Studies Of Resistance Molecular Mechanism Of CAA And Benzamide Fungicides In Phytophthora Sojae

Phytophthora sojae is a destructive oomycete pathogen which causes huge damages to soybean production worldwide.Application of chemical fungicides provides effective control against oomycete diseases.However,only a very few compounds are available for the control of oomycetes because of their phylogenetical distinctions from true fungi which can be controlled by many commercial fungicides.Carboxylic acid amide fungicides(CAAs)and benzamides are two types of effecitive fungicides against oomycetes.The research on the assement of the developing resistance risk of P.sojae to the two types of fungicides and their resistance molecular mechanism in P.sojae have rarely been reported.In this study,we attempted to fill the gap.First,the sensitivities of P.sojae to CAAs fungicides flumorph,dimethmorph,and benzamides fungicide zoxamide were detected,respectively.No spontaneous resistance to these fungicides was detected.The baseline sensitivity of P.sojae to flumorph,dimethmorph and zoxamide was estbablised with a mean EC50 of(0.54 ± 0.01)?g/ml,(0.16 ± 0.006)?g/ml and(0.05 ± 0.01)pg/ml,which can adopted for monitoring sensitivity changes in future.Five types of stable resistant mutatnts,exhibiting different resistance levels to CAA fungicides,were obtained by flumorph adaption.Type I with a low resistance level was screened out from the wild-type isolate Ps 13 through the adaption with a low concentration of flumorph.Types ?,Type ?,Type? and Type ?,with a higher resistance level to CAAs,were obtained from the Type ? by adapting Type ? on medium with a high concnetation of flumorph.In consideration of the fitness and the mutation frequency,the resistance risk of P.sojae developing to flumoph is supposed to be low to medium.The mutation I1027V was found in CesA3 in Type ?.Apart from I1027V,V1025L,G1020A,G1020S and Q992H were also detected in Type ?,Type ?,Type ? and Type ?,respectively.It is inferred that the resistance of P.sojae developing to flumorph should be a micro-evolutionary process.Two types of CAA-resistant mutants were obtained by dimethmorph adaption.Type ? with a G1020V mutation in CesA3 showed a high resistance level to all the tested CAAs;Type ? with a V1024L mutation in CesA3 showed a high resistance level to the other three CAAs except mandipropamid.In terms of the fitness parameters and the mutation frequency,the resistance risk of P.sojae developing to dimethmorph is assessed to be low to medium.Three stable zoxamide-resistant P.sojae mutants were obtained with a low frequency,but the biological characterization showed that there was no fitness penalty in the three mutants.The resistance risk of P.sojae developing to zoxamide is defined to be low to medium.The substitution C239S was found in the ?-tubulin of the resistant mutants,which is the same amino acid at position 239 in the ?-tubulin of Pythium aphanidermatum which is naturally resistant to zoxamide.Based on the mutation,two molecular detection methods AS-PCR and PCR-RLFP were constructed for rapid detection of zoxamide-resistant P.sojae isolates in field.The CRISPR/Cas9 technology was used in the study,which confirmed the above mutations in CesA3 confer the resistance to CAA fungicides,and the C239S mutation in ?-tubulin confer the resistance to the benzamide fungicide zoxamide.The study is the first application of the CRISPR/Cas9 technology in the research of fungicide resistance in oomycetes.