Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2014
Publication Date: 2/1/2015
Citation: Demers, J.E., Crouch, J., Castlebury, L.A. 2015. A multiplex real-time PCR assay for the detection of Puccinia horiana and P. chrysanthemi on chrysanthemum. Plant Disease. 99(2):195-200. Interpretive Summary: Rust fungi cause many serious diseases of ornamental and crop plants around the world. One of these, Puccinia horiana, causes a severe disease called chrysanthemum white rust, which can cause up to 80% losses in some varieties of chrysanthemums. This fungus is subject to strict quarantine regulations as it is not present in the United States. Another similar rust fungus, Puccinia chrysanthemi, which is already present in the United States, causes a mostly cosmetic disease with no serious losses. In this study, a diagnostic assay based on DNA sequences was developed that distinguishes between the two pathogens. In addition, images and descriptive information are included. Plant pathologists, quarantine officials and other scientists will be able to use this information to accurately distinguish between these two fungi and prevent the spread of chrysanthemum white rust to the United States and other countries where it does not yet occur.
Technical Abstract: Puccinia horiana, the cause of chrysanthemum white rust, is a regulated fungal plant pathogen in the United States, while Puccinia chrysanthemi, the cause of chrysanthemum brown rust, is a widespread but less destructive pathogen. Accurate identification of these pathogens is essential to correctly enforce quarantine measures, but the pathogens cannot be differentiated visually in the absence of mature spores or symptoms. A multiplexed 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. Detection of each fungus using the assay was compared to identifications based on fungal morphology for seven fresh samples and 270 herbarium specimens of chrysanthemum rust. Puccinia 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 of the fungi using the multiplexed assay and 7% were positive for both fungi. This multiplexed 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.