2453
J. gen. Virol. (1985), 66, 2453-2460. Printedin Great Britain
Key words: dsRNA/cryptie virus/protoplasts/vicia
Detection of dsRNA in Particles of Vicia Cryptic Virus and in Viciafaba
Tissues and Protoplasts
By M. A. A B O U - E L N A S R , t
A. T. J O N E S *
AND M. A. M A Y O
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, U.K.
(Accepted 25 July 1985)
SUMMARY
Isometric particles of vicia cryptic virus (VCV), purified from Viciafaba seedlings
infected through seed, had a buoyant density in CsCl of 1.37 g/ml. Analysis by P A G E
showed that VCV particles contained three species of d s R N A with tool. wt. of 1-37 ×
106, 1.26 × 106 and 1.21 x 106. These d s R N A species were also detected in extracts
from leaves, stems, roots, flowers, seeds and mesophyll protoplasts of V.faba; d s R N A
was readily detected in extracts from one leaflet (about 0.6 g). Slower-migrating species
of d s R N A occurred in many leaf extracts but were not obtained from VCV particles.
Electrophoresis of d s R N A extracts in gels that are then stained with silver appears to
be a simple and reliable method for detecting 'cryptic' viruses. Using this method,
VCV d s R N A was detected in 18 cultivars of V. faba, but not in cultivars Beryl and
Minica which were thought from previous work to be free from VCV. Neither VCV
particles nor VCV d s R N A were detected in VCV-free individual V. faba plants
inoculated with VCV mechanically or by dodder. VCV was not detected in leaves of
plants affected by cytoplasmic male sterility or in a maintainer line for this condition.
INTRODUCTION
Isometric virus-like particles (VLPs) about 30 nm in diameter have been found in low
concentrations in apparently healthy individuals of at least 15 plant species (Kassanis et al.,
1977; Jones, 1980; Koenig & Lesemann, 1980; Boccardo et al., 1983; Natsuaki et al., 1979,
1983a, b, 1984). These particles are considered to be those o f ' c r y p t i c ' (Kassanis et al., 1977) or
'temperate' (Natsuaki et al., 1979) viruses. These agents are transmitted to a large proportion of
the sexual progeny of infected mother plants but are not transmitted either by graft inoculation
or by sap inoculation. Where examined, 'cryptic' or 'temperate' viruses have been shown to
contain d s R N A (Lisa et al., 1981; Boccardo et aL, 1983; Natsuaki et al., 1983a, b, 1984).
VLPs found in several cultivars of Viciafaba were referred to as particles of vicia cryptic virus
(VCV) by Kenten et al. (1978) and Jones (1980). VCV was up to 88~o seed-borne in some V.faba
cultivars but was not transmitted to VLP-free plants by aphids or dodder, or by inoculating them
mechanically with infective sap or by grafting; it was not eliminated from seedlings by heat
treatment (Kenten et al., 1978, 1979, 1980, 1981). In field trials, VCV infection had little or no
effect on plant growth and yield (Kenten et aL, 1980, 1981). In this paper we show that VCV
particles contain d s R N A species and report the detection of these and other d s R N A species in
several cultivars of V. Jaba.
METHODS
Plant material. Duplicates of batches of V.faba leaf tissue from which VCV particles had been partially purified
by Jones (1980) were used after 4 to 5 years storage at - 15 °C. Other plants were grown from seed harvested from
field trials at this Institute or bought commercially, In addition, seed of a cytoplasmic male sterile (CMS)
maintainer line (line 6) and a CMS affected line (CMS 447x6) were kindly supplied by D. A. Bond, Plant Breeding
t Present address: Department of Microbiology, Faculty of Agriculture, Ain Shams University, Shobra, Cairo,
Egypt.
0000-6710 © 1985 SGM
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M.A.
ABOU-ELNASR, A. T. JONES AND M. A. MAYO
Institute, Cambridge, U.K. All plants were grown in soil-less compost and kept in aphid-proof glasshouses at 15 to
22 °C.
