Widely-based full-genome analyses enable development of universal and strain-specific PCR toolkit for wheat dwarf virus detection, revealing new alternative hosts and challenging strain-host specificity

Wheat dwarf virus (WDV) is a destructive cereal virus causing significant yield losses in wheat and barley. It is transmitted by the leafhopper Psammotettix alienus and can persist in wild grasses between growing seasons, making reliable detection and strain differentiation critical for disease management. We developed a comprehensive PCR toolkit for WDV by analysing 38 complete genome sequences, reviewing, validating, and upgrading existing primers and designing new primers spanning multiple viral genome regions. The primer toolkit achieved high diagnostic and analytical specificity as it consistently detected WDV in plants and insect vectors. This enabled the separation of WDV wheat- and barley-strains through a two-step workflow: screening with universal primers, and strain assignment with strain-specific primer pairs. Field testing across 13 Hungarian sites revealed barley strain dominance in the samples, infecting not only barley but also wheat and multiple grass species. Our surveys identified three previously undocumented reservoir grasses adding to the reviewed host range of 42 species. Complete genome sequencing of one wheat-strain and two barley-strain isolates confirmed > 99% intra-strain nucleotide identity but only ~ 85% between strains. Spatial mapping demonstrated virus concentration in grassy islands with declining titers toward cultivated areas, suggesting these serve as infection reservoirs. This validated primer panel provides a robust framework for studying WDV epidemiology and developing targeted management strategies for this economically important pathogen. Understanding this model of virus-vector system and the improvement of the presented methods are key factors to combat other similarly operating plant-vector-pathogen systems.