우리나라 토마토에 발생한 토마토황화잎말림바이러스(Tomato yellow leaf curl geminivirus)의 초간편 Virion Capture(VC)/PCR 진단법

조점덕,김태성,김주희,최국선,정봉남,최홍수,김정수 한국식물병리학회 2008 식물병연구 Vol.14 No.3

Tomato yellow leaf curl virus (TYLCV), a newly reported Geminivirus from tomato, generated recently large economic losses in Korea. Development of a fast and precise genetic diagnosis technique for detecting TYLCV which Agricultural research and extension services can utilize easy and handy is very important to prevent yield losses. Virion Capture (VC)/PCR is a simple, accurate and economical genetic detection method without any works or commercial kits for the extraction of the nucleic acid from the infected plants. Primers of twenty two for detection of TYLCV were designed and tested with extracted total DNA or crude sap from tomato leaf infected with TYLCV and healthy plant. Nine primers for total DNA using conventional PCR and another 9 primers for VC/PCR were selected eventually. Primers of six having same specificity were selected from the two methods and tested with other Geminivirus, Tobacco leaf curl virus (TLCV) by VC/PCR. Finally specific primers of four were selected for detection of TYLCV using VC/PCR, and Deng (540, 541), a degenerate primer for Geminivirus reported in 1996, was also developed for VC/PCR. Tomato yellow leaf curl virus (TYLCV), a newly reported Geminivirus from tomato, generated recently large economic losses in Korea. Development of a fast and precise genetic diagnosis technique for detecting TYLCV which Agricultural research and extension services can utilize easy and handy is very important to prevent yield losses. Virion Capture (VC)/PCR is a simple, accurate and economical genetic detection method without any works or commercial kits for the extraction of the nucleic acid from the infected plants. Primers of twenty two for detection of TYLCV were designed and tested with extracted total DNA or crude sap from tomato leaf infected with TYLCV and healthy plant. Nine primers for total DNA using conventional PCR and another 9 primers for VC/PCR were selected eventually. Primers of six having same specificity were selected from the two methods and tested with other Geminivirus, Tobacco leaf curl virus (TLCV) by VC/PCR. Finally specific primers of four were selected for detection of TYLCV using VC/PCR, and Deng (540, 541), a degenerate primer for Geminivirus reported in 1996, was also developed for VC/PCR.

Biological Characteristics and Nucleotide Relationships in Korean Tomato spotted wilt virus Isolates

조점덕,김정수,정봉남,김진영,최국선 한국식물병리학회 2009 Plant Pathology Journal Vol.25 No.1

Tomato spotted wilt virus (TSWV) was identified from seven plants at two areas, Anyang and Dangjin, in Korea. The isolates of TSWV were seven as TSWVKATm from tomato, TSWV-KAPo from potato, TSWVKABal from balsam, TSWV-KACTm from cherry tomato and TSWV-KAIxe from Ixeris dentata at Anyang area, and TSWV-KDSe from sesame and TSWV-KDRP from red pepper at Dangjin area. Pathogenicity of seven TSWV isolates was various on the assay plants, and could not be grouped definitely. Three isolates of TSWVKAIxe, TSWV-KACTm and TSWV-KABal had relatively narrower host ranges among the seven isolates. Percentage of nucleotide substitution in nucleotide sequences encoding nucleocapsid protein (NCP) was 1.2-1.7% among seven TSWV isolates and TSWV-KP. Korean TSWV isolates were divided into three groups by nucleotide homology or amino acid compositions. From the analysis of nucleotide sequences of Korean TSWV isolates compared with those of TSWV reported from other 5 countries including Japan, the Korean seven isolates of TSWV was grouped with German TSWV (D13926). No Korean TSWV isolates were grouped with those from The Netherlands, Brazil and USA.

