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本研究合成了苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的生物素标记cDNA探针,对斑点杂交和免疫印迹杂交检测这3种病毒的效果进行了分析。以含ACLSV和ASGV克隆片段的大肠杆菌菌液为样品时,斑点杂交检测灵敏度分别为80 cfu/μL和50 cfu/μL。以离体培养砂梨植株为材料,采用斑点杂交法检测砂梨离体培养植株粗提液中的3种病毒,均产生强的杂交信号,且特异性好。比较斑点杂交和ELISA检测病毒含量相对较低的热处理再生植株中ASGV和ACLSV,结果表明斑点杂交具有较高的灵敏度。组织印迹杂交检测砂梨离体植株ASGV和ACLSV的结果显示,这2种病毒在离体植株的各部位均有分布,自基部至茎尖各部位印迹均产生很强的杂交信号。 相似文献
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库尔勒香梨主要病毒多重RT-PCR检测技术研究 总被引：11，自引：1，他引：10
利用nad5基因作为内标系统,研究建立了库尔勒香梨上苹果茎痘病毒(ASPV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎沟病毒(ASGV)等的RT-PCR检测技术,在此基础上研究建立了库尔勒香梨多重RT-PCR内标检测系统。并对回收的特异片段进行了克隆、鉴定和测序。测序结果表明:库尔勒香梨上的ACLSV与GenBank中D14996序列(日本苹果上的ACLSV分离物)中的6860~7536bp片段有116个碱基的差异,序列相似性为83.75%;库尔勒香梨上的ASPV与GenBank中D21828序列(德国梨脉黄病毒相关分离物)中的8869~9238bp片段有72个碱基的差异,序列相似性为79.5%;库尔勒香梨上的ASGV与GenBank中AB004063序列(日本ASGV分离物)中的6039~6311bp片段有21个碱基的差异,序列相似性为92.3%。 相似文献
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Apple mosaic virus (ApMV, genus Ilarvirus) was detected in pears, a previously non-reported virus host. No symptoms were visible on the hosts leaves. Seventeen out of 22 randomly selected pear trees in Italy (Lombardy) and in three regions in the Czech Republic were ApMV-infected. All nine newly sequenced ApMV isolates from pears had a 15-nucleotide insertion in the capsid protein gene in identical position of that of apple isolates compared with isolates from hop and prunes. The insertion is the most prominent (but not essential) modification of the capsid protein gene, which results in a phylogenetic separation of ApMV isolates into three clusters. Sequence analysis data of an additional 15 isolates revealed a sequence correlation with kernelled fruit trees (apple and pear). 相似文献
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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
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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
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山楂是我国重要的果树,但山楂病毒的相关研究较少。本研究通过高通量测序及生物信息学分析首次在山楂样品中发现苹果茎痘病毒(apple stem pitting virus,ASPV)。利用RT-PCR和cDNA末端快速扩增技术对ASPV山楂分离物全长序列进行扩增,结果表明,ASPV山楂分离物基因组全长由9 290个核苷酸组成,包含5个开放阅读框(open reading frames,ORFs)。ORF1编码与病毒复制相关的蛋白-RNA依赖的RNA聚合酶(RNA dependent RNA polymerase,RdRp),其在氨基酸水平上与ASPV苹果和梨分离物的RdRp序列相似性分别为72.8%~80.9%和81.7%~92.8%;ORFs 2~4表达与病毒移动相关的三基因块(triple gene block 1~3,TGB 1~3),其与印度苹果N分离物(LM999967)的氨基酸序列相似性最高,为92.9%~98.2%;ORF5编码外壳蛋白(coat protein,CP),与苹果和梨分离物CP的序列相似性较低,分别为64.8%~88.4%和69. 8%~92.2%,高于目前凹陷病毒属病毒新种的划分界限。将ASPV山楂分离物的全长核苷酸序列与其他ASPV分离物的全长序列进行比对,构建系统发育树,结果表明,ASPV山楂分离物与巴西苹果分离物BR-Brae2的亲缘关系最近,但两者核苷酸序列相似性较低(81.5%),说明与其他分离物相比该分离物具有较大的变异性。综上,本研究初步验证了山楂是ASPV的重要自然寄主,并首次获得了ASPV山楂分离物的全长基因组序列,为山楂病毒病诊断和防控策略的制定提供参考。 相似文献
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In the last 10 years, more than a thousand almond trees have been analysed by the DAS-ELISA method in the Valencia region. The most frequent virus infecting unselected almond trees was prune dwarf ilarvirus (PDV) (62%), followed by prunus necrotic ringspot ilarvirus (PNRSV) (36%), apple mosaic ilarvirus (ApMV) (14%) and apple chlorotic leafspot trichovirus (ACLSV) (2%). Infection levels of selected trees were 26% for PDV, 15% for PRSV, 0% for ApMV and 5% for ACLSV. 相似文献
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E. Choueiri C. Haddad N. Abou Ghanem-Sabanadzovic F. Jreijiri S. Issa A. T. Saad B. Di Terlizzi V. Savino 《EPPO Bulletin》2001,31(4):493-497
