山东泰安甜樱桃“花叶病”树体症状观察及病毒检测

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。     

SYBR GreenⅠ实时定量RT-PCR技术在甜樱桃病毒定量分析中的应用

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。     

我国部分地区樱桃病毒病害初步调查和病原检测

对山东泰安、辽宁大连和北京的樱桃病毒病发生情况进行调查,发现8个果园/栽培区均有病毒病发生,主要症状为叶片皱缩、畸形、卷叶、花叶、植株矮缩等。采集20份样品,利用12种病毒的引物进行RT-PCR检测。结果表明,在样品中扩增出与樱桃病毒A(Cherry virus A,CVA)、李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),李矮缩病毒(Prune dwarf virus,PDV)、李树皮坏死与茎痘伴随病毒(Plum bark necrosis stem pitting-associated virus,PBNSPaV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃小果病毒-1(Little cherry virus-1,LChV-1)预期大小一致的目的片段;序列分析表明,与GenBank中注册所测的病毒核苷酸序列均具有较高的一致性。其中,大连、泰安和北京样品均检测到CVA;大连和北京样品中检测到PNRSV和PDV;北京样品中检测到PBNSPaV;大连苗木样品枝条中检测到CGRMV和LChV-1。这是在我国樱桃上首次检测到LChV-1。     

樱桃小果病毒1号胶体金免疫层析试纸的研发与应用

近年来，病毒病在甜樱桃主产区的发生呈上升趋势，制约了甜樱桃产业升级和发展。能够侵染甜樱桃的病毒种类中，樱桃小果病毒1号(little cherry virus 1,LChV-1)导致樱桃果实缩小，彻底丧失经济价值，对甜樱桃产量影响较大。LChV-1通常具有潜伏侵染的特性，在樱桃苗期难以通过症状进行判断。同时，多数品种对LChV-1敏感，因此该病毒的早期检测对甜樱桃的生产尤为重要。传统的病毒检测手段需要专业仪器，不能满足田间快速检测的需求。本研究获取了LChV-1外壳蛋白的多克隆抗体，并研发了一种能够准确检测LChV-1病毒的胶体金免疫层析试纸，确定了胶体金的最佳抗体标记浓度为0.24 mg/mL,检测线最佳重组蛋白浓度为0.25 mg/mL,质检线最佳羊抗兔IgG抗体浓度为0.04 mg/mL。运用该试纸检测只需10～15 min,检测时间短、成本低、结果容易判断，可用于田间快速检测。本研究为樱桃小果病的综合防控提供了有效的监测和检测手段。     

SYBR Green I实时定量RT-PCR技术在甜樱桃病毒定量分析中的应用

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。     

河南甜樱桃病毒病害调查及病原检测

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。     

甜樱桃李矮缩病毒(PDV) RT-PCR检测方法的优化与应用

以甜樱桃(Prunus avium L.)品种红灯的叶片为材料,提取总RNA,选用随机六聚体引物进行反转录合成cDNA,根据李矮缩病毒外壳蛋白基因设计2对特异引物,分别从感病样品中扩增出与预期片段大小相符的目的片段.通过对RT-PCR反应体系中引物、模板浓度和退火温度的优化,改进了现有的李矮缩病毒的RT-PCR检测方法,并成功用于山东泰安地区甜樱桃果园的病毒调查.另外,还可以扩增18 sRNA,实现对李矮缩病毒外壳蛋白基因表达的相对定量分析.     

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。     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

番茄斑萎病毒属6种病毒的多重RT-PCR检测

根据番茄斑萎病毒属(Tospovirus)中6种病毒S RNA上的N基因序列设计特异性引物,建立可同时检测这6种Tospovirus病毒的多重PCR体系。多重PCR扩增结果显示,番茄环纹斑点病毒(776 bp)、甜瓜黄斑病毒(505 bp)、鸢尾黄斑病毒(296 bp)、凤仙花坏死斑病毒(221 bp)、番茄斑萎病毒(175 bp)和番茄褪绿斑病毒(110 bp)均出现清晰的目标条带,各病毒的特异性引物不会对其他病毒产生非特异性扩增。本研究建立的6种Tospovirus病毒的多重PCR检测方法,有助于提高目标病毒的检测效率。     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

Determination of biological properties of cherry virus Trakiya and its incidence on sweet cherry (Prunus avium L.) in Bulgaria

Journal of Plant Diseases and Protection - The bi-cistronic virus cherry virus Trakiya was detected recently and is related to a group of unclassified viruses in the order Picornavirales, typically...     

