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.

中文翻译：

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开发基于 TaqMan 的实时 PCR 分析，用于检测和定量植物和土壤样品中的 Pythium aphanidermatum

摘要 土传卵菌病原体 Pythium aphanidermatum 导致多种经济重要作物和蔬菜的猝倒和根腐病。缺乏准确的病原体检测方法阻碍了有效的 P. aphanidermatum 疾病管理。我们开发了一种基于 TaqMan 的实时 PCR 工具，用于有效检测和量化植物和土壤样品中的 P. aphanidermatum 存在。基于生物信息学分析，选择真核肽链释放因子亚基1（eRF1）作为标记基因。P. aphanidermatum DNA 通过使用含有 eRF1 的质粒作为参考标准进行量化。这种方法足够灵敏，可以检测低至 10 fg 的 P. aphanidermatum 基因组 DNA 或 10 个参考质粒拷贝。在特异性评估中，8 个 P. 从44株植物病原菌中，可准确检测出丝兰分离株。此外，当完全没有症状出现时，在接种后 10 分钟的植物叶片中可以检测到 P. aphanidermatum DNA。同时，该测定成功地以 100 个游动孢子/g 的灵敏度水平检测到土壤中的 P. aphanidermatum 游动孢子，该密度足以抑制幼苗生长。总之，实时 PCR 工具对于田间传播的 P. aphanidermatum 的早期监测和量化可能很有价值。足以抑制幼苗生长的密度。总之，实时 PCR 工具对于田间传播的 P. aphanidermatum 的早期监测和量化可能很有价值。足以抑制幼苗生长的密度。总之，实时 PCR 工具对于田间传播的 P. aphanidermatum 的早期监测和量化可能很有价值。