Details of DPV and References

DPV NO: 42 June 1971

Family: Bromoviridae

Genus: Ilarvirus

Species: Tulare apple mosaic virus | Acronym: TAMV

Tulare apple mosaic virus

R. W. Fulton Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin, USA

Contents

Introduction

Described by Yarwood (1955).

Synonym

Apple mosaic virus (Rev. appl. Mycol. 35: 902)

An RNA-containing virus with isometric particles c. 33 nm in diameter. The virus is readily transmitted by inoculation of sap, but loses infectivity rapidly in sap unless a stabilizing agent is present. The virus has been found only once in nature, in an apple tree in Tulare County, California.

Main Diseases

Associated with a severe mosaic disease of apple (Fig. 1).

Geographical Distribution

Found only once in nature, in an apple tree in Tulare County, California. This tree no longer exists; as far as is known the virus exists only in experimental material.

Host Range and Symptomatology

In limited tests, 16 species in 9 dicotyledonous families were susceptible.

Diagnostic species

Nicotiana tabacum (tobacco). Necrotic lines and rings appear on inoculated leaves; necrotic lines and oak-leaf patterns appear on systemically infected leaves (Fig. 2). Infected tobacco ‘recovers’ and produces leaves free of symptoms, but containing virus.

Phaseolus vulgaris (French bean). On Bountiful and certain other varieties, small brown lesions appear 3-5 days after inoculation (Fig. 3).

Propagation species

Vinca rosea is suitable for maintaining cultures and Nicotiana tabacum is a good source of virus for purification.

Assay species

Phaseolus vulgaris cv. Bountiful or Pencil Pod.

Strains

None described.

Transmission by Vectors

No vectors known.

Transmission through Seed

None reported.

Transmission by Dodder

Transmitted by Cuscuta subinclusa and C. campestris (Yarwood, 1955).

Serology

The virus is a moderately good immunogen in rabbits. A course of 8-10 intramuscular injections, at 3-4 day intervals, each of 1 mg virus emulsified in Freund’s incomplete adjuvant, induces antiserum titres of 1/640 to 1/1280 (Fulton, 1967). Precipitates are granular; the virus reacts well in agar gel-diffusion tests, giving a single band of precipitate.

Relationships

None have been described. The virus does not cross protect against tobacco streak virus (Fulton, 1956) and there are no serological cross reactions, although the viruses are similar in properties and cause similar symptoms in some hosts.

Stability in Sap

The virus in undiluted sap has a half-life of about 5 min (Yarwood, 1955). Infectivity is stabilized in diluted sap by reducing agents (0.02 M) such as 2-mercaptoethanol and cysteine hydrochloride. Inactivation is caused by o-quinones formed in the sap by oxidation (Mink, 1965). When infectivity is stabilized, the thermal inactivation point (10 min) is between 60 and 62°C.

Purification

Two methods have been used:

Mink, Bancroft & Nadakavukaren (1963). Freeze infected leaves at -22°C, powder and pack to half fill a 600 ml beaker. Add a stabilizing solution of 0.02 M sodium diethyldithiocarbamate and 0.02 M cysteine-HCl (350-400 ml), and infiltrate into the tissue under reduced pressure. Homogenize the mixture mechanically for 1-2 min and express liquid through cheesecloth. Add n-butanol to the liquid to 8.5% (v/v), with stirring. Remove the precipitate by centrifuging cold for 10 min at 8000 g. Sediment the virus from the supernatant liquid by centrifuging 1.75 hr at 78,000 g. Resuspend the pellets in 2.5 ml of 0.15 M pH 5.0 acetate buffer, keep at 4°C for a few hours, then centrifuge at 3000 g for 10 min. Dialyse the supernatant liquid against 0.15 M pH 5.0 acetate buffer until a precipitate appears (1-2 days). Remove the precipitate by centrifugation; the purified virus remains in the supernatant liquid. Its stability is greatest at pH 5.0 in 0.01 M acetate buffer.

Another effective method is that used for tobacco streak virus (Fulton, 1967). Homogenize tissue cold in 1.5 ml of buffer/g tissue, plus Al₂O₃ equal to 15% of the tissue weight. The buffer is 0.02 M phosphate, pH 8.0, and contains 0.02 M 2-mercaptoethanol. After centrifuging 15-20 min at 1500 g, stir the supernatant liquid with hydrated calcium phosphate, 0.8 ml/g tissue, then centrifuge at 1500 g for 15-20 min. Virus is then sedimented from the supernatant liquid by centrifuging 3 hr at 78,000 g. Resuspend the pellets in 0.01 M disodium ethylene diamine tetraacetate, pH 6.0, adjust the pH to 4.8-5.0 with citric acid and remove the precipitate by centrifugation. Readjust the pH of the supernatant liquid to pH 6.0 and concentrate the virus by high speed centrifugation. When resuspended in distilled water and kept at 2°C for several months, purified virus loses little infectivity.

Properties of Particles

The virus has two kinds of nucleoprotein particles, with sedimentation coefficients (s_20,w) of about 85 and 91 S; only the latter are infective (Mink et al., 1963).

A₂₆₀/A₂₈₀ of the mixture of particle types: c. 1.36.

Particle Structure

Particles are isometric, c. 33 nm in diameter (Fig. 4). They disintegrate readily in phosphotungstate unless first fixed in glutaraldehyde.

Particle Composition

RNA: About 12% of particle weight for the mixture of particle types. Molar percentages of the nucleotides: G24; A24; C21; U31 (Barnett & Fulton, 1969).

Protein: Subunits have a M. Wt of about 19,000 and contain about 181 amino acid residues (Barnett & Fulton, 1969).

Relations with Cells and Tissues

No information.

Notes

Symptoms in apple are similar to those of common apple mosaic. The geographical designation was proposed by Gilmer (1958) to differentiate the disease from common apple mosaic. Apple is very difficult to infect by mechanical inoculation, but can be infected through dodder.

Figures

References list for DPV: Tulare apple mosaic virus (42)

1. Barnett & Fulton, Virology 39: 556, 1969.
2. Fulton, Phytopathology 46: 694, 1956.
3. Fulton, Virology 32: 153, 1967.
4. Gilmer, Phytopathology 48: 432, 1958.
5. Mink, Virology 26: 700, 1965.
6. Mink, Bancroft & Nadakavukaren, Phytopathology 53: 973, 1963.
7. Yarwood, Hilgardia 23: 613, 1955.