FACTORS EMUENCING SYMPTOM- EXPRESSION AND
MULTI‘PLICATEQN OF POTATO VIRUS X {N A.
POTATO VANETY HYPERSENSITWE TO YHE VERUS;

W» Eat the burn of PH. 52
MlthAN STATE UNIVERSITY
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Michigan State
University

    

This is to certify that the

thesis entitled
FACTORS INFLUENCING SYMPTOM EXPRESSION AND
MULTIPLICATION OF POTATO VIRUS X IN A
POTATO VARIETY HY PERSENS IT IVE
TO THE VIRUS

presented by
Hong-ji Su

has been accepted towards fulfillment
of the requirements for

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FACTORS INFLUENCING SYMPTOM EXPRESSION AND
MULTIPLICATION OF POTATO VIRUS X IN A
POTATO VARIETY HYPERSENSITIVE

TO THE VIRUS

by
Hong-ji Su
A Thesis submitted to the
Graduate Faculty in Partial Fulfillment of
the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major subject: Plant Pathology

Approved:

 

 

Guidance Committee:

5% AW rowan

 

%, j//. ”212/: «7/9.

 

/j/

Midhigan State University
1963

ABSTRACT

FACTORS INFLUENCING SYMPTOM EXPRESSION AND
MULTIPLICATION OF POTATO VIRUS X IN A
POTATO VARIETY HYPERSENSITIVE
TO THE VIRUS

by Hong-ji Su

This is a study of the environmental and physiological
factors influencing pathogenesis and multiplication of potato
virus (mix) in a hypersensitive potato variety. lpimre.

The Optimum post-inoculation tenpereture for disease
development was ISO—20°C, with considerable reduction in
severity of. systuic symptoms at 24°C. At 28°C local synptms
developed very slowly and systemic symptoms were precluded.
Disease severity of plants grown in continuous light at 16°-
24°C was generally higher than that or plants in 8 hour day.
At 28°C, disease severity was slightly greater in 8 hour day.

Pro-inoculation environment of 2 weeks at 28°C enhanced
susceptibility of plants grown at postaimculation tempera-
tures ranging from 16° to 28°C. A relatively short (2 days)
pro-inoculation treatment at 36°C was not effective in in-
creasing susceptibility. Susceptibility was decreased in
inoculated leaves treated within 30 minutes before or after
inoculation by a 60 second immersion in 50 c water.

Symptom severity and virus multiplication in inocul-
ated leaves were enhanced by growing plants in short photo-
periods especially at 20° or 28°C, and were retarded by

l

Hang-1i Bu
2
pre‘inoculetion incubation in continuous light especially at
15°c for 2 wears.

Symptom development and virus multiplication were
very rapid in old leaves. Infectivity of va in stem epi-
denaal tissue was higher in comparison with that in leaves
and maintained high levels of infectivity’cver a relatively
long period.

Virus infectivity in inoculated leaves was positiv~
sly correlated to symptom severity. Two types or virus
infectivity curves were obtained. i.e.. type A see an upward
curve flattened on top without or with a small downward drop
or infectivity, and type 3 curve was characterised by a rapid
rise and prompt decline in infectivity.

Type A curves were usually obtained in inoculated
leaves or plants kept at 16°C, or in young leaves, or stem
epidermal tissues. Type B curves were obtained in plants
kept at 20°C, in long days after inoculation, in leaves pro»
disposed at hdgh temperature (26°C). or predisposed.by short
days before inoculation, and in old leaves.

The re isolate of the virus caused slightly*more
rapid disease deveIOpmentuin_Epicure plants in comparison,
with I; isolate. although x8 isolate was a mild isolate on
some other potato varieties susceptible to PVX.

FACTORS INFLUENCING SYMPTOM EXPRESSION AND
MULTIPLICATION OF POTATO VIRUS X IN A
POTATO VARIETY HYPERSENSITIVE

TO THE VIRUS

BY

Hong—ji Su

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1963

 

a
pal;
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‘..-I~I

ACKNOWLEDGMENT

The writer wiShes to eXpress his sincere gratitude

to Dr. W. J. Hooker for his kind and careful direction and
encouragement during the course of this investigation and
his aid in preparation of this manuscript.

Thanks are also due to Dr. D. J. deZeeuw. Dr. H. H.
Murakishi, Dr. N. R. Thompson, and Dr. E. H. Barnes for

their kind guidance throughout the program.

IV.
v.

VI.

