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A Multiplex Real-Time PCR Assay for the Detection of Puccinia horiana and P. chrysanthemi on Chrysanthemum

February 2015 , Volume 99 , Number  2
Pages  195 - 200

Jill E. Demers, Jo Anne Crouch, and Lisa A. Castlebury, Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705

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Accepted for publication 28 August 2014.

Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while P. chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification is essential to enforce quarantine measures, but the two species cannot be differentiated visually in the absence of mature spores or symptoms. A multiplex real-time PCR assay was developed to detect and discriminate between P. chrysanthemi and P. horiana. Species-specific hydrolysis probes labeled with different fluorescent dyes were designed based on the rDNA internal transcribed spacer region. Seven fresh samples and 270 herbarium specimens of chrysanthemum rust were tested with the assay with results confirmed using spore morphology. P. horiana and P. chrysanthemi were accurately detected from all fresh samples, and as little as 1 pg of template DNA was reproducibly detected. Of the herbarium specimens, 99% were positive for at least one species using the multiplex assay with 7% positive for both species. This multiplex assay can discriminate between P. chrysanthemi and P. horiana and provides an additional tool for identification of P. horiana to ensure appropriate application of quarantine measures.

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2015.