식물병연구 Note Open Access Res. Plant Dis. 22(1): 44-49 (2016) http://dx.doi.org/10.5423/rpd.2016.22.1.44 Botryosphaeria dothidea 에의한오미자줄기마름병 Twig Blight on Chinese Magnolia Vain Caused by Botryosphaeria dothidea in Korea 박상규 1 ㆍ김승한 2 ㆍ이승열 1 ㆍ백창기 1,3 ㆍ강인규 4 ㆍ정희영 1,5 * 1 경북대학교응용생명과학부, 2 경상북도농업기술원, 3 농촌진흥청국립원예특작과학원원예특작환경과, 4 경북대학교원예과학과, 5 경북대학교식물의학연구소 *Corresponding author Tel: +82-53-950-5760 Fax: +82-53-950-6758 E-mail: heeyoung@knu.ac.kr Sangkyu Park 1, Seung-Han Kim 2, Seung-Yeol Lee 1, Chang-Gi Back 1,3, In-Kyu Kang 4, and Hee-Young Jung 1,5 * 1 School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea 2 Gyeongsangbuk-do Agricultural Research & Extension Services, Daegu 41404, Korea 3 Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Korea 4 Department of Horticultural Science, Kyungpook National University, Daegu 41566, Korea 5 Institute of Plant Medicine, Kyungpook National University, Daegu 41566, Korea Received December 17, 2015 Revised February 16, 2016 Accepted March 4, 2016 The twig blight symptoms were observed in Chinese magnolia vine (Schisandra chinensis) at Mungyeong city, Gyeongbuk province, Korea in June 2015. The typical symptoms of infected plant were shriveled and wilted in leaves which led to blight resulted in death. Based on the morphological characteristics, the isolate was suspected as Botryosphaeria sp. Inoculation of isolated pathogen was performed to identify its pathogenicity according to Koch s postulates. Re-isolated fungi from the inoculated stem was showed same morphological characteristics with original pathogen. Phylogenetic analysis was performed using combined sequence of rdna internal transcribed spacer region, EF1-α and β-tubulin gene. The isolated pathogen was identified to the B. dothidea by phylogenetic analysis. This is the first report of twig blight on S. chinensis caused by B. dothidea in Korea. Keywords: Botryosphaeria dothidea, Chinese magnolia vine, Combined sequence, Twig blight 오미자 (Schisandra chinensis Baill.) 는오미자과에속하는다 년생덩굴식물로, 동아시아지역에서오랜기간동안간질 환및심혈관질환의치료제로쓰이거나강장제로활용되 어왔다 (Samrat 등, 2015). 현대에들어서오미자의항산화및 항균효과에대한보고가이루어지면서약재뿐만아니라다 양한건강식품및미용제품으로서활용되고있다 (Nam 등, Research in Plant Disease The Korean Society of Plant Pathology pissn 1598-2262, eissn 2233-9191 2014). 이에따라우리나라에서도재배면적이해마다늘고있는추세이며, 2013년기준 2,155 ha의재배면적으로부터 9,575톤의오미자가생산되고있고지속적으로생산량이증가할것으로예상된다 (Ministry of Agriculture, Food and Rural Affairs, 2014). 2015년 6월, 경상북도문경시의오미자재배지역에서줄기마름증상이발생하였다. 줄기마름증상이관찰된식물은잎이위축되면서선단부위부터고사하기시작하였고, 증상이진행됨에따라줄기로부터식물전체로퍼져나가식물체 cc This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 44
45 Research in Plant Disease Vol. 22 No. 1 A C B Fig. 1. Symptoms of twig blight on Chinese magnolia vine caused by Botryosphaeria dothidea. (A) Infected plants (symptoms in circles). (B) Pycnidia on stem. (C) Conidia from pycnidia. A B C D Fig. 2. Morphological and cultural characteristics of Botryosphaeria dothidea. The front (A) and back (B) side of colony on potato dextrose agar plate. (C) Conidia ( 400). (D) Symptoms on the stem of Chinese magnolia vine by artificial inoculation. 가 고사하였다. 병반이 나타난 줄기는 짙은 갈색 또는 검은 으로 전염이 진행되는 것이 관찰되었다(Fig. 1). 색으로 변색되었고 검게 변색된 줄기의 표면으로부터 병자 줄기마름 증상을 일으키는 병원균을 동정하기 위해 병이 각이 형성되었다. 또한, 증상이 발생한 오미자로부터 주변 발생한 줄기로부터 병원균을 분리배양하였다. 분리된 병원