TABLE OF CONTENTS

INTRODUCTION AND LITERATURE REVIEW . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . .
EXPERIMENTAL RESULTS . . . . . . . . . . . . . .
1. Influence of Post-inoculation Environment

on Symptom Expression and Infective Virus

Content . . . . . . . . . . . . . . . . . .
2. Influence of Pro-inoculation Environment

on Symptom EXpression and Infective Virus

Content . . . . . . . . . . . . . . . . . .
3. Influence of Hot-water Treatment on

Symptom Expression . . . . . . . . . . . . .
4. Relation of Leaf Maturity on Disease

Severity and Infective Virus Content . . . .
5. Multiplication of PVX in Stem Epidermis

of Epicure Plants . . . . . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . . .

L ITEPATURE CITED 0 O O O O O O O O O O O O O O O

Page

11

ll

15

22

24

28

32

39
42

LIST OF FIGURES

Page

FIGURE

1.

A-D, Disease indices of inoculated Epicure

plants grown in an 8 hour day and in continuous
light at 4 different temperatures. E-H,

Relative infectivity on half leaves of g, globosa

in extracts from inoculated leaves of Epicure

plants grown under various photOperiods and
temperatures . . . . . . . . . . . . . . . . . . l2

A-C, Disease indices on plants predisposed

under different pre-inoculation temperatures.

D-F, Relative virus infectivity in inoculated

leaves of plants mentioned above . . . . . . . . l7

A-C, Disease indices of Epicure plants pre-

disposed under various pre-inoculation temper—
atures and photOperiods and grown at 22° C in a
glass house: D-F, relative virus infectivity

in inoculated leaves of plants mentioned above. . 20

A. Disease indices and B, relative virus
infectivity in Epicure plants predisposed at
36° and 200C and grown in 220 C glass house. . . 23

A. Disease indices on plants inoculated on

leaves heated in 50°C water for 60 seconds at
various time intervals either before, or B,

after inoculation . . .. . . . . . . . . . . . . 25

Susceptibility of leaves of different maturity

as measured by A, local symptom deve10pment and

B, by virus infectivity, and C, by local and
systemic symptoms . . . . . . . . . . . . . . . . 27

A. Relative infectivity of virus in stem

epidermal strips (l-SO dilution) or in leaves

(1—5 dilution), B, disease indices of plants
inoculated either on leaf or on stem surface. . . 30

I. INTRODUCTION AND LITERATURE REVIEW

Degreesof resistance to potato virus X differ widely
among the different potato varieties. The literature on the
subject has been reviewed.(Hooker g§_§l. 1954). Three rather
well-defined types of resistance have been identified as
susceptible or tolerant, hypersensitive or field immune, and
immune. Susceptibility or tolerance is characterized by the
plants being infected as more or less symptomless carriers,
and yet appearing to be healthy. Most commercial varieties
presently grown in the United States are tolerant to PVX.
These include Cobbler, Sebago, Katahdin, Green Mounéan, and
Triumph.

Potato varieties of hypersensitive type reapond to
inoculation by necrosis of the inoculated leaves and usually
infected plants develOp tap necrosis. In this type, resist-
ance is associated with extreme susceptibility or hyper-
sensitivity to the virus. This reaction is manifest either
as localized, necrotic lesions on inoculated leaves in whiCh
case the virus may not become systemic, or as systemic top
necrosis following inoculation either through leaves or with
X-infected scions. Only occasionally are diseased plants
observed.among the plants of this type in the field. Certain
varieties necrotic to PVX are Epicure, Arran Crest, King

Edward, Edgecote Purple, Ninetyfold, and Southesk.

1

The virus fails to multiply and survive in potato
plants of immune type. Immune types in this country have
been derived from S. 41956 which has not yet been typically
infected with any isolate of PVX. Following graft inocula-
tion of S. 41956, PVX'has been obtained apparently in very
low concentration from stems, roots (Benson and Hooker, 1960)
and leaves (Bagnell, 1961). Certain collections of an un-
cultivated potato Species, Solanum acaule Blossfeld, La Pena
blanca, and Bukasor, contain individuals immune to PVX.

Little work has been done on the nature of the hyper-
sensitivity or localized reaction of potato plant to PVX.

