Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

An electron microscopic study of the initial stages of infection of isolated tomato fruit protoplasts by tobacco mosaic virus

Summary Protoplasts were isolated from tomato fruit locule tissue and incubated with tobacco mosaic virus. Electron microscope observations on sections of suitably fixed and embedded material revealed that virus particles readily became attached to the plasmalemma, particularly in small invaginations in the surface of the protoplast. Virus particles were later observed in vesicles within the cytoplasm and it was clear that these vesicles were being formed as a result of pinocytic activity at the surface of the protoplast. Later, virus particles were observed near the nucleus. It is suggested that an initial attachment of the virus to the plasmalemma followed by a pinocytic uptake may represent the initial stages of virus infection of plant cells and that the pinocytic vesicle, containing virus, serves as the vehicle of cellular infection.     

RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts.     

感染大麦黄花叶病毒(BaYMV)的大麦细胞质内含体及细胞器病变的研究

超薄切片电镜观察表明,在感染大麦黄花叶病毒(BaYMV)的大麦(品种“早熟3号”)叶肉细胞中,液泡周围偶而可看到病毒颗粒束,在发病后期黄化或坏死的叶肉细胞中,可见到散布的病毒颗粒。在所有表现症状的病叶叶肉细胞,表皮细胞和木质部薄壁细胞中均可观察到风轮体、束状体、板状集结体以及膜状体等细胞质内含体,未见 卷简体和细胞核内含体。感病初期细胞中,细胞质丰富,核糖体数量增加,内质网肥大,随着病毒症状发喂,叶绿体、线粒体等细胞器逐渐肿大,外膜破裂直至解体。     

CYTOLOGICAL REACTIONS OF NORMAL AND TMV-INFECTED TOBACCO LEAF CELLS TO ACID AND ALKALINE SOLUTIONS

Acid and basic buffer solutions were applied on slides under cover glasses to thin, hand sections of leaf tissue from normal tobacco plants and equivalent plants infected with tobacco mosaic virus (TMV). The effects on living cells were observed and photographed through phase optics. First, reversible changes and, later, irreversible changes were produced in the cells. Movement of the cytoplasm in a cell could be stopped completely and started again by replacing the unfavorable buffer solution with a favorable medium. Of the organelles, plastids became static first, then mitochondria, and lastly spherosomes. Spherosomes often moved actively when all other organelles were still. Translucent virus monolayers, consisting of particles aggregated side by side, provided markers in the parietal cytoplasm of recently infected cells. Mitochondria generally moved across their surface on the side adjacent to the tonoplast, spherosomes across the surface facing the cell wall. Alkaline buffer solution caused little change of texture in nucleus and cytoplasm or change of form in mitochondria. Clear areas appeared in some plastids. The acid buffer emphasized a cytoplasmic network representing flow lines and eliminated many cytoplasmic vesicles; mitochondria became shorter or spherical in outline, grana of chloroplasts more obvious. Virus inclusions, even the fragile monolayers, were not greatly altered until irreversible changes began. In pH 11–treated cells, the final changes included violent bubbling of cytoplasm, and in pH 2.2–treated cells, snapping of flow lines and coagulation of the cytoplasm. In either case, disintegration of cell structure and virus inclusions was rapid.     

Electron microscopic study of the chitosan effect on the intracellular accumulation and the state of tobacco mosaic virus particles in tobacco leaves

Influence of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in the mesophyll cells of Nicotiana tabacum L. var Samsun leaves in early period of infection development (3 days after infection of leaves) has been studied. The virus accumulated in the cells of the leaves treated for 24 h before infection with chitosan to a lesser degree than in the control cells. The chitosan affected the formation of TMV-specific granular and tubular inclusions which are known to consist of the viral replicase components. Three days after infection of the leaves treated with the chitosan, a typical sign of the infection development was the predominant formation of granular inclusions which are known to appear at the early stages of TMV replication. The infected cells of the leaves untreated with chitosan contained mainly tubular inclusions which had been shown previously to be formed from granular ones at the last stages of the infection process. This indicates that chitosan treatment of the leaves leads to a delay of the development of infection. In phosphotungstic acid-stained suspensions obtained from the infected leaves, abnormal (swollen and "thin") TMV particles were observed along with normal ones. The appearance of abnormal virus particles seems to be caused by virus-induced activation of intracellular lytic processes. The most lytic activity in the infected cells as well as the highest number of abnormal viral particles was observed under the chitosan action. Therefore, it appears that chitosan-mediated stimulation of lytic processes causing destruction of TMV particles may be one of the protective mechanisms limiting virus accumulation in cells.     

