Universiẗat, Diss., 2002--Bonn.

Techn. Hochsch., Diss., 2004--Aachen.

Universiẗat, Diss., 2005--Bonn.

A macieira (Malus × domestica) possui importância mundial entre as frutíferas de clima temperado, e o Brasil se destaca entre os maiores produtores mundiais de maçã.À sarna da macieira, causada pelo fungo ascomiceto Venturia inaequalis, é uma das principais doenças nessa cultura. As duas cultivares mais consumidas no Brasil, Fuji e Gala, são altamente suscetíveis à sarna. O gene Vf2, derivado do clone 821 da espécie silvestre Malus floribunda, é capaz de conferir resistência à doença. O processo de melhoramento genético da macieira e a introgressão de novos genes derivados de germoplasma é um processo demorado e difícil devido ao longo tempo de geração das plantas e à autoincompatibilidade à autopolinização em Malus.Historicamente a introgressão recombinante de genes em vegetais baseou-se na transformação genética via Agrobacterium tumefaciens. Porém, novas técnicasestão sendo estabelecidas e vem revolucionado o campo da genômica funcional em plantas como a utilização de DNA epissomal em plasmídeos vegetais derivados de vírus. Estes vetores não se integram ao genoma da planta hospedeira e não são herdáveis. Os vetores consistem de vírus modificados capazes de espalhar-se por todos os tecidos da planta tratadasem causar doença e levando à expressão de genes de interesse sem a necessidade de obtenção de plantas transgênicas. Este trabalho tevecomo objetivo transferir a resistência aV. inaequalis, conferida pelo gene Vf2, para as cultivares de macieira Fuji e Gala, utilizando-seplasmídeos vegetais baseado nos vírus TYLCV (Tomato Yellow Leaf Curl Virus). O gene de resistência Vf2 derivado de Malus floribunda 821 foi clonado nosvetores de expressão e injetadoem plantas de macieira das cultivares Maxi Gala e Fuji Suprema.O DNA plasmidial foi detectado sistemicamente nas plantas tratadas e este se manteve estável durante os8 meses de testes. A expressão do gene Vf2 foi quantificada por RT-qPCR e diversas plantas apresentaram níveis elevados de expressão deste gene de resistência. Estas plantas foram desafiadas com esporos de V. inaequalis e a maioria das plantas tratadas apresentou fenótipo resistente à sarna. A introgressão recombinante baseada em plasmídeos vegetais demonstrou ser eficaz em transferir material genético de forma sistêmica nas duas cultivares de macieira testadas. O vetor plasmidial foi detectado nas plantas tratadas durante todo o período(8 meses) avaliado neste estudo, e a expressão do gene de resistência foi detectada em diversos indivíduos. Nosso estudo reforça a aplicabilidade desta técnica em plantas lenhosas perenes e abre a possibilidade para a introgressão rápida de outras características de interesse derivadas da expressão heteróloga de genes com potencial biotecnológico. / Apple (Malus x domestica) is an important crop among temperate fruit trees, and Brazil features among the world’s biggest apple producers. The apple scab, caused by the ascomycete fungus Venturia inaequalis, is a major disease of the culture. The two most consumed cultivars in Brazil, Fuji and Gala, are highly susceptible to apple scab. The Vf gene, derived from clone 821 of the wild species Malus floribunda, is capable of conferring resistance to the scab disease. The genetic improvement process of apple by introgression of new genes derived from germplasm is lenghty due to long generation time and self-incompatibility. Historically the recombinant introgression of genes in plantshas been based on genetic transformation emplyingAgrobacterium tumefaciens. However, new techniques have been developed and are revolutionizing plantfunctional genomics such as virus-derived plant episomalplasmids.These vectors do not integrate in to thehost plant genome and are not inheritable. The vectors consist of a modified virus capable of spreading to all tissues of thetreated plant without causing disease and leading to the systemic expression of the gene of interest without the need of obtaining transgenic plants.This workaimed to transfer the resistance against V. inaequalis, granted by the Vf2 gene, to Fuji and Gala apple suceptible cultivars, by using plant plasmids based on TYLCV (Tomato Yellow Leaf Curl virus).The resistance gene Vf2 derived from Malus floribunda 821 was cloned on expression vectors and injectedto Maxi Gala and Fuji Supremaplants.The plasmid DNA was systemically detected on treated plants and remained stable for at least8 months.The expression of theVfgene was quantified by RT-qPCR and several plants presented high levels of Vf2expression. These plants were challenged by spores of V. inaequalis and the majority of the treated plants displayed scab resistant phenotypes.The Vf2introgression based on plant plasmidswas shown to be effective on transferring genetic material sistemically to the apple cultivars tested. The plasmids weredetected on treated plants during the whole evaluated time of this study (8 months), and the resistance gene expression was detected on several individuals. Our study reinforces the applicability of such technique in woody perennial plants and givesthe possibility for fast introgression of other characteristics of interest dependent on genes with diverse biotechnological potential.