The top necrosis of potato due to viruses was first described
by Quanjer and Botjes (1929). Bawden (l936) made extensive
studies on top necrosis (acronecrosis) of potato plants caused
by viruses and stated that PVX and some other viruses induced
tap necrosis in the Epicure variety of potato. Cadman (1942)
showed that hypersensitivity for PVX in potato varieties was
inherited as a single dominant gene (Nx). Cocherham (1943b)
investigated the reaction of potato varieties to virus X,.A,
B, and C and considered hypersensitivity as "field immunity"
because under field conditions of infection with virus X,
potato varieties and seedlings with a localized reaction re-
mained free of this virus. He (1943a) also reported that

the hypersensitive reaction of potato plants to strains of
PVX, designated as either X3 or X? was governed by a single

pair of dominant genes (Nx and Nb), and emphasized the

particular value of variety stocks resPonding to PVX with
t0p necrosis in seeking control of PVX. {More recently,
Bagnall (1961) described the specific genetic nature of the
hypersensitive type of resistance to viruses A and x in
Canadian and American potato varieties. His data agree with
those of Cocherham that the reaction of varieties to virus
A and the 2 groups of PVX isolates (XX and Xb) was governed
in eaCh case by single allelic pairs of genes (Na, Nx, Nb).

Hutton and Wark (1952) discussed the inheritance of
resistance to PVX by the pattern of PVX development in inocul-
ated leaves of immune, localized reaction, and susceptible
phenotypes in potato. They indicated that the phenotypic
reaction of Epicure to PVX, giving a localized reaction,
could not be regarded as hypersensitive, the term resistance
should take its place. They further preposed that a common
virus-inactivation system determined resistance of both the
hypersensitive and the immune types. Kohler (1958) studied
the slope of the inactivation curve of HVX in inoculated
potato leaves of tolerant, hypersensitive, and immune types.
He used the data of Hutton and Wark (1952) for the interpre-
tation of the hypersensitive response. He believed that a
common virus-inactivating system determined the immunity of
S. 41956 and the localized resistance of certain other
potato varieties, such as Epicure.

The importance of air temperature in relation to

development of symptoms of PVX in potato was first recognized

4

by Jehnson (1922). He found Optimum temperature for the
disease development to be between 140 and 18°C. The reSponse
was further investigated by Tompkins (1926) and by Timian 33
a1. (1955). Pound and.HeLms (1955) showed that the vari-
ation in symptom expression of PVX in Nicotiana species at
different temperatures was correlated with Changes in virus
concentration. Multiplication of virus X was influenced by
host varieties as well as by temperature and season vari-
ation.

The extent of the stimulatory effect PVY infection
on P X multiplication varied with temperature (Stouffer and
Ross, 1961a). Concentration in both singly (PVX) and doubly
infected leaves (PVX and PVY) was higher at 190 than at 28°,
and higher at 28° than at 32°C. Ford and Ross (1962a) obtained
4.7 to 6.6 times as much infective PVX at 30°C, and 2.2 to
3.4 times at 20°C as mudh infective PVX in doubly (PVX and
PVY) infected leaves of Samsun NN tobacco as in the leaves
infected with PVX alone. Other factors also influenced the
concentration of PVX in doubly infected plants (Stouffer and
Ross, 1961b).

Ford and Ross (1962b) found that age of tobacco leaf
had no marked effect on the level of PVX concentration. In
leaves inoculated with both PVX and PVY, however, PVX con—

centration was higher in leaves 5 cm wide than in those that

were either 2 or 8 cm wide.

5

Mature plant resistance to PVX was demonstrated in
certain potato varieties such as Flava and COpella by Berks
(1951), and Bintje and Voran by Beemster (1957). It is pos-
sible that additional other physiological conditions influ-
ence the vaeEpicure interaction.

Except for the work of Hutton and Wark (1952), the
course of infection and subsequent localization of the virus
and death in hypersensitive potato varieties has received
little attention. It becomes important to determine the
degree of compatibility with the host-virus interaction under
different environmental conditions before a common virus in-
activating system as prOposed by Hutton and Wank (1952) can
be accepted. Information concerning the reaction of tolerant
potato plants to PVX under different environmental conditions
is available (Hooker and Kim, unpublished data). Numerous
attempts to infect immune S. 41956 and to recover PVX from
inoculated plants have failed to demonstrate virus multipli-
cation within the tissues (Kohler, 1958, Benson and Hooker,
1960, Bagnall, 1961). '

Kohler (1958) presented 3 different virus multipli-
cation curves in potato plants of the 3 resistance types,
i.e. the virus in immune potato plants was unable to multiply
and was inactivated very quickly: in susceptible varieties the

virus multiplied rapidly and virus titre was maintained at a

relatively high level over the period of observation, While

in hypersensitive varieties, the virus multiplied but declined

rapidly.

6

In preliminary trials with the Epicure variety, virus
infectivity curves were Obtained which were quite distinct
from those of Hutton and Wark (1952). Because of this dis-
crepancy the present investigation was made in order to
clarify the factors influencing pathogensis and multiplica-
tion of the virus in plants with the hypersensitive type of

resistance.

