应用实时荧光定量RT-PCR高效检测葡萄病毒B

 本研究建立了葡萄病毒B的 SYBR GreenⅠ实时荧光定量RT-PCR(RT-qPCR)检测技术。该技术标准曲线扩增效率102.4%,相关系数0.999,最低检测限达10^-4倍稀释cDNA,灵敏度为常规RT-PCR的100倍。重复性试验组内和组间变异系数分别为0.00%~0.65%和0.02%~2.00%,表明检测稳定性好。该技术对田间葡萄样品检测适用范围广,对枝条和老叶柄检测效果最好,冬季枝条和春夏秋季所有老叶柄样品检出率均为100%,与常规RT-PCR检测结果一致。对于其他季节或部位样品,RT-qPCR检出率(43% ~74%)则普遍高于常规RT-PCR(5% ~71%),特别是春季样品和春夏秋季所有嫩叶样品,检出率比常规RT-PCR分别高31% 和38%。对来自我国13个省21个品种的52份田间葡萄样品检测结果表明, RT-qPCR共检测到6个样品为阳性,检出率11.5%,为常规RT-PCR(检出率5.8%)的2倍。     

应用实时荧光定量RT-PCR高效检测葡萄病毒B

 本研究建立了葡萄病毒B的 SYBR GreenⅠ实时荧光定量RT-PCR(RT-qPCR)检测技术。该技术标准曲线扩增效率102.4%,相关系数0.999,最低检测限达10^-4倍稀释cDNA,灵敏度为常规RT-PCR的100倍。重复性试验组内和组间变异系数分别为0.00%~0.65%和0.02%~2.00%,表明检测稳定性好。该技术对田间葡萄样品检测适用范围广,对枝条和老叶柄检测效果最好,冬季枝条和春夏秋季所有老叶柄样品检出率均为100%,与常规RT-PCR检测结果一致。对于其他季节或部位样品,RT-qPCR检出率(43% ~74%)则普遍高于常规RT-PCR(5% ~71%),特别是春季样品和春夏秋季所有嫩叶样品,检出率比常规RT-PCR分别高31% 和38%。对来自我国13个省21个品种的52份田间葡萄样品检测结果表明, RT-qPCR共检测到6个样品为阳性,检出率11.5%,为常规RT-PCR(检出率5.8%)的2倍。     

应用常规RT-PCR和荧光定量RT-PCR检测柑桔衰退病毒

 根据柑桔衰退病毒(CTV)P20基因序列设计cquctv9/cquctv10特异引物对,以柑桔RNApolymeraseⅡ基因作为内参照,建立了柑桔衰退病的常规RT-PCR快速检测体系;依据柑桔衰退病毒P20基因序列设计cquctv1/cquctv2特异引物对和TaqMan探针cquctvp1,建立了柑桔衰退病荧光定量RT-PCR快速检测体系。常规RT-PCR检测下限是含有CTV的50pg总RNA,荧光定量RT-PCR法的检测下限是2fg纯CTV片段。荧光定量RT-PCR的灵敏度相对比常规RT-PCR高100倍。利用常规RT-PCR和荧光定量RT-PCR体系对从2005年3月到2006年7月采自田间的样品进行检测,结果表明,2种检测体系都有很好的特异性和准确性;对183个田间柑桔苗木样品带毒率检测结果表明,荧光定量RT-PCR检出率为(82.5%),比常规RT-PCR检出率(73.2%)高。     

