Development of a TaqMan-based real-time PCR assay for detection and quantification of <i>Pythium aphanidermatum</i> in plant and soil samples

The soilborne oomycete pathogen <i>Pythium aphanidermatum</i> causes damping-off and root rot in a wide range of economically important crops and vegetables. Effective <i>P</i>. <i>aphanidermatum</i> disease management is hindered by the lack of accurate pathogen detection methods. We developed a TaqMan-based real-time PCR tool for efficient detection and quantification of <i>P</i>. <i>aphanidermatum</i> presence in plant and soil samples. <i>Eukaryotic peptide chain release factor subunit 1</i> (<i>eRF1</i>) was selected as the marker gene based on bioinformatic analysis. <i>P</i>. <i>aphanidermatum</i> DNA was quantified by using a <i>eRF1</i>-containing plasmid as the reference standard. This approach is sensitive enough to detect as low as 10 fg of <i>P</i>. <i>aphanidermatum</i> genomic DNA or 10 copies of reference plasmid. In a specificity evaluation, eight <i>P</i>. <i>aphanidermatum</i> isolates can be precisely detected out of 44 plant pathogen isolates. Moreover, <i>P</i>. <i>aphanidermatum</i> DNA can be detected in plant leaves 10 min after inoculation when there is no symptom appearance at all. Meanwhile, this assay successfully detected <i>P</i>. <i>aphanidermatum</i> zoospores in the soil at the sensitivity level of 100 zoospores/g, a density sufficient for inhibiting seedling growth. Taken together, the real-time PCR tool could be valuable for the early surveillance and quantification of <i>P</i>. <i>aphanidermatum</i> spread in the field.