Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe.M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Effect of potato physiology on the interpretation of ELISA results for potato leafroll virus

This paper records the occurrence of an antigen produced in plants infected by potato leafroll virus (PLRV), which is copurified with the virus and induces an immune response in rabbits used for virus antiserum production. The antigen also appeared to be produced in uninfected but physiologically stressed potato plants. These plants reacted with antisera to PLRV in ELISA tests, thereby giving false positive results. We refer to the compound as virus-stress antigen (VSA). The importance of this finding, not only to serological testing for PLRV, but also as a possible explanation for some false-positive reactions that occur with other host-virus combinations, is discussed. The necessity of having detailed information on plants from which samples are taken for testing is emphasized.     

转二价核酶基因马铃薯及抗病性研究

 用克隆的特异性切割马铃薯卷叶病毒(Potato leaf roll virus,PLRV)复制酶基因负链RNA的二价核酶基因,构建植物表达载体pROKⅡ/DR,经土壤农杆菌(Agrobacterium tumefaciens)介导叶盘法转化马铃薯外植体,获得再生植株。PCR和Southern-blot检测,证明目的基因已成功地导入马铃薯再生植株,其转化率约为14.5%,并能够在无性繁殖后代植株中稳定存在。RT-PCR检测表明,再生马铃薯植株中的二价核酶基因可以转录表达。经病毒接种的转基因马铃薯株系L5、L7、L8和J-1的无性繁殖后代在继发感染中仍表现出较高的抗病性,为最终获得抗PLRV马铃薯新品系打下了基础。     

矿物油和维生素B1对马铃薯病毒病的防效研究

多数马铃薯病毒可以借助蚜虫传播, 并通过块茎世代积累, 导致马铃薯种性退化, 严重影响块茎的产量和品质?为了筛选新型?环保的马铃薯病毒病防治药剂, 本研究通过3个季节的田间试验, 对矿物油?维生素B1和杀虫剂吡虫啉在防治马铃薯病毒病中的效果进行了评价?结果表明, 通过马铃薯出苗后间隔10 d连续3次喷施, 矿物油能够控制马铃薯卷叶病(potato leaf-roll virus, PLRV)的发生, 对马铃薯M病毒(potato virus M, PVM)和马铃薯S病毒(potato virus S, PVS)的平均防效也分别达到66.72%和70.40%, 但对马铃薯Y病毒(potato virus Y, PVY)和马铃薯A病毒(potato virus A, PVA)在不同的年份和季节的防效不稳定, 平均防效为27.34%和65.02%?维生素B1对PLRV?PVM和PVS的防效也比较明显, 分别达83.36%?83.33%和73.32%, 而对PVY同样防效不稳定, 对PVA防效不明显?杀虫剂吡虫啉对PLRV?PVS和PVM的防效也不稳定, 且对PVY和PVA的防效均不显著?本研究中马铃薯X病毒(potato virus X, PVX)发生频率极低, 未进行病毒病的防效比较?综上所述, 矿物油和维生素B1对马铃薯主要病毒病的综合防效较吡虫啉好, 同时它们的增产效果更明显, 产投比高于化学药剂, 值得推广?     

二重RT-PCR快速检测马铃薯病毒的方法

本研究采用传统的蛋白酶K法和病毒RNA简易浸提法,从马铃薯块茎、茎干、叶梗、叶片中提取马铃薯X病毒,马铃薯Y病毒,马铃薯A病毒及马铃薯卷叶病毒RNA,并设计了4种马铃薯病毒引物,优化了二重RT-PCR反应条件,可以同步扩增出上述4种病毒,扩增产生的靶带分别为562bp(PVX)、480bp(PVY)、336bp(PLRV)、255bp(PVA).应用病毒RNA简易制样技术和优化的二重RT-PCR反应条件,可以同步快速检测田间自然感染的马铃薯病毒,此研究还可适合于检测马铃薯脱毒种薯及试管苗,对马铃薯病毒病早期监测有一定的作用.     

