BIOLOGIA PLANTARUM (PRAHA)

1 (2) : 126--134, 1959

The Behaviour of the S-strain of Tobacco Mosaic Virus

as Determined by Biological Tests

JARMILA SVOBODOV/[
Department of Plant Pathology, Institute of Biology, Czechoslovak Academy of Sciences, Praha
Received December 15, 1958

Souhrn

Byly pops~my p~iznaky, kter6 pfsobi nov~ vznikl~ S-kmenVTM (SvoBODOV~

1954, 1958, 1959) u dcseti hostitelfi a porovn~ny se symptomy, kter6 vytvhH
Al-kmen a obySejn~ zclen~ kmen tab~kov~ mosaiky. S.kmen vytvh~i na vtteeh
zkou~en~ch hostitelsk~ch rostlin~ch pHznaky od ostatnieh kmenfi odli~in$. Srov-
n~li inkubaSnl doby a ovlivn~nl rdstu na Nicotiana tabacum Samsun, chovh~i
na N. rustica a kvantitativnl testy na _N. glutinosa uk~zaly, ~e S-kmen je ze v~iech
srovn~van~ch kmenfi nejm6n~ par Podle v~sledk~ vltech biologick~ch
testfi je S-kmen bli~fhi obySejn~mu kmenu tabs mosaiky; pouze s6rologiek6
a interfcrenSnl zkou~tkysv~dSi o jeho bli~im pHbuzenstvl k Al-kmenu, ze kter6ho
vznikl. Chovg~l S-kmene VMT ve vytlaSen6 w je shodn6 s e h o v ~ m obySej-
n6ho zelen~ho kmene i ~Ll-kmene. Stejn~ tak okruh hostitel~ vw tH l~nen~ je
t ~ . DfferenSntm hostitelem do urSit6 miry je Physali~ alkelcengi a P. ~ranchetti.
Podle biologick~ch testfi je tedy S-kmen nov~m kmenem viru tab~kov~ mosaiky.

Summary

1. T h e s y m p t o m s c a u s e d by t h e S - s t r a i n (SVOBODOV~ 1954, 1958, 1959)

were d e s c r i b e d for t e n h o s t s a n d c o m p a r e d w i t h t h o s e c a u s e d b y t h e A l - s t r a i n
a n d t h e o r d i n a r y g r e e n s t r a i n o f t h e t o b a c c o m o s a i c v i r u s . I n all t h e h o s t
plants tested the S-strain produces s y m p t o m s different from those caused b y
other strains.
2. A c o m p a r i s o n of t h e i n c u b a t i o n p e r i o d a n d t h e effect on g r o w t h o f
N i c o t i a n a t a b a c u m S a m s u n , t h e b e h a v i o u r on N . rustica a n d q u a n t i t a t i v e
t e s t s on N . glutinosa s h o w e d t h a t t h e S - s t r a i n is t h e l e a s t p a t h o g e n i c o f all
the strains compared.

126
 T H E B E H A V I O U R OF T H E S-STRAIN TOBACCO MOSAIC VII~US 127

3. Results of biological tests show the S-strain to be most similar to the

ordinary green strain of tobacco mosaic virus, only serological and cross-
protection tests indicate a closer relationship to the Al-strain, from which it
developed.
4. The behaviour of the S-strain of TMV in expressed sap is similar to the
behaviour of the ordinary green strain and of the Al-strain. The range of
hosts for all three strains is also the same. Physalis alkekengi and P. Fran-
chetti are to a certain extent differential hosts.
Thus biological tests show t h a t the S-strain is a new strain of tobacco
mosaic virus.

Introduction

The S-strain of tobacco mosaic was obtained by influencing the metabolism

of the usual host plants of this virus. I t developed by the passaging of the
M-strain of TMV on the same varieties of tobacco and tomato plants. The
passaging was carried out alternately on normal and stunted plants which
had been cultivated under aseptic conditions on an agar medium in Erlen-
meyer flasks with limited air supply. I t was after two or three changes of
the environment of the virus brought about by transfer from a plant with
normal metabohsm to a plant with altered metabolism and the reverse (Svo-
BODOV~ 1954, 1958, 1959), t h a t this strain was obtained. The new strain has
now been maintained by continual passaging for six years. The symptoms
it causes clearly distinguish it from all other available strains. Its biological
characteristics have been ascertained in comparison with the characteristics
of its parent strain (Al-strain of TMV) and of the ordinary green strain of
tobacco mosaic virus.

