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Phytoplasma pp 123-138 | Cite as

Visualization of Phytoplasmas Using Electron Microscopy

  • B. Jean DevonshireEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 938)

Abstract

The use of electron microscopy, both transmission and scanning, provides reliable and accurate methods for detecting phytoplasmas in plants. Our understanding of these pathogens, their morphology, development, and intracellular location in plants and insect vectors has been greatly increased through the use of these instruments. Development of techniques such as immunolabeling, cryofixation with freeze substitution or plunge freezing with direct transfer to the microscope stage, together with advances in instrumentation is enabling us to study these pathogens under conditions close to their native state. The visualization of fine detail and ultrastructure, using modern and established techniques, can only be appreciated by the magnification and spatial resolution offered in the electron microscopes. Now that the full sequencing of four phytoplasma genomes (to date) has been achieved, electron microscopy can play an important role in identifying and understanding specific gene functions.

Key words

Cryofixation Electron microscopy Scanning electron microscopes Transmission electron microscopes Ultramicrotomy 

Notes

Acknowledgements

I would like to thank Prof. Phil Jones, former head of Bioimaging at Rothamsted, for his valued advice and from whom a version of the TEM protocol originated and the use of the TEM images. Also to Prof. John Lucas and Dr Allison van de Meene for helpful comments on the manuscript. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK.

References

  1. 1.
    Weintraub PG, Beanland LeA (2006) Insect vectors of phytoplasmas. Annu Rev Entomol 51:91–111PubMedCrossRefGoogle Scholar
  2. 2.
    Kawakita H et al (2000) Identification of mulberry dwarf phytoplasmas in the genital organs and eggs of leafhopper Hishimonoides sellatiformis. Am Phytopathol Soc 90:909–914CrossRefGoogle Scholar
  3. 3.
    Hull R, Horne RW, Nayar RM (1969) Mycoplasma-like bodies associated with Sandal Spike Disease. Nature 224:1121–1122CrossRefGoogle Scholar
  4. 4.
    Horne RW (1970) The ultrastructure of mycoplasma and mycoplasma-like organisms. Micron 2:19–38Google Scholar
  5. 5.
    Waters H, Hunt P (1980) The in vivo three dimensional form of a plant mycoplasma-like organism by analysis of serial ultrathin sections. J Gen Microbiol 116:111–131Google Scholar
  6. 6.
    Musetti R (2010) Biochemical changes in plants infected by phytoplasmas. In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CAB International, Wallingford, pp 132–146Google Scholar
  7. 7.
    Doi Y et al (1967) Mycoplasma—or PLT group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf, potato witches’ broom, aster yellows, or paulownias witches’ broom. Ann Phytopathol Soc Jpn 33:259–266CrossRefGoogle Scholar
  8. 8.
    Siddique ABM et al (2001) Electron microscopy and molecular characterization of phytoplasmas associated with little leaf disease of brinjal (Solanum melongena L.) and periwinkle (Catharanthus roseus) in Bangladesh. J Phytopathol 149:237–244CrossRefGoogle Scholar
  9. 9.
    Franova J et al (2003) Electron microscopy and molecular characterization of phytoplasmas associated with strawflower yellows in the Czech Republic. Eur J Plant Pathol 109: 883–887CrossRefGoogle Scholar
  10. 10.
    Kiernan JA (2000) Formaldehyde, formalin, paraformaldehyde and glutaraldehyde: what they are and what they do. Microsc Today 00–1:8–12Google Scholar
  11. 11.
    Sabatini DD, Bensch K, Barrnett MD (1963) The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19–58PubMedCrossRefGoogle Scholar
  12. 12.
    Dykstra MJ, Reuss LE (2003) Biological electron microscopy: theory, techniques and troubleshooting, vol 2. Springer Verlag GmbH, New York, pp 27–28Google Scholar
  13. 13.
    Musetti R et al (2002) Application of immunoelectron microscopy techniques in the diagnosis of phytoplasma diseases. Microsc Res Tech 56:462–464PubMedCrossRefGoogle Scholar
  14. 14.
    Arocha Y et al (2009) Detection of phytoplasma and potyvirus pathogens in papaya (Carica papaya L.) affected with ‘Bunchy Top Symptom’ (BTS) in eastern Cuba. Crop Prot 28:640–646CrossRefGoogle Scholar
  15. 15.
    Haggis GH, Sinha RC (1977) Scanning electron microscopy of mycoplasma like organisms after freeze fracture of plant tissues affected with Clover Phyllody and Aster Yellows. J Phytopathol 68:677–680Google Scholar
  16. 16.
    Poghosyan AV et al (2004) Possible phytoplasma disease in papaya (Carica papaya L.) from Baja California Sur: diagnosis by scanning electron microscopy. J Phytopathol 152: 376–380CrossRefGoogle Scholar
  17. 17.
    Studer D et al (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203:285–294PubMedCrossRefGoogle Scholar
  18. 18.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Bioimaging, Plant Pathogens and MicrobiologyRothamsted ResearchHarpenden HertsUK

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