Immunoelectron Microscopy of Plant Viruses and Mycoplasmas

Milne, Robert G.

doi:10.1007/978-1-4612-2910-0_9

Cited by 6 publications

(5 citation statements)

References 130 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Immunogold labelling, performed using a specific polyclonal antibody (Pab) previously tested in western blot analyses against the GPGV-CP (Gualandri et al 2015 ), supports our conclusion that the filamentous virus-like particles observed in grapevine BSCs are GPGV. The presence of gold in proximity of the particles could be due to the diffuse distribution of the CP in the infected cells and/or to the fact that, in ultrathin sections, elongated viruses are often detected as fragments of the whole particles, reducing the labelling accuracy (Milne 1992 ). The specificity of the Pab/virus reaction is here further supported by the absence of signal in cells different from that of BSCs, as well as in greenhouse-grown control grapevines.…”

Section: Discussionmentioning

confidence: 99%

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Tarquini

Ermacora

Bianchi³

et al. 2017

Protoplasma

View full text Add to dashboard Cite

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.Electronic supplementary materialThe online version of this article (10.1007/s00709-017-1198-5) contains supplementary material, which is available to authorized users.

show abstract

Section: Discussionmentioning

confidence: 99%

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Tarquini

Ermacora

Bianchi³

et al. 2017

Protoplasma

View full text Add to dashboard Cite

show abstract

“…Both the fixation and embedding procedures used (LR Gold and LR White protocols) maintained the typical phytoplasma ultrastructure well enough, while the morphology of plant cell organelles was not always good. Milne (1992) also observed that the ultrastruc-ture of the host was not optimally preserved, but the reaction antigen/antibody was highly specific using the mixture of 4% formaldehyde and 0.1% glutaraldehyde as fixative and LR Gold resin for the embedding. However, with the right Mab concentration the aim of labeling phytoplasmas in plant tissues by postembedding techniques could be followed up.…”

Section: Discussionmentioning

confidence: 99%

“…Phytoplasmas, prokaryotic pathogens of plants, lack cell walls and are pleomorphic in shape. This makes it impossible to use the traditional morphological methods to distinguish and classify the different phytoplasmas (Milne, 1992).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases

Musetti

Loi

Carraro

et al. 2002

Microscopy Res & Technique

View full text Add to dashboard Cite

An immunoelectron microscopy technique was applied to label Chrysanthemum leuchanthemum phytoplasma in infected leaf tissues of Chrysanthemum leuchanthemum L. and Catharanthus roseus L. plants. Specific monoclonal antibodies at different dilutions and secondary antimouse antibody conjugated with colloidal gold particles of different sizes were used. The monoclonal antibodies demonstrated their specificity against the antigen; immunocytological methods permitted the precise localization and identification of phytoplasmas in thin sections from infected tissues.

show abstract

“…Derrick and Brlansky (1976) demonstrated that corn stunt phytoplasma could be trapped using specific antibodies and visualized by immunosorbent electron microscopy (ISEM) technique. Later, similar approach was made for trapping phytoplasmas using specific antibodies by ISEM (Milne 1992). By using the homologous antiserum, aster yellows (AY) phytoplasma bodies could be trapped from partially purified extracts of AY-infected aster plants (Sinha and Benhamou 1983).…”

Section: Immunosorbent Electron Microscopy and Gold-labeled Antibody mentioning

confidence: 98%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

View full text Add to dashboard Cite

Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

show abstract

Immunoelectron Microscopy of Plant Viruses and Mycoplasmas

Cited by 6 publications

References 130 publications

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases

Detection of Bacterial and Phytoplasmal Pathogens

Contact Info

Product

Resources

About