Wound tumor virus
L. M. Black
Dept. of Botany, University of Illinois, Urbana, Illinois 61801, USA
- Clover wound tumor virus (Rev. appl. Mycol. 47: 461)
- Clover big vein virus (Rev. appl. Mycol. 23: 490)
- Aureogenus magnivena (Rev. appl. Mycol. 23: 490)
- Trifoliumvirus nervicrassans (Rev. appl. Mycol. 38:
An isometric virus with particles c. 70 nm in diameter containing
double-stranded RNA. Only six instances of sap transmission to plants have
been reported but the virus multiplies in certain agallian leafhoppers and is
transmitted by them to a wide range of host plants.
Wound tumor virus (WTV) was found in the vector. It has not been found causing
disease in any plant in the field.
Collected once in leafhoppers in the vicinity of Washington, DC, USA.
Host Range and Symptomatology
Species in at least 20 families of dicotyledonous plants are susceptible
Vein enlargement, not vein-clearing, is the first systemic symptom.
Root tumours are characteristic
stem tumours occur in a few species
The genetic constitution of individual plants
and plant hormones
(Black & Lee, 1957
influence the initiation
of tumours in infected plants; in sweet
clover, tumours are first detectable in the pericycle at the 4th or 5th
incipient lateral root
(Kelly & Black, 1949
- Trifolium incarnatum (crimson clover). Characteristic irregular vein
- Melilotus officinalis and M. alba (sweet clovers), and Rumex
acetosa (cultivated sorrel). Variable root tumour response among individual
plants. M. officinalis clone C10 develops many root tumours
- Medicago sativa cv. Grimm (alfalfa) is immune.
- Melilotus officinalis clone C10, by propagation of infected cuttings.
Virus is most concentrated in the tumours, about 1012 virions/g.
- Trifolium incarnatum is preferred for testing viruliferous insects.
Virus can be assayed by injection into vectors
(Maramorosch, Brakke & Black, 1949)
which are then tested for transmission of virus to plants or
for production of virus antigens
Reddy & Black, 1966).
Cell monolayer cultures from Agallia constricta provide the best assay;
a preparation containing c. 106 virions/ml or more is needed
to cause infection
(Gamez & Chiu, 1968).
Infected cells are detected by
staining with fluorescent antibody; the relation between virus concentration
in inoculum and number of infected cells is linear
(Chiu, Reddy & Black, 1966;
Chiu & Black, 1967,
Growth of the virus in sweet clover cuttings over a period of years without
transmission through vectors results in strains (subvectorial) that are
poorly transmissible and, eventually, in strains (exvectorial) that are not
transmissible by the vector
The subvectorial strains have low
specific infectivities for vector cell monolayers and the exvectorial strains
have no infectivity. Subvectorial strains produce less virus and more soluble
antigen in sweet clover tumours than does the wild (or vectorial) strain. The
strain, exvectorial 49, can no longer be purified from tumours
(Whitcomb & Black, 1969
Reddy & Black, 1969
Gamez & Black, 1969
Transmission by Vectors
The virus is transmitted by certain agallian leafhoppers, especially Agallia
and Agalliopsis novella
(Black & Brakke, 1952
Brakke, Vatter & Black, 1954
does not transmit. At 27°C the virus has
a minimum incubation period in the vectors of about 2 weeks
during which it infects the intestinal tract, haemolymph and salivary glands
It reaches a peak concentration of c.
virions per leafhopper (i.e.
per mg) in about 4 weeks
(Reddy & Black, 1966
Gamez & Black, 1968
Frequency of transovarial
transmission in A. constricta
is genetically controlled; this control is
independent of the genetic variation in ability to transmit
potato yellow dwarf virus
Nagaraj & Black, 1962
Sinha & Shelley, 1965
Transmission through Seed
Transmission by Dodder
Antisera react in precipitin, complement-fixation or neutralization tests with
virus or soluble antigen both in plant and in insect extracts
(Black & Brakke, 1954
The precipitin ring test or ring-time test has been used to
measure the increase of soluble antigen
(Whitcomb & Black, 1961a
and, indirectly, virus infectivity
(Reddy & Black, 1966
antibody may be used in haemolymph smear tests on individual insects
(Nagaraj, Sinha & Black, 1961
without killing them
(Sinha, Reddy & Black, 1964
The minimum concentrations of virions detectable by the ring test and by the
passive haemagglutination test were 7 x 109
/ml and 2.5 x
(Gamez & Black, 1967
Antisera to vectorial virus strains react with virus and soluble antigen of
subvectorial and exvectorial strains. WTV closely resembles the
vertebrates in particle morphology and in having several double-stranded RNA
components and a RNA polymerase. However, sensitive passive haemagglutination
tests failed to show relationships between WTV and three strains of reovirus
(Gamez, Black & MacLeod, 1967
Virion morphology, nucleic acid structure
and intracellular behaviour suggest that
maize rough dwarf
sugarcane Fiji disease
viruses may have affinities with WTV.
