|Marra, Robert - Connecticut Agricultural Experiment Station|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 7/1/2012
Publication Date: 7/1/2012
Citation: Crouch, J., Marra, R.E., Rossman, A.Y. 2012. Real-time PCR detection of the boxwood blight pathogen Calonectria pseudonaviculata. Phytopathology. 102(7):S4.26-S4.27.
Technical Abstract: Boxwood blight is a newly emergent, destructive disease of boxwood (genus Buxus), caused by the ascomycete fungus Calonectria pseudonaviculata. Initially identified in Europe in the mid-1990s, the disease was first reported in the U.S. in CT, NC and VA during October 2011. In less than four months, boxwood blight appeared in six additional states along the eastern seaboard and in the Pacific Northwest. Substantial losses due to boxwood blight have been reported from wholesale nurseries in the states of CT and NC. Current identification protocols for boxwood blight rely upon assessment of macroscopic symptoms and microscopic examination of the cylindrical asexual spores. Because fungicide treatments may suppress outward disease symptoms and fungal growth, rather than providing complete eradication of the causal fungus, the pathogen may elude detection. Therefore, to reliably identify the presence of C. pseudonaviculata in boxwood tissue, a real-time PCR assay based on a 74-bp amplicon in the beta tubulin (Tub2) region was developed. Assay specificity relied on a 5-carboxyfluorescein (FAM) fluorophore-labeled hydrolysis probe modified with four locked nucleic acid bases. Species-specific, reproducible identification of the fungus was made from as little as 0.0050 fg of DNA. This assay will provide a useful tool for monitoring the distribution and incidence of boxwood blight in the United States.