Fusarium graminearum est un champignon phytopathogène responsable de la fusariose de l’épi, maladie affectant les céréales telles que le blé ou le maïs. Le champignon peut également produire des métabolites secondaires toxiques ou mycotoxines, tels que les trichothécènes de type B qui sont stables d’un point de vue chimique et thermique. Ces mycotoxines peuvent contaminer les grains avant récolte. Aucun procédé à l’heure actuelle ne permet de les éliminer ou de limiter leur toxicité. Un moyen de lutte efficace est donc de limiter la biosynthèse de ces toxines. Cette voie implique les gènes Tri qui sont regroupés dans un cluster dont la régulation est complexe. Lors de l’infection de la plante hôte, le champignon se retrouve potentiellement en contact avec des molécules pro ou antioxydantes intervenant dans les mécanismes de défense de la plante. In vitro, ces molécules prooxydantesactivent la production de trichothécènes via la surexpression des gènes Tri alors que les composés antioxydants la répriment, via la répression des gènes Tri.Le facteur de transcription Fgap1, homologue au facteur Yap1 de levure impliqué dans la réponse austress oxydant, a été identifié chez F. graminearum et son rôle potentiel dans la régulation de labiosynthèse des trichothecenes a été étudié. Des souches recombinantes ont été construites et ontpermis de montrer l’implication de ce facteur non seulement dans l’activation de l’expression des gènes de détoxification, mais aussi dans la modulation de la production de trichothécènes en réponseau stress oxydant. Ce facteur n’intervient cependant pas dans la réponse aux composés antioxydants.Une analyse RNA-seq a été entreprise pour identifier plus globalement les réseaux de régulation impliqués en réponse aux variations redox. / Fusarium graminearum is a pathogenic fungus responsible for “Fusarium Head Blight”, a diseaseaffecting cereals, including wheat or maize. The fungus can produce toxic secondary metabolitesbelonging to the type B trichothecene family. These metabolites are heat and chemically stablemolecules. These toxins can be found in grains before harvest and no available decontaminationprocess can eliminate or detoxify trichothecenes. The best way to restrict their occurrence is to limittheir biosynthesis. The Tri genes involved in the biosynthetic pathway are clustered and theirregulation is complex. During plant/pathogen interaction, the fungus must cope with pro orantioxidants, involved in plant defense mechanisms. In vitro, prooxidant molecules stimulatetrichothecenes biosynthesis via Tri genes overexpression, whereas antioxidants compounds repress Trigenes expression and toxin accumulation.The transcription factor Fgap1, homologous to Yap1 in yeast and involved in response to oxidativestress, was identified in F. graminearum and its potential role in the regulation of trichothecenesbiosynthesis was investigated. Using recombinant strains, we demonstrated that, in response tooxidative stress, Fgap1 is not only involved in expression of detoxifying activities, but also in themodulation of trichothecene accumulation. Nonetheless, Fgap1 is not involved in response toantioxidant compounds. RNA-seq analysis has been initiated to identify regulatory network involvedin response to redox variations.

