Journal of Plant Pathology (2015), 97 (2), 321-326 Edizioni ETS Pisa, 2015 Cheong et al. 321 SHORT COMMUNICATION EVALUATION OF THE STATUS OF THE VIRUS AND VIROID INFECTION IN FLOWERING CHERRY (PRUNUS YEDOENSIS) COLLECTIONS IN KOREA AND THE U.S. E.J. Cheong1, C.S. Kim2, G. Kinard1 and R. Li1 1 National Germplasm Resource Lab, USDA, ARS, Beltsville, MD 20705, USA 2 Warm-Temperate, Subtropical Forest Research Center, KFRI, Jeju, Korea SUMMARY on the campus of American University in Washington, D.C. were planted in 1943 by a Korean statesman who The status of virus and viroid infections in flowering later became the first democratically elected President of cherry trees (Prunus yedoensis) in prominent ornamen- South Korea (American University-The Eagle, 1943). In tal collections in Korea (Seoul, Jinhae, Jeju) and the U.S. Korea, many of the flowering cherry trees in Seoul, Jinhae (Washington, D.C.) was investigated. A total of 344 trees and Jeju were planted using materials also imported from was tested by conventional RT-PCR for 13 viruses and 2 Japan in the early 1930s and again in the 1970s (Park, 1965; viroids. Eight viruses were detected in trees sampled from Kong, 2009). Although the species was unpopular in Korea Korea, and the same eight along with two additional vi- for several decades after World War II due to anti-Japan ruses were found in trees in U.S. Cherry Virus A (CVA) sentiment, evidence that P. yedoensis is native to Jeju Island was detected with the highest incidence in samples from (Park, 1965; Kim et al., 1997) has led to resurgence in its both countries, followed by Little Cherry Virus-2 (LChV-2). popularity. Many P. yedoensis trees have been propagated This study represents the first report of LChV-2, Plum bark from a few clones that were brought from Japan, and are necrosis stem pitting associated virus (PBNSPaV), and Amer- sold throughout the country by commercial nurseries. The ican plum line pattern virus (APLPV) in Korea. Almost all species is now the most popular ornamental tree in Ko- (96%) of the cultivated trees were infected with at least rea, and the numbers of trees planted have been rapidly one virus, and 73% were infected with 2-6 viruses. Espe- increasing (Korean Forest Service, 2012). cially interesting was the detection of three viruses (CVA, Flowering cherries belong to the genus Prunus, which LChV-2 and/or PBNSPaV) from three different wild trees includes production cherries, peaches and plums. Many on Jeju Island. All three infected trees were proximal to viruses and virus-like pathogens infect Prunus trees and cultivated flowering cherry trees that were infected with cause diseases affecting their growth, yields and flower/ at least two of these viruses, whereas ten other tested wild fruit quality (Hadidi et al., 2011). These pathogens can un- trees in more isolated locations were virus-free. It is pos- knowingly be distributed through infected clonally propa- sible that transmission occurred from the cultivated to the gated materials. Among the viruses infecting Prunus spe- wild trees. cies, eight viruses-Apple chlorotic leaf spot virus (ACLSV), Cherry rasp leaf virus (CRLV), Prunus necrotic ringspot virus Key words: Ornamental cherry, Prunus yedoensis, RT- (PNRSV), Little cherry virus 1 (LChV-1), Prune dwarf virus PCR, phytosanitary status, virus testing. (PDV), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), CVA and one vi- roid, Hop stunt viroid (HSVd), have been reported in sweet cherries, peaches and plums in Korea (Kim et al., 2009; Cho et al., 2011, 2012, 2013, 2014). These and numerous other viruses have been reported in Prunus species in the Prunus yedoensis is one of most popular ornamen- U.S. (Uyemoto and Scott, 1992; Bajet et al., 2008; Hadidi tal trees planted for spring blossoms in many countries et al., 2011; Li et al., 2012; Osman et al., 2012). All these including the Korea and the U.S. The flowering cherry pathogens are spread by grafting, and some such as PDV plantings around the Tidal Basin of the National Mall in and PNRSV are also transmitted by pollen (Kelly and Washington, D.C. were established using trees donated Cameron, 1986). The potential for natural spread (pollen, by Japan in the early 1900s (http://www.nps.gov/cherry/ insects, nematodes) for some viruses such as CVA and cherry-blossom-history.htm) (Table 1). Several historic trees LChV-1 is still unclear (Osman et al., 2012). Although phytosanitary surveys have been performed Corresponding author: R. Li E-mail: [email protected] for fruit bearing Prunus species in Korea, little attention has been given to ornamental Prunus despite the fact that 322 Virus and viroid