POSTERS: Fungicide and antibiotic resistance
Development of a rapid isothermal assays to detect QoI resistance in Erysiphe necator the causal agent of grape powdery mildew
Nancy Sharma - Michigan State University. Marivi Colle- Michigan State University, Timothy Miles- Michigan State University, Walter Mahaffee- USDA ARS, Tara Neill- USDA ARS
Quinone outside inhibitor fungicides (QoIs) are frequently applied for management of grape powdery mildew. However, repetitive use of QoIs can lead to development of resistance in E. necator populations as the result of a single nucleotide polymorphism (SNP). Currently, several allelic discrimination assays are available for this SNP, all of which require DNA extraction and costly laboratory instruments. Two isothermal approaches were designed to detect the SNP associated with QoI resistance, namely a recombinase polymerase amplification (RPA) assay and a loop-mediated isothermal amplification (LAMP) assay. These techniques are inexpensive, rapid, field-portable and fit for crude DNA. The RPA assay was sensitive to 1pg of crude DNA but was unable to discriminate the SNP. For LAMP, a set of four primers were designed to amplify the target DNA containing the SNP. The LAMP assay was able to discriminate this SNP and was optimized to improve the sensitivity and specificity by modifying, 1) primer concentration, 2) reagents, and 3) reaction temperatures. Results were compared with other allelic discrimination assays and amplifications were observed using multiple platforms (e.g. portable reader units and a qPCR machine). This assay was validated by testing outgroups of related powdery mildew species and QoI resistant and sensitive isolates of E. necator collected from different locations around the US. This technique provides growers with another tool to rapidly monitor the buildup of QoI resistance in E. necator populations.