ROLE OF SINGLE SITE-SPECIFIC ALLELE REPLACEMENT INTO SVHK1 LOCUS IN THE STUDY OF STEMPHYLIUM VESICARIUM DICARBOXIMIDE AND PHENYLPYRROLE FUNGICIDES RESISTANCE

Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to dicarboximide fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to dicarboximide and phenylpyrrole fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene [1]. The goal of this ongoing study is to define the role of SNPs in SvHK1 sequence on dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. A reference sensitive strain was selected through biological and molecular assays and DNA was properly extracted and Fusion PCR technique was used to build the linear disruption vector (KOSvHK1). Fungal protoplast were obtained by enzymatic lysis of cell wall and transformed. KOSvHK1 replacement of SvHK1 gene produced null mutants which were able to grow up on Hygromycin B. Transformants are currently screened for unique, complete and site-specific insertion of KOSvHK1 using PCR-based methods and Southern Blotting assays. Interesting mutants will be transformed with linear complementation vectors, and complemented strains will be tested for the expected acquired resistance level. Assessment of the role of SNP mutations in SvHK1 sequence in S. vesicarium resistant phenotypes to dicarboximides will allows us to develop a RealTime PCR assay to quickly determine resistant allele-frequency in monitored populations. The results obtained so far will increase the possibility of quantify, prevent and manage the iprodione and fludioxonil resistance risk in the field.