北京怀柔地区樱桃上发现李属坏死环斑病毒

 李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)属雀麦花叶病毒科(Bromoviridae)等轴不稳环斑病毒属(Ilarvirus),是我国二类检疫性有害生物。PNRSV主要危害李属和蔷薇属植物。如,樱桃、桃、李、月季等。春季感染PNRSV的樱桃早期幼叶会出现深棕色坏死斑和线纹,叶片展开期坏死斑脱落造成穿孔症状。受PNRSV危害后,果树树势减弱,产量降低,甚至死树。我国学者调查陕西、辽宁和山东等省的果树病害时发现并报道了该病毒^[1~3]。     

Serological differences between red currant spoon leaf virus,virus isolates from Eckelrade-diseased cherry trees and the Scottish raspberry ringspot virus

Antisera were made to red currant spoon leaf virus (SLV), an isolate of the Scottish raspberry ringspot virus (RRV), and two virus isolates from Eckelrade-diseased cherry trees (EV). Different virus isolates, including one from Belgium, were tested against these antisera. The results indicate that we are dealing with a group of virus isolates with different antigenic properties. SLV is very closely related to RRV, being undoubtedly a strain of this virus. Dutch EV isolates differ from SLV and RRV and from each other, the Belgian isolate being closely related to one of the Dutch EV isolates. The serological differences found do not correspond with the geographical distances between the localities where the viruses were collected.Samenvatting Antisera werden bereid tegen het lepelbladvirus van rode bes (SLV), een isolatie van het Schotse raspberry ringspot-virus (RRV) en twee virusisolaties uit kersebomen met Eckelraderziekte (EV). Met behulp van deze antisera werden verschillende virusisolaties getoetst, waaronder één uit België van kers met Eckelraderziekte. De resultaten zijn vermeld in de tabellen 1 en 2. Ze duiden erop, dat we te maken hebben met een groep van virusisolaties met verschillende antigene eigenschappen. SLV is zeer nauw verwant aan RRV en is ongetwijfeld een stam van dit virus. Nederlandse EV-isolaties verschillen zowel van SLV en RRV, als van elkaar. De Belgische isolatie is nauw verwant aan één van de Nederlandse EV-isolaties. De grootte van de gevonden verschillen correspondeert niet met de geografische afstand tussen de plaatsen waar de virusisolaties werden verzameld.     

The location of virus-like particles in the male gametophyte of birch, walnut and cherry naturally infected with cherry leaf roll virus and its relevance to vertical transmission of the virus

Virus-like particles (VLPs) in close-packed paracrystalline arrays were observed in cells forming the anther walls and in the sperm cell cytoplasm of immature pollen grains developing within cherry leaf roll virus (CLRV)-infected birch ( Betula pendula Roth.). VLPs within tubules, that were in some instances multiple and membrane bound, were also observed in anther cells and in pollen grains of CLRV-infected walnut ( Jugians regia L.). VLPs rarely coated the outer surfaces of developing grains. Washings from intact freshly collected pollen did not contain infective agents but pollen triturates were infectious after 12 months storage at-70°C. Purified CLRV (concentration 6·4 ng/ml) was readily detected by enzyme-linked immunosorbent assay (ELISA). CLRV-specific antigens (detected by ELISA) and VLPs (detected on grids coated with an antiserum prepared against CLRV) were readily removed by washing intact pollen grains from infected birch, walnut and cherry ( Prunus avium L. cv. F12/1). The antigen was less tenaciously held to the surfaces of anemophilous (birch and walnut) than entomophilous (cherry) pollen. Treatment of grains before ELISA testing with CLRV-specific γ globulin virtually eliminated the antigenicity of pollen washings whereas γ globulin from a pre-immune serum had no such effect. When anti-CLRV γ globulin-treated pollen grains were disrupted, CLRV-specific antigens were liberated. VLPs trapped on CLRV-antiserum coated grids to which pollen washings or extracts from disrupted grains had been applied were identified by decoration; a halo of antibody molecules enveloped VLPs treated with CLRV-antiserum but not those treated with antiserum prepared against poplar mosaic virus.