莴笋感染番茄斑萎病毒(TSWV)——病毒粒子分布与亚细胞病变特征

莴笋是西藏昌都市露地栽培的主要蔬菜种类之一,近年来斑萎病发生普遍,发病率达到了80%以上,引起严重的经济损失,前期研究表明发病植株受到番茄斑萎病毒(TSWV)侵染。为明确该区域莴笋感染TSWV不同时期病毒粒体分布与亚细胞病变的特征,为病毒病的精准诊断与绿色防控提供依据,本研究采集了西藏昌都市卡若区莴笋不同发病时期的样品,应用TSWV-N基因序列设计引物,通过RT-PCR扩增克隆与测序分析,根据N基因序列推导N蛋白氨基酸序列比对,明确采集样品受到TSWV侵染。采集不同发病时期的样品进行负染色和超薄切片制样透射电子显微镜观察,发现早期感病叶片(症状为轻微褪绿)中病毒粒体相对含量较少,TSWV粒体分布于细胞质中,叶绿体噬锇颗粒明显增加,线粒体嵴膜部分降解,少部分细胞的液泡内有较多TSWV粒体聚集于细胞质内的囊泡中;中期感病叶片(褪绿或黄化斑)中,病毒粒体相对含量较高,较多TSWV粒体聚集于细胞质内质网池中,叶绿体膜系统降解,噬锇颗粒增加,线粒体增多;后期感病叶片(坏死斑)中,病毒粒体相对含量高,叶绿体、线粒体等细胞器降解,有大量TSWV病毒粒体聚集于残存的细胞质内囊泡中。TSWV粒体相对含量...     

TSWV感染番茄叶片组织的BSE成像和精细定位

超薄切片制样透射电子显微镜观察病毒粒体在细胞中的聚集特征及其与亚细胞的相互作用是植物病毒诊断鉴定及致病性研究的基础,但存在视野小、因载网遮挡而信息不全的局限.本研究对目前蔬菜及其它重要经济作物生产上发生为害严重的番茄斑萎病毒(Tomato spotted wilt orthotospovirus,TSWV)侵染番茄叶片...     

感染番茄斑萎病毒和番茄环纹斑点病毒的植物叶片细胞病理变化观察

应用透射电子显微镜观察比较了番茄斑萎病毒（ TSWV）和番茄环纹斑点病毒（ TZSV）感病植物叶片的细胞超微结构,发现两种病毒在病变细胞内的形态及细胞病理变化特征相似,但病毒含量及细胞受损程度存在明显差异。与TSWV相比,TZSV侵染细胞的病变程度更为严重,叶绿体产生大的囊泡,片层结构被破坏,线粒体也发生囊泡化,而TSWV侵染的细胞内线粒体保存相对完好。高压冷冻－冷冻置换制备的样品细胞病理结构与常规化学固定的相似,但对膜结构的保存更加良好。该结果从细胞病理学上证明了TZSV在田间对农作物造成的危害更加严重。     

甘薯羽状斑驳病毒和甘薯褪绿矮化病毒复合侵染甘薯引起的细胞病理学研究

2011～2012年在浙江杭州连续发现感病甘薯植株具有甘薯病毒病SPVD的典型症状,透射电镜观察发现其叶片包含有长度为600—900nm,直径为12nm的线状病毒粒子。使用特异性引物进行RT—PCR扩增及测序,其病原为甘薯羽状癍驳病毒（Sweet potato feathery mottle virus,SPFMV）和甘薯褪绿矮化病毒（Sweet potato chlorotic stunt virus,SPCSV）复合侵染。运用透射电镜研究SPVD引起的细胞病理学变化,观察到叶肉细胞的细胞质中含有马铃薯Y病毒属特征性柱状内含体,其结构分类为Ⅳ型;线状病毒样粒子以束状平行排列成聚集体的方式存在于细胞质中;叶绿体伸出伪足状结构包裹线粒体。在维管束细胞的筛管和伴胞中观察到弯曲线状病毒样粒子形成的聚集体,其粒子排列交错,与叶肉细胞中平行排列的病毒样粒子聚集体不同,符合毛形病毒属特征。另外还在维管束和叶肉细胞结合的部位发现复合侵染的细胞病理学特征。本研究表明危害甘薯生产的SPVD已扩散蔓延到浙江省。     

