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1 식물병연구 Res. Plant Dis. 19(4) : (2013) Note Open Access Research in Plant Disease The Korean Society of Plant Pathology 국내양앵두나무에서발생한 Cherry green ring mottle virus 동정 조인숙 * 최국선 최승국농촌진흥청국립원예특작과학원원예특작환경과 Identification of Cherry green ring mottle virus on Sweet Cherry Trees in Korea In-Sook Cho*, Gug-Seoun Choi and Seung-Kook Choi Horticultural and Herbal Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon , Korea (Received on October 11, 2013; Revised on November 5, 2013; Accepted on November 5, 2013) During the 2012 growing season, 154 leaf samples were collected from sweet cherry trees in Hwaseong, Pyeongtaek, Gyeongju, Kimcheon, Daegu, Yeongju and Eumseong and tested for the presence of Cherry green ring mottle virus (CGRMV). PCR products of the expected size (807 bp) were obtained from 6 samples. The PCR products were cloned and sequenced. The nucleotide sequences of the clones showed over 88% identities to published coat protein sequences of CGRMV isolates in the GenBank database. The sequences of CGRMV isolates, KO 1 6 shared 98.8 to 99.8% nucleotide and 99.6 to 100% amino acid similarities. Phylogenetic analysis indicated that the Korean CGRMV isolates belong to the group II of CGRMV coat protein genes. The CGRMV infected sweet cherry trees were also tested for Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry mottle leaf virus (CMLV), Cherry rasp leaf virus (CRLV), Cherry leafroll virus (CLRV), Cherry virus A (CVA), Little cherry virus 1 (LChV1), Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) by RT-PCR. All of the tested trees were also infected with ACLSV. Keywords : CGRMV, RT-PCR, Sequence analysis, Sweet cherry 양앵두는크게단양앵두 (Sweet cherry, Prunus avium L.) 와신양앵두 (Sour cherry, Prunus cerasus L.) 로구분되는데국내에서재배되는거의대부분이단양앵두로써재배면적은약 130ha이고, 국내소비가급증함에따라재배면적또한꾸준하게증가하고있는추세이다 (Yoon 등, 2012). 전세계적으로양앵두나무에발생하는바이러스병은약 30여종이상이알려져있으며 (Mandic 등, 2007), 국내에서는최근 Apple chlorotic leaf spot virus(aclsv), Apple mosaic virus(apmv), Cherry necrotic rusty mottle virus(cnrmv), Cherry virus A(CVA) 4종이보고되었다 (Cho, 2011; Cho 등, 2013). Cherry green ring mottle virus(cgrmv) 는 Betaflexiviridae에속하지만아직완전 *Corresponding author Phone) , Fax) ) tuat@korea.kr 히분류되지않은바이러스로 1937년미국미시간에서처음보고된이후, 아프리카, 일본, 중국, 유럽, 북아메리카, 오세아니아등지에서발생하고있다 (Wang 등, 2013). CGRMV는양앵두, 벗나무, 복숭아, 살구나무등벗나무속에서발생되는데, 특히신양앵두나무에서황반엽과기형과등을일으켜경제적으로문제가되고있으며단양앵두, 복숭아, 살구나무에서는병징이나타나지않고잠복되어있는경우가일반적이다 (Zhang 등, 1998). 접목과삽목에의해전염되고매개곤충에의한전염은아직까지보고되지않았다. 과거에는목본식물검정으로 CGRMV 감염여부를판단하였지만최근분자생물학기술의발달로다양한기주의새로운분리주들에대한동정이활발히진행되고있다 (Li와 Mock, 2005; Wang 등, 2009; Wang 등, 2013). 본연구에서는국내양앵두나무에서발생하는 CGRMV

국내양앵두나무에서발생한 Cherry green ring mottle virus 동정 327 Fig. 1. Agarose gel electrophoresis of nested RT-PCR products for Cherry green ring mottle virus (CGRMV).

