烟草环斑病毒IC-RT-Realtime PCR检测方法研究

本研究首次应用免疫捕捉反转录实时荧光PCR技术(IC-RT-RealtimePCR)检测烟草环斑病毒,由于综合运用了抗原抗体特异性结合、核酸分子杂交、高灵敏度实时荧光PCR技术的优点,从而使病毒检测在特异性、灵敏度、稳定性等技术指标上比传统的DAS-ELISA方法都有显著提高,解决了烟草环斑病毒检测工作中由于隐症、病毒浓度低、干扰物质存在而影响检测结果的问题。     

烟草环斑病毒IC-RT-Realtime PCR检测方法研究

本研究首次应用免疫捕捉反转录实时荧光PCR技术(IC-RT-Realtime PCR)检测烟草环斑病毒,由于综合运用了抗原抗体特异性结合、核酸分子杂交、高灵敏度实时荧光PCR技术的优点,从而使病毒检测在特异性、灵敏度、稳定性等技术指标上比传统的DAS-ELISA方法都有显著提高,解决了烟草环斑病毒检测工作中由于隐症、病毒浓度低、干扰物质存在而影响检测结果的问题.     

烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

宁夏甜菜的烟草环斑病毒的鉴定

从宁夏患丛根病甜菜的病根中分离到一种球状病毒粒子,直径约为２８ｎｍ。提纯病毒的紫外扫描呈典型的核蛋白曲线,最大吸收为２６０ｎｍ,最小吸收为２４０ｎｍ,Ａ＿（２６０）／Ａ＿（２８０）＝１．３０。寄主范围广,能侵染茄科、豆科、藜科、葫芦科、番杏科等１７种植物。病毒的ＴＤＰ为６５℃,ＤＥＰ为１０￣（－３）,体外存活期６－７天。有较强的免疫原性。病毒的抗血清与烟草环斑病毒（ＴＲＳＶ）之间产生明显的沉淀线。经初步鉴定认为该分离物是烟草环斑病毒。     

Serological Strains of Tobacco Ringspot Virus Transmitted by Xiphinema americanum

Five serological strains of tobacco ringspot virus isolated from naturally infected tobacco in North Carolina, and a strain isolated from watermelon in the Rio Grande Valley of Texas were transmitted from cucumber to cucumber by mass-screened and handpicked Xiphinerna americanum from North Carolina. The Eucharis mottle strain from Peru was not transmitted, indicating that a specific strain-vector relationship may exist between the geographically isolated strains from North and South America.     

A Comparison of Some Serological and Biological Properties of Seven Isolates of Tobacco Ringspot Virus

Seven isolates of tobacco ringspot virus (TRSV) from grape, cherry, two cultivars of blueberry, tobacco, soybean and watermelon were compared in this study. The most virulent isolate was from‘Jersey’blueberry and the least virulent isolate was from soybean. The‘Jersey’blueberry isolate and the watermelon isolate could be distinguished serologically from all other isolates. The‘Jersey’blueberry isolate was different in electrophoretic mobility from the soybean and tobacco isolates. A unique technique for comparing the in vitro translation products of three of the TRSV isolates is described.     

Development of PCR Diagnostic System for Detection of the Seed-Transmitted Tobacco Ringspot Virus in Quarantine

Tobacco ringspot virus (TRSV) is a plant quarantine virus in Korea. As such, a TRSV examination is conducted when importing various crops. In this study, RT-PCR and nested PCR systems for TRSV detection in quarantine sites, and the modified-positive control plasmid for proving laboratory contamination and false positive reactions were developed. The developed diagnostic system was used to detect TRSV in the quarantine site. It revealed that from 2012 to August 2014, a total of 12 cases were detected in imported various crops. The system is expected to continue contributing to TRSV detection in plant quarantine.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0518-8) contains supplementary material, which is available to authorized users.     

Transmission of Tomato Ringspot Virus by Xiphinema americanum and X. rivesi from New York Apple Orchards

Populations of Xiphinema americanum and X. rivesi were collected from apple orchards in eastern and western New York and tested in the laboratory for ability to transmit tomato ringspot virus (TmRSV) to cucumber and dandelion. Populations varied in the frequency with which they transmitted TmRSV, but this variation did not correspond to variation in disease prevalence in the orchard. The lower prevalence of TmRSV-incited disease in apple trees in western New York cannot be attributed to inability of the local Xiphinema spp. to transmit TmRSV.     

