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Downy mildew of hops represents a serious disease affecting hops production in all growing regions. Disease management is primarily based on the application of fungicides at regular intervals based on a short-term forecasting methodology that is essential for evaluating the occurrence of theoretical infections. To enable a more reliable assessment of the pathogen’s presence in a given area, spore traps capturing airborne Pseudoperonospora humuli sporangia can be utilized. The use of quantitative real-time PCR (qRT-PCR) for the detection of sporangia collected by these traps allows for the elimination of laborious and time-consuming microscopic counting. Among four tested P. humuli-specific nuclear DNA sequences, an effective qRT-PCR detection method was developed based on the c127233.5e3 sequence. This detection approach was used for the quantification of sporangia from volumetric spore trap samples collected in situ under field conditions at three selected localities in Bohemia and Moravia during the 2021–2022 period. The obtained results were compared with the short-term forecasting method of the downy mildew (HDM) weather index (I) based on meteorological data. The overall course of the HDM weather index (I) closely correlated with the occurrence of sporangia: after reaching the maximum HDM weather index (I) value, the highest sporangium detection was observed with a time delay of 1–2 weeks at all the monitored sites. The results corresponded well with data obtained from volumetric spore traps in Germany, and the qRT-PCR method proved to be fully comparable to light microscopy. The combination of volumetric spore traps and qRT-PCR can significantly improve the precision of short-term forecasting systems for P. humuli infection, thereby enabling more efficient fungicide application programs in hops protection and contributing to a better understanding of the pathogen’s dispersal dynamics.