Mechanical Inoculation of Plant Viruses

Roger Hull1

1 John Innes Centre, Norwich Research Park, Colney, Norwich, United Kingdom
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16B.6
DOI:  10.1002/9780471729259.mc16b06s13
Online Posting Date:  May, 2009
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Abstract

This technique is for the mechanical inoculation of viruses to plants. It is used to diagnose a virus by its reactions in a variety of plant species, to test the infectivity of virus samples using local lesion hosts, and to propagate viruses. The virus preparation is rubbed onto the surface of the leaf in such a way as to break the surface cells without causing too much mechanical damage. The preparation of the virus sample, its application to the leaf, and the care of the plants before and after inoculation are described. Curr. Protoc. Microbiol. 13:16B.6.1‐16B.6.4. © 2009 by John Wiley & Sons, Inc.

Keywords: plant virus; mechanical inoculation; test plant; local lesion host; propagation plant

     
 
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Table of Contents

  • Introduction
  • Safety Considerations
  • Basic Protocol 1: Mechanical Inoculation of Plant Viruses
  • Alternate Protocol 1: Agroinoculation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Mechanical Inoculation of Plant Viruses

  Materials
  • Test plants, the choice of which depends on the objective of the experiment (see Section 16A, Propagation of major plant virus hosts)
  • Virus source material: this can be either (a) plant material known or suspected of being virus infected, (b) solution of virus preparation or (c) infectious clone
  • Liquid nitrogen, optional
  • Appropriate buffer: for many viruses 0.01 M phosphate buffer, pH 7.0 (see recipe), is satisfactory but some viruses are inactivated at this pH; if so, 0.01 M acetate buffer, pH 5.0, should be used
  • Additive to buffer overcome inhibitors present in the source material (the most common inhibitors are polyphenols, which can be overcome by a reducing/chelating agent such as 0.1% thioglycollic acid, 0.02 M sodium sulfite, or 0.01 M sodium diethyldithiocarbamate)
  • “Celite” (diatomaceous earth) or carborundum powder (600 mesh) available from laboratory chemical suppliers
  • Labels and a pencil
  • Pestle and mortar (sterilized)
  • Cheesecloth and scissors
  • Gloves
  • Glass spatula, optional
  • Squeeze bottle containing distilled water
  • Glasshouse or growth cabinet

Alternate Protocol 1: Agroinoculation

  Materials
  • Infectious clone of the viral genome
  • Agrobacterium tumefaciens or A. rhizogenes
  • Sterile distilled water
  • Hamilton micro‐syringe
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Figures

Videos

Literature Cited

   Boulton, M.I., Buchholz, W.G., Marks, M.S., Markham, P.G., and Davies, J.W. 1989. Specificity of Agrobacterium‐mediated delivery of maize streak virus DNA to members of the Graminae. Plant Molec. Biol. 12:31‐40.
   Dasgupta, I., Hull, R., Eastop, S., Poggi‐Pollini, C., Blakeborough, M., Boulton, M., and Davies, J.W. 1991. Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium‐mediated transfer. J. General Virol. 72:1215‐1221.
   Whitham, S.A., Yamamoto, M.L., and Carrington, J.C. 1999. Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 96:772‐777.
Key References
   Dijkstra, J. and de Jager, C.P. 1998. Practical Plant Virology: Protocols and Exercises. Springer‐Verlag, Berlin.
  This gives further background information and some variations to the technique.
   Hill, S.A. 1984. Methods in Plant Virology. Blackwell Scientific Publications, Oxford.
  This gives further background information and some variations to the technique.
   Walkey, D. 1991. Applied Plant Virology, 2nd Edition. Chapman and Hall, London.
  This gives further background information and some variations to the technique.
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