Made available in DSpace on 2017-07-10T17:37:39Z (GMT). No. of bitstreams: 1 Rafael_Hansen_Alves.pdf: 691556 bytes, checksum: 76623d639903f7b4404dca73bdfe4314 (MD5) Previous issue date: 2010-06-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Caused by several species of fungus, fusarium head blight (FHB) is a disease of great economic importance in wheat (Triticum aestivum L.), causing large losses in world grain production. In Brazil the main species of fungi responsible for disease is Gibberela zeae (Schwabe.), and its the anamorphic Fusarium graminearum (Schwabe). The control by chemical treatment of the disease with the use of fungicides has low efficiency due to the difficulty of identifying the exact time of application. Therefore, the use of resistant varieties is shown as a safe and economically viable alternative for the production of wheat scab-free. To contribute to the understanding of the behavior of some Brazilian wheat cultivars for FHB, the objectives were to assess the presence or absence of reaction to FHB resistance in wheat cultivars under inoculation in field conditions and greenhouse; and identify what kind of reaction that is acting (resistance type I or type II resistance). The proposed work was developed in the field, greenhouse and laboratory. Two methodologies inoculation were applied, depending on the type of resistance to be tested, and the targets of this work were to evaluate the resistance type I and type II resistance. Twenty eight genotypes with different responses to FHB resistance were used to both evaluations. The resistance of type I was measured by spraying the spikes with suspension. In the field, dispersal of inoculum in the area was used wheat grains colonized with perithecia previously inoculated. The assessment of disease severity in the 28 cultivars was made 21 days after inoculation (DAI), the green ear stage, taking on a note on a linear scale from zero to 100, based on the scale suggested by Stack and McMullen. The evaluation of resistance of type II was performed only in the greenhouse. Obtaining spore mass acquisition was performed by scraping the plates with PDA medium colonized by Fusarium graminearum. Conidia were washed with distilled water and received the inoculum. The concentration was adjusted to 5x104 spores per mL. The count was performed by an optical microscope with the aid of a Neubauer chamber. About 10μL of the suspension of spores (macroconidia) at a concentration of 5x104 mL-1 was injected between the lemma and palea of a spikelet located at the point of infection, on both sides of the ear, using a hypodermic syringe. Evaluations were performed at 7, 14 and 21 days after inoculation by counting the spikelets with symptoms of the disease, excluding the two infected spikelets. The FHB occurred in two periods in the field with greater variation among the 28 cultivars tested in the first season. For resistance type I, nine and nineteen cultivars had the lowest severity, respectively, in field and greenhouse conditions, showing that they are sources of resistance. In accessing the resistance of type II, BRS Umbu, CEP Nova Era, CEP Raizes, BRS 208 e Frontana not proved to be sources of resistance. Already BRS Guamirim, CD 120, Onix, Rubi, CEP 50, BRS 179, Pampeano, Abalone, CD 114, IPR 85, Safira, BRS Louro, CD 117, CDF 2002116, CD 115, BRS177, CD 0529 and BRS Camboim showed the lowest values for AUDPC thus demonstrating the potential for type II resistance to FHB / Causada por diversas espécies de fungo, a giberela e uma doença que possui grande importância econômica na cultura do trigo (Triticum aestivum L.), ocasionando perdas sensíveis na produção mundial de grãos. No Brasil a principal espécie de fungo responsável pela moléstia e Gibberela zeae (Schwabe.), sendo sua forma anamófica Fusarium graminearum (Schwabe). O controle através de tratamento químico da doença com o uso de fungicidas apresenta baixa eficiência devido a dificuldade de identificação do momento exato da aplicação. Portanto, a utilização de variedades resistentes mostra-se como uma alternativa segura e viável economicamente para a produção de trigo livre de giberela. Visando contribuir para o conhecimento do comportamento de algumas cultivares brasileiras de trigo em relação a giberela, os objetivos deste trabalho foram avaliar a presença ou ausência da reação de resistência a giberela em cultivares de trigo, sob inoculação em condições de campo e casa de vegetação; e identificar qual e o tipo de reação que esta atuando (resistência tipo I ou resistência tipo II). O trabalho proposto foi desenvolvido a campo, casa de vegetação e em