Sensitivity to fluopicolide of wild type isolates and biological characteristics of fluopicolide-resistant mutants in Pseudoperonospora cubensis

Cucurbit downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating disease that is distributed worldwide and affects cucumber in open fields and greenhouses. Fluopicolide, which was a novel systemic fungicide and was released in 2008, it is very effective in controlling downy mildew on cucumber and grape, potato late blight and pepper Phythophthora blight and reduces the loss caused by the diseases, but so far the potential for P. cubensis to develop resistance to fluopicolide has not been investigated. Hence, a laboratory study was undertaken to assess the risk of P. cubensis developing resistance to fluopicolide. Baseline sensitivity to fluopicolide was determined by using 75 P. cubensis isolates collected from cucumber-growing greenhouses in Hebei province, where no fluopicolide had been used for control of cucumber downy mildew before. Values of effective concentrations for 50% inhibition (EC₅₀) of sporulation ranged from 0.02 to 0.40 μg ml^?1 and were distributed as a unimodal curve, indicating that all 75 isolates were sensitive to fluopicolide. Sporangia of nine sensitive isolates were ultraviolet (UV)-irradiated, and four fluopicolide-resistant mutants were acquired at a mutation frequency of 7.4 × 10^?7. Seven mutants resistant to fluopicolide were obtained from seven isolates by sporangia adaptation on fluopicolide-treated leaves of cucumber. The EC₅₀ values for all eleven fluopicolide-resistant mutants ranged from 3.37 to 13.06 μg ml^?1 with mean resistance factors of 7.9–118.0. After 10 sporangia transfers on fungicide-free leaves of cucumber, all eleven resistant mutants remained resistant to fluopicolide with mean resistance factors of 8.2–81.3. Seven resistant mutants from the selection for resistance and one resistant mutant from UV mutagenesis exhibited stable resistance; however, the other three resistant mutants from UV irradiation became significantly less resistant. Compared to their respective sensitive parents, the eleven resistant mutants exhibited diversity in latent period, infection frequency, lesion extension and sporulation ability. Five out of the eleven resistant mutants exhibited prolonged latent period and three out of the eleven resistant mutants provided decreased infection frequency (IF) compared to their respective parents, indicating that in some cases, resistance mutation might affect the latent period and IF of P. cubensis. There were significant differences in pathogenicity and ability to produce sporangia, but this seemed not to be caused by resistance mutation. No cross-resistance was detected between fluopicolide and azoxystrobin, metalaxyl, dimethomorph, or cymoxanil. In all, there could be a moderate to high risk of field populations of P. cubensis developing resistance to fluopicolide, and populations of P. cubensis should be monitored regularly for their shift of sensitivity over years of application.