 

 

II. MATERIALS AND METHODS

The potato variety, Epicure, hypersensitive to the
virus Xx types of PVX (Cockerham, 1943), but tolerant to
isolates of the X? group, was used as a test plant in these
trials. Potato plants from tuber units were generally grown
in a green house at 22°C, although the green house tempera-
ture in summer could not be controlled. Plants were inoculated
on the lower 3-5 fully expanded leaves when the potato plants
were about 25 cm in height, 3-4 weeks old. Plants were grown
at pre-inoculation temperatures in prediSposition studies
10-14 days before inoculation.

Stock isolates of PVX, X and X8 (Timian 35 al. 1955a),

5
tested for freedom of XL by grafting to Katahdin, were passed
serially through the local lesion host Gomphrena globosa.L.,
and used as inocula. Stock cultures of the virus X isolates
were maintained in plants of Datura tatula L. Both isolates
were equally virulent on the Epicure variety of potato but
on 2. tatula isolate X5 produced severe symptoms of necrosis
and mottle while isolate X8 was symptomless. For the inoculum,
systemically infected leaves of 2. tatula were ground and the
juice diluted with water was used.

Potato leaves were dusted with 400emeSh carborundum
and inoculated with juice using a glass spatula. Inoculated

leaves were thoroughly rinsed with water following inoculation.

The inoculated plants were kept in separate houses

0

thermostatically controlled at 17°, 22 , and 28°C, under

normal winter illumination of 3,000 to 5,000 F.C. in bright

7

8

days. Plants were also grown in thermostatically controlled
temperature boxes set at 16°, 20°, 24°, and 28°C, with.con-
tinuous fluorescent artificial light totalling approximately
800 F.C. Short photOperiods (8 hour day) were accomplished
by placing a metal box over the plants. Temperatures inside
the boxes were the same as those of the incubator.

Disease records on the test plants kept under various
conditions were determined at 2 day intervals and the disease

index was calculated by the following method:

local symptoms

1 necrotic spots on inoculated leaves

2 severe necrotic Spots or/and vein necrosis
3 leaf yellowing

4 death of inoculated leaves

systemic symptoms
necrotic Spots on tOp young leaves

5
6 moderate tOp necrosis
7 severe top necrosis

8

death of top

Later separate disease indices were prepared, one for local
and one for systemic symptoms.

For determining relative virus infectivity in the
inoculated leaves at each.harvest date, leaf discs were

removed from each inoculated leaf with a 4 mm cork borer.

9

The collected leaf discs were placed in small vials contain~
ing 0.3 ml of phosphate buffer solution (0.1 M KH2P04
adjusted to pH 7 with 0.1 M NaOH) and stored in a deep
freeze. Sample weights were obtained by weighing these
vials before and after addition of leaf samples.

For assay, samples were then ground in a mortar and
diluted 1:5 with 0.1 M pH 7.0 phosphate buffer solution and
the homogenate was refrozen. This was thawed, and clari-
fied by: l) centrifuging at 12,800 g for 15 minutes:

2) heating the supernatant at 55°C in a water bath for 10
minutes; and 3) centrifuging as above and saving the super-
natant. These samples were stored in the refrigerator and
tested on g. globosa later the same day. One sample was
used at the 1-5 dilution and a second sample tested after
1-50 dilution by the 2-dilution method of Spencer and Price
(1943).

The uppermost pair of fully expanded leaves of
Gomphrena globosa.L. plants grown under uniform conditions
and selected for uniformity were used for assaying virus
infectivity. Thus on a given harvest date, all samples
were compared on a 2 dilution basis using the randomization
plan of Youden for half leaves (1937). Local lesions
were counted 5 days after inoculation. Relative infectivity

in discs of inoculated leaves collected at several intervals

10

of time were expressed by plotting logarithms (n+1) of
average lesion number (n) per half leaf, against time after

inoculation.

 

III. EXPERIMENTAL RESULTS
Environmental Factors
1. Influence of Post-inoculation Environment on Symptom

Expression and Infective Virus Content

Epicure plants inoculated in one test with X5 and

in another test with X8 were maintained at different temp-

O O
eratures l6 , 20°, 24 , and 28°C, and grown under Short day

(8 hours light and 16 hours dark) or in continuous light. Six
potted plants were used in each treatment, thus 48 plants
were used in each test. Plants were observed periodically
and the disease index was calculated as previously described.

