Plant Pathol. J. 26(2) : 130-138 (2010) The Plant Pathology Journal Mini-Review © The Korean Society of Plant Pathology Molecular Characterization and Survey of the Infection Rate of Orchid fleck virus in Commercial Orchids Sung Ryul Kim1,5, Ju-Yeon Yoon2,5, Gug Sun Choi1, Moo Ung Chang3, Jang Kyung Choi4 and Bong Nam Chung1* 1National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 440-310, Korea 2PVGB, Division of Environmental and Life Science, Seoul Women’s University, Seoul 139-724, Korea 3Department of biology, Yeungnam University, Gyongsan 712-749, Korea 4Department of applied biology, Kangwon National University, Chunchon 200-701, Korea (Received on February 1, 2010; Accepted on April 9, 2010) Orchid fleck virus (OFV) is an unassigned plant virus floricultural crop viruses (Lawson and Hsu, 1995). Thirty in the family Rhabdoviridae. OFV was isolated from kinds of viruses were reported on orchid worldwide, among Cymbidium sp. showing oval necrotic lesions on their them Odontoglossum ringspot virus (ORSV) and Cymbi- leaves in Korea, and designated as OFV-NHHS1. The dium mosaic virus (CymMV) are the most common viruses complete nucleotide sequence of the RNA1 (6,413 nt) (Lawson and Hsu, 1995). Since Orchid fleck virus (OFV) is (GenBank accession no. AB516442) and RNA2 (6,001 reported first in Cymbidium spp. with chlorotic or necrotic nt) (GenBank accession no. AB516441) was determined ring spots and fleck symptoms from Japan (Doi et al., in this study. RNA1 and RNA2 contained five and one ORF respectively. RNA1 encodes nucleocapsid (N) of 49 1969), it is recognized as an important viral pathogen of kDa, ORF2 of 26 kDa, ORF3 of 38 kDa, ORF4 of orchids infecting more than 6 orchid genera (Chang et al., 20 kDa and glycoprotein (G) of 61 kDa proteins, where- 1976). It has been reported in Australia, Brazil, Denmark, as RNA2 encodes a single polymerase of 212 kDa. OFV- Germany, Korea, USA and Costa Rica (Blanchfield et al., NHHS1 shared extremely high similarity of 98.6-100% 2001; Chang et al., 1991; Freitas-Astúa, 1999; Freitas- and 98.9-99.6% in nucleotide and amino acid sequences Astúa, 2002; Gibbs et al., 2000; Kitajima et al., 2001; with a Japanese isolate, OFV-so, respectively. However, Kondo et al., 2006). the N, G and L of OFV-NHHS1 revealed 6.9-19.3%, 7.3- OFV has non-envleoped, bacilliform particles of 40×150 12.0%, and 13.4-26.6% identities to those of 29 Rhabdo- nm in negatively stained preparations and is sap-trans- viruses, respectively. To survey the infection rate of missible to a few indicator species in the families Cheno- OFV in commercial orchids in Korea, 51 Cymbidium podiaceae, Solanaceae, Leguminosae and Aizoaceae (Chang sp., 10 Phalaenopsis sp., 22 Oncidium sp. and 21 Dendro- bium sp. plants that showed typical viral symptoms were et al., 1976; Doi et al., 1977). OFV is known to be trans- collected. RT-PCR with specific primers for detection of mitted by Brevipalpus californicus Banks (Kondo et al., Cymbidium mosaic virus (CymMV), ORSV and OFV 2003) and Brevipalpus phoenicis (Hogenhout et al., 2008) showed high infection rate by ORSV alone and double in a persistent manner (Kondo et al., 2003). It is composed infection by ORSV and CymMV. One of the orchids of negative sense, single stranded RNA molecules and tested was infected with OFV. This is the first report of bipartite; RNA 1 and 2 is 6,413 and 6,001 nucleotides long, the complete nucleotide sequences of OFV isolated in respectively. RNA1 consists of five open reading frames Korea. (ORF) of nucleocapsid (N), glycoprotein (G) and three uncharacterized ORFs, and RNA2 consists of a single ORF Keywords : Cymbidium sp., CymMV, Orchid fleck virus, of polymerase (L). The genome of OFV has common genes Rhabdovirus, ORSV, RT-PCR, sequence analysis in the same order with other viruses in the family Rhabdo- viridae, but is split between the G protein and L polymerase genes. Because of the bipartite genome, a new genus, Orchids are the largest family of flowering plants with more Dichorhabdovirus was proposed (Kondo et al., 2006). than 800 genera and over 25,000 species. It is one of the In Korea, OFV was first reported in 1991 but molecular most important potted floricultural crops accounting for characterization of OFV has never been studied (Chang et 25.8% in terms of cultivation area in Korea for the potted al., 1991). Therefore in this study we determined complete nucleotide sequences of OFV Korean isolate and compared *Corresponding author. Phone) +82-31-290-6236, FAX) +82-31-290-6259 them with those of OFV-so reported from Japan and other E-mail) [email protected] viruses in the family Rhabdoviridae. This result facilitated 5Equally contributed the detection of OFV by RT-PCR, which is sensitive, Determination of Complete Nucleotide Sequence of OFV 131 reliable and rapid method of detecting small amount of