Inhibitory Effect Of Prochloraz And Pyrimethanil On Penicillium Digitatum And Resistance Mechanisms

Green mold,caused by Penicillium digitatum,is one of the most important postharvest diseases of citrus.At present,the primary method to control green mold is to use fungicides.In this thesis,the inhibitory effects of the demethylation inhibitor?DMI?fungicide prochloraz and the anilino pyrimidines?AP?fungicide pyrimethanil on P.digitatum and their resistance mechanisms were studied.These studies have important implications for judicious application of prochloraz and pyrimethanil to control P.digitatum.By measuring the effective concentration causing 50%mycelial growth inhibition(EC₅₀)of prochloraz against 129 isolates of P.digitatum collected in 2017,a relative baseline sensitivity of P.digitatum to prochloraz was established.According to the distribution of EC₅₀values,112 isolates with EC₅₀values lower than 0.05?g/m L were tentatively considered to be sensitive to prochloraz,and the mean EC₅₀value was 0.0090±0.0054?g/m L?SD?.The 17 isolates with EC₅₀value higher than0.05?g/m L were tentatively considered to have reduced sensitivity to prochloraz,and the mean EC₅₀value was 0.108±0.095?g/m L?SD?.Prochloraz at 0.005?g/m L and0.01?g/m L reduced mycelial cell membrane permeability,destroyed spore cell membrane integrity,and reduced mycelial cell ergosterol content,but had no significant effect on the content of mannan in the cell wall of mycelia.Compared with prochloraz-sensitive isolates,c DNA sequence of CYP51B gene of the resistant isolate HBYC-A29R(EC₅₀=5.68?g/m L)had a point mutation at codon 405 GGT to AGT,leading to an amino acid substitution of G405S.Another resistant isolate SCMS-39R(EC₅₀=3.97?g/m L)had point mutations at codon 389 GGT to TGT and 390 TAT to TCT,leading to amino acid substitutions of G389C and Y390S,respectively.The promoter region of CYP51B gene of the resistant isolates had a 199 bp insertion sequence,and this might be the underlying molecular mechanism for the enhanced relative expression of CYP51B gene in resistant isolates compared with sensitive ones.Baseline sensitivity of P.digitatum to pyrimethanil was established.According to the distribution of EC₅₀values of 135 isolates,132 isolates of P.digitatum with EC₅₀values lower than 0.30?g/m L were tentatively considered to be sensitive to pyrimethanil,and the mean EC₅₀value was 0.134±0.034?g/m L?SD?.The 3 isolates with EC₅₀values higher than 0.30?g/m L were tentatively considered to have reduced sensitivity to pyrimethanil and the EC₅₀values of the three isolates were 0.318,0.363,and 0.436?g/m L.Pyrimethanil at 0.1 and 0.2?g/m L significantly reduced the activities of cellulase and pectinase and the methionine content in mycelia.Methionine in PDA at 100?g/m L could alleviate the toxicity of pyrimethanil at 0.1and 0.2?g/m L to sensitive isolates.Highly resistant isolates?Resistance Factor>1000?were attained after induction on pyrimethanil-amended PDA for 4 generations.Compared with sensitive isolates,pyrimethanil-resistant mutants had reduced pathogenicity and enhanced resistance to hydrogen peroxide.There was no cross-resistance between pyrimethanil and prochloraz?r=0.377,P=0.123?.No mutation was found in the coding region of the cystathionine gamma synthase genes?CGS1 and CGS2?of the pyrimethanil-resistant mutants compared to the sensitive isolates.The relative expression levels of the CGS1 and CGS2 genes of the resistant-mutants were decreased by 51.3%?P=0.235?and 62.8%?P=0.042?,respectively.These results indicated that the resistance of P.digitatum to pyrimethanil might be related to the reduced expression of CGS genes.