Resistance To SDHI Fungicides Of Double Mutations In Ccsdh In Corynespora Cassiicola

Corynespora leaf spot(CLS),caused by Corynespora cassiicola,has become one of the most important plant diseases.Due to the lack of resistant cultivars,SDHIs fungicides are the main fungicides to control Corynespora leaf spot.However,due to the extensive and repetitive application of SDHIs fungicides,the incidence of mutations in Ccsdh gene has gradually found in recent years,leading to a failure in the control of Corynespora leaf spot.Whether they are associated with SDHIs-resistance needs further verification.In our research,we analyzed the changes of resistance frequency and distribution of Ccsdh mutations from 2005 to 2020,demonstrated that the mutations in Ccsdh gene were responsible for the resistance to SDHIs,established a detection system to detect Ccsdh mutations,which provided guidance for the use of SDHIs fungicides.The main results are as follows:(1)The frequency of C.cassiicola isolates resistant to boscalid was rapidly increase in China,and the distribution and frequency of Ccsdh mutation vary in different years and regions.In our resistance monitoring,the frequency of resistant isolates rapidly increased from 9.68% to 85.88% in 2005-2020.In2014,the first resistant genotype,a histidine substitution to tyrosine at amino acid position 278 within subunit Sdh B of the SDH enzyme(B-H278Y),was detected as the dominant genotype.Over the next six years,eight genotypes were gradually detected,namely B-H278R/L,B-I280 V,C-N75 S,C-S73 P,D-D95 E,D-H105 R and D-G109 V,and the dominant genotype was change from B-H278 Y to D-D95 E and C-S73 P.We also found a tendency that Ccsdh genotype are more complex.There are differences in the dominant resistance genotypes in different regions,with C-S73 P predominating in Liaoning,B-H278 Y in Hebei and multiple Ccsdh resistance genotypes in Shandong.(2)Point mutations in the Ccsdh gene were responsible for resistance to SDHIs in Corynespora cassiicola.We confirmed,by site-directed mutagenesis of the Ccsdh B and Ccsdh D genes,that each of the mutations identified in the field strains of C.cassiicola conferred resistance to boscalid and,in some cases,cross-resistance to other SDHIs(fluopyram,carboxin and penthiopyrad).Analyses of the enzyme activity and Ccsdh B and Ccsdh D genes expression show that modifications(B-H278 Y and B-H105R)that result in a decline in SDH enzyme activity may be complemented by gene overexpression.The B-H278 Y,BI280V and D-H105 R mutants suffered large fitness penalties based on their biological properties,including conidia production and germination,mycelial growth,pathogenicity or survival abilities under environment stress.However,fitness cost was not found in the B-H278 R,D-D95 E and Sdh D＿G109V mutants.(3)Double mutations on the Ccsdh gene further increase the resistance to SDHIs in Corynespora cassiicola.We confirmed,by site-directed mutagenesis,that all double mutations(B-I280V+D-D95 E,BI280V+D-G109 V,B-I280V+D-H105 R,B-H278R+D-D95 E,B-H278R+D-G109 V,B-H278Y+D-D95 E,B-H278Y+D-G109V)conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation.Analyses of fitness showed that all double mutations had lower fitness than the wild type;most of double mutations suffered more fitness penalties than the corresponding single mutants.We also further found that double mutations(B-I280V+D-D95 E,B-I280V+D-G109 V,BI280V+D-H105R)containing low SDHI-resistant single point mutation(B-I280V)exhibited higher resistance to SDHI and low fitness penalty than double mutations(B-H278Y+D-D95 E,B-H278Y+DG109V)containing high SDHI-resistant single mutations(B-H278Y).Therefore,we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI.(4)A molecular detection system for Ccsdh mutations was established.Nine AS-PCR detection systems were respectively established for B-H278R/L/Y,B-I280 V,C-N75 S,C-S73 P,D-D95 E,D-H105 R and D-G109 V mutations,which show high sensitivity and specificity.To reduce detection time,a MASPCR assay was developed to simultaneously detect the D-D95 E,D-H105 R and D-G109 V mutant genotypes in a single PCR tube,and an AS-Realtime-PCR assay to quantify the frequency of B-H278 R mutation in field.In this study,we analyzed the frequency changes of Ccsdh mutation in C.cassiicola from 2005 to2020 and the dominant Ccsdh mutations among different regions,and established a molecular detection system for detecting Ccsdh mutations in field,which provided theoretical basis and technical support for guiding the rational use of SDHIs fungicides and the development of resistance management strategies.We also demonstrated that the point mutations in Ccsdh gene are responsible for the resistance of C.cassiicola to SDHIs,and showed that double mutations in Ccsdh gene can further enhance the resistance of C.cassiicola to SDHIs and reduce the fitness.Therefore,we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI.It plays important role in understanding the mechanism of variation and the evolution of resistance.