Early Detection of Both Pyrenophora teres f. teres and f. maculata in Asymptomatic Barley Leaves Using Digital Droplet PCR (ddPCR)

Efficient early pathogen detection, before symptom apparition, is crucial for optimizing disease management. In barley, the fungal pathogen <i>Pyrenophora teres</i> is the causative agent of net blotch disease, which exists in two forms: <i>P. teres</i> f. sp. <i>teres</i> (<i>Ptt</i>), causing net-form of net blotch (NTNB), and <i>P. teres</i> f. sp. <i>maculata</i> (<i>Ptm</i>), responsible for spot-form of net blotch (STNB). In this study, we developed primers and a TaqMan probe to detect both <i>Ptt</i> and <i>Ptm</i>. A comprehensive k-mer based analysis was performed across a collection of <i>P. teres</i> genomes to identify the conserved regions that had potential as universal genetic markers. These regions were then analyzed for their prevalence and copy number across diverse Moroccan <i>P. teres</i> strains, using both a k-mer analysis for sequence identification and a phylogenetic assessment to establish genetic relatedness. The designed primer-probe set was successfully validated through qPCR, and early disease detection, prior to symptom development, was achieved using ddPCR. The k-mer analysis performed across the available <i>P. teres</i> genomes suggests the potential for these sequences to serve as universal markers for <i>P. teres</i>, transcending environmental variations.