B I O L O G I A P L A N T A R U M (PRAHA)

1 (2) : 135--141, 1959

Cadmium Ions as lnhibitors of Tobacco Mosaic Virus

MARIE ULRYCHOv~-ZELINKOV~
D e p a r t m e n t of P l a n t Pathology, I n s t i t u t e of Biology, Czechoslovak Academy of Sciences, P r a h a

Received November 19, 1958

Souhrn

l~Sinek kademnat~-ch iontfl n a reprodukci viru tabAkov~ mosaiky byl studovAn

n a rostlinAch tabAku (N. tabacum Samsun), kter~ byly pitstov~ny ve skleniku
v ko~enA6ieh. Pokusn6 rostliny byly zal~vAny roztokem siranu kademnat~ho
o koncentraci 4 m g Cd~+[1 ml v r~zn~ch d~vkAeh. U ka~d~ rostliny byl oSkovAn
jeden list Al-kmenem VTM. OptimAlnl dAvka k a d e m n a t ~ c h lentil, kterA nemA
~Adn~ ~kodliv:~ vliv n a pokusn~ rostliny, je d~vka 160 mg Cd ~+ n a jednurostlinu
aplikovanA ve dvou z~livkAch, a to t~sn~ p~ed inokulacl a t~etl den po inokulaci.
Reprodukce VTM je inhibov~na ze 70 ~ podle testu n a N . glutinosa a hyper-
sensitivnlm hybridu. Inhibi~nl dSinnost kademnat:~ch iontfl znaSn~ poklesne p~i
zAlivce roztokem k a d e m n a t ~ c h iont~ 24 hodiny p~ed inokulaci. Z rozdilfi v prfl-
b~hu prim~rni infekce, sledovan6 jodovou zkouw jsou nApadn~ zvlA~it~ tyto
skuteSnosti: u pokusn:~eh rostlin se ztrAcl w z infikovan~ch listfi pomaleji,
v~voj chlorotick:~ch skvrn je zbrzd~n a jejich ohrani6enl a s t r u k t u r a nejsou ostr~.
Vyw d~vky kadmia (kolem 300 mg n a jednu rostlinu) vyvolAvaji n a n6kter~ch
listeeh mlad~ch rostlin tabAku (N. tabaeum Samsun) skvrny, kter~ p~ipominaji
nekrotick6 lese n a listech N. glutinosa po infekci VTM. P~edb6~n~ bylo zji~t~no,
~e kademnat~ ionty vyvolAvaji poruchy v metabolismu fosforu. V diskusi je
vyslovena domn~inka, ~e inhibi6nl d6inek kademnat:~ch iontfl n a reprodukci VTM
mfl~e spo61vat, analogicky k p~edstav6 vytvo~en6 v ~ivo6iw fysiologii, v inter-
akci iontfl zinku a k a d m i a v metabolismu nukleoproteinfl.

Summary

1. W h i l e s t u d y i n g t h e i n h i b i t o r y e f f e c t s o f c a d m i u m i o n s o n t h e r e p r o d u c -
t i o n o f t o b a c c o m o s a i c v i r u s i n v i v o i t w a s f o u n d t h a t a d o s e o f 1 6 0 m g . C d ~+
applied in two waterings to one plant (immediately before inoculation and

135
136 M. ULRYCHOVA-ZELINKOVi

the third day following inoculation) produced an average 70% inhibition,

without in any w a y injuring the plants themselves.
2. The inhibitory effect of cadmium ions decreased considerably when the
plants were watered with a cadmium ion solution 24 hours before inoculation.
3. The differences in the course of primary infection, as followed b y means
of iodine tests, were particularly apparent in the following respects: starch
is lost from the infected leaves of the experimental plants more slowly, the
development of chlorotic spots is slowed down and their delimitation and
structure are not so sharp.
4. On some leaves of young tobacco plants (N. tabacum Samsun) larger
doses of cadmium produce spots, which resemble the necrotic lesions on
leaves of N. glutinosa following infection with TMV.
5. Preliminary determination indicates that cadmium ions cause a dis-
turbance of phosphorus metabolism.
6. In the discussion it is suggested that the inhibitory effect of cadmium
ions on the reproduction of TMV may, analogically to the conception existing
in animal physiology, be connected with the interaction of zinc and cadmium
ions in nucleoprotein metabolism.