박과 작물 종자전염 바이러스 3종(CGMMV, ZGMMV, KGMMV)의간편한 동시진단 VC/RT-PCR 유전자 진단

조점덕,정봉남,김정수,이신호 한국식물병리학회 2007 식물병연구 Vol.13 No.2

Choi, G. S. 2001. Occurrence of two tobamovirus diseases incucurbits and control measures in Korea. Plant Pathol. J.17(5): 243-248.... , ... , ... , ... , ... . 2006. Tomato spottedwilt virus. .. ... .. .... .. ... : Virioncapture/RT-PCR (VC/RT-PCR). ..... 12(2): 139-143......... . 2004. ....... .James, D. 1999. A simple and reliable protocol for the detectionof apple stem grooving virus by RT-PCR and in a multiplexPCR assay. J. Virol. Meth. 83: 1-9.Kim, J. H., Choi, G. S., Kim, J. S., Le, S. H., Choi, J. K. and Ryu,K. H. 2006. Development of single-tube multiplex immuno-capture RT-PCR Assay for simultaneous detection of twopepper Tobamoviruses. Plant Pathol. J. 22(2): 164-167.Ko, S. J., Lee, Y. H., Cha, K. H., Lee, S. H., Choi, H. S., Choi, Y.S., Lim, G. C. and Kim, K. H. 2006. Incidence and distributionof virus diseases on cucumber in Jeonnam province during1999-2002. Plant Pathol. J. 2(2): 147-151.Li, R. and Mock, R. 2005. An improved reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection oftwo cherry flexiviruses in Prunus spp. J. Virol. Meth. 129:162-169.Nolasco, G. deBlas, C., Torres, V. and Ponz, F. 1993. A methodcombining immunocapture and PCR amplification in amicrotiter plate for the detection of plant viruses and subvirialphathogenes. J. Viro. Methods 45: 201-218.Ryu, K. H., Min, B. E., Choi, G. S., Choi, S. H., Kwon, S. B., Noh,G. M., Yoon, J. Y., Choi, Y. M., Jang, S. H., Lee, G. P., Cho, K.H. and Park, W. M. 2000. Zucchini green mottle mosaic virusis a new tobamovirus; comparison of its coat protein gene withthat of Kyuri green mottle mosaic virus. Arch. Virol. 145:2325-2333.Sharman, M., Thomas, J. E. and Dietzgen, R. G. 20. Developmentof a multiplex immunocapture PCR with colourimetric

고추에 엽맥퇴록병을 일으키는 오이 모자이크 바이러스(CMV-VCH)의 특징

조점덕,김현란,류기현,정봉남,이신호,김정수,최국선 한국식물병리학회 2006 식물병연구 Vol.12 No.3

Cucumber mosaic virus (CMV) was occurred on red pepper showing vein chlorosis or vein necrosis with the incidence rate of 52% from 62 specimens collected in natural fields. Among 32 samples infected with CMV, the specimens of 22 red pepper leaves showing vein chlorosis were infected singly with CMV-VCH. CMVVCH induced vein chlorosis on the inoculated leaves, and vein banding and vein necrosis on the upper leaves of Nicotiana glutinosa, and then killed after showing bud necrosis. The typical symptoms of vein banding, malformation and blister were produced on the upper leaves of Nicotiana benthamiana and N. tabacum ‘Ky- 57’ without symptoms on the inoculated leaves. The commercial cultivars of ‘Bugang’, ‘Manitta’ and ‘Gwariput’ were shown the typical symptom of vein chlorosis by the mechanical inoculation of CMV-VCH. CMV-VCH was detected specifically by RT-PCR. Virus particles of CMV-VCH were isometric shape having 30 nm diameter. Ultraviolet absorption of purified CMV-VCH was maximum at 260 nm and minimum at 242 nm. The ratio of A260/A280 was 1.71. CMV-VCH had the single nucleo-protein having the molecular weight of 24.5 kDa.

Symptom Variances in Mixed Infections of Six Turnip mosaic virus and One Ribgrass mosaic virus Isolates in Crucifers

조점덕,최홍수,김국형,Jeong-SooKim,Kyung-SooKim 한국식물병리학회 2003 Plant Pathology Journal Vol.19 No.2

