Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on quinone outside inhibitor (Q(O)I) fungicides. Despite the dependence on Q(O)Is for disease control for more than a decade, a comprehensive survey of Q(O)I sensitivity has not occurred in the sugar beet growing regions of France or Italy. In 2010, we collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the Q(O)I fungicide pyraclostrobin using a spore germination assay. In total, 213 isolates were identified with EC50 values greater than 1.0 mu g ml(-1) to pyraclostrobin, all of which originated from Italy. To gain an understanding of the molecular basis of Q(O)I resistance, we cloned the full-length coding region of Cbcytb, which encodes the mitochondrial Q(O)I-target enzyme cytochrome b in C. beticola. Cbcytb is a 1,162-bp intron-free gene with obvious homology to other fungal cytb genes. Sequence analysis of Cbcytb was carried out in 32 Q(O)I-sensitive (< 0.080 mu g ml(-1)) and 27 Q(O)I-resistant (> 1.0 mu g ml(-1)) isolates. All tested Q(O)I-resistant isolates harboured a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay developed to discriminate Q(O)I-sensitive and Q(O)I-resistant isolates based on the G143A mutation could detect and differentiate isolates down to approximately 25 pg of template DNA. Microsatellite analyses suggested that Q(O)I resistance emerged independently in multiple genotypic backgrounds at multiple locations. Our results indicate that Q(O)I resistance has developed in some C. beticola populations in Italy and monitoring the G143A mutation is essential for fungicide resistance management in this pathosystem.