A macieira (Malus × domestica) possui importância mundial entre as frutíferas de clima temperado, e o Brasil se destaca entre os maiores produtores mundiais de maçã.À sarna da macieira, causada pelo fungo ascomiceto Venturia inaequalis, é uma das principais doenças nessa cultura. As duas cultivares mais consumidas no Brasil, Fuji e Gala, são altamente suscetíveis à sarna. O gene Vf2, derivado do clone 821 da espécie silvestre Malus floribunda, é capaz de conferir resistência à doença. O processo de melhoramento genético da macieira e a introgressão de novos genes derivados de germoplasma é um processo demorado e difícil devido ao longo tempo de geração das plantas e à autoincompatibilidade à autopolinização em Malus.Historicamente a introgressão recombinante de genes em vegetais baseou-se na transformação genética via Agrobacterium tumefaciens. Porém, novas técnicasestão sendo estabelecidas e vem revolucionado o campo da genômica funcional em plantas como a utilização de DNA epissomal em plasmídeos vegetais derivados de vírus. Estes vetores não se integram ao genoma da planta hospedeira e não são herdáveis. Os vetores consistem de vírus modificados capazes de espalhar-se por todos os tecidos da planta tratadasem causar doença e levando à expressão de genes de interesse sem a necessidade de obtenção de plantas transgênicas. Este trabalho tevecomo objetivo transferir a resistência aV. inaequalis, conferida pelo gene Vf2, para as cultivares de macieira Fuji e Gala, utilizando-seplasmídeos vegetais baseado nos vírus TYLCV (Tomato Yellow Leaf Curl Virus). O gene de resistência Vf2 derivado de Malus floribunda 821 foi clonado nosvetores de expressão e injetadoem plantas de macieira das cultivares Maxi Gala e Fuji Suprema.O DNA plasmidial foi detectado sistemicamente nas plantas tratadas e este se manteve estável durante os8 meses de testes. A expressão do gene Vf2 foi quantificada por RT-qPCR e diversas plantas apresentaram níveis elevados de expressão deste gene de resistência. Estas plantas foram desafiadas com esporos de V. inaequalis e a maioria das plantas tratadas apresentou fenótipo resistente à sarna. A introgressão recombinante baseada em plasmídeos vegetais demonstrou ser eficaz em transferir material genético de forma sistêmica nas duas cultivares de macieira testadas. O vetor plasmidial foi detectado nas plantas tratadas durante todo o período(8 meses) avaliado neste estudo, e a expressão do gene de resistência foi detectada em diversos indivíduos. Nosso estudo reforça a aplicabilidade desta técnica em plantas lenhosas perenes e abre a possibilidade para a introgressão rápida de outras características de interesse derivadas da expressão heteróloga de genes com potencial biotecnológico. / Apple (Malus x domestica) is an important crop among temperate fruit trees, and Brazil features among the world’s biggest apple producers. The apple scab, caused by the ascomycete fungus Venturia inaequalis, is a major disease of the culture. The two most consumed cultivars in Brazil, Fuji and Gala, are highly susceptible to apple scab. The Vf gene, derived from clone 821 of the wild species Malus floribunda, is capable of conferring resistance to the scab disease. The genetic improvement process of apple by introgression of new genes derived from germplasm is lenghty due to long generation time and self-incompatibility. Historically the recombinant introgression of genes in plantshas been based on genetic transformation emplyingAgrobacterium tumefaciens. However, new techniques have been developed and are revolutionizing plantfunctional genomics such as virus-derived plant episomalplasmids.These vectors do not integrate in to thehost plant genome and are not inheritable. The vectors consist of a modified virus capable of spreading to all tissues of thetreated plant without causing disease and leading to the systemic expression of the gene of interest without the need of obtaining transgenic plants.This workaimed to transfer the resistance against V. inaequalis, granted by the Vf2 gene, to Fuji and Gala apple suceptible cultivars, by using plant plasmids based on TYLCV (Tomato Yellow Leaf Curl virus).The resistance gene Vf2 derived from Malus floribunda 821 was cloned on expression vectors and injectedto Maxi Gala and Fuji Supremaplants.The plasmid DNA was systemically detected on treated plants and remained stable for at least8 months.The expression of theVfgene was quantified by RT-qPCR and several plants presented high levels of Vf2expression. These plants were challenged by spores of V. inaequalis and the majority of the treated plants displayed scab resistant phenotypes.The Vf2introgression based on plant plasmidswas shown to be effective on transferring genetic material sistemically to the apple cultivars tested. The plasmids weredetected on treated plants during the whole evaluated time of this study (8 months), and the resistance gene expression was detected on several individuals. Our study reinforces the applicability of such technique in woody perennial plants and givesthe possibility for fast introgression of other characteristics of interest dependent on genes with diverse biotechnological potential.