Viruspurification. VCV particles were partially purified by grinding each gram of V.faba leaf and stem tissue in
4 ml 1% ascorbic acid in 0.5 M Sorensen's phosphate buffer pH 8, n-butanol and chloroform (2 : 1 : 1, by vol.). The
emulsion was centrifuged for 15 rain at 10000 g and the aqueous phase was left overnight at room temperature,
clarified by centrifugation for 15 min at 10000 g and then centrifuged for 2.5 h at 100000g. The resulting pellets
containing virus were resuspended in water adjusted to pH 8 with NaOH (SH). The virus was further purified by
centrifuging to equilibrium in CsCI in SH (initial density 1.37 g/ml) in Beckman SW50.1 tubes for 18 h at 40000
r.p.m, at 10 °C. Fractions were collected by upward displacement, monitored for absorbance at 254 nm and their
refractive index was determined at room temperature using a refractometer. Virus-containing fractions were then
dialysed against SH to remove the CsC1.
Nucleic acid extraction and purification of dsRNA. Extraction and purification of dsRNA largely followed
method 2 of Morris & Dodds (1979). Usually 2 to 3 g of fresh or frozen tissue was powdered in liquid nitrogen and
then ground in a pestle and mortar with a mixture (per g leaf) of 1 ml 0.2 M-glycine, 0.1 M-Na2HPO4, 0-6 M-NaCI
(pH 9-5), 0-1 ml 10% SDS, 0-0I mI thioglycerol, i ml water-saturated phenol containing 10% m-cresol and 0-1% 8hydroxyquinoline and 1 ml chloroform-pentanol (25 :l). The extract was centrifuged for 20 min at 10000g and the
aqueous phase was mixed per ml with 0.22 ml ethanol and 0-025 g cellulose powder (Cellex N-I, Bio-Rad). The
mixture was stirred for 10 to 30 min at 4 °C and centrifuged for 10 rain at 10000 g. The cellulose pellet was
collected, resuspended in 5 ml 0.1 M-NaCI, 0.05 M-Tris-HC1, 0-001 M-EDTA, pH 7.0 (STE) containing 18%
ethanol and the suspension placed in a small column. After the STE/ethanol had drained, the cellulose was washed
with 16 ml 18% ethanol in STE and then with 5 ml STE buffer alone to elute dsRNA. The dsRNA fraction was
made 0.03 M in MgC12 and incubated for 20 min at 30 °C with 10 ~tg/ml DNase and then mixed with 2.5 vol.
ethanol and stored at - 15 °C.
When required for nucleic acid extraction, purified virus particles were dialysed against 10 mM-Tris-acetate pH
7.2, 20 mM-sodium acetate, 0.5 mM-EDTA and then mixed with 0.1 vol. 10% SDS and extracted twice with an
equal volume of phenol :cresol (9:1, v/v) containing 0-1% 8-hydroxyquinoline. Nucleic acid was recovered by
adding 0.1 vol. 3 M-NaC1 and then 2.5 vol. ethanol.
Test for the ds nature ofRNA. In some tests, samples of dsRNA were made 0.2 Min NaC1 and incubated with 0.1
~tg/ml RNase A (Sigma) at 30 °C for 20 min. To remove residual RNase prior to electrophoresis, samples were
either treated with proteinase K or extracted twice with phenol.
Gel electrophoresis of RNA. Samples of dry RNA were resuspended in 10% sucrose in 36 mM-Tris, 30 mMNaH2PO~, 1 mM-EDTA, pH 7.8 (TPE) and centrifuged for 10 min at 10000 g. The supernatant fluid was
electrophoresed in gels of 7% acrylamide, 0.14% bisacrylamide in TPE at 50 V for 16 to 18 h. Gels were stained
with silver using the method of Sammons et al. (1981) as described by Igloi (1983). Molecular weights of
components were estimated by comparing their mobilities with those of the dsRNA species of maize rough dwarf
virus (MRDV) kindly supplied by A. F. Murant and using mol. wt. values given by Reddy et al. (1975).
