一种快速简便检测马铃薯病毒的方法——病毒细菌协同凝集法(VBA)的研究

 在国内、外首次选用金黄色葡萄球菌(Staphylococc aureus)的No.180菌株用于病毒细菌协同凝集试验(Virobacterial agglutination test简称VBA)检测了来自马铃薯的4种不同形态粒子的病毒:马铃薯X病毒(PVX)、马铃薯丫病毒(PVY)、烟草花叶病毒(TMV)和马铃薯卷叶病毒(PLRV),其灵敏度达2.7~6.1ng/ml,检出病汁液的最大稀释度达10⁴~10⁵(PLRV1:500),较国内、外采用的Cown I菌株的灵敏度提高5~10倍。与血清学方法相比,VBA在2~3分种内就可获得结果,无假阳性反应,其灵敏度显著高于间接酶联法和免疫电镜,而接近于A蛋白酶联法。经抗血清致敏的菌体在4℃下保存4个月,对VBA检测的灵敏度没有影响。用VBA对采自田间的93个马铃薯病样进行检测,它们大多受2~3种病毒(PVX、PVY及PLRV)的复合侵染;和用间接酶联法及鉴别寄主检测的结果趋向一致。室内和田间试验均表明VBA灵敏度高、特异性强、快速简便和经济,尤其适合在基层单位中推广应用。     

马铃薯种苗复合感染病毒多重RT-PCR同步快速检测

 基于马铃薯病毒ssRNA的快速制备方法,根据马铃薯病毒CP基因序列设计PVX、PVS、PVY和PLRV特异性引物对、P1基因序列设计PVA特异性引物对,建立了多重RT PCR快速检测体系,可以同步检出复合侵染马铃薯种苗主要病毒,灵敏度比传统的ELISA至少高100倍。结合生物活性稳定剂研制的固相化检测试剂盒,已用于四川和重庆等15个县市田间与苗圃248个马铃薯显症或无症样品的实际检测,表明四川和重庆地区2~3种马铃薯病毒往往复合侵染(PVX、PVA和PVS三种病毒复合侵染或PLRV和PVY二种病毒复合侵染)。     

一种快速简便检测马铃薯病毒的方法——病毒细菌协同凝集法(VBA)的研究

在国内、外首次选用金黄色葡萄球菌(Staphylococc aureus)的No.180菌株用于病毒细菌协同凝集试验(Virobacterial agglutination test简称VBA)检测了来自马铃薯的4种不同形态粒子的病毒:马铃薯X病毒(PVX)、马铃薯丫病毒(PVY)、烟草花叶病毒(TMV)和马铃薯卷叶病毒(PLRV),其灵敏度达2.7～6.1ng/ml,检出病汁液的最大稀释度达10~4～10~5(PLRV1:500),较国内、外采用的Cown I菌株的灵敏度提高5～10倍。与血清学方法相比,VBA在2～3分种内就可获得结果,无假阳性反应,其灵敏度显著高于间接酶联法和免疫电镜,而接近于A蛋白酶联法。经抗血清致敏的菌体在4℃下保存4个月,对VBA检测的灵敏度没有影响。用VBA对采自田间的93个马铃薯病样进行检测,它们大多受2～3种病毒(PVX、PVY及PLRV)的复合侵染;和用间接酶联法及鉴别寄主检测的结果趋向一致。室内和田间试验均表明VBA灵敏度高、特异性强、快速简便和经济,尤其适合在基层单位中推广应用。     

双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒

根据马铃薯A病毒全基因序列和马铃薯卷叶病毒衣壳蛋白区序列分别设计PVA和PLRV的特异性引物,应用双重RT-PCR同步检测马铃薯A病毒和马铃薯卷叶病毒,分别得到300bp和222bp大小的扩增片段.试验从反转录、PCR等方面对双重RT-PCR同步检测2种病毒进行了探索和优化.结果表明反转录反应中dNTPs浓度、2种病毒下游引物浓度比例以及PCR反应中Mg2+浓度对双重RT-PCR同时检测PVA和PLRV有较大的影响.     

多重RT-PCR同时检测6种马铃薯病毒及1种类病毒

病毒病是限制我国马铃薯生产的重要因子之一。本研究通过引物设计和体系优化建立了一套多重RT-PCR检测方法，可以在一个反应体系中同时检测6种马铃薯病毒和1种马铃薯类病毒以及1个马铃薯内参基因，包括马铃薯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X病毒(potato virus X, PVX)、马铃薯A病毒(potato virus A, PVA)、马铃薯卷叶病毒(potato leaf curl virus, PLRV)、马铃薯纺锤块茎类病毒(potato spindle tuber viroid, PSTVd)和细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,CoxⅠ)基因的特异片段，产物大小依次为181、226、275、565、630、681、359、500 bp,符合理论预期。采用建立的方法以相应病毒和类病毒的质粒作模板进行检测，其检测灵敏度在1.6×10^-3～1.8×10^-1 ...     

马铃薯卷叶病毒的RT-LDR-PCR检测

马铃薯卷叶病毒（Potato leaf roll virus,PLRV）属黄症病毒科Luteoviridae黄花病毒属Polerovirus,是目前严重影响马铃薯产量与品质的病毒,其分布局限于寄主植物的韧皮部,且含量非常低,难于提纯,这给PLRV检测带来很大的困难[1]。     

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.     