laboratório. Foram utilizadas duas metodologias de inoculação, conforme o tipo de resistência a ser testada, sendo que, foram alvo do presente trabalho a avaliação da resistência tipo I e resistência tipo II. Para ambas as avaliações foram utilizadas 28 genótipos de trigo com diferentes reações de resistência a giberela. A resistência do tipo I foi medida através da inoculação de plantas em casa de vegetação, através de borrifamento das espigas, com suspensão de esporos. No campo, para dispersão do inoculo na área foi utilizado grãos de trigo colonizados com peritécios previamente inoculados. A avaliação da severidade da doença nos 28 cultivares foi feita 21 dias apos a inoculação (DAI), no estádio de espiga verde, atribuindo-se uma nota em uma escala linear de zero a 100, baseada na escala sugerida por Stack e McMullen. A avaliação da resistência do tipo II foi realizada somente em casa de vegetação. A obtenção de esporos em massa foi realizada através da raspagem de placas com meio BDA colonizadas por Fusarium graminearum. Os conídios foram lavados com água destilada e obtido o inoculo. A concentração foi ajustada para 5x104 esporos por mL. A contagem foi realizada em microscópio óptico com o auxilio da Câmara de Neubauer. Aproximadamente 10μL da suspensão de esporos (macrogonídios) na concentração de 5x104 mL-1 foi injetada entre a lema e a palea de uma espigueta localizada no ponto de infecção, nos dois lados da espiga, utilizando uma seringa hipodérmica. As avaliações foram realizadas aos 7, 14 e 21 dias apos a inoculação, contando-se as espiguetas com sintomas da doença, excluindo as duas espiguetas que foram inoculadas. A giberela ocorreu nas duas épocas a campo com maior variação entre as 28 cultivares testadas na primeira época. Para a resistência do tipo I nove e dezenove cultivares obtiveram os menores valores de severidade, respectivamente, em campo e em casa de vegetação, demonstrando serem fontes de resistência. Na avaliação da resistência do tipo II, as cultivares BRS Umbu, CEP Nova Era, CEP Raízes, BRS 208 e Frontana não demonstraram serem fontes de resistência. Já as cultivares BRS Guamirim, CD 120, Onix, Rubi, CEP 50, BRS 179, Pampeano, Abalone, CD 114, IPR 85, Safira, BRS Louro, CD 117, CDF 2002116, CD 115, BRS 177, CD 0529 e BRS Camboim apresentaram os menores valores para AACPD demonstrando assim, potencial para a resistência do tipo II a giberela

Dentre os desafios do cultivo de trigo, encontram-se as doenças fúngicas e a presença de micotoxinas. Estima-se que as perdas anuais nesse setor sejam bilionárias. Além dos prejuízos econômicos, há preocupações quanto a segurança alimentar humana e animal. Com o objetivo de avaliar a micobiota e a ocorrência de micotoxinas em diferentes estádios de maturação do trigo foram realizados estudos morfológicos, utilizando métodos morfológicos clássicos e moleculares, identificação do perfil genotípico dos fungos produtores de tricotecenos, por PCR em tempo real e determinação de deoxinivalenol (DON) e zearalenona (ZEA), utilizando a Cromatografia Líquida de Alta Eficiência acoplada à Espectrometria de Massas (LC-MS/MS). Os dados obtidos também foram correlacionados com os fatores climatológicos. Os resultados revelaram a predominância do gênero Fusarium nas amostras das duas safras, seguido de Alternaria e Epicoccum. A atividade de água média para os dois anos foram, 0,608 e 0,886, consecutivamente. Dentro do gênero Fusarium constatou-se predomínio do complexo de espécies Fusarium graminearum (CEFG), identificando F. graminearum sensu stricto, F. meridionale, F. cortaderiae e F. austroamericanum. Os genótipos dos isolados foram, na sua maioria, 15-ADON para F. graminearum s.s, seguido de NIV para as espécies F. meridionale, F. cortaderiae e F. austroamericanum e 3-ADON para as duas últimas. Por PCR em tempo real, a determinação do genótipo nos grãos de trigo nas safras 2014/15 e 2015/16 demonstraram predomínio de 100% e 93% para 15-ADON, respectivamente. A quantificação de DNA dos isolados do CEFG demostrou que o perfil 15-ADON foi responsável por mais de 95% nas duas safras, com valores irrisórios, menores que 3% para 3-ADON e NIV. Os níveis de contaminação pela micotoxina DON nas amostras foi de 100% nas duas safras, com valores variando entre 24,3 a 5530 &#956g/kg. Para ZEA os valores foram 30% e 96%, nas duas coletas consecutivas, variação entre 26,0 a 4360 &#956g/kg. Ambas as micotoxinas foram detectadas em concentrações acima do limite estabelecido pela legislação brasileira, 3000 &#956g/kg para DON e 400 &#956g/kg para ZEA. Os fatores climáticos obtiveram uma correlação positiva tanto na contaminação fúngica quanto