应用高压冷冻-冷冻置换技术研究受Potyvirus侵染的寄主细胞超微结构

应用高压冷冻-冷冻置换技术（HPF-FS）制备了马铃薯Y病毒属的小西葫芦黄花叶病毒（ZYMV）和大豆花叶病毒（SMV）侵染寄主植物叶细胞的超薄切片样品,并与传统化学固定方法进行比较。透射电镜观察结果显示：病毒粒子和内含体的形态分布在两种方法处理的样品中无明显差异,但高压冷冻样品的细胞超微结构精细,细胞壁与细胞质之间边界清晰,质膜平滑而完整,细胞基质丰富;细胞核和叶绿体、线粒体等形态饱满,高尔基体潴泡结构及分泌小泡清晰,在ZYMV感染细胞的叶绿体中观察到囊泡结构。而化学处理的样品普遍存在细胞结构的收缩和变形,特别是质膜褶皱,与细胞壁分离,叶绿体呈梭形,片层结构发生改变,高尔基体潴泡结构及分泌小泡相当少见。实验结果有利于正确区别病毒所引起的细胞病理变化和化学处理而产生的人工假象。     

百合病毒病原的检测诊断

应用电镜和ELISA检测查明浙江丽水和杭州栽培的百合受到黄瓜花叶病毒(CMV)及多种线状病毒侵染。寄主反应测定显示侵染百合的植物病毒寄主范围都很窄,不能通过汁液摩擦接种侵染昆诺藜、苋色藜、普通烟、心叶烟等6科12种指示植物。在64个ELISA检测样品中有26个受到CMV侵染,侵染率达40．6％;在75个负染色检测样品中有58个观察到线状病毒,侵染率达77．3％;在40个被线状病毒侵染的样品中有27个观察到细胞质风轮状内舍体,表明受到马铃薯Y病毒属成员的侵染,侵染率达67．5％。从不同线状病毒粒子形态及所引起的细胞病理变化来看,可能分属于马铃薯Y病毒属、麝香石竹潜隐病毒属和马铃薯X病毒属。     

水稻不同品种叶表面硅质细胞的扫描电镜观察

应用扫描电子显微镜对8个不同品种的水稻（Oryza sativa L.）叶片表面硅质细胞的形态结构进行了观察,并首次用X射线能谱分析其硅含量.结果发现8个品种间叶片表面硅质细胞形态结构和硅元素含量差异明显,四倍体品种不但硅质细胞大,而且硅元素含量也较高,其中矮脚南特-4x叶片表面硅质细胞表面积最大,达301.40mm^2,硅元素相对含量也最高,为32.77 wt.%;广陆矮4号-4x硅质细胞表面积次之,为192.50mm^2,硅元素相对含量为25.80 wt.%.这2个四倍体品种的气孔硅乳突数都是8个,比二倍体品种多1倍.四倍体品种田间表现植株直立、基部通风性好、抗倒伏能力和抗病虫性均较强.二倍体品种零轮、P23、L202、D27 F2（野生稻杂种）、华粳籼74和E5等品种硅质细胞表面积明显比四倍体的小,平均小3.6倍;硅元素含量也较低,平均比四倍体的低1.4倍;抗性较2个四倍体水稻品种差.本文的结果初步说明水稻叶片硅质细胞大小和含量与抗性可能存在密切的相关.     