Stability in Sap
In vector extracts the thermal inactivation point (10 min) is 50-60°C;
infectivity survived in dried extracts for at least 1 year at 0°C
(Brakke, Maramorosch & Black, 1953
At 37°C WTV is most stable between pH 6.60
and 6.61 (Kimura & Black, unpublished).
From root tumors of sweet clover clone C10: tumor production is favoured by a
daily cycle of 16 h of light at 20°C and 8 h of darkness at 17°C
Purified by density gradient centrifugation and zonal electrophoresis in
(Brakke et al., 1954
Treatment with carbon tetrachloride
and Freon aids purification (Kimura & Black, unpublished). Virus can also be
purified from vectors.
Properties of Particles
The particles seem all of one type; the dilution curve indicates one particle
(Chiu & Black, 1969
Sedimentation coefficient (s20,w) at infinite dilution: c.
(Black, Hills & Markham, 1962)
or 514 ± 10 S
(Kalmakoff, Lewandowski & Black, 1969).
Molecular weight (daltons): 68 ± 2 x 106
(Kalmakoff et al., 1969).
Absorbance at 260 nm (1 cm light path): 1.0 for the hydrolysed RNA from
suspensions containing 10 x 1011 virions/ml
(Ahmed & Black, unpublished);
for the virus, an absorbance of 1.0 (after correction for light
scattering) = 8.8 x 1011 virions/ml (Kimura & Black, unpublished).
A260/A280: c. 1.55;
A260/A243: c. 1.16
(Black et al., 1962).
Particles are icosahedral, c.
70 nm in diameter
The virus seems
to have a membrane supporting 32 capsomeres and enclosing the double-stranded RNA
(Black et al., 1962
Streissle & Granados, 1968
Kimura & Black,
22% of the weight of the particle, double-stranded
containing 38% G + C
(Bils & Hall, 1962
Black & Markham, 1963
Gomatos & Tamm, 1963
Kleinschmidt et al., 1964
Tomita & Rich, 1964
Each particle contains about 15-16 x 106
daltons of nucleic acid
(Black & Markham, 1963
Kalmakoff et al., 1969
Wood & Streissle, 1970
which when separated from the particle is found to consist of 12 molecules
(Kalmakoff et al., 1969
or 15 molecules
(Wood & Streissle, 1970
mostly of different sizes. Isolated RNA is not infective.
Protein: 78% of the particle weight. The number and amounts of
different proteins in the particles is not known. A RNA polymerase is associated
with the virus (D. R. Black & C. A. Knight, personal communication).
Relations with Cells and Tissues
In plants, virus antigen is mostly restricted to the abnormal phloem where it
is concentrated in spherical bodies in the cytoplasm
(Nagaraj & Black, 1961
Perhaps these are the inclusions of
Littau & Black (1952)
Shikata & Maramorosch (1965)
Viruliferous vectors show no
macroscopic evidence of disease. Virions occur in the cytoplasm but not in the
nucleus of many kinds of vector cells, some of which show cytopathic changes
(Hirumi, Granados & Maramorosch, 1967
Maramorosch et al., 1968
Shikata & Maramorosch, 1967a
Fluorescent antibodies stain the
cytoplasm but not the nucleus of infected vector cells
(Chiu et al., 1970
Tobacco club root (Rev. appl. Mycol. 27
: 101) was listed as
another disease induced by wound tumor virus (Rev. appl. Mycol. 47
2420). The evidence is to the contrary
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- Black & Lee, Virology 3:146, 1957.
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Crimson clover, Trifolium incarnatum.
Upper surface of infected leaf showing depressed veins.
Crimson clover, Trifolium incarnatum.
Lower surface of infected leaf showing irregular vein enlargement.
Crimson clover, Trifolium incarnatum. Smooth upper
surface of healthy leaf (except for the mid-vein, veins on the lower surface
are no more prominent).
Agallian leafhoppers, (upper row) females, (lower
row) males. (Left to right) Aceratagallia sanguinolenta
(not a vector), Agallia constricta (a vector) and Agalliopsis
novella (a vector).
Stem tumours on infected sweet clover (Melilotus sp.).
Young infected root of Rumex acetosa. Tumours tend to arise
above, below, or at the side, but not opposite, the emergence points of lateral
Virions in phosphotungstate. Bar represents 150 nm. (Courtesy of I.
Double-stranded RNA filaments. Bar represents 150 nm. (Courtesy of
Kleinschmidt et al., 1964.)