Carnation cryptic virus
V. Lisa
Istituto di Fitovirologia applicata del C.N.R., Via O. Vigliani 104, I-10135 Torino, Italy
E. Luisoni
Istituto di Fitovirologia applicata del C.N.R., Via O. Vigliani 104, I-10135 Torino, Italy
R. G. Milne
Istituto di Fitovirologia applicata del C.N.R., Via O. Vigliani 104, I-10135 Torino, Italy
Contents
Introduction
Described by
Lisa et al. (1980).
A virus with isometric particles about 29 nm in diameter, containing three
major size classes of double-stranded (ds) RNA. It occurs symptomlessly in
carnation and is present in low concentration in plant tissues. It is apparently
not transmissible by sap inoculation or by grafting but is transmitted with high
frequency through the seed.
Main Diseases
Causes no symptoms in carnation (
Dianthus caryophyllus) and apparently
has no effect on those induced by any other virus when present
(
Lisa et al., 1981b).
Geographical Distribution
Found in carnation plants from Europe, Israel and USA
(
Lisa et al., 1981b).
Because of the extensive trade in carnation cuttings, the virus
is probably present in most carnation growing areas, worldwide.
Host Range and Symptomatology
Found in Mediterranean and miniature or spray carnations (
Dianthus
caryophyllus) and in commercial hybrids of
D. caryophyllus with other
Dianthus spp., such as ‘Chinese’ carnations; not found in eleven Sim
carnation cultivars. Experimental host range not determined because of lack of
transmissibility except through the seed
(
Lisa et al., 1981b).
- Diagnostic species
No diagnostic species known.
- Assay species
No assay species known.
- Propagation species
Any carrier carnation plant suitable for vegetative propagation, providing that
no other carnation virus is present.
Strains
Some isolates from Israel differed serologically from the original virus
described in Italy (see Relationships).
Transmission by Vectors
No vector known. An attempt to transmit the virus from carnation to carnation
by
Myzus persicae was inconclusive
(
Lisa et al., 1980).
Transmission through Seed
Transmitted with high frequency through seeds of Mediterranean and hybrid
carnations: the virus was detected in 90 to 100% of seedlings of Mediterranean
and hybrid ‘Chinese’ carnation cultivars. In the garden carnation
cv. ‘Chabaud’
the virus was found in
c. 10% of the seedlings checked. All the seedlings
tested (about 200) were from parents whose virus status was unknown
(
Lisa et al., 1981b).
Transmission by Grafting
Not transmitted from carnation to carnation either by grafting carrier
scions to non-carrier stocks or
vice versa, even when the grafts took
well and survived up to nine months
(
Lisa et al., 1981b).
Healthy
carnation plants grafted with scions carrying carnation cryptic virus and also
carnation mottle and
carnation etched ring viruses
had not acquired carnation
cryptic virus after 6 months although the two other viruses crossed the graft
union (V. Lisa, E. Luisoni & R. G. Milne, unpublished data).
Serology
The virus is a relatively good immunogen and antisera with titres of 1/512
in agar gel double diffusion tests have been obtained. Owing to the low virus
content in carrier plants, concentrated sap is required to obtain reliable
results with this test. Double antibody sandwich ELISA and immunoelectron
microscopy (immunosorbent electron microscopy (ISEM) plus decoration) detect
the virus in crude plant sap
(
Lisa et al., 1981b).
Relationships
Some isolates from Israel differed serologically from the original virus
described in Italy. In tests with antiserum to the original isolate, the isolates
from Israel gave very low ELISA values and low particle counts in ISEM (only
20% of those given by the homologous virus). In decoration tests, the titre of
the antiserum to the heterologous particles was two twofold dilution steps
lower than that to the homologous particles (J. Cohen, M. Alper, R. G. Milne
& P. Roggero, unpublished data, cited in
Boccardo et al., 1987).
The virus has many properties in common with other cryptic viruses
(
Francki et al., 1985;
Boccardo et al., 1987;
Descr. No. 298).
