Fusarium virguliforme (Aoki, O’Donnell, Homma & Lattanzi), the causal agent of sudden death syndrome (SDS) of soybean (Glycine max [L.] Merrill), is responsible for major soybean yield losses in North America and South America. Despite the importance of SDS, few agronomic practices have been used to manage SDS successfully. Understanding the pathogen and the mechanisms it uses to cause disease is vital to devise effective disease control strategies. However, our knowledge of the pathogenicity mechanisms used by F. virguliforme is limited. The identification of pathogenic genes will shed light on the molecular basis of the interaction between F.virguliforme and soybean, which may ultimately lead to better management of SDS. Therefore, the studies presented in this thesis were aimed at identifying and characterizing candidate pathogenicity genes in F. virguliforme.To fulfill this objective, 40 candidate pathogenicity genes of F. virguliforme were identified based on a combined approach, which included hands-on literature and database mining, functional genomics as well as transcriptome analyses. From these genes, the FvSNF1gene (a sucrose non-fermenting protein kinase 1 ortholog), the Fvstr1 gene (a striatin protein ortholog) were functionally characterized through a gene knock-out strategy. Targeted disruption of the FvSNF1 locus in wild type F. virguliforme strain reduced virulence significantly on soybean and abolished galactose utilization. In addition, the FvΔSNF1 mutant displayed significant reduction in expression of several CWDEs genes and was defective in colonizing the vascular system of the roots. To identify putative target genes regulated by FvSNF1, transcriptome analyses were performed in the FvSNF1 deletion mutant and in the wild-type. Disruption of FvSNF1 affected the level of transcription of 393 genes and a majority of the genes were involved in carbohydrate metabolism, lignin degradation, and cellular signaling pathway. The disruption of Fvstr1, a striatin ortholog in F. virguliforme, resulted in a complete loss of virulence as well as impaired conidiation, conidiophore development and pigmentation in the fungus. The FvΔstr1 mutant also failed to colonize the vascular tissues of roots of inoculated soybean plants. The results suggest that FvSNF1and Fvstr1 have critical roles in pathogenicity. Another part of the study was to investigate the efficacy of ILeVO®, a succinate dehydrogenase inhibitor fungicide from Bayer CropScience, against F. virguliforme. Our results showed that ILeVO® was very active against F. virguliforme in vitro and was very effective in minimizing F. virguliforme infection thus providing yet another tool to combat SDS.

Made available in DSpace on 2014-06-11T19:28:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-18Bitstream added on 2014-06-13T19:16:32Z : No. of bitstreams: 1 franco_hbj_me_jabo.pdf: 828158 bytes, checksum: 3da61c47398d1434dae4fea307ba8e5b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivou-se estudar diferentes metodologias de inoculação de Fusarium tucumaniae em folhas destacadas e em plântulas de soja, visando à caracterização da reação de resistência de cultivares de soja à F. tucumaniae. O trabalho foi desenvolvido em diferentes etapas relacionadas a seguir: obtenção e manutenção do inóculo; avaliação do desenvolvimento do patógeno em diferentes meios de cultura e diferentes luminosidades; avaliação de diferentes métodos de inoculação em folhas destacadas acondicionadas em papel de filtro e em solo, incubadas em duas temperaturas; avaliação de métodos de inoculação em plântulas cultivadas em casa de vegetação (primavera/verão e outono/inverno), reação de cultivares de soja a F. tucumaniae avaliadas em folhas destacadas, reação de cultivares de soja a F. tucumaniae avaliadas em plântulas (outono/inverno). As análises dos resultados permitiram concluir maior esporulação do fungo em meio de grãos de sorgo. As metodologias de inoculação de plântulas foram bem sucedidas na época de outono/inverno 2005, observando-se infecção das plântulas, principalmente de variedades suscetíveis, e os sintomas típicos da doença ocorreram em todos os métodos utilizados. FT-Cristalina apresentou maior nível de infecção da planta, para o método de grãos de sorgo (GS). O método de inoculação, em folhas destacadas, com grãos de aveia, promoveu maior infecção nas folhas e, o ambiente mais propício ao desenvolvimento dos sintomas da doença foi o solo a 23ºC. A cultivar FT-Estrela apresentou 90,2% de plântulas mortas, e CAC-1 46,9% (método GS). No método da folha destacada não foi possível caracterizar a reação das cultivares estudadas. / Detached leaves and seedlings of some cultivars were inoculated with F. tucumaniae using different methodologies in order to evaluated their resistance reaction. The conduction of the research work consisted of the following phases: obtainment and maintenance the isolate, evaluation of the pathogen growth and sporulation in different cultura media and luminosity; evaluation of different inoculation methods in detached leaves conditioned either in filter paper and soil being, incubated under two temperatures; evaluation of inoculation methods in soybean seedlings developed in greenhouse (Spring/Summer and Autumn/Winter); reaction of soybean cultivars to F. tucumaniae evaluated in detached leaves; reaction of soybean cultivars to F. tucumaniae evaluated in seedling (Autumn/Winter). The obtained results showed that the fungus sporulation was more intense when grown in a sorghum grain medium. Inoculation methodologies in seedlings showed best results when carried out during the 2005 Autumn/Winter period, because seedlings infection was observed, mainly in the susceptible cultivars, and all the employed methods allowed observation of the disease typical symptoms. FT-Cristalina was the cultivar in which the highest level of plant infection was observed, when the sorghum grain method (GS) was employed. The oat grain inoculation method in detached leaves, led to the highest leaf infection index, and the environmental condition which allowed the highest degree of leaf infection was in the soil under 23oC. FT-Estrela cultivar had 90.2% of their seedlings dead whereas for the CAC-1 cultivar that value was of 46.9% (method GS). Cultivars reaction could not be evaluated by means of the detached leaf method.

