Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis. / Ph. D.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-27T16:20:29Z No. of bitstreams: 1 Viviane Jurema Lopes Borges Rodrigues.pdf: 780077 bytes, checksum: c60257b425b95e0377eca176e4335872 (MD5) / Made available in DSpace on 2017-03-27T16:20:29Z (GMT). No. of bitstreams: 1 Viviane Jurema Lopes Borges Rodrigues.pdf: 780077 bytes, checksum: c60257b425b95e0377eca176e4335872 (MD5) Previous issue date: 2003-03-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cabbage (Brassica oleracea var. capitata ), cauliflower (Brassica oleracea var. botrytis), Chinese cabbage (Brassica pekinnensis) and broccoli (Brassica oleracea var. italica) have high economic expression in the Agreste region in the State of Pernambuco, Brazil. However, the occurrence of foliar diseases such as Alternaria black spot, caused by Alternaria brassicicola, represent great worry in farming systems areas. Thus, this research aimed to analyze the epidemiology of Alternaria black spot on Taibyo cultivar of Chinese cabbage in different farming systems (conventional, organic and protected); at four transplant seasons and two irrigation systems under organic farming; at three transplant spacing under conventional farming; as well as analyze the comparative epidemiology of Alternaria black spot in 22 brassica cultivars being nine broccoli, five Chinese cabbage, four cauliflower andfour cabbage, under conventional and organic farming systems. Evaluations of the beginning of initial symptoms (BIS) and severity of the disease on every leaf of 10 plants were made. Disease progress curves were ploted, and epidemiology were compared through by BIS, maximum disease severity (ymax), disease progress rate (DPR), and the area under disease progress curves (AUDPC) in all experiments. The October 2000 transplant season was the most propitious to Alternaria black spot, while the July 2001 transplant season was unfavorable to disease development, since it took longer for the symptoms to show, and the values of ymax and DPR were lower than in other periods. Comparing the three farming systems, the highest values of ymax, DPR and AUDPC were observed in organic farming, indicating that this condition was the most favorable to Alternaria black spot. Trickle irrigation compared to the sprinkle system showed lower development of Alternaria blackspot, represented by lower values of ymax, DPR and AUDPC. As far as spacing is concerned, the combination 0.33 x 0.50 m showed the lowest values of ymax and AUDPC. All brassicacultivars evaluated were susceptible to A. brassicicola. In most situations, significative difference was detected among cultivars grown in the same farming system. Higher disease intensity was obeserved in organic farming system. Chinese cabbage (Tropical Pride and Tropical Delight), cauliflower (Arfak), and cabbage (Fuyutoyo and Rubro) had the lowest disease levels. . On all situations, significative correlations were noted among the severity of Alternaria black spot disease and both the highest temperature average and the relative air humidity in the 14 days prior to the disease evaluation. / O cultivo de repolho (Brassica oleracea var. capitata ), couve-flor (Brassica oleracea var. botrytis), couve-chinesa (Brassica pekinnensis) e brócolis (Brassica oleracea var. italica) têm grande expressão econômica na região Agreste do Estado de Pernambuco. No entanto, a ocorrência de doenças foliares como a alternariose, causada por Alternaria brassicicola, representa grande preocupação ao sistema de produção das áreas de plantio. Neste contexto, este trabalho teve como objetivos analisar a epidemiologia da alternariose em couve-chinesa, cv. Taibyo, em diferentes sistemas de cultivo (convencional, orgânico e protegido), em quatro épocas de plantio e dois sistemas de irrigação no cultivo orgânico, em três espaçamentos de plantio no cultivo convencional, bem como analisar a epidemiologia comparativa da alternariose em 22 cultivares de brássicas, sendo nove de brócolis, cinco de couve-chinesa, quatro de couve-flor e quatro de repolho, nos sistemas de produção convencional e orgânico.Foram avaliados o início do aparecimento dos sintomas (IAS) da alternariose e a severidade da doença em todas as folhas de 10 plantas por parcela. Curvas de progresso da doença foram plotadas e as epidemias comparadas em relação ao IAS, severidade máxima (ymax), taxa estimada de progresso da doença (TPD) e área abaixo da curva de progresso da doença (AACPD) em todos os experimentos. Na comparação entre os três sistemas de cultivo, no orgânico foram observados os maiores valores de ymax, TPD e AACPD, indicando que este sistema propiciou condições mais favoráveis para a alternariose. O transplantio em outubro de 2000 foi o mais propício para a alternariose da couve-chinesa no cultivo orgânico, enquanto o realizado em julho de 2001 foi desfavorável ao desenvolvimento da doença, pois os sintomas apareceram mais tardiamente e os valores de ymax e TPD foram menores que nos outros períodos. No cultivo irrigado por gotejamento ocorreu menor desenvolvimento da alternariosedo que no irrigado por microaspersão, representado pelos valores inferiores de ymax, TPD eAACPD. Com relação ao espaçamento, na combinação 0,33 x 0,50 m foram registrados os menores valores de ymax e AACPD. Todas as cultivares de brássicas avaliadas foram suscetíveis a A. brassicicola, embora na maioria das situações tenha sido detectada diferença significativa entre as cultivares dentro de um mesmo sistema de cultivo. Em geral, foram constatados maiores níveis de intensidade da alternariose no sistema orgânico que no convencional. As cultivares de couve-chinesa Tropical Pride e Tropical Delight, de couve-flor Arfak e de repolho Fuyutoyo e Rubro merecem destaque pelos menores níveis de alternariose apresentados nos dois sistemas de cultivo. Em todas as situações analisadas foram constatadas correlações significativas entre a severidade da alternariose e as médias da temperatura e da umidade relativa do ar nos 14 dias que antecederam a avaliação da doença