Techn. Hochsch., Diss., 2004--Aachen.

Firethorns are usually planted in parks and gardens as an ornamental shrubs or hedges. Infestation of fruits by fungus V. inaequalis significantly decreases their aesthetic value and the fruits also lose atractivity. Venturia inaqualis is the most important pathogene of firethorn (Pyracantha coccinea M. J. Roemer). The aim of diploma thesis was to determine the options of firethorn scab regulation, to monitor and evaluate the frequency and intensity of pathogen occurrence on two sites (Šitbořice and Brno-Černá Pole), and to evaluate the efficiency of three fungicides with different mechanisms of action. It was proven a statistically significant influence of site on fruits infestation and also the influence of site on infestation of levels of shrub, from which the fruits were gathered. The least infested fruits were gathered from the top level of shrub. Highest efficiency on causative agent of firethorn scab was proved by fungicide Zato 50 WG, with the active substance trifloxystrobin (500 g/kg) and by fungicide Talent, with the active substance myclobutanil (200 g/l).

Made available in DSpace on 2014-08-20T13:42:10Z (GMT). No. of bitstreams: 1 Dissertacao_Joceani_Dal_Cero.pdf: 1522635 bytes, checksum: 6a5a31f6f6667ef5f524442ba0bb1e72 (MD5) Previous issue date: 2010-02-26 / Regulatory molecules, such as transcription factors, have been thoroughly investigated, especially in hormone-mediated responses that involve gene expression modulation. Frequently, the main determinant of gene expression is its transcriptional rate. Thus, molecular mechanisms underlying transcription regulation have become an important topic in genetic studies of ethylene signaling. The present work aimed to investigate the ERF (Ethylene Response Factor) family employing bioinformatic tools, integrating publicly available datasets from the model species Arabidopsis thaliana and phylogenetic analyses to help elucidating the biological roles of the family in apple. The preliminary survey of the ERF sequences in Malus has provided basic information to be incorporated in further studies of the functional role of ERFs in this perennial species. Expression analyses of MdERF1 and MdERF in apple fruits suggest that other factors, besides ethylene, are involved in their transcriptional regulation in Malus. The second chapter reports the investigation of the transcriptional profiling of those ERF genes in response to pathogen attack, using a biological assay of in vitro propagated plants inoculated with the fungus Venturia inaequalis (apple scab disease). The study has provided evidences of the involvement of MdERF1 in eliciting the plant response; whereas, MdERF2 does not appear to be participate in the pathogenesis. / Moléculas que participam dos processos regulatórios, como os fatores de transcrição, têm recebido atenção especial, pois uma das principais ações dos estímulos hormonais é a modulação da expressão gênica. Como a taxa de transcrição de um gene é o maior determinante da sua expressão, os mecanismos moleculares pelos quais a transcrição gênica é regulada têm se tornado um dos tópicos principais de estudos em genética molecular envolvendo o hormônio etileno. O objetivo deste trabalho foi realizar análises de bioinformática para a família ERF (Ethylene Response Factors), integrar bases de dados existentes na internet no modelo Arabidopsis, bem como análise filogenética que permitam avaliar os papéis dos diferentes membros da família. Este levantamento preliminar das seqüências ERF em Malus forneceu informação básica para estudos posteriores mais aprofundados, com relação aos mecanismos moleculares