从吐鲁番古葡萄树上检测到4种类病毒(AGVd、GYSVd-1、GYSVd-2、HSVd)

从约有150年树龄的新疆吐鲁番葡萄(品种无核白)叶子中抽提小分子RNA,经过RT-PCR、生物学检测及克隆测序分析,证明其中含有4种类病毒:AGVd、GYSVd-1、GYSVd-2和HSVd。通过二维电泳,正反向电泳及Northern印迹杂交,RT-PCR等分子生物学方法对接种的指示植物Suyo黄瓜中的子代类病毒进行了检测,结果仅仅检测到HSVd一种类病毒。对Suyo黄瓜中的HSVd子代类病毒进行克隆及序列分析,结果表明葡萄中亲代类病毒与黄瓜中的子代类病毒序列之间存在明显差异。     

桃树上啤酒花矮化类病毒(Hop stunt viroid)的检测及序列分析

 2005年8月和2006年2月从中国北京、陕西、河北、山东、广西等地共采集76个无明显症状的桃树样品,经斑点杂交、RT-PCR以及生物学鉴定检测,来自北京和陕西的11个样品中检测到啤酒花矮化类病毒(Hop stunt viroid,HSVd),总感染率达14.5%。上述3种方法检测桃树上的HSVd具有一致性。将5个样品中的HSVd进行克隆测序,得到12条不同HSVd核酸序列,与GenBank中D13764序列(日本桃果实HSVd分离物)同源性为93.29%~100%。可以看出,国内桃树HSVd分离物核酸序列变异比较小,地域和品种间核酸序列无明显差异。这是首次比较系统地检测中国桃树上HSVd发生情况的报道。     

山西省枣树上啤酒花矮化类病毒的检测及序列分析

［目的］ 从枣树样品中分离鉴定啤酒花矮化类病毒（HSVd）。［方法］ 从山西省农业科学院果树研究所国家枣种质资源圃采集70份枣树叶片样品,提取小分子RNA后通过Northern杂交、RT PCR进行检测,并对阳性样品中的类病毒进行克隆测序,利用生物学软件对所得序列进行分析。 ［结果］ 70份枣树样品中有1份样品感染HSVd,克隆测序后,共获得13条HSVd序列,它们与GenBank上首次报道的HSVd序列相似性为92.6%~92.8% 。［结论］ 本研究首次在国内报道了枣树上分离得到的HSVd序列,HSVd枣树分离物与已报道的HSVd分离物差异较大。     

法国进境葡萄砧木中啤酒花矮化类病毒的检测与序列分析

为检测法国进境葡萄砧木中啤酒花矮化类病毒（Hop stunt viroid,HSVd）,对其进行了RT-PCR检测及序列测定,并构建系统进化树比较了不同地域来源HSVd间的分子差异性。结果表明基于HSVd基因序列设计合成的1对引物能够扩增出302 bp大小的目标片段,而健康植株无此扩增产物。法国葡萄砧木中检出的HSVd分离物,与国内外已报道毒株的核酸序列同源性达90%~97%,与已报道的HSVd全基因组序列间差异较小,表明HSVd的序列变异与地域、寄主等无明显相关性。     

啤酒花矮化类病毒属属级寡核苷酸芯片筛查方法的建立

啤酒花矮化类病毒属是重要的植物类病毒属,目前尚无有效的筛查方法。通过对该属类病毒的核苷酸序列进行分析筛选,设计了8条用于该属类病毒筛查的属级特异性探针并制备了寡核苷酸芯片。应用啤酒花矮化类病毒标准样品对该芯片进行验证,结果表明所建立的属级芯片可以特异性检测啤酒花矮化类病毒,可检测到2ng/μL的总RNA。该芯片可用于啤酒花矮化类病毒属类病毒的筛查,为该属类病毒的检疫与防控提供技术支撑。     

啤酒花矮化类病毒实时荧光定量RT-PCR检测方法的建立与应用

为建立一种快速、灵敏、特异的定量检测啤酒花矮化类病毒(Hop stunt viroid,HSVd)的实时荧光定量RT-PCR (RT-qPCR)方法,设计了2对引物及特异探针,体外转录制备了RNA标准品,绘制标准曲线,并对该方法的特异性、灵敏度和重复性进行评估.建立的定量标准曲线Ct值与模板拷贝数对数之间呈良好的线性关系,相关系数R2为0.9988,扩增效率为95％;该方法的特异性好,与啤酒花潜隐类病毒(HLVd)、葡萄黄斑类病毒1(GYSVd-1)、葡萄黄斑类病毒2(GYSVd-2)和桃潜隐花叶类病毒(PLMVd)均无交叉反应;灵敏度为1.0×102拷贝/μL,比普通RT-PCR高10倍;试验内及试验间重复性试验的变异系数均小于3％.研究表明该方法适用于实际样品中HSVd的快速定量检测.     

