An experimental system for characterising isolates of Uncinula necator

Clonal lines of Uncinula necator were established using conidia from diseased Vitis vinifera leaves or berries collected from various Australian viticultural regions. Techniques for the establishment of single-conidial-chain isolates of U. necator on detached leaves and the subsequent maintenance of clonal lines on micropropagated grapevines in vitro are described. Conidia were successfully mass produced by the detached leaf technique and harvested efficiently using a cyclone separator device. Conidial yields were quantified for 14 clonal lines and ranged from 42 to 112 mg per 20 leaves at the first harvest. Nucleic acid extraction from conidia resulted in high-quality DNA suitable for restriction enzyme digestion and amplification by PCR, with yields ranging from 6 to 9 ng per mg conidia. This is the first report of a DNA extraction procedure for conidia of U. necator. Using the 18 base plant intron splice junction (ISJ) primer, R1, genetic variation among five South Australian clonal lines of U. necator was identified. Three of these clonal lines originated from vines grown within a 0.5 km radius. This preliminary identification of genetic variation in U. necator and the system for handling different clonal lines provide the essential tools for further development of DNA markers and the molecular characterization of this economically important pathogen.