식물병연구 Note Open Access Res. Plant Dis. 22(1): (2016) 서향에서분리한신종포티바이러스 (Daphne Mottle Virus) 의동정 Identi

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1 식물병연구 Note Open Access Res. Plant Dis. 22(1): (2016) 서향에서분리한신종포티바이러스 (Daphne Mottle Virus) 의동정 Identification of Daphne Mottle Virus Isolated from Daphne odora, a New Member of the Genus Potyvirus 박충열 1 ㆍ박정안 2 ㆍ이부자 2 ㆍ박상민 1 ㆍ이홍규 1 ㆍ김정선 3 ㆍ윤영남 4 ㆍ서상재 1 ㆍ이수헌 1,5 * 1 경북대학교응용생명과학부, 2 농림축산검역본부식물검역기술개발센터, 3 농촌진흥청국립농업과학원농업미생물과, 4 농촌진흥청국립식량과학원남부작물부, 5 경북대학교식물의학연구소 *Corresponding author Tel: Fax: suheon@knu.ac.kr These authors have contributed equally to this work. Chung Youl Park 1, Jungan Park 2, Boo-Ja Lee 2, Sangmin Bak 1, Hong-Kyu Lee 1, Jeong-Sun Kim 3, Youngnam Yoon 4, Sang Jae Suh 1, and Su-Heon Lee 1,5 * 1 School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea 2 Plant Quarantine Technology Center, Animal and Plant Quarantine Agency, Suwon 16686, Korea 3 Agricultural Microbiology Division, National Academy of Agricultural Science, Rural Development Administration, Wanju 55365, Korea 4 Functional Crop Resource Development Division, National Institute of Crop Science (NICS), Rural Development Administration, Miryang 50426, Korea 5 Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Korea Received November 15, 2015 Revised March 7, 2016 Accepted March 16, 2016 A new poty-like virus was isolated from plants of winter daphne (Daphne odora) that showed viruslike symptoms on leaves, from four regions of Korea during Filamentous-shaped particles were observed by transmission electron microscopy of preparations extracted from symptomatic leaves and examined by the direct negative stain method. RT-PCR assay showed that three samples were positive for both Cucumber mosaic virus and potyvirus, and only one sample was positive for potyvirus only. A BLAST comparison to partial sequences from helper-component proteinase, cylindrical inclusion and coat protein genes detected the highest nucleotide identity of 76%, 72%, and 72% with Daphne mosaic virus, respectively, levels below the potyvirus species discrimination threshold. The new potyvirus was isolated using indicator plants (Chenopodium amaranticolor), in which local lesions were produced. In this study, we identified a novel potyvirus from winter daphne, which we have named Daphne mottle virus (). Keywords: Daphne odora, Potyvirus, Tymelaeaceae 팥꽃나무과 (Tymelaeaceae) 에속하는서향 (Daphne odora) 은상록활엽관목으로늦겨울과이른봄에꽃을피워그향 기를천리까지보낸다고하여천리향이라는이름으로잘알 려져있다 (Garibaldi 등, 2009; Ro 등, 2009). 원산지가중국으로 Research in Plant Disease The Korean Society of Plant Pathology pissn , eissn 알려진서향은내한성이약하여제주도를포함하여일부남부지역에자생하고있으며 (Liang 등, 2010; Ro 등, 2011), 관상가치가뛰어나정원, 길거리및아파트조경용으로많이심겨지고있는정원수중하나이다 (Kwon 등, 2005). 또한, 한의학에서서향의뿌리와나무껍질은관절염, 고열, 피부염등의치료를위한한방재료로이용되기도한다 (Chen 등, 2009; Kim 등, 2005). 2009년, 국내에서재배되는서향에서바이러 cc This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 59

virus [WMV, Potyvirus]) 가동정되었다 (Lee와 Ryu, 2006, 2009). 동정된 5종바이러스에대한진단법개발, 염기서열분석, 게놈구조등의분자생물학적연구가수행되었다 (Lee 등, 2003, 2004, 2006). 국내에서재배되고있는서향은주로중국과일본에서수입되고있으며, 이와같은외래식물체의유입은철저한식물위생조치가요구된다.