Isolation ofprotoplasts. Plants with three or four fully developed leaves were kept for 2 to 3 days in a controlled
environment of 16 h photoperiod of 10000 Ix at 25 °C alternating with 8 h in darkness at 20 °C. Then wellexpanded leaflets were divided down the mid-rib and dsRNA was extracted from one half and mesophyll
protoplasts isolated from the other half. For protoplast isolation each half-leaf was abraded with Carborundum
(Beier & Bruening, 1975) and floated on 0.6 M-mannitol for 1 h. The mannitol solution was then replaced by 2%
cellulase R10 and 0.5 % Macerozyme R10 (each from Yakult Honsha Co., Nishinomiya, Japan) in 0.6 M-mannitol
and the leaf pieces were shaken gently at 30 °C for 2 to 3 h. The leaf debris was then removed by filtration and
protoplasts were recovered and washed three times by centrifugation from 0.6 M-mannitol. dsRNA was extracted
from pellets of washed protoplasts and also from the undigested halves of leaves used to prepare protoplasts.
Immunoelectron microscopy. Observations in the electron microscope of virus antigen and antibody reactions
were as described by Roberts & Harrison (1979) using antiserum to VCV kindly supplied by D. A. Govier and
A. J. Cockbain, Rothamsted Experimental Station, Harpenden, U.K.
RESULTS
Detection o f d s R N A in particles o f V C V
P a r t i c l e s o f V C V w e r e purified f r o m b a t c h e s o f 100 to 200 g o f l e a f a n d s t e m tissue o f V. faba
cv. M a r l s B e a d , Stella a n d T h e Sutton. P a r t i c l e s f o r m e d a single b r o a d l i g h t - s c a t t e r i n g z o n e
in CsC1 g r a d i e n t s w h i c h c o r r e s p o n d e d to a p e a k o f a b s o r b a n c e at 254 n m a n d to a d e n s i t y o f a b o u t
1.37 g/ml. F r a c t i o n s f r o m this z o n e c o n t a i n e d i s o m e t r i c p a r t i c l e s o f a b o u t 30 n m d i a m e t e r (Fig.
1 a). I m m u n o e l e c t r o n m i c r o s c o p i c tests s h o w e d t h a t p a r t i c l e s f r o m p l a n t s o f cvs M a r l s B e a d a n d
T h e S u t t o n could be c o a t e d w i t h a n t i b o d i e s to V C V (Fig. 1 b); n o serological test w a s m a d e w i t h
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2455
Vicia cryptic virus dsRNA
(a)
~)
Fig. 1
Fig. 2
Fig. 1. Particles of VCV purified from V. faba cv. The Sutton negatively stained with (a) 2%
phosphotungstic acid pH 7 and (b) uranyl formate/NaOH after coating with antibodies to VCV. Bar
marker represents 100 nm.
Fig. 2. Polyacrylamide gel electrophoresis of dsRNA from (a) particles of VCV purified by isopycnic
centrifugation in CsC1 from V. faba cv. The Sutton, and (b) particles of MRDV.
preparations from cv. Stella. When the same purification procedure was applied to tissue from
cv. Minica, no light-scattering zone was detected and no particles were found in regions of the
CsC1 gradient corresponding to a density of 1.37 g/ml.
When analysed by polyacrylamide gel electrophoresis, nucleic acid extracted from material of
density approx. 1.37 g/ml in preparations from cvs Maris Bead, Stella and The Sutton was
resolved into three bands. Treatment with DNase, or with RNase in STE containing 0.2 MNaC1, did not affect the occurrence of these bands but treatment with RNase in STE alone
greatly decreased their intensity of staining. VCV particles therefore contain three species of
dsRNA. The estimated mol. wt. of these species were 1.37 x 10 6, 1.26 × 106 and 1-21 x 10 6
(Fig. 2). No R N A was detected in fractions from CsC1 gradients of material purified from
Minica plants.
Detection of dsRNA in tissues of V. faba
d s R N A extracted from 5 g samples of frozen leaves, stems, roots and flowers of cv. Stella
contained bands of d s R N A similar in number, mol. wt. and in resistance to DNase and RNase
to those found in particles of VCV. Similar d s R N A components were also readily detected in
extracts of cotyledons and embryos of seed of cv. The Sutton that had been soaked in water for
24 h before sampling, but these components were barely detectable in extracts from 5 g of the
seed coat. The d s R N A species were readily detected in an extract made from one leaflet (0.6 g) of
cv. Stella.
