식물병연구 Res. Plant Dis. 25(1): (2019) Note Open Access Plasmodiophora brassicae Ocurrence of Clubroot Cau

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1 식물병연구 Res. Plant Dis. 25(1): (2019) Note Open Access Plasmodiophora brassicae Ocurrence of Clubroot Caused by Plasmodiophora brassicae on Kohlrabi in Korea *Corresponding author Tel: Fax: ORCID * Victor Galea 5 1, 2, 3, 4, 5 MinA Song 1, InYoung Choi 2 *, JeongHeub Song 1, KuiJae Lee 3, HyeonDong Shin 4, and Victor Galea 5 1 Eco-friendly Agricultural Research Department, Jeju-do Agricultural Research and Extension Services, Jeju 63556, Korea 2 Agricultural Environment Division, Jeollabuk-do Agricultural Research and Extension Services, Iksan 54591, Korea 3 Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 54596, Korea 4 Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Korea 5 School of Agriculture and Food Sciences, The University of Queensland, QLD 4343, Australia Received January 10, 2019 Revised February 7, 2019 Accepted February 8, 2019 From 2016 to 2018, approximately 15% of kohlrabi were observed displaying significant clubroot symptoms in farmer s fields in Jeju, Korea. The initial infection appeared as hypertrophy of root hairs, and as the disease progressed, galls formation occurred on the main roots, finally disease progress resulted in yellowing and wilting of leaves. Pathogenicity was proven by artificial inoculation of plants with resting spore suspension, fulfilling Koch s postulates. The resting spore is one-celled, spherical and subspherical, colorless, and 3-5 mm in diameter. On the basis of the morphological characteristics and phylogenetic analyses of internal transcribed spacer rdna, the causal agent was identified as Plasmodiophora brassicae. To our knowledge, this is the first report on the occurrence of P. brassicae on kohlrabi in Korea. Keywords: Clubroot, Kohlrabi, Plasmodiophora brassicae, Resting spore (Brassica oleracea var. gongylodes L.) (Brassicaceae) (Brassica).,., C,, Research in Plant Disease pissn , eissn (Lee, 2010). 120 ha, (Choi, 2015). Plasmodiophora brassicae. 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 34., (Lee, 2008; Suwabe, 2003)., (become reddened), (chlorotic), (necrotic), (abscise),,,,, (Kageyama Asano, 2009). P. brassicae (cortical stage) (Brassica oleracea), (,,,,, ), (, ), ( ),,,,,,, (Dixon, 2009). P. brassicae, (root hair infection), (cortical infection).. 1 (primary zoospore) (primary plasmodia)., (zoosporangia) (secondary zoospore). 2 (cortical infection). 2 (secondary plasmodia). (Devos, 2005; Kageyama Asano, 2009). P. brassicae,.,,,, (Kageyama Asano, 2009). P. brassicae 15-20, (half-life) ph., 3-4 (McGrann, 2016) ( ).. rdna Plasmodiophora brassicae,.,, , 3 (27.2%)., (Fig. 1A).,,, (Fig. 1B-C).,.,.,. (KUS-F30866).. Cho (2003), Williams (1966). 4 o C 2, 40 g 50 ml. 3, 2,500 rpm 5.,

Fig. 1. Typical clubroot symptoms naturally infected kohlrabi caused by Plasmodiophora brassicae. (A) The infected crop showing wilt symptoms and plants loss. (B) Galls produced on the roots.

Scanning electron and light micrographs of immature and mature resting spores in clubroot of kohlrabi. (A-E) Resting spores inside cell tissues in clubroot galls.