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

烟草环斑病毒(Tobaccoringspotvirus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大。本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT-PCR扩增得到长约1500bp的目的片段。将目的片段与质粒pET-22b( )连接,构建了含TRSVcp基因的融合蛋白原核表达载体pETRSV-CP。序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%~94.6%。将pETRSV-CP转入大肠杆菌,诱导表达。SDS-PAGE结果显示,表达的TRSVCP融合蛋白的相对分子质量约为58kDa。以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应。     

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

烟草环斑病毒(Tobacco ringspot virus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大.本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT PCR扩增得到长约1500bp的目的片段.将目的片段与质粒pET-22b(+)连接,构建了含TRSV cp基因的融合蛋白原核表达载体pETRSV-CP.序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%～94.6%.将pETRSV-CP转入大肠杆菌,诱导表达.SDS-PAGE结果显示,表达的TRSV CP融合蛋白的相对分子质量约为58kDa.以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应.     

番木瓜环斑病毒检测技术的研究

本研究在37℃条件下,以硝酸纤维素滤膜(NCM)为载体:3％酪蛋白为封闭剂、辣根过氧化物酶标记的A蛋白为酶标结合物(SPA-HRP)和以稀释100倍兔抗PRV-Ys的IgG为其工作浓度,成功地建立了检测PRV的间接Dot-ELISA。其检测灵敏度达到了较满意的ng水平。同时,本研究以PRV-Ys株系RNA作模板、oligo(dT)~(12-18)作引物、pUC19作cDNA克隆载体和以E.coli JM107作受体,采用缺口翻译(Nick Translation)方法成功地制备了pYs6 cDNA分子探针(插入片段约300bp)。其检测PRV-RNA的灵敏度达到了较理想的pg水平。     

番茄环斑病毒单克隆抗体的制备

以纯化的番茄环斑病毒(Tomato ringspot virus,ToRSV)为抗原,注射免疫BALB/c小鼠,将免疫小鼠脾细胞与小鼠骨髓瘤细胞Sp2/0进行融合,经多次细胞筛选及克隆化,获得3株(A8、B7和G9)可分泌抗ToRSV单克隆抗体的杂交瘤细胞株,并以之分别制备小鼠腹水单克隆抗体。经酶联免疫吸附试验检测表明,该3株杂交瘤细胞腹水抗体效价在10-5～10-6之间,且均具有与ToRSV反应的特异性。     

Detection of Tobacco Mosaic Virus Movement Protein in Association with Tobacco Nuclei Isolated from Intact and Detached Leaves

The movement protein (MP) of tobacco mosaic virus was observed to cofractionate with nuclei from intact and detached tobacco (Nicotiana tahacum cv. Xanthi-nn) leaves. When purified nuclei were treated with proteinase K, MP disappeared indicating that MP is localized close to but not inside nuclei. Moreover, the amount of MP was showti to increase greatly in nuclei isolated from detached leaves, thus facilitating the detection of MP. Accumulation close to nuclei was strongest early in infection, results indicating that it may be an early step in the pathway of MP from cell cytoplasm to plasmodesmata. Other TMV-specific proteins were not detected in the nuclei fraction.     

dsRNA介导的番木瓜环斑病毒（PRSV）的抗病性研究

以模式植物拟南芥（Arabidopsis thaliana）和烟草（Nicotiana tabacum）及PRSV寄主植物番木瓜（CaricapapayaL.）作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究。利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR（简称PHG12-CPIR）分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中。对转基因植株进行攻毒试验并分析了其抗病性。在接种3~7d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同程度的黄化、皱缩和枯斑等症状。在接种PRSV后,番木瓜和拟南芥转化植株表现症状的叶片的比例与对照相比,结果显著低于对照,而在烟草植株上症状表现的差异不明显。在3种植物上RT-PCR检测结果显示,在接种番木瓜环斑病毒PRSV后,野生型植株中有高浓度的病毒积累,而转pHG12-CPIR基因植株中几乎没有病毒积累,推测转pHG12-CPIR基因植株中瞬时表达系统已启动RNAi机制抑制了CP基因的表达。     

The Potential for Pollen-borne Virus Transfer in a Plum Orchard Infected With Prunus Necrotic Ringspot Virus

