Hae-Ryun Kwak

Phylogenetic analysis and inflow route of Tomato yellow leaf curl virus (TYLCV) and Bemisia tabaci in Korea

Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus of the family Geminiviridae, members of which are characterized by closed circular single-stranded DNA genomes of 2.7-2.8 kb in length, and include viruses transmitted by the Bemisia tabaci whitefly. No reports of TYLCV in Korea are available prior to 2008, after which TYLCV spread rapidly to most regions of the southern Korean peninsula (Gyeongsang-Do, Jeolla-Do and Jeju-Do). Fifty full sequences of TYLCV were analyzed in this study, and the AC1, AV1, IR, and full sequences were analyzed via the muscle program and bayesian analysis. Phylogenetic analysis demonstrated that the Korea TYLCVs were divided into two subgroups. The TYLCV Korea 1 group (Masan) originated from TYLCV Japan (Miyazaki) and the TYLCV Korea 2 group (Jeju/Jeonju) from TYLCV Japan (Tosa/Haruno). A B. tabaci phylogenetic tree was constructed with 16S rRNA and mitochondria cytochrome oxidase I (MtCOI) sequences using the muscle program and MEGA 4.0 in the neighbor-joining algorithm. The sequence data of 16S rRNA revealed that Korea B. tabaci was closely aligned to B. tabaci isolated in Iran and Nigeria. The Q type of B. tabaci, which was originally identified as a viruliferous insect in 2008, was initially isolated in Korea as a non-viruliferous insect in 2005. Therefore, we suggest that two TYLCV Japan isolates were introduced to Korea via different routes, and then transmitted by native B. tabaci.

Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates

Tomato spotted wilt virus (TSWV) infects numerous host plants and has three genome segments, called L, M and S. Here, we report the complete genome sequences of three Korean TSWV isolates (TSWV-1 to -3) infecting tomato and pepper plants. Although the nucleotide sequence of TSWV-1 genome isolated from tomato is very different from those of TSWV-2 and TSWV-3 isolated from pepper, the deduced amino acid sequences of the five TSWV genes are highly conserved among all three TSWV isolates. In phylogenetic analysis, deduced RdRp protein sequences of TSWV-2 and TSWV-3 were clustered together with two previously reported isolates from Japan and Korea, while TSWV-1 grouped together with a Hawaiian isolate. A phylogenetic tree based on N protein sequences, however, revealed four distinct groups of TSWV isolates, and all three Korean isolates belonged to group II, together with many other isolates, mostly from Europe and Asia. Interestingly, most American isolates grouped together as group I. Together, these results suggested that these newly identified TSWV isolates might have originated from an Asian ancestor and undergone divergence upon infecting different host plants.

2007?2011 Characteristics of Plant Virus Infections on Crop Samples Submitted from Agricultural Places

National Institute of Horticultural and Herbal Science, Horticultural and Herbal Environmental Division,Suwon 441-707, Korea(Received on September 11, 2012; Revised on October 11, 2012; Accepted on October 12, 2012)The total number of requests and associated specimens for the diagnosis of virus infection were 573 and 2,992,respectively, on crops from agricultural places of farmers, Agricultural extension services and so forth for 5years from 2007. The total number of virus tests was 13,325. The number of species of viruses infected on thesubmitted crops was 21 in 2007, 15 in 2008, 23 in 2009, 21 in 2010 and 17 in 2011. The newly recorded viruseswere Tobacco leaf curl virus (TbLCV) in 2007, Tomato yellow leaf curl virus (TYLCV) in 2008, Impatiencenecrotic spot virus (INSV) and Radish mosaic virus (RaMV) in 2009, and Beet western yellows virus (BWYV) in2010. Forty virus species including Alfalfa mosaic virus were detected over 5 years. The ten most frequentlydetected virus species were Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato leaf curlvirus (TYLCV), Cucumber green mottle mosaic virus (CGMMV), Broad bean wilt virus 2 (BBWV2), Zucchiniyellow mosaic virus (ZYMV), Melon necrotic spot virus (MNSV), Pepper mild mottle virus (PMMoV),Watermelon mosaic virus (WMV) and Pepper mottle virus (PepMoV). The types of crops submitted fromagricultural places were 51 in total and the ten most frequently submitted crops were red pepper, tomato,paprika, watermelon, melon, rice, cucumber, corn, radish and gourd. The total request rate for the top 10crops and top 20 crops was 81.6% and 94.2%, respectively. Eight pepper infecting virus species includedCMV, and the average infection rate was 24.6% for CMV, 18.9% for PMMoV and 14.7% for TSWV. Sevenkinds of double infection were detected in pepper including BBWV2+CMV at 14.7% on average, and fourtypes of triple infection including BBWV2+CMV+PepMoV at 0.9% on average. Six virus species detected ontomato including TYLCV, and the average infection rate was 50.6% for TYLCV, 14.5% for TSWV and10.9% for Tobacco leaf curl virus (TbLCV). The mixed infection of CMV+TSWV on tomato was 3.9% onaverage and of Tomato mosaic virus (ToMV)+TYLCV was 0.4% on average. Five viruses detected onwatermelon included MNSV and the average infection rate was 37.0% for MNSV, 20.4% for CGMMV,18.1% for ZYMV and 17.8% for WMV. The mixed infection rate on watermelon was CMV+MNSV andWMV+ZYMV having an average infection rate of 0.7% and 5.0%, respectively. The average infection rateson melon were 77.6% for MNSV, 5.6% for CMV and 3.3% for WMV. Mixed infections of CMV+MNSVoccurred on melon with an average infection rate of 13.5%. Keywords : BBWV2, BWVY, CGMMV, CMV, MNSV, Plant virus, TYLCV, ZYMV

