Factors affecting the detection of potato leafroll virus in potato foliage by enzyme-linked immunosorbent assay

Using antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme-linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10^-1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme. The detection end-point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10^-2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus-free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested. PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field-grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse-grown plants. In field-grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.     

Detection of six nepoviruses in their nematode vectors by immunosorbent electron microscopy

Particles of six nepoviruses were detected by immunosorbent electron microscopy (ISEM) in extracts of their respective vector nematodes. This technique was at least a thousand times more sensitive than conventional electron microscopy. It was also more rapid, reliable and sensitive than infectivity tests in which extracts of nematodes were inoculated to indicator plants. The viruses were detected in extracts of single nematodes, and in the roots and leaves of infector and bait plants.     

Application of enzyme-linked immunosorbent assay to the detection of potato leafroll virus in potato tubers

Factors affecting the detection of potato leafroll virus (PLRV) by enzyme-linked immunosorbent assay (ELISA) in tubers of field-grown potato plants with primary or secondary infection were studied. The reactions of extracts of virus-free potato tubers were minimised by pre-incubating the extracts at room temperature and by careful choice of the dilution of enzyme-conjugated globulin. PLRV was reliably detected in tubers produced by secondarily infected plants of all six cultivars tested. PLRV concentration was greater in heel-end than in rose-end vascular tissue of recently harvested tubers but increased in rose-end tissue when tubers stored at 4°C for at least 5 months were placed at 15–24°C for 2 wk. PLRV occurred at greater concentration in tubers from plants of cv. Maris Piper with natural or experimentally induced primary infection than in tubers from secondarily infected plants; again PLRV concentration was greater in heel-end than in rose-end vascular tissue. Plants whose shoots were infected earliest in the growing season were invaded systemically and produced the greatest proportion of infected tubers; plants infected late in the season also produced infected tubers but PLRV was not detected in their shoot tops. PLRV concentration in tubers from the earliest-infected plants was less than in tubers from later-infected plants. PLRV was detected reliably by ELISA in tubers from progenies that were totally infected but was not detected in all infected tubers from partially infected progenies. ELISA is suitable as a routine method of indexing tubers for PLRV, although the virus will not be detected in all infected tubers produced by plants to which it is transmitted late in the growing season.     

Determination of the mass of viruses by quantitative electron microscopy.

The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.     

Host range and some properties of potato mop-top virus

Potato mop-top virus (PMTV) was transmitted by inoculation of sap to twenty-six species in the Solanaceae or Chenopodiaceae and to Tetragonia expansa; species in eleven other plant families were not infected. The virus was cultured in inoculated leaves of Nicotiana tabacum cv. Xanthi-nc or in N. debneyi. Diagnostic local lesions were produced in Chenopodium amaranticolor. In winter, ten solanaceous species were slowly invaded systemically but the first leaves infected were those immediately above inoculated leaves. When transmitted to Arran Pilot potato by the vector Spongospora subterranea, PMTV induced all the main types of shoot and tuber symptoms found in naturally infected plants. Isolates of PMTV from different sources differed considerably in virulence. PMTV-containing tobacco sap lost infectivity when heated for 10 min at 80 °C, diluted to 10^-4, or stored at 20 °C for 14 weeks. Infectivity was partially stabilized by 0·02% sodium azide. When sap was centrifuged for 10 min at 8000 g, infectivity was mainly in the sediment. Infective sap contained straight rod-shaped particles about 20 nm wide, with lengths up to 900 nm and crossbands at intervals of 2·5 nm. Many of the particles were aggregated side-to-side, and the ends of most seemed damaged. The slight infectivity of phenol-treated leaf extracts was abolished by pancreatic ribonuclease. The present cryptogram of PMTV is R/*:*/*:E/E:S/Fu.     

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Detection of parvoviruses in wolf feces by electron microscopy

One hundred fifteen wolf (Canis lupus) feces were collected between 1980 and 1984 from northeastern Minnesota and were examined for canine parvovirus by negative contrast electron microscopy. Of these, seven (6%) samples revealed the presence of parvovirus. Some of these viruses were able to grow in cell cultures forming intranuclear inclusion bodies and giant cells.     

北京地区设施草莓8种病毒的酶联免疫吸附测定检测

[背景]病毒可以随同草莓无性繁殖材料传播扩散,导致产量和品质下降.选育无病毒种苗是草莓病毒病防治的主要措施,高效、灵敏的检测技术可为草莓病毒病防治提供技术保障.[目的]为明确8种能够侵染草莓的病毒在北京地区设施草莓上的发生情况,应用酶联免疫吸附测定(Enzyme-Linked Immunosorbent Assay,E...     

Enumeration and biomass estimation of planktonic bacteria and viruses by transmission electron microscopy.