2 60 Research in Plant Disease Vol. 22 No. 1 스병발생조사를수행한결과 5종의바이러스 (Cycas necrotic stunt virus [CNSV, Nepovirus], Cucumber mosaic virus [CMV, Cucumovirus], Daphne virus S [DVS, Carlavirus], Daphne virus Y [DVY, Potyvirus] 와 Watermelon mosaic virus [WMV, Potyvirus]) 가동정되었다 (Lee와 Ryu, 2006, 2009). 동정된 5종바이러스에대한진단법개발, 염기서열분석, 게놈구조등의분자생물학적연구가수행되었다 (Lee 등, 2003, 2004, 2006). 국내에서재배되고있는서향은주로중국과일본에서수입되고있으며, 이와같은외래식물체의유입은철저한식물위생조치가요구된다. 2014년, 수도권일대에위치한수입식물재배지에서재배중이던중국산서향묘목과대구의가정집에서관상용으로기르던서향에서전형적인바이러스병징을보이는식물체를채집하여감염된바이러스를동정한결과신종 Potyvirus가검출되었다. 본논문에서는이신종 Potyvirus 의형태적, 생물학적및분자생물학적특성에대하여기술하였으며, 본바이러스를 Daphne mottle virus () 로명명하고자한다. 시료채집. 2014년서울, 수원, 화성에위치한수입식물재배지와대구소재의가정집에서재배되고있는서향묘목을포함하여전체 4점의시료를채집하였다. 바이러스감염주로예상되는서향묘목의잎은괴저, 황화, 퇴록, 뒤틀림의병징을보였다 (Fig. 1A). 수도권일대에서채집한시료 3점은초저온냉동고 ( 80 o C) 에보관하였고, 대구에서수집한시료는온실에서유지하면서추가적인실험에이용하였다. 전자현미경검경. 서향에감염된바이러스입자를관찰 하기위하여 direct negative staining 방법을이용하였다. 병징을보이는잎 50 mg을절취하여인산완충용액 (phosphate buffer saline, ph 7.4) 과함께마쇄한뒤즙액을추출하였고, Park 등 (2014) 의방법으로바이러스입자를관찰하였다. 채집한서향 4점의시료에대하여전자현미경검경을실시한결과, nm 크기의사상형입자가관찰되었다 (Fig. 1B). 전체 RNA 추출및 PCR 진단. 서향에감염된바이러스를동정하기위하여 TRI Rea gent (MRC Inc., Cincinnati, OH, USA) 를이용하여제공사의매뉴얼에따라전체 RNA를추출하였다. 전체 RNA는랜덤프라이머 (RN 25 ) 와 TOPscript Reverse Transcriptase (GeneAll, Seoul, Korea) 를사용하여 cdna를합성하였다. 합성된 cdna를 PCR 증폭의주형으로이용하였으며, 국내이미보고된 5종바이러스 (CNSV, CMV, DVS, DVY, WMV) 에대하여 Lee와 Ryu (2009) 의방법으로진단을수행하였다. PCR 진단결과 DVY, WMV, CNSV, DVS 4종에서는음성반응을보였다. 반면에 CMV의경우수원, 화성, 대구에서채집한 3 점의시료에서는양성반응을보였으며, 서울에서채집한 1 점의시료에서만음성반응을보였다. 양성반응을보인 3점의 PCR 산물은정제후 direct seqeuncing을통하여 CMV의염기서열을확인하여최종동정하였다 (data not shown). 또한, 전자현미경검경결과를바탕으로 Potyvirus속을검출할수있는 universal primer (Table 1) 를이용하여 PCR을수행하였다 (Ha 등, 2008). 2쌍의 universal primer를이용하여 PCR을수행한결과전체 4점의시료에서 680 bp (helper-component proteinase, HC-Pro), 682 bp (cylindrical inclusion, CI) 크기의 DNA 단편을획득하였다. 추가적으로, 외피단백질 (coat A B Fig. 1. The symptoms on Daphne odora infected with the novel potyvirus (Daphne mottle virus) and electron micrograph of the virus particles. (A) D. odora showing chlorotic mottle, leaf distortion and yellowing symptoms. (B) Filamentous rod particles observed in sap from D. odora. Bar represents 200 nm.

