First Report of Tomato spotted wilt virus in Hoya carnosa in Korea

Abstract

Tomato spotted wilt virus (TSWV) is the type member of the Orthotospovirus genus, in the family Tospoviridae. TSWV has a wide host range that includes ornamentals and vegetable crops. It causes serious disease of many economically important plants over 35 plant families. Orthotosovirus including TSWV and Impatiens necrotic spot virus (INSV) have been identified from Hoya spp. (Baker et al. 2015; Hausbeck et al. 1992; Melzer et al. 2014). In 2016, virus-like symptoms including chlorosis, necrosis, and ringspots were observed on leaves of Hoya carnosa from a commercial greenhouse in Gyeonggi, South Korea. The disease incidence based on symptom observation was 10 to 50%. The symptomatic leaf samples reacted positively using a commercially immunostrip with TSWV (from Agdia). For further confirmation of TSWV and other viruses, total RNA was extracted from leaves of two H. carnosa plants using the total RNA extraction kit (Plant RNA Prep Kit, Biocubesystem, Korea) and tested in RT-PCR using specific primers of TSWV (TSWV-6F 5′-GAGATTCTCAGAATTCCCAGT-3′ and TSWV-6R 5′-AGAGCAATCGTGTCAATTTTATTC-3′, expected amplicon size 459 bp), INSV (INSV-1F 5′-ATCAATAGTAGCATTAAACAT-3′ and INSV-1R 5′-GACTCAATCTGATTCCTTAGA-3′, expected amplicon size 800 bp), and Tomato chlorotic spot virus (TCSV) (TCSV-F 5′-TCCCTTGGTTTCATTGACCAAACGC-3′ and TCSV-R 5′-ACATGACACTTGCAAATGAGACTCC-3′, expected amplicon size 590 bp). A 500-bp product of the expected amplicon size from the TSWV CP gene was obtained from these plants, whereas no amplification was obtained with INSV and TCSV. Complete nucleoprotein (N) gene of TSWV was amplified by RT-PCR using primers TSWV N gene-F 5′-CAGCTGCTTTTAAGCAAGTTCTG-3′ and J13-R 5′-CCCGGATCCAGAGCAAT-3′. The product was purified from the agarose gel using nucleic acid purification columns (GeneAll Expin Combo GP, GeneAll Biotechnology, Korea) and cloned into the pGEM-T Easy vector (Promega, Madison, WI). Three clones in each sample were sequenced in both directions by a commercial sequencing service (Geno Tech Corp., Daejeon, Korea). The amplified N gene of TSWV sequences contained 777 nucleotides (MG257896 and MG257897) and BLAST analysis of the consensus sequence showed that two isolates (TSWV-Hy1 and -Hy2) had 97 to 99% nucleotide sequence identity with TSWV-HJ (LC273307, Humulus japoncus, Korea), TSWV-Peperomia (LC167301, Baby Rubber plant, Korea), and 97% identity with a TSWV isolate MAF05 (KC494493, Hoya arnottiana, New Zealand). To our knowledge, this is the first report of TSWV on H. carnosa from Korea. Recently, severe damage in ornamental plants has been caused by TSWV after it was first detected on vegetables in South Korea in 2003 (Choi et al. 2004; Yoon et al. 2017). To prevent the spread of the virus disease, rapid detection and elimination of sources of virus is important.