J. gen. Virol. (1986), 67, 371-375. Printed in Great Britain
371
Key words: banana bunchy top/dsRNA/luteovirus
Double-stranded RNA in Banana Plants with Bunchy Top Disease
By J A M E S L. D A L E , * D E B R A A. P H I L L I P S t AND J U D I T H N. P A R R Y
Plant Pathology Branch, Department of Primary Industries, lndooroopilly, Queensland, Australia
(Accepted 21 October 1985)
SUMMARY
Double-stranded RNA (dsRNA) has been isolated from bananas infected with
banana bunchy top disease (BBTD) but not from healthy bananas of the same cultivar.
Four major dsRNA species were present in seven different extracts and these had
molecular weights of 4.40 x 10 6, 1.35 x 10 6, 0"50 × 106 and 0.48 x 106. This pattern
of dsRNA species is similar to that in extracts from plants infected with barley yellow
dwarf virus or beet western yellows virus strain ST9. The amount of extractable BBTDspecific dsRNA was affected by the temperature at which the infected plants were
incubated and the interval between infection and harvest. The maximum amount of
dsRNA was obtained from plants incubated at 30 °C and harvested 23 days after
inoculation.
Banana bunchy top disease (BBTD) is a severe disease of banana plants in many countries of
the Asian-Pacific Basin as well as North Africa and India. In Australia, BBTD virtually
destroyed the banana industry in the late 1920s and the industry now exists because of a vigorous
but expensive inspection and eradication scheme. The cause of BBTD is thought to be a virus,
probably a luteovirus (Matthews, 1982) because it is persistently transmitted by the banana
aphid, Pentolonia nigronervosa, and diseased plants have yellowed leaves and damaged phloem
(Magee, 1940). However, despite numerous attempts by many different workers to extract and
characterize virus particles from BBTD plants, this has not been accomplished. In this paper,
we report that we have isolated double-stranded RNA (dsRNA) from BBTD plants. This
evidence also indicates that BBTD is caused by a virus, most probably a luteovirus.
The isolate of BBTD used in this study was collected at Currumbin, Queensland, Australia
and was maintained, by aphid transfer using Pentolonia nigronervosa, in plantlets derived
vegetatively from a single cultured banana plant, Musa sapientum cv. Cavendish, regenerated
from callus. The method used to extract dsRNA from banana plants was similar to that of
Morris & Dodds (1979). Whole plants were harvested, frozen in liquid nitrogen and ground to a
fine powder. The ground tissue (100 g) was shaken for 1 h at 4 °C in 400 ml 50 mM-Tris-HC1
pH 7.0, 100 mM-NaC1 and 1 mM-EDTA (STE), 200 ml water-saturated phenol containing 0.1
8-hydroxyquinoline, 200 ml chloroform, 50 ml 10~ SDS and 5 ml 2-mercaptoethanol. The
mixture was centrifuged at 7000 g for 15 min and the aqueous phase was collected. Ethanol was
added to it while stirring to give a final concentration of 15~. Whatman CF-11 cellulose was
then added (0.15 g/g of original tissue) and the suspension was stirred under vacuum for 15 min
at room temperature. The suspension was then poured into a chromatography column and
washed with STE containing 15 ~ ethanol until the A zs4 of the eluate was that of STE containing
15 ~o ethanol. This normally required washing with 10 ml STE containing 15 ~ ethanol for each
gram of tissue extracted. Double-stranded RNA was then eluted by washing with STE alone and
precipitated by adding 2.5 vol. ethanol and keeping the mixture at - 2 0 °C overnight. The
precipitated dsRNAs were collected by centrifuging at 10000 g for 30 min, dried in vacuo and
resuspended in sterile distilled water.
~fPresent address: Queensland Health Department, Brisbane, Queensland, Australia.
0000-6725
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372
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
Fig. l
Fig. 2
Fig. 1. Ethidium bromide-stained polyacrylamide gel of dsRNA extracts of (a) FDV-infected
sugarcane, (b) fungal dsRNA markers, (c) BBTD-infected banana and (d) healthy banana.
