• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.145.1-145
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• 2003

This study was conducted to designing conserved regions of molecules for virus-derived resistance to transgenic Phlaenopsis orchids to protect against two major orchid viruses, Cymbidum mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Infected leaf samples of Phalaenopsis were randomly screened by the RT-PCR with specific primers to both of viruses. RT-PCR products of the viruses were cloned and their nucleotide sequences were determined. Multiple alignments of coat protein (CP) genes of the viruses revealed that over the 88 ％ and 94 ％ identities with CymMV and ORSV, respectively, were observed. These data can be useful for selection of highly conserved regions of CP gene of the viruses for transgenic orchid experiments.

• 한국식물병리학회지
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• 제14권2호
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• pp.130-135
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• 1998

This study was carried out to detect cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) in cultivated orchid plants in Korea. The standard double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) were carried out for detection of the viruses in the collected orchid samples. ELISA was suitable for massive-scale diagnostic method for virus detection in orchids. RT-PCR was rapid, time-saving and reliable detective method, and detection limit data showed that RT-PCR was 103 times more sensitive than ELISA. Of the 321 individual orchids representing 5 orchids genera tested by the ELISA, CymMV and ORSV were detected in 15.6% and 22.4%, and mixed infection of the both viruses with 4.9%, respectively. Of the Cymbidium plants tested, cultivated plants showed 52.5% virus infection rate with either CymMV or ORSV and both viruses.

• The Plant Pathology Journal
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• 제15권1호
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• pp.34-37
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• 1999

Cymbidium mosaic virus (CyMV) was purified from CyMV infected orchid plant leaves by Sephacryl S-500 HR column chromatography. Partial purification was done by solubilization with Triton X-100 (alkylphenoxypolyethoxy ethanol) and precipitation with polyethylene glycol (PEG 6,000) followed by ultracentrifugation on 30% sucrose cushion. Based on the spectrophotometric analysis, 33 mg of CyMV could be obtained form 100 g of CyMV-infected orchid plant leaves. The purified CyMV represented one distinct homogeneous band by SDS-PAGE, and electron microscopy revealed that it was highly homogeneous and not fragmented. Bioassay demonstrated that the purified CyMV had a normal infectivity to Chenopodium amaranticolor and orchid plants. Based on these results, the purification method in this work could be served as an improved method for the purification of CyMV and similar viruses with good yield, high purity and native integrity.

• 한국응용곤충학회지
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• 제15권3호
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• pp.137-145
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• 1976

우리나라에서의 바이러스 무감염란 생산에 필요한 기초자료를 얻기 위하여 Cymbidiun mosaic virus와 Tobacco mosaic virus를 중심으로 그 감염상 및 감염된 바이러스의 혈청학적 반응, 검정식물상의 반응, 물이적 성질, 형태등을 조사하여 다음과 같은 결과를 얻었다. 1. 란에 발생하는 바이러스병의 병징은 크게 1) 모자이크, 2) 괴저줄무늬 모자이크, 3) 괴원륜문, 4) 퇴록륜문 및 5) 괴달반점의 5군으로 분류되었다. 2. 수원근교의 난 재배농장에서 수집한 각종란의 바이러스 이병률을 조사한 결과 CyMV는 $45\%$의 감염율을 나타내었고, TMV는 한개체에서도 검출되지 않았다. 3. CyMV의 검정식물인 Chenopodium amaranticolor, Cassia occidentalis 및 Datura stramonium에 CyMV 병즙액을 접종한지 7-12일 후에 퇴록국부반점이 나타났다. 4. CyMV의 물리적 성질을 Chenopodium amaranticolor를 검정식물로 하여 조사한 결과, 내열성은$75-80^{\circ}C$ 내보존성은 8일, 내포석성은 $10^{-5}-10^{-6}$으로 나타났다. 5. CyMV의 혈정학적 한천내 확산반응에 사용된 0.1M 및 0.01M의 Phosphate, Imidazol 및 Tris 완충간에는 침강대 형성에 차이를 볼 수 없었다. 그러나 이들 완충액의 수소이온농도간에는 침강대 형성에 심한 차이를 나타내어, pH 7.0에서는 침강대가 뚜렷하였고 pH9.0에서는 미약하였다. 6. CyMV를 전자현미경으로 관찰한 결과 크기 460-580mu의 간상입자의 빈도가 제일 높았다.

• 한국식물병리학회지
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• 제14권6호
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• pp.559-562
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• 1998

Odontoglossum ringspot virus (ORSV) was finally purified from ORSV-infected orchid plants by diethylaminoethyl (DEAE) cellulose anion exchange column chromatography. The virus was reliably eluted by potassium chloride at the concentration from 0.1 M to 0.13 M. Partial purification was done by solubilization with Triton X-100 (allkylphenoxypolyethoxy ethanol) and precipitation with polyethylene glycol (PEG; MW 8,000). The finally purified ORSV represented one distinct homogeneous band and the molecular weight of its capsid protein was about 17,500 Dalton in electrophoretic analysis. Electron microscopy showed not only intact particles ranged from 280 nm to 340 nm in length, but also segmented particles that final 140 nm to 220 nm and even disks. Enzyme-linked immunosorbent assay (ELISA) showed that final yield was 12 mg/100 g of the infected leaves. Bioassay demonstrated that the purified ORSV had the normal infectivity to orchid plants and Nicotiana glutionsa. Based on these data, anion exchange column chromatography could be efficiently applied to the purification of ORSV and other viruses similar to ORSV.

