• 식물병연구
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• 제23권1호
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• pp.75-81
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• 2017

2016년 5월, 우리나라 복숭아의 주요산지인 경북 영천 지역에서 퇴록, 괴저반점, 엽맥퇴록, 황화와 같은 바이러스 병징과 이상증상을 보이는 복숭아 잎 24점을 채집하였다. 이 시료들의 바이러스 감염 여부를 확인하기 위해 RT-PCR 진단법을 이용하여 진단하였다. 진단 대상은 검역 병원체로 지정된 바이러스 또는 바이로이드를 중심으로, 국내외 연구를 참고하여 국내 발생가능성이 높거나 발생 시 위험도가 높은 종 17종이다. 진단결과, 국내에서 보고된 적이 없는 바이러스 1종(Apricot pseudo-chlorotic leaf spot virus, APCLSV)과 검역 바이로이드 1종(Peach latent mosaic viroid, PLMVd)을 포함하여, 총 7종의 바이러스와 바이로이드가 검출되었다. 바이러스를 동정하기 위해, RT-PCR 산물을 sequencing 하여 확인하였다. 또, 국내 미보고 종인 APCLSV가 검출된 시료를 이용하여 APCLSV의 외피단백질을 암호화하고 있는 염기서열을 증폭하여 결정하였다. 결정된 염기서열은 기 보고된 APCLSV 분리주와 97%의 상동성을 보였다. 이 외피단백질 염기서열을 Trichovirus속 바이러스들과 비교 분석하여, 최종적으로 APCLSV임을 확인하였다. 동정된 APCLSV를 Yeongcheon 분리주로 명명하고 결정된 외피단백질 염기서열은 NCBI GenBank에 등록하였다. APCLSV의 발생 양상을 보면, APCLSV가 검출된 시료들은 모두 Apple chlorotic leaf spot virus (ACLSV)가 복합감염되어 있었으며, APCLSV는 ACLSV와 유전학적 유연관계가 가까운 것으로 알려져 있다. ACLSV의 특성에 비추어 볼 때, APCLSV는 국내 과수 농가에 널리 퍼질 가능성이 높을 것으로 생각된다. 따라서, 추후 국내 농가에 미칠 영향 등에 관련된 연구가 필요하다.

• The Plant Pathology Journal
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• 제27권1호
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• pp.44-52
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• 2011

Field surveys were conducted in 45 stone fruit orchards in seven districts of Isparta Province located in western Mediterranean region of Turkey important for stone fruit production. Leaf samples were collected from 175 trees showing virus-like symptoms. These samples were first tested by ELISA for five different RNA viruses including Apple mosaic ilarvirus (ApMV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot trichovirus (ACLSV). While no ApMV and PPV infection was found, 46, 24 and 16 samples were tested positive for PDV, ACLSV and PNRSV, respectively, in ELISA showing about 45% of symptomatic trees in the region were infected with at least one of these viruses. In addition, it was found that nine sweet cherry trees were mixed infected with two or three of these viruses and PDV with an infection rate of 26.3% was the most widespread virus in symptomatic trees in western Mediterranean region. Thirty samples were selected and tested by a multiplex RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than half of the tested 20 samples were individually or mixed infected with ApMV, ACLSV, PNRSV and PDV. The mRT-PCR results were confirmed by detection of these viruses individually in some of the field samples using RT-PCR with primes specific to each virus. Comparison of ELSA and mRT-PCR results of 30 samples showed that numbers of infected and mixed infected samples as well as infection and mixed infection rates were significantly higher in RT-PCR (20 and 66.7%) than in ELISA (14 and 46.7%). The results confirm that mRT-PCR is more sensitive than ELISA.

• Journal of Plant Biotechnology
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• 제46권3호
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• pp.228-235
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• 2019