Detection of dsRNA in V. faba cultivars
Pooled samples were made of leaflets from three or four plants (1 to 2 g) of each of 22 V. faba
cultivars. Of these cultivars, nine had been reported to contain VCV or VCV-like particles and
two (Beryl and Minica) had been found to be free from such particles after extensive testing
(Kenten et al., 1978; Jones, 1980 and unpublished data; A. J. Cockbain, personal
communication). Table 1 shows that all nine cultivars known to contain particles also contained
d s R N A similar in mol. wt. to that found in particles of VCV; such d s R N A species were absent
from cvs Beryl and Minica (Fig. 3). Of the remaining 11 cultivars for which no tests for
VCV-like particles have been reported, extracts from all but cvs Banner and Optica contained
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2456
M. A. A B O U - E L N A S R ,
A. T. J O N E S
AND
M. A. M A Y O
M
M
(a)
(b)
(c)
(d)
(e)
(f)
M
(a)
(b)
i
(e)
(d)
.... 7
i
Fig. 3
Fig. 3. Polyacrylamide gel electrophoresis o f d s R N A from 1 to 2 g leaf from
Fig. 4
V.Jaba (a) cv. Strube,
(b) cv.
Marls Bead, (c) cv. Minden, (d) cv. Herra, (e) cv. Beryl and (f) cv. Minica. Lanes M contain MRDV
dsRNA as a marker. Arrows mark the positions of bands in group S, I and F.
Fig. 4. Polyacrylamide gel electrophoresis of dsRNA from equivalent samples of leaves (a, e) and
protoplasts (b, d) of V. faba cv. Marls Bead (a, b) and cv. The Sutton (c, d). M, dsRNA from MRDV
particles.
these d s R N A components. Although some extracts from cvs Banner and Optica gave very faint
bands in positions similar to VCV d s R N A , the low concentrations of the components separate
these two cultivars from those recorded as positive in Table 1. In addition to the d s R N A bands
associated with VCV infection, extracts of most cultivars produced other bands o f different
mobility but cultivars differed in the number and the intensity of staining of these bands (Fig. 3,
Table 1). They mostly contained much less material than the bands of VCV d s R N A and they
were not detected in all tests (Fig. 3). These additional d s R N A bands occurred as a group ofmol.
wt. 2.9 x 10 6 to 2.3 x 106 termed slow(S) and as a group o f m o l , wt. 2.0 x 10 6 to 1-5 x 10 6
termed intermediate (I) and were distinct from those of VCV d s R N A termed fast (F).
dsRNA patterns & ind&idual V. faba plants
To assess the relative extents of the variation in d s R N A patterns within and between
cultivars, d s R N A extracts were made from leaf samples (1 to 2 g) from each of ten plants of cvs
Marls Bead and Minica. These cultivars were chosen because of the presence and absence
respectively both of VCV particles and of VCV d s R N A bands (Table 1, Fig. 3). In these tests,
two of the ten Marls Bead plants contained group S bands only, one contained group F bands
only and seven contained group S and F bands. By contrast, no group S and F bands were
detected in the ten Minica plants but seven contained traces of group I bands. These results
confirm the absence of VCV-like d s R N A in cv. M i n i c a and show that some Marls Bead plants
lack detectable amounts o f V C V d s R N A . They also show the differences between individual
plants in occurrence and intensity of group S and I bands, suggesting that this m a y account for
the variation in occurrence of these bands in different cultivars and in different tests (Table 1,
Fig. 3). This variation was not observed with VCV d s R N A . The origin of group S and I bands is
not clear but their occurrence seems unrelated to the presence of VCV.
Occurrence of VCV dsRNA in protoplasts of V. faba
Fig. 4 shows that VCV d s R N A was approximately equally a b u n d a n t in extracts from
protoplasts or leaves from either cultivar. Light microscopy did not reveal any vascular tissue in
the protoplast preparations and it is most likely that VCV d s R N A was inside mesophyll cells of
intact leaves.
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Vicia cryptic virus dsRNA
T a b l e 1.