However
it was not serologically related to the following cryptic viruses:
beet cryptic 1 and 2,
poinsettia cryptic,
ryegrass cryptic,
white clover cryptic 1 and 2,
and the three cryptic viruses present in hop trefoil
(
Boccardo et al., 1987).
Cryptic viruses also share some properties with certain mycoviruses,
especially those of subgroup D
(
Buck et al., 1984).
No reaction was
found between carnation cryptic virus and 10 antisera to 15 mycoviruses (E.
Luisoni and R. G. Milne, unpublished data; antisera listed in
Boccardo et al., 1987).
There was also no serological relationship to any of 25
isometric viruses with single-stranded (ss) RNA genomes
(
Lisa et al., 1981b).
Stability in Sap
Thermal inactivation, longevity
in vitro and dilution end-point
have not been determined because infectivity tests are not feasible. The virus
is rather stable
in vitro in its serological and morphological properties
(
Lisa et al., 1981b).
Purification
The virus particles can survive different purification procedures, but the
yield may be only 50-100 µg per kg fresh plant tissue, because of the low
concentration of virus in the plants. A satisfactory purification procedure is
as follows: homogenize 500 g carnation leaves and shoots in 1.5 litres 0.5 M
potassium phosphate buffer, pH 7, containing 0.01 M sodium diethyldithiocarbamate,
0.02 M sodium sulphite and 0.005 M disodium ethylenediamine-tetraacetate, emulsify
the extract with an equal volume of chloroform, separate the phases by low speed
centrifugation and pellet the virus from the aqueous phase by ultracentrifugation.
Resuspend the sediments in 0.5 M potassium phosphate buffer, pH 7, containing
0.02 M sodium sulphite and repeat the chloroform treatment and the high speed
centrifugation. Resuspend the sediments in 0.05 M phosphate buffer, pH 7, and
centrifuge at low speed. To recover as much virus as possible, resuspend and
reprecipitate the low speed pellet several times, pooling the supernatant fluids,
to obtain a volume up to 1/100 that of the initial extract. Ultracentrifuge the
preparation for 2-4 h through a preformed 20-50% caesium sulphate density gradient
prepared in the same buffer as above. Collect the virus band or bands
(
Fig.1)
and remove the caesium sulphate by dialysis or ultracentrifugation
(
Lisa et al., 1981b
and unpublished data).
Properties of Particles
After isopycnic centrifugation in caesium chloride in 0.01 M phosphate
buffer, pH 7, the virus formed a major band with buoyant density 1.337 g/ml
plus other minor bands that varied in position and density in different
preparations
(
Lisa et al., 1981b).
Particle Structure
Purified virus particles negatively stained in uranyl acetate appear rounded
in profile, about 29 nm in diameter, and partly penetrated by the stain
(
Fig 2,
Fig.3).
No clear subunit structure is visible. The particles appear similar in other
negative stains such as neutral phosphotungstate or ammonium molybdate, pH 7
(
Lisa et al., 1981b).
Particle Composition
Nucleic acid. Three major size classes of dsRNA of M. Wt (x
10
6) 1.04, 0.95 and 0.84
(
Fig.6),
plus a minor component of M.
Wt 0.88 x 10
6, as determined by electrophoresis in 5% polyacrylamide
gels in non-denaturing conditions
(
Lisa et al., 1981a).
The minor
component was not detected in all preparations. Purified RNA preparations reacted
with a serum containing antibodies to dsRNA
(
Fig.5).
In the electron microscope
the dsRNA molecules appear linear under non-denaturing conditions, whereas the
ssRNA molecules of
carnation mottle virus
have obvious secondary structure
(
Fig.7,
Fig.8)
(
Lisa et al., 1981a).
RNA preparations from carnation cryptic
virus did not hybridize in Northern blot experiments with cDNA to RNA preparations
extracted from
white clover cryptic viruses 1, 2 or 3, or
beet cryptic virus 1 or
2 (G. Boccardo and P. Palukaitis, personal communication).
Protein: No information available.
Relations with Cells and Tissues
In thin sections of carrier carnation plants, virus particles were not seen,
possibly because of their low concentration and similarity to ribosomes. No
cytopathological disorders were associated with the presence of the virus
(
Lisa et al., 1981b).
