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

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 tissues