384 Research in Plant Disease Vol. 23 No. 4,, 1. (INSV),,, (vein clearing), (Tomato spotted wilt virus, TSWV),.,,,,,, (Brisco-McCann Hausbeck, 2016; S

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1 식물병연구 Note Open Access Res. Plant Dis. 23(4) : (2017) First Report of Impatiens necrotic spot virus in Hoya carnosa in Korea * 1, 2, 3 *Corresponding author Tel: Fax: hschoi@korea.kr These authors contributed equally to this work as co-first authors. Mikyeong Kim 1, Hyo-Jung Kang 2, Hae-Ryun Kwak 1, Jeong-Eun Kim 1, Jaedeok Kim 1, Jang-Kyun Seo 3, and Hong-Soo Choi 1 * 1 Crop Protection Division, National Institute of Agricultural Sciences, Wanju 55365, Korea 2 Chungcheongbuk-do Agricultural Research and Extension Services, Boeun 28902, Korea 3 Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea Received October 26, 2017 Revised October 31, 2017 Accepted November 1, 2017 In 2016, less than 30% of virus-like symptoms such as chlorosis, necrosis and ringspots were observed in Hoya carnosa from commercial greenhouse in Eumseong, Korea. A total of 6 samples from Hoya carnosa were collected both symptomatic and asymptomatic plants and tested for virus infection by RT-PCR of 3 viruses known to infect Hoya spp. including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Tomato chlorotic spot virus (TCSV). Three symptomatic samples were positives for INSV. Also, it was not the virus detected in three asymptomatic samples. To further confirm the presence of INSV, complete nucleocapsid (N) gene of the virus were amplified and sequenced from two samples. BLAST analysis of the consensus sequence showed that two isolates (INSV-Hy1 and -Hy2) shared nucleotide sequence identities of 99% with each other and 97-99% with other INSV isolates available in the GenbBank. Phylogenetic analysis showed that these isolates closely related to the INSV isolates from ornamental from China. This is the first report of INSV on Hoya carnosa from Korea. Keywords: Hoya carnosa, Impatiens necrotic spot virus, Orthotospovirus (Hoya carnosa) 100, 3-5 ( ). (Yang, 2009). (Impatiens necrotic spot virus, INSV) Tospoviridae, Orthotospovirus, (Frankliniella occidentalis), (F. intonsa) Research in Plant Disease pissn , eissn (Simko, 2017; Sakurai, 2004). 1990,,, 36 (Crosslin Hamlin, 2010; Daughtrey, 1997; De Avila, 1992; Kuo, 2014; Law Moyer, 1990) (Horita Sasaki, 2005), 2010 (Ding, 2011), 2009,,, ha, 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.

384 Research in Plant Disease Vol. 23 No. 4,, 1. (INSV),,, (vein clearing), (Tomato spotted wilt virus, TSWV),.,,,,,, (Brisco-McCann Hausbeck, 2016; Shin, 2013; Roggero, 1999; Vicchi, 1999).

2 384 Research in Plant Disease Vol. 23 No. 4,, 1. (INSV),,, (vein clearing), (Tomato spotted wilt virus, TSWV),.,,,,,, (Brisco-McCann Hausbeck, 2016; Shin, 2013; Roggero, 1999; Vicchi, 1999) RT-PCR, (INSV) ( 1,000 m 2, 15 ). 40 1%, 60 10%, 90 30%.,., 3 ( ) (Fig. 1). 7.. (Quick DIP) (2% PTA). 6,, nm. RNA RT-PCR , 3 1 3, 6, 7 ( mg), (BCS Plant RNA Prep Kit, BioCube System, Gwacheon, Korea) Total RNA. Orthotopsovirus (TSWV), (INSV) (Tomato chlorotic spot virus, TCSV) 3 RT-PCR (Table 1). 3 (INSV). Fig. 1. Disease symptoms induced in Hoya carnosa plants by Impatiens necrotic spot virus (INSV). (A) Chlorotic and necrotic ring spots on leaves in natural field. (B) Brown to dark brown, necrotic spotting in middle leaves of the plant. (C, D) Chlorotic spots and necrosis of leaves.

Research in Plant Disease Vol. 23 No. 4 385 Table 1.

3 Research in Plant Disease Vol. 23 No Table 1. Sequences of primers for detection of TSWV, INSV, TCSV, CMV and BBWV2 in this report Viruses a Name Sequence (5-3 ) Product size (bp) TSWV TSWV-6F GAGATTCTCAGAATTCCCAGT TSWV-6R AGAGCAATCGTGTCAATTTTATTC 459 bp INSV INSV-1F ATCAATAGTAGCATTAAACAT INSV-1R GACTCAATCTGATTCCTTAGA 800 bp TCSV TCSV-F TCCCTTGGTTTCATTGACCAAACGC TCSV-R ACATG ACACTTGCAAATGAGACTCC 590 bp CMV CMV DP u1 CGTCGTGGTTCCCGCTCCG CMV DP d2 AGCGCGCATCGCCGAAAGAT 473 bp BBWV2 BBWV2 1-1u AAACAAACAGCTTTCGTTCCG BBWV2 1R GCCATCTCATTGGCATGGA 380 bp a TSWV, Tomato spotted wilt virus; INSV, Impatiens necrotic spot virus; TCSV, Tomato chlorotic spot virus; CMV, Cucumber mosaic virus and BBWV2, Broad bean wilt virus 2. Table 2. Analysis of leaves of Hoya carnosa for five viruses using specific primers by RT-PCR 3 7 (TSWV), (INSV) (TCSV) (Cucumber mosaic virus, CMV) (Table 2). 3 (Ls), (La), (S), (R) (INSV),,,, (Fig. 2). (INSV) (Vicchi, 1999). Host a No. of positive samples b TSWV INSV TCSV CMV BBWV2 Hoya-S Hoya-A Pepper-S a Number of samples collected; S = symptomatic samples and A = asymptomatic samples. b Viruses: TSWV, Tomato spotted wilt virus; INSV, Impatiens necrotic spot virus; TCSV, Tomato chlorotic spot virus; CMV, Cucumber mosaic virus and BBWV2, Broad bean wilt virus 2; -, not tested. Fig. 2. Detection of Impatiens necrotic spot virus (INSV) in Hoya carnosa stocks by RT-PCR. Samples were collected from symptomatic leaf (Ls), asymptomatic leaf (La), stem (S) and root (R) from each of stocks (lanes 1,2 and 3). M, DNA ladder; N, negative; P, positive control.. RT-PCR (INSV) bp PCR direct sequencing. (National Center for Biotechnology Information, NCBI, USA) BLAST Genbank INSV 97-99%. (INSV) (INSV-Hy1,-Hy2) (Nucleocapsid gene) forward INSV-N (5 CACAACACAAAGCAAACCAAGC 3), reverse J13 (5 CCCGGATCCAGAGCAAT 3) (Cortez, 2001), PCR Expin TM ComboGP (GeneAll Bio., Seoul, Korea), pgem-t Easy vector (Promega, WI, USA). 3

