AN EXPERIMENTAL SYSTEM FOR CHARACTERIZING ISOLATES OF UNCINULA NECATOR

Clonal lines of Uncinula necator were established using conidia from d iseased Vitis vinifera leaves or berries collected from various Austra lian viticultural regions. Techniques for the establishment of single- conidial-chain isolates of U. necator on detached leaves and the subse quent maintenance of clonal lines on micropropagated grapevines in vit ro are described. Conidia were successfully mass produced by the detac hed leaf technique and harvested efficiently using a cyclone separator device. Conidial yields were quantified for 14 clonal lines and range d from 42 to 112 mg per 20 leaves at the first harvest. Nucleic acid e xtraction from conidia resulted in high-quality DNA suitable for restr iction enzyme digestion and amplification by PCR with yields ranging f rom 6 to 9 ng per mg conidia. This is the first report of a DNA extrac tion procedure for conidia of U. necator Using the 18 base plant intro n splice junction (ISJ) primer, RI, genetic variation among Eve South Australian clonal lines of U. necator was identified Three of these cl onal lines originated from vines grown within a 0.5 km radius. This pr eliminary identification of genetic variation in U.necator and the sys tem for handling different clonal lines provide the essential tools fo r further development of DNA markers and the molecular characterizatio n of this economically important pathogen.