Details of DPV and References

DPV NO: 109 October 1972

Family: Tombusviridae
Genus: Carmovirus
Species: Turnip crinkle virus | Acronym: TCV

Turnip crinkle virus

M. Hollings Glasshouse Crops Research Institute, Littlehampton, Sussex, England

Olwen M. Stone Glasshouse Crops Research Institute, Littlehampton, Sussex, England

Contents

Introduction

Described by Broadbent & Blencowe (1955), and Broadbent & Heathcote (1958).

An RNA-containing virus with isometric particles about 28 nm in diameter. It occurs in Brassica crops and is transmitted by flea-beetles and by inoculation of sap. It is not widespread or common, although it has a fairly wide experimental host range.

Main Diseases

Causes leaf crinkling, mottle and slight stunting in turnip, Brassica rapa (Fig. 4), or sometimes more severe distortion and rosetting. Other species of Brassica develop mild mottle (Broadbent, 1957).

Geographical Distribution

Recorded in Scotland, England and Yugoslavia.

Host Range and Symptomatology

It has a fairly wide host range, infecting species in about 20 dicotyledonous families. Readily transmissible by inoculation of sap.

Diagnostic species

Chenopodium amaranticolor. Numerous local chlorotic dots after 6 days, becoming necrotic later; no systemic infection (Fig. 5).

Chenopodium murale. Numerous local chlorotic dots after 10 days, expanding to large water-soaked rings; systemic chlorotic flecks after 3 weeks (Fig. 6).

Chenopodium quinoa. Local chlorotic lesions after 10-14 days; no systemic infection.

Datura stramonium. Local chlorotic spots after 10 days; no systemic infection.

Tetragonia expansa. Numerous local chlorotic dots after 10 days; symptomless systemic infection.

Brassica pekinensis (Pe-tsai, Chinese cabbage). Local chlorotic diffuse spots after 7 days; systemic light and dark green mottle and leaf crinkle (Fig. 1, Fig. 2).

Propagation species

Brassica pekinensis or B. juncea are good hosts for maintaining cultures and as sources for purification.

Assay species

C. amaranticolor is a good local lesion host. C. quinoa is less satisfactory; lesions are fewer, and take longer to appear.

Strains

None recorded; the virus reported by Verma & Varma (1961) was not proved to be turnip crinkle virus; the reactions in solanaceous plants suggest some other virus, and no serological or cross-protection tests were done using the type strain of turnip crinkle virus.

Transmission by Vectors

Transmitted by larvae and adult flea-beetles of the genera Phyllotreta (9 species) and Psylliodes (2 species). The virus was acquired within a few minutes and the beetles rarely remained infective for more than 1 day (Martini, 1958).

There are unconfirmed reports of transmission by larvae of the butterfly Pieris brassicae, leaf- mining larvae of the fly Phytomyza rufipes, hoppers of the locust Locusta migratoria and four species of aphid (Martini, 1958). Aphid transmission using Myzus persicae was not confirmed (Hollings & Stone, unpublished).

Transmission through Seed

No information.

Transmission by Dodder

No transmission by Cuscuta campestris (Hollings & Stone, unpublished).

Serology

The virus is a good immunogen; rabbits immunized by one intravenous plus two intramuscular injections gave antisera with specific titres in precipitin tube tests of 1/2048. The precipitates are granular (somatic). In gel-diffusion tests a single reaction line is formed; good reactions are obtained with crude sap. In immunoelectrophoresis, the single antigen component moves to the anode (Hollings & Thorne, 1969; Fig. 7).

Relationships

No serological relationships were found to any of twenty-seven other isometric viruses (Hollings & Stone, 1969) including carnation mottle, which McLeod & Markham (1963) suggested might be related (Tremaine, 1970).

Stability in Sap

In turnip or Brassica pekinensis sap the thermal inactivation point is 90-95°C, dilution end-point more than 10-6, and the virus survived at least 6 weeks at room temperature (c. 20°C). Lyophilized sap stored under vacuum at room temperature retained infectivity for at least 9 years.

Purification

The following methods are satisfactory. Preparations from Brassica pekinensis may yield 250 mg virus per kg leaf tissue.

1. Hollings & Stone (1969). Grind leaves at room temperature with 0.05 M phosphate buffer (pH 7.6) containing 0.1% thioglycollic acid (wt/vol=1/1.25). Squeeze out sap. Add n-butanol to 8.5% of the total volume and store at 2°C for 12-48 hr. Concentrate the virus by differential centrifugation. Further purification can be done by density gradient centrifugation.