사과(Malus pumila)는 국내에서 가장 경제적으로 중요한 과수 중의 하나이다. 하지만 사과 바이러스 감염은 생산량을 감소시키고 수확량 손실과 과일 품질 저하와 같은 심각한 문제를 야기한다. 국내에 감염된 사과 바이러스 및 비로이드 종류는 Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV)와 Apple scar skin viroid (ASSVd) 등이 알려져 있다. 사과는 바이러스나 비로이드에 감염되어 있어도 대체로 이상한 징후가 발견되지 않아 바이러스로 인해 피해가 많았다. 본 연구는 사과 왜성대목 M.9 및 M.26의 무독묘 생산을 위하여 고온처리($37^{\circ}C$, 6주), 화학처리(Ribavirin) 및 생장점 배양하여 바이러스 제거 처리를 하였다. 바이러스 검출에 일반적으로 사용되는 방법은 효소면역 측정법(ELlSA)과 중합효소연쇄반응(RT-PCR)을 이용하였는데, RT-PCR은 ELlSA방법보다 10 ~ 30% 더 민감하였다. 사과 왜성대목 바이러스 검정 결과, 바이러스 제거 효율은 생장점 배양이 가장 높았다. 생장점 배양 후 바이러스 무병묘의 획득율은 30 ~ 40%로 높게 나타났다. 생장점 배양에서 사과 왜성대목 M.9은 ACLSV, ASPV 및 ASGV의 비율이 각각 45%, 60%, 50%로 높았고, 사과 왜성대목 M.26에서는 ACLSV, ASPV 및 ASGV의 감염율은 각각 40%, 55%, 55%였다. 이상의 결과, 사과 왜성대목에서 무독묘를 생산할 수 있는 가장 효과적인 방법은 생장점 배양에 의한 것으로 판단되었다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.54-54
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• 2019

Apple (Malus domestica) is one of the most economically important fruits in Korea. But virus infection has decreased sustainable production of apple and caused the serious problems such as yield loss and poor fruit quality. Virus or viroid infection including Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple mosaic virus (ApMV) and Apple scar skin viroid (ASSVd) has been also reported in Korea. In many cases, apple is infected with virus and viroid with no specific symptoms, the damage caused by the virus are unaware significantly. In our research, we tried to eliminate viruses in the rootstock for the disease-free seedlings of the apple dwarfing rootstock M.9 and M.26. The method of virus elimination was meristem culture, heat($37^{\circ}C$, 6weeks) treatment and chemistry($Ribavirin^{(R)}$) treatment. The analytical methods commonly used for the detection of virus is Enzyme-linked Immuno-Sorbent Assay(ELlSA) and Reverse Transcription-polymerase Chain Reaction(RT-PCR). RT-PCR method was more 30% sensitive than ELISA method. Efficiency of method eliminate virus appeared meristem method > heat treatment > chemistry treatment. The higher acquisition rate of disease-free seedlings is 30~40% on meristem treatment. In meristem treatment, the apple dwarfing rootstock M.9 gained infection ratio of ACLSV, ASPV and ASGV were 45%, 60% and 50% respectively. In the apple dwarfing rootstock M.26, infection ratio of ACLSV, ASPV and ASGV were 40%, 55%, 55%, respectively. Based on our results, it was found that most effective method of disease-free seedlings apple dwarfing rootstocks was by meristem treatment than heat method and chemistry treatment.

• The Plant Pathology Journal
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• 제33권6호
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• pp.608-613
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• 2017

The full-length sequence of a new isolate of Apple chlorotic leaf spot virus (ACLSV) from Korea was divergent, but most closely related to the Japanese isolate A4, at 84% nucleotide identity. The full-length cDNA of the Korean isolate of ACLSV was cloned into a binary vector downstream of the bacteriophage T7 RNA promoter and the Cauliflower mosaic virus 35S promoter. Chenopodium quinoa was successfully infected using in vitro transcripts synthesized using the T7 promoter, detected at 20 days post inoculation (dpi), but did not produce obvious symptoms. Nicotiana occidentalis and C. quinoa were inoculated through agroinfiltration. At 32 dpi the infection rate was evaluated; no C. quinoa plants were infected by agroinfiltration, but infection of N. occidentalis was obtained.

• The Plant Pathology Journal
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• 제32권5호
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• pp.441-451
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• 2016

Deep sequencing has generated 52 contigs derived from five viruses; Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple green crinkle associated virus (AGCaV), and Apricot latent virus (ApLV) were identified from eight apple samples showing small leaves and/or growth retardation. Nucleotide (nt) sequence identity of the assembled contigs was from 68% to 99% compared to the reference sequences of the five respective viral genomes. Sequences of ASPV and ASGV were the most abundantly represented by the 52 contigs assembled. The presence of the five viruses in the samples was confirmed by RT-PCR using specific primers based on the sequences of each assembled contig. All five viruses were detected in three of the samples, whereas all samples had mixed infections with at least two viruses. The most frequently detected virus was ASPV, followed by ASGV, ApLV, ACLSV, and AGCaV which were withal found in mixed infections in the tested samples. AGCaV was identified in assembled contigs ID 1012480 and 93549, which showed 82% and 78% nt sequence identity with ORF1 of AGCaV isolate Aurora-1. ApLV was identified in three assembled contigs, ID 65587, 1802365, and 116777, which showed 77%, 78%, and 76% nt sequence identity respectively with ORF1 of ApLV isolate LA2. Deep sequencing assay was shown to be a valuable and powerful tool for detection and identification of known and unknown virome in infected apple trees, here identifying ApLV and AGCaV in commercial orchards in Korea for the first time.