46 Research in Plant Disease Vol. 22 No. 1 균은감자한천배지 (potato dextrose agar medium, PDA) 상에 서구름모양의흰색균사를형성하였으며, 배양후약 3 4 일 뒤부터 plate 뒷면의중심부가검은색으로변색되기시작하 Table 1. Morphological characteristics of the present isolate obtained from Schisandra chinensis Baill. and Botryosphaeria dothidea described previously Characteristic Present isolate B. dothidea* Conidia Shape Fusiform Fusiform Size (µm) 17.7 25.6 6.8 8.2 23 25 4 5 Colony color Black Black Setae Absent Absent *Described by Slippers et al. (2005) (Mycologia 97: 99-110). 였다. 또한동시에작고검은점들이나타나기시작하였다. 배양 7일후 PDA plate 전체로균사가생장하였으며, 이후약 14일만에뒷면이모두검은색으로변하였다 (Fig. 2A, B). 분생포자를관찰하기위해 14일간배양한 PDA의기중균사를모두제거하고, 가시광선을조사하였다. 가시광선의조사는기중균사를제거한 PDA를 12시간동안가시광선에노출시키고다시 12시간동안암조건에서배양하는것을반복하는방식으로수행하였다. 3주동안가시광선의조사를진행한결과배지의표면에검은색분생포자덩어리가나타났으며, 분생포자덩어리를희석하여광학현미경으로분생포자를관찰하였다. 분생포자는격벽이없는무색의타원형으로크기는 17.7 25.6 6.8 8.2 μm였으며, PDA상에서균핵이나강모는관찰할수없었다 (Fig. 2C). Table 2. Isolates of Botryosphaeria and Guignardia species considered in this study Isolates Identity Host Accession No. ITS β-tubulin EF1-α CMW991 Botryosphaeria dothidea Populus nigra AF241175 AY236924 AY236895 CMW7780 B. dothidea Fraxinus excelsior AY236947 AY236925 AY236896 CMW7999 B. dothidea Ostrya sp. AY236948 AY236926 AY236897 CMW8000 B. dothidea Prunus sp. AY236949 AY236927 AY236898 CMW9075 B. dothidea P. nigra AY236950 AY236928 AY236896 CMW9078 B. parva Actinidia deliciosa AY236940 AY236914 AY236885 CMW9079 B. parva A. deliciosa AY236941 AY236915 AY236886 CMW9080 B. parva P. nigra AY236942 AY236916 AY236887 CMW9081 B. parva P. nigra AY236943 AY236917 AY236888 CMW7054 B. ribis Ribes rubrum AF241177 AY236908 AY236879 CMW7772 B. ribis Ribes sp. AY236935 AY236906 AY236877 CMW7773 B. ribis Ribes sp. AY236936 AY236907 AY236878 CMW992 B. lutea A. deliciosa AF027745 AY236923 AY236894 CMW9076 B. lutea Malus domestica AY236946 AY236922 AY236893 CMW10309 B. lutea Vitis sp. AY339258 AY339266 AY339250 CMW6837 B. australis Acacia sp. AY339262 AY339254 AY339270 CMW9072 B. australis Acacia sp. AY339260 AY339252 AY339268 CMW9073 B. australis Acacia sp. AY339261 AY339253 AY339269 CMW10126 B. eucalyptorum Eucalyptus grandis AF283687 AY236921 AY236892 CMW6233 B. eucalyptorum E. nitens AY615138 AY615122 AY615130 CMW10125 B. eucalyptorum E. grandis AF283686 AY236920 AY236891 CMW7774 B. obtusa Ribes sp. AY236953 AY236931 AY236902 CMW7775 B. obtusa Ribes sp. AY236954 AY236932 AY236903 CMW7060 B. stevensii F. excelsior AY236955 AY236933 AY236904 UCP130 B. stevensii Citrus sp. JF271751 JF271769 JF271787 CMW9074 B. rhodina Pinus sp. AY236952 AY236936 AY236901 CMW10130 B. rhodina Vitex donniana AY236951 AY236929 AY236900 CMW7063 Guignardia philoprina Taxus baccata AY236956 AY236934 AY236905 PPL01* B. dothidea Schisandra chinensis LC120711 LC120690 LC120691 *The sequences obtained from this study. ITS, internal transcribed spacer; EF1-α, elongation factor 1-α.
