SUMMARY

Attachment of virus particles to antiserum-coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop-top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles.



PLRV particles (up to 50/μm2 of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus-carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae.



PMTV particles (up to 10/μm2 of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.

Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy

Publisher Summary This chapter provides an overview of immunosorbent electron microscopy in plant virus studies. An electron microscope (EM) support film is prepared on a specimen grid and is coated with antibody, and this coated film is used to trap the antigen (virus particles) from the suspension placed in contact with it. A 2- to 10,000-fold increase in particle numbers per grid area may be obtained, as compared with the results of methods not using antisera. After trapping, the preparation is rinsed and given contrast by shadowing, positive staining, or negative staining. The antigen is recognized in the EM by its morphology and staining properties and can be further labeled by a second incubation with a more concentrated antibody preparation, thus decorating each individual particle with an easily recognized halo of γ-globulin molecules. Standard methods for making support films are widely available. The support films generally used for ISEM are made of Formvar or a similar plastic, or of Parlodion, strengthened with a layer of evaporated carbon. Some antisera may contain virus-like isometric particles as contaminants. Therefore, a grid incubated with the diluted antiserum alone should be prepared as a control if isometric virus particles are to be trapped at very low concentrations.

Immunosorbent Electron Microscopy in Plant Virus Studies

Specificity among barley yellow dwarf viruses in enzyme immunosorbent assays.

YLM, a codaominant polymerase chain reaction (PCR) marker linked to Yd2, could substantially improve the precision and efficiency of barley yellow dwarf virus (BYDV) resistance breeding. The aim of this study was to assess the effectiveness of YLM in a marker-assisted introgression programme and to quantify associations between the presence of Yd2 and other agronomic and quality traits. The Yd2 gene was introgressed into a BYDV-susceptible background through two cycles of marker-assisted backcrossing. BC2 F2-derived lines, either carrying or not carrying the YLM allele associated with resistance, were compared in the presence and absence of BYDV. The YLM marker was shown to be effective in the introgression of Yd2. Lines carrying the YLM allele associated with resistance produced significantly fewer leaf symptoms and showed a reduction in yield loss when infected with BYDV. There were no deleterious effects associated with the introgression of Yd2 on grain yield, grain size or malting quality. The implications of marker-assisted selection for Yd2 on barley improvement are discussed.

Y. C. Paliwal

Papers

Rapid Diagnosis of Barley Yellow Dwarf Virus in Plants using Serologically Specific Electron Microscopy)1

Serological Relationships of Barley Yellow Dwarf Virus Isolates1)