Immunocytochemical investigation of the causal agent of cassava mosaic disease in southern Africa

Cassava mosaic disease (CMD) exists throughout Africa, and cassava latent virus (CLV) has been implicated as the etiological agent in Kenya and West Africa. However, in Southern Africa, the causal agent of CMD was not until recently associated with CLV, and the possibility of a second flexuous virus particle has not been ignored. Attempts to isolate and visualize CLV antigen have been successful with Nicotiana benthamiana, an indicator host plant of CLV, but all efforts to isolate and visualize particles in infected cassava plants have failed. Immunocytochemical studies were undertaken in an attempt to localize virus antigen in infected cassava tissue.Cytochemical staining (light microscope) of infected cassava leaf material revealed the presence of inclusion bodies in epidermal and palaside mesophyll cells, and in epidermal collenchyma and outer parenchyma cells from the petiole and stem. However, transmission electron-microscopical (TEM) investigations revealed electron dense bodies in the cytoplasm, and no characteristic CLV nuclear inclusion bodies were evident. Transmission experiments to N. benthamiana and N. tabacum were attempted and leaves, exhibiting symptoms, examined microscopically. The nuclei appeared swollen (in comparison to uninfected leaves), a characteristic of CLV- infected N. benthamiana. However at the TEM level, no characteristic fibrillar-ring inclusion bodies or particles, could be visualized.Further immunocytochemical investigations were initiated, employing antisera raised against CLV isolated from N. benthamiana, and antisera for cassava common mosaic virus (CCMV), cassava brown streak virus (CBSV) and cassava X virus (CsXV). Goat anti-rabbit IgG-gold was used as a direct stain. No labelling occurred with CCMV and CBSV antisera. Intense gold labelling was located in the cytoplasm of phloem, mesophyll and epidermal cells of infected cassava and to a lesser extent in N. tabacum and N. benthamiana using affinity chromatography purified CLV antiserum. Little labelling was observed in nuclei of infected cells. Inconclusive results were obtained with CsXV antiserum.Immunogold labelling located CLV viral antigens in infected cassava leaf tissue. This observation, together with positive ELISA, transmission and DNA hybridization experiments, proves conclusively that CLV viral antigen is present in infected cassava in Southern Africa. However, most viral antigen in infected cassava, unlike N. benthamiana (fibrillar and granular nuclear inclusions) appears to be in the cytoplasm. This may tentatively suggest that the CLV protein is synthesized in the cytoplasm of its natural host, cassava, even though the virus may assemble in the nucleus at the appropriate time. However, as yet no virus inclusions have been observed in nuclei of infected cassava. Due to previous isolation of a flexuous rod and ambiguous staining results, the possibility of two viruses in cassava cannot be ruled out.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

THE STRUCTURE OF CELLS DURING TOBACCO MOSAIC VIRUS REPRODUCTION : Mesophyll Cells Containing Virus Crystals

The submicroscopic organization of mesophyll cells from tobacco leaves systemically infected with tobacco mosaic virus (TMV) is described. After fixation with glutaraldehyde and osmium tetroxide the arrangement of the TMV particles within the crystalline inclusions is well preserved. Only the ribonucleic acid-containing core of the virus particles is visible in the micrographs. Besides the hexagonal virus crystals, several characteristic types of "inclusion bodies" are definable in the cytoplasm: The so-called fluid crystals seem to correspond to single layers of oriented TMV particles between a network of the endoplasmic reticulum and ribosomes. Unordered groups or well oriented masses of tubes with the diameter of the TMV capsid are found in certain areas of the cytoplasm. A complicated inclusion body is characterized by an extensively branched and folded part of the endoplasmic reticulum, containing in its folds long aggregates of flexible rods. Certain parts of the cytoplasm are filled with large, strongly electron-scattering globules, probably of lipid composition. These various cytoplasmic differentiations and the different forms of presumed virus material are discussed in relation to late stages of TMV reproduction and virus crystal formation.     

Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells

Accumulation of considerable amounts of viral particles has been demonstrated in parenchymal cells of young leaves in tobacco cultivar Samsun systemically infected with any of studied tobacco mosaic virus (TMV) strains isolated from pepper (TMV-p), tomato (TMV-t), and eggplant (TMV-e). Abnormal (swollen and thin) virions were found, which points to their destruction. Cell infection with all studied strains was accompanied by the activation of the lysosomal compartment manifested as formation of nascent dictyosomes, elements of smooth endoplasmic reticulum, cytoplasmic vacuoles, various vesicles, invaginated mitochondria, and multivesicular bodies. The studied viral strains could be arranged in the following sequence according to the degree of lysosomal compartment stimulation and induction of intracellular lytic processes mediating the destruction of viral particles and cell structures: TMV-p > TMV-e > TMV-t.     

芝麻花叶病的病毒病原鉴定

从患芝麻花叶病的病叶中提纯了一种线条状病毒颗粒,长700～800nm,宽13nm。经汁液摩擦接种可感染心叶烟、大豆,甜菜等9种植物,不感染西瓜,苋色藜、豇豆等。主要传毒介体是发生在芝麻田的桃蚜。病土、病种均不传病。该病毒与西瓜花叶病毒、芜菁花叶病毒及马铃薯Y病毒的抗血清无反应。超薄切片中可见到风轮形和纸卷形的圆柱状内含体以及结晶状内含体。结晶状内含体分布在细胞质和叶绿体中,其它内含体均见于细胞质中。同时,细胞质中还可见到大量聚集的线状病毒颗粒。初步认为此病毒可能是马铃薯Y病毒群中的一个新成员,暂称为芝麻花叶病毒。国内外均未见报道。     

Further Studies on a Virus Causing a Green Mosaic Disease of Eggplant in Nigeria

A virus reported earlier to cause a green mosaic disease of eggplant in Nigeria was studied in more detail. Its filamentous particles with a normal length of 820 nm reacted in immunoelectron microscopical tests strongly with the homologous antiserum and less strongly with antisera to dioscorea green banding mosaic, groundnut eyespot, zucchini yellow mosaic viruses and to a tomato potyvirus isolate from Taiwan. No reactions were seen with antisera to 25 other potyviruses. Several new host plants were identified. Infected cells contained cylindrical inclusions with scrolls and short curved laminated aggregates and clusters of small vesicles with electron-dense content. Host range and serological reactivities differentiate the virus for which the name eggplant green mosaic virus is suggested from all potyviruses so far known.     

Detection of Infectious Tobamoviruses in Forest Soils

Our objectives were to evaluate elution and bait plant methods to detect infectious tobamoviruses in forest soils in New York State. Soils were collected from two forest sites: Whiteface Mountain (WF) and Heiberg Forest (HF). The effectiveness of four buffers to elute tomato mosaic tobamovirus (ToMV) from organic and mineral fractions of WF soil amended with ToMV was tested, and virus content was assessed by enzyme-linked immunosorbent assay (ELISA). The effectiveness of Chenopodium quinoa (Willd.) bait plants to detect the virus also was tested. Both methods then were utilized to detect tobamoviruses in 11 WF and 2 HF soil samples. A phosphate buffer (100 mM, pH 7.0) eluted more ToMV from soil than the other buffers tested. Mineral soil bound more virus than organic soil. Virus recoveries from virus-amended organic and mineral soils were 3 and 10%, respectively, and the detection sensitivity was 10 to 20 ng/g of soil. Roots of bait plants grown in all virus-amended soils tested positive by ELISA, and virus concentrations averaged 10 ng/g. Both ToMV and tobacco mosaic tobamovirus (TMV) were transmitted to C. quinoa by elution from one of two HF soil samples but not from the WF soil samples. A tobamovirus was detected by bait planting in 12 of 73 (16%) root extracts representing 5 of 13 soil samples (38%). Tobamovirus-like particles were seen by transmission electron microscopy in 6 of 12 infected root extracts. Tobamoviruses occur in forest soils in New York State. Abiotic soil transmission to trees may permit localized spread and persistence of these viruses in forest ecosystems.     

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒ＣＭＶ不同分离物侵染寄主的细胞超微结构变化。来自一患红（Ｓａｌｖｉａｓｐｌｅｎｄｅｎｓ）的不含卫星ＲＮＡ分离物Ｍ－２２侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结果,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。