The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

Molecular Genetic Analysis of Cucumber mosaic virus Populations Infecting Pepper Suggests Unique Patterns of Evolution in Korea

Studying genetic structure and diversity of viruses is important to understand the evolutionary mechanisms that generate and maintain variations in viral populations. Cucumber mosaic virus (CMV) is endemic in most pepper fields in Korea. Currently, no effective methods for control of CMV are available due to many environmental and biological factors such as the extensive evolutionary capacity of CMV. Thus, analyzing the genetic structure of CMV populations may facilitate the development of strategies for the control of CMV. In this study, 252 pepper (Capsicum annuum) samples showing virus symptoms were collected by field surveys performed throughout Korea in 2007. Reverse-transcription polymerase chain reaction analyses revealed that, in total, 165 collected samples were infected with CMV. Forty-five CMV isolates were randomly selected within each regional subpopulation and analyzed by full-genome sequencing. Analyses of genetic diversity showed that the 2b gene of CMV is under weaker purifying selection than the other genes. Based on the phylogenetic analysis of RNA1, the CMV isolates from pepper were divided into three clusters in subgroup I. Our full-genome sequence-based molecular analyses of the CMV Korean population suggest that the subpopulations of CMV have been geographically localized in pepper fields in Korea.

Genetic Diversity of Sweet potato feathery mottle virus from Sweet Potatoes in Korea

Sweet potato feathery mottle virus(SPFMV) is one of the most prevalent viruses infecting sweet potatoes and occurs widely in sweet potato cultivating areas in Korea. To assess their genetic variation, a total of 28 samples infected with SPFMV were subjected to restriction fragment length polymorphism(RFLP) analysis using DNAs amplified by RT-PCR with specific primer sets corresponding to the coat protein(CP) region of the virus. The similarity matrix by UPGMA procedure indicated that 28 samples infected with SPFMV were classified into three groups based on the number and size of DNA fragments by digestion of CP-encoding regions with 7 enzymes including SalI, AluI, EcoRI, HindIII, FokI, Sau3AI, and DraI bands. Four primer combinations out of 5 designed sets were able to differentiate SPFMV and sweet potato virus G infection, suggesting that these specific primers could be used to differentiate inter-groups of SPFMV. Sequence analysis of the CP genes of 17 SPFMV samples were 97-99% and 91-93% identical at the intra-group and inter-groups of SPFMV, respectively. The N-terminal region of the CP is highly variable and examination of the multiple alignments of amino acid sequences revealed two residues(residues 31 and 32) that were consistently different between SPFMV-O and SPFMV-RC.

Molecular Characterization of Five Potyviruses Infecting Korean Sweet Potatoes Based on Analyses of Complete Genome Sequences

Sweet potatoes (Ipomea batatas L.) are grown extensively, in tropical and temperate regions, and are important food crops worldwide. In Korea, potyviruses, including Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), Sweet potato virus 2 (SPV2), and Sweet potato latent virus (SPLV), have been detected in sweet potato fields at a high (~95%) incidence. In the present work, complete genome sequences of 18 isolates, representing the five potyviruses mentioned above, were compared with previously reported genome sequences. The complete genomes consisted of 10,081 to 10,830 nucleotides, excluding the poly-A tails. Their genomic organizations were typical of the Potyvirus genus, including one target open reading frame coding for a putative polyprotein. Based on phylogenetic analyses and sequence comparisons, the Korean SPFMV isolates belonged to the strains RC and O with >98% nucleotide sequence identity. Korean SPVC isolates had 99% identity to the Japanese isolate SPVC-Bungo and 70% identity to the SPFMV isolates. The Korean SPVG isolates showed 99% identity to the three previously reported SPVG isolates. Korean SPV2 isolates had 97% identity to the SPV2 GWB-2 isolate from the USA. Korean SPLV isolates had a relatively low (88%) nucleotide sequence identity with the Taiwanese SPLV-TW isolates, and they were phylogenetically distantly related to SPFMV isolates. Recombination analysis revealed that possible recombination events occurred in the P1, HC-Pro and NIa-NIb regions of SPFMV and SPLV isolates and these regions were identified as hotspots for recombination in the sweet potato potyviruses.

