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 screening of metalaxyl tolerance in ®eld isolates of Plasmopara halstedii. High-pressure liquid chromatography (HPLC) was used to determine the fungicide concentration in the inoculation medium and in the incubated leaf tissue over the test period. These measurements revealed that the fungicide con- centration 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 application of the fungicide via seed dressing, the leaf disc method allows precise quanti®cation of the e€ective fungicide concentration at the site of infection and is less space and time consuming. Metalaxyl tolerance of P. halstedii isolates was gradually deter- mined according to the sporulation of the pathogen on sun¯ower leaf discs in the presence of increasing fungicide concentrations. Isolates collected in South Germany showed no tolerance and sporulation was prohibited when tests were carried out at 0.02 lg (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 lg (a.i.)/ml.