism Survey (FIGS) is a deep Hubble Space Telescope (HST) WFC3/IR (Wide Field Camera 3 Infrared) slitless spectroscopic survey of four deep fields. Two fields are located in the Great Observatories Origins Deep Survey-North (GOODS-N) area and two fields are located in the Great Observatories Origins Deep Survey-South (GOODS-S) area. One of the southern fields selected is the Hubble Ultra Deep Field. Each of these four fields were observed using the WFC3/G102 grism (0.8 mu m-1.15 mu m continuous coverage) with a total exposure time of 40 orbits (approximate to 100 kilo-seconds) per field. This reaches a 3 sigma continuum depth of approximate to 26 AB magnitudes and probes emission lines to similar to 10(-17) erg s(-1) cm(-2). This paper details the four FIGS fields and the overall observational strategy of the project. A detailed description of the Simulation Based Extraction (SBE) method used to extract and combine over 10,000 spectra of over 2000 distinct sources brighter than m(F105W) = 26.5 mag is provided. High fidelity simulations of the observations is shown to significantly improve the background subtraction process, the spectral contamination estimates, and the final flux calibration. This allows for the combination of multiple spectra to produce a final high quality, deep, 1D spectra for each object in the survey.

[EN] Disease problems are frequently observed in citrus orchards in Panama, but in most cases their aetiology is unknown. Between 2010 and 2013 a total of 85 plots cultivated with different citrus species of citrus were surveyed in the main production areas in Panamá. Greasy spot, caused by the fungus Zasmidium citri-griseum, was identified as the most prevalent citrus disease in the country. Other fungal pathogens causing foliar and fruit diseases were also identified by the first time in Panamá: melanose caused by Diaporthe citri, postbloom fruit drop and anthracnose caused by Colletotrichum acutatum, and scab caused by Elsinoë fawcettii. A collection of 42 isolates of Mycosphaerellaceae from different citrus-growing regions in Spain, Morocco, Ghana and Panama were characterized. Strain were identified based on morphological characteristics, growth at different temperatures, ITS sequencing and pathogenicity on citrus plants. The species Amycosphaerella africana was associated with greasy spot of citrus in Spain and Morocco and Z. citri-griseum was identified as the causal agent of greasy spot in Panama, being also associated with the disease in Ghana. The study greasy spot epidemiology in Panama showed that the greatest defoliation of trees orange 'Valencia' affected by the disease occurred in the dry season between December and April. In the study of inoculum dynamics in the leaf litter, the days until complete decomposition of the leaves was related in the model with the accumulated rainfall per week (mm), days with precipitation >1 mm per week (nº) and average relative humidity per week (%). Ascospores released from the leaf litter were related with days until complete decomposition of leaves, days with precipitation >1 mm per week (nº), weekly cumulative rainfall (mm) and average temperature (ºC). Furthermore, the experiment of airborne inoculum showed that the highest levels of Z. citri-griseum inoculum were recorded during April and May, coinciding with the onset of the rainy season. Likewise, the highest values of greasy spot incidence in trap plants were recorded during the months of higher inoculum availability. Disease control experiments confirmed the efficacy of the fungicide fenbuconazole to reduce the severity of greasy spot in grapefruit and sweet orange in Panama. However, harvested yield was not significantly affected by the application of fungicide sprays.
