식물병연구 Res. Plant Dis. 24(4): 302-307 (2018) Note Open Access https://doi.org/10.5423/rpd.2018.24.4.302 Botrytis cinerea Occurrence of Gray Mold Caused by Botrytis cinerea on Okra in Korea *Co-corresponding authors I.Y. Choi Tel: +82-63-290-6182 Fax: +82-63-290-6198 E-mail: choiiy21@korea.kr H.D. Shin Tel: +82-2-3290-3063 Fax: +82-2-925-1970 E-mail: hdshin@korea.ac.kr ORCID https://orcid.org/0000-0002-7778-2747 1 2 * 3 4 5 6 * Victor Galea 7 1, 2, 3, 4, 5, 6, 7 JangNam Choi 1, InYoung Choi 2 *, KuiJae Lee 3, JungNo Lee 4, SeongWan Cho 5, HyeonDong Shin 6 *, and Victor Galea 7 1 Department of Agricultural Biology, Chonbuk National University, Jeonju 54896, Korea 2 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 Department of Bionanometerials, Biocampus of Korea Polytechnics, Nonsan 32943, Korea 5 Department of Pharmaceutics & Biotechnology, Konyang University, Daejeon 35365, Korea 6 Division of Environmental Science and Ecological Engineering, Korea University, Seoul 02841, Korea 7 School of Agriculture and Food Sciences, The University of Queensland, QLD 4343, Australia Received August 26, 2018 Accepted September 14, 2018 From 2014 to 2016, approximately 5% of okra fruit were observed displaying gray mold symptoms at the research field of Jeollabuk-do Agricultural Research and Extension Services, Korea. The symptoms observed were water-soaked, brown or gray spots, and abundant mycelial with conidia appearing on the infected fruit. Initial infection commenced from the base of fruit and gradually moved to the pod, where it finally resulted in collapse. Colonies on potato dextrose agar were gray to grayish brown, felted and cottony expanding 65-80 mm after one week. The fungus formed several black sclerotia ranging 1.0-3.5 0.5-3.0 mm on the Petri dish after two weeks. The conidia were one-celled, ellipsoidal or ovoid, colorless or pale brown, and 6.2-15.4 5.0-10.4 mm. Conidiophores arose solitary or in groups, straight or flexuous, septate, with an inflated basal cell brown to light brown, and measured 85-450 10.0-40.0 mm. On the basis of the morphological characteristics and phylogenetic analyses of internal transcribed spacer rdna, the fungus was identified as Botrytis cinerea Pers. 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 B. cinerea on okra in Korea. Keywords: Abelmoschus esculentus, Botrytis cinerea, Gray mold, Okra [Abelmoschus esculentus (L.) Moench] (Malvaceae) Research in Plant Disease pissn 1598-2262, eissn 2233-9191 www.online-rpd.org.,,,,.,, The Korean Society of Plant Pathology 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.
Research in Plant Disease Vol. 24 No. 4 303 프 등에 넣어 먹는다(Salameh, 2014). 오크라는 아시아와 아프 확대되었으나 재배면적은 통계에 나타나 있지 않다(Ministry of 리카의 개발도상국에서 99% 이상 독점적으로 재배되고 있으 Agriculture, Food and Rural Affairs, 2017). -1 며, 전 세계의 연평균 생산량은 5.26 MT ha 로 183 만 ha에서 9.62 million metric tons (MMT)을 생산하고 있다. 특히, 인도의 Botrytis에 의한 잿빛곰팡이병은 기주범위가 넓어 초본류와 목본류에서 blight, blossom-end rot, gray mold, bulb rot, 생산성은 12.0 MT ha-1이며(faostat, 2014), 50만 ha 이상에서 blossom blight, damping-off 등을 유발하며 가장 일반적 6.3 MMT(세계 총 생산량 72%)의 생산량으로 세계 1위를 차지 인 종은 B. cinerea로 보고되고 있다(Plant Disease Diagnostic 하고 있다(Mishra 등, 2017). 우리나라의 경우 지구 온난화에 따 Clinic, 2011). 또한 B. cinerea에 의한 잿빛곰팡이병은 목화, 무 라 아열대작물의 재배 가능지역이 남부지역부터 점차 확대됨 궁화, 접시꽃 같은 아욱과에 주로 발병하는 중요한 식물병원균 에 따라 다양한 아열대작물이 국내에 도입되어 재배가 시도되 으로 235종의 식물에서 다양한 부위와 작기에 감염을 일으키 고 있다. 그 중 해외 수입에만 의존하던 오크라의 상업적인 재 며, 전형적인 사물기생균으로 식물세포를 죽이고 영양원으로 배가 2000년대부터 시작되어 제주, 전라, 경남 등의 지역으로 활용한다(Ten Have 등, 2010; Zhao 등, 2016). B. cinerea의 발육 Fig. 1. Typical symptoms of gray mold disease on naturally infected okra caused by Botrytis cinerea. (A) The fruit was infected, devastating the whole plant. (B) One-week-old colony of B. cinerea growing on a PDA (Potato Dextrose Agar). (C) Black sclerotia developed on PDA of two-week-old colony. (D) Conidia and conidiophores. (E) Conidia.