水稻细菌性条斑病的电镜观察

水稻细菌性条斑病是国内植物检疫对象之一,主要发生于东南亚及我国华南、华中和华东局部地区。在浙江一般在6～9月遇台风或洪涝容易流行,其病原为Xanthomonas eampestris pv.oryzicola。本文报道应用电镜技术研究感病稻侏叶片表面结构及病原菌在叶组织中的存在状态。一、材料和方法:采用盆栽的早稻。由于该病菌易从伤口侵入组织,可用针刺法人工接种经分离培养的细条病病原细菌。当叶片出现典型的暗绿色至黄褐色细条斑,并生有许多露珠状的蜜黄色菌脓时,取样进行常规超     

水稻施硅之抗虫机制的研究

从水稻施硅处理区和对照区分别采取叶片和叶鞘,进行相同部位的扫描电镜观察拍片,发现叶片上、下表皮之表面分布的硅粒,处理区明显比对照区突出而稍大;叶脉表面成对的硅化细胞扁平,排列紧密,在63.6μm长的视野内比对照的多出1对;表皮细胞外壁与硅化层、角质层的总厚度加厚O.6-O.7μm;叶脉维管束外的厚壁细胞壁较厚,排列行次及宽度均比对照的大。叶鞘切片也有类似的情况。因而处理区内稻纵卷叶螟和褐稻虱等害虫的种群密度分别比对照区下降5-6成。上述解剖形态的差异,也许就是水稻施硅后能抗虫的主要机制。     

海拔高度对淡黄香青叶片超微结构的影响

对于高海拔生境中植物的亚显微结构的研究国内尚无报道,我们对分布于马衔山不同海拔高度(2700米、3150米、3675米)处的淡黄香青(Anaphalis flavescens Hand-Mzt)的叶片超微结构与海拔高度的相关性作了分析,结果如下:1.叶肉细胞强烈液泡化,中央液泡随海拔升高而变小,但内含物浓度加大,细胞中除了大量的叶绿体外,还有丰富的脂滴、微体、过氧化物酶体,淀粉粒存在,在3675米处植株叶肉细胞中还有脂类小滴的团聚物。2.叶绿体中含有许多质体小球与淀粉粒,随海拔升高叶绿体体积变小,数量有所增加,结构简单化,其中基质与基粒片层的叠垛程度,以及基粒片层与基质片层的比率都有所下降,其中这个比率在栅栏细胞中最大,     

Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells

Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges.     

Three-dimensional architecture of virus-packed tubule

When rice dwarf virus (RDV), a member of the Reoviridae family, infects leafhopper cells, formation of protruding tubules composed of nonstructural viral protein Pns 10 can be observed. We examined the three-dimensional (3D) structure of these tubules containing RDV particles using electron tomography. The thin section of RDV-infected leafhopper vector cells in monolayers was subjected to double-tilt tomography. The tomographic 3D map provides a more reliable estimation of the real dimensions of the structure compared with the 2D image of the thin section. Docking of particle models made from atomic coordinates of RDV into the tomogram revealed that the inner diameter of the tubule was close to the outer diameter of the RDV particle. Fourier-transform of the reconstituted tubule image from the purified Pns 10 protein in vitro revealed a helical structure of the tubule.     

Ultra-high-resolution scanning electron microscopy of vaccinia virus and its recombinant carrying the gag gene of human immunodeficiency virus type 1.

FL cells infected with vaccinia virus or its recombinant carrying the gag gene of human immunodeficiency virus type 1 (HIV-1) were examined by ultra-high-resolution scanning electron microscopy. Virions, whether located extracellularly or intracellularly, had a brick-shaped or watermelon appearance as a whole. Extracellular virions observed on the surface of infected cells had variable surface ultrastructures depending on the manner in which particular virions were wrapped in cell membranes. Most of the intracellular naked virions adherent to the inner face of cell surface membranes clearly exhibited ridgy, rod-shaped or globular surface structures on their surface. HIV-like particles with a diameter of about 100 nm and virions of vaccinia virus were both observed distinctly on the surface of FL cells infected with the recombinant virus.     