Turnip mosaic Potyvirus (TuMV) and Ribgrass mosaic Tobamovirus (RMV) are major viruses infecting crucifer crops in Korea. RMV-FG22 was isolated from oriental cabbage. TuMV isolates were TuMV-CA7 from oriental cabbage, TuMV-TU and TuMV-TU2 from turnip, TuMVRA from rape, TuMV-ST from stock, and TuMV-R9 from radish. The six isolates of TuMV were classified by symptom expression in inbred lines of crucifers. TuMVCA7 and TuMV-TU isolates infected mostly oriental cabbages; TuMV-ST, TuMV-TU2, and TuMV-R9 infected radishes; and TuMV-RA infected both oriental cabbages and radishes. Crops used in six combinations of mixed infections were ‘Tambok’ cultivar resistant to TuMV, ‘SSD63’ susceptible inbred line of oriental cabbage, pure line of leaf mustard, and ‘Daeburyungyeorum’ cultivar of radish. External symptoms in ‘Tambok’ and radish by each of the six single infections of TuMV showed similar results by bioassay. Synergistic response of necrotic death occurred within 1 week after inoculation in all combinations mixed with TuMV and RMV-FG22 on leaf mustard. In oriental cabbage ‘SSD63’, synergism of necrosis occurred in four TuMV isolates, but not in TuMV-ST and TuMV-R9. In oriental cabbage ‘Tambok’, synergism was expressed only in two combinations of RMV-FG22+TuMV-CA7 and RMV-FG22+TuMV-TU, but other combinations had the same symptoms produced by RMV-FG22. In radish ‘Daeburyungyeorum’, only mild mosaic symptoms were induced by combinations of RMV-FG22+TuMVCA7, RMV-FG22+TuMV-TU, RMV-FG22+TuMVRA, and RMV-FG22+TuMV-R9. Mosaic and severe mosaic were induced in combinations of RMV-FG22 +TuMV-TU2 and RMV-FG22+TuMV-ST, respectively.

Strawberry mild yellow edge potexvirus from Strawberry in Korea

조점덕,최국선,정봉남,김정수,최홍수 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.2

Symptoms induced in the leaves of strawberry (Fragaria x ananassa Duch.), ‘Seolhyang’ and ‘Eyeberry’, were mosaic, distortion and black colored edge on leaves at Nonsan area, one of the important production areas in Korea. Electron microscopy by quick-dip revealed the flexuous rod-shape particles having about 550-600 nm length. Cytoplasmic inclusion bodies composed of aggregated virus particles were observed frequently in mesophyll parenchyma and epidermal cells for the leaves of strawberry. The specific primers amplifying products of 635 bp and 729 bp were developed for RT-PCR detection of Strawberry mild yellow edge virus (SMYEV). Nucleotide identity of the CP gene of SMYEV was 92.8-99.2% with those of other SMYEV isolates from Gen-Bank database.

채소(가지, 알타리무, 슈가로프)에 발생한 토마토반점위조바이러스 (Tomato spotted wilt virus) 발생과 병징 특성

조점덕,김진영,최홍수,최국선,김정수 한국식물병리학회 2010 식물병연구 Vol.16 No.3

Tomato spotted wilt virus (TSWV) was occurred on the three vegetables of egg plant (Solanum melongena),whole radish (Raphanus acanthiformis) and sugar loaf (Cichorium intybus) at Anyang area infested with TSWV. Whole radish was produced the symptoms of necrotic spots on the leaves, and necrosis and malformation on the roots by TSWV. Egg plant was induced the symptoms of typical multiple ring spots on the leaves and necrotic rings on the fruits. Sugar loaf was infected severely with the typical symptoms of ring spots on the leaves and stunt. The three isolates of TSWV could infect locally on the indicator plants of Chenopodium amaranticolor, C. quinoa and Nicotiana debney, and systemically on N. glutinosa, N. benthamiana and Datura stramonium. Two TSWV isolates from egg plant and sugar loaf were very similar in virulence. However, the virulence of TSWV from whole radish was very different as local infection on 5Nicotiana species including N. tabacum ‘Xanthi NC’. Tomato spotted wilt virus(TSWV)의 상습 발생지인 안양지역에서 가지, 알타리무, 슈가로프에서 TSWV가 발생하였다. 알타리무에서는 잎에 괴저반점과 뿌리에 괴저 병징이 나타났다. 가지에서는 잎에 전형적인 다중 원형반점을 나타냈으며 열매에 심한 괴저를 나타냈다. 슈가로프에서는 잎에 전형적인 원형반점과 심한 위축 병징이 나타났다. 가지, 알타리 무, 슈가로프에서 분리한 TSWV의 생물적 특성은 흰 명아주, 붉은 명아주, 담배(N. devney)에서는 국부 감염이었으며, 담배(N. glutinosa, N. benthamiana)와 독말풀(D. stramonium)에서는 전심감염이었다. 가지와 슈가로프에서 분리한 TSWV는 병원성이 유사하였으나, 알타리 무에서 분리한 TSWV는 N. tabacum ‘Xanthi NC’ 등 5종의 담배에서 국부 감염되어 병원성이 매우 상이하였다.