Bacteria and virus particles were harvested from water samples by ultracentrifugation directly onto Formvar-coated electron microscopy grids and counted in a transmission electron microscope. With this technique, we have counted and sized bacteria and viruses in marine water samples and during laboratory incubations. By X-ray microanalysis, we could determine the elemental composition and dry-matter content of individual bacteria. The dry weight/volume ratio for the bacteria was 600 fg of dry weight microns-3. The potassium content of the bacteria was normal compared with previous estimates from other bacterial assemblages; thus, this harvesting procedure did not disrupt the bacterial cells. Virus particles were, by an order of magnitude, more abundant than bacteria in marine coastal waters. During the first 5 to 7 days of incubation, the total number of viruses increased exponentially at a rate of 0.4 day-1 and thereafter declined. The high proliferation rate suggests that viral parasitism may affect mortality of bacteria in aquatic environments.     

Restricted multiplication of potato leafroll virus in resistant potato genotypes

Plants of a range of potato genotypes differing in rating for field resistance to potato leafroll virus (PLRV) were inoculated with the virus by grafting or by aphids (Myzus persicae). Plants of all genotypes tested became infected by each inoculation method and PLRV was detected by ELISA in the upper leaves of all genotypes within 26 days after grafting. Most genotypes with high resistance ratings developed only mild primary and secondary symptoms whereas those with low resistance ratings developed more pronounced symptoms. However, one genotype (G7461(4)) with a high resistance rating was very severely affected. The concentrations attained by PLRV in genotypes with high resistance ratings were only 1–10% of those in genotypes with low resistance ratings. These differences in virus concentration were found in young leaves of plants with primary or secondary infection, whether inoculated by grafting or by aphids and whether grown in the glasshouse or the field. In older leaves, differences in virus concentration between genotypes were at least as pronounced as those in younger leaves. In contrast, PLRV concentration in vascular tissue at the heel end of tubers of plants with primary infection was similar for all the genotypes tested. Although low PLRV concentration was consistently associated with high resistance rating it is not the only form of resistance to PLRV occurring in potato.     

Detection of epidermal growth factor receptor by scanning electron microscopy

A method of immunocytochemistry and low-voltage scanning electron microscopy (SEM) is described for visualization of the epidermal growth factor membrane receptor (EGFR). The specific labelling is achieved of antigenic sites on the surface of prefixed cells. The advantage of this approach over existing techniques is the capability for unlimited high-resolution surface examination at the single cell level. This is achieved by using low acceleration voltage (V ₀) and either very thin or no coating of the specimens to prevent the label from being masked. Furthermore, by using conventional field emission SEM and a highly sensitive detector for backscattered electrons, detection of the gold-conjugate (<10 nm in diameter) becomes possible even at low V ₀. A431 cells (human epidermoid carcinoma) show intercellular variability in their EGFR area density. Highest density was recorded upon cells in the mitotic stage of the cell cycle due to a decrease in the relative surface of rounded versus flattened cells. At the ultrastructural level a marked heterogeneity was also seen on the surface of contracted cells, where enhanced labelling could be observed only at the tips of microvilli. In contrast, spread cells displayed a homogeneous receptor distribution due to their smooth surface.     

Detection of single-stranded DNA in scrapie-infected brain by electron microscopy

The nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles from normal and scrapie-infected hamster brains were examined by electron microscopy. After spreading on collodion-coated grids circular molecules of approximately 15.7 kb corresponding in size to mitochondrial DNA (mtDNA) were observed both in normal and scrapie-infected brains. In nucleic acid preparations from scrapie-infected brains multimeric mtDNA and single-stranded DNA strands of about 0.49 X 10(6) daltons were also visualized. These findings demonstrate the presence of a single-stranded DNA in scrapie-infected brains and are consistent with previous data based on enzyme digestion of nucleic acids isolated from scrapie-infected brains.     

云南甘薯病毒的检测及主要病毒的多样性分析

[目的]明确云南甘薯病毒的种类,并对主要病毒进行遗传多样性分析.[方法]利用PCR/RT-PCR技术,对采自云南16个县、市的279个甘薯样品进行扩增、测序,对所得序列应用分子生物学软件MEGA 5进行系统发育分析.[结果]除普洱和祥云的样品中未检测到任何病毒外,其余14个县、市的123个甘薯样品中共检测到甘薯褪绿斑病毒(SPCFV)、甘薯羽状斑驳病毒(SPFMV)、甘薯卷叶病毒(SPLCV)、甘薯C病毒(SPVC)、甘薯G病毒(SPVG)和甘薯病毒2号(SPV2)等6种病毒.其中SPVG的检出率最高,达39.1％,为云南甘薯病毒的优势种,SPFMV和SPVC的检出率分别为26.9％和24.7％,而SPLCV检出率最低,仅为0.4％.在所检测的样品中未发现甘薯褪绿矮化病毒(SPCSV)和甘薯轻斑驳病毒(SPMMV).云南甘薯病毒多数为2-5种病毒复合侵染,占总样品数的31.9％,其中2-3种病毒复合侵染现象最为常见,单一病毒侵染占总样品数的12.2％.检出率比较低的SPCFV、SPLCV和SPV2未发现单独侵染现象.[结论]云南甘薯上发生的SPFMV分离物存在EA株系和O株系,未发现RC株系,另有两个分离物同EA、O、RC之间的亲缘关系均较远,有可能是一个新的株系;SPVC和SPVG分离物均可分为3个不同的组,大部分SPVG云南分离物属于Ⅰ组.     

