New Zealand Journal of Agricultural Research

ISSN: 0028-8233 (Print) 1175-8775 (Online) Journal homepage: http://www.tandfonline.com/loi/tnza20

Studies on a virus inhibitor from Chenopodium

leaves
A.D. Thomson & Barbara A. Peddie
To cite this article: A.D. Thomson & Barbara A. Peddie (1965) Studies on a virus inhibitor
from Chenopodium leaves, New Zealand Journal of Agricultural Research, 8:4, 825-831, DOI:
10.1080/00288233.1965.10423716
To link to this article: http://dx.doi.org/10.1080/00288233.1965.10423716

Published online: 16 Jan 2012.

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Date: 20 November 2016, At: 07:02

825

STUDIES ON A VIRUS INHIBITOR

FROM Chenopodium LEAVES
By A. D. THOMSON

AND

BARBARA A. PEDDIE

Plant Diseases Division,

Department of Scientific and Industrial Research,
Lincoln

(Received 29 July 1965)

ABSTRACT
Sap extracted from leaves of Chenopodium amaranticolor Costand Reyn. and C. album L. inhibited infection by tobacco mosaic virus
on Nicotiana glutinosa L., but only slightly on the species from which
the sap was extracted. Sap was still inhibitory after heating at 55c for
10 minutes and after storing at 20c for six days. No inhibitory activity
remained in sap heated at 100c for 10 minutes. Dilution of inhibitorvirus mixtures resulted in recovery of infectivity. Most inhibitor remained
in the supernatant fluid after centrifuging sap at 40,000 r.p.m. for four
hours in a Beckman/Spinco Model L ultracentrifuge (No. 40 rotor). Sap
was still inhibitory after dialysis against water. Nicotiana glutinosa leaves
sprayed with C. album sap were protected from infection by tobacco
mosaic virus for about three days after spraying.

INTRODUCTION

Chenopodium amaranticolor Coste and Reyn. is a useful test-plant

for plant viruses (Hollings 1956). Unfortunately viruses are not readily
transmitted from C. amaranticolor to other susceptible genera because of
the presence of a virus inhibitor in the Chenopodium sap. We have
studied the inhibitory activity of Chenopodium sap on tobacco mosaic
virus in an attempt to find ways of reducing the activity of the inhibitor
without diminishing virus transmission. Although numerous virus inhibitors have been described (Bawden 1954), there are few reports on the
use of these inhibitors to control viruses. We have therefore examined
the possibility of using the inhibitor in Chenopodium sap to control the
spread of mechanically-transmitted viruses.
MATERIALS AND METHODS
Sap was obtained by grinding leaves of C. amaranticolor and C.
album L. in a mincer and squeezing the sap through muslin. Sap was
then centrifuged at 5,000 X g for 10 minutes, and the resulting supernatant fluid provided the inhibitory material. The inhibitory effect of
Chenopodium sap was determined by comparing the infectivity of equal
volumes of tobacco mosaic virus and distilled water with equal volumes
N.Z. Jl agric. Res. 8: 825-31

826

Virus inhibitor from Chenopodium leaves

of tobacco mosaic virus and Chenopodium sap. The mixtures were

rubbed on leaves of Nicotiana glutinosa L. The preparation of tobacco
mosaic virus was infective sap diluted 10-2 with distilled water. A
diatomaceous earth was dusted on leaves before inoculation, and inoculations were made with the forefinger wet with inoculum. Each suspension
was rubbed on six or 12 whole or half leaves, which were distributed in
a regular manner to minimise errors caused by variations in susceptibility.
Experiments were made with sap from leaves of both C. amaranticolor and C. album. However, C. album was mostly used, since this plant
is a common weed, and large quantities of sap can be readily obtained.
Sap from Chenopodium leaves was also centrifuged at 40,000 r.p.m.
for up to four hours in a Beckman/Spinco Model L ultracentrifuge (rotor
No. 40). The supernatant fluid and the resuspended pemet were tested for
the presence of the inhibitor.
RESULTS
The powerful inhibitory effect of Chenopodium sap on tobacco
mosaic virus can be readily demonstrated, and the results of two experiments are shown in Table 1. Little inhibitory activity remains in sap
which had been diluted 10-3 with water (Table 2).
TABLE l.-EfJect of Chenopodium sap on infection by tobacco mosaic virus
Expt

Total No. lesions

produced by tobacco
mosaic virus on 12
Nicotiana glutinosa leaves

Tobacco mosaic virus

mixed with

------~------------------------~----~

IChenopodium amaranticolor sap

Chenopodium album sap

-- -"- - --- - ----~----

989

Distilled water

No significant reduction in effectiveness of the inhibitor resulted

from heating sap for 10 minutes at 55e, although no inhibitory activity
remained in sap whi.ch was heated at 100 e for 10 minutes (Table 2). Sap
was still inhibitory after storing at 20 e for six days (Table 2). Thus the
inhibitor appears to be relatively stable.
0