Methods

The following hosts were chosen for establishing and comparing the relationship of the new
strain to its parent strain (Al-strain) and the ordinary green strain of TMV: Nicotiana tabacum
Samsun, 17. tabacum White Burley, 17. tabacum Virginian golden, N. glutinosa, 17. rustica,
Physalis Franchetti, P. alkekengi, Capsicum annuum, Solanum lycopersicum. The symptoms
produced by systemic infection of leaves were also described for these hosts.
For Nicotiana tabacum Samsun the length of the incubation period for the different strains
was compared and their effect on the growth of the infected plants was observed. I n order to
evaluate biological activity, a qualitative and quantitative comparison of primary lesions
caused by inoculation of the leaves of 17. rustica was also made and the relative infectivity of
homogenates of systemically diseased plants was compared b y testing on 17icotlana 91utinosa.
The mutual relationship of the new strain with the two other strains was further determined
serologieally. (The drop method according to JEtC~OLJEV and HRUw 1947 and BAWDE~, 1950).
The identification of the new strain (S-strain) was completed by determination of the limiting
inactivation temperature of the virus in sap and by achieving infection from dried material.
128 J. S V O B O D O V ~

Results

1. l~eview of symptoms caused by individual strains on different host plants

is given in the table 1.
On all the host plants mentioned the S-strain produces symptoms which
differ both from the symptoms caused by the Al-strain and the ordinary
strain, and from those of all other strains. On all host plants these symptoms

Table 1.

Host plant Ordinary green Al-strain of TMV S-strain of TMV

strain of TMV

1.1Vicotiana Typical tobacco Dark-green, whitish, A t first yellow stripes

tabacum mosaic; green yellow, yellow-green along veins, later leaves
Samsun spots deforma- spots, blisters, deforma- with gre6n stripes, yellow
tion of leaves. tion. Y o u n g e s t leaves a n d green spots, finally
with strong s y m p t o m s . stripe p a t t e r n s . Young-
Some plants die, est leaves, a t least their
m a j o r i t y stop growing. apexes, w i t h o u t symp-
toms. Growth almost
completely non-inhibited.

2. N . tabacum The s a m e symp- The s a m e s y m p t o m s as The s a m e s y m p t o m s as

VChite t o m s as N. t, those caused b y Al-strain those caused b y S-strain
Burley Samsun. on ~'. t. Samsun. on N. t. Samsun, b u t
weaker.

3. N . tabacum T h e same s y m p - The s a m e s y m p t o m s as The s a m e s y m p t o m s as

Virginian t o m s as N. t. those caused b y Al-strain those caused b y S-strain
Golden Samsun on N. t. S a m s u n on N. t. S a m s u n ; the
reaction of this v a r i e t y
is more sensitive; de-
eolouration s y m p t o m s
are clearer at a time of
year when s y m p t o m s on
S a m s u n are n o t y e t
evident.

4. N . glutinosa P r i m a r y necrotic P r i m a r y necrotic lesions. P r i m a r y necrotic lesions.

lesions.

5. N . rustica Slight lesions, Slight lesions, larger Slight lesions.

often with yellow- areas of necrosis.
ish surroundings.
Leaves with m a n y
lesions often go
completely yellow.
Sometimes large
areas of necrosis.
F r e q u e n t large
yellow spots on
younger leaves.
 T H E B E H A V I O U R OF T H E S-STRAIN TOBACCO MOSAIC V I R U S 129

Tab. 1. contin.

Host plant Ordinary green Al-strain of TMV S-strain of TMV

strain of TMV

6. Physalis No symptoms. Dark-green whitish, Most often without

Franchetti yellow, yellow-green symptoms, or slight
and spots, blisters, deforma- clearing of veins, light
7. P. alkekengi tions. Youngest leaves yellowish decolouration,
show strong symptoms. youngest leaves stay
Some plants die, some green.
stop growing.

8. Capsicum Not determined. Following infection of The same symptoms, but

a~bTb~.~m cotyledons; cotyledons, considerably milder,
go yellow and plants die.
Older plants have yel-
lowish spots.