[ES] En las plantaciones de cítricos en Panamá se observan con frecuencia problemas de enfermedades, sin embargo en la mayoría de los casos se desconoce su etiología. Por tal motivo, durante los años 2010 a 2013 se prospectaron un total de 85 parcelas de diversas especies de cítricos en las principales zonas productoras del país. Se diagnosticó a la mancha grasienta, causada por Zasmidium citri-griseum, como la enfermedad más prevalente en los cítricos de Panamá. Se identificaron por primera vez en Panamá también otros patógenos fúngicos causantes de enfermedades foliares y de frutos como Diaporthe citri agente causal de la melanosis, Elsinoë fawcettii agente causal de la roña de los cítricos, Colletotrichum acutatum agente causal de la caída prematura de frutos y antracnosis. Se caracterizó una colección de 42 aislados de Mycosphaerellaceae procedentes de diferentes regiones productoras de cítricos en España, Marruecos, Ghana y Panamá. Los aislados se identificaron a nivel de especie a partir de sus características morfológicas, culturales (fisiológicas), moleculares (región ITS) y patogénicas. Se identificó la especie Amycosphaerella africana asociada a la mancha grasienta de los cítricos en España y Marruecos y Z. citri-griseum como agente causal de la mancha grasienta en Panamá, estando asociada también a la enfermedad en Ghana. En el estudio epidemiológico de la mancha grasienta en Panamá, mostró que la mayor defoliación de los árboles de naranja 'Valencia' afectados por la enfermedad ocurrió en la época seca entre los meses de diciembre a abril. En el estudio de dinámica de la producción de inóculo en la hojarasca, el modelo resultante para los días hasta la descomposición total de las hojas relaciono a las variables climáticas precipitación pluvial acumulada por semana (mm), días con precipitación pluvial >1 mm por semana (nº) y humedad relativa promedio por semana (%). En relación a las ascosporas liberadas a partir de la hojarasca el modelo resultante relaciono a las variables climáticas días hasta la descomposición total de las hojas, días con precipitación pluvial >1 mm por semana (nº), precipitación pluvial acumulada por semana (mm) y la temperatura promedio (ºC). Por otro lado, el experimento de seguimiento del inóculo en el aire, mostró que la mayor disponibilidad de inóculo de Z. citri-griseum ocurre durante los meses de abril y mayo cuando se inicia la estación de lluvias. De igual manera, la mayor incidencia de la mancha grasienta en las plantas trampa coincidió con los meses de mayor disponiblidad de inóculo. No obstante, se registraron infecciones también durante otras épocas del año. En los ensayos de control se confirmó la eficacia del fungicida fenbuconazol, que redujo significativamente la severidad de la mancha grasienta en pomelo y naranja en Panamá. Sin embargo, no se detectó un efecto significativo de los tratamientos sobre el peso de la cosecha de frutos.
[CAT] En les plantacions de cítrics en Panamà s'observen ben sovint problemes de malalties, no obstant això en la majoria dels casos es desconeix la seua etiologia. Per tal motiu, durant els anys 2010 al 2013 es visitaren un total de 85 parcel¿les de diverses espècies de cítrics en les principals zones productores del país. Es diagnosticà la taca greixosa, causada per Zasmidium citri-griseum, com la malaltia més àmpliament distribuïda en els cítrics de Panamà. Es van identificar per primera vegada en Panamà també altres patògens fúngics causants de malalties foliars i de fruits com Diaporthe citri agent causal de la melanosi, Elsinoë fawcettii agent causal de la ronya dels cítrics, Colletotrichum acutatum agent causal de la caiguda prematura de fruits i antracnosi. Es va caracteritzar una col¿lecció de 42 aïllats de Mycosphaerellaceae procedents de diferents regions productores de cítrics a Espanya, El Marroc, Ghana i Panamà. Els aïllats es van identificar a nivell d'espècie a partir de les seues característiques morfològiques, culturals (temperatures), moleculars (regió ITS) i patogèniques. Es va identificar l'espècie Amycosphaerella africana associada al la taca greixosa dels cítrics a Espanya i El Marroc i Z. citri-griseum com a agent causal de la taca greixosa en Panamà, estant associada també a la malaltia a Ghana. En l'estudi epidemiològic de la taca greixosa en Panamà, va mostrar que la major defoliació dels arbres de taronja 'València' afectats per la malaltia va ocórrer en l'època seca entre els mesos de desembre a abril. En el estudi de la dinàmica de la producció d'inòcul en la fullaraca, el model resultant per als dies fins a la descomposició total del les fulles es relacionà amb les variables climàtiques precipitació pluvial acumulada per setmana (mm), dies amb precipitació pluvial >1 mm per setmana (nº) i humitat relativa mitjana per setmana (%). En relació a les ascospores alliberades a partir de la fullaraca, el model resultant relacionà les variables climàtiques dies fins a la descomposició total de les fulles, dies amb precipitació pluvial >1 mm per setmana (nº), precipitació pluvial acumulada per setmana (mm) i la temperatura mitjana (ºC). D'altra banda, l'experiment de seguiment de l'inòcul en el aire, va mostrar que la major disponibilitat d'inòcul de Z. citri-griseum ocorregué durant els mesos d'abril i maig quan s'inicia l'estació de pluges. De la mateixa manera, la major incidència de la taca greixosa en les plantes trampa va coincidir amb els mesos de major disponibilitat d'inòcul. No obstant això, se van registrar infeccions també durant altres èpoques de l'any. En els assajos de control es va confirmar l'eficàcia del fungicida fenbuconazol, que va reduir significativament la severitat de la taca greixosa en pomelo i taronja en Panamà. No obstant això, no es va detectar un efecte significatiu dels tractaments sobre el pes de la collita de fruits.
