VIROLOGY 63, 476-478 (1973)

Differential Temperature Treatment of Plants Greatly Enhances

Multiplication Rates

W. 0. DAWSON’ AND D. E. SCHLEGEL

Department of Plant Pathology, University of California, Berkeley, California 9&?0

Accepted February 13, 197.3

Extremely rapid rates of virus multiplication have been achieved through the
manipulation of the temperature of different parts of the plants following inocula-
tion with either cowpea chlorotic mottle virus (CCMV) or tobacco mosaic virus
(TMV). Under the conditions of the treatment, TMV had a lag period of 15 hr and
reached a maximum yield after 5 days. The lag period for CCMV was 8 hr and maxi-
mum yield was reached by 24 hr. Under normal conditions lag periods and maximum
yields for TMV were l-2 days and lo-12 days, respectively, and for CCMV 1 day and
4-5 days, respectively.

The recent development of the proto-
plast system for study of plant virus mul-
tiplication (I) has made possible a whole
new series of studies on plant virus syn-
thesis. For the first time synchronous in-
fection of plant cells with viruses is possible.
While the protoplast system has many ob-
vious advantages over intact plant tissues,
it is still necessary to relate observations
made on protoplasts to the intact plant
tissues. This paper describes a system which
greatly enhanced the rate of virus multipli-
cat,ion and presumably the level of syn-
chrony of virus multiplication in intact
tissues.
Zech (2) reported that most of the cells
of young tobacco leaves systemically in-
fected with TMV appeared to be in the
same stage of infection. Using electron
microscopy, Nilsson-Tillgren et al. (3) con-
cluded that virus growth in the systemic
infection of young tobacco leaves was near
synchrony. Physiological studies of virus
replication in these leaves have recently
been reported (J-7). However, this system
has some distinct disadvantages: first, it
is not truly synchronous-the cells become
1 Present address: Department
thology, University of California,
92502.
Copyright @ 1973 by Academic Press, Inc.
All rights of reproduction in any form reserved.
infected over a l-2 day period; and, second,
there is no precise zero time from which
to measure events of replication-the pre-
cise time when the cells become infected is
not known.
We approached the problem of improving
the level of synchrony of plant virus multi-
plication through manipulation of the
temperature of different parts of the plant.
Fully expanded lower leaves were mechani-
cally inoculated and maintained at a tem-
perature which permitted rapid virus syn-
thesis. At the same time, the upper, non-
inoculated leaves were maintained at a
temperature which was too low to permit
detectable virus synthesis, but which al-
lowed movement of inoculum into t,hem.
After a suit.able incubation period during
which a substantial amount of virus syn-
thesis took place in the lower leaves, the
entire plant was transferred to a chamber
held at the optimum temperature for virus
synthesis in all leaves. The appearance of
infectivity in the upper leaves was then
monitored carefully.
Two dissimilar virus-host combinations
were tested using the above procedure:
of Plant Pa- TMV in tobacco [Nicotiuna tabacum (L.)]
Riverside CA and cowpea chlorotic mottle virus (CCMV)
in cowpea [Vigna sinensis (Turner) Savi].
476
 SHORT COMMUNICATIONS 477

For studies on TMV multiplication the extracted and assayed by the procedure of
lower four to six leaves of tobacco plants Schlegel (8).
20-30 cm in height were mechanically inocu- The infectivity curve of TMV in young
lated with TMV and the middle leaves tobacco leaves inoculated by the differential
longer than 5-7 cm were removed. The temperature treatment was determined
plants were immediately put into a styro- beginning at the time the plants were trans-
foam chamber (Fig. 1) with the uninocu- ferred to the 25 C chamber and compared
lated upper leaves extending out of the to both the virus infectivity curves of
chamber. The lower leaves inside the mechanically inoculated and systemically
chamber were at 28-30 C while the upper infected leaves (Fig. 2A). TMV infectivity
leaves were maintained at 3-5 C. After 7 began more rapidly, increased earlier, and
days of differential temperature treatment, exhibited a faster rate of increase in the
the plants were removed from the Styrofoam differential temperature inoculated (DTI)
box and transferred to a controlled en- leaves than in either mechanically inocu-
vironment chamber at 25 C. For CCMV
studies, excised cowpea plants with leaflets A
of the first two trifoliate leaves 2-5 cm
long were placed in test tubes containing
Hoagland’s nutrient solution or distilled
water. The primary leaves were mechani-
+/
cally inoculated and the plants were placed
in the Styrofoam chamber with the pri- 0
mary leaves at 28-32 C and the upper leaves
+
at lo-12 C. After 4 days, the whole plants
were transferred to a controlled environ- 0 +
ment chamber at 32 C. 3 +
Infectivity was determined by removing
discs from the leaves at intervals after
inoculation and storing them at -20 C 7
until all harvests could be assayed together.
TMV was assayed on Pinto bean [Phaseolus
vulgaris (L.)] and CCMV on Bragg soybean
[Glycine mczz (L.)]. Infectious RNA was

