Research in Plant Disease Vol. 23 No 요배추과등작물들을비롯한잡초들을포함한 300여종이상의식물들을감염시킬수있으며약 90여종의진딧물을통한비영속적전반으로포장에서퍼져나가는것으로알려져있다 (Choi와 Choi, 1992; Raybould

Size: px

Start display at page:

Download "Research in Plant Disease Vol. 23 No 요배추과등작물들을비롯한잡초들을포함한 300여종이상의식물들을감염시킬수있으며약 90여종의진딧물을통한비영속적전반으로포장에서퍼져나가는것으로알려져있다 (Choi와 Choi, 1992; Raybould"

재윤 대
6 years ago
Views:

1 식물병연구 Note Open Access Res. Plant Dis. 23(1): (2017) 순무모자이크바이러스에대한무육종계통저항성평가 Resistance Evaluation of Radish (Raphanus sativus L.) Inbred Lines against Turnip mosaic virus 윤주연 1 ㆍ최국선 1 ㆍ김수 2 ㆍ최승국 2 * 농촌진흥청국립원예특작과학원 1 원예특작환경과, 2 채소과 *Corresponding author Tel: Fax: viroid73@gmail.com Ju-Yeon Yoon 1, Gug-Seoun Choi 1, Su Kim 2, and Seung-Kook Choi 2 * 1 Department of Horticultural and Herbal Environment, National Institute of Horticultural and Herbal Science, Wanju 55365, Korea 2 Department of Vegetables, National Institute of Horticultural and Herbal Science, Wanju 55365, Korea Received September 12, 2016 Revised February 15, 2017 Accepted February 15, 2017 Leaves of twenties radish (Raphanus sativus L.) inbred lines were mechanically inoculated with Turnip mosaic virus (TuMV) strain HY to evaluate TuMV resistance of the radish inbred lines. The inoculated radish plants were incubated at 22 C±3 C and resistance assessment was examined using symptom development for 4 weeks. Based on the reactions of differential radish inbred lines, 16 radish lines were produced mild mosaic, mottling, mosaic and severe mosaic symptoms by TuMV infection. These results were confirmed by RT-PCR analysis of TuMV coat protein gene, suggesting that TuMV is responsible for the disease symptoms. Four resistant radish lines did not induce systemic mosaic symptoms on upper leaves and chlorosis in stem tissues for 4 weeks, showing they were symptomless by 8 weeks. Further examination of TuMV infection in the 4 radish lines showed no TuMV infection in all systemic leaves. These results suggest that the 4 radish lines are highly resistant to TuMV. Keywords: Breeding, Radish, Resistance, RT-PCR, Turnip mosaic virus 배추과 (Cruciferae family) 에속하는무 (Raphanus sativus) 는아시아및지중해연안이원산지로알려진한해살이또 는두해살이식물이다. 우리나라의 5 대채소가운데하나 이며우리나라전체채소생산량대비무는 10% 15% 로, 배 추와더불어우리의주요부식인김치, 단무지, 외식업체의 식재료로사용되는중요작물중하나이다 (Ku 등, 2006; Lee 등, 2008). 영양가치측면에서무는필수무기질들과칼슘, 비 타민등이풍부하여, 특히겨울철에무청을말린재료를활 용한다양한음식으로섭취함으로써한국인들에게는중 Research in Plant Disease pissn , eissn 요무기영양공급원작물이다. 최근많은양의화학비료를사용하는무의연작재배지에서각종생리적장해와바이러스병이점차증가하고있다. 특히무및배추등에서순무모자이크바이러스 (Turnip mosaic virus, TuMV) 를비롯한 5종바이러스들이보고되었으나 (Choi와 Choi, 1992; Chung 등, 2015; Ham, 1995; Kim 등, 2012), 순무모자이크바이러스에의한피해가우리나라뿐만아니라세계적으로도무재배지역에서가장크며지속적으로발생하고있다. 순무모자이크바이러스는 Potyviridae 과 ( 科, family) Potyvirus속 ( 屬, genus) 으로분류되며, 전세계적으로넓게퍼져있으며경제적피해를지속적으로끼치는중요바이러스중하나이다 (Provvidenti, 1996). 순무모자이크바이러스는주 The Korean Society of Plant Pathology 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. 60

