Search

Previous surveys conducted in commercial nurseries found that different wood fungi, namely Cylindrocarpon spp., Botryosphaeriaceae, Phomopsis viticola and Phaeomoniella chlamydospora infect grapevine cuttings. Two fi eld trials were carried out to evaluate the effectiveness of cyprodinil + fl udioxonil, pyraclostrobin + metiram, fl udioxonil and cyprodinil to prevent or reduce natural infections caused by such fungi. Rootstock and scion cuttings were soaked in fungicidal suspensions for 50 min prior to grafting. After callusing, the grafted cuttings were planted in two commercial fi eld nurseries with and without a previous history of grapevine cultivation. After nine months in the nursery, the plants were uprooted and analysed for the incidence and severity of the wood fungi. Plants uprooted from the fi eld without a previous history of grapevine cultivation were generally less strongly infected by wood fungi. Under this condition, only the mixture cyprodinil + fl udioxonil simultaneously reduced the incidence of Cylindrocarpon and Botryosphaeriaceae fungi, as well as the severity of Cylindrocarpon infections. Treatments did not produce signifi cant differences in the incidence and severity of P. viticola, and Pa. chlamydospora. For plants grown in the fi eld with a grapevine history, all fungicides except cyprodinil signifi cantly reduced the incidence and severity of Cylindrocarpon fungi. Also, the incidence and severity of Botryosphaeriaceae pathogens were signifi cantly decreased both by cyprodinil + fl udioxonil and by cyprodinil. No signifi cant differences were noticed for P. viticola incidence and severity, and Pa. chlamydospora was not detected again. These results suggest that the practice of soaking grapevine cuttings in selected fungicides prior to grafting signifi cantly reduces Cylindrocarpon spp. and Botryosphaeriaceae infections, thus improving the quality of planting material.

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.