Co-operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood

  1. Martos, S. 1
  2. Torres, E. 5
  3. El Bakali, M.A. 5
  4. Raposo, R. 2
  5. Gramaje, D. 4
  6. Armengol, J. 4
  7. Luque, J. 3
  1. 1 Universitat Autònoma de Barcelona
    info

    Universitat Autònoma de Barcelona

    Barcelona, España

    ROR https://ror.org/052g8jq94

  2. 2 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
    info

    Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria

    Madrid, España

    ROR https://ror.org/011q66e29

  3. 3 Institut de Recerca i Tecnologia Agroalimentaries
    info

    Institut de Recerca i Tecnologia Agroalimentaries

    Barcelona, España

    ROR https://ror.org/012zh9h13

  4. 4 Universidad Politécnica de Valencia
    info

    Universidad Politécnica de Valencia

    Valencia, España

    ROR https://ror.org/01460j859

  5. 5 Laboratori de Sanitat Vegetal DAR, Via Circulació Nord, Tram VI Carrer 3, Zona Franca, 08040 Barcelona, Spain
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Revista:
Journal of Phytopathology

ISSN: 0931-1785

Año de publicación: 2011

Volumen: 159

Número: 4

Páginas: 247-254

Tipo: Artículo

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DOI: 10.1111/J.1439-0434.2010.01758.X SCOPUS: 2-s2.0-79952307291 GOOGLE SCHOLAR

Otras publicaciones en: Journal of Phytopathology

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Resumen

The technique consisting of the co-operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co-operational PCR for P. chlamydospora and 17 additional grapevine-associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays. © 2010 Blackwell Verlag GmbH.