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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Disease and Pest Management Research Unit » Research » Publications at this Location » Publication #395344
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 from boxwood tissue for the detection of Calonectria pseudonaviculata, causal agent of boxwood blight

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

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2022
Publication Date: 5/4/2023
Citation: Ohkura, M., Scagel, C.F., Weiland, G.E. 2023. Rapid and scalable DNA extraction and real-time PCR assay from boxwood tissue for the detection of Calonectria pseudonaviculata, causal agent of boxwood blight. Plant Disease. 107(5):1279-1283. https://doi.org/10.1094/PDIS-06-22-1453-SR.
DOI: https://doi.org/10.1094/PDIS-06-22-1453-SR

Interpretive Summary: Boxwood blight is a serious plant disease affecting the $141 million US boxwood industry. Symptoms of the disease start out as leaf spots and stem lesions, and are usually followed by rapid leaf blight and significant defoliation, particularly during wet and warm weather. However, when the weather is dry, the symptoms may be very mild and difficult to detect. As a consequence, a greater number of plants must be screened in order to detect boxwood blight when symptoms are mild. A rapid boxwood blight detection assay is therefore needed to detect the pathogen from large amounts of plant tissue. To address this need, we developed a protocol to extract and amplify the DNA of the boxwood blight pathogen from large amounts of plant tissue. The protocol is specific for the boxwood blight pathogen and is sensitive enough to detect the pathogen from one infected leaf out of 1,000 to 10,000 healthy leaves or to detect up to 1000 to 10,000 spores in 1.2 of leaf tissue. This protocol will help detect the pathogen in a large scale boxwood blight survey being conducted in Oregon nurseries.

Technical Abstract: 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 rapid and scalable crude DNA extraction protocol and a Taqman real-time PCR assay to detect the boxwood blight pathogen, C. pseudonaviculata, from plant tissue. The DNA extraction protocol can process large volumes of tough boxwood tissue rapidly without using cetyl trimethylammonium bromide (CTAB) or phenol chloroform to remove inhibitors. The limit of detection was determined by diluting symptomatic boxwood leaves in non-diseased leaves and by adding spores to non-diseased 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 non-diseased leaves, and as low as 1,000 to 10,000 spores added to 1.2 g of non-diseased leaves. The method has successfully detected C. pseudonaviculata from Oregon nursery samples and the high sensitivity of the protocol allows processing large volumes of plant tissue.