J. Y. Yoon

First Report of Tomato spotted wilt virus in Brugmansia suaveolens in Korea

Brugmansia suaveolens, also known as angels trumpet, is a semi-woody shrub or a small tree. Because flowers of B. suaveolens are remarkably beautiful and sweetly fragrant, B. suaveolens is grown as ornamentals outdoors year-round in the tropics and subtropics, and as potted plants in temperate regions (1). In February 2013, virus-like symptoms including mosaic symptoms followed by distortion of leaves were observed in a potted B. suaveolens in a nursery in Chung-Nam Province, Korea. Symptomatic leaves were analyzed for the presence of several ornamental viruses including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato bush stunt virus (TBSV), and Tomato spotted wilt virus (TSWV) by immune-strip diagnostic kits that were developed by our laboratory. Positive controls and extract from healthy leaves of B. suaveolens as a negative control were included in each immune-strip assay. TSWV was detected serologically from the naturally infected B. suaveolens, but CMV, TBSV, and TMV were not detected from the B. suaveolens. The presence of TSWV (named TSWV-AT1) was confirmed by commercially available double-antibody sandwich (DAS)-ELISA kits (Agdia, Elkhart, IN). TSWV-AT1 was mechanically transmitted from the ELISA-positive B. suaveolens to Capsicum annuum and Nicotiana glutinosa, respectively. Inoculated C. annuum showed chlorotic rings in the inoculated leaves and inoculated N. glutinosa produced mosaic and systemic necrosis in the inoculated leaves after 7 days inoculation, respectively, which were consistent with symptoms caused by TSWV (2). To confirm further TSWV-AT1 infection, reverse transcription (RT)-PCR was performed using the One-Step RT-PCR (Invitrogen, Carlsbad, CA) with TSWV-specific primers, TSWV-NCP-For and TSWV-NCP-Rev (3), designed to amplify a 777-bp cDNA of the nucleocapsid protein (NCP) gene. Total RNAs from naturally infected B. suaveolens, symptomatic C. annuum, and N. glutinosa were extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Total RNAs obtained from a Korean isolate of TSWV (Accession No. JF730744) and healthy B. suaveolens were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic B. suaveolens, C. annuum, and N. glutinosa but not from healthy leaves of B. suaveolens. The amplified RT-PCR product from TSWV-AT1 was directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the TSWV-AT1 NCP sequence (AB910533) with NCP sequences of other TSWV isolates using MEGA5 software (4) revealed 99.0% aa identity with an Korean TSWV isolate (AEB33895) originating from tomato. These results provide additional confirmation of TSWV-AT1 infection. It is known that high-value ornamentals may act also as reservoirs for TSWV that can infect other ornamentals and cultivated crops, because TSWV has a very broad host range (2). Elaborate inspections for TSWV and other viruses are necessary for production of healthy B. suaveolens, since the popularity and economic importance of this ornamental plant is increasing. To our knowledge, this is the first report of TSWV in B. suaveolens in Korea. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) B.-N. Chung et al. Plant Pathol. J. 28:87, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

Complete Sequence Analysis of a Korean Isolate of Chinese Yam Necrotic Mosaic Virus and Generation of the Virus Specific Primers for Molecular Detection

