식물병연구 Note Open Access Res. Plant Dis. 24(1): (2018) Fusarium oxysporum Fusarium Wilt Caused by Fusariu

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1 식물병연구 Note Open Access Res. Plant Dis. 24(1): (2018) Fusarium oxysporum Fusarium Wilt Caused by Fusarium oxysporum on Passionfruit in Korea 1 2 * , 2, 3 *Corresponding author Tel: Fax: choiiy21@korea.kr Jae-Ho Joa 1, In-Young Choi 2 *, Min-Kyung Choi 2, Byong-Soo Heo 2, Jong-Ok Jang 1, and Hyeon-Dong Shin 3 1 Citrus Research Institute, National Institute of Horticultural and Herbal Science, Jeju 63607, Korea 2 Jeollabuk-do Agricultural Research and Extension Services, Iksan 54591, Korea 3 Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Korea Received November 6, 2017 Revised November 22, 2017 Accepted December 4, 2017 From 2014 to 2016, Fusarium wilt disease was found on fassionfruit in Iksan and Jeju, Korea. Symptoms included wilting of foliage, drying and withering of leaves, and stunting of the plants. The infected plants eventually died during growth. Colonies on potato dextrose agar were pinkish white, and felted with cottony and aerial mycelia with 35 mm after one week. Macroconidia were falcate to almost straight, thin-walled and usually 2-3 septate. Microconidia were usually formed on monophialides of the hyphae and were hyaline, smooth, oval to ellipsoidal, aseptate or medianly 1-septate, very occasionally 2-septate, slightly constricted at the septa, mm. On the basis of the morphological characteristics and phylogenetic analyses of two molecular markers, internal transcribed spacer rdna and translation elongation factor 1α, the fungus was identified as Fusarium oxysporum. Pathogenicity of a representative isolate was proved by artificial inoculation, fulfilling Koch s postulates. To our knowledge, this is the first report on the occurrence of F. oxysporum on fassionfruit in Korea. Keywords: Fusarium oxysporum, Fusarium wilt, Passiflora edulis, Passionfruit ( ; Passiflora edulis Sims) (Passifloraceae),.,,,, (Rural Development Administration, 2014). Research in Plant Disease pissn , eissn ha 920, Fusarium oxysporum f. sp. passiflorae (Silva, 2013). Fusarium, (Agrios, 2005) ,,,, The Korean Society of Plant Pathology This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 76 배 면적이 2014년도 9.4 ha에서 2015년도 44.4 ha에 달할 정도로 CMV), 등대풀잎말림바이러스(Euphorbia leaf curl virus, EuLCV), 파 급격히 증가하고 있다(Ministry of Agriculture, Food and Rural 파야잎말림광동바이러스(Papaya leaf curl Guangdong virus, PaL- Affairs, 2017). CuGdV) 및 토마토황화잎말림바이러스(Tomato yellow leaf curl 전 세계적으로 패션프루트에는 Fusarium에 의한 시들음병원균 virus, TYLCV)이 확인되었을 뿐이다(Hong, 2017). 으로 F. oxysporum, F. oxysporum f. sp. passiflorae, F. oxysporum 2014년부터 2016년까지 전라북도농업기술원(익산)과 국립원예 var. redolens 등이 기록되어 있다(Farr와 Rossma, 2017), 국내에서 특작과학원 온난화대응농업연구소(제주)의 시험연구포장에 식재 는 바이러스병 5종 - 동아시아시계초바이러스(East Asian Passiflora 한 패션프루트 Panama Red 에서 시들음병이 발생하였다. 병든 식 virus, EAPV), 오이모자이크바이러스(Cucumber mosaic virus, 물에서 병원균을 순수 분리한 후 단일균주를 확보하여 균학적 특 Fig. 1. Fusarium wilt caused by Fusarium oxysporum on passionfruit. (A) Occurrence of Fusarium wilt in a farm in July (B) The stem was infected, devastating the whole plant. The vascular system was blocked by the fungus. (C, D) Macroconidia and microconidia. (E) Monophialides with macroconidia on hypha. (F) Monophialides with microconidia on hypha. (G) Microconidia. (H) One-week-old colony of F. oxysporum growing on a potato dextrose agar.

