Occurrence of boscalid resistance in cucumber powdery mildew in Japan and molecular characterization of the iron-sulfur protein of succinate dehydrogenase of the causal fungus

A total of 74 mass isolates of cucumber powdery mildew fungus (Podosphaera xanthii) were collected from commercial greenhouses with a history of boscalid use in Ibaraki Prefecture, Japan, and tested in a leaf disk assay for their sensitivity to boscalid. The mildew development of 40 of 74 isolates and five sensitive reference isolates on the disks was completely suppressed at 5 mu g boscalid/ml. The minimum inhibitory concentrations (MIC) for the remaining 34 isolates were 50 mu g/ml or higher, and 21 of these isolates also grew well at 500 mu g/ml. Six single-spore isolates were resistant to boscalid with MIC values higher than 500 mu g/ml; four of these were moderately resistant (MR), and two were very highly resistant (VHR) isolates. The growth of MR isolates was almost completely suppressed at 500 mu g/ml, whereas two VHR isolates grew vigorously at 500 mu g/ml. In foliar inoculation tests of potted cucumber plants, the efficacy of boscalid (500 mu g/ml) against both MR and VHR isolates was very low. Partial DNA fragment of the iron-sulphur protein subunit (SdhB) gene of succinate dehydrogenase was PCR-amplified from five sensitive and five resistant isolates and directly sequenced, revealing that VHR isolates possess a substitution from a highly conserved histidine (CAT) to tyrosine (TAT) in a third cysteine-rich center of a putative SdhB, whereas MR isolates so far have not been found to have such substitution in SdhB.