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

곽해련,김재덕,김미경,서장균,정미남,김정수,이석찬,최홍수 한국식물병리학회 2015 Plant Pathology Journal Vol.31 No.4

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.

Complete Genome Sequences and Evolutionary Analysis of Cucurbit aphid-borne yellows virus Isolates from Melon in Korea

곽해련,이희주,김은아,서장균,김창석,이상규,김정수,최홍수,김미경 한국식물병리학회 2018 Plant Pathology Journal Vol.34 No.6

Complete genome sequences of 22 isolates of Cucurbit aphid-borne yellows virus (CABYV), collected from melon plants showing yellowing symptom in Korea during the years 2013-2014, were determined and compared with previously reported CABYV genome sequences. The complete genomes were found to be 5,680-5,684 nucleotides in length and to encode six open reading frames (ORFs) that are separated into two regions by a non-coding internal region (IR) of 199 nucleotides. Their genomic organization is typical of the genus Polerovirus. Based on phylogenetic analyses of complete nucleotide (nt) sequences, CABYV isolates were divided into four groups: Asian, Mediterranean, Taiwanese, and R groups. The Korean CABYV isolates clustered with the Asian group with > 94% nt sequence identity. In contrast, the Korean CABYV isolates shared 87-89% sequence identities with the Mediterranean group, 88% with the Taiwanese group, 81-84% with the CABYV-R group, and 72% with another polerovirus, M.. Recombination analyses identified 24 recombination events (12 different recombination types) in the analyzed CABYV population. In the Korean CABYV isolates, four recombination types were detected from eight isolates. Two recombination types were detected in the IR and P3-P5 regions, respectively, which have been reported as hotspots for recombination of CABYV. This result suggests that recombination is an important evolutionary force in the genetic diversification of CABYV populations.

국내 분리 토마토반점위조바이러스의 저항성 판별을 위한 생물검정법 개발

곽해련,최현용,홍수빈,허온숙,변희성,최홍수,김미경,Kwak, Hae-Ryun,Choi, Hyeon-Yong,Hong, Su-Bin,Hur, On-Sook,Byun, Hee-Seong,Choi, Hong-Soo,Kim, Mikyeong 한국환경생물학회 2021 환경생물 : 환경생물학회지 Vol.39 No.3

토마토반점위조바이러스(TSWV)는 고추, 토마토 등 경제적으로 중요한 작물에 심각한 피해를 주는 바이러스들 중 한 종이다. TSWV의 넓은 기주범위, 매개충인 총채벌레 방제의 어려움 및 TSWV의 효과적인 치료제가 없기 때문에, 저항성 품종을 사용하는 것이 TSWV를 예방하는 가장 효과적인 수단이 될 수 있다. 본 연구에서는 토마토에서 분리된 TSWV 분리주(SW-TO2)의 유전학적·생물학적 특성을 구명하고, 최근에 국내에서 분리된 구기자, 머위, 당귀 TSWV 분리주와 비교하였다. 순수분리된 SW-TO2는 28종의 지표식물 중 토마토를 포함한 17종에서 원형반점, 모자이크 증상 등 전신감염 증상을 보였다. SW-TO2의 유전자 계통분석 결과 국내에서 분리된 고추, 구기자 TSWV 분리주와 98~99%의 상동성을 보이며 같은 그룹에 속하였다. TSWV 저항성 평가를 위한 생물검정법을 확립하고, 시판되고 있는 고추와 토마토 품종을 대상으로 4종의 TSWV 분리주에 대한 저항성 평가를 검정하였다. TSWV 저항성 평가는 첫째, 접종엽에 괴사반점 증상이 나타나거나 병징이 없는 경우, 둘째, 상엽에 병징이 없는 경우, 셋째, 상엽을 RT-PCR 진단한 결과 음성이 나왔을 경우 등 3가지 조건이다 충족될 때 저항성으로 평가하였다. Tomato spotted wilt virus (TSWV) is one of the most destructive viruses worldwide, which causes severe damage to economically important crops, such as pepper and tomato. In this study, we examined the molecular and biological characterization of a TSWV isolate (SW-TO2) infecting tomato and compared it to the recently reported isolates from boxthorn, butterbur, and angelica plants. The phylogenetic analysis based on the complete genome sequences confirmed that SW-TO2 was clustered with those of isolates from boxthorn and pepper in Korea with the maximum nucleotide identities ranging from 98% to 99%. We developed the bioassay method for screening TSWV resistance and tested some commercial pepper and tomato cultivars for resistance evaluation of four isolates of TSWV. TSWV resistance was evaluated as TSWV resistance when all the following three conditions were satisfied: first, when symptoms of necrotic spots or no symptoms were present in the inoculated leaves; second, when there were no symptoms in the upper leaves; and third, when the upper leaves were negative as a result of RT-PCR diagnosis.