湖南省马铃薯病毒病发生情况调查

为了解湖南省马铃薯种薯质量和主要病毒病发生情况,2019年-2020年马铃薯秋作和冬作期间,对长沙、益阳、湘潭、澧临等马铃薯生产区的155个马铃薯样品,运用反转录-聚合酶链式反应(RT-PCR)和双抗体夹心酶联免疫吸附检测(DAS-ELISA)技术,筛查6种主要马铃薯病毒,包括马铃薯X病毒Potato virus X(PVX)、马铃薯Y病毒Potato virus Y(PVY)、马铃薯M病毒Potato virus M(PVM)、马铃薯S病毒Potato virus S(PVS)、马铃薯A病毒Potato virus A(PVA)、马铃薯卷叶病毒Potato leaf roll virus(PLRV)。检测结果表明:6种马铃薯病毒病在湖南均有不同程度的发生,单一和两种病毒复合感染植株占比最高,其次是3种病毒复合感染,存在极少数植株复合感染4～5种病毒病情况。在秋作马铃薯中,PVY检出率达到29.41%;PVS和PVA检出率均为27.94%;PVM、PVX、PLRV的检出率分别为20.59%、19.12%、17.65%。在冬作马铃薯中,PVX检出率最高,达到31.03%;其次是PLRV,...     

Localization of potato leafroll virus in leaves of secondarily-infected potato plants

Potato leafroll virus (PLRV) antigen was localized by immunogold labelling in semi-thin leaf sections of secondarily-infected potato plants cv. Bintje. Viral antigen was present in all cell types of the phloem tissue. but occurred most abundantly in the companion cells. Detectable amounts of PLRV antigen were found only in the sieve elements in veins with a large number of infected companion cells. Occasionally, parenchyma cells were also found to be infected. PLRV was not exclusively limited to the phloem tissue in the infected potato plants, but was also found in mesophyll cells neighbouring minor phloem vessles. Spread of virus from cell to cell in the mesophyll was not observed. The distribution of PLRV in the potato leaf tissue has implication on its availability, for acquisition by aphids.     

Molecular breeding for virus resistant potato plants

To engineer resistance against potato virus X (PVX), the viral coat protein (CP) gene has been introduced into two potato cultivars. Stable expression of the gene in transgenic clones throughout the growing season has been obtained and resulted in considerably increased virus resistance. With varying frequencies depending on the original cultivar used, true-to-type PVX resistant transgenic clones have been obtained. Since deviant light sprout characteristics were invariably associated with aberrations in plant phenotype, they can be used in procedures to early screen for deviations. Furthermore, it has been possible to unequivocally discriminate between the original untransformed and independent transgenic cultivars. Although no relation has been found between the presence, if any, of the CP of potato virus Y (PVY) or potato leafroll virus (PLRV) in CP gene transgenic potato, appreciable levels of resistance to these viruses has been obtained. This suggests that the mechanism by which a viral CP gene in the potato genome evokes resistance, differs amongst various viruses.     

Diagnosis and molecular analysis of Potato leafroll virus isolates in Tunisia

Potato leafroll virus (PLRV) is a major constraint to potato production in North Africa. Serological (sandwich and cocktail ELISA) and molecular (RT-PCR) tests were used to detect PLRV in 131 potato samples collected in different areas of Tunisia. RT-PCR proved to be usable as a routine diagnostic test for epidemiological purposes, being more sensitive and reliable, and less time-consuming, than serological tests. One RT-PCR-amplified portion of ORF3 (336 nt) was cloned and sequenced, and used for molecular characterization of Tunisian PLRV isolates. These showed high sequence identity with PLRV retrieved from GenBank.     

Hairy nightshade as a potential Potato leafroll virus (Luteoviridae: Polerovirus) inoculum source in Pacific Northwest potato ecosystems

