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Fungal Transmission of Plant Viruses

D'Ann Rochon1

1Agriculture and Agri‐Food Canada, Pacific Agri‐Food Research Centre, Summerland, British Columbia, Canada

Publication Name: 
Current Protocols in Microbiology
Unit Number: 
Unit 16B.4
DOI: 
10.1002/9780471729259.mc16b04s12
Online Posting Date: 
February, 2009
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Abstract

Fungal zoospores of Olpidium species transmit several viruses in the family Tombusviridae as well as in the Ophio- and Varicosavirus genera. This unit describes procedures for virus transmission by Olpidium sp. The method is useful for assessing fungal transmissibility of a given virus as well as for further studies on molecular and biological aspects of virus/vector interaction. Curr. Protoc. Microbiol. 12:16B.4.1-16B.4.17. © 2009 by John Wiley & Sons, Inc.

Keywords: Olpidium bornovanus; Olpidium brassicae; fungus transmission

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Initiating and Maintaining Fungal Cultures
  • Support Protocol 1: Storing Olpidium Cultures
  • Basic Protocol 2: Establishing Cultures from Infested Soil
  • Basic Protocol 3: “In Vitro” Virus Transmission
  • Support Protocol 2: Obtaining Virus-Free Olpidium Cultures
  • Basic Protocol 4: “In Vivo” Virus Transmission
  • Support Protocol 3: Growing Olpidium in Sand Culture
  • Support Protocol 4: Obtaining Zoospores Through “Root Washings”
  • Support Protocol 5: Observing and Counting Zoospores using Phase-Contrast Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Initiating and Maintaining Fungal Cultures

 Materials
  • Dried roots from an Olpidium-infested plant (Support Protocol 1)
  • Autoclaved H2O
  • Sterile sand (Support Protocol 3)
  • Host seeds or seedlings
  • 0.5× Hoagland's nutrient solution (see recipe)
  • 50 mM glycine, pH 7.6
  • Autoclaved forceps
  • Razor blades
  • 8.5-cm petri dishes
  • Pots for growth: 5- to 8-oz. (150- to 250-ml) colored plastic cups with drainage holes
  • Growth chamber or greenhouse with controlled conditions
  • Additional reagents and equipment for obtaining zoospores through “root washings” (Support Protocol 4) and observing/counting zoospores using phase-contrast microscopy (Support Protocol 5)

Support Protocol 1: Storing Olpidium Cultures

 Materials
  • Olpidium-infested roots
  • Deionized water
  • Small container with lid for drying and storing roots

Basic Protocol 2: Establishing Cultures from Infested Soil

 Materials
  • Olpidium-infested soil (air-dried or obtained directly from the field) and/or infested plants
  • Sterile potting soil
  • Sterile sand (Support Protocol 3)
  • Bait plants (see annotation to step 1)
  • Autoclaved water
  • 0.5× Hoagland's nutrient solution (see recipe)
  • 4- to 6-in. planting pots (purchase, e.g., from garden supply store)
  • Growth chamber or greenhouse with controlled conditions
  • Additional reagents and equipment for growth of Olpidium (Basic Protocol 1), obtaining zoospores through “root washings” (Support Protocol 4), observing/counting zoospores using phase-contrast microscopy (Support Protocol 5), and sand culture of Olpidium (Support Protocol 3)

Basic Protocol 3: “In Vitro” Virus Transmission

 Materials
  • Purified virus (Kakani et al., 2008)
  • 50 mM glycine, pH 7.6
  • Host seedlings
  • Sterile sand (Support Protocol 3)
  • 0.5× Hoagland's nutrient solution (see recipe)
  • 13- to 20-ml culture tubes
  • Pots for growth: 4-oz. plastic cups with drainage holes
  • Petri dishes
  • Growth chamber or greenhouse with controlled conditions
  • Additional reagents and equipment for obtaining zoospores through “root washings” (Support Protocol 4), observing/counting zoospores using phase-contrast microscopy (Support Protocol 5), and enzyme-linked immunosorbent assays (ELISA; Hornbeck, 1992)

