• The Plant Pathology Journal
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• v.36 no.5
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• pp.509-514
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• 2020

Satsuma dwarf virus (SDV) seriously damages citrus production by reducing the quality and yield of fruit. To avoid contamination with SDV, mother trees are checked to be SDV-free in advance of nursery tree distribution. In this study, we compared an immunochromatographic assay (ICA) kit with double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for large-scale diagnosis of SDV in orchardgrown trees in Shizuoka Prefecture, Japan. The two methods gave conflicting results for 11 of 1,705 samples, all of which were negative by DAS-ELISA but positive by ICA. The samples scored as positive by either DAS-ELISA or ICA were analyzed by reverse transcription polymerase chain reaction and all were confirmed to be positive. These results validate the use of ICA as a screening method for large-scale diagnosis. Strain discrimination revealed that 16 of 22 isolates belonged to SDV, while citrus mosaic virus (CiMV) infection only and co-infection (SDV and CiMV) were in a minority.

• The Plant Pathology Journal
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• v.22 no.1
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• pp.21-27
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• 2006

We immunized BALB/c mice with purified Cucumber green mottle mosaic virus isolate HY1 (CGMMV-HY1). Through the selection of positive clones that were grown on the HAT medium, four sensitive monoclonal clones (CG99-01, CG99-02, CG99-03, and CG99-04) were selected from 500 Hypoxanthine-guanine phosphoribosyltransferase positive hybridoma cells. Four sensitive clones of CGMMV-HYI were determined as IgM type of the subclass of mouse immunoglobulins Ig group. The titer of monoclonal antiserum against CGMMVHY1 was estimated 1:12,800 by the indirect ELISA. Although monoclonal antibodies (MAbs) from CG99-01 and from CG99-04 cross-reacted with Zucchini green mottle mosaic virus and Kyuri green mottle mosaic virus, MAb from the cell line CG99-03 was highly specific to CGMMV. No MAbs cross-reacted with Cucumber mosaic virus-Fny. Only CG99-04 reacted with Pepper mild mottle virus weakly and CG99-02 reacted with both CGMMV and KGMMV. CGMMV was detected from the rind of watermelon fruit by DAS-ELISA of CGMMV-HY1, but not from the flesh of watermelon. Average seed transmission rate of CGMMV in watermelon was $24\%$ from symptomatic watermelon collected from 5 regions of Gyeongnam province. CGMMV was detected by DAS-ELISA with specific MAb of CGMMVHY1 periodically from root stock, during the sequential process for nursery seedling in Haman. Necrotic spots on cotyledons of root stock seedling progressed to reveal the typical symptomatology on the primary leaves of scion upon grafting. Here, we have established MAb based ELISA system, which could accurately detect CGMMV from watermelon seeds, nursery seedlings, transplants and field samples from greenhouse or open out door field as well.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2005.04a
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• pp.57.2-58
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• 2005

• Research in Plant Disease
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• v.10 no.3
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• pp.194-197
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• 2004

Incidence of tobamovirus diseases was 100％ at late growth stage of green pepper on hydroponic systems in plastic house. Infection frequency of the diseases showed 34％ of Pepper mild mottle virus (PMMoV), 41.5％ of Tobacco mild green mottle virus (TMGMV), and 24.5％ of the co-infected viruses. The two viruses specifically reacted in DAS-ELISA prepared with each polyclonal antibody. A total of 77 pure tobamovirus isolates obtained from the crops was tested for pathotype determination. The isolation frequency of tobamovirus pathotype $P_{0}$ and $P_{1,2}$ was 61 ％ and 39％, respectively. All TMGMV isolates belonged to the pathotype $P_{0}$. In restriction enzyme analysis of the cDNAs synthesized with coat protein gene of PMMoV pathotype $P_{0}$ and $P_{1,2}$, the former had two TaqI sites but the later had one.one.

• Research in Plant Disease
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• v.16 no.3
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• pp.326-330
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• 2010

Three pome fruit viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASPV) and Apple stem pitting virus (ASGV) were detected in pear trees (Pyrus pyrifolia) using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in Ansung, Naju and Ulsan provinces of Korea. Infection rate of three viruses was 35.2% from 452 leaf samples of the three cultivars of pear trees. Also, each of three viruses was detected by reverse transcription polymerase chain reaction (RT-PCR) for a limited number of samples. Infection rate of three viruses was 86.3% from 233 leaf samples of the three pear cultivars. The virus infection rates by RT-PCR were much higher than ELISA. ASGV was prevailing on pear with 74.2%, whereas ASPV and ACLSV were found in 34.8% and 0.4% of tested samples, respectively. Symptoms caused by ASGV showed black spots of infected Niitaka cultivar leaves. The ACLSV, ASPV and ASGV isolates showed 83~94% sequence identity at a nucleotide level to other pome fruit virus isolates when analyzed by NCBI BLAST. Pome fruit viruses occurring in pear were ACLSV, ASPV and ASGV. This is the first report of pear trees infected ASPV in Korea.

