Leaf disc inoculation, a fast and precise test for the screening of metalaxyl tolerance in sunflower downy mildew

A new method, based on leaf disc inoculation, was developed for the screeni ng of metalaxyl tolerance in field isolates of Plasmopara halstedii. High-p ressure liquid chromatography (HPLC) was used to determine the fungicide co ncentration in the inoculation medium and in the incubated leaf tissue over the test period. These measurements revealed that the fungicide concentrat ion inside the leaf tissue within 24 h had adjusted to the concentration in the outer medium and remained constant for the time of cultivation over a period of more than 11 days. In contrast to whole seedling tests with appli cation of the fungicide via seed dressing, the leaf disc method allows prec ise quantification of the effective fungicide concentration at the site of infection and is less space and time consuming. Metalaxyl tolerance of P. h alstedii isolates was gradually determined according to the sporulation of the pathogen on sunflower leaf discs in the presence of increasing fungicid e concentrations. Isolates collected in South Germany showed no tolerance a nd sporulation was prohibited when tests were carried out at 0.02 mug (a.i. )/ml of metalaxyl or more. In contrast, a tolerant French isolate developed sporangia on leaf discs incubated in a metalaxyl solution of 100 mug (a.i. )/ml.