9. Solanum Not determined. Dark-green, whitish, and Yellow spots, rarely~

lycopersicum yellow spots, blisters and green blisters; disease
deformations. slight.

are very mild. As far as pathogenity, judged only b y symptoms, is concerned,

the S-strain resembles the ordinary tobacco mosaic more closely than its
parent strain. I t is, however, the mfldest of all three strains.

2. The range of host plants

As far as plants entered in the above table are concerned, all three strains
have the same range of host plants. Only Physalis Franchetti and P. alIcelcengi
form an exception as far as the symptoms of disease are concerned. Following
one infection with ordinary tobacco mosaic they show no symptoms; in order
to produce symptoms it is necessary to repeat infection of the same plant on
several consecutive days (BLATT~ 1958). The Al-strain attacks this plant
very strongly. The S-strain produces mild symptoms, which are sometimes
not very pronounced.

3. Comparison of the incubation period and the effect on the growth of

plants of Nicotiana tabacum Samsun
The comparison was carried out on plants of Nicotiana tabacum Samsun
at the stage when they had ~ 5 leaves. The plants were infected with
the three strains. There were 36 plants in each group. (The results are given
here for information only in order to fill in the results of the other experiments
in a general way.)
The first symptoms began to appear in plants infected with the Al-strain
and only at a later stage and less clearly in plants infected with the other
strains. Then, 7 days after infection, symptoms were observed as follows:
130 J. SVOBODOV~

in 83-3% of plants infected with Al-strain, in 63"8~/o of plants infected with

the ordinary green strain and in 38.8% of plants infected with the S-strain.
After a further 24 hours definite symptoms were observed in 94.4% of plants
infected with the Al-strain, in 94.4% of plants infected with the ordinary
green strain and in 66-6% of plants infected with the S-strain. During the
following days symptoms of systemic infection were shown by all plants infected
with the Al-strain and the ordinary green strain. I n the group infected with
the S-strain one plant remained without any signs of disease. The average
height one month after infection was for plants infected with the ordinary
green strain 7.6 em., for those infected with the S-strain 6.6 cm. and for
those infected with the Al-strain 3.6 cm.
With regard to the incubation period and the effect on the growth of the
host plant it appears t h a t the S-strain is more similar to the ordinary strain
of tobacco mosaic virus t h a n to its parent strain. Of all the strains compared
it is, however, the mildest in its effect on the host plant.

4. Behaviour on Nicotiana rustica

Groups of 5 plants with 4 leaves were infected with each of the strains
under comparison. The third leaf was infected with centrifuged, filtered sap
by means of a brush. Fifty-two days after the date of infection (the beginn-
ing of November) the average number of lesions on an inoculated leaf was
40-2 for the ordinary green strain, 10.6 for the Al-strain and 8.6 for the S-strain.
The average number of leaves showing signs of systemic infection was 2.4 for
the ordinary green strain, 1.8 for Al-strain and 1.2 for the S-strain. The
primary reaction to the ordinary green strain and to the Al-strain was
accompanied by several large areas of necrosis and chlorosis, while in the
primary reaction Nicotiana rustica infected with the S-strain showed only
slight lesions.
Thus the S-strain is seen to be the least pathogenic for plants. Similarly,
with regard to its effect on growth S-strain proved to be the least harmful.
Plants infected with S-strain reached an average height of 10.9 cm. (while
plants infected with the green strain attained 9.8 cm. and those infected with
the Al-strain only 8.8 cm.); the average number of leaves per plant was also
highest with the S-strain, i. e. 6.4, for the green strain it was only 5.6 and
for the Al-strain 5.2. These results are given for information. In the spring and
summer the differences between these strains are more pronounced.

5. Quantitative tests on Nicotiana glutinosa

Two quantitative tests were carried out. For the first time, in September
1956, Nicotiana tabacum Samsun plants were used, which had been infected
33 days previously and were of equal age and had been cultivated under the
same conditions. Sap was in every ease taken from only one plant of those
infected with each strain. After filtration and centrifuging this sap was
inoculated without dilution on to Nicotiana glutinosa by painting the leaves
with a brush, one half of each inoculated leaf blade being painted with sap
from plants infected with the S-strain and, in the first group, the other half
 THE BEHAVIOUR OF THE S-STRAIN TOBACCO MOSAIC VIRUS 131

with sap from plants infected by the Al-strain, in the second group with
sap from plants infected by the ordinary green strain. I n each group 12 leaves
were tested in this way. The sap was not washed off the leaves.
The results of this test are in agreement with those of a test carried out
in May of the following year (1957), in which plants of Nicotiana tabacum
Samsun infected 21 days previously were used. The leaves with petioles were
cut and twice the amount by volume of distilled water was added to the
amount by weight of the leaves taken from each plant separately. Following
homogenization the expressed sap was refiltered and centrifuged. Inoculation
was carried out as in the first test. Groups of t w e n t y leaves of Nicotiana
gIutinosa were used for testing. Results of both tests are given in the following
table:
Quantitative test on Nicotiana glutinosa