Introduction

The effects of many different substances on the reproduction of plant

viruses have been the subject of much work, which has been summarized for
instance b y MATHEWS and SMIT~ (1955). Recently the greatest attention has
been devoted to derivatives ofpurines and pyrimidines, which are also the only
substances for which the mechanism of the inhibitory effect has been explained
(MATHEWS and SMIT~ 1955). These derivatives block the biosynthesis of virus
nucleic acids b y becoming incorporated into the molecules in place of the
natural purines and pyrimidines.
Of the series of metal ions which have been tested as agents against plant
viruses (e. g. MATHEWS and SMITH 1955, RYZHKOV and MARCHENKO 1957),
the most significant inhibition was found with zinc (STODDARD1947, WEI-
TRAUB et. al. 1952, RuMEY and THOMAS 1951). The results are not, however,
quite consistent. YARWOOD (1953, 1954) came to the opposite conclusion and
demonstrated the stimulatory effect of zinc on the reproduction of tobacco
mosaic virus. This contradiction was explained b y H~.LMS and PounD's work
(1955); they found that zinc affects the reproduction of tobacco mosaic virus
in the same measure as the growth of the host. According to these authors,
increased doses of zinc increase virus accumulation insofar as they stimulate
the growth of the host.
The aim of the present work has been to find an effective chemotherapeutic
agent, the application of which would, at a later stage of this work, make it
possible to examine the mechanism of virus nucleoprotein biosynthesis. From
the start the use of purine and pyrimidine derivatives was excluded, since
these are substances the mode of action of which is known. On the basis of
 M. U L RYCH()V.~-Z E1,]NK()V.~

CAI)MIUM IONS AS INHIBITORS OF TOBACCO MOSAIC VIRUS

Fig. la Fig. Ib

Fig. 2a Fig. 2b
Course of p r i m a r y infection, followed by the iodine test, on leaves of' N . tabacum S a m s u n
after inoculation with Al-strain TMV and watering with c a d m i u m s u l p h a t e solution
(dose 160 rag. Cd ~ per plant) divided between two waterings: immediately before
inoculation and third day after inoculation.
Fig. 1. 3rd day after inoculation, a = control, b -- experiment.
Fig. 2. 4th day after inoculation, a = control, h = experiment.
 M. U L R Y C H O V ~ - Z E L I N K O V ~

CADMIUM IONS AS I N H I B I T O R S OF TOBACCO MOSAIC VIRUS

Fig. 3a Fig. 3b

Fig. 4
Fig. 3. 5th d a y after inoculation, a = control, b = e x p e r i m e n t .
Fig. 4. Necrotic s p o t s on leaves of y o u n g N. tabacum S a m s u n p l a n t s a f t e r w a t e r i n g w i t h
c a d m i u m s u l p h a t e solution (dose 320 m g . Cd 2+ p e r plant). T h e s p o t s r e s e m b l e lesions
on leaves of N. glutinosa a f t e r infection b y TMV.
 CADMIUM IONS AS I N H I B I T O R S OF TOBACCO MOSAIC V I R U S 137

considerations which will be mentioned in the discussion, the experimental

determination of the effect of cadmium ions on the reproduction of tobacco
mosaic virus was undertaken. So far as is known to the author, the effect of
this ion on the reproduction of plant viruses has not yet been studied.