Notes
Some clones of carnation contain a second dsRNA virus of similar size but
apparently serologically unrelated to carnation cryptic virus (V. Lisa, R. G.
Milne and G. Boccardo, unpublished data). Mediterranean carnations may be infected
by the ‘attenuated form’ of
carnation mottle virus. This occurs in
low concentration, like a cryptic virus, but may be distinguished by infectivity
and serological tests on concentrated preparations
(
Fig.4)
(
Luisoni et al., 1984).
Antisera to other carnation viruses, if prepared using carnation as virus
source, may contain antibodies to carnation cryptic virus and so give rise to
spurious results; the presence of dsRNA in the particles may elicit antisera
cross-reacting with other dsRNA viruses. These possibilities should be considered
when doing serological tests. The probably wide distribution of carnation cryptic
virus in carnations may also complicate screening for other viruses based on
detection of dsRNA in the plants.
Carnation cryptic virus was not eliminated from four Mediterranean carnation
cultivars by heat therapy (37°C for 4 weeks), followed by meristem tip
culture (A. Baratta, E. Luisoni & V. Lisa, unpublished data).
Carnation cryptic virus particles contain three major RNA size classes whereas
several other cryptic viruses contain only two. It is not yet determined whether
all three RNA species are genomic or whether one or more are satellites or
subgenomic sequences.
Morphologically, cryptic viruses can be divided into two types, those with
rather featureless particles 29-32 nm in diameter, and those with particles about
38 nm in diameter possessing prominent subunits
(Boccardo et al., 1987).
Carnation cryptic virus belongs to the first type.
References
- Boccardo, Milne, Luisoni, Lisa & Accotto, Virology 147: 29, 1985.
- Boccardo, Lisa, Luisoni & Milne, Adv. Virus Res. 32: 171, 1987.
- Buck, Ackermann, Bozarth, Bruenn, Koltin, Rawlinson, Ushiyama & Wood, Intervirology 22: 17, 1984.
- Francki, Milne & Hatta, Atlas of Plant Viruses, Vol. II, 284pp., CRC Press, Boca Raton, Florida, 1985.
- Lisa, Luisoni & Milne, Acta Hort. 110: 175, 1980.
- Lisa, Boccardo & Milne, Virology 115: 410, 1981a.
- Lisa, Luisoni & Milne, Ann. appl. Biol. 98: 431, 1981b.
- Luisoni, Milne & Boccardo, Virology 68: 86, 1975.
- Luisoni, Lisa & Roggero, Atti Giornate Fitopatol. 1984 3: 427, 1984.
Light-scattering band (arrow) formed by carnation cryptic virus after
4 h centrifugation at 149,800 g in a 20-50% preformed caesium
sulphate density gradient.
Purified particles of the virus in uranyl acetate. Bar represents
100 nm.
Particles of carnation cryptic virus (arrows) mixed with those of
tomato bushy stunt virus (diameter 34 nm). Bar represents 100 nm.
Concentrated preparation from carnations containing particles of
carnation cryptic virus (left) and the attenuated form of carnation
mottle virus (right) decorated with carnation mottle virus antibody.
Bar represents 100 nm.
Reaction of a maize rough dwarf virus antiserum containing antibodies
to dsRNA (Luisoni et al., 1975) with: (a) synthetic poly(I): poly(C),
(b) carnation cryptic virus dsRNA, (c) rice gall dwarf virus dsRNA; (d) shows
non-reaction with tobacco mosaic virus ssRNA.
Electrophoretic separation in a 5% polyacrylamide gel slab of dsRNA
species from maize rough dwarf virus (left), Bombyx mori cytoplasmic
polyhedrosis virus (middle), and carnation cryptic virus (right).
Nucleic acid from purified particles of carnation cryptic virus
spread under non-denaturing conditions and shadowed. Note the extended linear
molecules differing from those in Fig.8. Bar represents 100 nm.
Preparation as in Fig.7 of RNA from carnation mottle virus. Note
that the molecules are not extended but have a ‘collapsed’ appearance indicating
extensive secondary structure. Bar represents 100 nm.