T a b l e 2. A v e r a g e n u m b e r of lesions on one leaf:

Date S -- Z S -- A1 A1 - - Z n

14. 9. 1 9 5 6 49'3 83-9 62"9 146"7 12

27.5. 1957 9"1 19"3 7'7 36.1 41.3 33"2 20

Average 24"2 43"5 26"4 71"2

Statistical evaluation (by t-test) was carried out for the experiment of
14. 9. 1956 with a lower n (12) and here there were also smaller differences
in averages t h a n in the further comparative tests. I n the comparison of
S-strain with Al-strain there are statistically significant differences (I) ~ 0.01),
in the comparison of S and Z the result is somewhat below the limits of
significance ( t ~ ~ 1-902, 1) ~ 0-10 ~ 0-05).
I t m a y be said t h a t the least biological activity was shown by the S-strain;
the ordinary green strain of TMV showed a medium activity and the Al-strain
showed the highest activity. Thus the S-strain more closely resembles the
ordinary strain in its infectivity t h a n the Al-strain.

6. Cross-inoculation
Nicotiana tabacum Samsun was used for cross-inoculation. The plants were
infected with the second strain in each case three days after infection with
the first, t h a t is sooner t h a n any symptoms caused by the first strain could
appear. I t was found t h a t the S-strain protects tobacco plants against
secondary infection by the Al-strain, and alternatively t h a t the Al-strain
protects against the S-strain. These results are in agreement with BLATTN~'S
(1956) results and indicate a close relationship between the S-strain and the
Al-strain and a less pronounced similarity between these two strains on the
one hand and the green strain on the other hand.
132 J. SVOBODOVA

7. Serological tests
Two tests of serological relationship were carried out: antigen was similarly
prepared for both tests. The virus was isolated according to SIEGEL and
WILDMAN'S method (1954). Further purification was then carried out b y
three-fold coagulation at the isoelectric point according to BAWDEN and
PmIE (1943). Following dialysis the antigen was filtered through a bacterial
filter G5; for the second test lyophilized purificate was used. Immunization
was carried out as follows: on the first day rabbits were injected with 1 ml.
each of antigen, on the second day 1.5 ml., the fifth day 2 ml., the sixth d a y
2.5 ml., the seventh day 3 ml., the ninth d a y 3.5 ml., the tenth day 4 ml.
After an interval of one week the rabbits were starved for one day and were
then bled. The serum, after centrifuging, was p u t into ampules at a tempera-
ture of - - 1 0 ~ C.
In 1956 serological tests were carried out b y the drop method according
to JV,RMOLJEV and HRU~KA (1947) and in 1957 b y the classic method (in test
tubes) according to BAWDEN (1950). The different virus preparations were
diluted with physiological saline in geometric series. The strength of the
reaction was judged according to the dilution in which the virus preparation
being tested still reacted with the appropriate serum.
In the first and second tests the serum against the S-strain reacted more
strongly with the virus preparation of the Al-strain than with that of ordinary
tobacco
I
mosaic virus. The serum against the ordinary tobacco mosaic virus
reacted more strongly with the Al-strain than with the virus preparation of
the S-strain. I t can therefore be concluded from the results that all the strains
are serologically very closely related; a particularly close relationship exists
between the S-strain and the Al-strain; and also between Al-strain and the
ordinary strain. However, only small differences are involved. The results of
the serological tests are in agreement with those of the protection tests.

8. The behaviour of the strains under comparison in expressed sap

The S-strain, similarly to the Al-strain and the ordinary green strain of
tobacco mosaic virus, can stand a temperature of 90 ~ C for a period of 10 mlu.
without losing its activity. I t keeps its infectivity in dried sap and dry leaves.
Thus it behaves in the same w a y as the other two strains.

