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

Abstract

ABSTRACT The soilborne oomycete pathogen Pythium aphanidermatum causes damping-off and root rot in a wide range of economically important crops and vegetables. Effective P. aphanidermatum 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 P. aphanidermatum presence in plant and soil samples. Eukaryotic peptide chain release factor subunit 1 (eRF1) was selected as the marker gene based on bioinformatic analysis. P. aphanidermatum DNA was quantified by using a eRF1-containing plasmid as the reference standard. This approach is sensitive enough to detect as low as 10 fg of P. aphanidermatum genomic DNA or 10 copies of reference plasmid. In a specificity evaluation, eight P. aphanidermatum isolates can be precisely detected out of 44 plant pathogen isolates. Moreover, P. aphanidermatum DNA can be detected in plant leaves 10 min after inoculation when there is no symptom appearance at all. Meanwhile, this assay successfully detected P. aphanidermatum 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 P. aphanidermatum spread in the field.