3 Fig. 1. Typical clubroot symptoms naturally infected kohlrabi caused by Plasmodiophora brassicae. (A) The infected crop showing wilt symptoms and plants loss. (B) Galls produced on the roots. (C) Close-up of clubroot symptoms. Clubroot for 50 days after inoculation with resting spores suspension for pathogenicity test. 35 Fig. 2. Scanning electron and light micrographs of immature and mature resting spores in clubroot of kohlrabi. (A-E) Resting spores inside cell tissues in clubroot galls. (F) Resting spores released from cell tissues. 이 장착된 광학현미경(AXIO, Zeiss)의 관찰을 위해 -20oC로 설 포자를 세척하고자 30 ml 멸균수를 첨가하고 다시 원심 분리 정된 cryomicrotome (Leica cryostat CM3050S, Leica Micro- 를 반복하였다. 접종원으로 사용된 휴면포자 현탁액의 농도 systems, Wetzlar, Germany)을 이용하여 20 µm 두께로 절편을 8 는 spores/ml이었다. 병원성 검정을 위한 콜라비(품 제작한 후, 슬라이드 글라스에 올려놓고 400에서 1,000배로 관 종 아삭콜)는 72구 모종트레이에 종자를 파종하여 포트에서 찰하였다. 휴면포자의 형태와 크기(n=50) 등은 AxioVision LE 10일간 생육된 15개체를 사용하였다. 접종 방법은 포트에서 Module Interactive Measurement (Zeiss) 프로그램을 이용하 생육 중인 10개체의 뿌리 부분에 5-10 cm 정도 구멍을 내고 여 측정하였다. 접종원 20 ml를 부었으며, 대조구는 5개체에 멸균수를 동일 o 주사전자현미경으로 관찰된 병원균은 뿌리 세포조직 내에 한 방법으로 부었다. 접종한 모든 식물체를 C로 유지되 마치 마대 자루에 콩을 담아 놓은 것처럼 구형의 성숙되거나 미 는 유리온실에서 관리하였으며, 식물체가 마르지 않도록 정기 성숙된 휴면포자를 무수히 형성하였다(Fig. 2A-D). 미성숙된 휴 적으로 관수를 하였다. 휴면포자가 접종된 콜라비는 50일 이 면포자들 사이는 실타래로 서로 엉켜있는 것처럼 보였다. 광학 후부터 시들기 시작하였으며, 뿌리부분에는 포장에서 관찰된 현미경에서도 휴면포자가 세포조직 안에 빈 곳이 없을 정도로 모습과 동일한 혹이 형성되었고, 다량의 휴면포자가 관찰되었 무수히 관찰되었다. 휴면포자는 단세포로 무색, 원형 또는 타원 다. 그러나 대조구는 시들지 않았으며, 뿌리혹이 형성되지 않 형, 크기는 직경 3-5 µm이었다(fig. 2E-F). 았다. 따라서 콜라비에 뿌리혹을 일으키는 병원균은 병원성이 있는 것으로 판단되었다. 또한 형태적 특징을 기초로 한 병원균의 동정을 보완하기 위하여 염기서열 분석을 하였다. 뿌리혹이 형성된 콜라비 뿌 리 1 g을 액체질소를 넣고 마쇄한 후, DNeasy Plant Mini kit 병원균의 균학적 특징 및 rdna의 ITS 염기서열 분석. 병원 (QIAGEN, Valencia, CA)를 이용하여 genomic DNA를 추출 균의 형태적 특징은 뿌리혹이 형성된 콜라비 뿌리를 직접 절단 하였다. Ribosomal DNA (rdna)의 internal transcribed 하여 주사전자현미경(JSM-5410LV; JEOL, Japan)과 광학현미경 spacer (ITS) 영역을 분석하기 위해 ITS1/ITS4 primer를 사용 으로 관찰하였다. DIC (differential interference contrast) 모듈