consumed as a vegetable and as a source of herbal medicines in East Asia. In Korea, two virus species including Broad bean wilt virus 2 (BBWV2), and Chinese yam necrotic mosaic virus (CYNMV) have been reported to infect Chinese yam (Kang et al., 2003; Kwon et al., 2016). Infection of Chinese yam with CYNMV was the first report in Japan in 1978 (Fukumoto and Tochihara, 1978). Since then, CYNMV has been considered of important virus in Chinese yam because of serious yield loss (Kondo, 2001a; Kondo and Fujita, 2012; Kondo et al., 2007). While BBWV2 infection of Chinese yam was identified very recently in Korea, the infection of Chinese yam with CYNMV was reported in 2003 in Korea (Kang et al., 2003). CYNMV, a member of the genus Macluravirus in the Family Potyviridae, is a flexuous, filamentous virus with 660 nm in length (Fukumoto and Tochihara, 1978). CYNMV is easily transxadmitted by aphids in a nonpersistent manner but its host range is restricted to Dioscorea spp. (Fukumoto and Tochihara, 1978; Kondo et al., 2015). The size of the complete genome of CYNMV is approximately 8,230 nucleotides (nt). Currently, only two complete genome sequences of the CYNMV isolates (one from Japan and another from China) are available in the GenBank database. A previous study reported the 3’-proximal partial sequences of a CYNMV Korean isolate but no complete genome sequence of CYNMV isolated in Korea has been determined yet (Kondo et al., 2003). Therefore, we decided to determine the complete genome sequence of a CYNMV Korean isolate to examine molecular characteristics of the Korean isolate and to generate molecular detection primers specific for CYNMV Korean isolates.

First Report of Cucumber mosaic virus in Catharanthus roseus in Korea

Catharanthus roseus, commonly known as Madagascar rosy periwinkle (also called vinca), is a tropical perennial herb of the family Apocyanaceae. Periwinkle is a bedding plant widely used in Korea because of its drought tolerance, low maintenance, and varied flower colors. In May 2013, virus-like foliar symptoms, including a mosaic with malformation of leaves, were observed on a periwinkle plant in a greenhouse located in Chonbuk Province, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with an immune-strip kit developed by our laboratory. The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-Vin) caused necrotic local lesions on Chenopodium amaranticolor at 5 days-post-inoculation (dpi), while mild to severe mosaic was observed in Capsicum annuum, Cucumis sativus, Cucurbita pepo Cheonggobong, Nicotiana glutinosa, N. tabacumSamsun NN, Physalis angulate, and Solanum lycopersicum Pink-Top 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ~28 nm in diameter, respectively. To verify whether CMV was the causal agent for the disease symptoms observed in naturally infected periwinkle, virus-free periwinkle (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-Vin. At 6 weeks after inoculation, all plants produced systemic mosaic and distortion of leaves, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated periwinkle plants remained symptomless and virus-free. The presence of CMV-Vin in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with a RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CMVCPFor and CMVCPRev (1), which amplified the entire CP gene. RT-PCR products (657 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from a positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-Vin CP sequence (Accession No. AB910598) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-Vin infection. Being perennial, periwinkle plants could serve as a reservoir for CMV to infect other ornamentals and cultivated crops (2). To our knowledge, this is the first report of CMV infection on periwinkle in Korea. References: (1) S. K. Choi et al. Virus Res. 158:271, 2011. (2) P. Palukaitis et al. Adv. Virus. Res. 41:281, 1992.

Identification of rehmannia virus 1, a novel putative member of the genus Closterovirus, from Rehmannia glutinosa

Transcriptome sequencing analysis of a symptomatic Rehmannia glutinosa plant revealed a virome containing two known RNA viruses and one novel virus. In this study, we examined the molecular and biological characteristics of the novel virus. The complete genome of the novel virus is composed of monopartite single-stranded RNA of 15,322 nucleotides with 69% nucleotide sequence identity (with 68% coverage) to tobacco virus 1. Its genome organization is typical of the members of the genus Closterovirus, containing nine putative open reading frames. Molecular and phylogenetic analyses of the genome and encoded protein sequences strongly support that the identified virus is a new species of the genus Closterovirus in the family Closteroviridae. The name rehmannia virus 1 (ReV1) is proposed for this novel virus.