과일 괴저 병징의 단 고추에서 분류동정한 오이모자이크바이러스의 새로운 계통 CMV-NP 특성

조점덕,김정수,이중환,정봉남 한국식물병리학회 2008 식물병연구 Vol.14 No.2

A strain of Cucumber mosaic virus (CMV) was isolated newly from sweet pepper (Capsicum annuum var. angulosum) showing necrosis with large necrotic spots on fruits and vein banding with malformation on leaf at Cheongdo area in Gyeongsangbukdo. The new strain was designated as a CMV-NP and the shape of virus particles was isometric of 26 nm in diameter from the sweet pepper fruit by Dip method. The strain of CMVNP was identified genetically by VC/RT-PCR. CMV-NP could infect systemically on the 9 indicator plants including Cucumis sativus, but it could infect locally on Chenopodim amaranticolor and C. quinoa. CMV-NP induced the specific symptoms of necrotic rings on the inoculated and the upper leaves of N. rustica and Tetragonia expansa. On Cucumis sativus, the large chlorotic ring and vein chlorosis were produced on the upper leaves. CMV-NP had no virulence on Datura stramonium. A strain of Cucumber mosaic virus (CMV) was isolated newly from sweet pepper (Capsicum annuum var. angulosum) showing necrosis with large necrotic spots on fruits and vein banding with malformation on leaf at Cheongdo area in Gyeongsangbukdo. The new strain was designated as a CMV-NP and the shape of virus particles was isometric of 26 nm in diameter from the sweet pepper fruit by Dip method. The strain of CMVNP was identified genetically by VC/RT-PCR. CMV-NP could infect systemically on the 9 indicator plants including Cucumis sativus, but it could infect locally on Chenopodim amaranticolor and C. quinoa. CMV-NP induced the specific symptoms of necrotic rings on the inoculated and the upper leaves of N. rustica and Tetragonia expansa. On Cucumis sativus, the large chlorotic ring and vein chlorosis were produced on the upper leaves. CMV-NP had no virulence on Datura stramonium.

