Optimizing Molecular Detection for the Hop Downy Mildew Pathogen Pseudoperonospora humuli in Plant Tissue

Abstract

Downy mildew-free hop plantlets and rhizomes are essential to limit the introduction of this destructive pathogen, Pseudoperonospora humuli, into hopyards. The objective of this research was to determine which DNA-based diagnostic tools are optimal for P. humuli detection in plant tissue. Quantitative real-time PCR (qPCR) assays with TaqMan probes for nuclear ( c125015.3e1) and mitochondrial ( orf359) DNA loci were developed and tested side by side. A recombinase polymerase amplification (RPA) assay was designed based on the orf359 DNA locus. The mitochondrial qPCR assay had a 10-fold lower limit of detection (100 fg of genomic DNA) and was 60% more effective in detecting P. humuli in asymptomatic stems than the nuclear-based assay. Both qPCR assays had linear standard curves ( R2 > 0.99) but lacked the quantitative precision to differentiate leaf infections beyond 1 day postinoculation. A wide range of Cq values (≥4.9) in standardized tests was observed among isolates, suggesting that the number of mitochondria and nuclear DNA targets can vary. The absence of P. humuli DNA in symptomatic rhizomes was explained, in part, by the detection of Phytophthora DNA. However, the Phytophthora-specific atp9-nad9 assay cross-reacted with P. humuli, leading to false positive amplification. Sensitivity in the RPA assay was reduced by crude plant DNA extract. Improvements to the objectivity of calling positive amplifications and determining the onset of amplification from RPA fluorescence data were realized by applying the first and second derivatives, respectively. The orf359 qPCR assay is specific and sensitive, making it well suited for P. humuli diagnostics in plant tissue.