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Title: Real-time PCR detection of Puccinia chrysanthemi causing brown rust of chrysanthemum

item Demers, Jill
item Crouch, Jo Anne
item Castlebury, Lisa

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 10/25/2013
Publication Date: 3/1/2014
Citation: Demers, J.E., Crouch, J., Castlebury, L.A. 2014. Real-time PCR detection of Puccinia chrysanthemi causing brown rust of chrysanthemum [abstract]. American Phytopathological Society Annual Meeting. 104(S1):S1.2.

Interpretive Summary:

Technical Abstract: Fungi responsible for rust diseases are among the most challenging organisms to identify, as many identification keys are based on host identity. In the U.S., numerous rust fungi are quarantine-significant plant pathogens. As such, accurate identification is crucial to prevent the inadvertent introduction of destructive pathogens and to facilitate trade. Three species in the Pucciniales cause rust diseases of ornamental chrysanthemum, Phakopsora artemisiae, Puccinia horiana, and Puccinia chrysanthemi. P. horiana is a quarantine-significant pathogen causing white rust disease, which renders infected plants unmarketable and produces substantial economic losses. P. chrysanthemi, causing brown rust, is ubiquitous in the U.S., but losses due to this pathogen are trivial. P. artemisiae is known only from Japan and China. The objective of this study was to develop a real-time PCR assay for the detection of P. chrysanthemi, which can be difficult to distinguish from P. horiana in the early stages of infection. A fluorescence-labeled hydrolysis probe with four locked nucleic acids was designed from a region of the internal transcribed spacer region (ITS1) rDNA region. The probe region is unique to P. chrysanthemi, and falls within a 88 bp PCR amplicon. The assay was used to screen 206 specimens of rust fungi on chrysanthemum curated by the U.S. National Fungus Collections (Beltsville, MD). P. chrysanthemi was detected from 177 of 187 specimens (95%) previously morphologically identified as P. chrysanthemi, including specimens >100 years old. Subsequent morphological examination of specimens that yielded negative results showed that three specimens appeared to have insufficient material for determination, three specimens were P. horiana misidentified as P. chrysanthemi, and four specimens were mixed infections of P. horiana and P. chrysanthemi. Negative results were obtained from ten specimens of Phakopsora artemisiae and nine specimens of P. horiana. The P. chrysanthemi diagnostic assay will confirm the presence of P. chrysanthemi, which may aid in correct diagnosis of P. chrysanthemi or P. horiana in symptomatic plants.