J. gen. Virol. (1984), 65, 1741-1747. Printedin Great Britain
1741
Key words: Gaeumannomyces graminis/dsRNA/vegetath,e compatibility
Sequence Relationships between Virus Double-stranded RNA from Isolates
of Gaeumannomycesgraminis in Different Vegetative Compatibility Groups
By N. J A M I L , K. W. B U C K * AND M. J. C A R L I L E
Department of Pure and Applied Biology, Imperial College of Science and Technology, London
S 1417 2BB, U.K.
(Accepted 27 June 1984)
SUMMARY
Double-stranded RNA, from isolates of Gaeumannomyces graminis var. tritici in nine
vegetative compatibility groups, was separated by polyacrylamide gel electrophoresis
and transferred to aminophenylthioether-paper. Hybridization between these blots
and c D N A probes prepared to double-stranded R N A from viruses obtained from three
of the isolates, revealed several close relationships between double-stranded R N A from
isolates in different vegetative compatibility groups. The implications of this finding
for virus transmission in G. graminis are discussed.
INTRODUCTION
Virus infections of the wheat take-all fungus, Gaeumannomyces graminis var. tritici, are
common. Much variability has been found among viruses from different field isolates of G.
graminis, including isolates from the same field (Frick & Lister, 1978); 18 viruses from nine
isolates of this fungus have been classified into four groups (I to IV) based on their serological
and biophysical properties (Buck et al., 1981 ; McFadden et al., 1983 ; Buck, 1984; Jamil & Buck,
1984). Group I viruses had particles of 35 nm diameter with two to four dsRNA components in
the range 1.94 to 1.53 kbp and a single capsid polypeptide species of mol. wt. 54 × 103 to 60 x
10 3. Group II viruses had particles of 35 nm diameter with two or three dsRNA components in
the range 2.33 to 2.02 kbp and a single capsid polypeptide species of mol. wt. 68 x 103 to 73 x
103. Group III viruses had particles of 40 nm diameter with one or two dsRNA components in
the range 6.3 to 4.7 kbp and two or three capsid polypeptide species in the mol. wt. range 78 x
103 to 87 x 103. The only group IV virus to be described had particles of 29 nm diameter with a
single dsRNA of 1.80 kbp and a single capsid polypeptide species of mol. wt. 66 x 103. Viruses
within a group were related serologically to other members of the same group, but unrelated
serologically to members of other groups.
Double-stranded R N A mycoviruses do not have an extracellular phase in their replication
cycle and there is no evidence for transmission of viruses between individuals in a population
other than by vegetative (somatic) hyphal fusion or by mating (Lecoq et al., 1979). In G.
graminis, virus particles are usually eliminated in the sexual cycle (Rawlinson et al., 1973;
McFadden et al., 1983) and transmission via hyphal fusion may be the main method by which
viruses spread. Several fungi can be divided into numerous vegetative compatibility (v-c)
groups; hyphal fusions between strains within a group readily yield heterokaryons, but fusions
between strains from different v-c groups result in vegetative (somatic) incompatibility
reactions in which fusion is terminated by localized cell death (Lane, 1981; Todd & Rayner,
1980). These reactions are, in some fungi, visible to the naked eye as the formation between
incompatible colonies of a "barrage' (Esser, 1971), a clear zone containing few living hyphae and
often bounded on each side by a narrow zone of intense black pigmentation. A question that
arises is to what extent vegetative incompatibility in fungi may have evolved to limit the
transmission of viruses ? Indeed, Caten (1972) suggested that vegetative incompatibility in fungi
may have evolved to limit the transmission of potentially harmful cytoplasmic genetic elements,
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N. JAMIL, K. W. BUCK AND M. J. CARLILE
including viruses. If such incompatibility barriers are effective in G. graminis it might be
expected, given the known diversity of its viruses, that close relationships would be limited to
viruses within a v-c group. To determine if this is so we have investigated nucleotide sequence
relationships between dsRNA of viruses from G. graminis isolates in different v-c groups.
METHODS
The following methods were performed as described by Buck et al. (1981); gel immunodiffusion analysis;
electron microscopy; agarose gel electrophoresis of virus particles; SDS-PAGE of virus polypeptides.