First report of Zucchini yellow mosaic virus in Chayote (Sechium edule) in Korea

Chayote (Sechium edule) native originally to Mesoamerica is an edible plant that belongs to the family Cucurbitaceae. The chayote fruit is mostly consumed as food and the root, stem, seeds, and leaves are edible as well in the world. In recent years, chayote becomes economically important crops in Korea and its cultivation area is gradually increasing. In September 2016, virus-like symptoms including mosaic, chlorotic spots and vein chlorosis on leaves were observed in chayote plants in Iksan, South Korea. The disease Incidence in chayote field was about 70% by visual estimation. To identify a causal virus, 4 symptomatic leaf samples of chayote plants were analyzed by the transmission electron microscopy (TEM) in leaf dip-preparations. Potyvirus-like particles (about 680~730 x 11~13 nm) were observed from all the samples of chayote plants. To confirm the TEM result, the symptomatic leaf samples were further analyzed using the ImmunoStrip® Test Kits (Agdia, Elkhart, U.S.A.) for the presence of potyviruses,...

A Simple and Reliable Molecular Detection Method for Tomato yellow leaf curl virus in Solanum lycopersicum without DNA Extraction

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A Simple and Reliable Molecular Detection Method for Tomato yellow leaf curl virus in Solanum lycopersicum without DNA Extraction

First Report of Cucumber mosaic virus in Saintpaulia ionantha in Korea

African violet (Saintpaulia ionantha) is an ornamental species of the family Gesneriaceae and is characterized by fleshy leaves and colorful flowers. This popular, exotic ornamental, originally from Kenya and Tanzania, is vegetatively produced from cutting and tissue culture (1). In May 2013, virus-like foliar symptoms, including a mosaic with dark green islands and chlorosis surrounding the veins, were observed on an African violet plant in a greenhouse located in Icheon, Korea. Cucumber mosaic virus (CMV) was identified in the symptomatic plant by serological testing for the presence of CMV coat protein (CP) with a commercial immunostrip kit (Agdia, Elkhart, IN). The presence of CMV was confirmed by serological detection with a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia). Sap from the serologically positive sample was mechanically inoculated to test plants using 10 mM phosphate buffer (pH 7.0). The virus (named CMV-AV1) caused necrotic local lesions on Chenopodium amaranticolor at 5 days post-inoculation (dpi), while mild to severe mosaic was observed in Nicotiana glutinosa, N. tabacum Samsun NN, Cucurbita pepo Super-Top, Physalis angulate, and Solanum lycopersicum Unicorn 10 to 14 dpi. Examination of the inoculated plant leaves by DAS-ELISA and electron microscopy (leaf dips) showed positive reactions to CMV and the presence of spherical virions ∼28 nm in diameter, respectively. To verify whether CMV-AV1 is the cause of disease symptoms observed in African violet, virus-free African violet (10 plants) was mechanically inoculated by sap from local lesions on C. amaranticolor inoculated with CMV-AV1. At 8 weeks after inoculation, all plants produced systemic mosaic and chlorosis surrounding veins, resulting in strong DAS-ELISA reactions for CMV, whereas mock-inoculated African violet plants remained symptomless and virus-free. The presence of CMV-AV1 in all naturally infected and mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturers instructions. RT-PCR was carried out with the One-Step RT-PCR Kit (Invitrogen, Carlsbad, CA) using a pair of primers, CPTALL3 and CPTALL5 (2), amplifying the entire CP gene and part of an intergenic region and 3-noncoding region of CMV RNA3. RT-PCR products (960 bp) were obtained from all naturally infected and mechanically inoculated plants as well as from positive control (viral RNAs from virions), but not from healthy tissues. The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen) and sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the CMV-AV1 CP sequence (Accession No. AB842275) with CP sequences of other CMV isolates using MEGA5 software revealed that 91.8 to 99.0% and 71.0 to 73.0% identities to those of CMV subgroup I and subgroup II, respectively. These results provide additional confirmation of CMV-AV1 infection. CMV may pose a major threat for production of African violet since the farming of African violet plants is performed using the vegetative propagation of the African violet leaves in Korea. In particular, mosaic and chlorosis symptoms in African violet cause damage to ornamental quality of African violet. To our knowledge, this is the first report of CMV infection of African violet in the world. References: (1) S. T. Baatvik. Fragm. Flor. Geobot. Suppl. 2:97, 1993. (2) S. K. Choi et al. J. Virol. Methods 83:67, 1999.