Fig. 2. Ethidium bromide-stained polyacrylamide gel of RNase- and DNase-treated dsRNAs
extracted from BBTD-infected plants. (a) BBTD-specificdsRNA not treated with nuclease; (b) BBTDspecific dsRNA treated with RNase A in 0.1 × SSC; (c) as for (b) but in 2 × SSC; (d) BBTD-specific
dsRNA treated with DNase.
Double-stranded R N A was analysed by electrophoresis in polyacrylamide gels (14 cm × 14
cm x 0.75 mm) consisting o f a 1 0 ~ resolving gel and a 5 ~ stacking gel using the buffer system
of Laemmli (1970) but with SDS omitted from all buffers. The gels were electrophoresed at 150 V
for 16 h and then stained either with ethidium bromide (50 gg/ml for 10 rain) or with silver
(Merril et al., 1981). Molecular weights of the different d s R N A species were estimated by
comparison with the d s R N A of sugarcane Fiji disease virus (FDV) (Reddy et al., 1975) and
dsRNAs extracted from Helminthosporium maydis, Penicillium chrysogenum and Penicillium
stoloniferum kindly provided by Dr R. Bozarth.
Double-stranded R N A extracts from equal fresh weights of healthy banana plants and those
with bunchy top disease were compared by gel electrophoresis. A number of dsRNAs were
present in extracts from infected but not healthy plants (Fig. 1). These d s R N A s were in very low
concentrations and could only be detected when the total d s R N A s obtained from 75 to 100 g
infected tissue were electrophoresed in one lane of a 0.75 m m thick gel. Four major d s R N A
bands were present in all seven different extracts of infected bananas harvested within 5 days
after symptoms had arisen (Fig. 1). The largest of these, BT-1, had a molecular weight of 4.4 x
106 with BT-2 at 1'35 x 106 and a pair of bands at 0.50 x 106 (BT-3) and 0-48 x 106 (BT-4). Two
less-intense bands, BT-x (mol. wt. 0.86 × 106) and BT-y (mol. wt. 0.69 x 106) (Fig. 1) were
discernible in four of the seven extracts. Other very minor bands were present in some BBTD
d s R N A preparations.
That these nucleic acids were double-stranded R N A was proven by incubating d s R N A
preparations with ribonuclease A (1 ~g/ml) in 0.1 × SSC (1 x SSC --- 0.15 M-NaC1, 0.015
M-sodium citrate, pH 7.4), or in 2 × SSC or with deoxyribonuclease (10 ~tg/ml) in 50 mM-TrisHC1 pH 8.0 and 10 mM-MgC12. The nucleic acids were incubated with the nucleases at 37 °C for
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(a)
(b)
(c)
373
(d)
(e)
BT-1 - -
Fig. 3. Silver nitrate-stained polyacrylamide gels of dsRNA extracts from bananas infected with
BBTD incubated at different temperatures and harvested at different times. (a) Plants grown at 30 °C
and harvested after 23 days; (b) as for (a) but harvested after 29 days; (c) plants grown at 25 °C and
harvested after 23 days; (d) as for (c) but harvested after 29 days; (e) fungal dsRNA markers.
1 h, and then proteinase K was added to a final concentration of 20 gg/ml. Incubation was
continued for a further 30 rain, and then the mixture was shaken with phenol/chloroform (1:1)
and centrifuged. The dsRNAs of FDV were used as controls. All bands both major and minor
thought to be BBTD-specific d s R N A were resistant to RNase in 2 x SSC and DNase but not to
RNase in 0.1 x SSC (Fig. 2), demonstrating that these bands were dsRNA. There was some
high molecular weight material in the BBTD d s R N A control (Fig. 2a), but this was digested by
RNase in 0.1 x SSC and 2 x SSC (Fig. 2b, c) but not by D N a s e (Fig. 2d), suggesting that this
material was single-stranded RNA. Attempts to remove this material by a second cycle of CF-11
cellulose chromatography were unsuccessful.
