Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.

Extração de ácidos triterpénicos a partir da casca de eucalipto utilizando líquidos iónicos

Os recursos renováveis têm sido um forte alvo de investigação científica nos últimos anos, onde o aproveitamento de biomassa e seus resíduos para a obtenção de compostos de valor acrescentado, combustíveis e energia têm sido abordados no conceito de biorrefinaria integrada. As indústrias de papel geram quantidades significativas de resíduos, nomeadamente a casca de eucalipto que é atualmente queimada para a geração de energia. De forma a valorizar este resíduo, a presente dissertação teve como objetivo extrair compostos triterpénicos, a partir casca externa de Eucalyptus globulus, utilizando solventes de extração alternativos - soluções aquosas de líquidos iónicos (LIs) – para substituir os solventes orgânicos actualmente utilizados. Os ácidos triterpénicos apresentam um elevado interesse na indústria cosmética, farmacêutica e alimentar graças às suas propriedades antiinflamatórias, antitumurais, entre outras. Primeiramente, caracterizou-se a casca externa de Eucalyptus globulus, e posteriormente procedeu-se ao estudo de solubilidade de ácido ursólico (AU, utilizado como molécula modelo) a 25 ºC em soluções aquosas de LIs e surfactantes de modo a selecionar os solventes mais eficientes para a extração. Deste trabalho conclui-se que a capacidade surfactante das soluções aquosas de LIs, particularmente [C4C1im][C8H17SO4], [C16C1im]Cl e [C14C1im]Cl, desempenham um papel fundamental para a solubilização de AU em água, podendo aumentar quase 16000 vezes a sua solubilidade, e permitiu recuperar cerca de 89% deste composto com simples adição de água como anti-solvente. Por fim, compararam-se as quantidades de ácidos triterpénicos extraídas a partir da casca de eucalipto com soluções aquosas de [C14C1im]Cl, metanol e com extração em soxhlet com diclorometano.