Aguilera Cogley, VA. (2016). ENFERMEDADES FÚNGICAS DE LOS CÍTRICOS EN PANAMÁ. ESTUDIO PARTICULAR DE LA MANCHA GRASIENTA CAUSADA POR Mycosphaerellaceae [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61447
TESIS

This work describes the study chemical and pharmacological of the extracts and compounds isolated from two species of Rubiaceae (Sabicea grisea Cham. & Schltdl. var. grisea and Psychotria barbiflora DC.). Extracts and fractions of S. grisea, and fractions which showed positive reactions record to alkaloids of P. barbiflora, exhibited a significant reduction in the nociceptive response, with inhibition upper or comparable to the reference drug, indomethacin. The chemical study of fractions from leaves and stems of S. grisea, guided by pharmacological tests, resulted to the isolation of two saturated alcohols, octacosanol and hexacosanol, two pentacyclic triterpenes oleanane serie, siaresinolic and 3-O-α-L-rhamnopyranosyl-(1→2)-α-Larabinopyranosylsiaresinolic acids, vanillic and salicylic acids, scopoletin, ethyl caffeate and two phytosteroids, β-sitosterol and 3-O-β-D-glucopyranosylsitosterol. It is noteworthy that no previous reports about the presence of these compounds in the genus Sabicea. Moreover, no record was found on the 3-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-siaresinolic acid, named Gottliebside, an simple tribute to the greatest Natural Products Chemistry of the Brazil, Prof. Otto Richard Gottlieb, therefore, a new natural product. The chemical study of alkaloidal fractions from leaves and stems of P. barbiflora led to the isolation of two β-carboline alkaloids, harman and stricnosidinic acid, whose occurrences corroborate the inclusion of this species in the subgenus Heteropsychotria. In the pharmacological tests, octacosanol (10 and 1 mg/kg, i.p) and siaresinolic acid (1 and 0.1 mg/kg, i.p) showed peripheral antinociceptive effects mediated by α2-adrenergic receptors and channel K+ ATPdependent, respectively. Furthermore, these compounds inhibited the accumulation of inflammatory cells, as well as TNF-α in the model of carrageenan-induced inflammation, indicating an anti-inflammatory action. In addition, these compounds (≤200 μg/ml and ≤ 50 μg/ml, respectively) were unable to induce cell death in when evaluated in the reduction of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, demonstrating the absence of cytotoxicity of these compounds.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Este trabalho descreve o estudo químico e farmacológico dos extratos e compostos isolados de duas espécies de Rubiaceae (Sabicea grisea Cham. & Schltdl. var. grisea e Psychotria barbiflora DC.). Extratos e frações de S. grisea, bem como frações com reações positivas para alcaloides obtidas de P. barbiflora, exibiram redução significativa da resposta nociceptiva, com inibição superior ou comparável ao fármaco de referência, indometacina. O estudo químico das frações das folhas e caule de S. grisea, guiados pelos ensaios farmacológicos, resultou no isolamento de dois alcoóis saturados, octacosanol e hexacosanol, dois triterpenos pentacíclicos da série oleanano, ácidos siaresinólico e 3-O-α-L-rhamnopiranosil-(1→2)-α-Larabinopiranosilsiaresinólico, os