3 77 DNA F. oxysporum,.,, ( ) ( ) Panama Red 2 m 2 m. 60.,., (Fig. 1A)., (Fig. 1B)., , % mm 1% NaOCl (WA). 25 o C 3 (PDA)(Difco, MD, USA), 25 o C 7.. (Korean Agricultural Culture Collection) ( KACC48307),., Gardner (1989). (cotyledon stage) 5. 1 (scalpel) (10 6 conidia/ml). 5 (vermiculite), 5, o C 12 80%. 4,... 7 Stereo (SteREO Discovery V8, Zeiss) DIC (differential interference contrast) (AXIO, Zeiss, Oberkochen, Germany) 200-1,000., (n=30) AxioVision LE Module Interactive Measurement (Zeiss). 35 mm/7, - (Fig. 1H). monophialides,., monophialides,, -, 1, 2, m (Fig. 1C-1G).. 1 ribosomal DNA (rdna) internal transcribed spacer (ITS) Fusarium Translation Elongation Factor 1-alpha (EF-1 ) (Hsuan, 2011). Genomic DNA DNeasy Plant Mini kit (QIAGEN, Valencia, Canada), ITS1/ITS4 (White, 1990) EF-1/EF-2 (Geiser, 2004) primer PCR. PCR 1.5% agarose gel band, PCR purification kit (CoreoneTM, Core-bio, Seoul, Korea). autosequencer (ABI 3030), ITS EF-1 GenBank database (National Centre for Biotechnology Information [NCBI], US National Institute

4 78 Fig. 2. Phylogenetic analysis inferred by the neighbor-joining method comparing the internal transcribed spacer ribosomal DNA(rDNA) region of Fusarium oxysporum with those of other Fusarium species retrieved from GenBank. The numbers above the branches represent the bootstrap value. The fungi identified in this study are boldfaced. of Health Bethesda, Fusarium-ID ( GenBank ITS, EF-1, MEGA 6.0 neighbor-joining. Sequence distance Tajima-Nei parameter model (Saitou Nei, 1987; Tamura, 2013). KACC48307 ITS 544 bp NCBI GenBank MG NCBI Blastn search F. oxysporum GenBank accession number KP995455, KU901515, KJ475803, KF786127, KF671955, KF156304, KY >99%. EF bp GenBank MG356947, NCBI Blastn search F. oxysporum GenBank accession number KX431987, KJ127285, FJ538245, KJ699111, KX609708, KR >99%. Fusarium ID F. oxysporum ITS FD_0638, FD_0213, FD_0270, FD_0342, FD_0297 >99.5%, EF-1 FD_00793, FD_00788, FD_00741, FD_00718, FD_ %. ITS (MG356946) EF-1 (MG356947) F. oxysporum (Fig. 2, 3)., F. oxysporum E.F. Sm. & Swingle (Nelson, 1983; Lee Park, 2001). F. oxysporum,,,,, (Farr Rossman, 2017), (The Korean Society of Plant Pathology, 2009).,, ITS rdna, EF-1 F. oxysporum E.F. Sm. & Swingle. F. oxysporum complex. (recolonize) (Flores, 2012).

5 79 Fig. 3. Phylogenetic analysis inferred by the neighbor-joining method comparing the translation elongation factor 1α of Fusarium oxysporum with those of other Fusarium species retrieved from GenBank. The numbers above the branches represent the bootstrap value. The fungi identified in this study are boldfaced. Conflicts of Interest 요 약 2014년부터 2016년까지 전라북도농업기술원(익산)과 국립원예 특작과학원 온난화대응농업연구소(제주)에서 패션프루트 시들음 No potential conflict of interest relevant to this article was reported. 병이 관찰되었다. 병징은 생육 초기 건전주에 비해 생육이 더디고 새순부터 시듦 증상이 나타나기 시작하였으며, 오래된 잎은 마르 Acknowledgement 고 뒤틀리다가 더 이상 생육하지 않고 말라 죽었다. 감자한천배지 에서 균사 생육은 직경이 35 mm/7일이었으며, 핑크빛-흰색으로 양모상이었다. 병원균의 대형분생포자는 무색으로 투명하며, 길 이가 짧고 굴곡진 모양-일자형으로 세포벽이 얇고 주로 2-3개의 격벽이 있었다. 소형분생포자는 균사로부터 monophialides 위에 형성되었으며, 투명하고, 난형-타원형이며, 격벽이 없거나 1개인 데, 드물게 2개인 경우도 있었고, 크기는 μm이다. 균 학적 특징, 병원성 검정, ITS rdna, EF-1α 염기서열 비교분석 등의 결과를 바탕으로 이 병은 우리나라에서 지금까지 보고되지 않은 Fusarium oxysporum에 의한 패션프루트 시들음병 으로 명명하고 자 한다. This study was carried out with the support of Cooperative Research Program for Agricultural Science & Technology Development(Project No. PJ ) Rural Development Administration, Republic of Korea. References Agrios, G. N Plant Pathology. 5th ed. Elsevier Academic Press, Amsterdam, The Netherlands. Lee, Y. S. and Park, C. S Fusarium species of Korea. Plant Pathogens of Korea 5. National Institute of Agricultural Science and Technology, Suwon, Korea. 91 pp.

