Thesis (Ph. D.)--Colorado State University, 2004. / Includes bibliographical references.

No description available.

Using the genome database of the phytopathogenic fungus, Alternaria brassicicola, we identified a gene with high homology to the cell wall integrity-associated mitogen-activated protein (MAP) kinase, Slt2 in the yeast, Saccharomyces cerevisiae. This MAP kinase consists of a predicted 1,251-bp open reading frame, and encodes a 416-amino-acid protein weighing 47501 Da. This homolog was designated AbSlt2 (A. brassicicola Slt2) and gene disruption knockout (KO) mutants were generated in an A. brassicicola wild type background. Several altered phenotypes were found in the mutants compared to the wild type. During growth in various liquid and solid media, the abslt2 mutants displayed slightly aberrant hyphal growth and were unable to develop at the same rate as wild type. Furthermore, scanning electron microscopy (SEM) analysis revealed the abslt2 mutants showed decreased penetration ability, underdeveloped appresoria, and altered morphology on the leaf surface of the host plant, Brassica oleracea (cabbage) when compared to wild type. Abslt2 mutant hyphae exhibited slower growth in planta ultimately resulting in highly reduced virulence. Complementation of the disruption mutant with the wild type gene fully restored pathogenicity. Therefore, AbSlt2 is a new pathogenicity and developmental factor in A. brassicicola. / Master of Science

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.

La réussite de la mise en place d’une résistance efficace chez la plante hôte requiert la présence d’un dialogue entre les différentes voies signalétiques menant à l’induction de la défense. L’implication de l’extinction génique post-transcriptionnelle dans la résistance contre des agents pathogènes, autres que les virus, est une nouvelle avenue qui va permettre de disséquer le lien qui peut exister entre les deux voies immunitaires de la plante : celle qui est déjà importante pour lutter contre les bactéries et les champignons (voie induite à la suite de la reconnaissance des éliciteurs) et celle de l’extinction génique post-transcriptionnelle. Les résultats que nous avons obtenus ont une signification très importante concernant l’implication de l’extinction génique post-transcriptionnelle dans la résistance des plantes contre les agents pathogènes d’une manière générale et Botrytis cinerea, Alternaria brassicicola et Pseudomonas syringae en particulier. Mon travail de recherche a permis d’identifier le miR472 comme étant un acteur qui favorise la mise en place de la maladie causée par des agents pathogènes du type nécrotrophe et hémibiotrophe. De plus j’ai pu, en utilisant le modèle P. syringae pv tomato DC3000-Arabidopsis, disséquer dans le détail, comment la bactérie manipule la machinerie de la plante pour activer la voie des microARNs menant à l’accumulation du miR472, responsable de la suppression de la résistance basale et du PTI. Cette suppression passe par l’inhibition de sa cible, la protéine NB-LRR; RDS1 un nouveau régulateur impliqué dans le PTI induit par la Flg22. Cette étude permettra une avancée remarquable dans les connaissances reliées à l’immunité des plantes, ce qui constituerait une étape clé dans la découverte de nouvelles stratégies de lutte contre les agents phytopathogènes. Sur le plan fondamental, cette étude a permis de consolider le dialogue qui existe entre les voies immunes de la plante et de mettre en évidence un nouvel acteur impliqué dans la résistance basale. En matière d’application, comprendre les mécanismes de défense et de résistance des plantes aux agents pathogènes est primordial pour développer des plantes génétiquement résistantes et des stimulateurs de défense naturelle (Vaccin de plantes). Ceci constitue un enjeu majeur dans l'objectif de concevoir de nouveaux moyens de lutte phytosanitaire, à la fois plus respectueux de l’environnement et ayant moins d’impact sur la santé humaine.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-10T14:52:17Z No. of bitstreams: 1 Emanuel Feitosa de Assuncao.pdf: 874050 bytes, checksum: ab87c3228b1c082ff8bd7fcd620e23f2 (MD5) / Made available in DSpace on 2017-03-10T14:52:17Z (GMT). No. of bitstreams: 1 Emanuel Feitosa de Assuncao.pdf: 874050 bytes, checksum: ab87c3228b1c082ff8bd7fcd620e23f2 (MD5) Previous issue date: 2015-07-24 / The stain-de-alternaria caused by the fungus Alternaria brassicicola is considered one of the most common and destructive fungal diseases the brassica. The control of Alternaria is based on preventive sprays or after the appearance of first symptoms with fungicides. An alternative to controlling the disease is the use of biological control. In this context, yeasts emerge as a new class of biocontrol agents. This study aimed to: isolate, select and evaluate the potential of yeasts in biocontrol stain-of-alternaria, in kale; verify production Killer toxin, biofilm formation and production of volatile metabolites in vitro, as well as evaluate the yeast as resistance inducing agents in kale, through enzymatic analysis catalase and ascorbate peroxidase. Initially, 18 isolates of A. brassicicola were evaluated in greenhouse for their ability to cause disease in kale, being isolated 91 from the pathogen selected for biocontrol assays. 