no acúmulo de micotoxinas. Este estudo forneceu novas informações sobre a micobiota natural do trigo brasileiro, em diferentes etapas do cultivo, demonstrando a contaminação por DON e alta frequência de ZEA em amostras do provindas do Paraná. / The fungal diseases and the presence of mycotoxins in cereals cause annually, billionaire losses. In addition, there are concerns about human and animal food safety. Due to the wheat culture relevance in feed and foodstuff, the present work aimed to evaluate mycobiota and the occurrence of mycotoxins in different wheat maturation stages. In order to achieve these objectives, morphologic studies using classic and molecular methods, genotype profile and DNA quantification from trichothecene producers, by real-time PCR and determination of deoxynivalenol (DON) and zearalenone (ZEA), using High Performance Liquid Chromatography coupled to Mass Spectrometry (LC-MS/MS), were done. The obtained data were also correlated with the climatic factors. The results showed the predominance of the genus Fusarium followed by Alternaria and Epicoccum. The average water activity over two years were 0.608 and 0.886, consecutively. Within the genus Fusarium, it was identified a predominance of the Fusarium graminearum species complex (FGSC), identifying F. graminearum sensu stricto, F. meridionale, F. cortaderiae and F. austroamericanum. The genotypes of the isolates were 15-ADON for F. graminearum s.s, followed by NIV for the F. meridionale, F. cortaderiae and F. austroamericanum and 3-ADON for F. cortaderiae and F. austroamericanum. In real-time PCR, the determination of genotype in wheat grains in the 2014/15 and 2015/16 harvests showed a predominance of 100% and 93% for 15-ADON, respectively. The DNA quantification of the FGSC isolates showed that the 15-ADON profile was responsible for more than 95% in both harvests. The levels of DON mycotoxin contamination in the samples were 100%, with values ranging from 24.3 to 5530 &#956g/kg. For ZEA the values were 30% and 96%, in the two consecutive collections, ranging from 26,0 to 4360 &#956g/kg. Both mycotoxins were detected in concentrations above the limit established by Brazilian legislation, 3000 &#956g / kg for DON and 400 &#956g/kg for ZEA. The climatic factors obtained a positive correlation both in the fungal contamination and in the accumulation of mycotoxins. This study provided new information on the Brazilian native mycobiota to different stages of cultivation, demonstrating contamination by DON and high frequency of ZEA in samples from Paraná.

Pour lutter contre les nuisibles des cultures (herbivores et pathogènes), de nouvelles voies ont été explorées et en particulier la lutte biologique. Utilisé la nature et ses organismes pour réguler les populations de pathogènes, tel est le but de la lutte biologique aussi appelé biocontrôle. Parmi les organismes couramment retrouvés dans le biocontrôle se trouve les micro-organismes, que cela soit des champignons ou des bactéries. Ces micro-organismes possèdent des capacités très intéressante pour des agriculteurs et agronomes. En effet, des études ont montré qu’ils étaient capables d’interagir avec les plantes pour permettre une meilleure croissance et santé de celle-ci. Alors, dans cette thèse, nous nous sommes intéressés aux bactéries pour trouver des méthodes de luttes alternatives à Fusarium graminearum, un pathogène des céréales responsables de fortes pertes de rendements, en particulier grâce à la production de mycotoxines qui va rendre les grains impropres à la consommation humaine et animales. Par l’intermédiaire d’une approche original consistant à garder des souches non-inhibitrice in vitro pour des tests in planta, nous avons pu montrer les limites de la sélection in vitro. Grâce à l’exploration métaboliques d’une souche particulièrement efficace, nous avons aussi pu mettre en évidence de potentiels nouvelles molécules antifongiques. Par l’intermédiaire d’une souche modèle, nous avons explorer les mécanismes de la mise en place d’une résistance systémique chez le blé induite par les bactéries. Et enfin, nous avons exploré le potentiel des combinaisons de bactéries dans la protection du blé qui semble représenter un vrai futur dans le monde du biocontrôle. Les travaux effectués s’inscrivent dans les besoins de nouvelles ressources pour limiter l’utilisation des pesticides, mais aussi dans un besoin de mieux comprendre les interactions tripartite entre blé, pathogène et bactéries bénéfiques / To combat pests of crops (herbivores and pathogens) new pathways have been explored, in particular biological control. Used the nature and its organisms