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.

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

Magnaporthe oryzae est un champignon filamenteux responsable de la principale maladie du riz, la pyriculariose. Ce pathosystème est très étudié, notamment dans le but de contribuer à l’identification de facteurs pouvant permettre le développement de résistances efficaces. Si certaines hormones végétales, comme l’acide salycilique, sont requises pour la mise en place des défenses de la plante, d’autres sont impliquées dans des processus développementaux. Parmi elles, les cytokinines (CKs) sont des dérivés d’adénine décrites pour participer à la croissance et la différenciation de l’appareil aérien et racinaire. Elles contribuent à la répartition des nutriments et impactent également la viabilité des cellules, en retardant la senescence ou en induisant la mort cellulaire. Des études précédentes ont montré que les CKs pouvaient perturber la résistance de la plante hôte dans différents pathosystèmes. Chez le riz, les CKs agissent en synergie avec l’acide salicylique pour induire l’expression des gènes marqueurs des défenses. Cependant aucun phénotype de résistance associé aux CKs n’a été observé in planta. Mes travaux montrent qu’un apport exogène de CKs (kinétine, BAP) affecte la résistance du riz à Magnaporthe avant infection, de manière dose dépendante. Le phénotype de résistance observé est corrélé avec une plus forte expression des défenses pendant infection, limitant la pénétration et l’invasion du champignon. Des plantes de riz mutées pour une probable cytokinine UDP-glucosyl transferase (CK-UGT) ont été obtenues. Ces mutants ck-ugt sont affectés dans le métabolisme des CKs et sont également plus résistants à M. oryzae. Hors infection, une plus forte expression des gènes de défense a été mesurée chez les plantes mutantes, confirmant que les CKs endogènes affectent directement ou indirectement les défenses de l’hôte. En parallèle de ces analyses sur la plante, mes travaux ont aussi porté sur le rôle des CKs produites par M.oryzae. En effet, leur rôle dans l’interaction ainsi que la voie de leur biosynthèse chez le champignon n’était pas caractérisé. Conservées au sein des différents organismes, les tRNA-IPT (isopentenyl transferase) sont décrites pour participer à la biosynthèse de CKs. Un seul gène homologue a été identifié chez M. oryzae et nommé CKS1 car sa délétion abolit la production de CKs. Le mutant de Magnaporthe cks1 est moins virulent (pénétration et invasion in planta réduites) que la souche témoin complémentée. Il induit une plus forte accumulation des espèces actives de l’oxygène et une plus forte expression des défenses chez la plante. Les dosages des acides aminés et des sucres pendant infection ont montré que les concentrations de ces nutriments étaient différemment perturbées par la souche déficiente en CKs. Ces résultats suggèrent que les CKs fongiques pourraient être requises pour affecter la répartition des acides aminés et contribuer une accumulation progressive de sucres au cours de l’infection. Ainsi, chez un champignon qui n’induit pas de tumeurs, les CKs pourraient agir comme des effecteurs qui auraient une double fonction d’inhibition des défenses et de drainage des nutriments. Chez les champignons, ces hormones induisent également des réponses physiologiques comme la résistance à certains stress, les processus de nutrition et la reproduction sexuée. Ces effets ont été étudiés chez Magnaporthe dans différentes conditions de croissance in vitro plus ou moins stressantes. Les résultats indiquent que les CKs augmentent la tolérance au stress osmotique et oxydatif et suggèrent qu’elles affecteraient aussi l’absorption des nutriments ainsi que la reproduction sexuée. Comme le gène CKS1 est conservé, cette mutation peut être caractérisée chez d’autres organismes fongiques présentant des modes de vie différents de manière à mieux comprendre le rôle de ces hormones dans les interactions plante-microorganisme mais également au sein des interactions microbiennes. / The blast disease caused by Magnaporthe oryzae is one of the most devastating diseases on rice leading to important yield loss. Plant hormones, like salicylic acid, play a central role in plant resistance establishment. Among these hormones, cytokinins (CKs) are adenine derivatives well described to modulate root/shoot growth and differentiation, cell viability and nutrient distribution. Previous studies have shown that these hormonal compounds can also affect plant host resistance in different pathosystems involving monocot or dicot host plants and microbes (bacteria, oomycetes or fungi). In rice, CKs were described to act synergistically with the salicylic acid pathway to induce defense marker genes expression. However, no resistance phenotype associated with CKs was observed and the way that CKs could act in planta during infection is still unknown. In this work, a resistance phenotype induced by exogenous application of the CK kinetin was characterized and the role of endogenous CKs in rice resistance was investigated by phenotyping plant CK mutants. An exogenous supply of kinetin before infection led to a higher induction of defense marker genes that was associated with limited fungal penetration and invasion, suggesting. However the way CKs affected resistance or susceptibility (or virulence see below) depended on the timing at which they were applied (before or after inoculation). Rice lines mutated for a putative cytokinin- UDP-glycosyl transferase (CK-UGT) were produced. The ck-ugt mutants were more resistant, suggesting that endogenous CKs can also contribute to resistance. Defense marker genes were expressed higher in the absence of infection in the ck-ugt rice mutants, compared to the WT plants. In parallel of these analyses of CK on the plant side, we studied the possible role of CK produced by Magnaporthe. Indeed M.oryzae produces and secretes CKs. However, the way fungal CKs are involved in the rice blast disease development as well as the biosynthesis pathway in M.oryzae were not established. A putative tRNA-IPT (isopentenyl transferase) conserved across organisms was identified in M.oryzae. Mutant analysis of this gene confirmed that this enzyme, thus named CKS1, is required for CK production. Knock-out cks1 fungal mutants were less virulent on rice, affected in penetration and invasion compared to the control complemented strain. They triggered a stronger accumulation of reactive oxygen species and a higher expression of defense marker genes. Aspartate and glutamate, two amino acids important for M.oryzae growth, were differently affected at and around the infected zone by cks1 strain suggesting that fungal CKs could contribute to drain/consume nutrients during infection. Similarly, sugar accumulation was also differently disturbed, indicating that fungal-derived CKs may be required for maintaining a progressive sugar production during host invasion, probably by affecting photosynthesis process. Our results show that fungal CKs, in a non-gall forming fungal pathogen, could act as dual effectors by inhibiting defense and modifying nutrient fluxes. Furthermore, CKs are known to affect some physiological processes in fungi, like stress resistance, nutrition or sexual reproduction. In order to test whether CKs modulate Magnaporthe stress tolerance, the effect of CKs on the mycelial growth in different stressful conditions in vitro was tested. The results indicate that CKs increased osmotic and oxidative stress tolerance and suggest that they also affected nutrient acquisition as well as sexual reproduction. Since the CKS1 gene is highly conserved, the effect of the cks1 mutation could be studied in other fungi showing different lifestyles for improving our knowledge on the role these hormonal compounds play among microbes or in plant-microbe interactions.