39 yeast isolates obtained from leaves of healthy plants brassica were evaluated in greenhouse, being five isolates selected for the assays in vivo and in vitro. In the assay biocontrol, kale plants were sprayed with 15 mL of the suspension (1x107 cells mL-1) of yeasts (L32, L40, L44, L47 and L57) in the periods (72, 48, 24 hours before inoculation the pathogen), and immediate (followed of the pathogen inoculation). The yeast isolates (L46, L32, L44 and L40) were the most effective in reducing the severity of the stain-of-alternaria, accounting for severity index of 7,66%, 7,78, 8,13 e 9,07%, respectively. The periods 24 hours and immediate were the most significant in reduction disease. Among the evaluated yeasts, isolates L46 and L44 produced biofilm (weak 1+) and the others did not produce biofilm. As the Killer activity, the five evaluated yeasts produced the Killer toxin. It was also verified that the isolates (L46 and L40) inhibited in 60, 334% and 59,774%, respectively, growth in vitro of A. brassicicola and isolates (L32, L40, L44, L47 and L57) completely inhibited sporulation in vitro of the pathogen. The five isolates of yeasts evaluated provide kale plants higher levels of catalase enzyme in relation to the control treatment. The use of yeast proves to be a promising tool for the biological control of A. brassicicola in kale. / A mancha-de-alternaria causada pelo fungo Alternaria brassicicola é considerada uma das doenças fúngicas mais comuns e destrutivas das brássicas. O controle da alternariose baseia-se em pulverizações preventivas ou após o aparecimento dos primeiros sintomas com fungicidas. Uma alternativa para o controle da doença é o uso do controle biológico. Neste contexto, as leveduras surgem como uma nova classe de agentes de biocontrole. O presente estudo teve como objetivos: isolar, selecionar e avaliar o potencial de leveduras no biocontrole da mancha-de-alternaria, em couve-manteiga; verificar a produção de toxina Killer, formação de biofilme e produção de metabólitos voláteis in vitro, bem como, avaliar as leveduras como agentes indutores de resistência em couve-manteiga, através da análise enzimática da catalase e ascorbato peroxidade. Inicialmente, 18 isolados de A. brassicicola foram avaliados em casa de vegetação quanto à capacidade de causar doença em couve-manteiga, sendo o isolado 91 do patógeno selecionado para os ensaios de biocontrole. 39 isolados de leveduras obtidos de folhas de plantas sadias de brássicas foram avaliados em casa de vegetação, sendo cinco isolados selecionados para a realização dos ensaios in vivo e in vitro. No ensaio de biocontrole, plantas de couve-manteiga foram pulverizadas com 15 mL da suspensão (1x107 células mL-1) das leveduras (L32, L40, L44, L47 e L57), nos períodos (72, 48, 24 horas antes da inoculação do patógeno) e, imediato (seguido a inoculação do patógeno). Os isolados de leveduras (L46, L32, L44 e L40) foram os mais eficientes na redução severidade da mancha-de-alternaria, sendo responsáveis por índices de severidade de 7,66%, 7,78, 8,13 e 9,07%, respectivamente. Os períodos 24 horas e imediato foram os mais significativos na redução da doença. Dentre as leveduras avaliadas, os isolados L46 e L44 produziram biofilme (fraca 1+) e, os demais, não produziram biofilme. Quanto à atividade Killer, as cinco leveduras avaliadas produziram a toxina Killer. Também foi verificado que os isolados (L46 e L44) inibiram em 60, 334% e 59, 774%, respectivamente, o crescimento in vitro de A. brassicicola e, os isolados (L32, L40, L44, L47 e L57) inibiram completamente a esporulação in vitro do patógeno. Além disso, os cinco isolados de leveduras avaliados proporcionam nas plantas de couve-manteiga maiores níveis enzimáticos da catalase, em relação ao tratamento controle. O uso de leveduras demonstra ser uma ferramenta promissora para o controle biológico de A. brassicicola em couve-manteiga.