SET UP OF THE BEST DNA EXTRACTION METHOD AND THE BEST SAMPLING STRATEGY FOR STANDING VINES IN ORDER TO DETECT THE PRESENCE OF TRUNK DISEASES ASSOCIATED FUNGI BY MEANS OF A MICROARRAY TOOL (MYCORRAY).

Grapevine trunk diseases (GTD) including the whole Esca complex of diseases, Eutypa dieback, Botryosphaeria cankers and Black foot are widespread in all grapevine-growing areas causing losses in quality and quantity of the crop. Since different fungal species associated with GTD can be found in the woody tissues of the same vine, the possibility of their simultaneous detection would be an important goal both for vine growers and nurseries. With this purpose a microarray tool (named MYCORRAY) has been developed. In order to set up an efficient detection assay, the best DNA extraction method from woody tissues and the best sampling strategy are the starting points to be determined. Seven DNA extraction methods were compared to ensure maximum fungal DNA extraction and purification coupled with the highest sensitivity and minimum testing costs. Moreover to establish the best sample type, woody tissues surrounding the wounds were collected by drilling, i.e. using a non lethal method for standing vines. The wood sawdust obtained was analyzed for fungal detection by means of the nested-PCR protocols available for Phaeomoniella chlamydospora, Botryosphaeriaceae, Neofusicoccum parvum, Fomitiporia/Phellinus, Ilyonectria spp. and Eutypa lata. After the set up of the best DNA extraction protocol, to reliably assess the sanitary status of individual plants, single and pooled samples were tested and compared with traditional isolation techniques. Pooled samples included sampling from four points surrounding pruning wounds and samples from the rootstock and roots. This approach was shown to minimize the number of DNA extractions required being as efficient and reliable as many single samples.