Temperature had a greater influence on symptom
expression than did differences in length of eXposure to
light (Fig. l, A-D). Most rapid disease development occurred
in the plants kept at 20°C and symptoms appeared a little
earlier than in plants kept at the other temperatures.
Plants at 160 showed almost as severe symptoms as plants at
2000. At 240C, there was considerable reduction in severity
of systemic symptoms while local symptoms developed somewhat
slower than on plants at 160C. At 28°C, no systemic symptoms
develOped and disease develOped in severity on inoculated
leaves slower than at lower temperatures.

Disease indices of plants grown at 16°C, under con-

tinuous light were higher than those of the plants under

snort photOperiods. These differences were similar but less

11

 

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Day. after inoculation
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Days after inoculation Days after inoculation

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Days after inoculation

12 16 20 24 28 32 .6

Days after inoculation

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4 8 12 16 20

Days after inoculation

Fig. 1. AvD, Disease indices of inoculated Epicure plants
grown in an 8 hour day and in continuous light at 4 different
temperatures. EéH, Relative infectivity on half leaves of Q.

globosa in extracts from inoculated leaves of Epicure plants
grown under various photOperiods and temperatures.

13

pronounced at 20°C. At 24°C, early disease readings were
slightly more severe in continuous light than in the 8 hours
day but after approximately 12 days, disease was more severe
in the plants grown in 8 hour days. At 28cc, higher disease
indices were obtained in the 8 hour days than in continuous
light.

Generally, disease develOpment on the plants inoculated
wdth X8 isolate of PVX was slightly more rapid than that of

plants inoculated with X isolate. Differences between

5
these two isolates were greatest especially at temperatures
not Optimum for symptom develOpment. At the Optimum temper—
ature for symptom eXpression, disease severity with two
virus isolates were essentially similar (Fig. l, A-D).
Increase in virus infectivity in inoculated leaves
Of potato plants grown under different temperatures and
photo-periods was determined quantitatively as previously
described. Six potted plants were used in each treatment
and so 48 plants were used in each test. ~The leaf discs
were collected periodically. On each harvest date about 200
leaf discs were removed from the plants in each treatment
group. Relative infectivity was expressed in logarithms
(n+1) Of the average number of local lesions (n) on 7 half-
leaves Of g. qlobosa (Fig. l, EAH). Infectivity was deter-
mined by the 2-dilution method (Spencer and Price, 1943) at
1-5 and 1-50 dilutions. Since graphs were essentially simi-
lar at either dilution but infectivity was low at the 1-50

dilution, only data derived from 1-5 dilution are presented.

14
There was no evidence that at the 1-5 dilution, infectivity
had exceeded the level Of sensitivity Of the indicator
plant leaves.

NO infectivity was Obtained from leaves one day after
inoculation, but in a few instances infective virus was re-
covered two days after inoculation.

Essentially similar high levels of virus infectivity
develOped in inoculated leaves of plants at 16°, and 20°C,
and the initial rise Of the curves were quite similar at
both temperatures. Low levels of infectivity were Obtained
in inoculated leaves at 24°, and 28°C. Infectivity at 24°C
reached a low peak at about the same time, 4-10 days,as that
at 160 or 20°C. At 28°C, X5 failed to become infective and

infectivity of X slowly declined from a low level Of infect-

8
ivity.

After the peak had been reached, infectivity drOpped
rapidly at 29°C under both continuous light and Short day,
although the loss of infectivity with isolate X5 was more
rapid under long days and the loss of infectivity'wdth X8 was
more rapid under short days. At 24°C, loss Of infectivity
was relatively slow from the initial low peak, and the infect—
ivity was lower in short days than in long days. Relatively
little loss Of infectivity was Obtained in inoculated leaves
at 16°C in short days and the level of infectivity ranained
quite constant throughout the 20 days period of Observation.

This contrasts with the drOp in infectivity in continuous

light at 16°C.

15

Curves Of both virus isolates were essentially simi—
lar although infectivity Of X8 was consistently higher than
that Of X5. Higher infectivity was usually demonstrated dur-
ing the initial rise in inoculated leaves Of plants kept
under long photOperiOds than in plants in short photOperiOds.
Loss Of infectivity was more rapid at 16°C in continuous
light than in 8 hours day. At higher temperature, inactiva-
tion was somewhat more rapid in continuous light.

Severity of symptom develOpment was positively
correlated with virus infectivity in inoculated leaves. Both
were highest at 160 and 20°C, intermediate at 240 and lowest
28°C. At 16°C, both disease severity and the initial rise
in virus infectivity were slightly higher under continuous
light than in the 8 hour day. At 20°C, differences in
disease index and virus infectivity at the two photOperiOds
were less pronounced than at 16°C. In the early portion of

the Observation period at 240C, both infectivity and disease

index were higher in continuous light.