三种动物慢病毒形态及形态发生的比较研究

用电镜检查确定了不同感染时间的马传染性贫血病毒 (EIAV)、维斯纳 梅迪病毒 (MVV)和山羊关节炎脑炎病毒 (CAEV)的形态和形态发生。成熟的病毒粒子为电子致密的球形 ,有囊膜 ,直径约 10 0nm ,含有 1个 (有时有 2个或 2个以上 )的核芯。维斯纳 梅迪病毒和山羊关节炎脑炎病毒的核芯为球形 ,马传染性贫血病毒为杆形或锥形。在大部分病毒里能清晰地看到核芯壳 ,成熟的病毒从感染细胞的胞浆膜上出芽 ,在胞浆中还见到了花环状粒子和多板层结构     

Budding of fowlpox and pigeonpox viruses at the surface of infected cells

Chick embryo fibroblasts and chorioallantoic membranes infected with fowlpox virus (FWPV) or pigeonpox virus (PPV) were examined by transmission and scanning electron microscopy. Immature virus particles were observed in finely granular areas, i.e. virus factories, of the cytoplasm. These particles had various forms depending on their stages of development. Many tubular structures were also seen in these regions. Mature virus particles with ellipsoidal or brick-shaped forms enclosing electron-dense cores were detected throughout the cytoplasm. Notably, there was a high frequency of virus budding at the cell surface, but only occasional virus wrapping in the cytoplasm. Another remarkable feature of the infected cells was accumulation of many virions just beneath the plasma membrane, indicating that this phenomenon is closely related to virus budding. Based on the observed frequency of budding, this mechanism seems to be the predominant way for FWPV and PPV to exit the cell.     

侵染西瓜的黄瓜绿斑驳花叶病毒快速检测

黄瓜绿斑驳花叶病毒（ CGMMV）是危害西瓜生产的重要病害,造成西瓜果实腐烂,失去商业价值。应用透射电子显微镜和单抗dot?ELISA技术对采自浙江温岭的西瓜病叶进行快速病原检测,电镜负染色观察到病株汁液中存在大量刚直杆状的病毒粒子,超薄切片观察到细胞质内存在整齐排列的杆状病毒聚集体,与烟草花叶病毒属（ Tobamovirus）的形态特征相符;进一步采用dot?ELISA技术进行血清学检测,显示西瓜病叶与CGMMV特异性抗体呈阳性反应,检测结果表明感染西瓜的杆状病毒为CGMMV。从而为侵染西瓜的CGMMV提供快速检测途径。     

Electron microscopic study on the development of herpesviruses.

This review is concerned with comparative electron microscopic observations of in vitro cultured cells infected with twelve kinds of herpesviruses including six human herpesviruses. Morphological differences in intranuclear particles with cores of low density among these viruses mainly due to inherent differences in the ultrastructure of particular viruses, the morphological variety of intranuclear particles of herpes simplex virus due to their different developmental stages, and unusual structures observed in cells infected with some herpesviruses are described. Viral envelopment relevant to the final step of maturation of herpesviruses and their egress from infected cells are discussed.     

大蒜病毒原与其寄主细胞病理变化的电镜研究

电镜检查发现田间4个品种栽培大蒜各部位组织都含大量线形病毒粒子,长度为300nm～2200nm,但较多的集中在750nm和600nm两个范围内。感病大蒜叶组织细胞质大量存在着圆柱状内含体,内含体显然与内质网、质膜和胞间连丝有关。病毒粒子散布在细胞质中,有些附着在内含体上,未见病毒聚集体。病变细胞严重空泡化,有些细胞质中出现大量含纤维状物的小囊泡和类似内质网膨大的结构,同时伴随着大量病毒粒子。叶绿体和根细胞中的前质体产生特殊泡状腔结构,细胞核中未见内含体和病毒粒子。     

九种Potyviruses柱状内含体结构的比较研究

通过寄主叶肉细胞超薄切片的透射电镜观察，对马铃薯Ｙ病毒（ＰＶＹ），芋花叶病毒（ＤＭＶ），大蒜黄条病毒（ＧＹＳＶ），大豆花叶病毒（ＳＭＶ），蚕豆叶卷花叶病毒（ＰＬＲＭＶ），芜菁花叶病毒（ＴｕＭＶ），大丽菊上的马铃薯Ｙ组病毒（Ｐｏｔｙｖｉｒｕｓ－Ｄｈ），莴苣花叶病毒（ＬＭＶ）和大麦黄花叶病毒（ＢＹＭＶ）九种病毒共１２个病毒分离物所产生的柱状内含体的结构进行了比较研究，结果表明同一病毒的不同分离物所产生