2. Leberman (1966). Harvest plants 4-6 weeks after infection. Mince leaves and strain through muslin. Clarify by centrifuging at 12,000 g for 30 min. Add 1.34 g of a 20% solution of sodium dextran sulphate, 29 g of a 30% solution of polyethylene glycol 6000 and 5 g of 5 M NaCl to each 100 ml of clarified sap. Pour into separating funnel and leave to stand at 4°C overnight. Collect lower phase and interface, and centrifuge at low speed; discard supernatant fluid. Add 3 M KCl to the pellet, stirring until a thin creamy paste is obtained. Centrifuge at low speed; re-extract pellet with 0.5 M KCl if desired, and again centrifuge at low speed. Supernatant fluids are then centrifuged at high speed (30,000 rev/min) for 2 1/2 hr and the pellet resuspended in 0.l M disodium ethylene diamine tetraacetate, pH 7. The virus can be further purified by more cycles of high and low speed centrifugation, and resuspended in any of several buffers.

Properties of Particles

Sedimentation coefficient (s20, w) at infinite dilution 129 S.

Molecular weight (daltons): 8.0-9.0 x 106.

Electrophoretic mobility: In immunoelectrophoresis tests using 0.9% Ionagar in 0.03 M phosphate buffer, pH 7.6, the virus migrates as a single antigenic component towards the anode at a rate of 7.44 x 10-6 cm2 sec-1 volt-1.

A260/A280: 1.48.

Amax(260)/Amin(245): 1.27.

Particle Structure

Particles are isometric, about 28 nm in diameter in phosphotungstate, 33 nm in uranyl acetate. The intact virus is not penetrated by stain (Fig. 3). The particles contain 180 protein subunits of M. Wt 38,000 clustered in dimers in the 2-fold positions of a T=3 icosahedral lattice (Finch, Klug & Leberman, 1970). In addition it is suggested that there are 12 subunits of M. Wt 28,000 probably situated on the 5-fold axes and, possibly, a small amount of protein of M. Wt 80,000 which may represent the RNA polymerase (Butler, 1970). Small particles derived from the virus may represent inner cores (Haselkorn et al., 1961; Finch et al., 1970) but they can be produced by re-aggregation of the protein from degraded virus (Leberman & Finch, 1970).

Particle Composition

RNA: About 17% of particle weight, probably single-stranded. Molar percentages of nucleotides: G28; A26; C24; U22 (Symons et al., 1963).

Protein: About 83% of particle weight. The particles contain protein components of M. Wt 38,000 and 28,000, and possibly a third of M. Wt 80,000 (Butler, 1970). Amino acid compositions of the three proteins were determined by Butler (1970).

Relations with Cells and Tissues

All tissues are infected; no inclusion bodies reported, but plastid changes occur in some hosts (Stefanac, 1969).

Notes

Other beetle-transmitted isometric viruses infecting Cruciferae are turnip yellow mosaic, turnip rosette and radish mosaic. These three viruses cannot be reliably distinguished from turnip crinkle virus by the symptoms produced in Brassica species but they differ from turnip crinkle virus in that they do not infect species of Chenopodium or plants in families not close to the Cruciferae. They are all serologically distinct from turnip crinkle virus. In addition, preparations of turnip yellow mosaic and radish mosaic viruses separate into more than one component on analytical or density gradient centrifugation.

Figures

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Systemically infected plant of Brassica pekinensis.

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Systemically infected leaf of B. pekinensis showing crinkling and puckering.

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Virus particles from a purified preparation, stained with potassium phosphotungstate. Bar represents 100 nm.

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Systemically infected plant of B. rapa.

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Local chlorotic lesions in Chenopodium amaranticolor.

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Local chlorotic lesions in C. murale.

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Immunoelectrophoresis; migration of virus towards anode (0.9% agar in 0.03 M phosphate buffer, pH 7.6, 4 hr at set voltage, 200 V).

References list for DPV: Turnip crinkle virus (109)

  1. Broadbent, Investigation of viruses diseases of Brassica crops, Agric. Res. Council Rep. No. 14, Cambridge Univ. Press, 1957.
  2. Broadbent & Blencowe, Rep. Rothamsted exp. Stn. 1954: 87, 1955.
  3. Broadbent & Heathcote, Ann. appl. Biol. 46: 585, 1958.
  4. Butler, J. molec. Biol. 52: 589, 1970.
  5. Finch, Klug & Leberman, J. molec. Biol. 50: 215, 1970.
  6. Haselkorn, Hills, Markham & Rees, Abstr. Intern. Congr. Biophys. Stockholm 1961: 293, 1961.
  7. Hollings & Stone, Zentbl. Bakt. ParasitKde., Abt. II 123: 237, 1969.
  8. Hollings & Thorne, Rep. Glasshouse Crops Res. Inst. 1968: 109, 1969.
  9. Leberman, Virology 30: 341, 1966.
  10. Leberman & Finch, J. molec. Biol. 50: 209, 1970.
  11. McLeod & Markham, Virology 19: 190, 1963.
  12. Martini, Proc. 3rd Conf. Potato Virus Dis. Lisse-Wageningen 1957: 106, 1958.
  13. Stefanac, Acta biol. Iugoslav. Ser. B 6: 27, 1969.
  14. Symons, Rees, Short & Markham, J. molec. Biol. 6: 1, 1963.
  15. Tremaine, Virology 42: 611, 1970.
  16. Verma & Varma, Indian J. Microbiol. 1: 37, 1961.