304 Research in Plant Disease Vol. 24 No. 4 22-24 o C,. 2014 2016 ( )., DNA Botrytis cinerea,.,,. (2.3 10 5 conidia/ml). 5 10,. 95% 25 1 o C. 5...., 5%., Green Hope Red Hope.., (Fig. 1A).. 5 5 mm 1% NaOCl 1., 2-3 (PDA) (Difco, MD, USA), 22 1 o C 2-3, 2 (JARES71 JBARES72). 30 5. JARES71 2 (scalpel) rdna ITS. 7 (JARES71, JBARES72), Stereo (SteREO Discovery V8, Zeiss) DIC (differential interference contrast) (AXIO, Zeiss) 400-1,000., (n=50) AxioVision LE Module Interactive Measurement (Zeiss). 22 1 o C, 7 65-80 mm,, (Fig. 1B). 2, 1.0-3.5 0.5-3.0 mm (n=20) (Fig. 1C)., 6.2-15.4 5.0-10.4 mm (Fig. 1E).. 85-450 10.0-40.0 mm (Fig. 1D, Table 1). Table 1. Comparison of mycological characteristics of the present isolate obtained from okra (Abelmoschus esculentus) and Botrytis cinerea as previously described Conidia Characteristics Present isolate B. cinerea a B. cinerea b cell shape one-celled ellipsoidal or ovoid one-celled ellipsoidal or ovoid one-celled ellipsoidal or ovoid size (mm) 6.2-15.4 5.0-10.4 5.1-10.4 5.1-7.8 6.0-18.0 4.0-11.0 color colorless or pale brown dark brown colorless or pale brown Conidiophore size (mm) 85-450 10-40 110-406 10-27 16-30 2 Colony color brown to light brown brown to light brown grayish brown a Aktaruzzaman et al. (2018), b Ellis (1971).
