• 한국식물병리학회지
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• 제14권6호
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• pp.636-641
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• 1998

Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.243-250
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• 2005

한국 분리주 감자잎말림바이러스 (PLRV)로부터 627bp 크기의 외피단백질 유전자의 ORF (AF296280)를 분리하여 장려품종 감자인 '수미'를 형질전환 하였다. 17계통의 형질전환체를 선발하여 온실과 포장에서 5세대를 증식하면서 PLRV에 대한 저항성이 큰 5계통을 선발 하였다. 도입된 유전자들의 유전적인 안정성을 확인하기위해 PCR, Southern, 그리고 northern blot 분석을 수행하였다. 또한 클론으로 증식된 형질전환 감자들의 특성과 저항성도 검정하였다. 형질전환된 감자들에서 PLRV의 외피단백질 유전자는 안전적으로 발현되며 저항성을 유지하였으며, 유사도가 비교적 낮은 감자 바이러스 Y (PVY)에는 저항성을 나타내지 않았다. 따라서 이러한 저항성은 도입된 유전자와 유사성도가 높은 바이러스에 저항성을 나타내는 homology dependent gene silencing으로 판단되었다. 유망계통 형질전환 감자 계통들의 포장평가를 통해 PLRV에 대한 저항성을 제외한 주요한 농업적 특성과 식물학적 특성은 형질전환 되지 않은 감자와 큰 차이를 보이지 않았다.

• The Plant Pathology Journal
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• 제33권5호
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• pp.522-527
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• 2017

We determined the effects of atmospheric temperature ($10-30{\pm}2^{\circ}C$ in $5^{\circ}C$ increments) and carbon dioxide ($CO_2$) levels ($400{\pm}50ppm$, $540{\pm}50ppm$, and $940{\pm}50ppm$) on the infection of Solanum tuberosum cv. Chubaek by Potato leafroll virus (PLRV). Below $CO_2$ levels of $400{\pm}50ppm$, the PLRV infection rate and RNA content in plant tissues increased as the temperature increased to $20{\pm}2^{\circ}C$, but declined at higher temperatures. At high $CO_2$ levels ($940{\pm}50ppm$), more plants were infected by PLRV at $30{\pm}2^{\circ}C$ than at 20 or $25{\pm}2^{\circ}C$, whereas PLRV RNA content was unchanged in the $20-30{\pm}2^{\circ}C$ temperature range. The effects of atmospheric $CO_2$ concentration on the acquisition of PLRV by Myzus persicae and accumulation of PLRV RNA in plant tissues were investigated using a growth chamber at $20{\pm}2^{\circ}C$. The M. persicae PLRV RNA content slightly increased at elevated $CO_2$ levels ($940{\pm}50ppm$), but this increase was not statistically significant. Transmission rates of PLRV by Physalis floridana increased as $CO_2$ concentration increased. More PLRV RNA accumulated in potato plants maintained at 540 or $940{\pm}50ppm$ $CO_2$, than in plants maintained at $400{\pm}50ppm$. This is the first evidence of greater PLRV RNA accumulation and larger numbers of S. tuberosum plants infected by PLRV under conditions of combined high $CO_2$ levels ($940{\pm}50ppm$) and high temperature ($30{\pm}2^{\circ}C$).

• 한국응용곤충학회지
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• 제41권4호
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• pp.247-253
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• 2002

본 시험에서는 ´77년에서 2001년까지 대관령지역 비래진딧물의 발생을 조사한 성적을 바탕으로 진딧물의 비래변동을 분석하였고, 2000-2001년간의 비래진딧물의 PLRV 보독률을 구명하여 ´89-´91의 보독률과 대조하였다. 각 연도별 자료는 5년 단위로 묶어서 평균 비래량을 계산하였고, 2000년까지 비래량을 비교하였다. 최초 ´76-´80 시기의 비래량은 평균 575.2마리였으나, ´91-´95에는 2959.4마리, ´96-2000 시기에는 2281.6마리로 비래량이 증가하는 추세를 보였다. 특히 ´91-´95시기에 많은 비래량을 보인 것은 ´94, ´95년에 가뭄이 연속되어서 진딧물의 발생에 좋은 조건을 제공한 것으로 추측된다. 비래진딧물의 비래최성기는 6월 상-중순 사이로 아직까지 연도별로 유의성 있는 차이는 보이지 않고 있지만,비래최성기의 우점종은 연도별로 약간의 변화를 보여주고 있다. 1980년대 중반을 기점으로 그 이전에는 복숭아혹진딧물이 우점 비래하였으나, 그 이후부터는 목화진딧물의 비래가 가장 많았다. ´89-´91년에 수행한 PLRV 보독률 조사에서는 비래진딧물의 6.7-10.0%가 PLRV를 가지고 있는 보독진딧물로 밝혀진 바 있다. 그렇지만 2000년, 2001년 조사에서는 각각 10.1%, 11.0%로 조금 높게 나타났는데, 이는 과거 10년 전보다 진딧물의 비래량이 2배 이상 증가하고 있고 비래 진딧물의 종류도 다양해졌다는 사실을 감안할 때 보독률의 변동은 거의 없는 것으로 추정된다.

