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ARS Home » Northeast Area » Washington, D.C. » National Arboretum » Floral and Nursery Plants Research » Research » Publications at this Location » Publication #388671
Research Project: Evaluation and Genetic Improvement of Woody Ornamental Landscape Plants

Location: Floral and Nursery Plants Research

Title: High-resolution melting analysis enables efficient detection and differentiation of two boxwood blight pathogens by qPCR assays

Author
item Gouker, Fred
item Guo, Yonghong
item Pooler, Margaret

Submitted to: PhytoFrontiers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/2022
Publication Date: 3/20/2022
Citation: Gouker, F.E., Guo, Y.H., Pooler, M.R. 2022. High-resolution melting analysis enables efficient detection and differentiation of two boxwood blight pathogens by qPCR assays. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-21-0066-SC.
DOI: https://doi.org/10.1094/PHYTOFR-09-21-0066-SC

Interpretive Summary: Boxwood blight is a devastating fungal disease of boxwood plants that has caused economic losses to the entire horticultural chain, from growers to consumers, primarily in the U.S. and Europe. Identification and detection of this pathogen from infected plant material could play a significant role in breeding and selection of resistant cultivars and development of disease management strategies in the ornamental nursery industry. ARS Scientists from the US National Arboretum developed a fast, inexpensive, single tube method to extract DNA from boxwood leaves and cultures of the fungal pathogen. They then used a sensitive PCR method followed by a high-resolution melt analysis of the PCR products to detect and differentiate between the two species of the pathogen that causes boxwood blight. This method of rapid DNA extraction followed by reliable detection and differentiation of fungal species is a powerful tool in the quest to control boxwood blight in the landscape.

Technical Abstract: Boxwood blight is a devastating disease caused by fungal pathogens Calonectria pseudonaviculata (Cps) and Calonectria henricotiae (Che). Identification and detection of this pathogen from infected plant material could play a significant role in breeding and selection of resistant cultivars and development of disease management strategies in the ornamental nursery industry. We designed a simple, single tube method for extraction of PCR-amplifiable DNA from boxwood leaves and cultures of the Calonectria pathogen. Previously developed fungal-specific primers based on histone and calmodulin regions were used to detect and distinguish between Cps and Che using real-time PCR and high-resolution melt analysis, with clearly discernable melting temperature differences of 0.5 °C between amplified products. Here we describe this single-tube DNA extraction and qPCR-HRM assay as a suitable rapid method to detect and differentiate between Cps and Che species directly from plant tissue.