Revisiting the PCR-Based Molecular Approaches for Mycotoxigenic Fusarium Species Detection and Quantification in Wheat

Fusarium species cause yield losses in wheat production through fusarium head blight (FHB) and the associated contamination of regulated mycotoxins, such as trichothecenes and zearalenone. Despite the widespread use of PCR-based molecular approaches for Fusarium detection, quantification, and chemotyping, most primers were developed prior to both modern phylogenetic reclassification and the availability of high-quality genome assemblies, leaving their specificity and robustness largely untested. Existing PCR- and qPCR-based assays for Fusarium detection in wheat were reviewed and re-evaluated in silico using a curated genome panel. Of 53 species-specific primer pairs, 14 (26.4%) achieved high-specificity grades (A-B), whereas 25 (47.2%) were lower performing (D-E), mainly due to cross-reactivity or inconsistent target amplification. Chemotype assays targeting TRI and ZEN genes showed stronger agreement with reported chemotypes, especially for informative TRI loci such as Tri3, Tri7, and Tri12. To support improved qPCR assay design and reporting, we propose FusaMIQE, a Fusarium-adapted framework based on MIQE 2.0 guidelines, tailored to the specific challenges of Fusarium diagnostics in wheat. Together, this manuscript provides the first in silico assessment of PCR/qPCR primers as diagnostic tools for FHB pathogens and associated recommendations for good practice (FusaMIQE) of Fusarium diagnostics in wheat.