3 Research in Plant Disease Vol. 22 No protein, CP) 영역을증폭하기위하여 NIb2F/NIb3R (350 bp) (Zheng 등, 2008) 프라이머를이용하여 PCR을수행하였다. 획득한염기서열을이용하여 forward 프라이머 (NIb-F0: CTC TAT ACA GAG ATC ATA TAC AC) 를설계하여 oligo-dt anchor primer 와함께 PCR 증폭을수행하여최종적으로 CP 영역을결정하 였다. 염기서열결정및유연관계분석. 증폭된 PCR 산물은 Expin Combo GP (GeneAll) 를이용하여정제하였고, 이를클로닝하였다. 클로닝은 Lee 등 (2010) 의방법을이용하여수 Table 1. These primers were used for the detection of novel Potyvirus Primer name Sequence (5ʹ-3ʹ)* Size (bp) Target gene Reference CIFor GGIVVIGTIGGIWSIGGIAARTCIAC ~700 HC-Pro Ha et al., 2008 CIRev ACICCRTTYTCDATDATRTTIGTIGC HPFor TGYGAYAAYCARYTIGAYIIIAAYG ~700 CI Ha et al., 2008 HPRev GAICCRWAIGARTCIAIIACRTG HC-Pro, helper-component proteinase; CI, cylindrical inclusion. *I, deoxyinosine; R, A+G; V, A+C+G; W, A+T; Y, C+T. A B C CI HC-Pro CP IMV PepMoV PesMV J 90 J LMV PepMOV Pe Pe PepMOV J LMV Fig. 2. Maximum likelihood tree based on partial nucleotide sequences of cylindrical inclusion (CI; A), helper-component proteinase (HC-Pro; B), and coat protein (CP; C) region of closely related potyviruses. Phylogenetic tree generated using by DNAMAN software ver. 7.0 and bootstrap values applied from 1,000 replicates. Virus names and National Center for Biotechnology Information GenBank accession numbers were used in these analysis: (Bean common mosaic necrosis virus, NC004047), (Bean common mosaic virus, NC003397), (Brugmansia mosaic virus, NC020105), (Beet mosaic virus, NC005304), (Bean yellow mosaic virus, NC003492), (Cowpea aphid-borne mosaic virus, NC004013), (Celery mosaic virus, NC015393), (Clover yellow vein virus, NC003536), (Daphne mosaic virus, NC008028), J (Japanese yam mosaic virus, NC000947), LMV (Lettuce mosaic virus, NC003605), (Maize dwarf mosaic virus, NC003377), (Narcissus yellow stripe virus, NC011541), (Panax virus Y, NC014252), PepMoV (Pepper mottle virus, NC001517), Pe (Pepper severe mosaic virus, NC008393), (Plum pox virus, NC001445), (Potato virus Y, NC001616), (Sugarcane mosaic virus, NC003398), (Soybean mosaic virus, NC002634), (Turnip mosaic virus, NC002509), (Yam mild mosaic virus, NC019412), (Yam mosaic virus, NC004752).

62 Research in Plant Disease Vol. 22 No. 1 행하였고, 백색콜로니각각 8개를선발하여벡터의 T7과 SP6 영역을증폭하였다. 예상크기가일치하는콜로니는배양후플라스미드 DNA 추출후 EcoRI 제한효소로절단한후전기영동하여확인하였고, 솔젠트 (Solgent, Daejeon, Korea) 에염기서열분석을의뢰하였다.

획득한염기서열을 NCBI GenBank nucleotide BLAST를수행한결과 HC-Pro 단편염기서열은 Daphne mosaic virus () 와 76% (query cover: 97%; accession No.: DQ299908) 가장높은상동성을보였다. CI 영역의경우 와 72% (query cover: 89%; accession No.