to regulate the populations of pathogens, that is the goal of the biological fight also called biocontrol. Among the organisms commonly found in biocontrol are micro-organisms, be they fungi or bacteria. These microorganisms have very interesting capabilities for farmers and agronomists. Indeed, studies have shown that they are able to interact with plants to allow a better growth and health of it.Then, in this thesis, we were interested in bacteria to find alternative methods of struggle with Fusarium graminearum, a pathogen of cereals responsible for high yield losses, especially thanks to the production of mycotoxins that will make the seeds unfit for human and animal consumption.Through an original approach of keeping noninhibitory strains in vitro for in planta tests, we have been able to show the limitations of in vitro selection. Thanks to the metabolic exploration of a particularly efficient strain, we have also been able to highlight potential new antifungal molecules. Through a model strain, we explored the mechanisms of the establishment of systemic resistance in wheat induced by bacteria. And finally, we have explored the potential of bacterial combinations in wheat protection that seems to represent a real future in the world of biocontrol.The work done is in line with the needs of new resources to limit the use of pesticides, but also in a need to better understand the tripartite interactions between wheat, pathogen and beneficial bacteria

Made available in DSpace on 2016-12-08T16:44:32Z (GMT). No. of bitstreams: 1 PGPV07MA023.pdf: 3345243 bytes, checksum: 4a8cf32319e030bf77cdd4320f251d27 (MD5) Previous issue date: 2007-06-22 / The fungi Fusarium graminearum (teleomorph Gibberella zeae) is the etiological agent of scab wheat, one of the most important cereal s winter diseases in Brazil. Outbreaks are generally sporadic but in the last few years is frequently reported an increase in disease intensity in almost all the wheat producing areas around the world. Some studies demonstrate a high genetic diversity in F. graminearum from different geographic areas, as well as in isolates at the same locality. In filamentous fungi, as the F. graminearum, one of the main mutation s causes is the transposable elements or transposons which are capable to generate different types of mutations. In some cases, these mutations are involved with the resistance break, an important phenomenon for the occurrence of epidemics. The objectives of this study were to detect putative transposable elements sequences in the F. graminearum genome, as well as to develop and to analyze adequate procedures for co-transformation of brazilin isolates of this fungi with the vectors pNI160, which carry the transposon impala and pAN7.1, which code to hygromycin B resistance. To detect putative sequences of transposable elements in the F. graminearum genome, specifics oligonucleotides were constructed and 14 isolates, originated from the States of Rio Grande do Sul, Paraná and São Paulo were used. A total of 10 oligonucleotides pairs were constructed (6 oligonucleotides pairs were specific to transposase, 3 to transcriptase reverse and 1 to gene that code for GAG protein). Considering these 10 oligonucleotides pairs, the one that would amplify a transcriptase reverse region similar to a Magnaporthe grisea reverse transcriptase did not amplify any fragment in the isolates total DNA, and the pair that would amplify a transposase region similar to Metarhizium anisopliae originated many fragments of different sizes that do not show relation with 683 bp expected size fragment. Considering the eight oligonucleotides pairs remained, five of them amplified the expected fragments for transposase (715 bp region similar to F. oxysporum f. sp. ciceris transposase, 306 bp similar to Aspergillus awamori transposase, 556 bp similar to A. niger tansposase, 339 bp similar to Arabdopsis thaliana transposase and 554 bp similar to Cochliobolus carbonum transposase), two of them amplified the transcriptase reverse fragments (161 bp similar to A. thaliana reverse transcriptase and the 752 bp similar to Caenorhabditis elegans reverse transcriptase) and one oligonucleotide pair amplified an 581 bp expected size fragment similar to F. oxysporum gag gene. The amplification occurred, even for different oligonucleotides pairs, in all 14 analyzed isolates confirming the presence of transposable elements putative sequences in Brazilian isolates of F. graminearum. This also shows a diversity of groups of these putative elements, considering that some of the sequence amplified is characteristic of a transposable element specific