Molecular Characterization and Variation of the Broad bean wilt virus 2 Isolates Based on Analyses of Complete Genome Sequences

곽해련,최홍수,김미경,이예지,서장균,김정수,김국형,차병진 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.4

The full-genome sequences of fourteen isolates of Broad bean wilt virus 2 (BBWV2), collected from broad bean,pea, spinach, bell pepper and paprika plants in Korea during the years 2006−2012, were determined and analyzed comparatively along with fifteen previously reported BBWV2 genome sequences. Sequence analyses showed that RNA-1 and RNA-2 sequences of BBWV2Korean isolates consisted of 5950-5956 and 3568-3604nucleotides, respectively. Full-length genome sequencebased phylogenetic analyses revealed that the BBWV2Korean isolates could be divided into three major groups comprising GS-I (isolates BB2 and RP7) along with isolate IP, GS-II (isolates BB5, P2, P3 and RP3)along with isolate B935, and GS-III including 16 BBWV2Korean isolates. Interestingly, GS-III appears to be newly emerged and predominant in Korea. Recombination analyses identified two recombination events in the analyzed BBWV2 population: one in the RNA-1 of isolate K and another one in the RNA-2 of isolate XJ14-3. However, no recombination events were detected in the other 21 Korean isolates. On the other hand, out of 29 BBWV2 isolates, 16 isolates were found to be reassortants,of which each RNA segment (i.e. RNA1 and RNA2) was originated from different parental isolates. Our findings suggested that reassortment rather than recombination is a major evolutionary force in the genetic diversification of BBWV population in Korea.

Characterization of Melon necrotic spot virus Occurring on Watermelon in Korea

곽해련,김정수,조점덕,이중환,김태성,김미경,최홍수 한국식물병리학회 2015 Plant Pathology Journal Vol.31 No.4

Melon necrotic spot virus (MNSV) was recently identified on watermelon (Citrullus vulgaris) in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30‒65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSVSW) obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10‒40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28‒30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were 1 μl/ml and a 1:8,000‒1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb) as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

Genetic Diversity of Sweet potato feathery mottle virus from Sweet Potatoes inKorea

곽해련,김미경,Mi-Nam Jung,Su-Heon Lee,Sug-Ju Ko,최홍수,Jin-Woo Park,김국형 한국식물병리학회 2007 Plant Pathology Journal Vol.23 No.1

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 intergroups 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.

Virus Disease Incidences of Sweet Potatoes in Korea

곽해련,이수헌,김미경,박진우,김국형,Mi-Nam Chung,Hong-Soo Choi 한국식물병리학회 2006 Plant Pathology Journal Vol.22 No.3

In 2003, a survey of sweet potato virus disease was carried out in seed boxes as well as in various sweet potato fields. Virus infection rate was 5~100% and 100% at seed boxes and fields, respectively. No relationship of the disease incidence and severity was observed between sweet potato cultivating areas and cultivars. A total of 179 samples were collected and analyzed based on serological, electron microscopic and molecular properties. Field-grown sweet potatoes were examined to inspect 8 different viruses using NCM-ELISA, resulting that 30% of sweet potato was infected by one virus, whereas 70% was by more than 2 viruses. However, RTPCR using primers selected for seven viruses, such as Sweet potato feathery mottle virus (SPFMV) revealed that of one-hundred seventy-nine tested; 71 of SPFMV, 29 of SPGV, 19 of SPFMV+SPGV, 1 of SPFMV+ SwPLV, 1 of SPFMV+SPLCV, 2 of SPFMV+SPGV+ SwPLV, 6 of SPFMV+SPGV+SPLCV, 2 of SPFMV+ SPGV+SwPLV+SPLCV and 48 of unknown viruses were identified from the field samples. In root, viral diseases were severer in Yeoju than in Mokpo Experiment Station and infection rate was much different depending on sweet potato cultivars.

Genetic Compositions of Broad bean wilt virus 2 Infecting Red Pepper in Korea

곽해련,최홍수,김미경,남문,김정수,김국형,차병진 한국식물병리학회 2013 Plant Pathology Journal Vol.29 No.3

The incidence of Broad bean wilt virus 2 (BBWV2) on red pepper was investigated using the samples obtained from 24 areas of 8 provinces in Korea. Two hundred and five samples (79%) out of 260 collected samples were found to be infected with BBWV2. While the single infection rate of BBWV2 was 21.5%, the coinfection rate of BBWV2 with Cucumber mosaic virus,Pepper mottle virus, Pepper mild mottle virus and/or Potato virus Y was 78.5%. To characterize the genetic diversity of BBWV2 Korean isolates, 7 isolates were fully sequenced and analyzed. Phylogenetic analyses revealed that BBWV2 isolates could be divided largely into two groups as Group I and Group II. Based on the partial sequence analyses, 153 selected BBWV2 isolates were subgrouped into GS-I (21.6%), GS-II (3.9%) and GS-III (56.9%). BBWV2 GS-III, which was predominant in Korea, appears to be a new combination between Group I RNA-1 and Group II RNA-2. Viral disease incidence of BBWV2 on red pepper was under 2%before 2004. However, the incidence was increased abruptly to 41.3% in 2005, 58.2% in 2006 and 79% in 2007. These rapid increases might be related with the emergence of new combinations between BBWV2 groups