Field surveys were carried out in the main peach-growing areas of Lebanon to assess the presence and distribution of viruses and viroids in commercial orchards. Field inspections were made in spring and summer 2000 to observe symptoms of virus and viroid diseases respectively. In total, 950 trees in 95 commercial plantings from three different regions of Lebanon (Bekaa Valley, Mount Lebanon and north Lebanon) were surveyed and sampled. Immunoenzymatic tests (DAS-ELISA) were used to ascertain the presence of the following: Prunus necrotic ring spot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Apple mosaic ilarvirus (ApMV), Apple chlorotic leaf spot trichovirus (ACLSV), Plum pox potyvirus (PPV), Tomato ringspot nepovirus (ToRSV) and Strawberry latent ringspot nepovirus (SLRSV). Peach latent mosaic pelamoviroid (PLMVd) and Hop stunt hostuviroid (HSVd) were identified by molecular hybridization. About 25% of the tested samples were infected by one or more viruses. In particular, the prevailing virus was PNRSV (61.2% of infection), followed by ACLSV (27.1%), PDV (22.4%) and ApMV (2.1%). Mixed infections were about 13%. ToRSV, SLRSV and PPV were not found. HSVd was apparently absent, whereas PLMVd was identified in 34% of the samples examined. This viroid prevailed in certain areas of Mount Lebanon in both native and foreign cultivars. 相似文献
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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
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M. M. Mathioudakis V. I. Maliogka C. I. Dovas S. Paunovi&#; N. I. Katis 《Plant pathology》2009,58(2):228-236
In this study a spot nested RT-PCR assay was developed for the detection of Apple stem pitting virus (ASPV). A one step RT-PCR for the generic detection of foveaviruses using degenerate primers that target a conserved region of the RNA-dependent RNA polymerase (RdRp) gene was followed by a nested PCR that amplifies a 312 bp ASPV specific product. The method is rapid, simple and displays high sensitivity and broad detection range, overcoming the virus molecular variability. The optimum sampling conditions for reliable virus detection were also investigated. ASPV was detected throughout the year in different plant tissues of affected trees, thus the method could be used for routine screening and in certification schemes of pome fruits. ASPV was detected in quince orchards in Greece in all trees that were tested, showing a fruit deformation disorder. Sequencing and phylogenetic analysis of amplicons generated by RT-PCR from plant tissue affected with the deformation disease indicated that the agent responsible was a variant of ASPV. 相似文献
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Zhong-Bin Wu You-Xiu Zheng Chiou-Chu Su Chung-Jan Chang Fuh-Jyh Jan 《European journal of plant pathology / European Foundation for Plant Pathology》2010,128(1):71-79
A putative virus-induced disease of pear (Pyrus pyrifolia var. Hengshen) showing symptoms of reduced size of foliage and leaf distortion was observed in orchards in central Taiwan
in 2004. The sap of symptomatic leaf samples reacted positively to an antiserum against Apple stem grooving virus (ASGV). Two virus cultures, designated as TS1 and TS2, were isolated from symptomatic pears. Flexuous filamentous virions
of ∼ 12 × 600 nm were observed in symptomatic pear leaves and purified virus preparations. Results of back inoculation of
pear seedlings with TS1 revealed that ASGV was the causal agent of the disease. Sequence analyses of the cloned coat protein
(CP) genes of TS1 and TS2 shared 88–92.4% nucleotide and 90.7–97.1% amino acid identities with those of other ASGV isolates
available in GenBank. The polyclonal antibody generated against ASGV TS1 has been routinely used for the detection of the
ASGV-infection in the imported pear scions for quarantine purpose via enzyme-linked immunosorbent assays (ELISAs). One of
1,199 samples of pear scions imported from Japan during 2005–2007 was identified as ASGV-positive and the virus was designated
as AGJP-22. The CP gene amplified from this AGJP-22 shared 97.9–98.3% amino acid identities to those of the domestic isolates
and they were closely related phylogenetically. To date, these data present for the first time conclusive evidence revealing
that ASGV is indeed the causal agent of the pear disease displaying symptoms of reduced size of foliage and leaf distortion
in Taiwan. 相似文献
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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
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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