I+,;/,,,:,
1 4 5 6 7 9

FIG. 2. The growth curves of TMV at 25 C

FIG. 1. Tobacco plants in the differential tem-
perature chamber of l-in. styrofoam (36 X 60 X
30 cm). The top is sealed with parallel styrofoam
sheets with foam rubber glued to the edges to fit
around the tobacco stems. The inside chamber
temperature was regulated with two 60 W light
bulbs connected to a rheostat. The temperature
of the upper leaves was controlled by putting the
Styrofoam chamber containing the plants into a
controlled environment chamber.
(A) and CCMV at 32 C (B) infectivity of DTI
leaves (O-O), mechanically inoculated leaves
(+-+), and systemically infected leaves (@--
-0). Shown are typical experiments from four
replications of the TMV experiment and ten
replications of the CCMV experiment. Zero time
for DTI leaves was the time of transfer from the
low synthesis-limiting temperature. Zero time
for mechanicahy inoculated leaves was at the
time of inoculation. In the case of systemically
infected leaves, zero timeis the timeof inoculation
of the lower leaves which results in a shift of the
curve 2-3 days to the right on the graph.
478 SHORT COMMUNICATIONS
lated or systemically infected leaves. In the
differential temperature inoculated leaves,
the lag period for TMV was approximat’ely
15 hr, and the maximum concentration of
virus was attained after 5 days. With normal
mechanical inoculation, these values are
l-2 days for the lag period and lo-12 days
to reach maximum virus concentration.
The rate of increase in infectivity during the
period of most rapid virus increase was
much greater in the DTI leaves than in
either the mechanically inoculated or the
systemically infected leaves. One possible
explanation for the earlier and more rapid
synthesis in the DTI leaves is that more
cells were initially infected and that the
rapid phase of synthesis occurred in more
cells at the same time.
No TMV infectivity could be detected in
either expressed sap or phenol extracted
RNA from DTI leaves at the time they were
transferred to the 25 C chamber. The in-
fectivity growth curve in detached DTI
leaves was approximately the same as that of
DTI leaves left on intact plants which were
simultaneously moved to the 25 C chamber.
This demonstrates that the rapid increase
in infectivity which occurred in the DTI
leaves resulted from synthesis in these
leaves and not from translocation of TMV
synthesized in the lower leaves.
The results with CCMV were essentially
the same as those with TYIV. The lag period
in the DTI leaves was about 8 hr as com-
pared to about 24 hr in mechanically inocu-
lated leaves (Fig. 2B). The maximum virus
concentration was attained after 1 day
in the DTI leaves and 4-5 days in me-
chanically inoculated leaves. The maximum
rate of CCMV infectivity increase was much
greater in the DTI leaves than either the
mechanically inoculated or the systemically
infected leaves. No infectivity could be
detected in expressed sap or phenol ex-
tracted RNA of the DTI leaves at the time
they were removed from the low tempera-
ture treatment. As in the case of TMV,
with incubation the infectivity increased
at approximately the same rate in DTI
leaves which were detached at the time
they were transferred to a 25 C growth
chamber as in attached DTI leaves trans-
ferred to the same 25 C chamber.
To demonstrate that low temperatures
effectively inhibited synthesis of the viruses
and not just translocation, detached leaves
were mechanically inoculated and main-
tained 7 days at the low temperature after
which they were transferred to a chamber
at the higher temperature. Both Tb’IV and
CCJIV exhibited a lag period after the
temperature shift identical to that shown
by leaves mechanically inoculated at the
higher temperature.
The experiments outlined above show
greatly enhanced rates of virus synthesis in
young DTI leaves. We do not yet know
how near this system is to one of syn-
chronous virus multiplication. The lag
period before the rapid increase in infec-
tivity in DTI leaves was much shorter
than in mechanically inoculated leaves,
suggesting that many more cells are initially
infected in these leaves. This shorter lag
period was approximately the same as that
reported for TMV in tobacco protoplasts at
the same temperature (1). The greater
rate of infectivity increase during the
rapidly increasing phase of the infectivity
growth curve in DTI leaves as compared to
systemically infected leaves suggests that
it is much closer to synchrony than sys-
temically infected young leaves which
Nilsson-Tillgren et al. (S) propose is near
synchrony. Further studies are underway
on these observations and on a determina-
tion of the level of synchrony of infection
achieved in DTI leaves.
ACKNOWLEDGMENT
This work was supported in part by USPHS
Research Grant A103415 from the National In-
stitute of Allergy and Infectious Diseases.
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