group. To develop the genic inactivation protocol, we select the isolates F02 and F12 which are pathogenic for BR18-Terena cultivar. To evaluate the better medium for selection of chlorate resistant mutants, it was analyzed two different medium. This analysis showed that medium describe by Cove (1979) is more indicate for selected the chlorate resistant mutants. A total of 15 9 chorate resistant mutants were obtained, being five identified as nitrate reductase mutants (niaD), eight specific regulator mutants (nirA) and two permease mutants. Differences related to the number of mutants obtained and number inoculated plates, number of nitrate reductase mutants and macroconides production indicate that the choice of the isolate can influence the isolation of mutants and in protoplast production. Analysis with different hygromycin B concentrations revealed that doses up to 30 μg.mL-1 were enough to control the fungal growth. Two nitrate reductase mutants (M01 and M194) were select to protoplastization and co-transformation, once these two mutants in relation to others produce lower concentration of residual mycelium. Only one transformant were obtained and it was denominated T3, what shows that the transformation protocol needs to be modified to increase the number of transformants. If hybridization experiments confirm the transformation of T3 with only one copy of the impala element, it will be confirmed the transposition ability of impala in F. graminearum, and it will be useful in genetic inactivation studies, especially to genes involved in pathogenicity process / O fungo Fusarium graminearum (teleomorfo Gibberella zeae) é o agente etiológico da giberela do trigo, atualmente uma das principais doenças de inverno no Brasil. Epidemias ocorrem esporadicamente, embora nos últimos anos, registraram-se incrementos na intensidade da doença em quase todas as áreas produtoras de trigo no mundo. Vários estudos demonstram uma grande diversidade genética em isolados de F. graminearum de diferentes áreas geográficas, como também em isolados de uma mesma localidade. Em fungos filamentosos como o F. graminearum, uma das principais fontes de mutações capaz de gerar alta variabilidade genética são os elementos transponíveis ou transposons. Essas mutações algumas vezes estão envolvidas com a quebra de resistência, fenômeno importante para o surgimento de epidemias. Os objetivos deste trabalho foram de detectar putativas seqüências de elementos transponíveis no genoma deste fungo, bem como, desenvolver e analisar procedimentos e métodos adequados para a co-transformação de isolados brasileiros deste fungo com o plasmídio pNI160, o qual carrega o transposon impala, e o plasmídio pAN7.1, no qual esta inserido o gene de resistência a higromicina B. Para detectar putativas seqüências de elementos transponíveis no genoma deste patógeno foram construídos oligonucleotídeos específicos para amplificação, via PCR de seqüências características destes elementos transponíveis. Foram utilizados 14 isolados de F. graminearum, provenientes dos Estados do Rio Grande do Sul, Paraná e São Paulo e um total de 10 pares de oligonucleotídeos foram construídos (6 pares para genes que codificam a enzima transposase, 3 pares para genes que codificam a enzima transcriptase reversa e 1 par um gene que codifica a proteína GAG). Dos 10 pares de oligonucleotídeos utilizados, o par que amplificaria uma região de transcriptase reversa similar ao mesmo gene de Magnaporthe grisea não amplificou nenhum fragmento nos isolados utilizados, e o par que amplificaria uma região de transposase similar ao mesmo gene de Metarhizium anisopliae originou muitos fragmentos de diferentes tamanhos não condizentes com o tamanho esperado de 683 pb. Dos oito pares de oligonucleotídeos restantes, cinco amplificaram as regiões esperadas para a transposase (regiões de 715 pb similar a uma transposase de F. oxysporum f. sp. ciceris, 306 pb similar a uma transposase de Aspergillus awamori, 556 pb similar a uma transposase de A. niger, 339 pb similar a uma transposase de Arabidopsis thaliana e 554 pb similar a uma transposase de Cochliobolus carbonum), dois amplificaram as regiões de transcriptase reversa (regiões de 161 pb similar a uma transcriptase reversa de A. thaliana e 752 pb similar a uma transcriptase reversa de Caenorhabditis elegans) e um amplificou um fragmento de tamanho esperado de 581 pb similar ao gene gag de F. oxysporum. A amplificação ocorreu, mesmo que para diferentes pares de oligonucleotídeos, em todos os 14 isolados analisados, confirmando a presença de seqüências putativas de elementos transponíveis em isolados de F. graminearum