Studies On Resistance Risk And Mechanisms Of Penicillium Digitatum To Boscalid

Green mold,caused by Penicillium digitatum,is one of the most serious postharvest diseases of citrus.Green mold usually causes significant losses to citrus production.Due to the occurrence of resistance to demethylation inhibitor（DMI）fungicides,new fungicides with different modes of action are needed to control green mold.In this thesis,we studied bioactivities of the succinate dehydrogenase inhibitor（SDHI）fungicide boscalid to P.digitatum,and investigated resistance risk and resistance mechanisms of P.digitatum to boscalid.These studies have significant implications for the future application of boscalid to control citrus postharvest diseases.The baseline sensitivity of P.digitatum to boscalid was established by determining the median effective concentration（EC50）values of 189 isolates collected from five regions of China in 2017 and 2018.The frequency distribution of EC50 values was unimodal,and the mean EC₅₀ value was 0.099μg/m L±0.041（SD）.According to the EC50 values of the two fungicides for 120 isolates,there was no significant sensitivity correlation between boscalid and fludioxonil（r=0.114,P=0.338）.Similarly,thevalues of boscalid for mycelial growth did not correlate with those for spore germination（r=0.1258,P=0.168）.The preventive efficacies of boscalid at 100 and 200μg/m L against green mold were 67.3%and79.3%,respectively.Boscalid at 20μg/m L in potato dextrose agar（PDA）delayed the germination of spores.Furthermore,after the germinated spores were inoculated on fresh PDA without boscalid,the diameter of resulting mycelia colonies decreased by 77.4%.Similarly,after the germinated spores were inoculated on citrus fruit,pathogenicity decreased by 27.8%（P=0.002）.Boscalid at 0.03（P=0.001）and 0.12μg/m L（P<0.001）in PDA reduced spore production.However,the pathogenicity of these spores had no significant difference compared with the nontreated control.The lesion diameter and spore production on citrus fruit soaked with 50μg/m L boscalid decreased by 33.5%and 81.3%,respectively.After the spores produced on boscalid-treated fruit were inoculated on fresh fruit,pathogenicity and sporulation decreased by 30.1%and 70.5%,respectively.Boscalid in PDA caused more hyphae branched and twisted compared with the nontreated control.The activity of target enzyme succinate dehydrogenase（SDH）was decreased by 42.4%and61.9%,respectively,for treatments with boscalid at 0.03 and 0.12μg/m L.Boscalid at 0.12μg/m L reduced the activity of superoxide dismutase（SOD）and the content of hydrogen peroxideby 71.1%and 59.6%,respectively.In contrast,the activity of peroxidase（POD）and the content of superoxide anion（O2^-）were increased by 43.2%and 61.9%,respectively.Seven boscalid-resistant mutants of P.digitatum were attained by UV irradiation and chemical taming from wild-type isolates in the laboratory.The resistance of 5 mutants with EC₅₀values above 1000μg/m L was stable after consecutive transfers on PDA for 16generations.For the other two mutants,the EC₅₀ of HBWH20BR1 decreased from 193.2μg/m L to 21.7μg/m L,and the EC₅₀ of HBYCA44BR decreased from 14.5μg/m L to 4.3μg/m L.There was significant cross-resistance between boscalid and carboxin（r=0.817,P=0.002）,but no significant cross-resistance between boscalid and fludioxonil（r=0.372,P=0.260）or prochloraz（r=﹣0.467,P=0.147）was detected.Compared with their wild-type parental isolates,changes for the 7 resistant mutants in the mycelia growth,spore production,spore germination rate,pathogenicity,and sensitivities to Na Cl,salicylhydroxamic acid（SHAM）and H₂O₂ varied depending on mutants.Studies on resistance mechanisms showed that there was no point mutation in the Sdh A,Sdh C,and Sdh D subunits of Sdh in each resistant mutant,and Sdh B subunit of resistant mutants JXGZ96BR1,JXGZ96BR2 and HBYC51BR2 had the same point mutation H243Y.The expression levels of Sdh B and Sdh C varied greatly among different mutants.The sporulation of the resistant mutant HBYC51BR1 was inhibited on PDA amended with boscalid at 1000μg/m L.The molecular mechanism study showed that after boscalid treatment,the expression levels of sporulation-associated genes Brl A,Aba A and Wet A of the mutant HBYC51BR1 decreased by 99.3%98.4%and 80.8%,respectively,while only the expression of Wet A gene in the control mutant HBYC51BR2 decreased by 47.7%.