A virus with RNA-containing isometric particles c.
28 nm in diameter, which sediment as three components. The
virus occurs naturally in highbush blueberry and grapevine.
It is transmissible by inoculation of sap, but has a limited
herbaceous host range. The natural means of spread is
unknown. Found only in Michigan and New York States, USA.
Main DiseasesCauses a severe dieback of the main stems of highbush
blueberry (Vaccinium corymbosum) cvs. Rubel and
Jersey, with leaf mottling (Fig.1) and sometimes leaf
deformation (Fig.2). Symptoms are the most pronounced on cv.
Rubel (Ramsdell & Stace-Smith, 1979). In
Vitis labrusca cv. Concord, the virus causes delayed
bud break, irregular elongation of fruits, pale green foliage
and straggly fruit clusters (Uyemoto et al., 1977;
see also Description No. 186).
Geographical DistributionMichigan and New York States, USA.
Host Range and SymptomatologyTransmitted experimentally by inoculation of sap to a
narrow range of hosts comprising 23 species in seven
dicotyledonous families (Ramsdell & Stace-Smith, 1979;
Uyemoto et al. , 1977); symptomless infection
occurred in nine of these species (Ramsdell &
Stace-Smith, 1979). When purified virus preparations were
used as inoculum, the MI (blueberry) strain infected
seedlings of cv. Rubel highbush blueberry (Fig.3) but the
NY (grapevine) strain did not (Ramsdell & Stace-Smith,
1979). A low percentage of seedlings of American grape
(Vitis labrusca) cv. Niagara were infected by
inoculation with the MI strain (Ramsdell &
- Diagnostic species
- Chenopodium quinoa. In inoculated leaves,
chlorotic local lesions are formed within 7-10 days
(Fig.4). Symptoms consist of mottling and epinasty of
terminal leaves, followed by death of the apex.
- Nicotiana clevelandii. Necrotic ringspots form
in inoculated leaves. Younger leaves develop localized
systemic necrotic spotting (Fig.5). Symptoms occur in
- Propagation and assay species
- Both C. quinoa and N. clevelandii are
good propagation hosts. Only C. quinoa is
satisfactory as a local lesion host
StrainsTwo strains have been distinguished. The MI strain,
found in blueberry in Michigan (Ramsdell & Stace-Smith,
1979), has slight serological differences from the NY
strain, found in grapevine in New York State (Uyemoto
et al., 1977). Although the two strains may differ
in ability to infect blueberry and grapevine (see Host Range
and Symptomatology) they cannot be distinguished on
Transmission by VectorsAlthough the virus resembles nepoviruses in many
properties, no success was obtained in attempts to
transmit the MI strain by hand-picked Xiphinema
americanum to C. quinoa or
N. clevelandii test plants, whether the nematodes
were from a Michigan source (T. Vrain & D. C. Ramsdell,
unpublished data) or from an Arkansas source (J. McGuire
& D. C. Ramsdell, unpublished data). Under the same
conditions, the Arkansas nematodes transmitted tobacco
ringspot virus. Uyemoto et al. (1977) found no
transmission of the NY strain to bait seedlings of
C. quinoa or cucumber planted in
X. americanum-infested soil taken from beneath a
diseased Concord grapevine.
Transmission through SeedThe NY strain is transmitted through seeds to seedlings of
Vitis labrusca (c. 5%) and C. quinoa (12%)
(Uyemoto et al. , 1977). The MI strain is seed-borne
in 20% of C. quinoa seeds (by sap indexing) and 29%
of blueberry seeds (by ELISA) (A. M. Childress-Roberts
& D. C. Ramsdell, unpublished data). It is not known
if the virus is transmitted to the seed through pollen.
SerologyThe virus is an excellent immunogen. A rabbit given
intramuscular injections of 0.5 to 1.0 mg virus (MI strain)
