Skip to main content
ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Disease and Pest Management Research Unit » Research » Publications at this Location » Publication #393128
Research Project: Development of Knowledge-based Approaches for Disease Management in Small Fruit and Nursery Crops

Location: Horticultural Crops Disease and Pest Management Research Unit

Title: Rapid and scalable dna extraction and real-time pcr assay for the detection of the boxwood blight pathogen calonectria pseudonaviculata

Author
item OHKURA, MANA - Oregon State University
item Scagel, Carolyn
item Weiland, Gerald - Jerry

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 8/7/2022
Publication Date: 11/1/2022
Citation: Ohkura, M.M., Scagel, C.F., Weiland, J.E. 2022. Rapid and scalable DNA extraction and real-time PCR assay for the detection of the boxwood blight pathogen Calonectria pseudonaviculata. Phytopathology. 112:S3.60.

Interpretive Summary:

Technical Abstract: Production and export of boxwood, the most valuable broadleaf evergreen nursery plant produced in the USA, can be severely hindered by Boxwood blight caused by Calonectria pseudonaviculata (Cp). The pathogen can be challenging to detect in the field, especially when symptoms are mild requiring large numbers of plants to be screened. Therefore, a rapid and sensitive diagnostic assay that can detect the pathogen from large amounts of plant tissue would be useful for growers and regulatory agencies. We developed a rapid and scalable crude DNA extraction protocol and a real-time Taqman assay to detect the boxwood blight pathogen, Cp, from tough boxwood leaf and stem tissues. DNA extraction from plant tissue occurs in 15 or 50 ml centrifuge tubes, which enables larger volumes of plant tissue to be processed compared to routine DNA extraction kits. In addition, the protocol does not include labor intensive steps that require use of liquid nitrogen for homogenization, or cetyltrimethylammonium bromide or phenol chloroform for removal of inhibitors. The protocol limit of detection was determined by diluting leaves displaying boxwood blight symptoms with non-diseased leaves and by diluting Cp spores in non-diseased leaves to simulate diagnostic scenarios. The assay detected Cp in symptomatic leaves diluted up to 1×104 fold with non-diseased leaves, and as low as 1000 spores in 1.2 g of non-diseased leaves. The protocol has been successfully used to detect boxwood blight from Oregon nurseries.