‘阳光玫瑰’葡萄病毒小RNA测序鉴定及RT-PCR检测

 ‘阳光玫瑰’是我国从日本引进的葡萄优良品种。为了明确我国‘阳光玫瑰’葡萄病毒病的病原,本研究采用小RNA测序技术对2株显症和无症状的‘阳光玫瑰’葡萄样品进行病毒鉴定结果显示:显症样品中测定到8种病毒,其中包含葡萄蚕豆萎蔫病毒(Grapevine fabavirus, GFabV)和灰比诺葡萄病毒(Grapevine Pinot gris virus, GPGV);无症状样品中测定到3种葡萄病毒。对46个‘阳光玫瑰’样品进行14种葡萄病毒的RT-PCR检测,结果表明:‘阳光玫瑰’葡萄带毒率较高,病毒复合侵染情况普遍;显症样品中,GFabV检出率为88.2%,GPGV和葡萄浆果内坏死病毒(Grapevine berry inner necrosis virus,GINV)检出率为64.7%和29.4%,均明显高于无症状样品(13.8%和10.3%)。本研究旨在探明‘阳光玫瑰’葡萄携带病毒的种类和侵染状况,为其病毒病防控及病毒脱除奠定基础。     

‘阳光玫瑰’葡萄病毒小RNA测序鉴定及RT-PCR检测

 ‘阳光玫瑰’是我国从日本引进的葡萄优良品种。为了明确我国‘阳光玫瑰’葡萄病毒病的病原,本研究采用小RNA测序技术对2株显症和无症状的‘阳光玫瑰’葡萄样品进行病毒鉴定结果显示:显症样品中测定到8种病毒,其中包含葡萄蚕豆萎蔫病毒(Grapevine fabavirus, GFabV)和灰比诺葡萄病毒(Grapevine Pinot gris virus, GPGV);无症状样品中测定到3种葡萄病毒。对46个‘阳光玫瑰’样品进行14种葡萄病毒的RT-PCR检测,结果表明:‘阳光玫瑰’葡萄带毒率较高,病毒复合侵染情况普遍;显症样品中,GFabV检出率为88.2%,GPGV和葡萄浆果内坏死病毒(Grapevine berry inner necrosis virus,GINV)检出率为64.7%和29.4%,均明显高于无症状样品(13.8%和10.3%)。本研究旨在探明‘阳光玫瑰’葡萄携带病毒的种类和侵染状况,为其病毒病防控及病毒脱除奠定基础。     

我国部分地区沙地葡萄茎痘相关病毒分离物外壳蛋白序列变异分析

为明确我国葡萄中沙地葡萄茎痘相关病毒（GRSPaV）的感染情况及病毒外壳蛋白（coat protein,CP）基因的变异特点,从而为其致病性、病害的防治以及抗病毒基因工程等研究提供依据,本研究对采自我国16个省市自治区的65个葡萄品种305株葡萄样品中的GRSPaV进行RT-PCR检测,根据地区与品种差异选取了24个阳性样品进行cp基因克隆与测序分析,并对不同RNA提取方法进行了比较。结果显示,114株样品被GRSPaV侵染,平均带毒株率为37.4%;分离物间及同一分离物不同克隆间的序列差异较大,从24个分离物克隆获得的37条cp基因序列与来源于不同国家的12个GRSPaV分离物的核苷酸序列同源性为80.5%~99.7%,氨基酸序列同源性为88.8%~100%;各个分离物的遗传距离无明显地域差异;SiO₂吸附法比SDS法和CTAB法更适宜葡萄样品RNA的提取。     

柑橘黄化脉明病毒巢式RT-PCR检测方法的建立及应用

为探索柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)引起的柑橘新病害—柑橘黄脉病的早期快速检测技术,针对CYVCV核酸结合蛋白基因的保守序列设计2对特异性引物,通过优化退火温度,建立了CYVCV巢式RT-PCR检测方法,并对采自不同柑橘品种的54个CYVCV疑似样品进行了检测。结果表明,CYVCV巢式RT-PCR检测中,以55℃和60℃分别作为第1轮和第2轮扩增的退火温度时检测效果最佳;该方法检测样品中病毒总核酸的最低浓度为2.40μg/L,灵敏度较RT-PCR提高100倍。在CYVCV疑似样品检测中,巢式RT-PCR和RT-PCR的阳性检出率分别为59.26%和57.41%,前者更适用于检测不同来源的CYVCV。当尤力克柠檬、锦橙北碚-447、天草和台湾椪柑嫁接接种CYVCV后,巢式RT-PCR比RT-PCR提前10~30 d检测出病毒。表明所建立的CYVCV巢式RT-PCR检测方法适用于田间病树的早期诊断。     