Hairy nightshade, Solanum sarrachoides, is a solanaceous weed found abundantly in Pacific Northwest potato ecosystems. It serves as a reservoir for one of the important potato viruses, Potato leafroll virus (PLRV) (Luteoviridae: Polerovirus), and its most important vector, the green peach aphid, Myzus persicae (Homoptera: Aphididae). Laboratory research indicated an increased green peach aphid settling and performance on S. sarrachoides than on potato. It also revealed that green peach aphids transmitted PLRV more efficiently from S. sarrachoides to potato than from potato to potato. To test the efficiency of S. sarrachoides as an inoculum source in the field, a two season (2004 and 2005) trial was conducted at Kimberly, Idaho. Two inoculum sources, PLRV-infected potato and PLRV-infected S. sarrachoides, were compared in this trial. Green peach aphid density and temporal and spatial PLRV spread were monitored at weekly intervals. Higher densities of green peach aphids were observed on plots with S. sarrachoides and inoculum sources (PLRV-infected S. sarrachoides and potato) than on plots without S. sarrachoides and inoculum sources. PLRV infection in plots with PLRV-infected S. sarrachoides was similar to or slightly higher than in plots with PLRV-infected potato as an inoculum source. Temporal and spatial PLRV spread was similar in plots with either inoculum source. Thus, S. sarrachoides is as efficient as or a better PLRV inoculum source than potato.     

Molecular breeding for virus resistant potato plants

To engineer resistance against potato virus X (PVX), the viral coat protein (CP) gene has been introduced into two potato cultivars. Stable expression of the gene in transgenic clones throughout the growing season has been obtained and resulted in considerably increased virus resistance. With varying frequencies depending on the original cultivar used, true-to-type PVX resistant transgenic clones have been obtained. Since deviant light sprout characteristics were invariably associated with aberrations in plant phenotype, they can be used in procedures to early screen for deviations. Furthermore, it has been possible to unequivocally discriminate between the original untransformed and independent transgenic cultivars. Although no relation has been found between the presence, if any, of the CP of potato virus Y (PVY) or potato leafroll virus (PLRV) in CP gene transgenic potato, appreciable levels of resistance to these viruses has been obtained. This suggests that the mechanism by which a viral CP gene in the potato genome evokes resistance, differs amongst various viruses.     

基于小RNA深度测序和RT-PCR检测侵染广东省冬种马铃薯的病毒

为明确侵染广东省冬种马铃薯的病毒种类及优势病毒,结合小RNA深度测序技术及RTPCR检测方法,对采集于广东省冬种马铃薯7个主产区的189份疑似病样进行检测分析。结果表明,经小RNA深度测序技术检测马铃薯病毒病混合样,发现存在马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)和马铃薯卷叶病毒(Potato leaf-roll virus,PLRV)3种病毒。进一步设计3种病毒的特异性引物并利用国内已报道的其它5种马铃薯病毒的特异性引物进行RT-PCR检测,发现189份马铃薯病毒病疑似病样中仅检测到PVY、PVS和PLRV这3种病毒,检出率依次为75.13%、10.05%和4.76%,且3种病毒在马铃薯上还存在复合侵染,复合侵染率为14.19%,其中PVY在各马铃薯产区均可检测到。表明侵染广东省冬种马铃薯的病毒为PVY、PVS和PLRV,其中PVY是优势病毒。     

甘肃省马铃薯主要病毒病发生情况调查

2015年-2016年,在甘肃省10个地市24个马铃薯主栽县(区)146个生态区域(乡镇)采集了757份具有典型症状的马铃薯样品,应用DAS-ELISA法进行检测,筛查6种主要病毒(PVX、PVY、PLRV、PVA、PVS和PVM)。结果表明:631份样品检测到病毒,PVS的检出率最高,达47.03%,PVY次之,为33.82%,PVA最低,只有0.63%;发生复合侵染的病毒主要为PVY+PVS,复合侵染率达到10.13%,三种病毒复合侵染主要是PVY+PVS+PVM;病毒种类和感病程度与品种、地域有关。     

利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯

为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。     

Serological reaction of beet western yellows and potato leafroll viruses in ELISA

The detection of potato leafroll virus (PLRV) and beet western yellows virus (BWYV) and their serological relatedness were investigated by the double sandwich ELISA (DS). In both pure preparations and crude plant extracts, an unequivocal detection of each of these viruses was obtained by DS, provided the homologous antisera were used. No detection was achieved when the heterologous antisera were used, although purified virus could be detected when using the heterologous antisera for coating, providing the homologous antisera were used as conjugates. Therefore, for routine screening of BWYV or PLRV infection in plants, the DS method with homologous antisera can be used. However, it does not seem that BWYV infection in potato could be detected by the routine DS aimed for PLRV screening. In immunosorbent electron microscopy, PLRV and BWYV could be easily detected by coating grids with either homologous or heterologous antiserum. This and the possibility of trapping each virus in ELISA plates with the heterologous antiserum indicate serological cross-reactivity between PLRV and BWYV.     