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

곽해련,김미경,신준철,이예지,서장균,이형은,정미남,김선형,최홍수 한국식물병리학회 2014 Plant Pathology Journal Vol.30 No.4

Sweet potato is grown extensively from tropical to temperateregions and is an important food crop worldwide. In this study, we established detection methodsfor 17 major sweet potato viruses using single and multiplexRT-PCR assays. To investigate the current incidenceof viral diseases, we collected 154 samples of varioussweet potato cultivars showing virus-like symptomsfrom 40 fields in 10 Korean regions, and analyzed themby RT-PCR using specific primers for each of the 17viruses. Of the 17 possible viruses, we detected eight inour samples. Sweet potato feathery mottle virus (SPFMV)and sweet potato virus C (SPVC) were most commonlydetected, infecting approximately 87% and 85% ofsamples, respectively. Furthermore, Sweet potato symptomlessvirus 1 (SPSMV-1), Sweet potato virus G (SPVG),Sweet potato leaf curl virus (SPLCV), Sweet potato virus2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV),and Sweet potato latent virus (SPLV) were detectedin 67%, 58%, 47%, 41%, 31%, and 20% of samples,respectively. This study presents the first documentedoccurrence of four viruses (SPVC, SPV2, SPCFV, andSPSMV-1) in Korea. Based on the results of our survey,we developed multiplex RT-PCR assays for simple andsimultaneous detection of the eight sweet potato viruseswe recorded.

당귀에서 발생한 토마토반점위조바이러스의 감염 첫 보고

곽해련(Hae-Ryun Kwak),홍수빈(Su-Bin Hong),최현용(Hyeon-Yong Choi),박고수(Gosoo Park),허온숙(On-Sook Hur),변희성(Hee-Seong Byun),최홍수(Hong-Soo Choi),김미경(Mikyeong Kim) 한국식물병리학회 2021 식물병연구 Vol.27 No.2

2019년 6월 충남 논산의 당귀 재배 농가에서 원형반점, 괴사 반점, 황화, 모자이크 등의 증상을 보이는 당귀잎을 채집하였 고, 이들 시료에 대한 바이러스 감염 여부를 확인하기 위하여 전자현미경 검경과 감염우려가 있는 바이러스 3종(cucumber mosaic virus, broad bean wilt virus 2, tomato spotted wilt virus [TSWV])에 대해 reverse transcription polymerase chain reaction 진단을 수행한 결과 TSWV에 대해 양성반응을 보였다. 당귀 TSWV의 생물학적 특성을 구명하기 위해 순수분리하여 5 과 28종의 지표식물과 일당귀에 즙액접종하여 기주범위와 병 원성을 확인하였다. TSWV의 3 segment (L, M, S)의 전체 염기서 열을 결정하고 기존에 보고된 TSWV 분리주와 계통분석을 한 결과, 당귀 TSWV 분리주(NS-AG-28)는 논산지역의 머위 분리주 (TSWV-NS-BB20)와 가장 유사한 것으로 나타났다. TSWV는 넓 은기주범위를가지고있고특히고추,토마토등가지과작물 에 큰 피해를 주고 있기 때문에, 당귀가 TSWV의 중간기주로서 작용하지 않도록 주의하고, 당귀의 바이러스병 피해를 예방하 기 위하여 지속적인 모니터링이 요구된다. In June 2019, Angelica acutiloba plants showing virus-like symptoms such as chlorotic local lesion and mosaic on the leaves were found in a greenhouse in Nonsan, South Korea. To identify the causal virus, we collected 6 symptomatic A. acutiloba leaf samples and performed reverse transcription polymerase chain reaction (RT-PCR) analysis using specific detection primers for three reported viruses including tomato spotted wilt virus (TSWV). RT-PCR results showed that five symptomatic samples were positive for TSWV. Mechanical sap inoculation of one of the collected TSWV isolate (TSWV-NS-AG28) induced yellowing, chlorosis and mosaic symptoms in A. acutiloba and necrotic local lesions and mosaic in Solanaceae species. Phylogenetic analysis based on the complete genome sequences showed that TSWV-NS-AG28 had a maximum nucleotide identity with TSWV- NS-BB20 isolated from butterbur in Nonsan, South Korea. To our knowledge, this is the first report of TSWV infection in A. acutiloba.