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1.
为了探明山东泰安患有"花叶病"的甜樱桃树体内感染病毒的种类,本研究以嫁接在3种不同砧木上的甜樱桃品种‘红灯’叶片为试验材料,提取植物样本总RNA,采用随机六聚体引物反转录,选取10种甜樱桃病毒作为检测对象,根据各病毒基因组序列设计特异引物,进行RT-PCR检测。结果显示,10种甜樱桃待检病毒中,5种病毒检测结果呈阳性,分别为PNRSV(Prunus necrotic ringspot virus,PNRSV)、PDV(Prune dwarf virus,PDV)、CVA(Cherry virus A,CVA)、CGRMV(Cherry green ring mottle virus,CGRMV)、LChV-2(Little cherry virus-2,LChV-2),各病毒检出率较高。各"花叶病"甜樱桃样品均至少同时感染2种病毒,多病毒复合感染比例较高。 相似文献
2.
侵染肥城桃的病毒和类病毒的分子检测与鉴定 总被引：1，自引：0，他引：1
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。 相似文献
3.
河南甜樱桃病毒病害调查及病原检测 总被引：1，自引：0，他引：1
在河南省郑州市、巩义市、荥阳市、新郑市选择具有代表性的甜樱桃生产园对病毒病发生情况进行调查,采集表现为疑似病毒病症状的样本65份,利用7种病毒引物进行RT-PCR检测。5种病毒检测结果呈阳性,分别是李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)、李矮缩病毒(Prune dwarf virus,PDV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃坏死锈斑病毒(Cherry necrotic rusty mottle virus,CNRMV)及樱桃病毒A(Cherry virus A,CVA);序列分析结果表明,5种病毒扩增片段与GenBank中注册的相应病毒核苷酸序列均具有较高的一致性;样本病毒检出率为100%,其中13份样本为单独侵染,其余52份样本均为多病毒复合侵染,占比高达80%,复合侵染比例随着侵染病毒种类的增多逐渐降低;病毒侵染组合与叶片表型症状无明显对应关系。 相似文献
4.
为了探讨SYBR Green Ⅰ实时定量RT-PCR技术在甜樱桃病毒粒子定量分析中的应用前景,以复合感染李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)、李矮缩病毒(Prune dwarf vi-rus,PDV)、樱桃病毒A(Cherry virus A,CVA)、樱桃小果病毒-2(Little cherry virus-2,LChV-2)的甜樱桃"红灯"Prunus avium cv.Red Lamp植株为研究对象,采用相对定量方法,分析各病毒的外壳蛋白基因的表达,用以指示病毒的增殖量。在花、幼叶、功能叶、衰老叶中均能检测到4个基因,但各基因表达量在各器官中存在差异。PNRSV-CP与CVA-CP表达模式相似,功能叶中明显高于其它器官,衰老叶中急剧降低。PDV-CP与LChV2-CP表达模式类似,幼叶中的表达量较高,功能叶片中较低。PNRSV-CP在花、功能叶中的表达显著高于其它3个病毒基因。LChV2-CP在各器官中的表达量均低于其余3个基因。该方法适用于植物组织内多种甜樱桃病毒增殖量的分析。 相似文献
5.
两种主要甜樱桃病毒RT-PCR检测方法的改进 总被引：5，自引：2，他引：3
李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)和李矮缩病毒(Prune dwarf virus,PDV)是较为常见且危害严重的两种植物病毒,1992年被我国列为进境检疫危险性有害生物,主要危害李属和蔷薇属植物.反转录-聚合酶链式反应检测法(RT-PCR)因操作简便且灵敏度高而备受青睐.利用该方法已分别从北京怀柔[1-2]及大连地区[3]栽培的桃、樱桃中检测到PNRSV和PDV两种病毒.由于PNRSV和PDV同属雀麦花叶病毒科等轴不稳环斑病毒属,且传播方式相似,往往同时发病.已有的RT-PCR检测方法,一次反转录操作仅能检测一种病毒,耗时长、效率低,急需建立新的RT-PCR检测体系.本试验采用随机六聚体引物进行反转录,对RT-PCR检测方法进行了改进. 相似文献
6.