Research in Plant Disease Vol. 22 No. 1 47 분리배양한병원균의병원성을검증하기위해병원균을배양한 PDA를 5 mm 지름의원형으로잘라내어균총디스크를제작하였다. 건전한오미자식물체의줄기를 70% 알코올로닦아표면을소독하고, 멸균한바늘로상처를낸부위와상처를내지않은부위에각각균총디스크를접지한뒤수분증발을막기위해랩으로밀봉하였다. 24시간동안접지한뒤균총디스크를제거하고온실에서배양하여접종부위에서이상증상이나타나는지관찰하였다. 접종 10일후상처를낸줄기로부터최초병원균을분리한줄기에서나타난병징과동일한병징을관찰할수있었으며, 나타난병반으로부터다시병원균을분리배양한결과동일한배양적, 형태적특징을가지는균이분리되었다 (Fig. 2D). 병원균의배양적, 형태적특징을통해해당병원균은 Botryosphaeria sp. 로 판명되었으며, Slippers 등 (2005) 이보고한균학적특징과일치하였다 (Table 1). 분리한병원균의정확한동정을위해염기서열분석을실시하였다. PDA에서 1주일간배양한병원균으로부터 genomic DNA를추출하고곰팡이의동정에널리쓰이는유전자의염기서열을분석하기위해 polymerase chain reaction (PCR) 을수행하였다. 분석의정확성을높이기위해일반적으로사용되는 internal transcribed spacer (ITS) 영역과 Botryosphaeria 속의동정에사용되는 β-tubulin, translation elongation factor 1-α (EF1-α) 의유전자를함께사용하였으며, 각각 ITS1F/ITS4 (White 등, 1990), Bt2a/Bt2b (Glass와 Donaldson, 1995), EF1-728F/EF1-986R (Carbone 등, 1999) primer set을 PCR 에사용하였다. 증폭한 PCR 산물을 1.5% agarose gel에전 Botryosphaeria parva CMW9080 94 B. parva CMW9081 B. parva CMW9079 B. parva CMW9078 B. parva B. ribis CMW7772 B. ribis CMW7054 B. ribis CMW7773 B. ribis B. lutea CMW992 96 99 B. lutea CMW10309 B. lutea CMW9076 B. australis CMW9072 B. australis CMW9073 B. australis CMW6837 B. lutea B. australis 91 86 B. eucalyptorum CMW10125 B. eucalyptorum CMW6233 B. eucalyptorum CMW10126 B. eucalyptorum B. dothidea CMW8000 B. dothidea CMW7999 96 B. dothidea CMW7780 B. dothidea CMW991 B. dothidea 89 B. dothidea CMW9075 B. dothidea PPL01-Schisandra* 97 0.1 B. obtusa CMW7774 B. obtusa CMW7775 B. stevensii CMW7060 B. rhodina CMW9074 B. rhodina CMW10130 B. stevensii UCP130 B. obtusa B. stevensii B. rhodina Guignardia philoprina Fig. 3. Phylogenetic tree of Botryosphaeria species using combined sequence of internal transcribed spacer region, β-tubulin and elongation factor 1-α gene. The tree was constructed based on neighbor-joining method with 1,000 replicates. *The fungi isolated in this study.
48 Research in Plant Disease Vol. 22 No. 1 기영동하여확인하였고 ExoSAP-IT를이용하여정제한뒤염기서열을분석하였다. 분석한염기서열은 BLAST search 를통해 National Center for Biotechnology Information (NCBI) 의 GenBank database에저장되어있는염기서열들과비교하여병원균의종을확인하였다. 분석결과, 분리한병원균의 ITS 영역, β-tubulin 및 EF1-α 유전자의염기서열을모두 NCBI GenBank database에각각등록하였으며 (accession No. LC120711, LC120690, LC120691), 분석한유전자의염기서열들은모두 Botryosphaeria dothidea로등록되어있는균주들의염기서열과 >99% 일치하는것으로확인되었다. 계통수의분석은 Botryosphaeria속에속하는여러종의 ITS 영역, β-tubulin, EF1-α 유전자의염기서열들을 NCBI의 Genbank database 로부터조사하여수행하였다. NCBI의데이터베이스및본실험의염기서열분석으로부터얻은 ITS 영역, β-tubulin, EF1-α 유전자의염기서열들을순서대로연결하여결합염기서열을작성하고이를계통수의작성에사용하였다 (Table 2). DNA Data Bank of Japan (DDBJ) 홈페이지 (http://www.ddbj.nig.ac.jp) 의염기서열분석기능을사용하여결합염기서열들을정렬하고 TreeView 프로그램 (Page, 1996) 을사용하여 neighbor-joining 방법으로계통수를작성하였다. 분석결과, 오미자줄기마름병의병원균은명확하게 B. dothidea와같은계통군에속해있었으며, Botryosphaeria속의다른종들과구분되는클러스터를형성하고있었다. 이분석결과를통해해당병원균이 B. dothidea에속함을확인할수있었다 (Fig. 3). B. dothidea는국내에서사과나무, 복숭아나무, 배나무, 포도나무등의여러과수에열매썩음병과줄기마름병을유발하는것으로알려져있으며, 해외에서는유칼립투스와피스타치오에서도발병이보고되어있다 (Ma 등, 2001). 