• Journal of Plant Biotechnology
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• 제49권3호
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• pp.222-229
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• 2022

사과는 국내 과수산업에서 가장 많이 재배되고 있는 과종이다. 하지만 apple mosaic virus (ApMV), apple stem grooving capillovirus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), apple scar skin viroid (ASSVd)와 같은 바이러스 및 바이로이드에 감염되면 과실의 수확량 감소 및 품질 저하를 야기시킨다. 본 연구에서는 국내 사과 농가에서 가장 많이 감염되어 있는 ASGV 바이러스를 제거하기 위한 효율적인 무병화 시스템을 확립하고자 하였다. ASGV에 감염된 폿트묘를 36℃, 38℃, 40℃가 유지되는 항온·항습장치에서 4주간 열처리를 수행하였으며, 신초 생장율과 바이러스 제거율을 조사하였다. 신초 생장률은 36℃ 처리구에서 가장 높았으며 신초의 중간부와 상단부는 바이러스가 제거되었으나 하단부는 바이러스가 제거되지 않았다. 38℃, 40℃ 처리구는 신초의 모든 구간에서 바이러스가 제거되지 않았으며, 40℃ 처리구는 신초의 생장 없이 열처리 3주 후 고사되었다. 36℃ 온도에서 열처리된 폿트묘의 경정을 절취하여 기외에서 접목하였으며 94%의 생존율과 20%의 바이러스 제거율을 보였다. 따라서 열처리 및 경정접목을 통해 무병묘 생산이 가능할 것으로 판단되었다.

• 농업과학연구
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• 제43권1호
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• pp.21-27
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• 2016

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) have been known to induce top working disease causing economical damage in apple. Occurrences of these three viruses in pome fruit trees, including apple, have been reported around the world. The transmission of the three viruses was reported by grafting, and there was no report of transmission through mechanical contact, insect vector, or seed except some herbaceous hosts of ASGV. As RNA extraction methods for fruit trees, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and multiplex RT-PCR techniques have been improved for reliability and stability, and low titer viruses that could not be detected in the past have become detectable. We studied the seed transmission ability of three apple viruses through apple seedling diagnosis using RT-PCR. Nineteen seeds obtained from commercially grown apple were germinated and two of the resulting plants were ASGV positive. Seven clones of the amplified ASGV coat protein (CP) genes of these isolates were sequenced. Overall sequence identities were 99.84% (nucleotide) and 99.76% (amino acid). Presence of a previously unreported single nucleotide and amino acid variation conserved in all of these clones suggests a possible association with seed transmission of these 'S' isolates. A phylogenetic tree constructed using ASGV CP nucleotide sequences showed that isolate S sequences were grouped with Korean, Chinese, Indian isolates from apple and Indian isolates from kiwi.

• The Plant Pathology Journal
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• 제20권2호
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• 식물병연구
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• 제19권4호
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• pp.288-293
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• 2013

국내외적으로 ACLSV, ASGV, ApMV, ASSVd와 같은 바이러스 및 바이로이드 병의 발생으로 사과 과실의 생산량 감소와 기형적인 외형 등 많은 문제점들이 보고되었다. 하지만 사과 바이러스의 감염에 대한 방제 대책은 거의 알려진 바가 없는 실정이다. 따라서 본 논문에서는 사과 신품종인 '단홍', '홍안', '새나라', '썸머드림'을 분양하기에 앞서 바이러스 무병묘를 생산하는 시스템을 확립하고자 하였다. $37^{\circ}C$가 유지되는 항온 항습장치에서 4주간 열처리를 하였으며 기내에서 경정 배양을 하였다. 열처리된 각각의 사과 신품종들은 바이러스 진단 프라이머를 통해 RT-PCR을 수행하여 바이러스 진단을 수행하였다. 결과적으로 '단홍'은 28%의 바이러스 무병묘를 확보할 수 있었으며 '홍안'은 16%, '새나라'와 '썸머드림'은 12%의 확률로 바이러스 무병 사과를 확보할 수 있었다. 본 연구결과는 열처리 및 경정배양을 통해 사과 신품종에서 바이러스 무병묘 생산 시스템 구축이 가능함을 보여주었다.