41
June 1971
Family: Alphaflexiviridae
Genus: Potexvirus
Species: White clover mosaic virus
Acronym: WClMV


White clover mosaic virus

R. Bercks
Biologische Bundesanstalt für Land- und Forstwirtschaft, Braunschweig, Germany

Contents

Introduction
Main Diseases
Geographical Distribution
Host Range and Symptomatology
Strains
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Serology
Nucleic Acid Hybridization
Relationships
Stability in Sap
Purification
Properties of Particles
Particle Structure
Particle Composition
Properties of Infective Nucleic Acid
Molecular Structure
Genome Properties
Satellites
Relations with Cells and Tissues
Ecology and Control
Notes
References
Acknowledgements
Figures

Introduction

Described by Pierce (1935).

Selected synonyms

Clover mosaic virus (Rev. appl. Mycol. 39: 232)
Trifolium virus 1 (Rev. appl. Mycol. 19: 210)
White clover virus 1 (Rev. appl. Mycol. 15: 418)

An RNA-containing virus with elongated particles 480 x 13 nm. It has a fairly narrow host range, and is readily transmitted by inoculation of sap, but normally not by vectors. World-wide distribution.

Main Diseases

Causes mosaic and mottle of different degrees of severity in various clover species, and vein-clearing, light green mottle and sometimes small light yellow spots in pea.

Geographical Distribution

North America, Europe and New Zealand.

Host Range and Symptomatology

Most of the known hosts are legumes; many legumes are susceptible. A strain from Indiana, USA, infected plants from four other families (Bancroft, Tuite & Hissong, 1960).

Diagnostic species

Trifolium spp. (clovers). A weak and diffuse, sometimes irregular mosaic (Fig.7), occasionally latent, sometimes giving necrotic flecks.

Phaseolus vulgaris (French bean). Chlorotic spots in the inoculated leaves, often forming necrotic patches in leaf veins (Fig.1, Fig.2). Chlorotic discolorations of the veins of systemically infected leaves.

Vicia faba (broad bean). Diffuse ring-like or necrotic local lesions; mild systemic mosaic, sometimes accompanied by necrosis.

Vigna sinensis (cowpea). Small necrotic lesions or chlorotic spots or rings in inoculated primary leaves. Systemic mosaic, sometimes with vein-banding.

Pisum sativum (pea). Wilting of the inoculated leaves; occasionally develops local lesions. Systemically infected leaves show vein-clearing initially, then diffuse mottling (Fig.6), sometimes with mild necrotic flecks. If the wilt progresses upwards (Fig.5) the plants die (Bos, Delevic & van der Want, 1959).

Cucumis sativus (cucumber). Inoculated cotyledons develop yellow-green spots or indistinct white local lesions; diffuse yellow spots appear in systemically infected leaves (Fig.3).

Propagation species

Phaseolus vulgaris is suitable both for maintaining cultures and as a source of virus for purification.

Assay species

Vigna sinensis is a good local lesion host; some varieties of Phaseolus vulgaris give lesions with some strains.

Strains

Minor variants can be distinguished on the basis of symptom differences in various hosts. A Californian isolate causes local lesions in Gomphrena globosa; Dutch isolates produce systemic mosaic in cowpea. An isolate from Indiana produces local lesions in Ipomoea purpurea, Datura inoxia, D. stramonium, and an exceptionally severe local lesion reaction in inoculated leaves of bean and cowpea (Bancroft et al., 1960).

Transmission by Vectors

Goth (1962) reported a low percentage of transmission of one virus strain by Acyrthosiphon pisum (Macrosiphum pisi) to a clonal line of Ladino clover (Trifolium repens). However, other authors have failed to transmit the virus using various insects, including A. pisum.

Transmission through Seed

Recorded (6%) in Trifolium pratense (Hampton, 1963).

Transmission by Dodder

Dodder can become infected and transmit the virus (Bos et al., 1959). Failure to transmit has also been reported (Bancroft et al., 1960).

Serology

The virus is moderately to strongly immunogenic. It reacts with antiserum to form specific flocculent precipitates in liquid media. It is most simply assayed by the slide-precipitin test. It gives specific precipitates in agar gel-diffusion tests with virus fragments but such tests are much less sensitive than precipitation tests with intact virus particles (Koenig, 1969).

Relationships

Only minor serological differences, if any, exist between different strains. There is partial or complete protection between strains in plant protection tests. The virus is distantly related serologically to hydrangea ringspot, potato X, cactus X and clover yellow mosaic viruses, all of which have particles with the same or similar normal lengths. These viruses form the potato virus X group (Brandes & Bercks, 1965).

Stability in Sap

The thermal inactivation point is about 60°C (for a strain from Indiana 75-80°C), dilution end-point usually 10-5-10-6, longevity in vitro at room temperature 10-99 days.

