Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/25/2012
Publication Date: 11/1/2012
Citation: Birla, K., Rivera-Varas, V., Secor, G.A., Khan, M.F.R., Bolton, M.D. 2012. Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance. European Journal of Plant Pathology. 134(3):475-488. Interpretive Summary: 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 (QOI) fungicides, which are also known as strobilurins. European sugar beet growers have relied on strobilurins for more than a decade, yet no strobilurin sensitivity surveys have been carried out. In 2010, we collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed isolate sensitivity to pyraclostrobin, a commonly used strobilurin to control CLS. Although all isolates from France were sensitive to pyraclostrobin, over 200 isolates from Italy were resistant. Since strobilurin fungicides target the fungal enzyme cytochrome b, we cloned the cytochrome b (Cbcytb) gene from C. beticola. We sequenced the full-length gene from sensitive and resistant isolates to find that all resistant isolates harbored a mutation that caused an amino acid exchange in the cytochrome b protein. Since all resistant isolates harbored the mutation, we designed a set of primers that specifically amplified either the sensitive or the resistant version of the gene. These primers will be useful tools for screening field isolates for strobilurin resistance. We carried out additional molecular analyses that suggest that resistance occurred independently at several locations, as opposed to resistance occurring at one location and spreading from there. Our results indicate that strobilurin resistance can be found in Italy and is associated with a mutation in the Cbcytb gene. Careful monitoring of resistance is vital to ensure the efficacy of strobilurins in Europe.
Technical Abstract: 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 (QOI) fungicides. Despite the dependence on QOIs for disease control for more than a decade, a comprehensive survey of QOI 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 QOI fungicide pyraclostrobin using a spore germination assay. In total, 213 isolates were identified with EC50 values greater than 1.0 µg ml-1 to pyraclostrobin, all of which originated from Italy. To gain an understanding of the molecular basis of QOI resistance, we cloned the full-length coding region of Cbcytb, which encodes the mitochondrial QOI-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 QOI-sensitive (<0.080 µg ml-1) and 27 QOI-resistant (>1.0 µg ml-1) isolates. All tested QOI-resistant isolates harbored 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 QOI-sensitive and QOI-resistant isolates based on the G143A mutation could detect and differentiate isolates down to approximately 25 pg of template DNA. Microsatellite analyses suggested that QOI resistance emerged independently in multiple genotypic backgrounds at multiple locations. Our results indicate that QOI resistance has developed in some European C. beticola populations in Italy and monitoring the G143A mutation is essential for fungicide resistance management in this pathosystem.