ácidos salicílico e vanílico, a escopoletina, o cafeato de etila, além dos fitoesteroides, o β-sitosterol e 3-O-β-D-glicopiranosilsitosterol. Não há relatos sobre a presença destes compostos no gênero Sabicea e não foi encontrado registro sobre o ácido 3-O-α-L-rhamnopiranosil-(1→2)-α-Larabinopiranosilsiaresinólico, nomeado de Gottliebsídeo, uma singela homenagem ao maior Químico em Produtos Naturais do Brasil, Prof. Otto Richard Gottlieb, sendo, portanto, um novo produto natural. O estudo químico das frações alcaloídicas das folhas e caule de P. barbiflora conduziu, pela primeira vez, ao isolamento de dois alcaloides β-carbolínicos, harmana e ácido estrictosidínico, cujas ocorrências corroboram com a inclusão desta espécie no subgênero Heteropsychotria. Nos ensaios farmacológicos, o octacosanol (10 e 1 mg/kg, i.p) e o ácido siaresinólico (1 e 0,1 mg/kg, i.p) apresentaram efeitos antinociceptivos periféricos mediado por receptores α2-adrenérgicos e canais de K+ATP-dependente, respectivamente. Além disso, estes compostos inibiram o acúmulo de células inflamatórias, bem como os níveis de TNF-α no modelo de inflamação induzida por carragenina, indicando uma ação anti-inflamatória. Em adição, o octacosanol (≤ 200 μg/ml) e o ácido siaresinólico (≤ 50 μg/ml) não foram capazes de induzir morte nas célula quando avaliados no teste de redução do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT), revelando ausência de citotoxicidade destes compostos.

The pig production in Sweden is divided into conventional and organic production, with the organic production divided into EU organic and KRAV production. Pigs are divided into different age categories: weaned pigs, growing pigs, fattening pigs, gilts/sows before farrowing and dry sows. Roundworm, nodular worm, whipworm, coccidia, red stomach worm, threadworm and lungworm are common parasites in pigs affecting growth, feed conversion and economy. Organic pigs are more exposed to parasites because of outdoor stay and deworming could help if used with care. The aim of this project was to investigate parasites in pig herds with organic production. The investigation would increase the knowledge of parasites in different age categories in currently used production systems and contribute to good recommendations about parasite control and treatment. A modified McMaster technique was used to identify and quantify parasite eggs with microscope. Larval culturing and microscopy were used to distinguish eggs from nodular worm and red stomach worm. Roundworm, nodular worm, whipworm and coccidia were found in all age categories. Threadworm was only identified in dry sows and gilts/sows before farrowing. Nodular worm and coccidia showed highest quantity of positive samples at herd and sample level followed by roundworm and whipworm. Roundworm and nodular worm had highest quantity of eggs per gram faeces followed by whipworm and threadworm. The results corresponded to previous studies in parasite occurrence despite new conditions. More samples, herds and environmental factors should be investigated in conjunction with parasite occurrence to get broader knowledge and to give good recommendations in parasite control and treatment.

