Molecular Diagnostics of the Wheat Leaf Spot Complex Using the β-Tubulin 1 Gene

The wheat leaf spot complex is a globally pervasive foliar disease caused by multiple fungal pathogens: <i>Pyrenophora tritici-repentis</i> (tan spot), <i>Parastagonospora nodorum</i> and <i>Parastagonospora pseudonodorum</i> (septoria nodorum blotch), <i>Zymoseptoria tritici</i> (septoria tritici blotch), and <i>Bipolaris sorokiniana</i> (spot blotch). Diagnostic challenges arise from overlapping symptoms and similar morphologies. We evaluated previously released molecular diagnostic tools and found that they either lacked specificity or failed to detect all species in the complex. Existing methods target different multicopy genomic regions and lacks validation against other wheat-associated pathogens. To overcome these limitations, we developed a detection tool targeting a single copy, conserved gene (β-tubulin 1, <i>tub1</i>) across all species. Species-specific primers were designed for multiplex PCR (mPCR) and TaqMan-based real-time quantitative PCR (qPCR), enabling sensitive, specific, and simultaneous quantification. The qPCR accurately quantified pathogen biomass with detection limits down to 0.04 pg of fungal DNA. We further showed that assays were highly accurate and species-specific when tested on wheat tissues inoculated under controlled conditions with defined single-species or mixed infections, as well as on naturally infected samples. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with selected restriction enzymes further distinguished species with unique cleavage patterns, providing an easy to use and clear identification system. Moreover, we confirmed that the assays do not cross detect barley-associated species such as the barley leaf spot pathogen <i>Pyrenophora teres</i>, ensuring robustness for use where host overlaps occurs. This comprehensive diagnostic provides a rapid and reliable detection, quantification of these pathogens, supporting improved disease diagnosis and enhance breeding for resistance.