Orientador: Maria Aparecida Pessoa da Cruz Centurion / Banca: Vanoli Fronza / Banca: Antonio Orlando Di Mauro / Resumo: Objetivou-se estudar diferentes metodologias de inoculação de Fusarium tucumaniae em folhas destacadas e em plântulas de soja, visando à caracterização da reação de resistência de cultivares de soja à F. tucumaniae. O trabalho foi desenvolvido em diferentes etapas relacionadas a seguir: obtenção e manutenção do inóculo; avaliação do desenvolvimento do patógeno em diferentes meios de cultura e diferentes luminosidades; avaliação de diferentes métodos de inoculação em folhas destacadas acondicionadas em papel de filtro e em solo, incubadas em duas temperaturas; avaliação de métodos de inoculação em plântulas cultivadas em casa de vegetação (primavera/verão e outono/inverno), reação de cultivares de soja a F. tucumaniae avaliadas em folhas destacadas, reação de cultivares de soja a F. tucumaniae avaliadas em plântulas (outono/inverno). As análises dos resultados permitiram concluir maior esporulação do fungo em meio de grãos de sorgo. As metodologias de inoculação de plântulas foram bem sucedidas na época de outono/inverno 2005, observando-se infecção das plântulas, principalmente de variedades suscetíveis, e os sintomas típicos da doença ocorreram em todos os métodos utilizados. FT-Cristalina apresentou maior nível de infecção da planta, para o método de grãos de sorgo (GS). O método de inoculação, em folhas destacadas, com grãos de aveia, promoveu maior infecção nas folhas e, o ambiente mais propício ao desenvolvimento dos sintomas da doença foi o solo a 23ºC. A cultivar FT-Estrela apresentou 90,2% de plântulas mortas, e CAC-1 46,9% (método GS). No método da folha destacada não foi possível caracterizar a reação das cultivares estudadas. / Abstract: Detached leaves and seedlings of some cultivars were inoculated with F. tucumaniae using different methodologies in order to evaluated their resistance reaction. The conduction of the research work consisted of the following phases: obtainment and maintenance the isolate, evaluation of the pathogen growth and sporulation in different cultura media and luminosity; evaluation of different inoculation methods in detached leaves conditioned either in filter paper and soil being, incubated under two temperatures; evaluation of inoculation methods in soybean seedlings developed in greenhouse (Spring/Summer and Autumn/Winter); reaction of soybean cultivars to F. tucumaniae evaluated in detached leaves; reaction of soybean cultivars to F. tucumaniae evaluated in seedling (Autumn/Winter). The obtained results showed that the fungus sporulation was more intense when grown in a sorghum grain medium. Inoculation methodologies in seedlings showed best results when carried out during the 2005 Autumn/Winter period, because seedlings infection was observed, mainly in the susceptible cultivars, and all the employed methods allowed observation of the disease typical symptoms. FT-Cristalina was the cultivar in which the highest level of plant infection was observed, when the sorghum grain method (GS) was employed. The oat grain inoculation method in detached leaves, led to the highest leaf infection index, and the environmental condition which allowed the highest degree of leaf infection was in the soil under 23oC. FT-Estrela cultivar had 90.2% of their seedlings dead whereas for the CAC-1 cultivar that value was of 46.9% (method GS). Cultivars reaction could not be evaluated by means of the detached leaf method. / Mestre