Fungal isolates. Thirty-one isolates of G. graminis from wheat crops in Highfield, Rothamsted Experimental
Station, Harpenden, U.K. were kindly provided by Mr D. B. Slope.
Assignment ofJungal isolates to v-c groups. Petri dishes of potato glucose agar (Potato Dextrose Agar, Difco) were
inoculated with two pairs of plugs of agar carrying mycelium from different isolates. Incubation at 25 °C resulted
in four colonies which came into contact, giving test encounters and also control encounters between colonies of
the same isolate. Those between some isolates showed vegetative incompatibility in the form of a striking barrage,
a clear zone with adjacent pigmented zones, whereas encounters between other isolates did not differ in
appearance from the control encounters between colonies of the same strain. Isolates were assigned to v-c groups
on the basis that within each group no incompatibility reactions were seen, but any pair from different groups
yielded barrage reactions.
Isolation ofdsRNA. G. graminis isolates were grown for 10 to 14 days at 24 °C in shaken flasks in a medium
containing 1 °.o (w/v) glucose monohydrate, 3 % (w/v) corn steep liquor, 0.1% (w/v) KH,POa, 0.05 % (w/v) MgSO,,
0.001% (w/v) FeSO~. 7H20 (adjusted to pH 6.0 with NaOH). Mycelium was harvested by filtration and disrupted
by grinding in liquid nitrogen. The dsRNA was then isolated by phenol extraction and cellulose chromatography
as described by Morris & Dodds (1979).
Isolation and purification ofcirus. G. graminis isolates were grown in 60 litre fermenters for 3 days at 24 °C in the
glucose corn steep liquor medium described above using the methods described by Banks et al. (1971).
Preparation and purification of virus by polyethylene glycol precipitation, ultracentrifugation and sucrose densitygradient centrifugation were carried out as described by Buck et al. (1981). The two viruses in isolate 87-1 were
separated by CsCl density gradient centrifugation as described by Jamil & Buck (1984).
Polyacrylamide gel electrophoresis ofdsRNA. About 1 rag dsR N A was put in each slot of vertical slab gels (20 x
20 cm) containing 4% (w/v) acrylamide and 0-04~ N,N'-methylenebisacrylamide. Electrophoresis was at 5 V/cm
for 18 h in 0.04 M-Tris-acetate buffer pH 8.0, containing 1.6 raM-sodium EDTA. After electrophoresis gels were
stained in ethidium bromide (1 gg/ml) for 15 rain and washed with water. The dsRNA bands were detected by u.v.
transillumination. Sizes of dsRNA were determined by co-electrophoresis with virus dsRNA from G. graminis
(Buck et al., 1981), Aspergillusfoetidus (Buck & Ratti, 1977) and Penicillium stoloniferum (Bozarth & Harley, 1976).
Preparation ofcDNA probes. The dsRNA was obtained from virus particles by phenol extraction and ethanol
precipitation, and, after denaturation by heating for 1 to 5 min in 0-1 mM-sodium EDTA, pH 7.0, used as a
template for synthesis of 32p-labelled cDNA by reverse transcription using random oligonucleotide primers
(Taylor et al., 1976). When cDNA probes from individual RNA components were needed, RNA was first
separated by PAGE and then electroeluted and purified by precipitation with cetyltrimethylammonium bromide
(Schuerch et al., 1975).
Gel transfer hybridization. After separation by PAG E, R N A was transferred electrophoretically to aminophenylthioether (A PT)-paper by the method of Seed (1982) and hybridized with cDNA probes as described by Thomas
(1980). Two conditions of stringency were used for hybridizations, 50% formamide (high stringency) and 25%
formamide (low stringency).
RESULTS
Assignment o f G. g r a m i n i s isolates to v-c groups and analysis o f their d s R N A contents
F o l l o w i n g tests o f all p o s s i b l e p a i r w i s e c o m b i n a t i o n s , 31 isolates o f G. graminis f r o m a single
field at R o t h a m s t e d E x p e r i m e n t a l S t a t i o n were classified i n t o 18 v-c groups, N o d s R N A c o u l d
b e d e t e c t e d in 10 o f t h e s e isolates a n d only t r a c e a m o u n t s in a f u r t h e r s e v e n isolates. T h e d s R N A
in t h e r e m a i n i n g 14 isolates, w h i c h b e l o n g e d to n i n e o f t h e v-c g r o u p s , w a s a n a l y s e d by P A G E .