2. Influence Of Pre-inoculation Environment on Symptom

Expression and Infective Virus Content.

The influence Of pre—inoculation temperatures on
predisposition of Epicure plants to PVX and virus multiplica-

tion within such plants was determined at different post-

inoculation temperatures. In three preliminary trials, two

groups Of uniform and healthy plants were grown in 170 and

16
28°C glass houses respectively for 2 weeks. Then all of the

plants were inoculated with PVX on the fourth and the fifth

5
leaves from the tOp of every plant. Oneehalf of the 8 plants
in eaCh group were kept at 28°C and the other half Of the
plants were kept at 17°C after inoculation. Disease develOp-
ment was Observed periodically. Data from these 3 prelimin-
ary trials were in agreement and were similar to results
from an additional more extensive trial (Fig. 2).
Approximately 3—week-Old plants were uniformly

0, and 28°C

divided into 3 groups and were grown in 17°, 22
glass houses respectively for 2 weeks. Eadh Of 5 well-
developed leaves of these plants was inoculated with PVXB.
Then the plants Of each group were separated into 3 groups

Of similar plants and were kept at the 3 different tempera-
tures reSpectively. The disease develOpment was Observed
periodically and leaf discs were collected from inoculated
leaves at time intervals Of 4, 8, 12, 16, and 20 days after
inoculation. Relative virus concentration in these leaf
samples was detenmined by assay on 5 half leaves of g. globosa
as previously described.

Similar results were obtained in the 4 preliminary
trials. Symptom expression and virus multiplication in
inoculated leaves were influenced by post-inoculation tempera-
tures similarly to those shown in Fig. 1. The Optimum tempera—

ture for local and systemic symptom development was 22°C.

Both symptom types develOped slightly later at 17°C. At 28°C,

Dieeaee index

P‘.

U

-

N
I

dyeteeic invaeion
a—e

‘

 

17

11° C.P°It-inoc. tenp. B. 22° C.p0It-1n°¢- telp. C- 230 C' p°"'m°c' “'9‘
a--— 173 c, pre-ingc. teen.

0' ..
20C. "

 

 

 

N

 

Local invaeion
_ H

Log relative infectivity

 

 

 

 

 

 

 

 

 

 

 

 

A

 

5 10 15 20 5 1o 15 20 5 10 15 20
Days after inoculation Days after inoculation Days after inoculation

Fig. 2. A-C, Disease indices on plants predisposed under

different pro-inoculation temperatures. D—F, Relative virus
infectivity in inoculated leaves of plants mentioned above.

18
local symptoms were retarded and systemic symptoms almost
precluded.

Pre-inoculation temperatures modified symptom expres-
sion in inoculated leaves at any post-inoculation temperature
(Fig. 2, A-C). Local symptom development was increased at
each post-inoculation temperature by high temperature (28°C)
incubation before inoculation, and was decreased by pre-
inoculation incubation at 17°C at eaCh Of the 3 post-inocula-
tion temperatures. Plants grown at 22°C were intermediate
and those grown at 17°C were most retarded in symptom
expression. Systemic invasion was not consistently influenced
by pre-inoculation temperatures. This may have been due in
part to recovery from the influence of the predisPosition
environment during the 12 days required for develOpment Of
systemic symptoms following inoculation and transfer to the
post-inoculation environment.

Infectivity Of virus from the treated plants (Fig. 2,
D-F) kept at these 3 post-inoculation temperatures were in
essential agreement with those previously presented. The
influence Of pre-inoculation temperatures on virus infect-
ivity on potato plants was marked. The virus multiplied
more rapidly, readhed a peak more quidkly and declined.more
rapidly in the leaves Of the plants grown at 28°C, than
within groups Of plants kept at any temperature after inocula-
tion. This was the only treatment in whidh inactivation Of

the virus in inoculated leaves was rapid and pronounced.

19

In contrast, virus multiplied slower in the leaves of plants
grown at 17°C before inoculation, and virus concentration
continued to increase at all post-inoculation temperatures
up to the end of observation period. Virus multiplication

in inoculated leaves of plants prediSposed at 22°C was inter-
mediate between that of plants predisposed at temperatures

of 16° and 28°C.

To determine the prediSposing influence of length of
time of illumination, 9 uniform Epicure plants were grown
under 8 hour illumination or continuous light at 16°, 20°,
or 28°C. After 2 weeks, these treated plants were inoculated

on 5 well-expanded leaves with PVX and then all of the

8'
plants were kept in a 22°C glass house, about 10 hours day.
Disease indices were determined periodically by symptom
severity in inoculated leaves and by systemic symptoms. Leaf
discs were collected for virus infectivity measurement from
inoculated leaves at intervals of 3, 6, lO, and 20 days after
inoculation and the relative infectivity of virus was deter-
mined as previously described (Fig. 3).