6 80 Farr, D. F. and Rossman, A. Y Fungal Databases, Systematic Mycology & Microbiology Laboratory, ARS, USDA. United States Department of Agriculture, Washington, DC, USA. [cited 2017 Oct 10]. URL Flores, P. S., Otoni, W. C., Dhingra, O. D., de Souza Diniz, S. P. S., Dos Santos, T. M. and Bruckner, C. H In vitro selection of yellow passion fruit genotypes for resistance to Fusarium vascular wilt. Plant Cell Tissue Organ Cult. 108: Gardner, D. E Pathogenicity of Fusarium oxysporum f. sp. passiflorae to banana poka and other Passiflora spp. in Hawaii. Plant Dis. 73: Geiser, D. M., Jimenez-Gasco, M. D., Kang, S. C., Makalowska, I., Veeraraghavan, N., Ward, T. J., Zhang, N., Kuldau, G. A. and O Donnell, K FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. European J. Plant Pathol. 110: Hong, S. B Development of a Multiplex PCR System for diagnosis of Major Passionfruit Viruses and Their Epidemiology in Korea. The graduate school dissertation. Chonbuk National University, Korea. 88 pp. Hsuan, H. M., Salleh, B. and Zakaria, L Molecular identification of Fusarium species in Gibberella fujikuroi species complex from rice, sugarcane and maize from peninsular Malaysia. Int. J. Mol. Sci. 12: Ministry of Agriculture, Food and Rural Affairs Statistics of Cultivation and Production of Industrial Crops in Korean Ministry of Agriculture, Food and Rural Affairs, Sejong. Nelson, P. E., Toussoun, T. A. and Marasas, W. F. O Fusarium Species: an Illustrated Manual for Identification. pp Rural Development Administration New Subtropical and Tropical Fruit. Jeonju, Korea. 187 pp. (In Korean) Saitou, N. and Nei, M The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: Silva, A. D. S., Oliveira, E. J. D., Haddad, F., Laranjeira, F. F., Jesus, O. N. D., Oliveira, S. A. S. D., Costa, M. A. P. D. C. and Freitas, J. P. X. D Identification of passion fruit genotype resistant to Fusarium oxysporum f. sp. passionflorae. Trop. Plant Pathol. 38: Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: The Korean Society of Plant Pathology List of Plant Disease in Korea. 5th ed. 853 pp. White, T. J., Bruns, T., Lee, S. and Taylor, J Amplification and direct sequencing of fungi 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. Academic Press, San Diego, CA, USA.

28 Research in Plant Disease Vol. 21 No 년 4월에 전라북도농업기술원의 노지 시험연구포장에 정식한 콜라비(품종 아삭콜)에서 처음으로 시들음병이 발생 하였다. 이병주에서 병원균을 순수분리하여 균주를 확보하 였다. 균학적 특성 및

28 Research in Plant Disease Vol. 21 No 년 4월에 전라북도농업기술원의 노지 시험연구포장에 정식한 콜라비(품종 아삭콜)에서 처음으로 시들음병이 발생 하였다. 이병주에서 병원균을 순수분리하여 균주를 확보하 였다. 균학적 특성 및 식물병연구 Note Open Access Res. Plant Dis. 21(1) : 27-31(2015) http://dx.doi.org/10.5423/rpd.2015.21.1.027 Fusarium oxysporum First Report of Fusarium Wilt Caused by Fusarium oxysporum on Kohlrabi in Korea