2 국내양앵두나무에서발생한 Cherry green ring mottle virus 동정 327 Fig. 1. Agarose gel electrophoresis of nested RT-PCR products for Cherry green ring mottle virus (CGRMV). Lane designations indicate sweet cherry trees examined in this study: 1 5 and 6 were collected from the Hwaseong and Yeongju, respectively. A black arrow head indicates the position of the expected products specific to CGRMV (807 bp). 를신규양앵두재배과원을중심으로조사하고, 외피단백질유전자분석을통하여국내 CGRMV 분리주들을동정한결과를보고하고자한다. CGRMV 유전자검정. 2012년화성, 평택, 경주, 김천, 대구, 영주, 음성 7지역, 특히경기지역의신규양앵두재배과원을위주로양앵두나무 154주를무작위로선발하고잎을채집하여 CGRMV 유전자검정에사용하였다. 검정할시료의핵산 (RNA) 은 NucliSens Extractor(Biomerieux) 를이용하여추출하였으며, 프라이머는 CGRMV 외피단백질유전자에특이적으로반응하는 CGRMV1/2와 nested 프라이머 CGRM-CPF/CGRM-CPR를사용하였다 (Fiore 등, 2013; Li 등, 2005). RT-PCR 검정은 CGRMV1/2 프라이머로 1차 PCR을한다음, 이 PCR 산물들을 10배희석한것을주형으로해서 nested PCR을수행하였다. 그결과전체시료 154점중 6점의시료에서 PCR 증폭산물이검출되었다 (Fig. 1). PCR 산물을정제하고클로닝하여염기서열을분석한결과 807bp 크기의 CGRMV 외피단백질유전자가확인되었다. CGRMV 발생. 조사된양앵두나무 154주중 6주에서 CGRMV가검출되어 3.9% 의감염률을보였으며, CGRMV 에감염된 6주중 5주는화성지역에서발생되었고, 1주는영주지역에서발생되었다 (Table 1). CGRMV에감염된양앵두나무는육안상특이한바이러스증상이관찰되지않았으며이것은 CGRMV가단양앵두기주에서병징을나타내지않는다는기존보고와도같았다 (Zhang 등, 1998). 중국에서는양앵두나무뿐만아니라복숭아나무에 서도 CGRMV 발생이보고된바있어 (Zhou 등, 2011), 국내복숭아나무에서의 CGRMV 발생유무를추가적으로검정해보았다. 그결과국내복숭아나무에서는중국과는달리 CGRMV가전혀검출되지않았다 ( 자료생략 ). 복숭아나무시료는음성, 춘천, 충주 3지역에서무작위로채집한 298주의잎을사용하였다. CGRMV 외피단백질유전자분석. 양앵두나무에서검출된 CGRMV는외피단백질유전자염기서열분석을통해분리주 6개 (KO 1 6) 를확인하였으며 GeneBank 에등록된외국의 CGRMV 분리주들과염기서열을비교한결과 88% 이상의염기서열상동성을나타내었다 ( 자료생략 ). 이들국내 CGRMV 분리주들간에는 % 의염기서열및 % 의아미노산서열상동성을나타내었다 (Fig. 2). 기존에보고된외국의 CGRMV 분리주 12개와국내분리주들의외피단백질유전자계통도분석으로국내분리주들은기존에분류된 CGRMV I, II, III 그룹 (Zhang, 2000) 중 II 그룹에모두속하는것으로나타났다 (Fig. 3). CGRMV 감염주의단독감염여부를확인하기위해양앵두에서발생하는 10종의바이러스, ACLSV, ApMV, Cherry mottle leaf virus(cmlv), CNRMV, Cherry rasp leaf virus(crlv), Cherry leafroll virus(clrv), CVA, Little cherry virus1(lchv1), Prune dwarf virus(pdv), Prunus necrotic ringspot virus(pnrsv) 에대해서 RT-PCR 을검정을수행하였다. 각바이러스진단에사용한프라이머와 RT-PCR은기존에보고된방법에준하여실시하였다 (Cho, 2011; Isogai 등, 2004; Li 등, 2008). RT-PCR 검정결과모든시료에서 ACLSV가검출되어 CGRMV 와 ACLSV가복합감염된것을확인하였다 (Table 2). 국내양앵두나무의 CGRMV는 6주가검출되었고, 특히단양앵두나무에서는병징이나타나지않아현재로서는국내에서 CGRMV는크게문제가되지않는다고볼수있다. 그러나앞으로새로운바이러스병의출현과신양앵두나무의수요및재배가늘어난다면 CGRMV 피해가발생할가능성도배제할수없는부분이다. 본연구는최근양앵두신규재배과원을중심으로발생한 CGRMV를유전자검정에의해동정하였으며, 실험에사용한바이러스검정기술과이들바이러스분리주들의유전자분석자료는금후양앵두나무에발생하는 Table 1. Detection of Cherry green ring mottle virus (CGRMV) by RT-PCR from sweet cherry trees in different areas No. of samples Infection No. of infected samples/tested samples Infected Tested rate (%) Hwaseong Pyeongtaek Gyeongju Kimcheon Daegu Yeongju Eumseong /64 0/41 0/19 0/9 0/7 1/5 0/9

3 328 조인숙 최국선 최승국 Fig. 2. Nucleotide sequence (A) and amino acid sequence (B) alignments of coat protein genes of Cherry green ring mottle virus (CGRMV) isolates, KO 1 6 obtained from CGRMV infected sweet cherry trees. The asterisk (*) indicates the stop codon.