2 Research in Plant Disease Vol. 23 No 요배추과등작물들을비롯한잡초들을포함한 300여종이상의식물들을감염시킬수있으며약 90여종의진딧물을통한비영속적전반으로포장에서퍼져나가는것으로알려져있다 (Choi와 Choi, 1992; Raybould 등, 1999; Shukla 등, 1994). 순무모자이크바이러스는병리학적으로 4개그룹으로분류되는데, 첫번째로배추과작물을감염하지만병징을발현하지않으며무는감염하지못하는 TuMV (B) 계통, 두번째로배추과작물을감염하며모자이크등병징을발현하나무는감염하지못하는 TuMV B계통, 세번째로배추과작물을감염하며모자이크등병징을발현하며무역시감염시키나병징을발현시키지않는 TuMV B(R) 계통, 네번째로배추과작물및무를감염시키며모자이크등병징을발현시키는 TuMV BR계통으로구분할수있다 (Ohshima 등, 2002, 2007). 또한분자계통학적연구에의해순무모자이크바이러스는 4개계통, 즉 basal-b (Brassica), basal-br (Brassica/ Raphanus), Asian-BR 및 World-B 그룹으로구별할수있음이보고되었다 (Ohshima 등, 2007). 순무모자이크바이러스병을예방하기위한가장효율적이며현실적인방법들로는진딧물의철저한포장에서의구제, 저항성배추및무품종을재배하는것이다. 최근에는많은나라들이농약을가급적사용하거나무농약으로재배하는친환경농업정책을펼치고있어, 채소작물들에대한저항성품종을개발하기위한연구가활발히진행되고있다. 순무모자이크바이러스에대한새로운저항성유전자원선발및저항성품종의육종이필요하며, 이를위해순무모자이크바이러스에극도로저항성을가지는무육종계통들에대한저항성조사를이번연구에서수행하였다. 식물재배및접종. 저항성평가용순무모자이크바이러스계통으로서병원성과유전적특성을분석하기위하여 2014년도 1월제주도무에서분리하여국립원예특작과학원에서보존하고있는순무모자이크바이러스 HY계통 (TuMV-HY) 을공시바이러스로사용하였다. 순무모자이크바이러스 HY계통의생물학적기주범위조사결과배추에서모자이크, 황화병징및무에서모자이크및위축등전신감염을시킬수있는계통으로조사되었다 (Table 1). 기존순무모자이크바이러스계통들에서연구되어발표된방법들인 RT-PCR법, 클로닝및생물정보학 (Tomimura 등, 2003) 을이용하여순무모자이크바이러스 HY 전체염기서열을결정하였다. 염기서열분석결과순무모자이크바이러스 HY는 9,798개염기들로구성되어있었으며 (accession no. LC215859), 외피단백질상동성분석결과기존의 BR 계통으 로알려진 TuMV FD27J 계통 (accession no. AB093618) 과아미 노산서열이 100% 일치하였다. 또한 FD27J 계통과 polyprotein 상동성도가장높은것으로분석되었다. 무에대한저항성평가를위해순무모자이크바이러스 HY 계통을무품종 ( 보석알타리 ; Dongbu Farm Hannong, Seoul, Korea) 에서 10 mm 인산완충액 (ph 7.0) 을이용하여즙액접 종으로증식시켜서모자이크등병징이발현된잎을이후 저항성평가조사에사용하였다. 다양한상업용 F1 품종을 육성하는데사용되는무육종계통은포장에서순무모자 이크바이러스에대하여저항성을가질것으로예측된 20 개 무육성계통들 (radish inbred lines) 을선택하였다. 위에서기 술된순무모자이크바이러스 HY 계통이전신감염된무잎 을 10 mm 인산완충액 (ph 7.0) 을 3 대 1 비율 (1 g 감염잎 /3 ml 인산완충액 ) 로첨가후막자사발에서갈아즙액을추출하 였다. 20 개무육종계통들의종자를상토 ( 바로커 ; Seoul Bio, Eumseong, Korea) 에파종후본잎이 4 장이나온잎들을대 상으로각계통당 6 개개체를선택하여 mesh 600 크기연마 제 (Carborundum ; Thermo Scientific, Waltham, MA, USA) 를뿌 린후, 순무모자이크바이러스 HY 계통이감염된즙액으로 면봉을사용하여접종하였다. 저항성비교평가를위하여 바이러스가없는 10 mm 인산완충액 (ph 7.0) 을연마제가뿌 Table 1. Symptoms of plant species inoculated with Turnip mosaic virus (TuMV)-HY Plant species Reactions* Brassica campestris subsp. pekinensis Y & mm /Ch & M Brassica juncea -/- Brassica rapa M/M Capsicum annuuum cv. Cheong-Yang -/- Chenopodium quinoa NLL/NLL Cucurbita pepo -/- Datura stramonium -/- Nicotiana tabacum cv. Samsun -/NLL Physalis floridana -/SM & St. Raphanus sativus cv. Bosuk-Altari -/SM & St. R. sativus cv. Cheongun-mu -/SM & St. R. sativus cv. Seoho-mu -/SM & St. R. sativus cv. Matdong-mu -/SM & St. *Symptoms are briefly shown as symbols: Y, yellowing; mm, mild mosaic; Ch, chlorosis; M, mosaic; -, no infection confirmed using RT-PCR analysis; NLL, necrotic local lesion; SM, severe mosaic; St., stunnting. Inoculated leaves. Upper leaves.

