Inhibitory Effects Of Fludioxonil On Penicillium Digitatum And Penicillium Italicum

Green mold and blue mold,caused by Penicillium digitatum and Penicillium italicum,respectively,are the most serious postharvest diseases of citrus,which seriously affect the yield and quality of citrus.In China,the demethylation inhibitors(DMI)fungicide resistance of P.digitatum and P.italicum has been reported.Therefore,it is of great significance to study the biological activities of fungicides with different action mechanisms for control of P.digitatum and P.italicum.According to our previous studies,the phenylpyrrole fungicide fludioxonil has an excellent inhibitory effect on P.digitatum.In this thesis,we studied the inhibitory effect of fludioxonil on P.digitatum and P.italicum,evaluated the resistance risk of P.digitatum and P.italicum to fludioxonil,and explored its resistance mechanisms.The main results are as follows:1.From 2020 to 2021,81 strains of P.digitatum and 89 strains of P.italicum were collected and isolated from citrus orchards in five regions of China.The median effective concentration(EC50)of fludioxonil for inhibition of mycelial growth was determined and the sensitivity baseline of P.digitatum and P.italicum to fludioxonil were established.The range of EC50 values for 81 strains of P.digitatum was 0.0052-0.0336 μg/m L with an average EC50 value of 0.0127 μg/m L±0.0066(SD).The range of EC50 values for 89 P.italicum strains was 0.0075-0.0486 μg/m L,and the average EC50 value was 0.0216 μg/m L ± 0.0084(SD).2.The preventive efficacies of fludioxonil at 200 μg/m L against citrus green mold and blue mold were 46.3% and 54.1%,respectively,which were significantly lower than those of prochloraz.The indoor preventive efficacies of fludioxonil at 200 μg/m L against prochloraz-resistant strains of P.digitatum and P.italicum were 26.5% and 51.5%,respectively,which were higher than those of prochloraz.These results indicate that fludioxonil can be employed to manage prochloraz resistance in these two pathogens.3.Resistant mutants of P.digitatum and P.italicum were obtained by UV induction and chemical taming for successive 3 or 4 generations from wild-type isolates in the laboratory(Resistance factor > 9000).Resistance of P.italicum resistant mutants could be stably inherited,and the mycelial growth,spore production and pathogenicity of the mutants decreased significantly.The spore production and pathogenicity of resistant mutants of P.digitatum also decreased significantly.P.digitatum had no cross-resistance to fludioxonil and prochloraz(R=0.222,P=0.159)but had cross-resistance between fludioxonil and dimethachlone(R=0.993,P<0.001).P.italicum showed the same resistance pattern.The tolerance of resistant mutants of P.digitatum and P.italicum to NaCl,glucose,and sorbitol was significantly lower than that of their parent strains(P<0.05).The intracellular glycerol content of resistant mutants was significantly higher than that of sensitive isolates in the absence of fludioxonil.4.Two-component histidine protein kinase gene PdHK,mitogen activated protein kinase gene PdHog1,mitogen activated protein kinase kinase gene PdPbs2 and mitogen activated protein kinase kinase kinase gene PdSsk2 in the two-component signal transduction system of P.digitatum were amplified and sequenced.Compared with sensitive strains,point mutations occurred in the PdHK gene of 8 P.digitatum resistant mutants.The amino acid mutation sites of 7 strains were all located in the HAMP domain(N501I,T508 I,N546I,N546 H,L691P).The amino acid mutation site of 1 strain was located in the HATPase domain(S955L).A synonymous nucleotide point mutation in the PdHog1 gene of a resistant mutant was found.None of the 8 resistant mutants was found to harbor any mutations in PdPbs2 or PdSsk2.It was speculated that the high level of resistance to fludioxonil was caused by the amino acid mutation of two-component histidine protein kinase.5.The relative expression levels of four key kinase genes(PdHK,PdHog1,PdPbs2 and PdSsk2)of 3 P.digitatum resistant mutants and their respective parent isolates before and after fludioxonil treatment were determined by qRT-PCR.Compared with the sensitive strains,the expression of the 4 genes in the resistant mutant 20HB-WH-Pd19-BR increased significantly,but there was no significant change in the other two mutants.The results of gene expressions and mutations indicated that the resistance mechanism may be different among different mutants.