T h e sizes o f t h e d s R N A c o m p o n e n t s w h i c h w e r e d e t e c t e d are g i v e n in T a b l e 1.
Isolation and characterization o f virus particles
Isolates 87-1, 74 and 38 were selected to represent the range of dsRNA sizes in virus groups I
to IV. To confirm the grouping of the viruses from which the dsRNA components arose,
purified virus preparations from each of these isolates were examined by electron microscopy
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Related dsRNA in G. graminis v-c groups
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Table 1. The dsRNA components from G. graminis isolates in nine ~,-c groups
Sizes of dsRNA components within virus groups
(
Isolate v-c Group III range
no.
group
(6.3-4.7 kbp)
3O
A
38
A
62
A
43
B
73
C
75
C
82
C
37
D
39
D
74
E
87-1
F
6.1
83
G
6.1
16
H
86
J
Ungrouped*
3.3
3.3
3.3
3.3
Group II range
(2.33-2.02 kbp)
Group I/IV range (1.94-1.53 kbp)
and below (1.48-1.35 kbp)
1-94 1.85, 1.82, 1.73, 1.64, 1.62
1-94 1.85, 1.82, 1.73, 1-64, 1.62
2.33, 2.24, 2-08, 2.04 1.85 1.66
1.94 1.85, 1.73, 1.66, 1.63, 1-59
1-85 1.73, 1.66
2.24
1.85 1.76, 1-73, 1.66, 1.60
2.24
1.85 1.73, 1.62
2.18, 2.11
1-82 1-73, 1-67, 1-63, 1.48, 1.35
2.33, 2-24
t-85 1-73, 1-63
2.33, 2.24
1.85, 1.76, 1.48
1.85, 1.76, 1.63, 1-48
2.33, 2.30
1.85, 1.76, 1.67, 1.63, 1.48, 1.44
1.85, 1.73, 1-62
* No virus particles associated with this size of dsRNA have, as yet, been isolated.
and their d s R N A and polypeptide components were analysed by P A G E . Isolate 87-1 has been
shown previously (Jamil & Buck, 1984) to contain two viruses, 87-1-L (group I, d s R N A s of 1.85,
1.76 and 1.48 kbp) and 87-1-H (group III, d s R N A of 6.1 kbp). Isometric virus particles from
isolate 74 had properties characteristic of group II viruses, namely diameter about 35 nm, a
single capsid polypeptide species of mol. wt. 73000 and d s R N A components of 2.33 and 2.24
kbp. Isometric virus particles from isolate 38 had properties characteristic of group I viruses,
namely diameter about 35 nm, a single capsid polypeptide species of mol. wt. 55 000 and d s R N A
components of 1.94, 1.85, 1.82, 1.73, 1.64 and 1.62 kbp. In agarose gel electrophoresis, intact virus
particles from this isolate gave two closely spaced bands which stained with both Coomassie
Brilliant Blue and ethidium bromide and may comprise a mixture of two similar viruses.
Gel transfer hybridizations
The d s R N A s from all the G. graminis isolates in Table 1 were separated by P A G E and the gels
were stained with ethidium bromide and photographed on a u.v. transilluminator. The R N A
was then transferred to APT-paper and hybridized (in separate experiments) to d s R N A from
the viruses obtained from isolates 87-1, 74 and 38.
Using a c D N A probe prepared from d s R N A from unseparated viruses 87-1-L and 87-1-H, an
additional d s R N A band of 3.3 kbp was detected in the homologous hybridization which was
barely visible in the ethidium bromide-stained gel (Fig. 1 a, lane 1). Hybridization was clear
between the 87-1 c D N A and d s R N A s of 1.85, 1.76 and 1.48 kbp in isolates 83 and 16 and
d s R N A s of 6.1 and 3.3 kbp in isolate 83 (Fig. 1 a, b : lanes 2 and 3). Further hybridizations were
carried out with probes synthesized from electrophoretically separated d s R N A s of 1.85, 1.76
and 1.48 kbp from virus 87-1-L and d s R N A of 6.1 kbp from virus 87-1-H purified in CsC1. It was
found that c D N A prepared from each R N A hybridized only with the R N A of the same size
from isolates 83 and 16. No hybridization could be detected under conditions of high or low
stringency between any of these c D N A s and the remaining d s R N A components of isolates 87-lH, 83 and 16 or d s R N A from any of the other isolates in Table 1. The absence of relationship
between the individual d s R N A components within each of the viruses from isolates 87-1, 83 and
16 is consistent with solution hybridization and fingerprinting studies of another group I virus
(Romanos et al., 1981).