Prunus necrotic ringspot virus (PNRSV) is borne in stonefruit pollen. Previous work has shown that virus particles can enter cucumber seedlings when virus-bearing pollen grains contact puncture holes made in plant cells by thrips feeding. Stonefruit plant parts on which pollen is deposited and thrips spend considerable time feeding, are likely sites of PNRSV inoculation. The principal agents of pollen deposition may therefore play a key role in PNRSV epidemiology. We determined the principal sites of pollen deposition on Japanese plum trees and the major pollen depositing agents in a PNRSV-infected orchard in southeastern Queensland. Plum pollen was deposited mostly onto flowers, with few grains being found on leaves or stems. Within the flowers, pollen grains were distributed mostly on the petals, but some were found on the sepals, filaments and carpels. Honey bees were the most frequent visitors to plum flowers and they deposited more than any other insects. Flies visited flowers at low frequencies and also deposited pollen. Significant amounts of pollen were deposited onto flowers by unidentified nocturnal agents. Thrips were not observed during the study period, although they were present in the orchard. Previous studies have assumed that thrips carry virus-bearing pollen as well as bring it into proximity of feeding wounds. Our results suggest that, although thrips carriage of pollen may occur, it is not necessary for PNRSV spread in stonefruit orchards.     

Ultrastructure of Tobacco Ringspot Virus-Induced Local Lesions in Lima Bean Leaves

Lignin is hypothesized to be a factor in the ability of cucurbits to resist fungal infection. To determine if lignin was present around sites of infection, susceptible cucumber plants and cucumber plants with systemic induced resistance were inoculated with Colletotrichum lagenarium, and the ultrastructure of plant and pathogen was examined 24 to 72 hours later. Conidia produced germ tubes with appressoria within 24 hours of inoculation on both control and induced plants. Penetration of plant epidermal cell walls occurred by 48 hours, and papillae formed at the site of penetration. Both elemental bromine and potassium permanganate were used to histochemically stain for lignin. Potassium permanganate, used together with energy dispersive X-ray microanalysis (EDS), indicated the presence of lignin in papillae and in electron-dense areas of plant cell walls directly beneath the appressoria. Using EDS, silicon was shown to be localized in the electron-dense areas of cucumber leaf cells. This is the first report of silicon in induced cucumber plants. There were no qualitative differences between the resistance reactions of induced and control plants.     

番木瓜环斑病毒弱株系保护作用的研究

本文报道了在番木瓜环斑病毒（ＰＲＶ）弱株系保护作用中,保护接种与攻击接种的深度,以及攻击接种的部位等因素对ＨＡ５－１弱株系保护作用效果的影响结果。通过分析弱株系和强株系在保护接种后攻毒的植株体内的病毒浓度,表明ＨＡ５－１弱株系对Ｓｍ株系的保护作用的崩溃是由于强株系在植株顶端叶片听病毒浓度越来越高所致。本文还就ＰＲＶ弱株系在田间的应用技术作了一些讨论。     

Molecular Characterization and Detection of African Oil Palm Ringspot Virus

African oil palm ringspot virus (AOPRV) had been previously described as a fovea‐like virus associated with a lethal disease of African oil palm (Elaeis guineensis) in South America. The original report was based on partial sequence and a distant relationship between AOPRV and Apple stem pitting virus, Apricot latent virus and Grapevine rupestris stem pitting‐associated virus, definitive species of the genus Foveavirus, family Flexiviridae. We report the full sequence of the RNA genome of AOPRV, and demonstrate that this virus is more closely related to two unassigned virus species of the family Flexiviridae (Cherry green ring mottle virus and Cherry necrotic rusty mottle virus) than to any definitive species of the genus Foveavirus. Thus, AOPRV should be considered as a new species of the Flexiviridae until the International Committee on Taxonomy of Viruses (ICTV) resolves the taxonomic status of the increasing number of unassigned species in this family. The molecular characterization of AOPRV has provided a highly sensitive and reliable RT‐PCR assay for the early detection of AOPRV in different genotypes of African, American (E. oleifera) and hybrid oil palms.     

Detection of Tomato Ringspot Nepovirus and a Clostero-like Virus in French Hybrid Vidal 256 Grapevines

Eight- to ten-year old French hybrid Vidal 256 grapevines in southern Maryland produced berries about one-third normal size but did not express any obvious leaf symptoms. Electron microscopy of negatively stained tissue-dip preparations and sectioned material from such vines showed individual and membrane-associated 28 nm spherical virus-like particles and closteroviruslike particles. The spherical particles were characterized as an isolate of tomato ringspot virus (TomRSV-G) that infected a wide range of herbaceous hosts by mechanical inoculation, but did not infect tomato, bean or petunia plants susceptible to the type strain of TomRSV. The closterovirus-like particles did not react, by immunosorbent electron microscopy, with antisera to grapevine virus A (grapevine stem-pitting associated virus of Conti et al. 1980) or the 2200 nm Swiss grapevine leafroll closterovirus (Gugleri et al. 1984).