Magnaporthe grisea est un champignon ascomycete responsable de la principale maladie du riz la pyriculariose. Notre projet de recherche est d'isoler par clonage positionnel, les genes d'avirulence avrku86-1, avrmednoi-1 et avrirat 7-1, genetiquement independants et precedemment identifies dans le croisement entre isolats guy11 et 2/0/3 (silue et al. 1992). Des marqueurs rapd lies aux genes d'avirulence en segregation ont ete tout d'abord identifies en utilisant la methode du bulk segregant analysis. La grande majorite de ces marqueurs (7/11) correspond a des fragments de jonction entre sequences repetees de type transposon et des sequences non caracterisees chez m. Grisea. Les marqueurs rapd lies ont ete caracterises et ont servi de points de depart de marches chromosomiques vers les 3 genes d'avirulence. Deux banques cosmidiques, celle realisee avec l'isolat guy 11, et celle du descendant 96/0/76 construite lors de cette etude ont ete criblees par hybridation ou par rapd-based screening. Cette derniere methode consiste a identifier par une analyse rapd, le cosmide contenant le marqueur lie au gene d'avirulence. La caracterisation des cosmides, a l'image de celle des marqueurs rapd lies, revele une abondance de sequences repetees dispersees nouvelles et deja caracterisees (mgr 583, mgr768 et pot2). Les cosmides d31c12 et g08e2 situes au niveau du locus avrirat7-1 transforment l'isolat virulent 2/0/3 en un isolat avirulent. Ils contiennent le gene d'avirulence avrirat7-1. Le cosmide d31c12 a ete aussi utilise pour transformer l'isolat ph19. Les transformants ph19 et 2/0/3 du cosmide ont ete inocules sur des cultivars et lignees isogeniques de riz. Le gene de resistance interagissant avec avrirat7-1 est present dans la lignee isogenique c101lac et dans les cultivars dj8-341, carreon, ir1529, bw100, ir64. Ce dernier cultivar a ete utilise en croisement avec le cultivar sensible azucena pour construire une carte genetique du riz. Il est donc possible d'envisager la cartographier, puis le clonage positionnel du gene de resistance interagissant avec avrirat7-1. Le cosmide d31c12 a ete sous clone en quatre fragments chevauchants, dans le but d'identifier les sequences necessaires a l'expression du gene d'avirulence. Les trois sous clones testes ne complementent pas l'isolat avirulent 2/0/3. Le gene serait donc probablement localise dans le quatrieme sous clone bgli / noti de 6,5 kb. Ce sous clone a deja ete introduit dans l'isolat virulent 2/0/3 et les transformants obtenus sont actuellement testes en inoculation sur le cultivar de riz irat7. L'identification du sous clone portant avrirat7-1, puis de la sequence de ce gene permettra de rechercher de la fonction de ce gene.

En agriculture, les ravages causés par les champignons pathogènes sont les plus répandus et les plus dommageables. Au cours du développement parasite, ils présentent un besoin en méthionine et cystéine. Notre étude vise à mieux comprendre le rôle clé de la méthionine dans la physiologie du champignon phytopathogène Magnaporthe grisea. Nous sous sommes focalisés sur deux mutants de délétion obtenus par remplacement de gène : le mutant du gène codant pour la méthionine synthase (mutant ΔMS) et celui du facteur de transcription MgMetR (mutant ΔMgMetR). Une étude globale a été entreprise pour chacun des mutants en utilisant les techniques de transcriptome et de métabolome. Les signatures métaboliques des mutants ont été déterminées par HPLC. Le mutant ΔMS est caractérisé par une accumulation d’homocystéine et de SAM et le mutant ΔMgMetR présente une réduction drastique de son taux de glutathion. L’analyse en transcriptome du mutant ΔMS montre un nombre important de gènes différentiellement exprimés. Plusieurs voies métaboliques se sont vues affectées dans le mutant ΔMS : le métabolisme des folates, la biosynthèse des acides aminés et l’homéostasie du fer ont fait l’objet d’une étude particulière. Les premières analyses bioinformatiques du transcriptome du mutant ΔMgMetR ont confirmé le rôle de MgMetR dans le contrôle de l’expression des gènes de la voie d’assimilation du sulfate. L’ensemble de ces résultats a permis de mettre en évidence de nouvelles connections métaboliques et ainsi, facilite la compréhension du métabolisme du champignon phytopathogène M. grisea afin de trouver de nouvelles voies d’étude pour la protection des cultures