Material and Methods

All experiments were carried out with tobacco plants (Nicotiana tabacum Samsun) at various
stages of growth, cultivated in pots in the glasshouse.
Cadmium ions were applied in the form of sulphate. The experimental plants were watered
with a solution of concentration 4 rag. Cd ~+ in 1 ml. Doses of cadmium were tested within the
range of 20 to 400 rag. Cd ~+ to one plant. Watering with the experimental solution was carried
out immediately and 24 hours before inoculation. I n some of the experimental series plants
were watered with the cadmium sulphate solution for a second time, on the third day following
inoculation.
The )A-strain of tobacco mosaic virus (BT,A~r~ 1955) was used for inoculation because of
its short incubation period. One leaf of each plant was inoculated, b y the usual technique with
carborundum. There were t w e n t y plants in each variant of the experiment. The experiments
were carried out from May to October 1958.
Following inoculation the course of primary infection was followed b y means of the iodine
test on starch (HoLM~,S 1931), in each variant on five leaves. The inoculated leaves were cut
off, killed by plunging into boiling water, chlorophyll was extracted from t h e m b y boiling in
96% alcohol and they were then submerged in a dilute solution of iodine in potassium iodide.
On about the seventh day after inoculation, when the youngest leaves of the control plants
began to show signs of systemic disease, the tips of the plants were cut off, homogenized and
the relative virus concentration was determined by testing on Nicotiana glutinosa and on
a hypersensitive hybrid1), which has been evolved b y Kv~ta Sehwammenh5ferovs
The content of free and labile phosphorus in tobacco leaves following watering with cadmium
sulphate solution was determined orientationally. Phosphorus was determined by the modified
method according to Po~s and GUT~RIE (1946) and ZELLER 0949). Plant homogenates were
deproteinized with 0.25 M triehloracetie acid.

Results

Figures 1 to 3 show the differences in the course of primary infection between

experimental and control plants from the June series. I t is clear from the
figures that cadmium ions delay the reproduction of tobacco mosaic virus.
The following facts are particularly striking: in comparison with the control
the leaves of experimental plants lose starch more slowly, the development
of chlorotic spots is slower and their delimitation and structure are not sharp.
The results of quantitative tests on N. glutinosa and the hybrid with
necrotic reaction are summarized in table 1. This table shows that the optimal
dose of cadmium ions, which has no injurious effect on the plants themselves,
is a dose of 160 mg. Cd 2+ to one plant, applied in two waterings, one ira-

1) Hybrid: Trapezond 161 8/37 (8/37--new selection V I ~ T P - - B~b pri Seredi). This
material was used for the quantitative testing of TMV for the first time. I t was subjected to
preliminary testing and results compared with those from _N'. glutlnosa.
138 M. ULBYCHOVA_-ZEL~NKOVA

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 C A D M I U M I O N S AS I N H I B I T O R S OF TOBACCO MOSAIC VIRUS 139

mediately before inoculation and the other on the third day after inoculation.
Inhibition of tobacco mosaic averaged 700/0. I t is interesting that the in-
hibitory effect of cadmium ions was found to decrease considerably when the
experimental plants were watered with cadmium sulphate solution 24 hours
before inoculation.
Larger doses of cadmium ions, about 250 mg. to one plant, inhibit growth
slightly. Naturally, a very important factor is the age of the plant. Following
watering with cadmium ion solution containing a 320 nag. dose, necrotic spots
(fig. 4) appear on the leaves of young tobacco plants (N. tabacum Samsun).
These resemble the necrotic lesions on the leaves of N. glutinosa following
inoculation with TMV.
The results also show that tobacco plants can stand a relatively high dose
of cadmium without injury if the solution is applied b y watering. The question
of the amount of cadmium ions that get into the plant remains open.
When the leaves are sprayed necrosis appears on them even with con-
centrations of 1.5 #g. Cd ~+ in 1 ml. This fact also did not permit direct proof
to be given that inhibition of the virus in the inoculum is not involved. The
results obtained while following the course of primary infection are regarded
as sufficient in this respect (fig. 1 to 3). The number of chlorotic spots on the
inoculated leaves of the experimental plants was the same as in the control.
In preliminary experiments changes in the content of inorganic and labile
phosporus in tobacco leaves following watering with cadmium sulphate solu-
tion were followed. Although the methodic aspect of these experiments is
very difficult, because the age of the plants must be taken into account
and watering does not allow of exact dosing with cadmium, these orienta-
tional experiments seem to indicate that the smaller doses of cadmium ions
increase the content of inorganic phosphorus; on the contrary, larger doses,
which delay plant growth, decrease the free phophorus content to nearly one
half. I t will be possible to arrive at a final conclusion on this question only
after a thorough analysis has been made.