4 62 Research in Plant Disease Vol. 22 No. 1 행하였고, 백색콜로니각각 8개를선발하여벡터의 T7과 SP6 영역을증폭하였다. 예상크기가일치하는콜로니는배양후플라스미드 DNA 추출후 EcoRI 제한효소로절단한후전기영동하여확인하였고, 솔젠트 (Solgent, Daejeon, Korea) 에염기서열분석을의뢰하였다. 최종결정된염기서열은 National Center for Biotechnology Information (NCBI) BLAST를통하여이미보고된바이러스와의상동성을확인하였고, 이미보고된 Potyvirus 23종을이용하여유연관계분석을 Nam 등 (2012) 의방법에따라서수행하였다. 획득한염기서열을 NCBI GenBank nucleotide BLAST를수행한결과 HC-Pro 단편염기서열은 Daphne mosaic virus () 와 76% (query cover: 97%; accession No.: DQ299908) 가장높은상동성을보였다. CI 영역의경우 와 72% (query cover: 89%; accession No.: DQ299908) 상동성을보였다. 신종 potyvirus 여부를결정하기위하여 CP 영역에대한 BLAST를수행한결과뉴클레오타이드 72% (query cover: 91%; accession No.: DQ299908), 아미노산 74% (query cover: 99%; accession No.: YP611120) 상동성을보였다. 23종의 Potyvirus들과염기서열유연관계를분석한결과, HC-Pro, CI 그리고 CP 유전자에서는 와가장유사함을나타냈다 (Fig. 2). 따라서, 천리향으로부터검출된본바이러스는이미보고된종들과 80% 미만의상동성을보였고, CP 영역의아미노산의경우 80% 미만의상동성을보였으므로, International Committee on Taxonomy of Viruses (ICTV) 의국제적기준에부합하여 Potyvirus속의신종으로동정하였다. 바이러스분리. 서향에감염된바이러스 ( 가칭 Daphne mottle virus) 를분리하기위하여명아주과 (Chenopodiaceae) 를포함하여콩과 (Leguminosae), 가지과 (Solanaceae) 속의지표식물 10종 (Chenopodium amaranticolor, C. quinoa, Physalis floridana, Nicotiana debney, N. turkish, N. tabaccum cv. KY57, N. tabaccum cv. Samsun, N. tabaccum cv. Xanthi, Vigna sinensis, Glycine max cv. Wiliams 82) 을이용하여즙액접종하였다. 병징을보이는서향의잎을 0.1 M 인산완충용액과함께마쇄하여즙액을추출하였고, Silicon carbide (Sigma-Aldrich, St Louis, MO, USA) 를이용하여지표식물에접종하였다. 명아주과의 C. amaratnicolor에서는국부병반을형성하였고, C. quinoa의접종엽에서퇴록반점을보였다 (Fig. 3). 이외에나머지 8종의지표식물에서는병징이발현되지않았다. 국부병반을보이는 C. amaranticolor는순수분리하기위하여단병반을분리를 3회수행하였고, 최종적으로분리된병반은 C. quinoa에접종하여증식하였으며, RT-PCR과 direct sequencing을수행한후접종원과염기서열이일치하는것을확인하였다 ( 자료미제출 ). Potyvirus의경우진딧물에의해서쉽게전파되는것으로알려져있으나 (Gibbs 등, 2008), 외래식물체에서동정된 Daphne mottle virus에대한전염방법, 생물학적특성조사등을통하여국내농작물에미칠위험성에대한연구가추가적으로필요할것으로생각된다. 요약 2014 년, 국내 4 개지역에서바이러스와같은병징을보이 A B Fig. 3. Symptoms of indicator plants inoculated mechanically with novel potyvirus. (A) Chenopodium amaranticolor observed showing local lesions on inoculated leaves. (B) Chenopodium quinoa produced with chlorotic local lesions on inoculated leaves.