环渤海湾地区甜樱桃小果病毒及樱桃病毒A的鉴定与调查 总被引：1，自引：0，他引：1
为了解环渤海湾地区甜樱桃[Cerasusavium (Linn) Moench]中樱桃病毒的发生情况,选取该地区代表性樱桃示范园中甜樱桃品种‘红灯’为研究对象,以生长期功能叶片为材料,根据樱桃小果病毒-1(Little cherry virus-1,LChV-1)、樱桃小果病毒-2(Little cherry virus-2,IChv-2)和樱桃病毒A(Cherry virus A,CVA)基因组序列设计特异性检测引物进行RT-PCR扩增,结果扩增出与LChV-2、CVA预期片段337 bp和652 bp大小相符的目的片段,测序结果与基因组(基因登录号NC-005065、NC-003689)对应片段一致性分别为89.0％、98.2％,未检测到LChV-1.本研究完成了对环渤海湾地区甜樱桃‘红灯’样品LChV-2、CVA的检测调查,LChV-2检出率43.1％,CVA检出率60.8％;在该地区首次发现LChV-2、CVA复合侵染植株,复合检出率37.3％0.结果表明该地区CVA发生较为普遍,可与LChv-2等其他病毒共同加重对甜樱桃的危害. 相似文献
7.
为了探讨SYBRGreenI实时定量RT—PCR技术在甜樱桃病毒粒子定量分析中的应用前景,以复合感染李属坏死环斑病毒（Prunusnecrotic ringspot virus,PNRSV）、李矮缩病毒（Prune Dwarf vi—rus,PDV）、樱桃病毒A（CherryvirusA,CVA）、樱桃小果病毒一2（Little cherry virus一2,LChV－2）的甜樱桃“红灯”PrunusaviumCV．RedLamp植株为研究对象,采用相对定量方法,分析各病毒的外壳蛋白基因的表达,用以指示病毒的增殖量。在花、幼叶、功能叶、衰老叶中均能检测到4个基因,但各基因表达量在各器官中存在差异。PNRSV-CP与CVA—CP表达模式相似,功能叶中明显高于其它器官,衰老叶中急剧降低。PDV-CP与LChV2一卯表达模式类似,幼叶中的表达量较高,功能叶片中较低。PNRSV-CP在花、功能叶中的表达显著高于其它3个病毒基因。LChV2一cP在各器官中的表达量均低于其余3个基因。该方法适用于植物组织内多种甜樱桃病毒增殖量的分析。 相似文献
8.
<正>樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)和桃潜隐花叶类病毒(Peach latent mosaic viroid,PLMVd)是桃树上的主要病原。在检测中发现,我国桃树病毒多以CGRMV与PLMVd复合侵染为主,侵染率较高。通过分子克隆,串联几个病毒核酸序列合成多聚探针,能同时检测多个病毒和类病毒(Torchetti et al.,2012)。目前尚无关于CGRMV单一探针和同时检测CGRMV与PLMVd二聚探针的报道。本研究通过设计带酶切位点的特异引物制备二聚cRNA探针,以期建立适用于田间果树样品中CGRMV和PLMVd的快速检测技术,为桃树病毒病害的防治提供理论基础。 相似文献
9.
北京月季病原病毒的高通量测序鉴定和RT-PCR检测 总被引：1，自引：1，他引：0
本研究利用高通量测序技术对北京地区的月季染病样品进行了病毒鉴定,通过序列比对和拼接获得了李属坏死环斑病毒(prunus necrotic ringspot virus,PNRSV)、苹果茎沟病毒(apple stem grooving virus,ASGV)、柑橘碎叶病毒(citrus tatter leaf virus,CTLV)、月季黄叶病毒(rose yellow leaf virus,RYLV)、月季黄花叶病毒(rose yellow mosaic virus,RoYMV)、月季潜隐病毒1(rose cryptic virus1,RCV1)和玫瑰黄脉病毒(rose yellow vein virus,RYVV)等7种已知病毒和2种未知病毒的序列信息。分别对PNRSV,ASGV和RYLV园博园分离物的外壳蛋白基因进行系统进化分析,结果表明PNRSV-YBY归属于PV32组,ASGV-YBY归属于Ⅰ组,RYLV-YBY为玫瑰叶畸形病毒(Rosa rugosa leaf distortion virus, RrLDV)的一个新的分离物。对中国农业大学校园、北京植物园、北京动物园和北京园博园的37份样品进行了PNRSV、ASGV和RYLV的RT-PCR检测,检出率分别为41.7%、44.4%和10.8%。本研究初步明确了侵染北京月季的病毒种类和侵染情况,为月季病毒病的检测和防控提供参考。 相似文献
10.
