Molecular characterization and development of diagnostic methods for the rapid detection of Cercospora beticola isolates with resistance to fungicides

Cercospora leaf spot (CLS), caused by Cercospora beticola Sacc., is the most destructive foliar disease of sugar beet (Beta vulgaris L.) and various strategies are used for disease management such as the application of fungicides. Fungicide resistance has been reported in C. beticola and was associated with either mutations in the beta-tubulin and cytochrome b genes or an overexpression of the C14alpha--demethylase (Cyp51) gene. Molecular characterization was conducted on various fungicide-targeted sites to develop assays for the rapid detection of C. beticola resistance. A PCR-RFLP assay was developed for the rapid detection of benzimidazole resistance in C. beticola isolates due to a single nucleotide polymorphic (SNP) site on the beta-tubulin gene. The assay involving an in vitro digestion with a BstUI restriction enzyme found ~37% C. beticola benzimidazole-resistant isolates from the High Plains. A baseline analysis was performed on 66 C. beticola isolates to determine the genetic diversity of the Cyp51 gene and found three mutations that were unrelated to DMI resistance. We investigated the potential role of a silent mutation at codon 170 as a mechanism of C. beticola DMI resistance. However, results did not support the hypothesis that the silent mutation at codon 170 was associated with C. beticola DMI resistance mechanism. Molecular analysis of two C. beticola DMI-resistant isolates found no mutation suggesting DMI resistance may not be directly caused by a mutation in the Cyp51 gene. Genetic analysis of the cytochrome b gene (the site of action for quinone outside inhibitor fungicides) found identical partial sequences from 84 C. beticola isolates. Two C. beticola QoI-resistant isolates had a SNP in the cytochrome b gene and was predicted to lead to an amino acid substitution at position 143 (G143A). A PCR-RFLP assay was developed involving an in vitro digestion with a Fnu4HI restriction enzyme for the rapid detection of C. beticola QoI-resistance. Molecular analysis of 173 C. beticola isolates for mating-type idiomorphs distribution revealed ~80% had a MAT1-1 idiomorph and ~20% had a MAT1-2 idiomorph. This was a departure from an equal proportion of mating-type genes as reported for isolates from the Red River Valley region of the United States, Western Europe, parts of Iran, and New Zealand. Results indicated a sexual cycle was likely absent in the C. beticola population from the High Plains. Linkage disequilibrium was present between mating-type idiomorphs and C. beticola benzimidazole sensitivity (resistant or sensitive), indicating asexual reproduction may be dominant in the High Plains region.