5 Research in Plant Disease Vol. 22 No 는서향잎에서신종 potyvirus 를분리하였다. 병징을보이는 잎에서추출한즙액을 DN 법 (direct negative stain) 으로전자 현미경을이용해관찰한결과사상형형태의입자가관찰되 었다. RT-PCR 검출결과 3 점의시료에서 Cucumber mosaic virus 와 potyvirus 에대하여양성반응을보였으며, 1 점의시료에 서는 potyvirus 양성반응을보였다. HC-Pro, CI, CP 유전자일부 를 BLAST 분석한결과각각 Daphne mosaic virus 와 76%, 72%, 72% 의높은뉴클레오티드상동성을보였다. 신종 potyvirus 는국부병반을나타내는지표식물 ( 붉은명아주 ) 을이용하여 분리하였다. 본논문에서는서향에서신종 potyvirus 를동정 하였으며, 본바이러스를 Daphne mottle virus () 로명 명하였다. Acknowledgement This research was supported by a grant from the Animal and Plant Quarantine Agency (QIA), Ministry of Agriculture, Republic of Korea (Project Code No. Z ). References Chen, R. J., Cao, S. W. and Ruan, Z Isolation of chemical constituents from Daphne odora var. Margirmt by high-speed counter-current chromatography. Chem. Nat. Compd. 45: Garibaldi, A., Bertetti, D. and Gullino, M. L First report of collar and root rot caused by Phytophthora nicotianae on Daphne odora in Italy. Plant Dis. 93: 848. Gibbs, A. J., Ohshima, K., Phillips, M. J. and Gibbs, M. J The prehistory of portviruses: their initial radiation was during the dawn of agriculture. PLoS One 3: e2523. Ha, C., Coombs, S., Revill, P. A., Harding, R. M., Vu, M. and Dale, J. L Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Arch. Virol. 153: Kim, G. H., Hur, J. S., Choi, W. B. and Koh, Y. J Fusarium wilt of winter daphne (Daphne odora Thunb.) caused by Fusarium oxysporum. Plant Pathol. J. 21: Kwon, J. H., Jee, H. J. and Park, C. S Phytophthora blight on Daphne odora caused by Phytophthora nicotianae. Res. Plant Dis. 11: (In Korean) Lee, B. Y., Choi, S. H. and Ryu, K. H Characterization of the 3ʹ-terminal nucleotide sequence of two Korean isolates of Daphne virus S support its placement as a distinct species of the genus Carlavirus. Arch. Virol. 148: Lee, B. Y., Ha, J. H. and Ryu, K. H Daphne-infecting viruses in Korea and properties of Daphne virus S. Acta Hortic. 722: Lee, B. Y., Min, B. E., Ha, J. H., Lee, M. Y., Paek, K. H. and Ryu, K. H Genome structure and complete sequence of genomic RNA of Daphne virus S. Arch. Virol. 151: Lee, B. Y. and Ryu, K. H Identification and molecular detection of Cycas necrotic stunt virus from Daphne odora plants in Korea. Hort. Environ. Biotechnol. 47: Lee, B. Y. and Ryu, K. H Incidence of virus diseases and RT- PCR detection of Daphne-infecting viruses in Korea. Eur. J. Plant Pathol. 124: Lee, J. H., Park, S. J., Nam, M., Kim, M. J., Lee, J. B., Sohn, H. R., Choi, H. S., Kim, J.-S., Lee, J.-S., Moon, J.-S. and Lee, S.-H Identification of a new potyvirus, Keunjorong mosaic virus in Cynanchum wilfordii and C. auricilatum. Res. Plant Dis. 16: (In Korean) Liang, S., Shen, Y. H., Feng, Y., Tian, J. M., Liu, X. H., Xiong, Z. and Zhang, W. D Terpenoids from Daphne aurantiaca and their potential anti-inflammatory activity. J. Nat. Prod. 73: Nam, M., Park, S. J., Kim, Y. J., Kim, J. S., Park, C. Y., Lee, J. S., Choi, H. S., Kim, J. S., Kim, H. G. and Lee, S. H First report of Peanut stunt virus on Glycine max in Korea. Plant Pathol. J. 28: 330. Park, C. Y., Kim, B. S., Nam, M., Lee, M. A., Baek, D. S., Bae, Y. S., Park, E. H., Kim, J. S., Choi, J. Y., Lim, S. M., Moon, J. S. and Lee, S. H Characterization of Brugmansia mosaic virus Isolated from Brugmansia spp. in Korea. Res. Plant Dis. 20: (In Korean) Ro, N. Y., Ko, H. C., Hur, O. S., Kang, M. J., Oh, S. J. and Huh, Y. C Rooting performance using cuttings and analysis of light and soil environmental characteristics for indoor plants of winter daphne (Daphne odora Thunb). J. Bio-Environ. Control 20: Ro, N. Y., Song, E. Y., Kim, S. C. and Kang, K. H Characteristics of cutting in Daphne kiusiana Miq. and Daphne odora Thunb. Poster session presented at the meeting of The Plant Resources Society of Korea, Wonju, Korea. 77 pp. Zheng, L., Wayper, P. J., Gibbs, A. J., Fourment, M., Rodoni, B. C. and Gibbs, M. J Accumulating variation at conserved sites in potyvirus genomes is driven by species discovery and affects degenerate primer design. PLoS One 13: e1586.

01-15(3)-12(최장경).fm

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