• The Plant Pathology Journal
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• 제26권2호
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• pp.130-138
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• 2010

Orchid fleck virus (OFV) is an unassigned plant virus in the family Rhabdoviridae. OFV was isolated from Cymbidium sp. showing oval necrotic lesions on their leaves in Korea, and designated as OFV-NHHS1. The complete nucleotide sequence of the RNA1 (6,413 nt) (GenBank accession no. AB516442) and RNA2 (6,001 nt) (GenBank accession no. AB516441) was determined in this study. RNA1 and RNA2 contained five and one ORF respectively. RNA1 encodes nucleocapsid (N) of 49 kDa, ORF2 of 26 kDa, ORF3 of 38 kDa, ORF4 of 20 kDa and glycoprotein (G) of 61 kDa proteins, whereas RNA2 encodes a single polymerase of 212 kDa. OFV-NHHS1 shared extremely high similarity of 98.6-100% and 98.9-99.6% in nucleotidle and amino acid sequences with a Japanese isolate, OFV-so, respectively. However, the N, G and L of OFV-NHHS1 revealed 6.9-19.3%, 7.3-12.0%, and 13.4-26.6% identities to those of 29 Rhabdoviruses, respectively. To survey the infection rate of OFV in commercial orchids in Korea, 51 Cymbidium sp., 10 Phalaenopsis sp., 22 Oncidium sp. and 21 Dendrobium sp. plants that showed typical viral symptoms were collected. RT-PCR with specific primers for detection of Cymbidium mosaic virus (CymMV), ORSV and OFV showed high infection rate by ORSV alone and double infection by ORSV and CymMV. One of the orchids tested was infected with OFV. This is the first report of the complete nucleotide sequences of OFV isolated in Korea.

• The Plant Pathology Journal
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• 제17권6호
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• pp.325-328
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• 2001

Gelatin particle agglutination test (GPAT) was used to detect Cymbidum mosaic virus (CymMV) in Cattleya plants. Gelatin particles were coated with purified anti-CymMV immunoglobulin of 25-100 $\mu\textrm{g}$/ml and were subjected to several different concentrations of purified CyMfV as well as varying dilutions of orchid leaf extracts. The GPAT detected purified CymMV up to a minimum concentration of 10 $\mu\textrm{g}$/ml. CymMV was detected from crude sap extract of infected Cattleya leaves and roots up to 1:51,200 and 1:25,600 dilutions, respectively. However, the optimum range of leaf and root sap dilutions was between 50-100. Non-specific reactions were not encountered from any of the healthy orchid plants tested. The entire GPAT process was completed within 2-3 hours. This test was found to be very useful for the detection of CymMV in orchids because it is sensitive, economical, and easy to perform.

• Journal of Plant Biology
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• 제37권3호
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• pp.349-355
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• 1994

Viral RNA was extracted from a purified orchid strain of tobacco mosaic virus (TMV-O) from Cymbidium "Grace Kelly". Polyadenylated viral RNAs were primed with Not I-oligo (dT) primer-adapter. First-strand cDNAs were reversely transcribed by Moloney murine leukaemia virus reverse transcriptase (RNAse H-), and then second-strand cDNAs were synthesized by RNase H and DNA polymerase I. The resulting double-stranded cDNAs were ligated into pSPORT1 vector and transformed into competent E. coli strain JM109 cells. The size of cDNAs within the recombinant plasmids was ranging from 0.9 to 3.9 kb. Among the selected clones, pTMO-0205 and -0210 covered the 3' half and the 5' half of the viral genomic RNA, respectively, which were covering more than 99% of the viral genemo size based on sequencing analysis. Two cDNA fragments which were 3.1 kb BamHI and NotI fragement released from pTMO-0.205 and 3.3 kb SalI and BamHI fragment released from pTMO-0210 were ligated with T4 DNA ligase. The clone was almost entire length, lacking only 31 nucleotides from the 5' terminus based on the sequencing result. This method was shown to be efficiently applicable to other plant viral gnomic RNA for the construction of cDNA.n of cDNA.

• The Plant Pathology Journal
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• 제38권6호
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• pp.665-672
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• 2022

Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39℃) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.

• Journal of Plant Biology
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• 제38권2호
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• pp.129-136
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• 1995

Complementary DNA of the genomic RNA of odontoglossum ringspot virus Cymbidium strain (ORSV-Cy) was synthesized from polyadenylated viral RNA and cloned. Selected clones containing the viral RNA-dependent RNA polymerase gene of the virus has been sequenced by automated sequencing system. The complete nucleotide sequence of an open reading frame is 1377 base pairs in length, and encodes a protein of 458 amino acids about 52, 334 D. The 52 kD protein of ORSV shares four sequence motifs characteristic of viral RNA-dependent RNA polymerase. Comparison of the ORSV 52 kD protein sequence with that of other five viruses in tobamovirus group showed 76.0 to 60.7% homologies at the amino acid level and the conservation of the four motifs betwen the viruses.