4 36 Fig. 3. Phylogenetic tree obtained through the neighbor-joining method using the MEGA 6.06 program based on the internal transcribed spacer ribosomal DNA (rdna) sequence of our isolate of Plasmodiophora brassicae, and that of other fungi retrieved from GenBank. The numbers above the nodes are the bootstrap values obtained from 1,000 replicates. The isolates obtained in this study are shown in boldface. PCR (White, 1990), 1.5% agarose gel PCR purification kit (Core-one TM, Core-bio, Korea). ABI 3130 auto sequencer ITS1 ITS4, ITS rdna GenBank database (National Centre for Biotechnology Information [NCBI], US National Institute of Health Bethesda, nih.gov/blast.cgi). MEGA 6.06 program neighbor-joining, Tamura-Nei parameter model, bootstrap analysis 1,000 (Tamura, 2013). P. brassicae ITS 547 bp NCBI GenBank MH NCBI Blastn search (Canola) (Sinapis alba) P. brassicae GenBank accession number KY628944, KY628950, KY628953, EF %. (,,,,,, ) P. brassicae GenBank accession number KX KX430462, KX KX %. ITS (MH ), P. brassicae (Fig. 3)., Plasmodiophora brassicae (Buczacki, 1979; Kim, 2009). Plasmodiophora brassicae (clubroot, ), Peronospora brassicae (downy mildew, ), Rhizoctonia solani (damping-off, ), Alternaria brassicae (black spot, gray spot, ), Fusarium oxysporum (wilt, ) (Farr Rossman, 2019)., P. brassicae,,,,,,,, (Farr Rossma, 2019). Fusarium oxysporum (Choi, 2015; The Korean Society of Plant Pathology, 2009).,, Plasmodiophora brassicae ( 27.2%).,,. P. brassicae 50.,,, 3-5 µm. ITS rdna, P. brassicae.,, ITS rdna Plasmodiophora brassicae. Conflicts of Interest No potential conflict of interest relevant to this article was reported.

5 37 Acknowledgment 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 Buczacki, S Plasmodiophora brassicae. [Descriptions of Fungi and Bacteria]. URL: [19 March 2019]. Cho, W. D., Kim, W. G. and Takahashi, K Occurrence of clubroot in cruciferous vegetable crops and races of the pathogen in Korea. Plant Pathol. J. 19: Choi, I. Y., Kim, J., Ju, H. J., Park, J. H. and Shin, H. D First report of Fusarium wilt caused by Fusarium oxysporum on kohlrabi in Korea. Res. Plant Dis. 21: Devos, S., Vissenberg, K., Verbelen, J. P. and Prinsen, E Infection of chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance. New phytol. 166: Dixon, G. R The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. J. Plant Growth Regul. 28: Farr, D. F. and Rossman, A. Y Fungal Databases. U.S. National Fungus Collections, United States Department of Agriculture. URL: [19 March 2019]. Kageyama, K. and Asano, T Life cycle of Plasmodiophora brassicae. J. Plant Growth Regul. 28: Kim, W. G., Moon, M. H., Kim, J. H., Choi, H. W. and Hong, S. K Occurrence of clubroot on Pak-Choi caused by Plasmodiophora brassicae. Mycobiology 37: Lee, J. W., Lee, D. Y., Cho, J. G., Back, N. I. and Lee, Y. H Isolation and identification of sterol compounds from the red kohlrabi (Brassica oleracea var. gongylodes) sprouts. J. Appl. Biol. Chem. 53: Lee, S. O., Choi, G. J., Choi, Y. H., Jang, K. S., Park, D. J., Kim, C. J. et al Isolation and characterization of endophytic actinomycetes from Chinese cabbage roots as antagonists to Plasmodiophora brassicae. J. Microbiol. Biotechnol. 18: McGrann, G. R., Gladders, P., Smith, J. A. and Burnett, F Control of clubroot (Plasmodiophora brassicae) in oilseed rape using varietal resistance and soil amendments. Field Crops Res. 186: Suwabe, K., Tsukazaki, H., Iketani, H., Hatakeyama, K., Fujimura, M., Nunome, T. et al Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L. Theor. Appl. Genet. 107: 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. The Korean Society of Plant Pathology, Suwon, Korea. 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 and Applications - A Laboratory Manual, eds. by M. A. Innis, D. H. Gelfand, J. J. Sninski and T. J. White, pp Academic Press, San Diego, CA, USA. Williams, P. H A system for the determination of races of Plasmodiophora brassicae that infect cabbage and rutabaga. Phytopathology 56:

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