Journal of 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 ( 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 (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 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, 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 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 . 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 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). The virus origin of each sequence Journal of Plant Pathology (2015), 97 (2), 321-326 Cheong et al. 323

Table 2. Primers, sequences and product sizes of the target viruses and viroids.

Target virus/viroid Primer ID Sequence (5’-3’) Product size (bp) Reference ALCSV ACLSV7 AAGATCGCAGAAGGGGATATTCC 1180 Li et al., 2012 ACLSV8b TGTTCGCGAAGATGGYCTCCAG APLPV APLPV1 GACGAGGCTGATGAAGGGAA 501 Li et al., 2008 APLPV2 ACCCACAGAAGGACCTACCA APV APV2F CCTAGAACCGTGGAGAGGCCT 749 Li et al., 2012 APV2R GTACGGTCTGTCAGCGAACAC CGRMV/ CNRMV* CGRMV1 CCTCATTCACATAGCTTAGGTTT 958/959 Li and Mock, 2005 CGRMV2 ACTTTAGCTTCGCCCCGTG CNRMV CNRMV3 GACATACTGCGAGCCAGAAG 632 Li et al., 2012 CNRMV4 TGAATTTGGCCAGTCCTGCC CR LV FAVR1-7Fm TGACTTTCCCAAGGATGAGAAG 452 James and Upton, 2005 FAVR1-8R m CACTTGTGACATACCATAGATCC CVA CVA13 TTGGCTTTGCTGGCTCTGGTAAA 269 Li et al., 2012 CVA14 CAAATCAATGAAGCCAGGTGGGT LChV-1 LCV1-3EC GCTCTAGAGGCACCTTTTATTTTTTATATATGC 276 Bajet et al., 2008 LCV1-16659 GTTATAGAATTCACTGCAAGTG LChV-2 LCV2-1 TTCAATATGAGCAGTGTTCCTAAC 781 Li et al., 2012 LCV2-2 ACTCGTCTTGTGACATACCAGTC PBNSPaV PBNSPaVF TACCGAAGAGGGTTTGGATG 400 Al Rwahnih et al., 2007 PBNSPaVR AGTCGCACCACCAGTCTTCT PDV PDV1 GGAAAACCTACTGCCCGTTC 540 Li et al., 2008 PDV4 ATCGAGTGTTGGAGGTACTGAGT PNRSV PNRSV10F CTTGAAGGACCAACCGAG 348 Massart et al., 2008 PNRSV10R ATCTGCTAACGCAGGTAAG ToRMV ToRSV5 AGGTAGGACGCYATTGTTCCAGG 433 Li et al., 2012 ToRSV6 AGTCTCAACTTAACATACCACTAC HSVd VP19 GCCCCGGGGCTCCTTTCTCAGGTAAG 300 Astruc et al., 1996 VP20 CCCGGGGCAACTCTTCTCAGAATCC PLMVd PLMVd1 ACCTCTCAGCCCYTCCACCTT 240-250 Li et al., 2012 PLMVd2b AGAGACTCATCAGTGYGCTWAGC * Primers (CGRMV1/2) detect both CGRMV and CNRMV; CNRMV was confirmed by primers (CGRMV3/4). was confirmed by a BLAST search of the GenBank the Jeju 2 group. The planting in Seoul was mainly in- Databases. fected with CVA (84% incidence) with one occurrence of Eight viruses-APLPV, CGRMV, CNRMV, CRLV, CVA, PBNSPaV. LChV-1, LChV-2 and PBNSPaV were detected from the Among the U.S. ornamental trees tested from the Na- ornamental