2457
Detection of virus-like particles and dsRNA species in 22 cultivars of V i c i a f a b a
V. Jctba
cultivar
Aqua Dulce
Banner
Beagle
Beaver
Beryl
Blaze
Darius
Deiniol
Diana
Giza
Herra
Hertz Freya
Kristal
Marls Bead
Minica
Minden
Optica
Stella
Strube
The Sutton
Wierboon
Zwoon
VCV
particles
detected*
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
* Data from Kenten
No. dsRNA bands detected
with mol. wt. ( × 10-6) of
r
~
2.9-2.4
2.0-1.5
1.4-1-2
(S)
(I)
(F)
2
0
1
2
0
2
0
2
2
2
2
2
0
3
0
2
1
1
2
2
0
0
0
3
3
0
3/4
0
3
1
1
0
0
1
0
3
2/3
0
>3
0
1
2
1
0
2/3
(3)
3
3
0
3
3
2
2
1
3
2/3
2
3
0
3
(3)
3
3
3
2
2
et al. (1978) and Jones (1980 and unpublished data).
Attempts to transmit V C V to VCV-free V. faba plants
Seedlings of cvs Beryl, Marls Bead and Minica, that had been found not to contain VCV
dsRNA, were manually inoculated with sap from plants of cv. The Sutton known to contain
relatively high concentrations of VCV dsRNA. Inoculated leaves and uninoculated tip leaves of
the test plants were harvested 1, 4, 8 and 15 days, and 8 and 15 days after inoculation respectively
and their dsRNA content was determined. None of the samples was found to contain VCV-like
dsRNA bands.
In other transmission tests, dodder seeds were sown in pots containing the individual Marls
Bead and Minica seedlings whose dsRNA contents had been assessed in an earlier experiment.
After the dodder had established on plants, dodder strands were trained to form a bridge
between single VCV-infected plants of Marls Bead and those of uninfected Minica. Four pairs
of plants were used. Six to 8 weeks after the dodder links had established between plant pairs,
dsRNA was extracted from leaves of the Minica plants. None of the four Minica plants
contained VCV dsRNA species and VCV dsRNA was not detected in extracts from the
connecting dodder tissues, but it was readily detected in the Marls Bead source plants.
dsRNA extracts from V. faba lines containing a CMS factor
CMS in V. faba has been reported to be associated with a high mol. wt. dsRNA which is
transmissible from plant to plant using dodder (Grill & Garger, 1981). Although the reported
mol. wt. of this dsRNA is several times higher than that in VCV particles, d s R N A analysis was
made from a CMS-affected and a CMS maintainer line of V.faba, to determine whether VCV is
involved in CMS. In several tests, no VCVqike dsRNA bands were produced by these extracts
suggesting that VCV is not involved in CMS of V. faba.
DISCUSSION
Our results show that VCV resembles other 'cryptic' or 'temperate' viruses (Boccardo et al.,
1983; Lisa et al., 1981 ; Natsuaki et al., 1983a, b, 1984) in having particles that contain dsRNA.
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2458
M.A.
ABOU-ELNASR,
A. T. JONES A N D M. A. MAYO
Table 2. d s R N A components of 'cryptic' viruses
No. of main dsRNA
components
2
3
4
5
Virus
Mol. wt. ( x 10-6) of
R N A species
Reference*
Alfalfa cryptic
Hop trefoil cryptic
Red clover cryptic
Ryegrass cryptic
White clover cryptic
White clover temperate
Alfalfa temperate
Carnation cryptic
Radish yellow edge
Spinach temperate
Vicia cryptic
Beet temperate
Festuca cryptic
1.27,1.17
1.30,1.07,(1.22)t,(1"17)
1.56,1.51
1.03,0.92
1.26,1.03,(1.59),(1-47)
1.50,1.25,(2.6),(2-5),(1-75),(1.65)
1.58,1.55,1.48
1.04,0.95,0.84
1.30,1.25,1.21 ,(1.14),(1.09)
1.31,1.21,1.10
1.37,1.26,1.21
1-45,1-28.0.65,0-56
1.32,1.27,1.22,0.95,0.90
1
1
1
1
1
5
5
2
3
4
6
4
1
* 1, B o c c a r d o e t a L (1983); 2, Lisa etal. (1981); 3, Natsuaki etal. (1983a); 4, Natsuaki etal. (1983b); 5, Natsuaki
et al. (1984); 6, this paper.
i Values in parentheses are mol. wt. of minor species.
A compilation of the reports of the numbers and mol. wt. of the main dsRNA species obtained
from particles of these viruses indicate that they do not form a homogeneous group (Table 2).
