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Development of a rapid and sensitive ddPCR method for detection of Cytospora leucostoma in peach orchards

Jorge Ibarra Caballero: Department of Bioagricultural Sciences and Pest Management, Colorado State University

<div>Colorado peach production is ranked 6th highest in the U.S., accounting for 75% of fruit produced in Colorado and contributing greatly to the state’s $41 billion dollar agricultural industry. Peach production is threatened by changing environmental conditions and pest and disease problems. The main pathogen species causing losses in peach production is <em>Cytospora leucostoma</em>, which causes Cytospora canker disease. It is currently thought that <em>C. leucostoma</em> is spread via rainsplash, however dispersal by insects and wind has not been thoroughly studied. Study of the insect and wind dispersal mechanisms has been hampered by the low quantities of DNA present in these types of samples. Therefore a rapid and sensitive assay for detection of <em>C. leucostoma</em> will be essential for epidemiological studies of the disease. Herein we present the development of a ddPCR method that can selectively detect <em>C. leucostoma</em>, from its non-pathogenic congeners. Diagnostic genomic regions were selected using the assembled genomes of four <em>C. leucostoma</em> isolates and one of <em>C. parasitica</em>. Primers and probes were then designed to selectively amplify <em>C. leucostoma</em>, using ddPCR. These primers were then validated using field collected spore samples. This work will help identify dissemination mechanisms used by <em>C. leucostoma</em>, thereby supporting the development of best management practices.</div>