The influence of pre-inoculation temperatures on
symptom eXpression (Fig. 3, A-C) at post-inoculation temp-
erature of 22°C, and virus concentration was similar to that

observed earlier. Symptom severity was highest in inoculated

leaves of plants grown in an 8 hours day at 28°C, and 20°C

were essentially similar, and was retarded at 16°C pre-

inoculation temperature. Continuous light at all temperatures

20

‘ I-
A. 16° C, pro-lace. tap. 8. 20° C, pre-inoc. to». C, 28° C, pre-tnoc. temp.

   
 
   

""" I hour light.pre-inoc
— conti. light, "

 

3
3 : 18: C. prczlnoc.
s 0 3° g. "
.
u~2
3
0
CI.
8b
“
.
Us
a

 

 

Dina-o index
.

 

 

 

 

 

 

 

 

     

 

 

8
v.
2%
>
a
v.
H
a
§2
1
2 P
D
l
E f
2‘ I‘
5 I
E l
o l
)1 p ‘
I: I
2 I
3 {
§ l
l
l
l
l
l
l
0 A A A l_‘ A A A fie v A
5 10 15 20 5 10 15 2O 5 10 15 20
Days alter inoculalinn Days after inoculation

Day:. 3! [er mncu lat rm

Fig. 3. A=C, Disease indices of Epicure plants predis-
posed under various pre-inoculation temperatures and photo-
periods and grown at 22° C in a glass house: D-F, relat ve
virus infectivity in inoculated leaves of plants mentioned

above.

21

before inoculation retarded symptoms in inoculated leaves and
slightly enhanced symptom severity in systemically invaded
leaves at 16° and 20°C pre-treatment. Plants predisposed at
28°C in either day length develOped less severe systemic
symptoms than plants predisposed at 16° or 20°C (Fig. 3, A-C).

Infectivity of leaf disc homogenates (Fig. 3, D-F)
from these plants was similar to those in previous trials.
The level of infectivity remained relatively constant in
plants predisposed at 16° and 20°C in continuous light and at
16°C in an 8 hours day. Loss of infectivity was most rapid
When plants were predisposed in short days at either 20° or
28°C, and was delayed but pronounced with long day 28°C
pre-treatment.

Six 4 week-old uniformly selected plants grown in a
22°C glass house were pre-treated at either 20° or 36°C in
continuous light for 2 days before inoculation, and inoculated
with isolate X5 (Fig. 4). Then the plants were transferred
to a 22°C glass house. Disease indices were calculated and
leaf discs were collected for infectivity assay at time
intervals of 2, 3, 5, 8, 12, 16 and 20 days after inoculation.
Local symptoms develOped more quidkly on inoculated leaves of
plants pre-inoculation treated at 36°C than at 20°C, while
severity of systemic symptoms was retarded by the high temp-
erature pre-treatment. Virus infectivity increased someWhat
more rapidly in the inoculated leaves of plants prediSposed

at 36°C than in the control plants. Differences in this

22
trial were relatively small due possibly to the short pre-
treatment period. Since potato plants tolerate high temper-
ature poorly, a longer pre-treatment period did not seem

advisable.

3. Influence of Hot-water Treatment on Symptom Expression.

The influence of hot-water treatment of inoculated
leaves on the pathoqenesis of PVX was determined in 2 trials.
In one preliminary test, uniform Epicure plants were
separated into 2 groups. The 6 plants of one group were
inoculated on the leaves soon after heating in 50°C water
for 60 seconds, and the other 6 plants inoculated on the non-
heated leaves with PVX5 were served as control. All of
these plants were kept in a 22°C glass house under sun
light in winter. The disease index of the plants inoculated
soon after heating was lower than that of the control plants.
The systemic symptoms occurred on the plants inoculated on
non-heated leaves as early as 12 days after inoculation,
While plants with inoculated heated leaves develOped systemic
spots on the upper young leaves at least 2 days later.