3 62 Research in Plant Disease Vol. 23 No. 1 려진각계통당본잎 4 장이나온 3 개무식물들에위에서기 술한방법과동일하게면봉으로문질러접종하였다. 접종 후전신감염병징을관찰하기위하여 22 C±3 C, 주간 16 시 간야간 8 시간으로조절되는식물생장상에서 28 일간재배 하였다. 순무모자이크바이러스를접종한후 28 일에접종하 지않은무유묘상엽의병징을육안으로조사하였으며, 정 확한순무모자이크바이러스를무상엽에서조사하기위하 여전체 RNA 를식물 RNA 추출 kit (Qiagen, Hilden, Germany) 를이용하여, 제조회사에서제공된방법을이용하여추출 하였다. 추출된 RNA 는 TuMV-CP-For (5 -gcaggtgaaacgcttgatgcaggtttg-3 ), TuMV-CP-Rev (5 -taaccccttaacgccaagtaagttatg-3 ) 라명명된프라이머들과함께 One-Step SuperScriptIII 역전사중합효소 kit (Thermo Scientific) 를이용하여 65 C, 10분 (1 Table 2. Resistance of radish inbred lines against Turnip mosaic virus (TuMV) Name Score* TuMV-inoculated radish Mock-inoculated radish Symptom RT-PCR Symptom RT-PCR Rad-V1 S mm (6/6 ) O - (0/3) X Rad-V2 S M (6/6) O - (0/3) X Rad-V3 S M (6/6) O - (0/3) X Rad-V4 S M (6/6) O - (0/3) X Rad-V5 S M (6/6) O - (0/3) X Rad-V6 S M (6/6) O - (0/3) X Rad-V7 S M (6/6) O - (0/3) X Rad-V8 S SM (6/6) O - (0/3) X Rad-V9 S SM (6/6) O - (0/3) X Rad-V10 R - (0/6) X - (0/3) X Rad-V11 R - (0/6) X - (0/3) X Rad-V12 R - (0/6) X - (0/3) X Rad-V13 S M (6/6) O - (0/3) X Rad-V14 S M (6/6) O - (0/3) X Rad-V15 S M (6/6) O - (0/3) X Rad-V16 S M (6/6) O - (0/3) X Rad-V17 S mm (6/6) O - (0/3) X Rad-V18 S M (6/6) O - (0/3) X Rad-V19 S M (6/6) O - (0/3) X Rad-V20 R - (0/6) X - (0/3) X *S, susceptible; R, resistant. mm, mild mosaic; M, mosaic; SM, severe mosaic; -, no symptom. Number of plants developed systemic symptoms/number of plants inoculated with TuMV. O, detection of RT-PCR product for TuMV; X, no detection of RT- PCR product for TuMV. 회 ), 얼음에 5분간방치후, PCR 기계를이용하여 55 C, 30분및 95 C, 2분 ( 각 1회 ), 94 C, 20초, 55 C, 30초, 72 C, 60초 (35회반복 ), 72 C, 10분 (1회) 증폭반응을시켰다. 반응후 1.2% 아가로스겔전기영동 (agarose gel electrophoresis) 으로산물을확인하였다. 무육종계통들의저항성평가. 무저항성판정을위한지표로서강한저항성계통을선발하기위하여, 순무모자이크바이러스의병징이나타나지않으며순무모자이크바이러스를접종하지않은상엽에서역전사중합효소연쇄반응으로검출되지않는상태를기준으로정했다. 접종후 28일후병징을조사한결과, 16개무육종계통들에서순무모자이크바이러스에대한약한모자이크에서부터심한모자이크병징을나타내었으며생육이불량해지고위축이되는병징이관찰되고무기형이관찰되었으나, 대조구로사용된완충액이접종된무계통들에서는순무모자이크바이러스에의한병징이관찰되지않았다 (Table 2). 4개무육성계통들에서순무모자이크바이러스가접종되었으나, 접종되지않은상엽에서완충액이접종된대조구와동일하게모자이크등순무모자이크바이러스병징이나타나지않았다 (Fig. 1). 순무모자이크바이러스를접종하거나완충액을접종한 20개무육종계통들에서순무모자이크바이러스의외피단백질유전자를검출할수있는특이적프라이머들을이용한역전사중합효소연쇄반응으로조사한결과, 병징이관찰된 16개계통들 (Rad-V1 Rad-V9, Rad-V13 Rad- V19) 에서모두순무모자이크바이러스외피단백질유전자에대한증폭산물이검출이되었으나, 대조구로사용된 16 개계통들의각 3개체들에서는증폭산물이검출되지않았다. 또한순무모자이크바이러스가접종되었으나전신감염병징이관찰되지않은 4계통 (Rad-V10 Rad-V12, Rad-V20) 들에서모두순무모자이크바이러스외피단백질유전자에대한증폭산물이검출이되지않았다. 이 4계통들의대조구로사용된각 3개체들에서는증폭산물이검출되지않아 4계통은순무모자이크바이러스에대한전신감염을억제하는강한저항성을가지고있는것으로판단된다. 일부순무모자이크바이러스계통들 ( 예를들면, TuMV UK1 계통 ; Tomimura 등, 2003; Walsh, 1989) 에서보고된것처럼, 배추들은매우잘감염되지만무는드물게 TuMV에의해서감염이되며바이러스는분명히상엽에존재하며감염이되더라도무에서아무런병징을나타내지못하는순무모자이크바이러스계통그자체의특이적현상때문이아닌것으로차별성이있다. 또한위의결과는 Tan 등 (2005) 이순무모자이크