cherry trees in Korea (Table 3). These same tional Mall and American University, CVA was detected eight viruses were all also detected in the U.S. samples, with the highest incidence (80%) along with CNRMV and along with two more viruses (PDV and ToRSV) that were LChV-2 (both 34%). There were some different infection detected only in the groups recently planted on the Na- patterns between the AU and NM samples. LChV-2 was tional Mall (NM 3) or at American University (AU 2). detected in all three AU trees planted in 1943 but was not ACLSV, APVs, PNRSV, HSVd or PLMVd were not de- detected in the recently planted AU 2 collection. In con- tected in samples from either country. The greatest virus trast, CGRMV was not detected in the AU 1 trees but was incidence in trees sampled from both countries was CVA prevalent in AU 2 (69%). The highest incidence of virus (79% incidence), followed by LChV-2 (44%), PBNSPaV in AU 2 was CNRMV (90%) whereas there was only one (28%), CNRMV (22%), LChV-1 (11%) and CGRMV incidence of this virus among all the National Mall groups. (11%) (Table 3). The incidences of virus infection differed CVA was dominant in the NM groups (96%), followed among groups in Korea. The viruses detected in the Jeju by LChV-2 (48%) and APLPV (17%). CGRMV was not and Jinhae landscape plantings were similar; however, detected, and CNRMV and PBNSPaV were less common the incidences of CVA (100%), PBNSPaV (82%) and in the National Mall trees. The PBNSPaV infection was APLPV (17%) were higher in Jinhae than in Jeju (74%, associated with severe stem pitting and gummosis, but not 28% and 7%, respectively). Incidences of viruses in Jeju 4 all trees with stem pitting were infected with PBNSPaV were unique among Jeju groups where LChV-1 was com- in the NM 1 and NM 2 groups. Among the more recently mon (59%), while it was sporadic in all other groups. On planted NM 3 trees, CVA was most frequently detected the other hand, PBNSPaV was detected in only two Jeju although other viruses occurred, including the only inci- 4 samples (5%), compared to much higher incidences in dence of PDV and ToRSV among the National Mall trees Jeju 2 (34%) and Jeju 3 (44%). The only incidence of CRLV sampled. All of these different infection patterns are prob- in trees tested from either country was from one tree in ably explained by different locations and/or nurseries from 324 Virus and viroid infections in flowering cherry Journal of Plant Pathology (2015), 97 (2), 321-326