Even though the conditions of P A G E and of the dsRNA markers used may differ between
laboratories, many of the viruses clearly differ both in the number and mol. wt. of dsRNA
species. However, as with dsRNA-containing mycoviruses (Buck, 1983), it is possible that one
or more dsRNA components of any of these viruses, including VCV, are defective copies of
larger species, or are satellite species. Nevertheless, all the 'cryptic' viruses examined previously
contain two or more major dsRNA species of mol. wt. 0.8 x 106 to 1.6 × 106 (1-25 to 2.5 kbp)
(Table 2), as also does VCV.
dsRNA extracted from infected plant tissue, but not that from VCV particles, contained
species (S and I) that migrated more slowly than VCV RNA. It seems unlikely that these species
are related to VCV RNA as they were present in some VCV-free plants. Possibly they are
dsRNA of another virus which either does not co-purify with VCV particles or forms few or
no virus particles in V. faba tissue.
Our estimate of the density in CsC1 of VCV particles is similar to that of Kenten et al. (1979)
although we did not observe the minor less-dense component reported by them. Similar values
for particle density are reported for beet cryptic, carnation cryptic, beet temperate, alfalfa
temperate and radish yellow edge viruses (Kassanis et al., 1977 ; Lisa et al., 1981 ; Natsuaki et al.,
1983a, b, 1984). The figure of 1-37 g/ml suggests that VCV particles contain 20 to 30% R N A
(Sehgal et al., 1970). It seems unlikely that all three species ofVCV dsRNA (total tool. wt. 3.8 x
106) could constitute 20 to 30% of a single 30 nm diameter particle. It is more likely that each
RNA species is encapsidated separately. If this is so, the minor differences in buoyant density
between particle types might not be adequately resolved by preparative isopycnic centrifugation
in CsC1, but may account for the broad band of absorbance observed.
'Cryptic' viruses resemble some mycoviruses in the size and buoyant density in CsC1 of their
particles and in containing several dsRNA species (Buck, 1983) and this has raised the
possibility that 'cryptic' viruses are viruses of fungi present in the plant tissues. However, our
results reinforce the arguments against this idea (Boccardo et al., 1983) because the amounts of
VCV dsRNA recovered from intact leaf tissue were similar to those recovered from isolated
washed mesophyll protoplasts; protoplast isolation would be expected to remove most surface
and intercellular microflora. Although we cannot exclude the possibility that intracellular fungi
were isolated with the protoplasts, these results, together with the detection of VCV dsRNA in
different tissues and in a high proportion of seedling progeny, make this possibility very unlikely.
In addition, the detection of VCV dsRNA in bean mesophyll protoplasts suggests that VCV
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Vicia cryptic virus dsRNA
2459
particles are not confined to vascular tissue, as has been suggested for the particles of some other
'cryptic' viruses (Natsuaki et al., 1979; Boccardo et al., 1983).
Our results are the first report of the detection of 'cryptic' virus d s R N A in extracts of plant
tissues. We found 18 of 22 V. faba cultivars examined to be infected with VCV or VCV-like
viruses; only two cultivars (Beryl and Minica) were clearly uninfected and possibly are immune.
Moreover, VCV dsRNA was detected in all types of tisssue examined and in most (but not all)
individuals of cv. Maris Bead tested. Thus, the extraction and electrophoretic examination of
dsRNA proved to be a reliable, rapid and sensitive test for VCV. We found R N A analysis more
sensitive and reliable than either electron microscopy of concentrated sap extracts or
immunoelectron microscopy, the only other methods used previously to detect VCV (Kenten et
al., 1981; Jones, 1980).
Because 'cryptic' viruses occur in low concentrations and induce few or, more typically, no
symptoms in plants, they represent a hazard in virological studies. Thus, antibodies to 'cryptic'
viruses may occur in antisera produced against unrelated viruses if these viruses were
propagated unwittingly in plants containing 'cryptic' virus particles. The use of such bivalent
antisera could complicate the interpretation of some types of serological assay. Our results with
V.faba suggest that a test for dsRNA in apparently healthy tissue is a worthwhile precaution and
that, when the very sensitive technique of silver staining is used, only small quantities of leaf
tissue are needed.
We thank I. M. Roberts for immunoelectron microscopy and A n n e Jolly for technical assistance. This work was
done while one of us (M.A.A-E.) was in receipt of a N O R A D research fellowship as part of the E G N O project.
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