Grapevine yellows: Comparison of different procedures for DNA extraction and amplification with PCR for routine diagnosis of phytoplasmas in grapevine

Authors

  • E. Boudon-Padieu
  • A. Béjat
  • D. Clair
  • J. Larrue
  • M. Borgo
  • L. Bertotto
  • E. Angelini

DOI:

https://doi.org/10.5073/vitis.2003.42.141-149

Keywords:

phytoplasma, grapevine yellows, Flavescence dorée, Elm yellows, 16SrV, diagnosis, DNA extraction, nested-PCR, ribosomal DNA, non-ribosomal DNA, Vitis vinifera, Catharanthus roseus

Abstract

In order to devise a procedure to be used as reference for detection of grapevine phytoplasmas and monitoring of Flavescence dorée, 12 combinations comprising three methods of plant DNA extraction and 4 procedures for amplification in polymerase chain reaction of phytoplasma DNA were examined in parallel using the same plant tissues infected with phytoplasmas. In a first series tissues of periwinkles (Catharanthus roseus) infected with phytoplasma isolates of the Elm yellows group (16SrV) and maintained in the greenhouse, were used. In a second series tissues of grapevines (Vitis vinifera) naturally infected with Flavescence dorée or Palatinate grapevine yellows phytoplasma were used. The DNA preparations obtained with each of the three extraction procedures were used undiluted or serially diluted, as target DNA in the 4 nested-polymerase chain reactions. The results showed differences in the efficiency among different methods of extraction as well as in the sensitivity among the DNA amplification procedures, which improved when DNA extracted from field grapevines was diluted. After additional comparative validation on numerous field-collected samples of GY-affected grapevines, the quickest extraction procedure was selected for use in routine diagnosis, with nested-PCR amplification either of ribosomal DNA or of the FD9 DNA fragment specific for Flavescence dorée and other 16SrV group phytoplasmas.


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Published

2015-04-21

Issue

Vol. 42 No. 3 (2003): Vitis

Section

Article

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