4 386 Research in Plant Disease Vol. 23 No. 4 Fig. 3. Phylogenetic analysis by Maximum likelihood method showing the relationship of Impatiens necrotic spot virus (INSV) isolates from various hosts and countries based on an alignment of complete nucleocapsid (N) gene sequences. The percentage of trees in which the associated texa clustered together is shown next to the branches. The Tomato spotted wilt virus (TSWV), Zucchini lethal chlorosis virus (ZLCV) and Tomato chlorotic spot virus (TCSV) were used as an outgroup. (Genotech., Daejeon, Korea). BLAST Genbank INSV 97-99%. INSV-HDL (GU112504,, ), INSV J (AB109100,, ) 99%. MEGA 7, (outgroup) Orthotospovirus (TSWV), (TCSV), (Zucchini lethal chlorosis virus, ZLCV). Clustal W.,,, (INSV), (GQ336989) (GU112504) (Fig. 3) %,. 6, (TSWV), (INSV) (TCSV) 3 RT-PCR., 3 (INSV), 3. 2 (INSV) (N),. (INSV) Hy-1 Hy-2 BLAST 99%, Genbank (INSV) 97-99%.. (INSV). Conflicts of Interest No potential conflict of interest relevant to this article was reported. Acknowledgement 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.

5 Research in Plant Disease Vol. 23 No References Brisco-McCann, E. I. and Hausbeck, M. K Handbook of Florists Crops Diseases, pp Cortez, I., Saaijer, J., Wongjkaew, K. S., Pereira, A. M., Goldbach, R., Preters, D. and Kormelink, R Identification and characterization of a novel tospovirus species using a new RT-PCR approach. Arch Virol. 146: Crosslin, J. M. and Hamlin, L. L First report of Impatiens necrotic spot virus infecting greenhouse-grown potatoes in Washington state. Plant Dis. 94: Daughtrey, M. L., Jones, R. K., Moyer, J. W., Daub, M. E. and Baker, J. R Tospoviruses strike the greenhouse industry: INSV has become a major pathogen on flower crops. Plant Dis. 81: De Avila, A. C., Haan, P. D., Kitajima, E. W., Kormelink, R., Resende, R. D., Goldbach, R. W. and Peters, D Characterization of a distinct isolate of Tomato spotted wilt virus (TSWV) from Impatiens sp. in the Netherlands. J. Phytopathol. 134: Ding, M., Yin, Y., Fang, Q., Li, S. and Zhang, Z First report of Impatiens necrotic spot virus in Gentiana macrophylla in China. Plant Dis. 95: Horita, H. and Sasaki, J Necrotic spot of lisianthus caused by Impatiens necrotic spot virus in Hokkaido. Jpn. J. Phytopathol. 71: 83. (Abstract, In Japanese) Kuo, Y. W., Gilbertson, R. L., Turini, T., Brennan, E. B., Smith, R. F. and Koike, S. T Characterization and epidemiology of outbreaks of Impatiens necrotic spot virus on lettuce in coastal California. Plant Dis. 98: Law, M. D. and Moyer, J. W A tomato spotted wilt-like virus with a serologically distinct N protein. J. Gen. Virol. 71: Shin, Y.-G List of plant quarantine viruses in Korea newly revised in Res. Plant Dis. 19: Roggero, P., Ciuffo, M., Dellavalle, G., Gutta, P., Gallo, S. and Peters, D Additional ornamental species as hosts of Impatiens necrotic spot tospovirus in Italy. Plant Dis. 83: 967. Sakurai, T., Inoue, T. and Tsuda, S Distinct efficiencies of Impatiens necrotic spot virus transmission by five thrips vector species (Thysanoptera: Thripidae) of tospoviruses in Japan. Appl. Entomol. Zool. (Jpn.) 39: Simko, I., Richardson, C. and Wintermantel, W Variation within Lactuca spp. for resistance to Impatiens necrotic spot virus. Plant Dis. doi: /PDIS RE. (In press) Vicchi, V., Fini, P. and Cardoni, M Presence of Impatiens necrotic spot tospovirus (INSV) in horticultural crops in Emilia-Romagna. Informatore Fitopatologico 4: Yang, D. S., Pennisi, S. V., Son, K. C. and Kays, S. J Screening indoor plants for volatile organic pollutant removal efficiency. HortScience 44:

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