Discussion

A number of investigations have been carried out on the effect of cadmium

on higher plants (e. g. SCHARRERand ScHRoPP 1934, SPENCER 1937, SCHARRER
1941). Most of these have been carried out from the point of view of plant
nutrition, but none of them deals with the physiological effects of this element.
All authors agree that cadmium ions are definitely more poisonous than zinc
ions.
PARiZEX (1956, 1957) found that cadmium is a very strong poison for male
gonads, i. e. organs with a very intensive nucleoprotein biosynthesis and with
relatively high zinc content. In the view of the above author, cadmium injures
the testes because it excludes zinc, which is linked to nueleoprotein metabolism
as a biogenic element. Following the application of cadmium, intensive de-
composition of nucleotides takes place and phophorus is freed and washed
out of the bodies of the experimental animals.
140 M. U L R Y C H O V . ~ - Z E L I N K O V f l -

Very little is know as yet about the biological function of zinc ions in plants
(ScK~R~ER 1941, CHESTERS and ROLI~SO~ 1951). The most typical symptoms
of zinc deficiency are disturbances in chlorophyll synthesis and dwarf growth.
Recently FuJII (1956) found t h a t some cells, e. g. the staminal hair cells of
Tradescantia, contain a relatively high content of zinc at the prophase. The
last-mentioned facts would seem to indicate t h a t zinc in plants is intimately
connected with growth and cell division and that it is most probably involved
in nucleoprotein metabolism, and therefore also in virus biosynthesis. This
assumption is finally supported by the work of ttV.LMS and POUND (1955),
as mentioned in the introduction. They show the dependence of growth of
the host and virus reproduction on dosing with zinc.
The results obtained so far from experiments on the inhibitory effect of
cadmium ions on TMV reproduction do not permit the author to draw any
conclusions regarding the mechanism of the inhibitory effect of these ions.
One of the possible hypotheses, which might be adopted, is an analogy with
the conception put forward in animal physiology that cadmium displaces
zinc, which is linked with nucleoprotein metabolism. The alternative cannot,
however, be excluded, i. e. that cadmium as a chemically related element can
to a certain extent compensate for or take the place of zinc.
In conclusion it m a y be said t h a t further study under more exact conditions
of the already established inhibitory effect of cadmium ions on the reproduc-
tion of tobacco mosaic virus could contribute not only to a deeper understand-
ing of the mechanism of the biosynthesis of virus nucleoproteins, but also to
the explanation of the physiological effect of cadmium ions and of the bio-
logical function of zinc ions.

Referenees

BLATTN~', C.: A l k e - k m e n v i r u oby6ejn6 tab~kov4 m o s a i k y (VTM). (Al-strain of t h e o r d i n a r y

tobacco mosaic virus.) - - Sbornik prs o t a b a k u . 231--264, 1955.
F u J I I , T. : Presence of zinc in nucleoli a n d its possible role in mitosis - - N a t u r e 174 : 1108--1109,
1956.
CHESTE~S, C. G. C. a n d RoLr~soN, G. N.: T h e role of zinc in p l a n t m e t a b o l i s m - - Biol. R e v s .
Cambridge Phil. Soc. 26 9 239--252, 1951.
HELMS, K . a n d POUND, G. S." Zinc n u t r i t i o n of N i c o t i a n a t ~ b a c u m L. in relation t o multiplica-
t i o n of t o b a c c o mosaic virus. - - Virology 1 9 408---423, 1955.
HOLMES, F. O.: Local lesion of mosaic in Nicotiana t a b a c u m L. - - Contrib. Boyce T h o m p s o n
I n s t . 3 : 165--172, 1931.
HRUB~, K.: Variabilita a korelace v biologii. (Variation a n d correlation in biology.) - - Roz-
p r a v y I I . t~. ~. Akad. (Praha) 66 (17) : 1--98, 1950.
MATHEWS, R . E . F . a n d S ~ T ~ , J. I).: The c h e m o t h e r a p y of viruses. A d v a n c e s Virus Res. 3 :
49--148, 1955.
PA~iZ~.X, J.: Effect of c a d m i u m salts o n testicular tissue. - - N a t u r e | 7 7 : 1036--1037, 1956.
PAI%iZEK, J . a n d Z~HO~, Z.: Effect of c a d m i u m salts on testicular tissue. - - N a t u r e 177 : 1036,
1956.
PA~fZEK, J." K a s t r a c e k a d m i e m . (Castration b y m e a n s of cadmium.) - - P r a h a 1957.
PoNs, W. A. a n d GU~rHlaIE, J . D.: D e t e r m i n a t i o n of inorganic p h o p h o r u s in p l a n t materials. - -
I n d . E n g . Chem., Anal. Ed. 18 : 184--186, 1946.
Ru~:EY, G. E. a n d THOYlAS, W. D.: The i n a c t i v a t i o n of the c a r n a t i o n mosaic virus. - - P h y t o -
p a t h o l o g y 41 : 301--303, 1951.
 C A D M I U M I O N S AS I N H I B I T O R S OF TOBACCO MOSAIC VIRUS 141