da família nesta importante cultura perene. A análise da expressão de MdERF1 e MdERF2 em frutos de maçã indica que outros fatores além do etileno estão envolvidos na regulação da transcrição dos ERF em Malus. O segundo capítulo refere-se à resposta dos ERF frente ao ataque de patógenos. Para isso, foram infectadas plantas de macieira provenientes de cutivo in vitro com o fungo Venturia inaequalis (sarna da maçã). As evidências desses estudos sugerem o envolvimento do gene MdERF1 no processo de patogênese, enquanto que o gene MdERF2 parece não estar envolvido no processo.

In 1992, a research program was initiated to select suitable antagonists against the saprophytic (or winter) phase of the apple scab pathogen, Venturia inaequalis. An improved method for the mass screening of a vast collection of fungi was developed for this purpose. Some of the previously reported criteria such as leaf rheology and overwintering structure production proved unreliable or fastidious and cannot be used for in vitro antagonist selection. The main antagonism selection criterium retained was the in vitro inhibition of ascospore formation. To measure ascospore production, a simplified method of in vitro pseudothecia production was devised. This new method eliminates the need for prior conidia production by using a mycelial suspension and greatly reduces the risks of sterile mating by using a cocktail of Venturia inaequalis strains of different origins. Finally, the production cycle duration was reduced by varying the incubation temperature during the simulated winter. Moreover, a quick and efficient method of ascospore collection was developed. Ascospores were forcibly ejected in a large scale bubbler type apparatus in only one hour. This bubbler can be used for other studies including axenically produced ascospores. This new method compared favourably to the previously reported method and was used to screen a collection of about forty-two fungi. Six proved to significantly reduce the ascospore production of Venturia inaequalis. Two were as effective as Athelia bombacina, a previously reported antagonist of pseudothecia formation and inhibited over 98% of the ascospore production. These new organisms are now available for future field tests. Future selections from a large collection of saprophytes can now be based on a reliable and simple in vitro screening methodology.

Venturia inaequalis, the causal agent of apple scab, overwinters in apple leaves on the orchard floor. To develop a control strategy based on the prevention of the maturation of overwintering pseudothecia, a sampling of fungi colonizing dead apple leaves was conducted from different orchard floors in Quebec during the spring and fall of 1993. A total of 345 different isolates were obtained, from which fifteen genera have never been previously recorded as colonizers of apple leaves in North America. Small differences were detected in genera richness among orchards but the fungal composition of each orchard was fairly unique. Different tests on growth on amended media and leaf decomposition demonstrated that leaf degradation is not a reliable parameter alone to screen antagonist against V. inaequalis. No significant relation between growth on amended media, leaf rheology and ascospore inhibition was detected. However, 40 fungi reduced significantly ascospore production more than 87% compared to the control (V. inaequalis only). Of these antagonists, 30% decomposed apple leaves, suggesting that competition for the substrate is involved in the mode of action of at least one third of the antagonits detected. Other possible modes of antagonism are discussed.