three or four times at 7-14 day intervals yielded antiserum
samples with titres of 1/512-1/1024 in agar double diffusion
tests (Ramsdell & Stace-Smith, 1979). The virus, in
crude sap from C. quinoa , forms a single precipitin
band in agar gel-diffusion tests. It is readily detected
in blueberry tissue by ELISA (A. M. Childress-Roberts &
D. C. Ramsdell, unpublished data).
RelationshipsThe biological and physicochemical properties of
blueberry leaf mottle virus place it in the nepovirus group
(Ramsdell & Stace-Smith, 1981). In agar gel double
diffusion tests, purified preparations of the MI strain did
not react with antisera to the following nepoviruses:
arabis mosaic, artichoke Italian latent, cherry leaf roll
(cherry and elderberry strains), cherry rasp leaf, grapevine
fanleaf, peach rosette mosaic, raspberry ringspot,
strawberry latent ringspot, tobacco ringspot, tomato black
ring and tomato ringspot viruses (Ramsdell &
Stace-Smith, 1979). However, purified preparations of the
MI and NY strains reacted weakly with antiserum to
grapevine Bulgarian latent virus, the heterologous titres
for both strains being 1/128, whereas the homologous titre
was 1/2048; antiserum to the MI strain had a homologous
titre of 1/1024, and heterologous titres of 1/512 against
the NY strain and 1/128 against grapevine Bulgarian latent
virus. In tests with sap from infected C. quinoa,
the MI and NY strains showed reactions of identity against
antiserum to the MI strain, but against antiserum to the NY
strain the NY strain formed a slight spur with the MI
strain (Ramsdell & Stace-Smith, 1979). These results
indicate that the MI and NY strains are almost identical
and that the NY strain is too distantly related to
grapevine Bulgarian latent virus to be considered merely
a strain of that virus, as suggested by Uyemoto
et al. (1977) and in Description No. 186.
Stability in SapThe MI strain was infective in sap of C. quinoa
held at room temperature (c. 20°C) for 5 but
not 7 days, at 4°C for 14 but not 21 days, or after
heating for 10 min at 65°C but not 70°C.
Infectivity in C. quinoa sap survived dilution to
10-4, but not 10-5 (Ramsdell &
PurificationThe MI and NY strains required different purification
methods for maximum yield (Ramsdell & Stace-Smith,
1981). All steps should be performed at 0-4°C.
MI strain. Harvest well-infected C. quinoa
leaves 7-10 days after inoculation. Homogenize in 2 ml/g
cold 0.05 M boric acid-borax buffer (pH 7), containing
0.1 % (w/v) each of sodium thioglycollate (TGA) and sodium
diethyldithiocarbamate (DIECA). Squeeze extract through
cheesecloth, freeze overnight, and thaw at 4°C.
Centrifuge thawed extract at low speed for 15 min,
retaining supernatant fluid. Add chloroform and
n-butanol, each to 10% (v/v), and stir for 15 min.
After low-speed centrifugation, collect the aqueous
supernatant fluid. Add polyethylene glycol (PEG, M. Wt
6000) to 8% (w/v) and NaCl to 1% (w/v). After the PEG
dissolves, centrifuge the mixture at low speed for 15 min
and resuspend the pellet in 10% of the starting volume
with 0.05 M Tris-HCl buffer, pH 7.4. Allow suspension to
stand several hours. Centrifuge at low speed for 15 min
and retain the supernatant fluid. Centrifuge at 137,000
g for 90 min. Resuspend pellet overnight in
0.05 M Tris-HCl buffer, pH 7.4. Further purify by
centrifugation in a 5-30% sucrose gradient in a Beckman
SW 41 rotor for 90 min at 38,000 rev./min. Collect the
combined middle (M) and bottom (B) components (usually
poorly separated), dilute three or four-fold with buffer
and centrifuge at 137,000 g for 3 h.
Resuspend pellet in 0.05 M Tris-HCl buffer, pH 7.4. Average
yield is c. 0.5 mg/100 g infected C. quinoa
NY strain. Extract C. quinoa leaves in 2 ml/g
0.5 M boric acid-borax (pH 7), containing 0.1% (w/v) each
of TGA and DIECA. Freeze extract overnight and after
thawing at 4°C, centrifuge at low speed. Add ammonium
sulphate to the supernatant fluid to 20% (w/v) and stir
for 6 h. Centrifuge at low speed for 15 min. Discard the
sediment and centrifuge the supernatant fluid at 108,000
g for 2.5 h. (Note: ammonium sulphate
apparently interferes with precipitation of the virus by
PEG; therefore, ultracentrifugation is necessary at this
stage). Resuspend pellet overnight in 0.05 M Tris-HCl
buffer, pH 7.4. Centrifuge at low speed for 15 min.
Further purify by passing through sucrose gradients as
Properties of ParticlesThe virus particles sediment as three components
(T, M and B) in sucrose density gradients (Ramsdell &
Stace-Smith, 1979, 1981).
Sedimentation coefficient, s20,w
(svedbergs): 53 (T), 120 (M), 128 (B). The MI strain has
consistently more M component than B component, whereas
the converse is true for grapevine Bulgarian latent virus
(Fig.6). Particles of the NY strain consistently fail to
resolve into M and B components in sucrose gradients
Isoelectric point: pH 7.5.
