A robust and specific real-time PCR tool for the detection of Phytophthora lateralis in plant tissues

Phytophthora lateralis is an oomycete responsible for Port-Orford Cedar ( Chamaecyparis lawsoniana ) dieback and mortality since the 1920s in western North America. It is recommended for quarantine regulation by EPPO (A2 list) and has been recently detected in Europe. In order to implement efficient control of the disease, a sensitive real-time PCR assay was developed to detect the presence of P. lateralis in plant tissues. In this study, we used the species-specific polymorphisms within the RAS-related protein gene Ypt 1 to design a primer pair and a hydrolysis probe targeting the pathogen. The tool proved to be specific and inclusive, based on in silico and in vitro assessments, and could detect as few as 47 copies of target DNA. In addition, the test demonstrated its robustness and remained highly sensitive and specific under deliberately modified experimental conditions such as the hybridization temperature, or the reaction and DNA template volumes. The DNA extraction step from diseased tissues was also optimized, and the reliability of the results was ensured by a set of controls and a test targeting plant DNA that enabled the assessment of the quality of the extracts.