J. gen. ViJvL (I978), 39, 19x-I94
I9I
Printed in Great Brim&
Inhibition of the Multiplication of Tobacco Mosaic Virus
by Methyl Benzimidazol-2-yl Carbamate
(Accepted I I November r977)
SUMMARY
Methyl benzimidazol-2-yl carbamate (MBC, carbendazim) is the fungitoxic
principle of the systemic fungicide benomyl. It reduces the severity of symptoms
produced by tobacco mosaic virus infection of tobacco plants (Tomlinson et aL
I976). Measurements of the accumulation of TMV RNA in MBC-treated and control plants showed that MBC treatment significantly reduced the accumulation
of virus, by 2o ~ in lower leaves and by up to 9o ~ in upper leaves. No phytotoxic
effects of MBC were observed at the concentrations effective against virus
multiplication. Possible modes of action of MBC are discussed.
Methyl benzimidazol-z-yl carbamate (MBC, carbendazim) is the water decomposition
product and fungitoxic element of the systemic fungicide benomyl. Recently, Tomlinson
et al. (I976) showed that MBC reduces the severity of disease symptoms caused by tobacco
mosaic virus (TMV) in tobacco plants. In this paper we report that MBC inhibits the
multiplication of TMV in systemically-infected tobacco plants.
Nicotiana tabaeum L. cv. White Burley plants were grown in John Innes No. 2 compost
in I2"5 cm diam. pots. The plants were grown under natural lighting conditions in a glass
house at 2o to 25 °C. Plants with three fully expanded leaves were watered with coo ml
of a 5 g/l suspension of 'Bavistin' (BASF 346oF), a commercial formulation containing
5o ~o, w/w, MBC and 5o ~ non-active filler. Seven days later, a second application of
Bavistin was made: each plant thus received a total of I g of MBC. There was no sign of
any toxic effects of MBC treatment. Plants of the same size treated with up to ten times
the dose also showed no ill effects or inhibition of growth.
Two days after the second application of MBC, the two highest fully-expanded leaves
on each plant were dusted with 4oo mesh Carborundum and inoculated by rubbing with
a o.I ~, w/v, solution of TMV strain 'Grapevine' in 5o raM-sodium phosphate buffer,
pH 7"o. Control plants which had not received MBC were also inoculated.
TMV multiplication was measured by the accumulation of TMV RNA. Samples of
o'5 to I.O g of infected leaf were homogenized in IO ml extraction medium [e ~o, w/v,
sodium tri-isopropyl naphthalene sulphonate; 5o mM-tris-HCl, pH 7"8; ~o mM-NaC1;
o-z ~, v/v, z-mereaptoethanol; o.2 ~, v/v, silicone DC antifoam emulsion (Dow Corning)]
in an Ultra Turrax homogenizer. Protein was removed from the homogenate by treatment
with phenol-chloroform-m-cresol (Fraser, I975). Total nucleic acid was precipitated with
ethanol and further purified by dialysis against o'5 ~, w/v, sodium dodecyl sulphate;
I5O mM-sodium acetate at pH 6.o (Fraser, I969). Samples of IO to I5/zg total nucleic acid
were fractionated by electrophoresis on 2"2 ~oo polyacrylamide gels (Loening, I969) for 3 h
at 8 V/cm. The gels were scanned for absorbance at 265 nm in a Joyce Loebl Gel Scanner.
TMV RNA was detected as a prominent peak running between the DNA and the larger
ribosomal RNA. No such peak was detected on gels of nucleic acids from healthy leaves.
x3
VIR
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39
I92
Short communications
1.0
1
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I
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(~)
I
(b)
=_
0.5
.©
<
J
0
I
10
I
20
30
0
10
Distance along gel (ram)
I
I
20
30
40
Fig. I. Polyacrylamide gel electrophoresis of nucleic acids from tobacco leaves infected with
TMV. The ultraviolet absorption peaks are D N A at I t ram; T M V R N A at zo to 22 mm and the
larger (25S) ribosomal R N A at 37 mm. The nucleic acids were extracted from the systemicallyinfected ninth leaves above the inoculated leaves, z8 days after inoculation. The scans represent
electrophoresis of nucleic acids from equal weights of leaf material: (a) control, (b) MBC-treated.
