Rapid and Scalable DNA Extraction and Real-Time PCR Assay from Boxwood Tissue for the Detection of Calonectria...

Boxwood blight can be challenging to detect in the field, especially when symptoms are mild, thus requiring large numbers of plants to be screened. Therefore, a rapid diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful. Here, we present a crude DNA extraction protocol that is rapid and scalable. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyltrimethylammonium bromide or phenol-chloroform to remove inhibitors. Additionally, to detect the boxwood blight pathogen Calonectria pseudonaviculata, we developed a TaqMan probe to use with previously described PCR primers for a real-time PCR assay. The assay's limit of detection was determined by diluting symptomatic boxwood leaves in nondiseased leaves and by adding spores to nondiseased leaves to simulate diagnostic scenarios. The assay was able to detect the pathogen in symptomatic leaves diluted up to 1 × 10⁴- to 1 × 10⁵-fold in nondiseased leaves and from as low as 1,000 to 10,000 spores added to 1.2 g of nondiseased leaves. The ability to extract DNA from large volumes of plant tissue facilitates screening more plant tissue using the real-time PCR assay without increasing the number of samples to process in the lab.