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 Ascomycete fungus Magnaporthe oryzae is the causative agent of Rice Blast, and has become the predominant model organism for study of fungal phytopathogens and other appressorium-forming fungi. I identified and attempted to elucidate the function of the six aquaporin genes in the M. oryzae genome. These small membrane channels have been implicated in the transport of water, glycerol, and a variety of other small molecules. We speculated that these functions might be important in the formation of the appressorium – a specialised infection structure that relies on the generation of turgor. The role of these proteins in general fungal biology is poorly understood, and this project endeavoured to correct this gap. A phylogenetic analysis of aquaporins in extant fungi revealed the significant expansion of this gene family in Pezizomycete and Basidiomycete fungi, with up to 17 genes in some species. I characterised the expression pattern of the six identified aquaporins in the M. oryzae genome during pathogenic development and found there to be substantial up-regulation co-incident with appressorial turgor generation. Single knock-outs of each aquaporin gene were fully pathogenic, with normal infection-related development and axenic growth. I speculated that the absence of a strong phenotype may have been caused by functional redundancy, and used qRT-PCR investigate the incidence of transcriptional up-regulation of remaining family members in the knock-out backgrounds. This uncovered a putative reciprocal relationship between two genes MoAQP1 and MoAQP2, suggesting that deletion of one results in up-regulation of the other. I subsequently tested the substrate permeability of this gene pair and found them to be permeable to hydrogen peroxide. However, deletion of one of these genes did not appear to affect the rate of flux of hydrogen peroxide across the plasma membrane. My attempt to characterise the cellular localisation of these proteins using GFP fusions was largely unsuccessful with the exception of a single gene, MoAGP2, which appears to be located on the endoplasmic reticulum.