provenientes de diferentes regiões do Brasil, além de demonstrar uma diversidade de classes de putativos elementos, uma vez que cada seqüência é característica de um determinado grupo de elementos transponíveis. Para o desenvolvimento do protocolo de inativação gênica foram 7 selecionados os solados F02 e F12 patogênicos a cultivar BR18-Terena. Um teste para avaliação do meio de cultura para seleção dos mutantes resistentes ao clorato demonstrou que o meio descrito por Cove (1979) é o mais indicado para os isolados selecionados. Um total de 15 isolados mutantes resistentes ao clorato foi obtido, dos quais 5 foram identificados como mutantes para o gene nitrato redutase (niaD), 8 mutantes para um regulador específico (nirA) e 2 mutantes para permease. Diferenças quanto ao número de mutantes selecionados pelo número de placas inoculadas, número de mutantes nitrato redutase e produção de esporos assexuais, indicam que a escolha do isolado pode influenciar na obtenção de mutantes e na produção de protoplastos. Testes com diferentes concentrações de higromicina revelaram que doses acima de 30 μg. mL-1 são suficientes para o controle do crescimento do fungo. Dois mutantes nitrato redutase (M01 e M194) foram selecionados para a protoplastização e cotransformação, com base na menor produção de micélio residual em relação aos demais. Apenas um transformante foi obtido sendo denominado T3, mostrando a viabilidade do protocolo, mas também, que mais estudos devem ser realizados para se aumentar o número de transformantes. Se confirmada, através de hibridização, a transformação de T3 com uma única cópia do elemento impala, este poderá além de confirmar a capacidade de transposição deste elemento em F. graminearum, também servir em estudos de expressão gênica, principalmente àqueles genes envolvidos no processo de patogenicidade

Dans ce travail, nous avons étudié la contribution que les inhibiteurs de glicosidases ont dans la réponse de défense du blé à Fusarium graminearum. Nous avons démontré que les inhibiteurs de xylanases ont la capacité à la fois de contenir l'activité de dégradation de xylanases sécrétées par l'agent pathogène, et de limiter la possibilité de provoquer nécrose dans les tissus du blé. Nous avons démontré que l’expression de la PvPGIP2 dans la lemme, la paléa, les anthères et le rachis détermine une réduction des symptômes de la fusariose de l’épi, au même niveau de l’expression constitutive de cet inhibiteur. Inversement, l'expression de la PvPGIP2 dans l'endosperme ne détermine pas une réduction des symptômes de la maladie. Cela indique que, lorsque l'agent pathogène a atteint ce tissu, l'activité de polygalacturonases de l'agent pathogène n’est pas indispensable pour la propagation fongique. Enfin, la combinaison des différents inhibiteurs de glicosidases, qui renforcent différentes parties de la paroi cellulaire dans le même génotype, a été efficace pour réduire les symptômes de la fusariose, par rapport aux génotypes qui présentent seulment un type d’inhibiteur. Nous avons démontré cet aspect à travers les plantes qui expriment la PvPGIP2 et le TAXI-III. Au contraire, les génotypes qui expriment la PvPGIP2 et l’AcPMEI n’ont pas montré un effect additif sur la résistance, probablement parce que ils renforcent la même partie de la paroi cellulaire, c’est-à-dire la pectine. / In this work we studied the contribution of glycosidase inhibitors in the defense response of wheat against Fusarium graminearum. We demonstrated that xylanase inhibitors are able to limit both the degrading activity of the xylanases secreted by the pathogen and to limit their ability to induce necrosis in wheat cell suspensions and tissues.We demonstrated that the expression of PvPGIP2 in lemma, palea, anthers and rachis causes a reduction in Fusarium head blight symptoms, at the same level of the constitutive expression of this inhibitor. On the contrary, the expression of PvPGIP2 in the endosperm did not result in a reduction of disease symptoms, suggesting that once the pathogen has reached the endosperm, the activity of polygalacturonases secreted by the pathogen is not essential for the progression of symptoms.The pyramiding of glycosidase inhibitors in the same genotype is effective in reducing FHB symptoms although it depends on the specific combination. Pyramiding of PvPGIP2 and AcPMEI does not enhance further wheat resistance against FHB, possibly because they target the same virulent component secreted by the pathogen, that is PG. Conversely, the pyramiding PvPGIP2 and TAXI-III supports a further improvement of resistance compared to plants carrying only PvPGIP2 or TAXI-III.