Support Protocol 2: Obtaining Virus-Free Olpidium Cultures

 Materials
  • Virus/Olpidium-infested roots
  • 20% (w/v) Na3PO4 or 5% (v/v) HCl
  • 0.5 M sodium phosphate buffer, pH 7.2 (appendix 2A)
  • 0.5× Hoagland's solution (see recipe)
  • 100- to 200-ml beaker
  • Additional reagents and equipment for production and passaging of fungal zoospores (Basic Protocol 1), ELISA (Hornbeck, 1992), PCR (Kramer and Coen, 2001), sand culture of Olpidium (Support Protocol 3), obtaining zoospores through “root washings” (Support Protocol 4), and observing/counting zoospores using phase-contrast microscopy (Support Protocol 5)

Basic Protocol 4: “In Vivo” Virus Transmission

 Materials
  • Lettuce seedlings
  • Olpidium brassicae zoospores (Basic Protocol 1 and Support Protocol 2)
  • Sterile sand (Support Protocol 3)
  • 600-mesh carborundum (available from machine shop suppliers; see, e.g., http://www.msdiscount.com)
  • 10 mM potassium phosphate buffer, pH 7.0 (appendix 2A)
  • Pots for growth: 5- to 8-oz. (150- to 250-ml) colored plastic cups with drainage holes
  • Growth chamber with controlled conditions
  • Additional reagents and equipment for sand culture (Support Protocol 3), enzyme-linked immunosorbent assays (ELISA; Hornbeck, 1992), and obtaining zoospores through “root washings” (Support Protocol 4)

Support Protocol 3: Growing Olpidium in Sand Culture

 Materials
  • Sand (free of clay and silt; sieve size, 20 to 40; grit size 0.85-0.425 mm; http://TargetProducts.com)
  • 0.5× Hoagland's nutrient solution (see recipe)
  • Pan (for baking sand)
  • Pan lid or aluminum foil
  • Drying oven
  • Pots for growth: colored, opaque 4-to 8-oz. plastic cups
  • Petri dish lids

Support Protocol 4: Obtaining Zoospores Through “Root Washings”

 Materials
  • Olpidium-infested plants
  • Autoclaved distilled water
  • 50 mM glycine, pH 7.6
  • Razor blades, sterile
  • Small plastic basin or bucket
  • 100- to 200-ml glass beaker
  • Cheesecloth
  • Additional reagents and equipment for growing Olpidium in sand culture (Support Protocol 3) and observing/counting zoospores by phase-contrast microscopy (Support Protocol 5)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 16B.4.1
    Zoospores of O. bornovanus and O. brassicae. Fluorescence images demonstrating features of (A) O. bornovanus and (B) O. brassicae zoospores. Note the single, posteriorly placed whiplash flagellum. The O. bornovanus zoospore has an ellipsoid body that is ~4.5 × 8 µm, whereas zoospores of O. brassicae are more spherical and ~4.5 µm in diameter. Zoospores were labeled with FITC–concanavalin A and observed using a Zeiss Axiophot epifluorescent microscope. Magnification bars are 10 µm.