500
L
I0
400
-
-
200Ca0
100
-
-
I
14
22
Time after primary inoculation (days)
30
Fig. e. Changes in T M V R N A content per g fresh weight with time in the systemically-infected
fifth leaf above the inoculated leaves. © - - © , Control; 0 - - 0 , MBC-treated. Day o is the
time of inoculation of the lower leaves.
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Short communications
The identity of the TMV RNA peak was confirmed by co-electrophoresis with authentic
TMV RNA prepared from purified virus. The TMV RNA content of the leaf was calculated
from the area of the TMV RNA ultraviolet absorption peak: peak area is linearly proportional to weight of RNA in the peak (Fraser, I97I).
The nucleic acid extraction and purification method used gave yields of over 9o ~ of
the total leaf nucleic acid content, measured chemically as explained elsewhere (Fraser,
I97I). Determinations of TMV RNA content on replicate samples taken from the same
batch of infected leaf material indicated that reproducibility was high: all individual
determinations were within + IO ~ of the mean.
Fig. I shows the nucleic acids extracted from comparable systemically-infected upper
leaves of control and MBC-treated plants, 28 days after primary infection. The amount
of TMV RNA in the MBC-treated leaf was only about Io ~o of that in the control: MBC
therefore strongly inhibited virus multiplication. In contrast, there was no significant
effect of MBC on the accumulation of the host nucleic acids: the peak areas of the DNA
and 25S ribosomal RNA were similar in gels of nucleic acids from control and MBCtreated leaves (Fig. I). In these upper leaves, most of the growth and nucleic acid accumulation occurred during the time the plants were exposed to MBC. E. M. Faithfull (personal
communication) has shown that MBC persists in White Burley tobacco leaves for 45 days
after application.
The extent of inhibition of TMV multiplication by MBC treatment depended on the
height of the leaf on the plant. The 9o 700 inhibition of TMV RNA synthesis shown in
Fig. I occurred in the ninth leaf above the upper inoculated leaf. In the fifth leaf above the
inoculated leaf, MBC treatment inhibited TMV multiplication by about 6o ~ (Fig. 2).
In the inoculated leaves themselves, TMV multiplication was inhibited only 2o to 3o
by MBC treatment. The different levels of inhibition of TMV RNA accumulation in upper
and lower leaves were probably not caused by differences in MBC concentration in different
parts of the plant. J. A. Tomlinson (personal communication) has shown that the highest
concentrations of MBC in the plant occur in the lower leaves.
The inhibition of TMV RNA accumulation in the upper leaves by MBC treatment was
long lasting. Fig. 2 shows that the rate of TMV RNA accumulation in treated leaves
decreased earlier than in control leaves. The TMV RNA content remained fairly constant
for a period of at least IO days following the phase of virus RNA accumulation in MBCtreated leaves. Tomlinson et aI. (1976) showed that 30 days after inoculation, the infectivity
of the non-dark green areas of infected control leaves was higher than that of leaves from
MBC-treated plants. One hundred days after inoculation, however, they found similar
levels of infectivity in the uppermost systemically infected leaves of MBC-treated and
control plants.
The means by which MBC treatment restricts TMV multiplication are not known. Fig. 2
shows that TMV RNA synthesis was measureable in systemically-infected leaves at the
same time after inoculation of lower leaves in control and MBC-treated plants. It is thus
unlikely that MBC is slowing the systemic spread of virus through the host. Other
benzimidazole derivatives have been shown to inhibit animal virus RNA polymerase
(Tamm & Eggers, i963) and initiation of heterogeneous nuclear RNA synthesis by RNA
polymerase II in HeLa cells (Sehgal et al. I976): it is conceivable that MBC inhibits
TMV RNA synthesis directly. MBC also exhibits cytokinin-like activity (Thomas, T974):
it is possible that inhibition of virus RNA synthesis is mediated through growth regulator
metabolism. Effects of cytokinins on infectivity and local lesion size have been reported
(Milo & Srivastava, 1969, Aldwinckle, I975).
13-2
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We thank John Tomlinson for useful discussions and Su Loughlin for capable technical
assistance.
Biochemistry Section
National Vegetable Research Station
Wellesbourne
Warwick CV35 9EF
R . S . S . FRASER
R . J . WHENHAM
REFERENCES
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(Received 28 September I 9 7 7 )
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