Research in Plant Disease Vol. 24 No. 4 305 Fig. 2. 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 isolates of Botrytis cinerea, and that of other Botrytis spp. 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.., DNeasy Plant Mini kit (QIAGEN, Valencia, CA) genomic DNA. Ribosomal DNA (rdna) internal transcribed spacer (ITS) ITS1/ITS4 primer PCR (White, 1990), 1.5% agarose gel PCR purification kit (Core-one TM, Core-bio, Korea). ABI 3130 auto sequencer ITS1 ITS4, ITS rdna sequence data GenBank database (National Centre for Biotechnology Information [NCBI], US National Institute of Health Bethesda, http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Phylogenetic analysis MEGA 6.06 program neighbor-joining, sequence distance Tamura-Nei parameter model, bootstrap analysis 1,000 (Tamura, 2013). B. cinerea JARES71, JBARES72 ITS 539 bp NCBI Gen- Bank (JBARES71) MH763647. NCBI Blastn search B. cinerea GenBank accession number KY817370, MH458888, KX229751, KR055052, KU570456, KP165493, KX443701 >99%. JARES71, JBARES72 ITS Botrytis spp. B. cinerea (Fig. 2).,
306 Research in Plant Disease Vol. 24 No. 4 Botrytis cinerea Pers. (Aktaruzzaman, 2018; Ellis, 1971). 230 Botyrtis cinerea (Farr Rossman, 2018). Podosphaera xanthii (Choi, 2018) Choanephora cucurbitarum Choanephora rot (Park, 2015) (The Korean Society of Plant Pathology, 2009).,, ITS rdna Botrytis cinerea Pers.. B. cinerea, 1,. 95%, (Plant Disease Diagnostic Clinic, 2011).. 2014 2016 ( ) 5%.,. 65-80 mm/7,. 2, 1.0-3.5 0.5-3.0 mm., 6.2-15.4 5.0-10.4 mm.. 85-450 10.0-40.0 mm.,, ITS rdna Botrytis cinerea Pers.. Conflicts of Interest No potential conflict of interest relevant to this article was reported. Acknowledgement This study was carried out with the support of Coopera tive Research Program for Agricultural Science & Technology Development (Project No. PJ0124192018) Rural Develop ment Administration, Republic of Korea. References Aktaruzzaman, M., Afroz, T., Kim, B. S., Choi, I. Y. and Shin, H. D. 2018. First report of gray mold caused by Botrytis cinerea on strawflower in Korea. Plant Dis. 102: 452. Choi, I. Y., Kim, J. H., Uhm, M. J., Cho, S. E. and Shin, H. D., 2018. First report of powdery mildew caused by Podosphaera xanthii on okra in Korea. Plant Dis. 102: 1663. Ellis, M. 1971. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK. 608 pp. FAOSTAT. 2014. FAOSTAT URL http://faostat.fao.org/ [8 February 2017]. Farr, D. F. and Rossman, A. Y. 2018. Fungal Databases, Systematic Mycology & Microbiology Laboratory. URL http://nt.ars-grin. gov/fungaldatabases/ [14 November 2018]. Ministry of Agriculture, Food and Rural Affairs. 2017. Statistics of cultivation and production of industrial crops in 2015. Korean Ministry of Agriculture, Food and Rural Affairs, Sejong, Korea. Mishra, G. P., Singh, B., Seth, T., Singh, A. K., Halder, J., Krishnan, N. et al. 2017. Biotechnological advancements and begomovirus management in okra (Abelmoschus esculentus L.): status and perspectives. Front. Plant Sci. 8: 360. Park, J. H., Cho, S. E., Choi, I. Y. and Shin, H. D. 2015. First report of Choanephora rot of okra caused by Choanephora cucurbitarum in Korea. J. Phytopathol. 163: 503-506. Plant Disease Diagnostic Clinic. 2011. Botrytis blight: Botrytis cinerea; Botrytis spp.. Cornell University, Department of Plant Pathology and Plant-Microbe Biology Report. URL http://plantclinic.cornell.edu/factsheets/botrytisblight.pdf [14 November 2018]. Salameh, N. 2014. Genetic diversity of okra (Abelmoschus esculentus L.) landraces from different agro-ecological regions revealed by AFLP analysis. Am-Euras. J. Agric. Environ. Sci. 14: 155-160. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729. ten Have, A., Espino, J. J., Dekkers, E., Van Sluyter, S. C., Brito, N., Kay, J. et al. 2010. The Botrytis cinerea aspartic proteinase family. Fungal
Research in Plant Disease Vol. 24 No. 4 307 Genet. Biol. 47: 53-65. The Korean Society of Plant Pathology. 2009. List of plant disease in Korea. 5th ed. The Korean Society of Plant Pathology, Suwon, Korea. 853 pp. (In Korean) White, T. J., Bruns, T. D., Lee, S. B. and Taylor, J. W. 1990. Amplification and di rect 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. 315-322. Academic Press, San Diego, CA, USA. Zhao, Q., Chai, A. L., Shi, Y. X., Xie, X. W. and Li, B. J. 2016. First report of grey mould disease on Abelmoschus esculentus caused by Botrytis cinerea in China. J. Phytopathol. 164: 354-357.