Discussion

The S-strain is most closely related to the ordinary green strain of tobacco
mosaic virus both in its pathogenity and its behaviour in the host plant. On
the other hand, interference and serological tests indicate a closer relationship
to the Al-strain from which the S-strain originated. The similarity to the
ordinary green strain of tobacco mosaic is particularly remarkable in view
of the fact that KAUSCH~, and STUBBE's strain TM44, which the S-strain
resembles in certain respects (SvoBODOVX 1958, 1959), arose b y mutation
 THE BEHAVIOUR OF THE S-STRAIN TOBACCO MOSAIC VIRUS 133

directly from the green strain of ordinary tobacco mosaic. TM44, however,
acts more pathogenically than the ordinary strain of TMV, while the S-strain
is the least pathogenic of all the strains which have been compared. Although
both these strains produce similar symptoms, their pathogenesis is dia-
metrically opposed. This indicates some kind of mutual independence in the
production of symptoms by the virus and by the plant. This observation
coincides to a certain extent with the occurrence of a large quantity of virus
in carriers which do not show symptoms.
T h e writer wishes to e x p r e s s h e r t h a n k s to Dr. BrShk for his h e l p in c o n n e c t i o n w i t h t h e
q u a n t i t a t i v e t e s t s o n /V. glutinosa, to I n g . P o z d ~ n a for h e l p w i t h t h e serological t e s t s a n d to
J. L i m b e r k for h e l p w i t h t h e t e s t s o n N . tabacum S a m s u n .

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Address: In~. J ~ r m i l a S v o b o d o v s I n s t i t u t e of B i o l o g y of t h e C z e c h o s l o v a k A c a d e m y
of Sciences, D e p a r t m e n t o f P l a n t P a t h o l o g y , N a K a r l o v e e 1, P r a h a 6, Dejvice.
134 J. SVOBODOVA

IIoBe~eHHe ~-mTaMMa BTM, yCTaHO~eHHOe ~HO~IOrH~eCKHMHMeTO~aMH

}~PMrI~IA CBOBO~I~OBA

Pe3mMe

t . O n ~ c a u u npnanatcH, BuauaaeMue S-IUTaMMOM (SVOBODOVk t954, 1958, 1959) y ~ecaTrt

pacTeHHfi-xoaaeB H conocTaB~ieHbt C~MnTOMbI, n u a u B a e ~ u e AI-mTaM~tOM ~ oSuq~thtM 3e:ie-
HhIM IIITaMMOM B~pyca TaSaq~ofi MoaamcH. S-IIITaMM oSpaayeT Ha Bcex HO~OIIhITHI)IX pacTe-
n u 9 x - x o a g e a a x npHanaKK, oTn~tqammHecR OT ~ p y r n x II[TaMMOB.
2. CpasHenHe BpeMeHH HH~ySatlHn H B:m~H~e u a pOCT Ha Nicotiana tabacum S a m s u n ,
IIOBe~eHHe Ha AT. rustica H ROJIHqeCTBeHHhIe TeCTbI Ha N. glutinosa noRa3aJiiL ~tTO S-IIITaMM
o S ~ a ~ a e T CaMOH HH3KOH naTorenHocvb~) c p e n n cpaa~KBaeMbIX IIITaMMOB.
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IIlTaMMy TaSaqnofi MOaaH~n; TOn~,Ir cepo~or~qec~cHe H n H T e p ~ e p e i ~ O H n ~ i e O~tUTU C B ~ e -
TeJIhCTByIOT O ero 63IH3ROM pO~CT~e c AI-IIITaMMOM, H3 t~OTOpOro BOaHHI~.
4. IIoaeReHHe S-IIITaMMa B T M n oTnpeccoBannoM comte COOTBeTCTByeTnoaeReHn~O oS~iq-
Horo 3eJienoro rt il-mTaMMa. T o m e RnanaaoH pacTeHufi-xo3aeB Bcex Tpex IIITaMMOB TOT ~I{e.
~ n ~ e p e u t t ~ p y i o m ~ M xoa~HOM ~0 onpe)ie~iennofi cTenenn ~B:i~evc~ Physalis alkekengi"
P. Franchetti.
IIo pe3y~IbTaTaM 514OXgMHgeCK~IX TeCTOB S-IIITaMM gBJIi~eTC~I HOBI)IM IIITaMMOM B~pyca
TaSaqHofi Mo3aHKH.