A Quantitative PCR Assay for Detection and Quantification of Fusarium sambucinum

Abstract

Fusarium sambucinum is an ascomycete that has been isolated from a broad range of plant hosts, including hop (Humulus lupulus L.), where it acts as a causal agent of Fusarium canker, a disease that can impact cone quality and yield in severe cases. Current diagnostic methods rely on isolation of the fungus from plant tissue, a time- and resource-intensive process with limited sensitivity, complicated by the potential presence of other Fusarium spp. that have been reported on hop. Our objective was to develop a rapid and sensitive diagnostic tool to detect and quantify F. sambucinum in plant tissues. Using a modified random amplified polymorphic DNA PCR assay, we identified a F. sambucinum-specific marker that serves as the target in a TaqMan (hydrolysis) probe quantitative PCR (qPCR) assay that can be used to detect F. sambucinum DNA in a background of plant DNA. When used to screen 52 isolates of F. sambucinum and isolates representing 13 other Fusarium spp., the assay was robust in detecting F. sambucinum while discriminating between F. sambucinum and closely related Fusarium spp., including F. venenatum. Furthermore, this assay reliably detects as little as 1 pg of F. sambucinum DNA in a background of total DNA from plant tissue. Within-sample comparisons of this qPCR assay with traditional cultural isolation methods demonstrated the greater sensitivity of the qPCR-based method for detection of F. sambucinum. When used to screen 220 asymptomatic stem samples, the qPCR assay detected F. sambucinum in 100 samples (45.5%); by comparison, F. sambucinum was detected in only 3 samples (1.4%) by culturing methods. Moreover, quantification of F. sambucinum DNA was possible for 60 of these samples, indicating the utility of the qPCR assay for early detection. This assay should be useful in diagnostic and epidemiological applications to detect and quantify F. sambucinum from multiple hosts and environmental samples.