    View Image

Literature Cited

Literature Cited
    Adams, M.J. 1991. Transmission of plant viruses by fungi. Ann. Appl. Biol. 118:479-492.
    Campbell, R.N. 1962. Relationship between the lettuce big-vein virus and its vector, O. brassicae. Nature (Lond.) 195:675-677.
    Campbell, R.N. 1985. Longevity of Olpidium brassicae in air-dried soil and the persistence of the lettuce big-vein agent. Can. J. Bot. 63:2288-2289.
    Campbell, R.N. 1988. "Cultural characteristics and manipulative methods". In Viruses with Fungal Vectors (J.I. Cooper and M.J.C. Asher, eds.) Association of Applied Biologists, Wellesbourne, U.K.
    Campbell, R.N. 1996. Fungal transmission of plant viruses. Annu. Rev. Phytopathol. 34:87-108.
    Campbell, R.N., Grogan, R.G., and Purcifull, D.E. 1961. Graft transmission of big vein of lettuce. Virology 15:82-85.
    Campbell, R.N. and Grogan, RG. 1964. Acquisition and transmission of lettuce big-vein virus by O. brassicae. Phytopathology 54:681-690.
    Campbell, R.N. and Fry, P.R. 1966. The nature of the associations between Olpidium brassicae and lettuce big-vein and tobacco necrosis viruses. Virology 29:222-233.
    Campbell, R.N., Lecoq, H., Wipf-Scheibei, C., and Sim, S.T. 1991. Transmission of cucumber leafspot virus by Olpidium radicale. J. Gen. Virol. 72:3115-3119.
    Campbell, R.N., Sim, S.T., and Lecoq, H. 1995. Virus transmission by host specific strains of Olpidium bornovanus and Olpidium brassicae. Eur. J. Plant Pathol. 101:273-282.
    Fry, P.R. and Campbell, R.N. 1966. Transmission of a tobacco necrosis virus by Olpidium brassicae. Virology 30:517-552.
    Hornbeck, P. 1992. Enzyme-linked immunosorbent assays. Curr. Protoc. Immunol. 1:2.1.1-2.1.22.
    Hui, E. and Rochon, D. 2006. Evaluation of specific regions of the Cucumber necrosis virus coat protein arm on particle accumulation and fungus transmission. J. Virol. 80:5968-5975.
    Kakani, K., Sgro, J.Y., and Rochon, D. 2001. Identification of cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment. J. Virol. 75:5576-5583.
    Kakani, K., Robbins, M., and Rochon, D. 2003. Evidence that binding of cucumber necrosis virus to vector zoospores involves recognition of oligosaccharides. J. Virol. 77:3922-3928.
    Kakani, K., Reade, R., and Rochon, D. 2004. Evidence that vector transmission of a plant virus requires conformational change in virus particles. J. Mol. Biol. 338:507-517.
    Kakani, K., Reade, R., Katpally, U., Smith, T., and Rochon, D. 2008. Induction of particle polymorphism by cucumber necrosis virus coat protein mutants in vivo. J. Virol. 82:1547-1557.
    Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1-15.1.14.
    Lot, H., Campbell, R.N., Souche, S., Milne, R.G., and Roggero, P. 2002. Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and their roles in Lettuce Big-Vein etiology. Phytopathology 92:288-293.
    Matthews, R.E.F. 2002. Plant Virology, 4th ed. Academic Press, London.
    Robbins, M.A., Reade, R.D., and Rochon, D.M. 1997. A cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain. Virology 234:138-146.
    Rochon, D. 2007. "Molecular insights into plant virus-vector interactions". In Biotechnology and Plant Disease Management (Z.K. Punja, S. DeBoer, and H. Sanfaçon, eds.). CAB International, Wallingford, U.K.
    Rochon, D., Kakani, K., Robbins, M., and Reade, R. 2004. Molecular aspects of plant virus transmission by Olpidium and plasmodiophorid vectors. Ann. Rev. Phytopath. 42:211-241.
    Smith, P.R., Campbell, R.N., and Fry, P.R. 1969. Root discharge and soil survival of viruses. Phytopathology 59:1678-1687.
    Teakle, D.S. and Gold, A.H. 1964. Prolonging the motility and virus-transmitting ability of Olpidium zoospores with chemicals. Phytopathology 54:29-32.
    Teakle, D.S. and Thomas, B.J. 1985. Effect of heat on zoospore motility and multiplication of O. radicale and O. brassicae. Ann. Appl. Biol. 107:11-15.
    Teakle, D.S. 1988. "The effect of environmental factors on fungus-transmitted viruses and their vectors". In Viruses with Fungal Vectors (J.I. Cooper and M.J.C. Asher, eds.). Association of Applied Biologists, Wellesbourne, U.K.
    Westerlund, F.V., Campbell, R.N., Grogan, R.G., and Duniway, J.M. 1978. Soil factors affecting the reproduction and survival of Olpidium brassicae and its transmission of big vein agent to lettuce. Phytopathology 68:927-935.
 Key References
    Campbell, 1988. See above.

Provides an overview of Olpidium transmission of plant viruses and of methods used to isolate and maintain Olpidium cultures.

    Campbell, 1996. See above.

Provides a comprehensive overview of fungally transmitted plant viruses and their modes of transmission.

    Rochon et al., 2004

Provides a comprehensive review of molecular aspects of several fungally transmitted viruses and their modes of transmission. Also provides a description of molecular aspects of the interaction between CNV and O. bornovanus zoospores.

     
 
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