乳胶溶液电镜在检测线状和棒状植物病毒中的应用

建立了一种检测线状或棒状植物病毒的新的电镜技术。用聚苯乙烯乳胶预先包被铜网用以测定烟草花叶病毒,马铃薯X、Y病毒,玉米矮化花叶病毒表明,铜网上沾附的病毒数量与免疫电镜法相似,比直沾法多几倍至十几倍。乳胶溶液电镜法不需制备特异抗血清,方法简便快速,特别适合于植物样品的初次病毒检测工作。     

Inhibitory effect of fucoidan from brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus in tobacco leaves of two cultivars

The effect of fucoidan from the brown alga Fucus evanescens on the spread of infection induced by tobacco mosaic virus (TMV) was investigated in the leaves of tobacco (Nicotiana tabacum L.) of two cultivars (Ksanti-nk and Samsun). In the leaves of cv. Ksanti-nk inoculated with a mixture of TMV preparation (2 μg/ml) and fucoidan (1 mg/ml), the number of local necrotic lesions induced by the virus decreased by more than 90% as compared with the leaves inoculated with the virus alone. In tobacco leaves of cv. Samsun, virulence and the concentration of the virus 3 days after inoculation with the same mixture of TMV and fucoidan were by 62 and 66%, respectively, lower than in the leaves inoculated with TMV alone. As the infection spread, the inhibitory effect of fucoidan decreased. When the leaves were treated with fucoidan before and after the inoculation with TMV, its antiviral activity was less pronounced than when a mixture of the virus and the polysaccharide was used as inoculum. Electron microscopic investigation of TMV mixed with fucoidan often showed agglutinated virions. The highest virulence of the mixture (TMV preparation, 12 μg/ml, plus fucoidan, 1 mg/ml) was observed upon its twofold dilution, and after that it decreased. It was concluded that, when the leaves were inoculated with the mixture of TMV and fucoidan, the latter affected not only the plant but the virus as well. Treatment of tobacco leaves, cv. Ksanti-nk, with actinomycin D (10 μg/ml) 24 h before the inoculation with TMV almost completely suppressed the effect of fucoidan, indicating that fucoidan acted at a gene level.     

应用斑点法检测植物病毒的研究

应用斑点法检测了病叶粗汁液中的芜菁花叶病毒(TuMV)、大豆花叶病毒(sMV)和黄瓜花叶病毒(CMV),病叶粗汁液可被检测的最大稀释度分别为1:5120、1:2560和1:1280。提纯的大豆花叶病毒和黄瓜花叶病毒可检测的最低限量分别为1.7ng和1.2ng。以牛血清白蛋白、吐温和聚乙烯吡咯啉酮作封闭液,均可获得满意的结果。应用斑点法检测芜菁花叶病毒和大豆花叶病毒时,其抗血清稀释1:500倍可获得满意效果,稀释2000倍仍可用于检测。     

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Homology between the proteins encoded by tobacco mosaic virus and two tricornaviruses

Summary A comparison was made of the amino acid sequences of the proteins encoded by RNAs 1 and 2 of alfalfa mosaic virus (A1MV) and brome mosaic virus (BMV), and the 126K and 183K proteins encoded by tobacco mosaic virus (TMV). Three blocks of extensive homology of about 200 to 350 amino acids each were observed. Two of these blocks are located in the A1MV and BMV RNA 1 encoded proteins and the TMV encoded 126K protein; they are situated at the N-terminus and C-terminus, respectively. The third block is located in the A1MV and BMV RNA 2 encoded proteins and the C-terminal part of the TMV encoded 183K protein. These homologies are discussed with respect to the functional equivalence of these putative replicase proteins and a possible evolutionary connection between A1MV, BMV and TMV.     

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。     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.     

Acquisition and Retention of Potato virus Y Helper Component in the Transmission of Potato aucuba mosaic virus by Aphids

Potato aucuba mosaic virus (PAMV) is transmissible by aphids in a non-persistent manner but only in the presence of a Potyvirus helper component (HC) such as that of Potato virus Y (PVY) that must be acquired beforehand or simultaneously. To compare the acquisition and persistence of PAMV and PVY-HC, the PVY-HC was acquired first and then PAMV. The acquisition access and post-acquisition fasting periods were varied. The results show that PAMV and PVY particles, and also PVY-HC could be acquired in 10 s. Some PVY-HC activity could be detected in fasting aphids at least 6 h after acquisition whereas PAMV infectivity was retained for only 2 h. These findings are compatible with the non-persistent transmission concept being controlled by enzymatic release of PAMV particles bound to aphid mouthparts by PVY-HC.