4种葡萄卷叶伴随病毒多重RT-PCR检测

 葡萄受卷叶伴随病毒侵染后,树势减弱,抗逆性变差,果穗着色不良,成熟期推迟,含糖量降低。目前已报道11种葡萄卷叶伴随病毒(Grapevine leafroll-associated virus,GLRaV)。为提高检测效率,降低检测费用,本文在研究单个卷叶伴随病毒RT-PCR检测技术基础上,对4种葡萄卷叶伴随病毒的多重RT-PCR模板浓度、引物浓度和退火温度进行优化,建立了同时检测葡萄卷叶伴随病毒-1(GLRaV-1)、葡萄卷叶伴随病毒-3(GLRaV-3)、葡萄卷叶伴随病毒-4(GLRaV-4)和葡萄卷叶伴随病毒-5(GLRaV-5)的多重RT-PCR技术体系。模板浓度、引物浓度、Taq DNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变延伸时间和dNTP浓度对检测结果影响较小。对4种葡萄卷叶伴随病毒的PCR产物进行克隆和测序,扩增基因片段与GenBank中登录的基因序列同源性为95%~99%。所建立的多重RT-PCR技术检测田间样品效果良好。     

新疆三种葡萄病毒RT-PCR检测及序列分析

为研究新疆葡萄中沙地葡萄茎痘伴随病毒(Grapevine rupestris stem pitting associated virus,GRSPa V)、葡萄斑点病毒(Grapevine fleck virus,GFk V)及葡萄病毒A(Grapevine virus A,GVA)的发生情况和新疆分离株系统进化关系,分别克隆3种病毒新疆分离株部分基因区域,应用RT-PCR对新疆64份葡萄样品中上述3种病毒进行检测,并进行系统进化分析。结果显示,GRSPa V、GFk V和GVA的检出率分别为31.3%、62.5%和25.0%。新疆GRSPa V分离株(KJ801847)与美国GRSPa V分离株(AY368590)同源性达96.59%;新疆GFk V分离株(KJ801846)与日本GFk V分离株(AB222861)及中国辽宁GFk V分离株(JF927942)的同源性分别为91.70%和91.03%;新疆GVA分离株(KJ801845)与波兰GVA分离株(JN860997)同源性为93.88%,与中国四川GVA分离株(HQ671655)及辽宁GVA分离株(FJ445220)的同源性分别为92.92%和89.53%。表明3种葡萄病毒在新疆发生比较普遍,且新疆分离株与国内其它地方的分离株存在较大差异。     

葡萄卷叶伴随病毒1号和3号宁夏分离物部分基因序列分析

为明确葡萄卷叶伴随病毒(GLRaVs)在宁夏贺兰山东麓酿酒葡萄上的侵染状况,采用RT-PCR技术对40份酿酒葡萄样品中的GLRaV-1~GLRaV-5进行了外壳蛋白(CP)、复制酶(RdRp)和热激蛋白(HSP70)基因序列的克隆和分析。检测结果表明,在所检测的5种病毒中,除GLRaV-2和GLRaV-4未检测到外,GLRaV-1和GLRaV-3的检出率最高,分别为20.0%和32.5%,GLRaV-5的检出率仅为5.0%;有6个样品存在GLRaV-1和GLRaV-3两种病毒复合侵染。序列分析表明,GLRaV-1宁夏分离物的部分CP基因序列长度为232nt,其两种分离物间的核苷酸序列同源率为90%,与已报道的国内外其他分离物CP基因序列相比,其同源率为90%~99%;GLRaV-3宁夏分离物的CP基因序列长度为942nt,其两种分离物间的核苷酸序列同源率为40%,与已报道的国内外其他分离物CP基因序列相比,其同源率为40%~99%;GLRaV-3宁夏分离物的RdRp基因序列长度为683nt,其各分离物间的核苷酸序列同源率为90%以上,与已报道的国内外其他分离物RdRp基因序列相比,其同源率为90%~99%;GLRaV-3宁夏分离物的HSP70基因序列长度为546nt,其两种分离物间的核苷酸序列同源率为96%,与已报道的国内外其他分离物HSP70基因序列相比,其同源率为96%~99%。     

