ALLELE-SPECIFIC REAL-TIME PCR FOR QUANTIFICATION AND DISCRIMINATION OF STEROL 14 α-DEMETHYLATION-INHIBITOR-RESISTANT GENOTYPES OF MYCOSPHAERELLA GRAMINICOLA

The level of resistance of Mycosphaerella graminicola to sterol 14 alpha-demethylation inhibitors (DMIs) is characterised by point and deletion mutations in the CYP51 gene that encodes sterol 14 alpha-demethylase, Rapid and pre-symptomatic detection of these mutations is required for effective control by fungicides. In this study, an allele-specific real-time PCR method was developed. An additional mismatched nucleotide at the third position from the single nucleotide polymorphism at the 3-prime end of each allele-specific primer stops non-specific PCR amplification. Minor groove binding specific probes were designed to quantify general strains of M. graminicola and strains that contain isoleucine at position 381 of the CYP51 protein sequence. Specific amplification of the target alleles was reproducible. A high level of discrminination between genotypes using pure fungal DNA was confirmed in vivo, based on leaf samples collected from different wheat growing regions in France. A high level of I381V-genotypes (>70%) was found in all samples. The results showed that allele-specific real-time PCR allows pre-symptomatic and accurate quantitative detection of DMI-resistant genotypes of M. graminicola, with a shorter turnaround time compared to conventional methods. The simplicity and effectiveness provided by intentional mismatch primers offer a broad range of applications for laboratory and field analysis.