目前, 我国梅树上的病毒种类及发生情况仍不完全清楚。本研究从北京、武汉、南京和无锡的梅园中采集了64份疑似感染病毒的叶片样品, 通过RT-PCR和斑点杂交, 对7种病毒和2种类病毒进行了检测。共检测到6种病毒和1种类病毒。其中, 李属坏死环斑病毒(prunus necrotic ringspot virus, PNRSV)和桃潜隐花叶类病毒(peach latent mosaic viroid, PLMVd)为我国梅树上的首次检出。PNRSV、亚洲李属病毒2号(Asian prunus virus 2, APV2)、桃叶痘伴随病毒(peach leaf pitting-associated virus, PLPaV)的检出率高于30%。综合考虑病毒的分布及检出率, PLPaV、APV2、PNRSV和李树皮坏死茎痘伴随病毒(plum bark necrosis stem pitting-associated virus, PBNSPaV)是武汉、南京和无锡梅树上的主要病毒。此外, 通过克隆和测序, 获得了PLMVd和梅树病毒A(mume virus A, MuVA)的基因组, PLPaV的RNA1组分和PNRSV外壳蛋白(CP)基因序列。序列比较分析显示, 我国PLMVd梅分离物和PNRSV梅分离物与我国桃分离物亲缘关系最近, 表明PLMVd和PNRSV可能在梅和桃树间交互侵染;我国MuVA梅分离物序列与日本梅分离物序列的相似性高达98.56%;PLPaV梅分离物与我国桃分离物之间序列变异较大。上述结果不仅进一步明确了我国梅树上的病毒及类病毒种类和分布情况, 而且有助于深入了解它们的流行与传播。 相似文献
11.
A. Bouani M. Al Rwahnih N. Abou Ghanem-Sabanadzovic I. Alami M. Zemzami A. Myrta V. Savino 《EPPO Bulletin》2004,34(3):399-402
Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting. 相似文献
12.
Field surveys were carried out in the main stone-fruit-growing areas of East Anatolia (Turkey) to assess the sanitary status of varietal collections, mother blocks and commercial orchards. The presence of virus and virus-like diseases was ascertained by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, graft transmission to peach cv. GF305 and molecular hybridization tests. A total of 1019 samples was tested by ELISA (859 apricot, 120 cherry, 21 almond and 19 peach). The sanitary status of apricot was extremely satisfactory, as the infection level was less than 0.3%. Cherry and almond, however, showed 21% and 33% infection respectively. The viruses identified were apple chlorotic leaf spot trichovirus (ACLSV), prune dwarf ilarvirus (PDV) and prunus necrotic ringspot ilarvirus (PNRSV). The commonest virus was PDV. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV) and the nepoviruses tomato black ring (TBRV), raspberry ringspot (RpRSV), strawberry latent ringspot (SLRV), cherry leaf roll (CLRV), arabis mosaic (ArMV) and tomato ringspot (ToRSV) were not encountered. Peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) were not detected either. 相似文献
13.
Viruses and viroids of stone fruits in Syria 总被引：1，自引：0，他引：1
F. Ismaeil A. Myrta N. Abou Ghanem-Sabanadzovic S. Al Chaabi V. Savino 《EPPO Bulletin》2002,32(3):485-488
Field surveys were carried out in the main stone fruit-growing areas of Syria to evaluate the sanitary status of mother blocks, varietal collections and commercial orchards. The presence of virus and virus-like diseases was checked by enzyme-linked immunosorbent assay (ELISA), sap transmission to herbaceous hosts, testing on the woody indicators Prunus persica cv. GF 305 and Prunus serrulata cv. Kwanzan and dot-blot hybridization tests. A total of 1337 samples was tested by ELISA (444 apricot, 283 peach, 246 cherry, 222 almond and 142 plum). The overall mean infection rate was 13%, and the percentage infection level of single species was: peach 24%, cherry 16%, almond 13.5%, apricot 6%, plum 5%. The following viruses and viroids were detected: PNRSV, PDV, ACLSV, PPV, ApMV, PLMVd and HSVd 1 . 相似文献
14.