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-20T15:01:07Z No. of bitstreams: 1 Luiz Augusto Martins Peruch.pdf: 694087 bytes, checksum: 27f67eb702a71e64c5b03780e75fe68e (MD5) / Made available in DSpace on 2017-03-20T15:01:07Z (GMT). No. of bitstreams: 1 Luiz Augusto Martins Peruch.pdf: 694087 bytes, checksum: 27f67eb702a71e64c5b03780e75fe68e (MD5) Previous issue date: 2004-02-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The brassica cultivation has been of high importance in Brazil. However, the profitable crop production can be limited by occurrence of diseases such as Alternaria black spot, caused by Alternaria brassicicola and/or Alternaria brassicae, and black rot, caused by Xanthomonas campestris pv. campestris. In that context, the present study had as objectives to evaluate the intensity of Alternaria black spot and of black rot diseases on brassica under organic cultivation in the states of Pernambuco and Santa Catarina, and the spore production longevity of A. brassicicola in leaf debris of broccolis. The intensity of the diseases was evaluated from November 2001 to February 2002, in 103 fields under organic farming systems, including broccoli, Chinese cabbage, cauliflower, kale and cabbage. High prevalence of the diseases was registered in both states, except on Chinese cabbage in Santa Catarina. Prevalence of Alternaria black spot was 100% on broccoli fields in Pernambuco, as well as on cauliflower in the two states, while the black rot reached that level on broccoli and cauliflower fields in Santa Catarina. In the average of the different brassica species, the diseases were more prevalent in Pernambuco than in Santa Catarina. However, when considered severity averages of each disease, no significant differences were verified between the two states, although the climatic conditions have been sharply different. The levels ofdisease severity were low, considering that 98% of the fields presented severity lower than 5%. The Alternaria black spot severity varied among the brassica species in Pernambuco, being higher on Chinese cabbage and lower on kale. In Santa Catarina no significant differences were verified among the brássicas species. In relation to the black rot, only in Santa Catarina there was difference in the disease severity among the brassicas, being registered the lowest level on Chinese cabbage. No significant correlations were verified between severity levels of Alternaria black spot and black rot, neither between disease severity and total number of plants or plant age. The spore production longevity of A. brassicicola was investigated in leaves debris of broccoli, considering different depths of debris incorporation, periods of the year, and systems of soil handling, in two experiments carried out in Urussanga city, State of Santa Catarina, Brazil. Infected leaves debris of broccoli were conditioned in bags of polyethylene and distributed in portions in the field, on the soil surface and at depths of 5 and 10 cm. Periodically, the debris in the bags were collected and conidia concentrations were quantified for longevity of spore production andextinction rate. In both experiments there was higher spore production longevity in leaves debris in the period I, characterized by lighter temperatures, higher air relative humidity and lower pluvial precipitation than in the period II. Depth of debris incorporation influenced directly on A. brassicicola spores production, being lower when incorporation was made at 10 cm depth. In relation to soil handling systems, when the inoculum source was deposited on the soil surface there was not difference in the rate of spore production extinction between the soils under conventional and organic handling. However, at depths of 5 and 10 cm, the rate was significantly higher in the soil under conventional handling. The management of Alternaria black spot on broccoli at the area under study can be accomplished by integrated control, including the incorporation of infected leaves debris in the soil, at least 10 cm depth, along with crop rotation, for no less than a 60-day interval in subsequent brassica plantings. / O cultivo de brássicas tem destacada importância na olericultura brasileira. No entanto, a produção dessas olerícolas pode ser limitada pela ocorrência de doenças, dentre as quais se destacam a alternariose, causada por Alternaria brassicicola e/ou Alternaria brassicae, e a podridão negra, causada por Xanthomonas campestris pv. campestris. Nesse contexto, o presente estudo teve como objetivos realizar o levantamento da intensidade da alternariose e da podridão negra em cultivos orgânicos de brássicas nos Estados de Pernambuco e Santa Catarina, bem como avaliar a longevidade da esporulação de A. brassicicola em restos foliares de brócolis. Os levantamentos foram realizados no período de novembro de 2001 a fevereiro de 2002, num