Digital PCR assays for quantifying trichothecene-producing Fusarium species, including Fusarium langsethiae, F. poae, and F. sporotrichioides, in oats

Fusarium fungi cause Fusarium head blight (FHB) in oats, reducing yield and contaminating grains with harmful trichothecene mycotoxins. FHB symptoms in oats are often not visually distinct, necessitating alternative detection methods. We developed digital PCR (dPCR) assays as the most accurate DNA-based method to detect trichothecene-producing Fusarium species commonly found in oats. Building on existing quantitative PCR (qPCR) assays, we developed dPCR assays targeting all trichothecene producers (the Tri5 gene), or specific to F. langsethiae (Fl), F. poae (Fp), and F. sporotrichioides (Fs). All targeted single copy genes, except F. poae which targeted rDNA which is a variable and multi-copy target (and hence not as reliable as the other assays for quantification). Optimized dPCR assays showed excellent linearity (R² = 0.99) and greater resilience than qPCR to varying oat DNA concentrations. Overall, when comparing assay sensitivity using both fungal and field oat DNA extracts, dPCR assays were superior to qPCR for Tri5, Fl, and Fs, but the converse was true for Fp. Performance comparisons using field samples showed moderate to perfect agreement between qPCR and dPCR for Tri5 and Fl (κ = 0.5 and 0.86) and poor agreement for Fp (κ = 0.00). Strong correlations were observed between the methods for Tri5, Fl, and Fp (r = 0.88-0.97), but unlike dPCR, qPCR did not detect Fs in any of the field samples. We conclude that the dPCR assays for Tri5, Fl, and Fs offer a reliable method for quantification while that for Fp is reliable for fungal detection but less reliable for quantification of the pathogen in field samples.