• The Plant Pathology Journal
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• 제18권1호
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• pp.1-5
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• 2002

The availability of nucleotide sequences of the coat protein gene of Potato leafroll virus (PLRV) permitted the construction of DNA primers that were utilized for cDNA synthesis. Polymerase chain reaction (PCR) products of a 487 bp. and approximately 500 bp DNA fragments were amplified from nucleic acid extracts of PLRV-infected tissue and strawberry mild yellow-edge (SMYE) diseased strawberry tissue, respectively. The amplified DNA fragments were further differentiated by hybridization analysis with a CDNA probe for the coat protein gene of PLRV and restriction fragment length polymorphism (RFLP) analysis. These results suggest that a luteovirus is associated with the SMYE disease.

• The Plant Pathology Journal
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• 제27권4호
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• pp.385-389
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• 2011

A new reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the Potato leafroll virus (PLRV) was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR) to address its advantages over RTPCR. RT-LAMP primers were designed from the open reading frame 3 (ORF3) sequence of PLRV. The RT-LAMP reactions were conducted without or with a set of loop primers. By real-time monitoring using Turbimeter, the RT-LAMP (with loop primers) detects PLRV in less than 30 min, compared to 120 min of RT-PCR. By adding fluorescent reagent during the reaction, final products of the RT-LAMP were fluorescently visualized under UV light or could be differentiated by naked-eye inspection under normal light. The RT-LAMP was extremely sensitive, about 2000-fold more sensitive than RT-PCR. This study presents great potential of the RT-LAMP for diagnosis and PLRV epidemiology because RT-LAMP method is speedy, sensitive, inexpensive, and convenient.

• 한국식물병리학회지
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• 제11권2호
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• pp.101-106
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• 1995

Deep blue fluorescence of resorcin blue-stained callose was observed only in the potato leafroll virus (PLRV)-infected potato plants, but not in other potato viruses investigated. The plant sections stained with aniline blue showed non-specific fluorescence regardless of PLRV infection, which means that aniline blue is not suitable for the staining of callose induces by PLRV infection. The fluorescence of resorcin blue-stained callose was more easily detectable than autofluorescence by a direct fluorescence detection method because of its deep blue color. The lateral branch of lower leaves was turned out to be the best material for fluorescence observation of all plant parts tested. In comparison of diagnostic efficacy of this technique to enzyme-linked immunosorbent assay (ELISA), PLRV infected potato plants showed corresponding increment of the fluorescence of resorcin blue stained callose as absorption values in ELISA increased. In the future, the criteria measuring the fluorescence objectively are thought to be determined for the practical application to the diagnosis of potato leafroll disease.

• 한국작물학회지
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• 제59권4호
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• pp.498-504
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• 2014

Potato virus Y (PVY) and potato leafroll virus (PLRV) are among the most damaging potato viruses and prevalent in most potato growing areas. In this study, cryopreservation was used to eradicate PVY and PLRV using two cryogenic methods. Potato shoot tips proliferated in vitro were cryopreserved through droplet-vitrification and encapsulation-vitrification using plant vitrification solution 2 (PVS2; 30% glycerol + 15% dimethyl sulfoxide + 15.0% ethylene glycol + 13.7% sucrose) and modified PVS2. Both cryogenic procedures produced similar rates of survival and regrowth, which were lower than those from shoot tip culture alone. The health status of plantlets regenerated from shoot tip culture alone and cryopreservation was checked by reverse transcription-polymerase chain reaction. The frequency of virus-free plants regenerated directly from highly proliferating shoot tips reached 42.3% and 48.6% for PVY and PLRV, respectively. In comparison, the frequency of PVY and PLRV eradication after cryopreservation was 91.3~99.7% following shoot-tip culture. The highest cryopreserved shoot tip regeneration rate was observed when shoot tips were 1.0~1.5 mm in length, but virus eradication rates were very similar (96.4~99.7%), regardless of shoot tip size. This efficient cryotherapy protocol developed to eliminate viruses can also be used to prepare potato material for safe long-term preservation and the production of virus-free plants.