Virus inhibitors may act either directly on the virus or indirectly by

affecting the susceptibility of the host. The latter type of inhibitors are
more common (Bawden 1954). Two kinds of evidence indicate that the
inhibitor in Chenopodium sap affects the susceptibility of the host plant
to tobacco mosaic virus. Firstly, the degree of inhibition depended on the

A. D.

THOMSON AND BARBARA

A.

827

PEDDIE

TABLE 2.-EDect of dilution, heat, and agIng on the virus inhibitor present in
Chenopodium album sap
Expt

Tobacco mosaic virus

mixed with

, Chenopodium album
sap heated at 55
for 10 minutes '

Dilution
of sap

10-'
10-2
10-'

Water

2
10

334
387

10

234
369

498

Chenopodium album
sap heated at 100
for 10 minutes

940

Unheated S"P

975

Water

----'--------

Total No. lesions

on Nicotiana
glutinosa leaves'

I
----------]--I

10-'
10-2
10-'

Unheated sap

-------;------'----------

Chenopodium album
sap kept at 20
for six days

989

Freshly extracted sap

Water

, In heating experiments, sap was centrifuged at 5,000 X g after heating, and

the supernatant fluid was mixed with tobacco mosaic virus.
2

Six leaves in experiment 1 and 12 leaves in experiments 2 and 3.

identity of the host on which the inhibitor-virus mixtures were assayed;

there was less inhibition on Chenopodium species than on N. glutinosa
(Table 3). Indeed, one characteristic of virus inhibitors from plants is
that they have little inhibitory effect ~hen used on the species from which
they were extracted (Bawden 1954). Secondly, dilution of the inhibitorvirus mixture resulted in the elimination of the inhibitory effect (Table 4).
We have made some observations on the nature of the inhibitor in
C. album sap. Sap was centrifuged at 40,000 r.p.m. (No. 40 rotor) for
four hours, and the resulting pellet was resuspended in a volume of
distilled water equal to the original volume of sap. The supernatant fluid
contained most of the inhibitor, although some was present in the
resuspended pellet (Table 5). Sap which had been dialysed for 24 hours
at room temperature against running tap water retained the inhibitor
(Table 5). In another experiment, the inhibitor was concentrated as

828

Virus inhibitor from Chenopodium leaves

TABLE 3.-1nfectivity of inhibitor-virus mixtures inoculated to different hosts

Tobacco mosaic virus
mixed with

Expt

Total number
of lesions on
12 leaves

Assay host

Chenopodium album sap

Nicotiana glutinosa

Water

Nicotiana glutinosa

1,391

Chenopodium album sap

Chenopodium album

272

Water

Chenopodium album

620

Chenopodium
amaranticolor sap

Nicotiana glutinosa

Water

Nicotiana glutinosa

Chenopodium
amaranticolor sap

Chenopodium
amaranticolor

Water

Chenopodium
amaranticolor

0
639

300
700

TABLE 4.-Effect of diluting inhibitor-virus mixtures on lesion numbers

Tobacco mosaic virus
mixed with

Expt

Chenr>podium album sap

I
I

Water
,

Total No. of lesions on 12 Nicotiana

glutinosa leaves after diluting mixture
0

10-'

10-'

10-3

10-"

10-'

15

47

70

38

1,478

680

284

36

28

"------~.-

Chenopodium album sapl

Water

---1\

498

52

57

40

follows. Sap was extracted, centrifuged at 5,000 X g for 10 minutes and

then centrifuged at 30,000 r.p.m. for 90 minutes (No. 30 rotor). Ten ml
of the supernatant fluid was reduced in volume to 1 ml by evaporation in
a dialysis tube at 20. This procedure resulted in an approximately
lO-fold increase in concentration of the inhibitor (Table 5).
We have examined the possibility of using Chenopodium sap as
a spray to control the transmission of tobacco mosaic virus. Nicotiana
glutinosa leaves were sprayed with sap from C. album, and control leaves
were sprayed with water. At intervals after spraying, the leaves were

A. D.

THOMSON AND BARBARA

A.