S. Massart Y. Brostaux L. Barbarossa V. César M. Cieslinska O. Dutrecq F. Fonseca R. Guillem A. Laviña A. Olmos S. Steyer T. Wetzel J. Kummert M. H. Jijakli 《European journal of plant pathology / European Foundation for Plant Pathology》2008,122(4):539-547
The operational capacity of a duplex RT-PCR method for simultaneous detection of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) has been established by nine European laboratories. A total of 576 samples from Prunus trees with known sanitary status, corresponding to 32 samples in two repetitions for each laboratory, were analysed. The
level of sensitivity achieved by the method was 98.3% for PDV and 90.4% for PNRSV. The specificity was 87.4% for PDV and 94.3%
for PNRSV. The unilateral 95% confidence intervals were calculated for all these values. Cohen’s Kappa coefficient of repeatability
and reproducibility of the technique indicated a strong agreement between data. Likelihood ratios were 7.50 (positive) and
0.02 (negative) for PDV. For PNRSV, the positive likelihood ratio was 15.00 while the negative likelihood ratio was 0.11.
In addition, post-test probabilities of infection were calculated to manage the risk associated with the routine use of this
method. This allows an accurate test result interpretation to facilitate the integration of this new technique into a certification
scheme. 相似文献
15.
M.E. Rott W. Jelkmann 《European journal of plant pathology / European Foundation for Plant Pathology》2001,107(4):411-420
For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus 相似文献
16.
Investigations were carried out in the main stone-fruit growing areas of Lebanon to assess the phytosanitary condition of commercial orchards. The presence of virus and virus-like diseases and their identification was ascertained through: (i) field surveys, (ii) sap transmission to herbaceous hosts, (iii) graft transmission to woody indicators; and (iv) ELISA and IEM tests. The mean infection level was 25%. ranging from 5% in apricot to 45% in cherry. The following viruses were identified: apple chlorotic leaf spot trichovirus (ACLSV), prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. Plum pox potyvirus (PPV), apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered. 相似文献
17.
Surveys were carried out in the main stone-fruit growing areas of Albania to assess the phytosanitary status of Prunus in conimercial orchards and varietal collections. The presence of virus and virus-like diseases and their identification was ascertained through field observations, sap transmission to herbaceous hosts, graft transmission to woody indicators, ELISA and IEM tests. The mean infection level was 42%. In particular, infections in apricot and almond were 12 and 16%, respectively, i.e. lower than in plum and cherry (47 and 56%, respectively). The following viruses were identified: plum pox potyvirus (PPV). apple chlorotic leaf spot trichovirus (ACLSV). prunus necrotic ringspot (PNRSV) and prune dwarf (PDV) ilarviruses. PPV infection was very severe in plum, and limited in apricot and peach. Apple mosaic ilarvirus (ApMV), and six nepoviruses tested for (SLRV, TBRV, RRV, CLRV, ArMV and ToRSV) were not encountered in Primus. 相似文献
18.
Field surveys were carried out in the main stonefruit-growing areas of Jordan to assess the sanitary status of varietal collections, mother plant blocks and commercial orchards. The presence of virus and virus-like diseases was determined by ELISA, sap transmission to herbaceous hosts, graft transmission to Prunus persica cv. GF 305 and P. serrulata cv. Kwanzan, and molecular hybridization tests. A total of 1312 samples was tested by ELISA (531 peach, 361 plum, 218 apricot, 135 almond and 67 cherry trees). The overall mean level of infection was about 14%, indicating an acceptable sanitary status as a whole, considering that no sanitary selection has ever been carried out in Jordan. The infection level of different species was: peach (18%), cherry (15%), almond (14%), apricot (11%) and plum (10%). The following viruses and viroids were identified: Plum pox potyvirus (PPV), Prunus necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Apple mosaic ilarvirus (ApMV), Apple chlorotic leaf spot trichovirus (ACLSV), Hop stunt viroid (HSVd) and Peach latent mosaic viroid (PLMVd). Most of these agents (ApMV, ACLSV, PLMVd and HSVd) are reported for the first time from Jordan. 相似文献