2006년과 2007년 상주와 구례에서 발생한 오이 바이러스병의 병징 특성

조점덕,김정수,이중환,고숙주,최홍수,이수헌,최국선 한국식물병리학회 2011 식물병연구 Vol.17 No.2

Virus diseases occurring on cucumber was surveyed at main plantation areas of ‘Sangju’ and ‘Gurye’ in 2006and 2007. Viral infection rate on cucumber was ranged from 14% to 90% in fields and the average infection rate was 46% at Sangju area. Cucumber cultivated at Gurye area had viral incidence ranged 9−100% and averaged 48%. The majorly infected viruses were Cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, Papaya ring spot virus and Watermelon mosaic virus-2, and their infection rates were 23.5%,13.0%, 9.0% and 2.0%, respectively in 2006 and 2007. Mixed infection rate of duplex, triplex and tetraplex was 31.5%, 7.5% and 2.5%, respectively. The rate of infection type was 47.7% by single infection, 31.5% by double infection and 88.7% by both infection types. Important viruses involved in mixed infection types were Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, Papaya ring spot virus and Broad been wilt virus2, orderly. Symptom in single infection was almost same on ‘Dadagi’ line and ‘Chicheong’ line of cucumber cultivars. Cucumber green mottle mosaic virus produced various symptoms of chlorotic spot, vein chlorosis and vein wrinkle and so on. Main symptoms of vein chlorosis, and severe mosaic and malformation were induced by Zucchini yellow mosaic virus. Mild symptoms were occurred relatively by Papaya ring spot virus and Watermelon mosaic virus2. 오이 주요재배지인 상주와 구례에서 오이에 발생하는 바이러스병을 2006년과 2007년에 조사하였다. 경상북도 상주 지역 오이의 병징 발생률은 농가 포장에 따라서 14%에서 90%이었으며 평균 발생률은 46%이었다. 전라남도 구례 지역의 경우 농가 포장에 따라서 9%에서 100%이었으며 평균 발병률은 48%이었다. 주요 발생 바이러스는 CGMMV, ZYMV, PRSV와 WMV2의 4종류였다. 이러한 바이러스는 단독감염과 복합감염 형태로 발생하였다. 2006년과 2007년의 평균 감염률을 보면 단독감염의 경우 ZYMV가 23.5%로 가장 많이 발생하고 있었으며, PRSV가 13.0%, CGMMV가 9.0%, WMV2가 2.0% 순서이었다. 복합감염의 경우에는 2종 바이러스 복합감염이 31.5%, 3종 복합감염이 7.5%, 4종 복합감염이 2.0% 발생하였다. 오이에 발생하고 있는 바이러스 감염형태는 단독감염과 2종 복합감염이 각각 47.7%와 31.5%로 총 88.7%로 대부분을 차지하였다. 오이에 발생한 바이러스의 복합감염으로 많이 발생되는 바이러스로는 ZYMV, CGMMV, PRSV, BBWV2의 순서이었다. 단독감염된 오이의 병징은 다다기 계통이나 취청 계통 오이 모두 비슷한 병징이 나타났으나, CGMMV의 경우 퇴록 반점, 엽맥 퇴록, 엽맥 쭈그러짐 등 매우 다양한 병징이 나타났다. ZYMV는 엽맥 퇴록 병징을 위주로 심한 모자이크 및 기형 병징이 나타났다. 그 이외의 PRSV, WMV-2의 병징은 CGMMV와 ZYMV에 의한 병징에 속하는 비교적 약한 병징이었다.

Ultrastructural Aspects of Mixed Infections with Turnip mosaic virus(TuMV-AC18 and -C5) and Ribgrass mosaic virus (RMV-CA1) in Oriental Cabbage

조점덕,최홍수,나용준,Jeong-SooKim,Kyung-SooKim 한국식물병리학회 2002 Plant Pathology Journal Vol.18 No.4

Turnip mosaic virus (TuMV) in the family Potyviridae and Ribgrass mosaic virus (RMV) in the genus Tobamovirus, were studied ultrastructurally on oriental cabbage. TuMV-AC18 (alpine isolate in Korea) induced chlorotic spots on inoculated leaves of both ‘SSD63’ inbred line known as susceptible to TuMV, and ‘Tambok’ commercial cultivar, known as resistant to the virus, in the early stages of infection. TuMV-C5 (Taiwan isolate) caused severe mosaic and malformation on the upper leaves of ‘SSD63’, and necrotic spots in both inoculated and upper leaves of ‘Tambok’. RMV-CA1 (oriental cabbage isolate from alpine in Korea) induced vein chlorosis, leaf malformation, and midrib necrotic streak in the upper leaves of both ‘SSD63’ and ‘Tambok’. Both oriental cabbages infected with a combination of TuMV-AC18 and RMV-CA1 showed synergistic symptoms of severe yellowing, severe mosaic, and necrotic spot or vein necrosis on their leaves. A combination of TuMV-C5 and RMV-CA1 produced synergistic symptoms only in ‘SSD63’. In ‘Tambok’ infected with the combination of TuMV-C5 and RMV-CA1, the number of necrotic spots on the inoculated leaves was one half lesser than that on singly infected with TuMV-C5. A few necrotic spots progressed systemically. In cells infected with a combination of TuMV-AC18 and RMV-CA1 or TuMV-C5 and RMV-CA1, the particles of the two viruses made nonagon-like rings (NLR); one TuMV particle was surrounded loosely by nine RMV particles. Two unrelated viruses of TuMV and RMV were compacted in the central part of the spiral aggregates (SA) that was induced strikingly in cells by the mixed infections. The SA showed NLR in its center of the cross-sectioned side. Many particles of RMV of Tobamovirus were closely associated with Potyvirus-characteristic cylindrical inclusions. The SAs in the mixed infections were formed easily by the Potyvirus of TuMV-AC18 or -C5 isolates.