Filamentous fungi cause devastating diseases in agricultural crops. Recent studies highlighted a need for methionine and cysteine during the infectious process. Our goal is to understand the role of methionine biosynthesis in the plant pathogenic model Magnaporthe grisea. To this end, we focused our study on two null mutants for the genes encoding methionine synthase which catalyzes the last step for methionine biosynthesis (ΔMS), and the sulphur regulator MgMetR which controls sulphate assimilation pathway (ΔMgMetR). A global analysis was carried out for each mutant by using transcriptomic and targeted metabolomic approaches. Metabolic signatures were determined by HPLC. ΔMS mutant was characterized by an increase in homocysteine and S-adenosylmethionine levels whereas ΔMgMetR mutant displayed a drastic reduction in glutathione content. Microarray analyses of ΔMS mutant highlighted an important molecular perturbation since 507 to 1565 genes were differentially expressed in ΔMS mutant compared to the wild type strain depending on our experimental conditions (from starvation to excess of methionine). Our analyses focused on different metabolic pathways: folate metabolism, amino acid biosynthesis and iron homeostasis which were subject to molecular and biochemical validations. Microarray analyses of ΔMgMetR mutant confirmed the transcriptional control of the expression of the genes involved in sulphate acquisition and assimilation. The set of results obtained during this work gets insight into new metabolic interplays between methionine biosynthesis and M. grisea general metabolism and reveals new research areas for antifungal molecules with the aim of crop protection

La perception de l’environnement extérieur est importante pour des interactions efficaces entre plantes et champignons. Chez les champignons filamenteux, l’information associée au pH extracellulaire est transmise via une voie de signalisation conservée et impliquant sept protéines. Parmi ces protéines, la protéine transmembranaire PalH est un récepteur présumé initier la réponse aux pH neutre ou alcalin. Le facteur de transcription PacC, présent sous forme inactive dans le cytoplasme de la cellule fongique, est clivé en réponse à l’activation de la voie, et migre dans le noyau où il active la transcription des « gènes alcalins » et réprime la transcription des « gènes acides ». Chez Magnaporthe grisea, un Ascomycète responsable de la principale maladie du riz, le rôle de cette voie dans la physiologie du champignon est encore inconnu. Les deux homologues des gènes PACC et PALH ont été identifiés. Dans le but d’analyser le rôle des deux protéines PacC et PalH chez M. grisea, la délétion de ces gènes a été réalisée. Plusieurs phénotypes ont été analysés chez les deux souches mutantes, notamment la croissance, la sporulation et le pouvoir infectieux. Ceci a permis d’étudier le rôle de la signalisation liée au pH extracellulaire dans le cycle de développement de M. grisea. De plus, une analyse du profil des gènes exprimés chez le mutant ΔpacC a été initiée. Les résultats obtenus indiquent que cette voie est importante pour l’adaptation du champignon à un environnement alcalin et qu’elle joue un rôle dans la pathogénicité du champignon
Perception of external environment is important for successful infection of plants by fungi. In these organisms, the information about extracellular pH is provided to the cell by a conserved signalling pathway that involves seven proteins. Among these proteins, the transmembrane protein PalH is the putative receptor which would initiate the pH response. The transcription factor PacC, existing in an inactive form in the fungus cell cytoplasm, is activated through proteolysis in response to the pathway activation, and migrates into the nucleus where it activates the « alkaline » genes transcription and represses that of the « acidic » genes. In Magnaporthe grisea , an ascomycete responsible for the main rice disease, the role of this pathway is still unknown. In this work, the PACC and PALH genes have been identified. In order to analyse the role of the two corresponding proteins PacC et PalH, the deletion of these two genes has then been performed. Several phenotypes were studied in the two mutant strains, including growth rate, conidiation and ability to infect host plants. This enabled the investigation of the involvement of the pH signalling pathway in the M. grisea development cycle. Furthermore, a gene expression profiling analysis of the ΔpacC mutant has been undertaken and revealed the multiple cellular responses to pH changes. Taken all together, the results collected in this work indicate that the pH signalling pathway is important for M. grisea's adaptation to an alkaline environment and that it plays a significant role in the fungus pathogenicity