Phylogenetic lineage of Tobacco leaf curl virus in Korea and estimation of recombination events implicated in their sequence variation

New strains of Tobacco leaf curl virus (TbLCV) were isolated from tomato plants in four different local communities of Korea, and hence were designated TbLCV-Kr. Phylogenetic analysis of the sequences of the whole genome and of individual ORFs of these viruses indicated that they are closely related to the Tobacco leaf curl Japan virus (TbLCJV) cluster, which includes Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and TbLCJV isolates. Four putative recombination events were recognized within these virus sequences, suggesting that the sequence variations observed in these viruses may be attributable to intraspecific and interspecific recombination events involving some TbLCV-Kr isolates, Papaya leaf curl virus (PaLCV), and a local isolate of Tomato yellow leaf curl virus (TYLCV).

Characteristics of Cucumber mosaic virus isolated from Zea mays in Korea

A virus causing mottle and stunt symptom on Zea mays was observed around Ulleng-do, Korea and identified as Cucumber mosaic virus (CMV-ZM) based upon biological, serological, and molecular characteristics. In host range studies, the CMV-ZM isolate produced local lesions on Datura stramonium, Vigna unguiculata, Cucurbita moschata, Chenopodium amaranticolor, Ch. quinoa, whereas this isolate produced systemic mosaic on Nicotiana tabacum cv. ‘Xanthi-nc’, Capsicum annuum, Solanum lycopersicum, Solanum melongena, Cucurbita pepo, and Z. mays. In addition, chlorotic local rings on inoculated leaves along with severe mosaic, malformation, and fern leaf symptoms on upper systemic leaves were shown in N. glutinosa plants. Complete nucleotide sequences of each genomic RNA segment was determined and compared to those of the other CMV strains. Comparison of the nucleotide sequence of 1a open reading frame (ORF) revealed approximately 89.2– 92.4% sequence identity with each CMV subgroup IA and IB strain, while showing only 78% sequence identity with CMV subgroup II. Nucleotide sequence analysis of RNA2 ORFs revealed 85.3–97.6% sequence identity with subgroup I. In ORFs of RNA3, levels of nucleotide sequence identities were higher than 92– 99.2% with CMV subgroup I and lower than 82% with CMV isolates of subgroup II. These results suggest that CMV-ZM isolate is more closely related to subgroup I than subgroup II and therefore, CMV-ZM isolate might be classified into as CMV subgroup I based on biological and molecular analysis.

Isolation and Characterization of Pepper mottle virus Infecting Tomato in Korea

A peculiar virus-like disease of tomato showing yellow mosaic and necrotic spots on leaves and necrosis on veins, petioles and stems was observed at the Tomato Experimental Station (TES), Buyeo, Chungcheongnamdo, Korea. The disease incidence at TES fields ranged from 21 to 35% infecting different tomato cultivars. For this reason, to identify the virus infecting tomato and to characterize the virus based on biology, serology, cytology and at molecular level. Here, leaf samples were randomly collected from different infected tomato cultivars at TES fields and greenhouses and tested by ELISA using Pepper mottle virus (PePMoV) and Tomato mosaic virus (ToMV) antisera. Infected saps were mechanically inoculated in different host plants to test for pathogenicity, symptomatology and host ranges. Infected tissues and ultrathin sections were examined by electron microscopy. Finally, putative coat protein and 3`-untranslated region (CP/3`-UTR) fragment was amplified and cloned for sequence determination and analyzed its genetic relationship to existing PepMoV and PVY sequences at the Genbank. Results showed 69% of the samples were positive with PepMoV, 13% with ToMV and 19 % were doubly infected with PepMoV and ToMV. Symptoms greatly varied from different host plants inoculated with tomato leaf sap infected with PepMoV alone and discussed in detailed in this paper. Electron microscopy from infected tissues showed filamentous particles of 720-750nm in length, a typical morphology and size of PepMoV. In addition, cylindrical inclusion bodies, pinwheels, scrolls and laminates with masses of fibrillar inclusions were also found in ultrathin sections. Alignment of the sequences of the CP/3`-UTR revealed >96% sequence identity with PepMoV and only