Studies On Risk Of Resistance In Pseudoperonospora Cubensis To Fluopicolide

Cucumber downy mildew, caused by the oomycete pathogen Pseudoperonosporacubensis, is a devastating and worldwide-distributed disease on cucumber, and responsiblefor serious losses of yield and quality in the production of cucumber. At present, thecontrol of the disease relies mainly on using synthetic fungicides. A new fungicidefluopicolide belongs to the benzamide class, and was recently registered for control of theoomycete pathogens. To clarify the risk of P. cubensis developing resistance to fluopicolide,The following studies were carried out (i) to determine the sensitivity of P. cubensis tofluopicolide;(ii) to determine whether cross-resistance relationship exists betweenfluopicolide and several oomycete fungicides;(iii) to select mutants resistant tofluopicolide by adaptation and UV irradiation, and to study the biological characteristics ofthe resistant mutants of P. cubensis;(iv) to study inheritance of resistance to fluopicolidein P. cubensis;(v) to determine effects of fungicides with mixture application on resistancedevelopment of P. cubensis; and (vi) establish the methold of preservation of P. cubensis.The main results are summarized as follows:1.105isolates of Pseudoperonospora cubensis collected from Hebei and Shandongprovinces were tested for the sensitivity of P. cubensis to fluopicolide by leaf disc floatingassay. The results showed that sensitivities to fluopicolide of the isolates of P. cubensisfrom different regions were very different in sensitivity to fluopicolide, and all thoseisolates from eight regions of Hebei and Shandong were sensitive to fluopicolide. With thefrequency of sensitivity following a normal distribution curve, the mean EC50of62strainscollected from the fields where benzamide fungicides had never been used was（0.1498±0.1090）μg/mL, which could be regarded as the baseline sensitivity of P.cubensis to fluopicolide for field resistance monitoring.2.4mutants resistant to fluopicolide were acquired from9sensitive isolates by UVirradiatiing sporangia with a mutantion frequency (7.4×10–7),7mutants resistant tofluopicolide were obtained from7isolates by sporangia adaptation on fluopicolide-treatedleaves of cucumber. Resistance factors of the11fluopicolide-resistant mutants ranged from8.0to114.9. Resistance was stable through10sporangia transfers on fungicide-free leavesof cucumber. Compared with their sensitive parents,11resistant mutants exhibiteddiversity in infection frequency, pathogenicity and sporulation ability, with few exceptions, the resistant mutant LT-3-1UVwas stronger in pathogenicity and sporulation than its parentisolate LT-3-1. No cross-resistance relationship was found between fluopicolide and theother four fungicides with the different mechanisms of action. To sum up, there could be amoderately risk of field populations of P. cubensis developing resistance to fluopicolide.3、The Pseudoperonospora cubensis was tamed for a total of seven generations byusing fluopicolide, pyraclostrobin, propamocarb hydrochloride and two kinds of mixtures,respectively. The sensitivity of tamed P. cubensis for3,5,7th generations to fluopicolide,pyraclostrobin and propamocarb hydrochloride were tested by leaf disc floating method.The results showed that the resistance developed slowly when the P. cubensis was inducedby mixture application of fluopicolide and pyraclostrobin or propamocarb hydrochloridethan three single fungicides. The resistance factors of the mutants induced by two kinds ofmixtures to fluopicolide, pyraclostrobin and propamocarb hydrochloride were up to13.49(12.06),9.63and4.20fold at the7th generation, respectively. However, the mutantsinduced by three single fungicides showed16.38,11.64and5.14fold resistance factors.The results suggested that the mixture application of fungicides could delay thedevelopment of resistance of the cucumber downy mildew to fluopicolide.4. The isolates of Pseudoperonospora cubensis were preserved under-20℃temperature on the detached leaves of cucumber and the sporangia of the preserved andunpreserved strains were tested for the sensitivity to fluopicolide by leaf disc floating assay.The fitness of the preserved isolates of P. cubensis was tested on the detached half-leavesand on the leaf discs. The results showed that the sporulating leaves under-20℃temperature conditions could be kept for up to3months, and use10%dimethyl sulfoxideplus10%skim milk as preservation solution frozen sporangia can be kept for up to6months under-20℃temperature conditions. Compared with their parent isolates, theisolates preserved under-20℃significantly decreased in pathogenicity and fitness, thelatent period became longer after cryopreservation. No significant difference insensitivities to fluopicolide existed between the frozen isolates and their parent isolates. Tosum up, it can be used as a long-term preservation method of P. cubensis by in vitro leaffreezing test and sporangium freezing assay.5. Crossing between sensitive (s) and fluopicolide-resistant (r) isolates of oppositemating types, A1and A2, yielded abundant oospores of P. cubensis. The sensitivity tofluopicolide of single-oospore F1-progeny isolates was tested by leaf disc floating method.The results showed that the sensitivity of all single-oospore F1-progeny isolates of theresistance-phenotype between two parental isolates to fluopicolide, suggesting thatfluopicolide resistance may be controlled by semidominant genes.