• The Plant Pathology Journal
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• 제32권4호
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• pp.321-328
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• 2016

We examined the effects of temperature on acquisition of Potato virus Y-O (PVY-O), Potato virus A (PVA), and Potato leafroll virus (PLRV) by Myzus persicae by performing transmission tests with aphids that acquired each virus at different temperatures. Infection by PVY-O/PVA and PLRV increased with increasing plant temperature in Nicotiana benthamiana and Physalis floridana, respectively, after being transmitted by aphids that acquired them within a temperature range of $10-20^{\circ}C$. However, infection rates subsequently decreased. Direct qRT-PCR of RNA extracted from a single aphid showed that PLRV infection increased in the $10-20^{\circ}C$ range, but this trend also declined shortly thereafter. We examined the effect of temperature on establishment of virus infection. The greatest number of plants became infected when N. benthamiana was held at $20^{\circ}C$ after inoculation with PVY-O or PVA. The largest number of P. floridana plants became infected with PLRV when the plants were maintained at $25^{\circ}C$. PLRV levels were highest in P. floridana kept at $20-25^{\circ}C$. These results indicate that the optimum temperatures for proliferation of PVY-O/PVA and PLRV differed. Western blot analysis showed that accumulations of PVY-O and PVA coat proteins (CPs) were lower at $10^{\circ}C$ or $15^{\circ}C$ than at $20^{\circ}C$ during early infection. However, accumulation increased over time. At $25^{\circ}C$ or $30^{\circ}C$, the CPs of both viruses accumulated during early infection but disappeared as time passed. Our results suggest that symptom attenuation and reduction of PVY-O and PVA CP accumulation at higher temperatures appear to be attributable to increased RNA silencing.

• 한국응용곤충학회지
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• 제57권4호
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• pp.415-423
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• 2018

진딧물은 감자에 다양한 바이러스병을 옮기는 대표적인 매개충이다. 따라서 감자밭으로 날아오는 진딧물을 방제하는 것은 매우 중요하다. 비래진딧물의 종류, 비래시기와 비래량, 바이러스 보독률 및 진딧물의 겨울철 월동기주를 구명하기 위하여 강원도 고랭지의 씨감자 생산지역 3곳을 중심으로 진딧물 비래양상을 조사하였고, 비래초기부터 6월 하순까지 날아오는 진딧물의 잎말림바이러스(PLRV) 보독 여부를 조사하였다. 또한 진딧물의 이동방향을 따라 57종의 수목류 가지와 수피를 채집하여 월동중인 진딧물 알의 존재 여부를 조사하였다. 강릉 왕산 지역과 홍천 내면 지역의 여름기주 비래최성기는 모두 6월 중순이었고 평창 횡계 지역은 5월 하순으로 나타났다. 겨울기주로 날아가는 비래최성기는 3개 지역 모두 10월 상순이었다. 비래진딧물의 2.8%가 PLRV를 갖고 있었으며, 진딧물 종류별 보독률은 복숭아혹진딧물 15.4%, 감자수염진딧물 9.1%였다. 시기별로는 5월 하순부터 PLRV 보독 진딧물이 비래하였고 6월 중순에 비래한 진딧물의 바이러스 보독률이 10.4%로 가장 높게 나타났다. 고로쇠나무(Acer pictum subsp. mono (Maxim.)), 당단풍나무(Acer pseudosieboldianum (Pax)) 등 17종의 수목류에서 진딧물알이 월동하였다. 월동 진딧물은 복숭아혹진딧물(Myzus persicae), 홉사마귀진딧물(Phorodon humuli) 등 14종으로 동정되었다. 특히 자작나무진딧물(Betula platyphylla var. japonica), 단풍알락진딧물(Yamatocallis hirayamae) 등은 아직까지 감자에서 바이러스를 옮기는지 여부는 알려진 바 없지만, 넓은 지역에 걸쳐 분포하는 기주식물에서 월동하기 때문에 앞으로 바이러스 매개 능력에 대한 연구가 필요하다.