Fusarium head blight (FHB) caused by Fusarium graminearum and Septoria tritici blotch (STB) caused by Septoria tritici are economically important wheat diseases in Ontario. Both reduce yield, FHB is associated with mycotoxin accumulation including deoxynivalenol (DON). Different F. graminearum chemotypes produce either DON/15-acetyldeoxynvialenol (ADON) or DON/3-ADON. The majority (97.5%) of F. graminearum isolates collected from commercial fields across Ontario were 15-ADON chemotype, the remaining were 3-ADON. In inoculated field experiments 3-ADON chemotypes were more aggressive and toxic compared with 15-ADON chemotypes as measured by FHB symptoms and DON content. In inoculated field experiments with a population derived from ‘RCATL33’ and ‘RC Strategy’ soft red winter wheat parents, genetic resistance was more effective than fungicide application at controlling FHB. Field trials with the hard red winter wheat population derived from ‘Maxine’ and ‘FTHP Redeemer’ parents revealed that STB and FHB phenotypic resistance had no negative impact on grain yield in the absence of disease.

The introduction of Quality Protein Maize (QPM), hard endosperm opaque-2 maize, into northern temperate maize growing areas is a desirable breeding objective. In topcrosses with opaque-2 testers, in diallel combination, as inbreds per se, and in inbred disease screening nurseries, some QPM lines performed better than or equal to the best local checks. In general, while agronomic potential is high for some lines and gains from selection are statistically possible, longer days to flowering intervals and higher levels of moisture at harvest than check hybrids indicated a need to improve adaptation for the locations studied. Methodology experiments indicated that detasselling of check hybrids is a suitable experimental method to facilitate the inclusion of normal endosperm local checks into QPM performance tests. The screening for Fusarium graminearum resistance in the seedling stage has not been proven to be a viable alternative to field scale ear inoculation screening procedures. (Abstract shortened by UMI.)

Plant growth regulators and nitrogen fertilization have been associated with the increased incidence of fusarium head blight, a destructive disease of wheat (Triticum aestivum L.). In Canada, the major causal organism of this disease is Fusarium graminearum Schwabe, the conidial state of Gibberella zeae (Schw.) Petch. Most studies concerning the effect of plant growth regulators on fusarium head blight were conducted in fields with natural infection. The objective of this research was to evaluate the effect of growth regulators and nitrogen fertilizer on the incidence of fusarium head blight of wheat with artificial inoculations. / A survey conducted in a field trial testing the effect of the plant growth regulator Cerone on the yield components of several cultivars of spring wheat showed that Cerone treatments increased Fusarium infection only in cultivar Columbus. Further research was conducted using cultivar Max, a cultivar susceptible to fusarium head blight, which is widely grown in Quebec. In controlled-condition greenhouse trials, the growth regulators Cycocel and Cerone, as well as nitrogen fertilization did not influence the disease progress. In the 1991 field experiment, the highest incidence of seed infection was observed in Cycocel treatments when the macroconidia of F. graminearum were directly applied to the heads, but not significantly different from the non-treated control. None of the nitrogen levels affect the incidence of seed infection. In the 1992 field trial, the plots were treated with macroconidia of F. graminearum applied to the heads or with Fusarium-colonized corn applied to the rows. Both Cycocel and Cerone significantly increased the incidence of spikelet only in the colonized corn treatments. Cycocel also increased the incidence of seed infection, but only in colonized corn treatments. Cycocel also increased the incidence of seed infection in the non-inoculated treatments. Growth regulators had no effect on the disease when heads were inoculated directly with macroconidia.

The characteristics and inheritance of maize silk resistance to Fusarium graminearum ear rot were investigated. In an in vitro test, genotypic differences in the degradation of detached silk tissue by F. graminearum were correlated to field evaluations of resistance. Susceptibility to infection decreased with silk age. Total phenolics of silk channel silk tissue increased in response to infection in resistant inbreds but decreased in susceptible inbreds. The flavones iso-orientin, iso-vitexin, maysin, luteolin, and apigenin were identified in the silk. No significant genotype by isolate interaction effects were found when 13 inbred lines were inoculated with three F. graminearum isolates. Simple models of quantitative and qualitative inheritance were not adequate to explain the inheritance of resistance. Disease severity ratings were bimodally distributed in the F$ sb1$, F$ sb2$, and backcross generations. In a complete diallel cross among 12 inbred lines, general and specific combining ability effects were significant for both disease incidence and disease severity. A screening of 12 accessions of exotic maize germplasm with resistance to either Aspergillus flavus or Heliothis zeae, identified several possible new sources of resistance to F. graminearum. Visual evaluations of resistance were correlated to deoxynivalenol levels of the ear.