829

PEDDlE

TABLE 5.-Some properties of the inhibitor in Chenopodium album sap

Tobacco mosaic virus

Total No. of lesions on six Nicotiana
mix~wi~ _ _ _ __ l!!~tirl~~~e:v~ after inhibitor diluted

Expt

--I
0

Chenopodium album sap

Supernatant fluid'

63

Water
-

- - - -

--_.-

Unconcentrated supernatant fluid

Concentrated supernatant fluid'

Water
iUndialysed
IChenopodium album sap
IDialysed sap'
IWater

10-'
---

10

234

369

58

214

648

322

536

866

----

Chenopodium album sap

10-3

10-'

498
--

---'--------------

10-'

Sediment'

---

------- ---

80

328

302

23

260

380

19

251

318
- - - - - _ . - - _ . _ _. _ -

_. _ _._...L ___ _
I

o
o
975

, Sap was centrifuged at 40,000 r.p.m. for four hours in Beckman/Spinco rotor
No. 40.
2 Sediment from above resuspended in a volume of water equal to the original
volume of sap.
, Concentrated IO-fold by evaporation in dialysis tube.
, Dialysed for 24 hours at room temperature against running tap water.

rubbed with tobacco mosaic virus. The results shown in Table 6 indicate
that the inhibitor gave protection for about three days, but protection
was greatly reduced after seven days. Some damage was observed on
the leaves sprayed with Chenopodium sap, although plants recovered
rapidly. However, no damage-:was observed on N. tabacum L. (var.
White Burley) seedlings sprayed with sap.
DISCUSSION
The powerful inhibitory effect of Chenopodium sap on tobacco
mosaic virus has been demonstrated. Blaszczak, Ross, and Larson (1959)
examined the inhibitory activity of sap from C. amaranticolor and C.
album on potato virus X, and some of their results are similar to those
recorded in the present paper. They showed that Chenopodium sap
could be diluted 1/10, heated at 60 for 10 minutes, and stored at 4c
for 77 days without loss of activity. They also stated that heating sap at

Virus inhibitor from Chenopodium leaves

830

TABLE 6.-lnhibitory effect on tobacco mosaic virus of Chenopodium album sap

sprayed on Nicotiana glutinosa leaves
Treatment
Sap and TMV inoculated together

Leaves sprayed with sap and

inoculated after one day

Leaves sprayed with water and

inoculated after one day
Sap and TMV inoculated together

Total No. of lesions on

N. glutinosa leaves!

Leaves sprayed with sap and

inoculated after three days

512
6
34

Inoculated after seven days

1,313

Leaves sprayed with water and

inoculated after seven days

1,817

12 and six N. giutillos(l leaves in cxper'mcnts I and 2 respectively.

lOOoe for 10 minutes resulted in a slight loss of inhibitory activity. It is

clear that the virus inhibitor in Chenopodium sap is extremely stable.

Like most virus inhibitors, the inhibition seems to be produced by

affecting the susceptibility of the host to virus, rather than by any direct
effect on the virus. Since some inhibitor sediments after high-speed centrifugation, this could mean that more than one inhibitor occurs in sap. On
the other hand, the inhibitor may be adsorbed to large particles in the sap
and sedimented with them. The inhibitor should be amenable to chemical
analysis using, for example, heat-clarified, centrifuged sap. Unfortunately
our results give no clue to methods which could be used to reduce the
influence of the inhibitor on the transmission of viruses from C. amaranticolor to other genera.
Little use has been made of the $reat variety of plant virus inhibitors
which have been described, as a practical means of controlling saptransmissible viruses such as tobacco mosaic virus. Since C. album is
readily available in New Zealand and the inhibitor is stable, it might
provide a method for reducing the spread of tobacco mosaic virus in
tobacco and tomato crops. The present results show that the inhibitor
protects leaves for about three days after spraying, and there is little
damage on sprayed leaves. The addition of a compound to increase
adherence of the sap to the leaf surface might improve the amount of
protection. Another possible use for the inhibitor is in the control of
non-persistent aphid-transmitted viruses. In a recent paper Shanks and
Chapman (1965) have, in fact, demonstrated some reduction in the
spread of certain non-persistent viruses with virus inhibitors.

A. D.

THOMSON AND BARBARA

A.

PEDDlE

REFERENCES
F. C. 1954: Inhibitors and plant viruses. Adv. Virus Res. 2:
31-57.

BAWDEN,

W.; Ross, A. F.; LARSON, R. H. 1959: The inhibitory activity

of plant juices in the infectivity of potato virus X. Phytopathology
49: 784-91.

BLASZCZAK,

M. 1956: Chenopodium amaranticolor as a test plant for plant

viruses. Pl. Path. 5: 57-60.

HOLLINGS,

C. H.; CHAPMAN, R. K. 1965: The use of antiviral chemicals to

protect plants against some viruses transmitted by aphids. Virology

SHANKS,

25: f13-7.

831