马铃薯4种病毒多重PCR检测体系的建立

我国马铃薯病毒主要有马铃薯Y病毒(PVY)、马铃薯X病毒(PVX)、马铃薯S病毒(PVS)、马铃薯卷叶病毒(PLRV),常发生复合侵染。根据GenBank中4种马铃薯病毒的外壳蛋白(coat protein,CP)基因全长设计引物,通过RT-PCR扩增得到4种病毒CP基因全长片段,测序结果显示序列同源性96%以上;针对4种病毒CP基因的保守序列分别设计引物,在一个PCR体系中同步对4种病毒进行扩增,得到421、202、516、330bp的特异性条带,优化建立了能同步检测PVY、PVX、PVS和PLRV的多重RT-PCR检测体系。检测结果证明优化后的多重RT-PCR体系能在田间样品中快速、高效地检测出4种病毒。     

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。     

Transmission by aphids of potato spindle tuber viroid encapsidated by potato leafroll luteovirus particles

Potato spindle tuber viroid (PSTVd) was transmitted by Myzus persicae to Physalis floridana from P. floridana plants that also were infected with potato leafroll luteovirus (PLRV), whereas it was not transmitted by aphids from plants infected with PSTVd alone. Dot-blot hybridisation was used to detect PSTVd. The results indicate that PLRV can assist PSTVd in its transmission by M. persicae. Doubly infected, aphid-inoculated P. floridana plants from the previous experiment were used as the source plants in aphid transmission tests to the tomato cv. Rutgers, P. floridana and Datura stramonium. PSTVd was detected in 17 of 30 plants of tomato. The viroid was not detected by dot-blotting in any plant of P. floridana and D. stramonium in this experiment, but it was recovered from some plants by sap inoculation of the Rutgers plants. Treatment with RNase A of PLRV preparations purified from doubly infected plants indicated that PSTVd was encapsidated by PLRV particles.     

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.     

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.     

Exclusion of viroids from potato resources and the modified use of a cDNA probe1

Threats from potato spindle tuber viroid (PSTV) to potato breeding and centralized elite seed-tuber production have been identified in world potato genetic resources. In the UK effective diagnostic testing has proved essential in preventing acquisition. Inoculation of potato nucleic acids to tomato and subsequent viroid detection by polyacrylamide gel electrophoresis (PAGE) has proved a sensitive, but cumbersome, test over 8 years. Additionally, over 2 years, ³²P-labelled PSTV cDNA was used to probe denatured sap and nucleic acid extracts: 10^-4 of peak viroid concentrations in tissue could be detected. Spurious positives were seen in particular circumstances, but could be avoided. Probing of non-denatured samples was not as sensitive. Tubers became infected and PSTV was readily detected by PAGE in leaves of potato experimentally inoculated and maintained below 20°C, but the cDNA probe could not detect infection in tuber sprouts growing at 8–10°C in darkness. Otherwise similar green-leaved sprouts were faintly positive. Detection for all sprouts was unproblematic after movement to 25°C and light for 10 days.     

Transmission of Tomato chlorotic dwarf viroid by Myzus persicae assisted by Potato leafroll virus

Journal of Plant Diseases and Protection - In this study, we have shown Myzus persicae transmissibility of Tomato chlorotic dwarf viroid (TCDVd) by exposing Potato leafroll virus (PLRV) plus TCDVd...     

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。     

马铃薯疮痂病菌在植株和田间的分布与动态分析

为探明马铃薯疮痂病菌在植株和土壤中的分布情况及种群动态变化特点,利用常规PCR和定量PCR(qPCR)技术对不同环境的马铃薯疮痂病株和田间植株不同生育期的土壤样品进行病原菌的定性定量检测.结果 表明,病田、温室盆栽和微型薯苗床中马铃薯疮痂病重度发病植株的根、匍匐茎、块茎、地上茎、叶片等组织样品均可检测到184 bp的疮...     

The movement of potato virus Y (PVY) in the vascular system of potato plants

Potato virus Y (PVY) is responsible for major viral diseases in most potato seed areas. It is transmitted by aphids in a non-persistent manner, and it is spread in potato fields by the winged aphids flying from an infected source plant to a healthy one. Six different PVY strains groups affect potato crops: PVY^C, PVY^N, PVY^O, PVY^N:O, PVY^NTN, and PVY^N-Wi. Nowadays, PVY^NTN and PVY^N-Wi are the predominant strains in Europe and the USA. After the infection of the leaf and accumulation of the virus, the virus is translocated to the progeny tubers. It is known that PVY^N is better translocated than PVY^O, but little is known about the translocation of the other PVY strains. The translocation of PVY occurs faster in young plants than in old plants; this mature plant resistance is generally explained by a restriction of the cell-to-cell movement of the virus in the leaves. The mother tuber may play an important role in explaining mature plant resistance. PVY is able to pass from one stem to the other stems of the same plant through the vascular system of the mother tuber, but it is unknown whether this vascular link between stems is permanent during the whole life of the plant. Two greenhouse trials were set up to study the spread of PVY in the vascular system of the potato plant. The PVY-susceptible cultivar Charlotte was used for both trials. It was demonstrated that all stems growing from a PVY-infected tuber will become infected sooner or later, and that PVY^N-Wi translocates more efficiently to progeny tubers than PVY^NTN. It was also demonstrated that the progressive decay of the mother tuber in the soil reduces the possibility for virus particles to infect healthy stems through the vascular system of the mother tuber. This new element contributes to a better understanding of the mechanism of mature plant resistance.