Testing of Transmission of Tobraviruses by Nematodes

Stuart A. MacFarlane1, Roy Neilson1

1 Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16B.5
DOI:  10.1002/9780471729259.mc16b05s12
Online Posting Date:  February, 2009
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Virus diseases often are spread between plants by vector organisms, some of which live below ground (e.g., fungi and nematodes) and feed on the plant root system. Tobraviruses are one of only two groups of plant viruses that have nematode vectors. They are primarily viruses of weed plants but can cause significant economic damage in a range of cultivated crops including potato, peas, beans, and many ornamental species. Identifying these viruses and their nematode vectors is a very important part of the struggle to combat disease in crop plants, and requires specialized techniques that will be discussed further in this unit. Curr. Protoc. Microbiol. 12:16B.5.1‐16B.5.16. © 2009 by John Wiley & Sons, Inc.

Keywords: virus transmission; tobraviruses; trichodorid nematodes; TRV; PEBV; PepRSV; Trichodorus; Paratrichodorus

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Table of Contents

  • Introduction
  • Basic Protocol 1: Viruliferous Status of Field Populations of Nematodes
  • Basic Protocol 2: Virus Acquisition by Nematodes
  • Basic Protocol 3: Virus Transmission by Nematodes
  • Basic Protocol 4: RT‐PCR Amplification of TRV RNA from Infected Plants
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Viruliferous Status of Field Populations of Nematodes

  • Silica‐based river sand
  • Field soil containing a population of a target trichodorid nematode species
  • 2‐week‐old Petunia grandiflora (or other species, see Background Information) seedlings
  • Corundum
  • Virus indicator plants, e.g., Chenopodium amaranticolor and C. quinoa
  • Plastic pots (6‐cm diameter)
  • Glasshouse, temperature controlled
  • Mortar and pestle

Basic Protocol 2: Virus Acquisition by Nematodes

  • Air‐dried silica‐based river sand
  • Steam‐sterilized loam mixture (particle size between 250 and 1500 µm)
  • 2‐week‐old P. grandiflora seedlings
  • 25‐ml plastic pots
  • Standard vented plastic glasshouse seed propagator (available from any garden center)
  • Glasshouse cubicle at 15° to 20°C
  • Pasteur pipets
  • Low‐power binocular microscope

Basic Protocol 3: Virus Transmission by Nematodes

  • Nematodes with acquired virus (see protocol 2)
  • Air‐dried silica‐based river sand
  • 1‐week‐old P. grandiflora seedling
  • Standard potting compost
  • Corundum or similar abrasive powder
  • C. amaranticolor and C. quinoa virus indicator plants
  • 0.5‐ml Beem capsules (size 00, TAAB Laboratories Equipment)
  • 10‐ml conical centrifuge tubes
  • Fine needles
  • Low‐power binocular microscope
  • 18.5 × 12.5 × 7.5–cm plastic container
  • Pasteur pipets
  • Well‐vented seed propagators (available from any garden center)
  • Room at 20°C with 2000 Lux lighting
  • 7.5‐ml glass bottles
  • Counting dishes
  • Microscope slides
  • Coverslips
  • ≤60°C heating block
  • Seed tray with individual compartments
  • Mortar and pestle

Basic Protocol 4: RT‐PCR Amplification of TRV RNA from Infected Plants

  • Washed roots of a bait plant
  • RNA extraction kit (e.g., Qiagen RNeasy plant mini kit)
  • RNase‐free water (e.g., DEPC‐treated water, Ambion)
  • Liquid nitrogen
  • TLES extraction buffer (see recipe)
  • Buffer‐saturated phenol, pH 5.0 (BioGene)
  • Chloroform
  • 4 M lithium chloride (LiCl)
  • 70% ethanol (diluted with water)
  • 1 µg/µl first‐strand cDNA oligonucleotide primer in water (5′‐GGGCGTAATAACGCTTACG‐3′; store at –20°C)
  • 200 U/µl Moloney Murine Leukemia Virus reverse transcriptase (M‐MLV RT; Promega) or equivalent
  • 5× M‐MLV reverse transcriptase reaction buffer (see recipe)
  • 2 mM and 10 mM 4dNTP mix (see recipe)
  • 40 U/µl RNase inhibitor (e.g., RNasin, Promega)
  • 10× Taq DNA polymerase reaction buffer with MgCl 2 (Roche)
  • 20 µM oligonucleotide PCR primer A (5′‐CAGTCTATACACAGAAACAGA‐3′, at 20 pmol/µl in water; store at –20°C)
  • 20 µM oligonucleotide PCR primer B (5′‐GACGTGTGTACTCAAGGGTT‐3′, at 20 pmol/µl in water; store at –20°C)
  • 5 U/µl Taq DNA polymerase (Roche)
  • Loading buffer (see recipe)
  • 1.2% agarose gel
  • TBE electrophoresis buffer (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Plastic, disposable pestle (Fisher Scientific)
  • Vortexer
  • Centrifuge
  • 42° and 70°C heating blocks or water baths
  • 200‐µl thin‐walled PCR tubes
  • Thermal cycler
  • Gel electrophoresis apparatus
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Literature Cited

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