Development of a Multiplex High-Throughput Diagnostic Assay for the Detection of Strawberry Crown Rot Diseases Using High-Resolution Melting Analysis.

Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp., and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To inform strawberry growers in a timely fashion of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the abovementioned pathogens. The multiplex HRM assays using three predesigned primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10-µl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration and target DNA dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost effective, and reliable for the timely detection of strawberry crown rot pathogens.