4 국내양앵두나무에서발생한 Cherry green ring mottle virus 동정 329 요약 Fig. 3. Phylogenetic analysis based on nucleotide sequences of coat protein genes from isolates of KO 1 6 and other 12 Cherry green ring mottle virus (CGRMV) isolates available in nucleotide database (California isolates: F and N; Washington isolates: Wp1, Wp2 and Ww; Michigan isolate: MI; British Columbia, Canada isolate: BC; Italy isolates: Ita1, Ita5, Ita6 and Ita7; Lebanon isolate: Leb). The tree was generated by the neighbor-joining method with 1000 bootstrap replicates using MEGA 5.0. Table 2. RT-PCR analysis for the presence of 10 viruses from Cherry green ring mottle virus (CGRMV) infected sweet cherry trees Tested viruses KO 1 KO 2 Host of CGRMV isolate KO 3 KO 4 KO 5 KO 6 ACLSV ApMV CMLV CNRMV CRLV CLRV CVA LChV1 PDV PNRSV CGRMV을지속적으로모니터링하고연구하는데기초자료로활용될수있을것으로본다. 2012년국내양앵두나무에서발생하는 CGRMV를조사하기위해화성, 평택, 경주, 김천, 대구, 영주음성의양앵두재배과원 7개지역에서잎시료 154점을채집하였다. 채집한시료에대해 CGRMV 유전자검정을수행한결과 6점의시료에서 807bp 크기의 PCR 증폭산물이검출되었다. 이들 PCR 증폭산물은클로닝과유전자염기서열분석결과 GeneBank에등록된외국의 CGRMV 분리주들과 88% 이상의외피단백질유전자염기서열상동성을보였다. 국내양앵두나무에서분리된분리주들, KO 1 6 간에는 % 의염기서열및 % 의아미노산서열상동성을나타내었다. 국내 CGRMV 분리주들은외피단백질유전자계통도분석에서기존에분류된 I, II, III 그룹중 II 그룹에속하였다. 또한 CGRMV 가감염된국내양앵두나무이병주들은 ACLSV 등 10종바이러스에대해서도 RT-PCR을수행한결과모든시료에서 ACLSV가검출되어 CGRMV와 ACLSV가복합감염된것을확인하였다. Acknowledgement This research was carried out with the support of research fund (Project No. PJ006358) from Rural Development Administration, Korea. References Cho, I. S Development of diagnostic and control techniques for horticultural diseases and insect pests. In: Res. Rept. RDA (NIHHS), pp (In Korean) Cho, I. S., Choi, G. S., Choi, S. K., Seo, E. Y. and Lim, H. S First report of Cherry necrotic rusty mottle virus infecting sweet cherry trees in Korea. Plant Dis /PDIS PDN. Fiore, N. and Zamorano, A First report of Cherry green ring mottle virus and Cherry necrotic rusty mottle virus in sweet cherry (Prunus avium) in Chile and South America. Plant Dis. 97: Isogai, M., Aoyagi, J., Nakagawa, M., Kubodera, Y., Satoh, K., Katoh, T., Inamori, M., Yamashita, K. and Yoshikawa, N Molecular detection of five cherry viruses from sweet cherry trees in Japan. J. Gen. Plant Pathol. 70: Li, R. and Mock, R An improved reverse transcriptionpolymerase chain reaction (RT-PCR) assay for the detection of two cherry flexiviruses in Prunus spp. J. Virol. Meth. 129:

5 330 조인숙 최국선 최승국 Li, R., Mock, R., Huang, Q., Abad, J., Hartung, J. and Kinarda, G A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. J. Virol. Meth. 154: Mandic, B., Matic, S., Al Rwahnih, M., Jelkmann, W. and Myrta, A Viruses of sweet and sour cherry in Serbia. J. Plant Pathol. 89: Wang, L. P., Hong, N., Wang, G. P., Michelutti, R. and Zhang, B. L First report of Cherry green ring mottle virus in plum (Prunus domestica) in North America. Plant Dis. 93: Wang, L., Jiang, D., Niu, F., Lu, M., Wang, H. and Li, S Complete nucleotide sequences of two isolates of Cherry green ring mottle virus from peach (Prunus persica) in China. Arch. Virol. 158: Yoon, I. K., Nam, E. Y., Chung, K. H. and Yun, S. K Cherry cultivation manual. RDA, pp (In Korean) Zhang, Y. P., Kirkpatrick, B. C., Smart, C. D. and Uyemoto, J. K cdna cloning and molecular characterization of Cherry green ring mottle virus. J. Gen. Virol. 79: Zhang, Y. P., Kirkpatrick, B. C., Terlizzi, B. D. and Uyemoto, J. K Comparison of Cherry green ring mottle virus strains using RT-PCR and coat protein sequence phylogeny. J. Plant Pathol. 82: Zhou, J. F., Wang, G. P., Kuang, R. F., Wang, L. P. and Hong, N First report of Cherry green ring mottle virus on cherry and peach grown in China. Plant Dis. 95: 1319.

01-15(3)-12(최장경).fm

01-15(3)-12(최장경).fm Res. Plant Dis. 15(3) : 165-169 (2009) Research in Plant Disease The Korean Society of Plant Pathology GM s ü š w Cucumber mosaic virus v y 1 Á y Á»x 1 Á * w w, 1 w y w Comparative Analysis of Coat Protein