Fusion proteins of single-chain variable fragments derived from phage display libraries are effective reagents for routine diagnosis of potato leafroll virus infection in potato

ABSTRACT A panel of 11 different single-chain variable fragment antibodies (scFv) that bind to potato leafroll virus (PLRV) has been studied to assess each one's suitability as practical diagnostic tools. The scFv, previously obtained from naive phage display libraries, were expressed in Escherichia coli as fusion proteins. The fusion proteins comprised scFv joined to either the human light chain kappa constant domain (C(L)), an amphipathic helix (Zip), a combination of C(L) and Zip, or alkaline phosphatase (AP/S). The fusion proteins were tested for their ability to detect, or trap on enzymelinked immunosorbent assay (ELISA) plates, PLRV in extracts of infected potato leaves. The tests done with the different scFv fusion proteins were compared with a standard triple-antibody sandwich (TAS)-ELISA that employs a rabbit polyclonal antibody preparation to coat microtiter plates and a monoclonal antibody, SCR3, to detect PLRV. Of 11 scFvC(L) fusion proteins, 7 detected PLRV as readily as SCR3 when used as detecting antibodies in TAS-ELISA. The limit of detection of purified PLRV for the different scFvC(L) fusion proteins ranged from 250 to 5 ng/ml; that for SCR3 is 5 ng/ml. Of the 11 scFv, 4 cross-reacted with some other luteoviruses. Several scFvC(L) and scFvC(L)Zip fusion proteins trapped PLRV from extracts of infected potato leaves as effectively as the polyclonal antibody preparation. Four scFv fusion proteins were used in a stem print assay to detect PLRV, and the results were similar to those obtained in tests using SCR3. The scFvC(L) fusion proteins retained activity for at least 6 months at 4 degrees C, and all scFv fusion proteins were fully active on reconstitution after lyophilization. A fully recombinant ELISA was devised that detected PLRV in extracts of infected potato, with results comparable to those obtained using the standard TAS-ELISA. The advantages of using scFv fusion proteins for the routine detection of plant viruses include the ability to produce large quantities of reagents cheaply in bacterial fermenters and to incorporate them into standardized tests.     

Increased susceptibility of potato to <Emphasis Type="Italic">Rhizoctonia</Emphasis> diseases in <Emphasis Type="Italic">Potato leafroll virus</Emphasis>-infected plants

In Hokkaido potato fields, tubers produced from the plants with leaf curl symptoms caused by potato leaf roll virus (PLRV) were noted to be more densely covered with Rhizoctonia sclerotia. This observation led us to hypothesize that potato infected with PLRV would have an increased susceptibility to Rhizoctonia solani. To test this hypothesis, in a pot experiment, we inoculated PLRV-infected mother tubers with Rhizoctonia. As a result, PLRV-infected plants produced significantly fewer and smaller tubers than virus-free plants did, suggesting that PLRV-infected plants are more susceptible than virus-free plants to R. solani. Virus-free seed tubers should thus be used to reduce Rhizoctonia diseases.     

福建省福清地区马铃薯病毒病病原的分子检测

2017年调查福建福清地区马铃薯病毒病的发生情况,以明确该地区马铃薯主要病毒病原。共采集了46份疑似感染病毒的马铃薯植株,提取总RNA,利用RT-PCR技术进行分子检测,结果表明,福清地区危害马铃薯的病毒有马铃薯Y病毒Potato virus Y(PVY)、马铃薯卷叶病毒Potato leaf roll virus(PLRV)、马铃薯S病毒Potato virus S(PVS),检出率分别为56.52%、17.39%和10.87%,以PVY检出率最高,说明PVY是危害该地区马铃薯样品的主要病毒病原。通过病毒复合侵染进行分析,发现该地区存在病毒复合侵染马铃薯现象。研究结果可为福清地区马铃薯种薯的引进和病毒病害防治提供参考依据。