• The Plant Pathology Journal
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• 제18권5호
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• pp.244-250
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• 2002

Grapevine leafroll-associated 3 virus (GLRaV-3) and Grapevine fanleaf virus (GFLV) are important viral diseases of grapevine in the world. In this study, the most reliable woody indicator plants were selected for virus indexing. Two grapevines, LN33 (Couderc 1613x vitis berlandieri) and Vitis riparia Gloire, were selected for CLRaV-3 and CFLV graft indexing, respectively. The specific characteristics of Closterovirus isolated from grapevines cultivated in Korea were identified. filamentous virus-like particles only existed in the phloem parenchyma cell. In particular, the vesiculation of mitochondria was observed. This mitochondrial vesicu-lation was considered to be one of the most reliable cytopathic features of Closterovirus. During observation of GFLV-infected Chenopodium quinoa sections, virus-like particles arranged consistently were found forming several layers in cytoplasm. Moreover, virus-like particles in tubules were observed and were associated with plasmodesmata in cytoplasm. This is the first report on cytopathological characteristics of Closterovirus and Nepovirus identified from grapevines in Korea.

• The Plant Pathology Journal
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• 제32권4호
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.138.2-139
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• 2003

Grapevine leafroll-associated 1 virus (GLRaV-1) and Grapevine leafroll-associated 3 virus (GLRaV-3), member of the genus Ampelovirus, are important viral disease of grapevine in the world. these viruses transmitted only dicotyledonous host by vectors such as mealybugs and there is no suitable herbaceous host for virus. The diseased leaves turn yellowish or reddish depending on cultivars and viruses. Viruses are existed at low concentration and ununiformly distribution in grapevine. Using small-scale double-stranded RNA (dsRNA) extraction method, reverse transcription and polymerase chain reaction (RT-PCR) product of 1Kb long which encoded of coat protein (CP) gene for both viruses was successfully amplified with a specific primers. The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined from selected recombinant cDNA clones. Sequence analysis revealed that the CP of GLRaV-1 consisted of 969 nucleotide, which encoded 323 amino acid residues and CP of GLRaV-3 consisted of 942 nucleotide, which encoded 314 amino acid residues. The CP of GLRaV-1 and GLRaV-3 has 93.8％ and 98.7％ amino acid sequence identities, respectively.

• Journal of Plant Biotechnology
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• 제30권2호
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• pp.155-160
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• 2003

Grapevine leafroll-associated virus 3(GLRaV-3) is one of the most severe pathogens for viral diseases found in Korea. This study was conducted to establish the virus-free stock production system for the virus disease control. The effects of thermotherapy, merestem culture and chemotheratpy to eliminate the GLRaV-3 in gratevines were tested. Thermotherapy at 37$\pm$2$^{\circ}C$ for 6∼8 weeks combined with 0.5∼1.0mm size of meristem culture method was the most effective for virus elimination. Thermotherapy alone was not effective. In chemotheratpy, DHT and Amantadine (20, 40mg/L) treatment in medium was more effective than Ribavirin to eliminate the GLRaV-3 in grapevine. However, Ribavirin spraying to potted was not available for virus elimination. Therefore, virus-free stock production system using the thermotherapy combined with shoot apical meristem culture was the most effective in grapevine.

• The Plant Pathology Journal
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• 제33권6호
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• pp.561-571
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• 2017

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. 'Hencha' were successfully induced from filament, when cultured on $Ch{\acute{e}}e$and Pool (1987). based-medium, enriched with $2mg1^{-1}$ of 2,4-dichlorophenoxyacetic acid and $2.5mg1^{-1}$ of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPaV as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% 'Hencha' somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination.

• The Plant Pathology Journal
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• 제33권1호
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• pp.34-42
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• 2017

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a worldwide-distributed pathogen in grapevines with a high genetic variability. Our study revealed differences in the complexity of GRSPaV population in a single host. A single-variant GRSPaV infection was detected from the SK30 grapevine plant. On the contrary, SK704 grapevine was infected by three different GRSPaV variants. Variant-specific RT-PCR detection protocols have been developed in this work to study distribution of the three different variants in the same plant during the season. This study showed their randomized distribution in the infected SK704 grapevine plant. Comparative analysis of full-length genome sequences of four Slovak GRSPaV isolates determined in this work and 14 database sequences showed that population of the virus cluster into four major phylogenetic lineages. Moreover, our analyses suggest that genetic recombination along with point mutations could play a significant role in shaping evolutionary history of GRSPaV and contributed to its extant genetic diversification.