Table 3. Viruses, number of trees infected, and infection rates a in each group.

Country No. of Virus Group samples APLPV CGRMV CNRMV CRLV CVA LChV-1 LChV-2 PBNSPaV PDV ToRSV Korea Jeju 1 13 0 0 0 0 3 0 2 1 0 0 Jeju 2 32 3 5 6 1 31 3 31 11 0 0 Jeju 3 55 5 5 16 0 29 2 29 24 0 0 Jeju 4 41 1 6 15 0 30 24 23 2 0 0 Jeju 5 2 0 0 0 0 2 0 1 1 0 0 Jinhae 1 40 2 0 2 0 40 1 10 26 0 0 Jinhae 2 10 2 1 7 0 10 0 10 10 0 0 Jinhae 3 20 5 1 2 0 20 0 15 16 0 0 Seoul 45 0 0 0 0 38 0 0 1 0 0

Subtotal 258 18 (7) 18 (7) 48 (19) 1 (0.4) 203 (79) 30 (12) 121 (47) 92 (36) 0 0 U.S. AU 1 3 0 0 2 0 2 0 3 0 0 0 AU 2 29 0 20 26 0 15 0 0 0 0 1 NM 1 15 0 0 1 0 15 9 5 1 0 0 NM 2 25 7 0 0 0 23 0 18 3 0 0 NM 3 14 2 0 0 0 14 0 3 0 1 3 Subtotal 86 9 (10) 20 (23) 29 (34) 0 69 (80) 9 (10) 29 (34) 4 (5) 1 (1) 4 (5) Total 344 27 (8) 38 (11) 77 (22) 1 (0.3) 272 (79) 39 (11) 150 (44) 96 (28) 1 (0.3) 4 (1) a Infection rate percentages indicated in parentheses. * ACLSV, APV, PNRSV, HSVd and PLMVd were not detected in the 344 trees tested. which the trees originated. For example, LChV-2 was com- (> 98%) with an American isolate of APLPV (AF235166), mon in the NM 1 group but was not detected in the NM 2 a Chinese isolate of LChV-2 (HQ412772) and PBNSPaV trees. Similarly APLPV was detected in NM 2 but not in (JF810192), respectively. Two of these viruses, LChV-2 and NM 1. The sources of the trees Japan donated in 1910 and PBNSPaV, have been reported in neighboring Japan and/ 1912 were different, the former being from Tokyo and the or China (Isogai et al., 2004; Rao et al., 2011; Cui et al., latter from Ekita (Jefferson and Fusonie, 1977). 2011; Zhou et al., 2011). With the exception of the native group in Jeju (Jeju 1), This work also provides anecdotal information about nearly all of the trees were infected with one or more vi- the sources of infections including the possibility of natu- ruses in the Korean groups. More than 73% were infected ral spread of some viruses. The most interesting obser- with two or more viruses, and up to six different viruses vation was the detection of viruses (CVA, LChV-2 and (CGRMV, CNRMV, CVA, LChV-2, PBNSPaV, APLPV or PBNSPaV) in three wild native trees on the Korean island LChV-1) were detected as mixed infections in four trees of Jeju (Table 1; Table 3). All three infected trees were prox- from Jeju 3 and one tree from Jinhae 3, respectively. The imal to introduced trees, whereas no virus was detected average number of viruses per infected tree varied from in more isolated trees. The 70-years-old wild tree infected two to four in all groups except Seoul and Jeju 1. Mixed with all three viruses was approximately three meters from infections were also common in the U.S. groups. Among a roadside landscape tree in the Jeju 5 group that was in- the 81 infected trees, 62 were infected with at least two fected with the same three viruses. The other two infected viruses, mainly CVA and/or LChV-2 in combination with wild trees (ca. 150 years old) were in an old growth for- other viruses. est preserve adjacent to a camping area (2-3 meters apart), This study represents the first report of APLPV, which contained more recently introduced ornamental LChV-2 and PBNSPaV in Korea. BLAST search of the ob- Prunus species (ca. 5 years old). One of the two wild trees tained nucleotide sequences confirmed the virus origin of was infected with CVA, and another was co-infected with each amplicon. The two APLPV (accession Nos KP410828 CVA and LChV-2. It is possible that the viruses spread and KP41082), LChV-1 (accession Nos KP410830 and from cultivated landscape trees to the native ones by root- KP410831) and PBNSPaV (accession Nos KP410832 and grafting and/or insect transmission. The other ten trees KP410833) isolates were almost identical in their sequenc- of Jeju 1 group, from which no viruses were detected, are es and shared the highest nucleotide sequence identities scattered in the mountainside forest and relatively distant Journal of Plant Pathology (2015), 97 (2), 321-326 Cheong et al. 325 from landscape trees and other flowering cherry species. Hymes and Sam Grinstead (U.S. Department of Agricul- All of the wild trees are ungrafted and growing on their ture), Mary Willeford Bair (U.S. National Park Service) own roots as observed. Although there is no definitive evi- and Gene Y. Suk for sample collection and technical dence of natural transmission for any of these three wild assistance. trees, the possibility exists and is augmented by other re- ports of transmission of LChV-2 (Raine et al., 1986; Rott and Jelkmann, 2001), CVA (James and Jelkmann, 1998; REFERENCES Kirby et al., 2001; Isogai et al., 2004; Sabanadzovic et al., 2005; Svanella-Dumas et al., 2005; Noorani et al., 2010; Al Rwahnih M., Uyemoto J.K., Falk B.W., Rowahni A., 2007. Crimová et al., 2010; Li et al., 2012) and PBNSPaV (Osman Molecular characterization and detection of Plum bark ne- et al., 2012; Cui et al., 2011). crosis stem pitting-associated virus. Archives of Virology 152: 2197-2206. 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Received September 17, 2014 Accepted May 28, 2015