Purification

The following method is satisfactory. Add ascorbic acid (to 0.2%) and sodium sulphite (to 0.2%) to sap of infected Phaseolus vulgaris, shake with an equal volume of ether, centrifuge at low speed and discard the ether phase, shake the aqueous phase for 5 min with an equal volume of carbon tetrachloride, centrifuge at low speed and discard the carbon tetrachloride phase; repeat the treatment with carbon tetrachloride, and store the clarified sap at 4°C overnight. Sediment and clarify by two cycles of high and low speed centrifugation, resuspending the pellets obtained at high speed in 0.01 M phosphate buffer, pH 7 (Wetter, 1960).

An alternative method is to mince frozen infected pea plants, express the sap after thawing, extract the fibre with 0.1 M K2HPO4 and mix the extract with the sap. Clarify and sediment by three cycles of low and high speed centrifugation, resuspending the pellet obtained at high speed in distilled water. Further purify the preparation by precipitating the virus with a half volume of saturated ammonium sulphate or by adjusting to pH 4.5 with 1 N acetic acid (Fry, Grogan & Lyttleton, 1960).

Properties of Particles

The virus sediments as a single component in the analytical ultracentrifuge. Sedimentation coefficient at infinite dilution (s20,w): 119 S (Varma, Gibbs & Woods, 1970).

Isoelectric point: about pH 4.5.

Electrophoretic mobility: -7 x l0-5 cm2 sec-1 volt-1 in phosphate buffer pH 7.5, ionic strength 0.1.

A260/A280: 1.2.

Absorbance at 260 nm (1 mg/ml, 1 cm light path): about 3.6 (Fry et al., 1960).

Particle Structure

Particles are flexuous helically constructed filaments (Fig.4), normal length about 480 nm, diameter about 13 nm (Brandes, 1964), axial canal about 3.5 nm in diameter, pitch of helix 3.4 nm, perhaps 11 subunits per turn (Varma et al., 1968, 1970).

Particle Composition

RNA: probably single stranded. M. Wt 2.4 x 106 (Koenig, 1971). Molar percentages of nucleotides: G 15.5; A 31.8; C 26.9; U 25.7. RNA is about 6% of particle weight (Varma et al., 1970).

Protein: Miki & Knight (1967) found the protein subunit to have a molecular weight of 1.4 x 104, whereas Koenig et al. (1970) report a value of 2.0 x 104. The amino acid composition of the protein (moles percent) is: ala 14.4; arg 4.0; asx 9.3; cys 1.3; glx 6.9; gly 5.5; his 1.6; ile 6.1; leu 7.5; lys 5.8; met 1.5; phe 4.2; pro 6.1; ser 7.6; thr 8.4; trp 1.6; tyr 1.9; val 5.4 (calculated from Miki & Knight, 1967).

Relations with Cells and Tissues

No information.

References

  1. Bancroft, Tuite & Hissong, Phytopathology 50: 711, 1960.
  2. Bos, Delevic & van der Want, Tijdschr. PlZiekt. 65: 89, 1959.
  3. Brandes, Mitt. biol. BundAnst. Ld- u. Forstw. 110, 130 pp., 1964.
  4. Brandes & Bercks, Adv. Virus Res. 11: 1, 1965.
  5. Fry, Grogan & Lyttleton, Phytopathology 50: 175, 1960.
  6. Goth, Phytopathology 52: 1228, 1962.
  7. Hampton, Phytopathology 53: 1139, 1963.
  8. Koenig, Phytopath. Z. 65: 379, 1969.
  9. Koenig, J. gen. Virol. 10: 111, 1971.
  10. Koenig, Stegemann, Francksen & Paul, Biochim. biophys. Acta 207: 184, 1970.
  11. Miki & Knight, Virology 31: 55, 1967.
  12. Pierce, J. agric. Res. 51: 1017, 1935.
  13. Varma, Gibbs & Woods, J. gen. Virol. 8: 21, 1970.
  14. Varma, Gibbs, Woods & Finch, J. gen. Virol. 2: 107, 1968.
  15. Wetter, Arch. Mikrobiol. 37: 278, 1960.

Acknowledgements

Photographs except Fig.4: courtesy of Dr L. Bos, Wageningen.


Figure 1

Primary leaf of Phaseolus vulgaris cv. Beka, 5 days after inoculation.

Figure 2

Primary leaf of Phaseolus vulgaris cv. Beka, 14 days after inoculation.

Figure 3

Systemic diffuse yellow spots in Cucumis sativus.

Figure 4

Shadowcast virus particles from a purified preparation. Bar represents 250 nm.

Figure 5

Wilting in Pisum sativum cv. Eroica.

Figure 6

Diffuse mottle in Pisum sativum cv. Fertilas.

Figure 7

Mosaic symptoms in leaves of Trifolium pratense.