Electrophoretic mobility: 0.168 x 10-5
cm2 sec-1 volt-1 at
pH 7.0 in 0.02 M Na2HPO4, 0.02 M
Tris-citrate buffer. Determinations were made in 0.7%
agarose slab gels. The virus migrates as a single
electrophoretic component (D. C. Ramsdell &
J. M. Gillert, unpublished data).
Absorbance at 260 nm (1 mg/ml, 1 cm light path):
10.0 (M & B components combined).
A260/A280 (unfractioned virus)
= 1.69 (not corrected for light-scattering).
Buoyant density in CsCl (g/cm3): (M) 1.471;
Particle StructureIsometric particles c. 28 nm in diameter (Fig.8)
(Ramsdell & Stace-Smith, 1979). T particles are
penetrated by negative stain.
Particle Composition(Ramsdell & Stace-Smith, 1981).
Nucleic acid: RNA, single-stranded. Two species,
of M. Wt 2.15 x 106 and 2.35 x 106
(determined under non-denaturing conditions in 2.4%
polyacrylamide gels) are found in M and B components,
respectively. Unfractionated RNA preparations have a
Tm of 60°C and show 15.4% hyperchromicity
when heated from 30 to 99°C in 1 x SSC buffer,
pH 7.0. M and B components contain 37 and 40-41% RNA,
respectively, based on calculations from buoyant density in
CsCl (Sehgal et al. , 1970).
Protein: Subunits have M. Wt of c. 54,000,
estimated by electrophoresis in 5.0% SDS-polyacrylamide
Relations with Cells and Tissues(D. C. Ramsdell & R. Stace-Smith, unpublished data).
In diseased blueberry the virus is present in leaves and
blossoms throughout the bush. Roots have not been tested.
The virus was readily detectable by ELISA in pollen from
infected bushes (A. M. Childress-Roberts &
D. C. Ramsdell, unpublished data). In infected
C. quinoa leaves (Fig.7), virus-like particles
are found in aggregates in the cytoplasm of parenchyma
cells, in close proximity to vesicles of the endoplasmic
reticulum but not in association with other cell organelles.
NotesThe vector of blueberry leaf mottle virus remains
unknown. It is suspected that the virus spreads in
blueberry fields via pollen. The pattern of spread is not
patchy like that of a typical nepovirus, but rather more
random. In preliminary tests in which virus-free
2-year-old blueberry plants were caged with established
infected plants in the field together with honeybees, a
few berries (seeds) on the healthy test plants contained
blueberry leaf mottle virus at harvest, as assayed by
ELISA, but the pedicels did not (A. M. Childress-Roberts
and D. C. Ramsdell, unpublished data).
- Ramsdell & Stace-Smith, Acta Horticulturae 95: 37, 1979.
- Ramsdell & Stace-Smith, Proc. 7th Meeting Int. Counc. for Study of Viruses and Virus-like Dis. of Grapevine, Niagara Falls, Canada, 1980: 119, 1980.
- Ramsdell & Stace-Smith, Phytopathology 71: 468, 1981.
- Sehgal, Jean, Bhalla, Soong & Krause, Phytopathology 60: 1778, 1970.
- Uyemoto, Taschenberg & Hummer, Pl. Dis. Reptr 61: 949, 1977.
Mottled leaves of infected cv. Rubel highbush
Leaf malformation of infected cv. Rubel highbush
Mottle symptom on cv. Rubel seedling inoculated
with purified preparation of isolate MI.
Yellowish local lesions in inoculated leaves of
Chenopodium quinoa 7 days post inoculation.
Necrotic ringspots in inoculated leaves of
Nicotiana clevelandii and pin-point necrotic lesions
in uninoculated terminal leaves 4 days post-inoculation.
Linear-log sucrose density gradient profiles of
a) strain MI,
b) grapevine Bulgarian latent virus,
c) strain NY. Equal amounts of purified virus were applied
to gradients and centrifuged in a Beckman SW41 rotor at
4°C, 38,000 rev./min for 90 min.
Cross-section through parenchyma cell of a
C. quinoa leaf infected with isolate MI. Virus-like
particles (VLP) occur in the cytoplasm in aggregates
amongst endoplasmic reticulum vesicles (ERV), and are not
associated with other sub.cellular components.
Bar represents 500 nm. (Courtesy of B. Schroeder, Agriculture
Canada, Vancouver, British Columbia, Canada.)
Virus particles in 2% uranyl acetate.
Bar represents 100 nm. (Courtesy of B. Schroeder, Agriculture
Canada, Vancouver, British Columbia, Canada.)