We studied the effect of growth temperatures and time after inoculation on the amount of
extractable dsRNA. Inoculated banana plants were incubated at either 25 °C or 30 °C and
harvested either 23 days or 29 days after inoculation. After 23 days, plants at each temperature
showed BBTD symptoms but only in the youngest emerged leaf. After 29 days, plants grown at
30 °C showed symptoms in the two youngest emerged leaves, but those grown at 25 °C had not
yet produced a second symptom-bearing leaf. The amount of d s R N A extracted varied
enormously between the four treatments. Most was obtained from plants grown at 30 °C and
harvested 23 days after inoculation; extracts from these produced very dense bands of BT-1 from
100 g of infected tissue (Fig. 3a). Very faint BT-1 bands were produced by plants grown at 30 °C
and harvested 29 days after inoculation or grown at 25 °C and harvested 29 days after
inoculation (Fig. 3b, d). The least was obtained from plants at 25 °C and 23 days after
inoculation; extracts from these produced BT-1 bands that were only just visible (Fig. 3 c). N o
BBTD-specific d s R N A was detected in inoculated plants before BBTD symptoms appeared.
The results presented here are additional evidence that banana bunchy top disease is caused
by a virus. The presence of d s R N A in infected but not healthy plants as well as the fact that the
concentration of extractable d s R N A in infected plants is related specifically to the time of
infection and incubation temperature indicates that a virus is responsible for the disease. There
have been some reports of dsRNAs being isolated from healthy plants either as small
heterogeneous molecules (Ikegami & Fraenkel-Conrat, 1979) or as large homogeneous
molecules consistently associated with one particular cultivar (Grill & Garger, 1981; Dodds et
al., 1984); however, in our work a single cultivar of banana was used throughout and
homogeneous high molecular weight dsRNAs were never found in healthy plants.
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374
Table 1. A comparison of numbers and molecular weights of major dsRNA species extracted
from BBTD and the luteoviruses B YDV and B W Y V
BYDV
BWYV§
A
Band
1
2
3
4
5
6
7
A
BBTD*
4"4011
Group 1t
3.60
2.00
Group 2:~
3.80
1.60
SCTFL
3.60
ST9
3.60
2.20
1.35
0.86
0.69
0.50
0.48
1.20
1.20
1.40
1.40
0.55
0.50
0.55
0-46
* Present study.
t Gildow et al. (1983) strains MAV, PAV and SEV.
:~Gildow et al. (1983) strains RPV and RMV.
§ Falk & Duffus (1984).
J]Molecular weight x 10-6.
The d s R N A pattern from BBTD-infected plants is similar to that of group 1 strains of barley
yellow dwarf virus (BYDV) (Gildow et al., 1983) and also the ST9 strain of another luteovirus,
beet western yellows virus (BWYV) (Falk & Duffus, 1984) both in the n u m b e r and molecular
weight of the d s R N A species isolated. A comparison of the numbers and sizes of dsRNAs from
the three viruses (Table 1) shows that the largest d s R N A of each, probably the replicative form
of the genomic RNA, are similar; apart from being different viruses the differences in size may
be because different molecular weight markers and electrophoresis systems were used in
different studies. Also both BBTD and BYDV group 1 extracts contain a d s R N A doublet with a
molecular weight of about 0.5 × 106 whereas BYDV group 2 and BWYV strain ST9 extracts
contain a single band. The BWYV strain STFL (Falk & Duffus, 1984) d s R N A extracts appear
to be the most different in that they do not contain any bands with a molecular weight of about
0.5 × 106.
We could isolate only very small amounts of d s R N A from BBTD-infected plants and
normally it was necessary to load d s R N A from 75 to 100 g of infected material before the
dsRNAs could be tested. By contrast, a m i n i m u m of 6 to 10 g of tissue was required with BWYV
(Falk & Duffus, 1984) and 10 to 25 g with BYDV (Gildow et al., 1983) both of which are
considered 'low concentration' viruses. This, coupled with the great difference in the
concentration o f d s R N A extracted from BBTD-infected plants grown at different temperatures
at different times after infection, could easily explain the failure to find and characterize virus
particles in BBTD-infected plants, and thus prove that the disease is caused by a virus. This
explanation is likely as d s R N A can be extracted in sufficient quantity for analysis from less than
1 g of tissue infected with viruses such as tobacco mosaic virus and cucumber mosaic virus
(Dawson & Dodds, 1982; Morris & Dodds, 1979).
This research was supported by a grant from the Queensland Banana Industry Protection Board.
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