福建三明地区柑橘病毒类病原种类的鉴定及检测

为了明确福建省三明地区柑橘病毒类病原(病毒和类病毒)种类,利用RT-PCR技术对其进行了鉴定和检测,并对其检出率进行了分析。结果表明,从207份柑橘叶片样品中检出柑橘衰退病毒(citrus tristeza virus, CTV)、柑橘黄化脉明病毒(citrus yellow vein clearing virus, CYVCV)、柑橘叶斑驳病毒(citrus leaf blotch virus, CLBV)和蚜虫致死麻痹病毒(aphid lethal paralysis virus, ALPV)等4种病毒以及柑橘曲叶类病毒(citrus bent leaf viroid, CBLVd)、啤酒花矮化类病毒(hop stunt viroid, HSVd)、柑橘矮化类病毒(citrus dwarfing viroid, CDVd)、柑橘类病毒Ⅴ(citrus viroidⅤ, CVdⅤ)和柑橘类病毒Ⅵ(citrus viroidⅥ, CVdⅥ)等5种类病毒。其中,CTV、CYVCV、CLBV和ALPV的检出率分别是71.01%、66.67%、0.97%和6.28%,CBLVd、HSVd、C...     

侵染肥城桃的病毒和类病毒的分子检测与鉴定

为明确山东肥城桃种植区桃树上主要存在的病毒和类病毒及其发生情况,采集具有花叶、斑驳和皱缩典型症状的肥城桃样品,提取叶片总RNA后,分别选用桃树上已报道的啤酒花矮化类病毒Hopstuntviroid(HSVd)、桃潜隐花叶类病毒Peach latent mosaic viroid(PLMVd)、苹果褪绿叶斑病毒Apple chlorotic leaf spot virus(ACLSV)、樱桃锉叶病毒Cherry rasp leaf virus(CRLV)、桃花叶病毒Peach mosaic virus(PMV)、李属坏死环斑病毒Prunus necrotic ringspot virus(PNRSV)、李痘病毒Plum pox virus(PPV)、李矮缩病毒Prunus dwarf virus(PDV)、樱桃绿环斑驳病毒Cherry green ring mottle virus(CGRMV)、杏假褪绿叶斑病毒Apricot pseudo-chlorotic leaf spot virus(APCLSV)、李树皮坏死茎纹孔伴随病毒Plum bark necrosis stem pitting-associated virus(PBNSPaV)和小樱桃病毒1号Little cherry virus 1(LchV1)的特异性引物进行RT-PCR检测。PCR结果显示仅HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV的扩增产物中得到了预期大小的目的片段,将目的片段克隆测序后,经NCBI BLAST比对发现,山东肥城桃分离物HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV与GenBank已报道分离物序列一致性均达90%以上。表明山东肥城桃已感染HSVd、PLMVd 2种类病毒和ACLSV、PNRSV、PBNSPaV 3种病毒。     

白术矮化病毒病病原的分子鉴定和部分序列分析

 本试验在生物学接种的基础上,利用非序列依赖性PCR扩增(sequence-independent amplification,SIA)对白术矮化病毒病病原进行了分子鉴定,序列测定及分析。结果发现,具有矮化症状的白术为黄瓜花叶病毒(Cucumber mosaic virus,CMV)所侵染。为明确CMV白术分离物(CMV-Am)的分类地位,本研究进一步克隆了CMV-Am的外壳蛋白基因(CP)和移动蛋白基因(MP)全序列。序列比对分析表明,CMV-Am与CMV亚组ⅠB中株系XJ2序列同源性最高,核苷酸、氨基酸序列同源性分别为98.9%、99.1%。氨基酸序列同源性聚类分析表明,CMV-Am与中国大多数CMV分离物一样,属于CMV亚组ⅠB。     

Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.     

Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.     

Peach red marbling and peach sooty ringspot, two new virus-like degenerative diseases of Prunus

More than 600 Prunus samples were examined by using a nonradioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd). Prunus salicina and Prunus armeniaca appeared to be better hosts than Prunus persica . The weak viroid concentration in flowers and young leaves of peach trees growing in the field did not permit its detection in such samples. The diagnosis was more reliable (about 85%) with bark and leaves aged 4 months and more, from regrowths of GF 305 peach seedlings inoculated and kept in the greenhouse. Detection of HSVd in leaves and bark of apricot and Japanese plum plants aged 3 months or more also proved reliable (about 80% and 90%, respectively). HSVd could be transmitted in apricot, peach and plum nucleic acid preparations to GF 305 peach seedlings by repeated stem slashing, and to cherries ( Prunus avium and Prunus serrulata ) by approach grafting with an infected P. salicina source. The viroid was eliminated from 18% of the clones obtained after thermotherapy.
In the course of this study, 25 selected Prunus accessions suspected to be infected by unusual diseases were analysed by hybridization with a HSVd-specific probe and by indexing on GF 305 peach seedlings in the greenhouse. Fifteen of these accessions were found to be infected by HSVd, 19 induced reddish marbling, and four induced small blackish spots on the leaves aged about 4 months. Repeated assays showed that these foliar symptoms were not caused by the viroid. Peach red marbling (PRMa) has not been associated with any known virus and seems to be caused by an infectious agent not yet described. That could also be the case with the agent of peach sooty ringspot (PSRS). PRMa and PSRS symptoms were reproduced by grafting and indexing, and their causal agents eliminated by thermotherapy in a significant fraction of the treated plants. They behave like viral agents and can infect the different Prunus species studied.     

Molecular Characterization of an Almond Isolate of Hop Stunt Viroid (HSVd) and Conditions for Eliminating Spurious Hybridization in its Diagnosis in Almond Samples

Hop stunt viroid (HSVd) has a wide range of hosts including herbaceous and woody plants. Recently, HSVd was demonstrated to infect almond trees (Astruc et al., 1996). In this work, we present the molecular characterization of an almond HSVd isolate and report on the problems encountered in the diagnosis of HSVd in almond tissue through dot-blot non-radioactive hybridization procedures. False positives were eliminated through incubation of the membranes with RNase at high ionic strength after hybridization. Further experiments which included Northern hybridization, RT-PCR coupled to Southern hybridization, and cloning and sequencing suggested that spurious hybridization signals were due to host RNAs with sequence similarity to HSVd. Genetic characterization of the almond isolate demonstrated the existence of two new HSVd sequence variants named HSVd.alm1 and HSVd.alm2. The changes of HSVd.alm1 and HSVd.alm2 were located at residues variable among HSVd sequences, in loops on the left part of the rod-like molecule that includes the pathogenic (P) domain. Multiple alignments with all the available HSVd sequences and subsequent phylogenetic analyses revealed that the two new almond sequence variants were included in the previously described Prunus group of HSVd sequences.     

豇豆退绿斑驳病毒(CCMV)突变体的混合侵染研究

 本研究采用PCR点突变的方法,对豇豆退绿斑驳病毒(CCMV) RNA3的cDNA克隆进行突变,突变体TP7是在外壳蛋白基因之前突变形成一个独特的Sal I酶切点;TP8是在3a基因之前突变形成一个独特的Bam H I酶切点;AG 1是在外壳蛋白基因之后突变形成一个独特的Not I酶切点。TP7、TP8、AG1体外转录产物分别与CCMV RNA1、RNA2的c DNA克隆的体外转录产物混和后接种豇豆,结果表明,接种后第6 d TP7植株首先表现出症状,9d后TP8、AG1和野生型CCMV接种的植株表现出症状,TP7的症状发展最快。
采用TP7、TP8、AG1接种20 d的毒源植株,按TP7+TP8、TP7+AG1、TP8+AG1等量混合汁液接种豇豆,接种20 d,通过RT-PCR分析接种植物,在TP7+TP8和TP7+AG1混合侵染中,TP7在混合侵染中占优势,在TP8+AG1的混合侵染中,TP8和AG1的种群十分接近。     

Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.