RYZHKOV, V. L. a n d MARSHENKO, N. K . : Vliyanie k a t i o n o v n e k o t o r y l d a m e t a l o v no, r a z n m o z h c n i e

v i r u s a m o z a y c h n o y b o l e z n i t a b a k a (VTM). [ T h e effect of c e r t a i n m e t a l c a t i o n s u p o n m u l t i -
p l i c a t i o n of t h e t o b a c c o m o s a i c virus.] - - Mikrobiologiya 26 : 3 8 0 - - 3 8 5 , 1957.
SCH~.~, K . : B i o c h e m i e d e r S p u r e n e l e m e n t e . - - B e r l i n 1941.
S e ~ A ~ E ~ , K . a n d SCHIZOid, W . : S a n d - u n d W a s s e r k u l t u r v e r s u c h e fiber die W i r k u n g d e s Zink-
u n d K a d m i u m - I o n s . - - Z t s c h r . 1)flErn/~hr. Diing. B o d e n k d e (A) 34 : 1 4 - - 2 9 , 1934.
S~ENCE~, E. L.: F r e n c h i n g of t o b a c c o a n d t h a l l i u r a t o x i c i t y . - - A m e r , J . B e t . 24 : 1 6 - - 2 4 , 1937.
STODDA_~D, E . M.: T h e X - d i s e a s e of p e a c h a n d i t s c h e m o t h e r a p y . Conn. Agr. E x p t . Sta. Bull.
506 : 1947. Cir. a f t e r Y a r d w o o d 1954.
UTECH, M. :N. a n d JOHNSOn, J . : T h e i n a c t i v a t i o n o f p l a n t v i r u s e s b y s u b s t a n c e s o b t a i n e d f r o m
b a c t e r i a a n d fungi. - - ~ h y t o p a t h o l o g y 40 : 2 4 7 - - 2 6 5 , 1950.
WEr~TX~A~rB, M., GXL~AT~t~CK, J . D. a n d WxI~SON, R . C.: T h e effect o f c e r t a i n w a t e r - s o l u b l e
c o m p o u n d s o n v i r u s infection. - - F h y t o p a t h o l o g y 42 : 4 1 7 - - 4 1 9 , 1952.
YA-~WOOD, C. E., HAL~, A. P . a n d NELSON, M. N.: N u t r i t i v e v a l u e of r u s t - i n f e c t e d leaves. - -
Science 117 : 3 2 6 - - 3 2 7 , 1953.
Y~WOOD, C. E.: Zinc i n c r e a s e s s u s c e p t i b i l i t y of b e a n leaves to t o b a c c o m o s a i c virus. - - P h y t o -
p a t h o l o g y 44 : 2 3 0 - - 2 3 3 , 1954.
ZE~LE~, E. A.: t~ber P h o s p h a t a s e n I. A p p a r a t z u r B e s t i m m u n g y o n a n o r g a n i s c h e n P h o s p h a t . - -
Helv. c h i m . A c t a 32 : 2 5 1 2 - - 2 5 1 9 , 1949.

Address: Dr. M a r i e U l r y c h o v s I n s t i t u t e of Biology o f t h e Czechoslovak 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 c e 1, P r a h a 6, Dejvice.

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