total de 103 cultivos orgânicos, incluindo brócolis, couve-chinesa, couve-flor, couve-manteiga e repolho. Foram registradas elevadas prevalências das doenças nos dois Estados, com exceção em couve-chinesa em Santa Catarina. A prevalência da alternariose foi de 100% nos cultivos de brócolis em Pernambuco, bem como em couve-flor nos dois Estados, enquanto a podridão negra atingiu esse nível nos cultivos de brócolis e couve-flor em Santa Catarina. Na média das diferentes espécies de brássicas, as doenças foram mais prevalentes em Pernambuco que Santa Catarina. Entretanto,quando consideradas as médias de severidade de cada doença no conjunto das brássicas, não foram constatadas diferenças significativas entre os dois Estados, embora as condições climáticas tenham sido nitidamente distintas. Os níveis de severidade das doenças foram baixos, tendo em vista que 98% dos cultivos apresentaram severidade inferior a 5%. A severidade da alternariose variou entre as espécies de brássicas em Pernambuco, com maior severidade registrada em couve-chinesa e menor em couve-manteiga, enquanto em Santa Catarina não foram constatadas diferenças significativas entre as brássicas. Em relação à podridão negra, somente em Santa Catarina houve diferença na severidade entre as brássicas, sendo registrados os menores níveis em couve-chinesa. Não foram constatadas correlações significativas entre os níveis de severidade da alternariose e da podridão negra, bem como da severidade destas com o número total de plantas e a idade das plantas nos cultivos. Foi também investigada a longevidade da esporulação de A. brassicicola em restos foliares de brócolis, considerando diferentes profundidades de incorporação dos restos, períodos do ano e sistemas de manejo do solo, em dois experimentos realizados em Urussanga - SC. Restosfoliares de brócolis infectados pelo patógeno foram acondicionadas em sacolas de polietilenoperfuradas e distribuídos em parcelas no campo, na superfície do solo e nas profundidades de 5 e 10 cm. Nos dois experimentos foi verificada uma maior longevidade da esporulação nos restos foliares no período I, caracterizado por temperaturas mais amenas, maior umidade relativa do ar e menor precipitação pluvial que no período II. A profundidade de incorporação dos restos influiu diretamente na esporulação de A. brassicicola, que foi inferior quando a incorporação foi efetuada a 10 cm de profundidade. Em relação aos sistemas de manejo, quando a fonte de inóculo foi depositada na superfície do solo não houve diferença na taxa de extinção da esporulação entre os solos sob manejo convencional e orgânico. No entanto, nas profundidades de 5 e 10 cm, essa taxa foi significativamente superior no solo sob manejo convencional. O manejo da alternariose em brócolis na região do estudo pode ser realizado pela combinação de vários métodos de controle, inclusive pela incorporação dos restos foliares infectados no solo, à profundidade mínima de 10 cm, combinado com rotação de culturas, visando um intervalo mínimo de 60 dias entre cultivos de brássicas.

L'induction par un hôte d'un stress oxydatif chez un parasite représente l'une des stratégies de défense chimique les plus répandues. Chez les Brassicacées, famille de plantes comprenant des espèces à fort intérêt agronomique comme le chou ou le colza, ou d'intérêt scientifique comme Arabidopsis thaliana, le stress oxydatif peut être généré soit par des formes actives d'oxygène libérées au niveau de la zone d'agression lors du burst oxydatif soit par des molécules issues du métabolisme secondaire de la plante telles que les isothiocyanates (ITC). Bien que fortement exposé à un tel stress lors de l'infection, Alternaria brassicicola, champignon pathogène nécrotrophe inféodé aux Brassicacées, est capable d'accomplir son cycle parasitaire et donc de s'y adapter. Cette thèse met en évidence que la tolérance du champignon aux stress oxydatif est partiellement dépendante du métabolisme du mannitol qui participe à la résistance des conidies lors du burst oxydatif in planta ou à la protection contre les dommages intracellulaires des FAO générées par les ITC lors des phases précoces de l'interaction. Les travaux réalisés montrent également que la charge fongitoxique des dérivés des glucosinolates peut être inhibée par leur conjugaison au glutathion via des Glutathion-Stransférases (GST) spécifiques. L'altération du métabolisme du mannitol ou l'inactivation de ces GST diminue significativement le pouvoir pathogène d'A. brassicicola et la régulation de ces voies métaboliques est donc à considérer dans l'optique de développer de nouvelles stratégies de protection des cultures.

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.