Use of PCR-RFLP Analysis to Monitor Fungicide Resistance in Cercospora beticola Populations from Sugarbeet (Beta vulgaris) in Michigan, United States

Genetic resistance to Quinone outside inhibitor (QoI) and benzimidazole fungicides may be responsible for a recent decline in efficacy of chemical control management strategies for Cercospora leaf spot (CLS) caused by Cercospora beticola in Michigan sugarbeet (Beta vulgaris) fields. The target genes and fungicide resistance mutations are known for these two fungicides. Based on this, two standard polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays were developed to detect the G143A and E198A point mutations in the fungal mitochondrial cytochrome b and the beta-tubulin genes, respectively. These mutations confer a high level of resistance to either QoI or benzimidazole fungicides. The presence of the G143A and E198A mutations was monitored within C. beticola populations recovered from Michigan sugarbeet production fields collected in 2012. Both the QoI-resistant cytochrome b allele and the benzimidazole-resistant beta-tubulin allele were detected directly from leaf tissue following a PCR-RFLP assay. Using either detection assay, the G143A and E198A mutations were detected in over 90% of the 118 field samples originating from Michigan sugarbeet production under fungicide management programs for CLS control. Monitoring of the G143A and E198A mutations in fields located in 9 counties and 58 townships indicated that the mutations were widespread in Michigan sugarbeet production areas. The PCR-based assays used and developed in this study were effective in detecting the presence of the G143A and E198A mutations in C. beticola field populations from Michigan.