Research in Plant Disease Vol. 23 No. 1 63 A B C Fig. 1. Symptoms of radish inbred lines infected with Turnip mosaic virus (TuMV)-HY isolate 28 days post-inoculation.

(C) Radish Rad-V2 line (susceptible to TuMV) inoculated with TuMV-HY isolate as a control, showing mosaic symptoms in the upper leaves.

) 육종계통들에대한순무모자이크바이러스 (Turnip mosaic virus, TuMV) 저항성을평가하기위하여, 20개무육종계통들의잎에순무모자이크바이러스 BR 병원성을가지는국내분리계통을즙액접종하였다. 순무모자이크바이러스를접종한무개체들은 22 C±3 C에서재배하였으며 4주동안바이러스병징발현을육안으로조사하여저항성을평가하였다.

4 Research in Plant Disease Vol. 23 No A B C Fig. 1. Symptoms of radish inbred lines infected with Turnip mosaic virus (TuMV)-HY isolate 28 days post-inoculation. (A) Radish Rad-V10 line (resistant to TuMV) inoculated with TuMV-HY isolate showed no symptoms in the upper leaves. (B) Radish Rad-V10 line inoculated with 10 mm phosphate buffer (ph 7.0). (C) Radish Rad-V2 line (susceptible to TuMV) inoculated with TuMV-HY isolate as a control, showing mosaic symptoms in the upper leaves. 바이러스 BR계통 ( 배추와무를모두감염시킬수있는계통 ) 을사용하여무를감염시켰을때 100% 감염률이조사되었는데, 이번연구에사용된순무모자이크바이러스 HY계통이 BR계통그룹에속하는결과와일치하였으며, 공시된순무모자이크바이러스계통특이적병원성결여에의하여저항성이발현되는것은아님을시사해준다. 요약 무 (Raphanus sativus L.) 육종계통들에대한순무모자이크바이러스 (Turnip mosaic virus, TuMV) 저항성을평가하기위하여, 20개무육종계통들의잎에순무모자이크바이러스 BR 병원성을가지는국내분리계통을즙액접종하였다. 순무모자이크바이러스를접종한무개체들은 22 C±3 C에서재배하였으며 4주동안바이러스병징발현을육안으로조사하여저항성을평가하였다. 순무모자이크바이러스감염에의해서무육종계통들의다른병징발현에의해분석한결과, 16개무계통은약한모자이크, 모틀링에서심한모자 이크전신병징을보였으며감수성으로판별되었다. 이러한결과는순무모자이크바이러스외피단백질유전자에대한특이적역전사중합효소연쇄반응에의하여확인되어, 순무모자이크바이러스가병징을발현시킨원인이었다. 이와는다르게 4개무육종계통들에서는모자이크등전신감염병징이발현되지않았으며, 동일한무육종계통들의개체들에서 8주동안병징이관찰되지않았다. 역전사중합효소연쇄반응으로조사한결과 4개무육종계통들의상엽들에서순무모자이크바이러스가검출되지않았다. 이런결과는 4개무육종계통들이순무모자이크바이러스에대한강한저항성을가지고있음을예시해준다. Conflicts of Interest No potential conflict of interest relevant to this article was reported.