• The Plant Pathology Journal
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• 제32권5호
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• pp.452-459
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• 2016

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-${\beta}$-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

• 식물조직배양학회지
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• 제28권6호
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• pp.335-339
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• 2001

우리나라 포도원에서 가장 많이 발생하여 피해를 주고 있는 grapevine leafroll-associated 3 Closterovirus (GLRaV-3)를 대상으로 기내 배양묘를 이용한 바이러스 순화 및 진단효율성을 검토하였다. 바이러스 감염주의 절간을 기내배양하여 증식시킨 기내 배양묘를 1개월 간격으로 계대배양하면서 배양묘를 ELISA 검정한 결과 고농도의 바이러스가 검출되었으며 배양묘의 부위별로 잎, 줄기 및 줄기 유래의 callus 조직에서 모두 고농도로 검출되었다. 또한 포장에서 재배되고 있는 포도나무의 잎이나 엽병조직에 비해 기내 배양묘의 조직을 사용하였을 때 높은 농도의 정제 바이러스를 얻을 수 있었으며, 전자현미경에 의한 dip 검경에서도 사상형 바이러스 입자가 관찰되었다. RT-PCR 진단에서는 기내 배양묘 조직을 사용하였을 때 포도 유엽조직과 엽병+중륵조직에 비해 검출효율이 높았다. 기내 배양묘의 잎조직을 이용하여 ELISA와 RT-PCR 검정감도를 비교한 결과 ELISA에 비해 RT-PCR 검정이 약 1,000배 감도가 높았다.

• The Plant Pathology Journal
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• 제20권2호
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• pp.127-130
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• 2004

Grapevine leaf roll-associated virus 3 (GLRaV-3) is one of the most important viral diseases of grapevine in the world. In this study, GLRaV-3 Ampelovirus was identi-fied from grapevines in Korea by analyzing viral coat protein size, nucleotide, and amino acid sequences. The molecular weight of viral coat protein from virus-infected in vitro plantlets was determined by western blot using a commercial GLRaV-3 polyclonal antibody. Western blot analysis showed a coat protein of about 43 kDa. RT-PCR product of about 942 bp which encoded the coat protein (CP) gene was amplified with specific primers. When the viruses existed at low titers in the host plant, the dsRNA had very specific template in RT- PCR amplification of fruit tree viruses. Especially, small-scale dsRNA extraction method was very reliable and rapid. Sequence analysis revealed that the CP of the GLRaV-3 Ko consisted of 942 bp nucleotide, which encoded 314 amino acid residues. The CP gene of GLRaV-3 Ko had 98.9% nucleotide sequence and 98.7% amino acid sequence identities with earlier reported GLRaV-3. This is the first report on molecular assay of GLRaV-3 Ampelovirus identified from Korea. The GLRaV-3 Ko CP clone would be very useful for breeding of virus resistant grapevines.

• Journal of Plant Biotechnology
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• 제44권4호
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• pp.401-408
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• 2017

본 연구진은 세계 최초로 GFkV에 단독 감염된 포도 품종 거봉의 휴면아로부터 직접 경정 분열조직을 절취하여 배양함으로써 별도의 열처리나 화학처리 없이 GFkV가 제거된 신초 재생에 성공하였다. GFkV는 포도 식물체의 체관에만 한정적으로 존재하고 접목으로 감염되는 포도 바이러스이다. 경정조직은 $4^{\circ}C$에서 일정기간 저장된 1년생 경화가지의 휴면아로부터 0.3 mm (73 절편체)와 0.8 mm (5절편체)를 절취하였는데, 절취 부위는 생장점(apical meristem)을 포함하여 2 ~ 5개의 엽원기(leaf primordia)와 미분화 원기(uncommitted primordia) 1 ~ 4개를 포함하였다. 절취된 경정조직을 BA 3.0 mg/L와 IBA 0.1 mg/L가 혼합된 배지에 16주간 배양한 결과, 0.3 mm와 0.8 mm의 경정조직에서 각각 4.1%와 40.0%의 신초 재생률이 관찰되었고, 재생된 신초에서의 바이러스 제거율(재생된 신초수에 대한 RT-PCR negative 신초수의 백분율)은 0.3 mm의 경정조직에서는 100%를, 0.8 mm에서는 50%를 나타내었다. 신초를 증식시킨 후 감염바이러스를 다시 진단한 결과, 신초가 처음 재생되었을 때의 진단결과와 동일하였다. 휴면아로부터 분열조직을 배양하여 바이러스가 제거된 신초를 확보한 예는 세계적으로 보고된 바가 없는 것으로서, 본 연구진의 새로운 바이러스 제거방법은 향후 포도 뿐만 아니라 과수를 포함한 낙엽성 수목의 무병묘 생산에 매우 유용하게 활용될 수 있을 것이다.