viral manufacture’s instructions. RNA. Primer design. To determine the complete sequence of Materials and Methods OFV-NHHS1 RNA1 and 2, sixteen pairs of primers were designed on the basis of OFV-so sequences (GenBank Source of virus and plants. OFV-infected Cymbidium sp. accession no. AB244417 and AB244418), and expected was collected from a market selling orchids in Gyeongbuk size of amplified each fragment is listed in Table 2. To Province, Korea. They showed oval necrotic lesions or survey the incidence of viral disease in orchids collected, necrotic line patterns on leaves (Fig. 1). Those leaves, three specific primer pairs were designed for detection of neither infected with CymMV nor with ORSV (data not CymMV, ORSV and OFV on the basis of GenBank shown), were used as a source of nucleotide sequencing accession no. of NC_001812, DQ915440 and AB516441, and host range test of virus, and they were designated as respectively. OFV-NHHS1 in this study. To survey the incidence of The sequence of the CymMVK-F was homologous to CymMV, ORSV and OFV in commercial orchids in Korea, nucleotides 5462 to 5486 (5'-ACAATAATTTGAAATAAT- 51 Cymbidium, 10 Phalaenopsis, 22 Oncidium and 21 CATGGGA-3') of the NC_001812 and the sequence of Dendrobium plants showing typical viral symptoms were CymMVK-R was complementary to nucleotides 6156 to collected from Gyeonggi, Chungnam and Gyeongbuk pro- 6180 (5'-AAAACCACACGCCTTATTAAGTTTG-3') of vinces. the NC_001812. The sequence of the ORSVK-F was homologous to nucleotides 26 to 49 (5'-ACGCACAAT- Host range test. Eight indicator plants including Tetra- CTGATTCGTATTGAA-3') of the DQ915440 and the gonia expansa were inoculated with sap of source orchid of sequence of ORSVK-R was complementary to nucleotides OFV, NHHS1 in 0.05 M sodium phosphate buffer, pH 7.0. 530 to 553 (5'-TATCAACGTTATTTTCCTAAATAT-3') of Three independent inoculation tests were conducted in each the DQ915440. The sequence of the OFVK-F was homo- time course of May and September. Five plants were logous to nucleotides 3758 to 3780 (5'- TACTGATGCTG- replicated with every indicator plant in every test. Symptom ATGCCACTCTTT-3') of the AB516441 and the sequence was determined at 15-20 days after inoculation and con- of OFVK-R was complementary to nucleotides 530 to firmed by RT-PCR using a primer pair specific to OFV. 553 (5'-ACCCAACTGGGAGAGACTCTATT-3') of the AB516441. Extraction of viral RNA. Viral RNAs were extracted from leaf tissue of orchids showing viral symptoms with RNeasy RT-PCR. Ten ng of RNA in 9 μl of nuclease free water and Plant Mini Kit (QIAGEN, Germany) according to the 1 μl of 10 pm reverse primer was heated at 70 oC for 5 min followed by adding 4 μl of 5× reaction buffer, 2.5 mM MgCl2, 0.25 mM of each dNTP, 1 μl of ImProm II reverse transcriptase (Promega, USA) and 1 μl of RNase inhibitor (1 U/μl) on ice, and incubating at 37 oC for 1 hr. PCR amplification was performed in a 50 μl containing 20 μl of cDNA solution, 0.2 mM of each dNTP, 2 mM MgCl2, 10 pM of each primer, 2.5 units of GoTaq DNA polymerase (Promega, USA), and 1× PCR buffer. Thirty five PCR cycles were conducted in PTC-0220 Perlitier Thermal Cycler (MJ Research, MA, USA). The thermal conditions were as follows: denaturation at 94 oC for 30 sec (2 min for the first cycle), annealing at 50 oC for 1 min and extension at 72 oC for 1 min, and final extension at 72 oC for 10 min. For detection of CymMV and ORSV, duplex RT-PCR was conducted. Determination of complete nucleotide sequences of OFV. The amplified PCR products of the expected length Fig. 1. Symptoms induced by natural infection with OFV- were eluted and cloned into the pGEM-T easy vector. The NIHHS1 on Cymbidium. (A) Oval necrotic flecks; (B) Necrotic ligation mixture was used to transform competent cells of line pattern on leaves. Escherichia coli JM109. Nucleotide sequences of the 132 Sung Ryul Kim et al. Table 1. Response of eight indicator plants to OFV-NHHS1 cloned PCR products were determined using ABI PrismTM infection in comparison with OFV-so Terminator Cycle Sequencing Ready Reaction Kit and ABI Indicator plants OFV-NHHS1 OFV-soa Prism 377 Genetic Analyzer (Perkin Elmer, USA). Tetragonia expansa NS/−b LL/− Chenopodium amaranticolor −/− LL/− Sequence comparison and phylogenetic tree analysis. C. quinoa −/− cLL/− The nucleotide sequences were assembled and analyzed Nicotiana clevelandii nLL, Y/− nt with Lasergene package version 7 (DNAstar Inc., USA). N. glutinosa −/− LL/− Databases were searched using the Blast suite of programs N. tabacum cv. White Burley −/− LL/− from NCBI. Multiple sequence alignments and phyl- N. tabacum cv. Xanthi-nc −/− LL/− Vigna unguiculata nLL/− nt ogenetic analysis were done using Clustal W of MegAlign program (DNAstar Inc., USA) based on its alignment a Referred to Chang et al., 1976, Kondo et al., 2003, ICTV dB descrip- tions.