Field and glasshouse experiments on the control of potato mop-top virus

Field observations during 3 yr on a stock of potato cv. Red Craigs Royal partially infected with potato mop-top virus (PMTV) confirmed that the virus was passed by an infected mother plant to only a proportion of its progeny tubers, and showed that in this cultivar symptomless plants gave rise only to symptomless progeny. The elimination of PMTV from stocks can therefore be greatly accelerated by removing symptom-bearing plants. Infected potato tubers were not freed from PMTV by treating them at 37 °C for up to 8 wk. Treating ‘seed’ tubers bearing powdery scabs that contain PMTV-carrying resting spores of Spongospora subterranea with formaldehyde or organo-mercurial fungicide greatly decreased PMTV establishment when the tubers were planted in previously uninfective soil, but fumigation with 2-aminobutane was ineffective. Decreasing the pH of infective soil to 5-0 by applying sulphur greatly decreased the infection of potato cv. Arran Pilot with PMTV and S. subterranea in field experiments, but this treatment did not eliminate either; when the pH of treated soil was raised the transmission of PMTV resumed. Treating infective soil with a range of fungicides greatly decreased the infection of Nicotiana debneyi bait seedlings in glasshouse experiments but only calomel at 75 kg/ha controlled spread of PMTV and 5. subterranea to potato in field experiments. In other field experiments, applying zinc frit, zinc sulphate or zinc oxide to infective soil greatly decreased the spread of both to potato. The amount of zinc required increased with increase in clay content of the soil. However, treatment with zinc compounds did not eliminate PMTV-carrying vectors from soil, and when treated soil was diluted with autoclaved soil many of the bait seedlings planted in the mixture became infected. The zinc frit was phytotoxic because of its boron content but zinc sulphate and zinc oxide caused little or no decrease in tuber yield. The zinc content of potato tubers was increased but not doubled in zinc-treated plots, and during the first year after treatment the zinc content of topsoil decreased greatly. The zinc content of ryegrass grown after potatoes was greater than of potato tubers but did not reach a level considered dangerous to livestock. Treatment of soil with sulphur, zinc oxide or calomel may be useful for small plots used in the early stages of propagation of virus-tested potato clones where there is risk of infection with PMTV.     

Factors affecting the development of spraing in potato tubers infected with potato mop-top virus

A larger proportion of tubers of Arran Pilot potato growing at the surface of soil infested with potato mop-top virus (PMTV) showed spraing symptoms (brown rings) at harvest than of tubers from below the surface. Infected tubers with or without spraing developed a spraing ring when stored in darkness, first for 1–2 wk at 18 d?C and then for 1–2 wk at any constant temperature between 5 and 13 d?C. Only a faint surface ring developed when either of these periods was decreased to 1 day; 4-day periods were needed to induce distinct symptoms. Internal tuber symptoms developed more slowly than surface symptoms, and their formation was favoured by cutting the tubers in half. Additional pigmented surface rings were produced outside the first ring by successive cycles of treatment at 18 and 9d?. Spraing did not develop when the first stage of treatment was at 22–25d?, when the tubers were kept first at 10d? and then at 5d?, when the treatment at 5–13d? preceded that at 18d?, or when the tubers were kept at constant temperatures ranging from 5 to 25d?. When tubers of six potato varieties were grown in PMTV-infested soil and then stored at temperatures designed to induce symptoms, the varieties known to be the most susceptible in the field were those which had the greatest tendency to develop spraing during storage. When infected tubers were exposed to light, typical spraing symptoms were not induced, but greening of the tuber surface was much delayed in localized ring-shaped areas, so that pale weals appeared. Spraing symptoms were produced, in favourable conditions, by the reaction of cells at the periphery of the PMTV-invaded zone. Internal spraing did not prevent PMTV invading tissue outside the brown arcs; its rate of spread was about 10 μm/h at 14–18d?.     

Detection of surface-exposed epitopes on Chlamydia trachomatis by immune electron microscopy

The cell surfaces of two Chlamydia trachomatis serovars were explored by immune electron microscopy with monoclonal antibodies that recognize a number of chlamydial outer-membrane components. Species, subspecies and serovar-reactive epitopes on the major outer-membrane protein (MOMP) of a lymphogranuloma venereum biovar strain, L2/434/Bu, and a trachoma biovar strain, F/UW-6/Cx, were exposed on the surfaces of both elementary bodies (EBs) and reticulate bodies (RBs). Three epitopes on MOMP were inaccessible on EBs and RBs of both strains. These included a genus-reactive, species-reactive, and a subspecies-reactive epitope. In contrast, genus-specific epitopes on lipopolysaccharide (LPS) were not detected on the EB surface, but were clearly expressed on RBs of both L2/434/Bu and F/UW-6/Cx chlamydiae. Antibodies specific for the 60 kDa and 12 kDa 'cysteine-rich' outer-membrane proteins did not react with surface epitopes on either EBs or RBs. These data provide evidence that MOMP is a major surface antigen of both morphological forms, whereas some portions of the LPS molecule are exposed on the RB surface but become inaccessible to antibody after conversion to the infectious EB form.