Department of Biology, Wingate University, Wingate, NC, USA Detection of Puccinia pelargonii-zonalis-infected Geranium Tissues and Urediniospores Erika A. Scocco 1 , Ronald R. Walcott 2 , Steven N. Jeffers 3 and James W. Buck 1 Authors’ addresses: 1 Department of Plant Pathology, University of Georgia, Griffin, GA, 30223, USA; 2 Department of Plant Pathology, University of Georgia, Athens, GA, 30602, USA; 3 School of Agricultural, Forest, and Environmental Sciences, Clemson University, Clemson, SC, 29634, USA (correspondence to J. W. Buck. E-mail: jwbuck@uga.edu) Received October 30, 2012; accepted December 7, 2012 Keywords: geranium rust, PCR, ornamental Abstract The occurrence of geranium rust (caused by Puccinia pelargonii-zonalis) in commercial greenhouses can result in unmarketable plants and significant eco- nomic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii- zonalis-infected tissues or urediniospores on green- house-grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii- zonalis. The primers amplified a 131-bp product from genomic DNA from each isolate of P. pelargonii- zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii-zonalis DNA in conventional PCR and at 1 pg/ml using real-time PCR. The detection threshold was 10 2 urediniospores/ ml for real-time PCR and 10 4 urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii- zonalis DNA was amplified by real-time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non-inoculated leaves. Conventional and real-time PCR did not detect P. pelargonii-zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conven- tional and real-time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subse- quent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses. Introduction The US floriculture industry, including bedding and garden plants, potted herbaceous perennials, flowering and foliage plants and cut flowers, was valued at US $4.08 billion in 2010 (USA Department of Agriculture National Agriculture Statistics Service 2012). Vegeta- tive zonal geranium (Pelargonium 9hortorum) cuttings used to produce bedding plants sold as flats or hanging baskets were valued at US $29 million in a 15-state survey of US growers (USA Department of Agriculture National Agriculture Statistics Service 2012). The pres- ence of geranium rust, caused by Puccinia pelargonii- zonalis, in commercial greenhouses can result in unmarketable plants and major economic losses (Jeffers 1998; Buck 2007). The first report of geranium rust in the United States occurred in 1967 in New York followed by occurrences in other states during the 1970s (Dimock et al. 1968; Wehlburg 1970; Nichols and Forer 1972). Currently, P. pelargonii-zonalis is endemic in California and is con- sidered a quarantine pathogen in Ohio (Ohio Depart- ment of Agriculture 2010). Consequently, any zonal geranium plant material shipped from California to Ohio must be certified disease free. Occasional geranium rust outbreaks have also occurred in the southeastern United States (Jeffers 1998). Accidental introductions of exotic fungal pathogens, including Puccinia horiana (chrysanthemum white rust) and Uromyces transversalis (gladiolus rust), are of major concern to growers of ornamental plants in the United States (Wise et al. 2004; Rossman 2009). Mandatory federal quarantine and eradication measures to eliminate these fungi are expensive and occasionally fail (Wise et al. 2004). This was the case with daylily rust in the United States, where a federal quarantine eventually failed due to rapid and widespread movement of infected plants that resulted in establishment of the disease over a large geographical area (Wise et al. 2004; Buck and Ono 2012). J Phytopathol 161:341–347 (2013) doi: 10.1111/jph.12072 © 2013 Blackwell Verlag GmbH