infections in flowering cherry Journal of Plant Pathology (2015), 97 (2), 321-326 Table 1. Locations, number of trees sampled, and year of planting of Prunus yedoensis in each group. Country Groups Location Number of trees Year of planting Korea Jeju 1a Mt. Halla, Jeju 13 N/A Jeju 2b Seguipo, Jeju 32 1980s Jeju 3 b Jeju University, Jeju 55 1980s Jeju 4 b Musuchun, Jeju 41 1980s Jeju 5 b,c Sanchundan, Jeju 2 1970s Jinhae 1 b Yojwachun, Gyungsangnam-do 40 1976 Jinhae 2 d Yojwachun, Gyungsangnam-do 10 2000s Jinhae 3 b Mt. Jangbok, Gyungsangnam-do 20 1966 Seoul b Yoeuido, Seoul 45 1930s-70s 258 U.S. AU 1 e American University, DC 3 1943 AU 2 f American University, DC 29 2011 NM 1 b National Mall, DC 15 1910 NM 2 b National Mall, DC 25 1912 NM 3 g National Mall, DC 14 1980s 86 a 40 to 200-year old trees native on Mt. Halla. b Trees were donated by Japan and planted in known locations according to city records. c Trees planted along roadside and located next to wild tree. d Trees were clonally propagated by commercial nurseries in Korea using trees from Jinahe 3. e Trees are historic treasures of the University obtained from a commercial nursery in Philadelphia. f Trees were obtained from a commercial nursery in New Jersey. g Trees were propagated in the U.S. from NM 2. trees are being widely planted and they are natural hosts in Washington, D.C. in April, 2010 and at American for many viruses. There has been some examination of University in Washington, D.C. in April 2012 (Table 1). virus infections of ornamental Prunus in the U.S. (Li et With exception of the trees from the Jinhae 2, AU 1, AU 2 al., 2012; Osman et al., 2012). Such studies are relevant and NM 3 groups, all other trees were imported directly not only for producing healthy ornamentals, but also for from Japan as indicated in Table 1. Samples of plant tis- protecting fruiting species. Researchers in Hungary rec- sues (young shoots, leaves, buds, stems and/or petioles) ognized that ornamental trees in public areas may play were collected from several positions of each tree. The a role in spreading pathogens to production orchards. trees were randomly selected and did not have apparent Sebestyen et al. (2008) detected Plum pox virus (PPV) in disease symptoms on the new shoots. Some old trees in ornamental Prunus species in public areas. Ornamental the National Mall showed stem pitting and gummosis. trees and shrubs in Hungarian botanical gardens and an Total nucleic acids were extracted from pooled tissues of arboretum were also infected with viruses such as PPV, leaves, buds, petioles and bark by a CTAB-based method PDV, PNRSV and CRLV, raising the possibility that these (Li et al., 2008). Testing for 13 viruses and 2 viroids was trees could play a role in spreading the viruses to orchards performed by conventional RT-PCR as summarized in (Németh et al., 2010). Similar surveys were conducted in Table 2. The pathogens included in the evaluation were New Zealand (Everett et al., 1993) and China (Rao et al., ACLSV, APLPV, Asian prunus viruses (APVs), CGRMV, 2011). Therefore, it is important to understand the phy- CNR MV, CR LV, CVA, LChV-1, LChV-2, PBNSPaV, PDV, tosanitary status of flowering cherry trees to reduce the PNRSV, Tomato ringspot virus (ToRSV), HSVd and Peach possibility of spreading pathogens to related fruiting and/ latent mosaic viroid (PLMVd). RT-PCR was performed us- or native species. This study investigated the virus infec- ing a SuperScriptTM III One-Step RT-PCR system (Invit- tion status of flowering cherry trees in several important rogen, Carlsbad, CA, USA) following the manufacturer’s plantings in Korea and the U.S. instruction. The RT-PCR program varied in the anneal- A total of 344 trees (13 wild and 331 cultivated) was ing temperature and extension time according to primer sampled from the two countries. Two hundred fifty eight combination and length of the amplicon. The general ther- samples were collected in April 2012 from nine distinct mal cycling conditions were 50°C for 45 min and 95°C for locations in Seoul, Jinhae and Jeju, Korea that feature 15 min, 35 cycles of 94°C for 45 s, 55-58°C for 45 s and large cherry blossom tree plantings (Table 1). One group 72°C for 1 min with one final extension at 72°C for 7 min. was comprised of 13 trees of P. yedoensis on Jeju (Jeju 1) To confirm the infections of APLPV, LChV-2 and that are native to the island (Kim et al., 1997). Eighty- PBNSPaV in Korea, RT-PCR amplicons from two iso- six samples of five groups were collected from the U.S., lates of each virus were directly sequenced (MCLAB, San based on location and date planted, at the National Mall Francisco, CA, USA).