Master of Science / Department of Plant Pathology / Erick D. DeWolf / Wheat blast, caused by Magnaporthe oryzae pathotype triticum, has emerged as a serious problem for wheat production in South America and recently emerged as a threat to wheat production in Bangladesh. To prepare for the possible introduction of wheat blast in to the United States, it would be helpful to identify areas of the country most at risk for blast epidemics. Because wheat blast occurs primarily in tropical and subtropical regions of the world, cold winter temperatures may restrict the establishment of the blast pathogen in the United States. Therefore, the first objective of this research was to quantify the freeze-thaw tolerance of the wheat blast pathogen in naturally infected wheat rachises from Bolivia and to measure the viability of the conidia after exposure to various treatments. The results indicate that exposing the fungus in moist residue to multiple freeze-thaw cycles is more damaging than exposing the fungus in moist residue to longer, single freezes. When in dry residue, the fungus was not harmed by the freeze-thaw cycles. Freezing and thawing of the wheat blast fungus in moist residue significantly affected its ability to produce viable conidia. The second objective of this research was to identify environmental conditions that could be conducive for wheat blast epidemics by examining historical epidemics of rice blast, caused by Magnaporthe oryzae pathotype oryza. The dataset used in this analysis consisted of 60 site-years of historical observations of rice blast levels from Arkansas, Louisiana, and Texas. These observations were coupled with monthly and weekly summaries of hourly weather variables based on temperature, relative humidity, precipitation, and regional moisture indices. Classification trees and logistic regression were used to identify variables associated with rice blast epidemics. The results indicate that rice blast epidemics are favored by cooler April temperatures and higher levels of precipitation in June. Preliminary models for rice blast based on these variables were able to correctly classify epidemic years with >75% accuracy. In the future, the results of this project will be used as part of a risk assessment for a wheat blast introduction and establishment in the United States.

The ascomycete fungus Magnaporthe oryzae, causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and M. oryzae is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made M. oryzae an ideal model system for understanding general plant-pathogen interactions as well. The objective of this dissertation was to characterize the M. oryzae homologs of Histoplasma capsulatum RYP (Required for Yeast Phase) genes that are required for transition to the parasitic phase. H. capsulatum is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37°C, the host body temperature. Four H. capsulatum RYP genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. RYP1 is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. RYP2 and RYP3 are homologs of vosA and velB, members of the Velvet family, best characterized in Aspergillus nidulans, where they coordinate morphological differentiation with secondary metabolism. RYP4 is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the M. oryzae RYP1 homolog, RIG1 (Required for Infectious Growth), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. Δrig1 was blocked in the transition to infectious hyphal growth, similar to H. capsulatum ryp1, which could not transition to the yeast phase. Deletion mutants of M. oryzae RYP2, RYP3, and RYP4 homologs were similar to the wild type in somatic growth and pathogenicity indicating that although RIG1 is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the RYP pathogenicity genes identified in H. capsulatum. Δrig1 is the first M. oryzae mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.

An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity.

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.

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