또한, 국내에서지금까지오미자에병해를끼치는것으로보고된병원균으로는 Botrytis cinerea, Microsphaera schizandrae, Penicillium sp., Pestalotiopsis guepinii, Phoma sp. 가있고잎이나열매에주로병징을나타내는것으로알려져있다 (The Korean Society of Plant Pathology, 2009). 그러나, B. dothidea에의해나타나는오미자의줄기마름병은지금까지보고된바가없다. 따라서향후오미자의재배에있어 B. dothidea에의한줄기마름병의방제대책이필요할것으로예상되며, 지금까지보고되지않았던 B. dothidea에의한오미자줄기마름병을국내처음으로보고하고자한다. 요약 2015 년 6 월에경북문경시에서재배하는오미자에서줄 기마름증상이발생하였다. 발병초기에는잎의선단부위부 터위축되면서고사현상이발생하였고, 병이진행됨에따 라줄기를거쳐식물체전체가고사하였으며주변으로전 염되었다. 감염된줄기에서병원균을분리배양하여배양적, 형태적특징을관찰한결과해당병원균은 Botryosphaeria 속 에속하는것으로나타났으며, 인공접종에의한병원성검 정에서도동일한병징을나타내었다. 병원균의정확한동정 을위해 ITS 영역과 β-tubulin, EF1-α 유전자의염기서열을복 합적으로사용한염기서열분석을수행하였으며, 분석결과 오미자의줄기마름증상을일으키는원인균은 B. dothidea 로 동정되었다. 지금까지국내에서 B. dothidea 에의한오미자 의병이보고되지않았기에본연구를통해국내처음으로 B. dothidea 에의한오미자줄기마름병을보고한다. Acknowledgement This research was supported by Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ010481032015) funded by Rural Development Administration, Republic of Korea. References Carbone, I. and Kohn, L. M. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553-556. Glass, N. L. and Donaldson, G. C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61: 1323-1330. Ma, Z., Boehm, E. A. W., Luo, Y. and Michailides, T. J. 2001. Population structure of Botryosphaeria dothidea from Pistachio and other hosts in California. Phytopathology 91: 665-672. Ministry of Agriculture, Food and Rural Affairs. 2014. 2013 Industrial crop production statistics. Ministry of Agriculture, Food and Rural Affairs, Sejong, Korea. 51 pp. (In Korean) Nam, S. Y., Lee, J. Y., Ko, J. S., Kim, J. B., Jang, H. H., Kim, H. R. and Lee, Y. M. 2014. Changes in antioxidant and antimicrobial activities of Schizandra chinensis Baillon under different solvent extraction. Korean J. Int. Agric. 26: 513-518. (In Korean) Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Applic. Biosci. 12: 357-358.
Research in Plant Disease Vol. 22 No. 1 49 Samrat, B., Pariyar, R., Yoon, C. S., Yun, J. M., Jang, S. O., Kim, S. Y., Oh, H. C., Kim, Y. C. and Seo, J. W. 2015. Effects of Schisandrae fructus 70% ethanol extract on proliferation and differentiation of human embryonic neural stem cells. Korean J. Pharmacogn. 46: 52-58. (In Korean) Slippers, B., Johnson, G. I., Crous, P. W., Coutinho, T. A., Wingfield B. D. and Wingfield, M. J. 2005. Phylogenetic and morphological re-evaluation of the Botryosphaeria species causing diseases of Mangifera indica. Mycologia 97: 99-110. The Korean Society of Plant Pathology. 2009. List of Plant Diseases in Korea. 5th ed. The Korean Society of Plant Pathology, Suwon, Korea. 273 pp. (In Korean) White, T. J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications, eds. by M. A. Innis, D. H. Gelfand, J. J. Sninski and T. J. White, pp. 315-322. Academic Press, San Diego, CA, USA.