5 64 Research in Plant Disease Vol. 23 No. 1 Acknowledgements We thank Dr. Han-Yong Park (Sejong University, Seoul, Korea) for his helpful comments. This work was financially supported by a grant of Basic Research Projects of NIHHS (Project No. PJ010247) funded by Rural Development Administration, Republic of Korea. References Choi, G. S. and Choi, J. K Biological properties of two isolates of turnip mosaic virus isolated from Chinese cabbage and radish in Korea. Korean J. Plant Pathol. 8: (In Korean) Chung, J., Han, J. Y., Kim, J., Ju, H., Gong, J., Seo, E. Y., Hammond, J. and Lim, H. S Survey of viruses present in radish fields in Res. Plant Dis. 21: (In Korean) Ham, Y. I Recent occurrence of TuMV disease on radish and Chinese cabbage in alpine region, Kangwon province. Plant Dis. Agric. 1: (In Korean) Kim, J. S., Lee, S. H., Choi, H. S., Kim, M. K., Kwak, H. R., Kim, J. S., Nam, M., Cho, J. D., Cho, I. S. and Choi, G. S characteristics of plant virus infections on crop samples submitted from agricultural places. Res. Plant Dis. 18: (In Korean) Ku, K. H., Lee, K. A., Kim, Y. L. and Lee, M. G Effects of pretreatment method on the surface microbes of radish (Raphanus sativus L.) leaves. J. Korean Soc. Food Sci. Nutr. 3: (In Korean) Lee, H. C., Lee, Y. J. and Yang, D. C Genetic characterization of mitochondrial DNA in novel CMS radish line. Bull. Nat. Sci. 22: Ohshima, K., Tomitaka, Y., Wood, J. T., Minematsu, Y., Kajiyama, H., Tomimura, K. and Gibbs, A. J Patterns of recombination in turnip mosaic virus genomic sequences indicate hotspots of recombination. J. Gen. Virol. 88: Ohshima, K., Yamaguchi, Y., Hirota, R., Hamamoto, T., Tomimura, K., Tan, Z., Sano, T., Azuhata, F., Walsh, J. A., Fletcher, J., Chen, J., Gera, A. and Gibbs, A Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread. J. Gen. Virol. 83: Provvidenti, R Turnip mosaic potyvirus. In: Viruses of Plants, eds. by A. A. Brunt, K. Crabtree, M. J. Dallwitz, A. J. Gibbs and L. Watson, pp CAB International, Wallingford, UK. Raybould, A. F., Maskell, L. C., Edwards, M. L., Copper, J. I. and Gray, A. J The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea. New Phytol. 141: Shukla, D. D., Ward, C. W. and Brunt, A. A The Potyviridae. CAB International, Wallingford, UK. Tan, Z., Gibbs, A. J., Tomitaka, Y., Sánchez, F., Ponz, F. and Ohshima, K Mutations in Turnip mosaic virus genomes that have adapted to Raphanus sativus. J. Gen. Virol. 86: Tomimura, K., Gibbs, A. J., Jenner, C. E., Walsh, J. A. and Ohshima, K The phylogeny of Turnip mosaic virus; comparison of 38 genomic sequences reveal a Eurasian origin and a recent emergence in east Asia. Mol. Ecol. 12: Walsh, J. A Genetic control of immunity to turnip mosaic virus in winter oilseed rape (Brassica napus spp. oleifera) and the effect of foreign isolates of the virus. Ann. Appl. Biol. 115:

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