Detection of Cylindrocarpon Black-Foot Pathogens in Grapevine by Nested PCR

Cecìlia Rego, Helena Oliveira, Teresa Nascimento


Black-foot disease of grapevine has been associated with two closely related species of the genus
Cylindrocarpon, C. destructans and C. obtusisporum. However, only C. destructans isolates could be obtained from
young vines and diseased grapevine rootstock nurseries in Portugal. In the present study, an alternative to traditional
methods of detection of Cylindrocarpon spp. fungi from infected grapevine is described. In 1996, Hamelin et al.
designed species-specific primers (Dest1 and Dest4) for detection of C. destructans ITS variants from conifer seedlings.
With these primers, a DNA fragment of 400 bp was produced by direct PCR using DNA extracted from cultures
of C. destructans (60 isolates) obtained from grapevine plants. Whereas no fragments were detected when cultures of
common wood grapevine fungi were analysed, an amplicon of the same size was obtained for isolates of C. obtusisporum
revealing the failure of these primers to distinguish Cylindrocarpon species. The 400 bp fragment mentioned could
also be produced by direct PCR after adding C. destructans to healthy grapevine tissue (cv. Periquita), followed by
DNA extraction using frozen plant tissue in liquid nitrogen and polyvinylpyrrolidone (PVP) treatment. However,
amplification failed to detect C. destructans in artificially inoculated grapevine plants (cv. Periquita). Consequently,
a nested PCR was carried out by modifying the procedure described by Hamelin et al. The universal primer ITS4 and
the fungus-specific primer ITS1F were used in a first-stage fungus-specific amplification, followed by a second-stage
amplification with the primers Dest1 and Dest4 using the PCR products from stage one. This approach was found to
be a simple and reliable method for collective detection of Cylindrocarpon spp. directly from infected grapevine

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