5.5.4
DETECTION OF DMI FUNGICIDE RESISTANT STRAINS OF UNCINULA NECATOR IN AUSTRALIAN VINEYARDS

S SAVOCCHIA1, BE STUMMER1, DL WHISSON2, TJ WICKS2 and ES SCOTT2

Cooperative Research Centre for Viticulture: 1Department of Crop Protection, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia; 2South Australian Research and Development Institute, Waite Precinct, GPO Box 397, Adelaide, SA 5001, Australia

Background and objectives
Powdery mildew of grapevine, caused by the obligately biotrophic fungus, Uncinula necator, reduces grape quality and yield. U. necator reproduces both asexually and sexually, however, the relative importance of sexual reproduction in contributing to variation in Australia is unknown. A functional sexual stage creates significant opportunity for variation in U. necator, such as the development of new pathotypes and reduced sensitivity to demethylation inhibiting (DMI) fungicides.
DMIs inhibit one metabolic site within the organism, and can be applied as protective sprays when sulphur applications are inadvisable. Lack of disease control due to resistance to DMIs has been reported in Europe and USA and there is circumstantial evidence that resistance is occurring in Australia. We are developing a bioassay for fungicide resistance to determine sensitivity of U. necator isolates from different viticultural regions of Australia to DMIs such as Bayfidan (triadimenol), Rubigan (fenarimol) and Topas (penconazole). Isolates characterised in the bioassay will be used in the identification of molecular markers for detection of fungicide resistance.

Materials and methods
A collection of 85 powdery mildew isolates is presently maintained on micropropagated grapevines in vitro. Additional isolates are being collected from vineyards in different viticultural regions where DMIs have been used and where powdery mildew control has been inadequate.
Mass-produced conidia were bulked on detached leaves [1] prior to testing for fungicide sensitivity. Discs were cut randomly from disease-free, surface-sterilised Cabernet Sauvignon leaves. Technical grade triadimenol (Bayer Australia Ltd) was diluted to 10.0 to 0.01 mg/L [2]. Leaf discs were placed on filter paper and imbibed with fungicide solution or sterile distilled water, then transferred into multi-well dishes containing sterile distilled water agar amended with antibiotics. Each plate was inoculated with one isolate of U. necator. After 12 days the percentage of leaf surface infected was determined. The data were graphically presented using the logistic method and EC50 (concentration inhibiting development of fungus by 50%) determined for each isolate and each fungicide.

Results and conclusions
Preliminary results show that isolates collected from the Adelaide Hills and Southern Vales regions of South Australia have reduced sensitivity to triadimenol. Isolates representing the extremes of resistance and sensitivity, as determined in the bioassay, will be characterised using existing RFLP and PCR markers [1]. Additional markers will be developed if existing markers show no association with resistance. The information gained from these studies will provide insight into the development of DMI resistance in Australian vineyards.

References
1. Evans KJ, Whisson DL, Scott ES, 1996. Mycological Research 100, 675-80.
2. Erickson EO, Wilcox WF, 1997. Phytopathology 87, 784-91.