Molecular diagnostic for detecting the cytochrome b G143S – QoI resistance mutation in Cercospora beticola

The mutation G143S has been associated with high-level strobilurin resistance in laboratory mutant strains of Cercospora beticola, one of the most destructive pathogens in sugar beet plants. By using allele specific primers (PASA-PCR) and agarose gel visualization, a molecular diagnostic was developed for the detection of the G143S resistance mutation. This assay is simple and applicable in low tech laboratory settings, with high reliability when a relatively large proportion of mutated mitochondrial alleles are present in the resistant strains. To achieve detection of resistant alleles at low frequencies, a more sensitive Real Time PCR based assay capable of discriminating resistant (S143) genotypes in frequencies as low as 1:10,000 resistant:sensitive alleles was developed. Both diagnostics were successfully validated in laboratory strains. Subsequently, a large number of C. beticola isolates from QoI-treated sugar beet experimental fields in Greece were screened for resistance to Qo fungicides using these diagnostics and classic bioassays. No proportion of the 143S resistant allele was detected in all field isolates tested, which was in agreement with the phenotypes revealed by the biotests confirming that the efficacy of QoIs against C. beticola has been sustained in Greece 7 years after their introduction.