Viruses and virus diseases of grapevine in Tunisia

Mature canes were collected from vines in the main grapevine-growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother-plant plots, to assess the presence of virus and virus-like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll-associated 3 closterovirus (GLRaV-3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll-associated 1 closterovirus (GLRaV-1, 36.8%), grapevine leafroll-associated 2 closterovirus (GLRaV-2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll-associated 7 closterovirus (GLRaV-7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother-plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother-plant plots, the prevailing viruses were GLRaV-3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV-1 (17%) and GLRaV-2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

Grapevine leafroll-associated viruses and grapevine virus A in selected Vitis vinifera cultivars in northern Italy

The occurrence of grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine virus A (GVA) was demonstrated in a viticultural region of northern Italy (Emilia-Romagna) using immunoelectron microscopy. Virus incidence was subsequently assessed using ELISA. A total of 60.6% of the 150 clone selections tested, from 18 local Vitis vinifera cultivars, were found to be infected. ELISA did not reveal the presence of grapevine leafroll-associated virus 2 (GLRaV-2) or grapevine leafroll-associated virus 5 (GLRaV-5). GLRaV-1, GLRaV-3 and GVA were found individually and in various combinations. The most common findings were GLRaV-1 alone (25.3%) and associated with GVA (33%). Serological data confirmed that the majority (91%) of the clones known to be affected by grapevine leafroll (GLR), on its own or in association with rugose wood (RW), contained viruses. On the other hand, where the RW phenomenon was present on its own, only 40% of these clones were ELISA-positive. The implications for the biology of GLR and RW are discussed and the complex aetiology of these grapevine diseases is confirmed.     

葡萄卷叶伴随病毒2号和3号辽宁分离物部分基因组的序列分析

 将采自辽宁兴城地区在生长期间具典型卷叶病症状的金星无核(Venus Seedless)葡萄品种休眠枝条,用RT-PCR检测4种葡萄卷叶伴随病毒(Grapevine leafroll-associated viruses,GLRaVs),扩增得到了葡萄卷叶伴随病毒2号(GLRaV-2)和葡萄卷叶伴随病毒3号(GLRaV-3)两种病毒的主要外壳蛋白(major coat protein,CP)基因的完整序列(GenBank登录号分别为FJ786017和FJ786016)。这表明该葡萄植株受到了GLRaV-2和GLRaV-3辽宁分离物(GLRaV-2-LN和GLRaV-3-LN)的复合侵染。根据检测结果,克隆了GLRaV-2-LN基因组3'端CPm (minor capsid protein)、p19(19-kDa protein)和p24(24-kDa protein)基因(GenBank登录号分别为FJ786018、FJ786019和FJ786018)。序列分析表明,GLRaV-3-LN的CP基因全长942 nt,与已报道的国内外其它分离物CP基因全序列相比,核苷酸序列同源性为89.8%~91.8%,由此推导的氨基酸序列同源性为94.9%~97.4%。GLRaV-2-LN的CP、CPm、p19和p24基因全长分别为597 nt、672 nt、486 nt和618 nt。与国外报道的几个分离物的相应蛋白基因全序列相比,核苷酸序列同源性分别为88.3%~100.0%、78.7%~99.9%、75.1%~99.4%和87.5%~99.5%;由此推导的氨基酸序列同源性分别为92.9%~100.0%、89.2%~100.0%、73.9%~99.4%和89.3%~99.0%。