With c D N A probes synthesized from the two d s R N A s of virus particles from isolate 74, no
hybridization with d s R N A from any of the isolates in Table l could be detected under
conditions of high or low stringency.
Probes of c D N A synthesized from the mixture of virus d s R N A s (1,94 to 1.62 kbp) from
isolate 38 did hybridize with d s R N A s from isolates 30 and 43 under conditions of high
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(a)
N. J A M I L , K. W. BUCK AND M. J. C A R L I L E
87-1
83
16
87-1
83
16
Fig. 1. Relationships between dsRNAs from isolate 87-1 and those from isolates 83 and 16. R N A was
separated by PAGE and stained with ethidium bromide (a). It was then transferred to APT-paper and
hybridized with a 3-'P-labelled c D N A probe prepared by reverse transcription of denatured d s R N A
from virus particles obtained from isolate 87-1. Electrophoresis was from top to bottom and hybridizing
bands were detected by autoradiography (b). Sizes of d s R N A are in kbp.
(a)
38
43
30
(b)
38
43
30
(c)
38
@
43
• ,~
~
30
i
1.73
Fig. 2. Relationships between d s R N A from isolate 38 and those from isolates 43 and 30. Methods were
as in the legend to Fig. 2. (a) Ethidium bromide-stained gel; (b, c) autoradiographs: (b) Probe
synthesized from 1.94 kbp dsRNA of isolate 38 ; (c) probe synthesized from 1.85 to 1.62 kbp d s R N A of
isolate 38. Sizes of dsRNA are given in kbp.
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Related dsRNA in G. graminis v-c groups
1745
stringency, but not with dsRNAs from any of the other isolates in Table 1 under conditions of
high or low stringency. A cDNA probe made from electrophoretically purified dsRNA of 1.94
kbp from isolate 38, hybridized only with dsRNAs of the same size in isolates 30 and 43 (Fig. 2a,
b). cDNA probes to the remaining dsRNAs from isolated 38 virus (1-85 to 1.62 kbp) hybridized
with three bands of RNA from isolates 38, 30 and 43 (Fig. 2e). These corresponded to (1.85 +
1.82), 1.73 and (1.64 + 1.62) kbp dsRNAs in isolates 38 and 30 and 1.85, 1.73 and (1.66 + 1.63)
kbp dsRNAs in isolate 43 (closely spaced dsRNA components could not be resolved after the
transfer of RNA from polyacrylamide gel to APT-paper and the hybridization treatments).
DISCUSSION
Most of the dsRNA components detected in the field isolates ofG. graminis fell within the size
ranges of the previously described virus groups I to IV (see Table 1). However, four isolates
contained a dsRNA of 3-3 kbp which is, as yet, ungrouped because no virus particles
corresponding to this size of dsRNA have been isolated. Some isolates contained dsRNA
components in the range 1.48 to 1.35 kbp, which is slightly below the group I range, and these
may be satellites (Romanos et al., 198l) associated with viruses from one of the groups.
In the hybridization experiments no relationships were detected between dsRNA in different
virus groups. Furthermore, relatively few relationships were detected between dsRNA
components within a virus group. For example, virus 87-1-L dsRNA was related to dsRNA in
the group I range in only two out of 10 isolates and dsRNA from isolate 74 was not related to
dsRNA in the group II range from seven other isolates. In previous studies with different
isolates (Buck et al., 1981) no serological relationships were found between viruses in different
groups and considerable serotype diversity was found between viruses within a group. It is not
known whether serological relatedness parallels dsRNA relatedness. However, it is noteworthy
that the hybridization results indicate divergence of the entire genomes of viruses, whereas
serological diversity indicates differences in virus epitopes which represent only a fraction of the
virus genome.
