Development of qPCR Assays to Detect and Quantify Fusarium spp. on Hemp (Cannabis sativa) Flowers

Fusarium head blight (FHB) is one of the most important diseases affecting hemp. Multiple Fusarium spp. have been reported to be associated with FHB on hemp. The most commonly isolated species were those that potentially produce mycotoxins, such as F. graminearum (FG) and F. sporotrichioides (FS). Specific and sensitive detection assays of these species can assist in disease management and prevention of mycotoxin contamination. This study aimed to develop species-specific quantitative PCR (qPCR) assays for detection and quantification of FG and FS on hemp. The specificity of previously designed primers from several studies targeting various Fusarium genes were evaluated toward a range of Fusarium spp. and other pathogens isolated from hemp. The potential of the qPCR assays to detect and quantify FG and FS in hemp were evaluated using inoculated hemp. Specific amplification was observed with qPCR assays using primers based on the 3^’-coding regions of the galactose oxidase (gaoA) gene and the translation elongation factor 1 alpha (TEF1) gene for FG and FS assays, respectively. The effective dynamic range for the FG assay was 0.4 pg to 0.4 ng with 5 orders of magnitude, and the range for the FS assay was 4.0 pg to 0.4 ng with 4 orders of magnitude. The ability of the assays to reliably quantify fungal DNA in hemp tissue was demonstrated. The level of specificity and sensitivity of the developed assays reflects their potential to detect and quantify the pathogens in field samples, which will potentially have significant implications for pathogen monitoring and disease management.