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.

Evolução da linha de costa da praia da Vagueira

As costas arenosas estão actualmente sujeitas a intensos processos erosivos, resultado tanto de actividades humanas como de processos naturais. Na costa portuguesa, este aspecto é bem evidente pelo recuo da linha de costa, pela diminuição da largura das praias, pela degradação dos sistemas dunares e principalmente, pela proliferação de obras fixas de protecção costeira ao longo da linha de costa. Desde a última metade do século passado que a Vagueira tem sido submetida a um intenso processo erosivo, resultando num acentuado recuo da linha de costa. A principal causa apontada para este facto, é a retenção de sedimentos por parte do molhe Norte do porto de Aveiro, impedindo-os de serem transportados pela deriva litoral. Existem também outras causas que contribuem para o acelerar deste processo, tais como a diminuição da quantidade de sedimentos fornecida pela deriva litoral e a retenção de sedimentos por parte das obras transversais de defesa costeira. Nos dias de hoje, a erosão costeira é um verdadeiro risco, e mesmo com a existência de obras fixas de protecção costeira, o avanço do mar é uma constante. Na Vagueira, a pressão urbana sobre o ecossistema costeiro, alterando as suas morfologias e interferindo no seu dinamismo, tem sido um factor interveniente no processo da erosão neste sector costeiro. Desde 1958, tem-se observado um recuo efectivo da linha de costa na Praia da Vagueira, e hoje em dia já se verifica a quase completa destruição do cordão dunar frontal e a construção de diques arenosos na tentativa de impedir a abertura de um novo canal de ligação entre o canal de Mira e o oceano e de proteger valores naturais irrecuperáveis. A construção da cartografia para cada ano em estudo permitiu a quantificação dos valores médios e absolutos do recuo da linha de costa na Vagueira desde 1958 a 2002. A monitorização da praia da Vagueira entre Outubro de 2002 e Outubro de 2003 permitiu a caracterização da praia emersa e apontar o comportamento geral da área em estudo.