The dsRNA components from isolates 30 and 38 were of the same size and appeared to be
closely related in the hybridization tests; these two isolates, which are in the same v-c group, may
be identical. However, several relationships were found between dsRNAs from isolates in
different v-c groups all under high stringency conditions, namely group I dsRNA components
from isolate 38 were related to those of similar size in isolate 43 and dsRNA components from
isolate 87-1-H were related to those of the corresponding size in isolates 83 and 16. Making the
assumptions used by Street et al. (1982) in similar studies with rotavirus dsRNA, an
approximate estimate of the maximum base sequence mismatch which would be tolerated under
these conditions is 10~. More precise quantification would require solution hybridization
studies.
Such close similarities between dsRNAs from isolates in different v-c groups suggest that
virus transmission between isolates in different v-c groups could have occurred, particularly as
the isolates all came from the same geographical location. Anagnostakis & Day (1979) and
Anagnostakis (! 981 ) reported transmission of dsRNA between isolates of Endothia parasitiea in
different v-c groups. From more extensive tests it was concluded that the efficiency of
transmission was inversely dependent on the number of v-c gene differences and on the relative
strengths of individual v-c genes (Anagnostakis, 1983). Over 75 v-c groups have been found in E.
parasitiea, and these are estimated to be determined by at least seven nuclear genes
(Anagnostakis, 1982). The detection of 18 v-c groups in 31 isolates of G. graminis from a single
site suggests that in this fungus also v-c groups and v-c genes are numerous. It is likely that in
nature any strain has a considerably greater chance of meeting a strain in a different v-c group
than it does one in its own v-c group.
Two other possible explanations for the occurrence of closely related and possibly identical
dsRNAs in G. graminis isolates of different v-c groups deserve comment. First, virus infection
could predate the evolution of the v-c groups. If a strain remained infected, subsequent evolution
would be expected to result in virus diversification, as is found. Nevertheless, if the number of vc groups is large and the number of allowable virus mutations is limited by structural and
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1746
N. JAMIL, K. W. BUCK AND M. J. CARLILE
replication constraints, closely related viruses may still persist in some isolates of different v-c
groups. However, the low efficiency of virus transmission into ascospores (McFadden et al.,
1983) and the absence of virus from most G. graminis isolates in first-year cereal crops
(Rawlinson et al., 1973) argues against infections which persist over long time periods in this
fungus.
The second alternative explanation is suggested by the recent finding that virus 87-1-L is
apparently identical to virus 2-2-A from an isolate of Phialophora sp. (lobed hyphopodia), a
weakly virulent cereal parasite believed to be the anamorph of G. graminis var. graminis (Jamil &
Buck, 1984). It was postulated that this Phialophora sp., which does not generally form
ascospores, might act as a reservoir of viruses for subsequent infection of virus-free, ascoporederived, isolates of G. graminis. Since we have shown here that dsRNAs of virus 87-1-L are
closely related and possibly identical to dsRNAs from two other G. graminis isolates (83 and 16),
each in a different v-c group, the possibility must be considered that these three isolates could
have become infected with virus 2-2-A by direct transmission from Phialophora sp. (lobed
hyphopodia). The above Phialophora isolate, when grown on solid medium alongside isolates of
G. graminis, including isolate 87-I, has been observed to give 'barrage' reactions along the
interface of the two colonies, which resemble those formed between colonies of G. graminis
isolates in different v-c groups (McGinty, 1981 ; N. Jamil, unpublished results). Transmission of
virus from Phialophora sp. (lobed hyphopodia) to G. graminis may, therefore, also occur across
vegetative incompatibility barriers.
We have showed that gel transfer hybridization is a useful technique for investigating
relationships between dsRNAs both from different viruses and from the same virus. It should be
a valuable addition to serology in studying the epidemiology of fungal virus infections about
which little is known. Although our results suggest that virus transmission may occur between
isolates of G. graminis in different v-c groups, they do not imply that this occurs generally.
Further progress in our understanding of the effect of vegetative incompatibility on virus
transmission in this fungus will require investigations of the numbers and relative strengths of
different v-c genes and of virus transmission between fungal strains of defined genotype in a
range of v-c groups.
We wish to thank the Pakistan Ministry of Education and the U.K. Overseas Research Students' Fees Support
Scheme for financial support for N. Jamil.
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