• The Plant Pathology Journal
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• v.31 no.1
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• pp.41-49
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• 2015

Sweet cherry is an important fruit crop with increasing economical value in Turkey and the world. A number of viruses cause diseases and economical losses in sweet cherry. Prune dwarf virus (PDV), is one of the most common viruses of stone fruits including sweet cherry in the world. In this study, PDV was detected from 316 of 521 sweet cherry samples collected from 142 orchards in 10 districts of Isparta province of Turkey by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA). The presence of PDV in ELISA positive samples was confirmed in 37 isolates by reverse transcription- polymerase chain reaction (RT-PCR) method. A genomic region of 862 bp containing the coat protein (CP) gene of PDV was re-amplified from 21 selected isolates by RT-PCR. Amplified DNA fragments of these isolates were purified and sequenced for molecular characterization and determining genetic diversity of PDV. Sequence comparisons showed 84-99% to 81-100% sequence identity at nucleotide and amino acid level, respectively, of the CP genes of PDV isolates from Isparta and other parts of the world. Phylogenetic analyses of the CP genes of PDV isolates from different geographical origins and diverse hosts revealed that PDV isolates formed different phylogenetic groups. While isolates were not grouped solely based on their geographical origins or hosts, some association between phylogenetic groups and geographical origins or hosts were observed.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.2
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• pp.89-95
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• 2006

Potato plants carrying the Ry gene are extremely resistance to a number of potyviruses, but it is not known which variety expressed the resistance. In this investigation, combined classical and molecular techniques were used to identify virus resistance potatoes. Mechanical inoculation of 32 varieties of Korean potato cultivars, with potato virus Y (PVY), induced various symptoms, such as mosaic, yellowing, necrosis, mottle, vein clearing and vein bending. Different virus spreading patterns were observed, such as highly sensitive, moderate and resistant to $PVY^o$ inoculated leaves in different cultivars. From the results of double antibody sandwich-enzyme links immunosorbant assays (DAS-ELISA), coupled with reverse transcription polymerase chain reaction (RT-PCR), Winter valley and Golden valley were found to be highly susceptible and resistant cultivars to $PVY^o$ respectively. TEM was used as a complementary method to conform the localization of the virus in leaf tissues. TEM detect virus particles in Golden valley, where, ELISA and RT-PCR were unable to detect the CP gene. However, the interior part of the tissues was severely deformed in $PVY^o$ infected Winter valley, than Golden valley The Ry gene is involved in an induced response in $PVY^o$ infected Golden valley plants. The methods described in this study could be applied for the screening and development of antiviral potatoes.

• The Plant Pathology Journal
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• v.25 no.3
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• pp.225-230
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• 2009

Lily mottle virus (LMoV) causes flower quality reduction in Lilium spp. The coat protein gene was RT-PCR-amplified from total RNA extracted from infected lily leaves and the amplified fragment was cloned into the pRSET expression vector tagged with a His-MBP. The plasmid of recombinant coat protein was used to transform an Escherichia coli strain pLysS and was expressed. The coat protein was purified by affinity chromatography using a Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE. The in vitro-expressed protein was used for immunization of mice. The polyclonal and monoclonal antibodies reacted specifically for the detection of LMoV in lily extracts in Western blot. Moreover the monoclonal antibodies reacted with lily extracts in DAS-ELISA with no unspecific or heterologous reactions against other non-serologically related viruses, but the polyclonal antibodies revealed a weak reaction against both infected lily and healthy control.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.140.2-141
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• 2003

Among over-wintering small brown planthoppers, population of the rice stripe virus (RSV)-viruliferous insects was surveyed throughout the country in late April of 2003 by using DAS-ELISA. Averaged population of the RSV-viruliferous insects in this year was 2.1％, which was lower than that of last year of 3.7％. However, the insect population in Seoul, Incheon and Kyeonggi areas were relatively high showing 6.7％, 6.2％ and 2.6％, respectively. Based on the survey results, it was expected that overall occurrence of RSV on rice could be decreased in this year, except certain areas. Ovarial transmission rate of RSV by the insects on diseased rice samples collected from 10 areas ranged from 22.2％ to 77.8％. Among 35 graminous weed species collected from rice fields in Ganghwa and Kimpo in 2002 and 2003, common reed and formosens were found to be infected by RSV. The result indicates that those weeds are potential alternative natural hosts of the RSV Further studies on ecological and pathological impacts of the alternative natural host of RSV are being processed.

• The Plant Pathology Journal
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• v.19 no.3
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• pp.166-170
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• 2003

Ninety samples showing mosaic symptoms on soybean (Glycine max) cv. Sukryangputkong were collected from the Cheongsongkun area, Kyungbuk province in Korea. Initially, DAS-ELISA was conducted far detection of Soybean mosaic virus (SMV). Negative samples were chosen at random and mechanically inoculated on soybean cv. Buffalo, which reported not to produce mosaic symptoms when mechanically inoculated with SMV. An isolate of SMV, designated as B-1, from Buffalo showing mosaic and mottle symptoms was used for identification and biological characterization of the causal vim. The purified B-1 isolate had spherical particles of approximately 24nm. It positively reacted with the antiserum against Cowpea mosaic virus (CPMV) but not with Cucumber mosaic virus (CMV) and SMV antisera. CPMV was newly isolated from soybean and had been characterized by host range and by serological and electron microscopic methods. Results of this study suggest that CPMV is the possible cause of mosaic disease in vegetable soybean and that based on sympto-matology, a difference between the typical mosaic and rugose symptoms caused by SMV and CPMV was observed. This is first report of CPMV from soybean in Korea.