In an additional test (Fig. 5) uniform plants were
divided into 7 groups of 6 plants each and PVX5 was used as
inoculum. The fifth and sixth leaves from the tOp of eadh
plant were heated similarly either before or after inocula—
tion at the following time intervals: 1) heated and
inoculated as soon as possible after heating, 2) heated and

then inoculated 30 minutes later, 3) heated and inoculated

23

 

 

 

 

 

4
Mg. 4. A
'—— 30: C. pre-inoc. telp.

3 t . C, "

3 2

--a

=

E“ 4

2 ,x‘
~51 ’ // :‘
8: ° :
an 3
s ‘
§= .
a; El

(3 1

E 2

.4 g

3:» *

 

’-
fl

 

 

 

 

 

 

 

5 10 15 g ‘0 1‘5
Days liter inoculntion Days aner muculnrmn

Fig. 4. A. Disease indices and B, relative virus
infectivity in Epicure plants predisposed at 360 and 200 C
and grown in 220 C glass house.

24
15 hours later, 4) inoculated and heated at once, 5) inoculated

and heated 30 minutes later, 6) inoculated and heated 15
hours later, and 7) inoculated control, no heat treatment.
All of the inoculated plants were kept in 22°C glass room in
early Spring.

Plants inoculated on leaves heated before or after
inoculation develOped relatively low disease indices in
comparison with the control plants inoculated without heating
or when heating preceded or followed inoculation by 15 hours
(Fig. 5). Pre—inoculation heat treatment was slightly more
effective in reducing disease indices than was post-inocula-
tion treatment. Slightly lower indices were obtained on the
plants inoculated on the leaves 15 hours after heating in

comparison with those unheated plants.

4. Relation of Leaf Maturity on Disease Severity and Infec-

tive Virus Content.

In earlier trials there was some evidence that PVX
induced severe and rapid disease develOpment and multiplied
more rapidly in the old leaves than in the young leaves.

Ten Epicure plants of equal maturity were used in eaCh of 2
trials. In one trial, 7-week-old plants were inoculated on
4 young leaves (approximately 2—week-old) and 4 old leaves
(approximately 6—week—old) with X5 and in another trial, 10-
week-old plants were inoculated on 5 young leaves, approx-

imately 2 weeks old, and 5 old leaves, approximately 8 weeks

25

 

Fig. 5.1 Pre-inoc .henteMSOOC)

A--- 0 ninuten
.—— 30 ninuten
._._ 15 hours
’ ._ non-heated

   
   

Diunu index
b

N
T

 

 

 

 

 

A A A

A A A

5 10 15 20 25 30 5 10 15 20 25 :50
Day. after inoculation Days after inoculation

 

Fig. 5. A. Disease indices on plants inoculated on
leaves heated in 500 C water for 60 seconds at various time
intervals either before, or B, after inoculation.

26

old, with X8. All of the inoculated plants were kept in a
22°C glass house. Disease develOpment on both old and
young inoculated leaves was observed and leaf discs were
also collected periodically.

Similar results were obtained in each test involving

the 2 virus isolates, X and X8 (Fig. 6, A). Old leaves

5
inoculated with X5 became yellow on the fifth day and died

7 days after inoculation. Infectivity of the virus in
these leaves reached a peak on the third day, declined very
quickly and was inactive by the eighth day after inocula-
tion. The symptoms on inoculated young leaves develOped
more gradually, and leaves were still alive by the end of
observation period. The virus in young leaves (Fig. 6, B)
multiplied more slowly than in old leaves, and reached
highest infectivity on the eighth day. Infectivity remained
high throughout the observation period. In the trial with

X essentially similar results were obtained. In another

8!

trial with X curves of virus infectivity in inoculated

5:
leaves grown at 22°C in the glass house were similar to
those of Fig. 6, B. However, the drOp in infectivity in
the old leaves was not as rapid because in the previous
test (Fig. 6, B) 6-8 week-old mature leaves had been used

and, in the present test, 3-4 week-old leaves were used as

mature leaves.

27

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2
4 .V: .A
l //r E
‘5. / g
:‘3 ’ V C
. / ,r"‘fl 7“
'2 45/0 ' El
35 1/ =
. j? E
“R / ' 1
z 3
'g 4 old leaf,xb
" °—'- old leaf.XB
81) t—— young leaf,xs
: .--— young 1mm,
3.
c:
A A o
5 10 15
Day: after inoculation Dh>s filler Innculatron
Fig.6.C
n-—-—— inoculated on old leaves
3, .__ inoculated on young leaves‘,
1’
n
Efll
u
n
>.
a
3
I ll»
1:0
on
can
W h
a:
o
a
E
35 A //‘>
H
/
2"‘°’
: . / ‘
,.. / .
I
3 ./
.J / e
7/
15 20

 

 

5
Days after inoculation

Fig. 6. Susceptibility of leaves of different maturity
as measured by A, local symptom develOpment and B, by virus
infectivity, and C, by local and systemic symptoms.

i i
;_ .. _ . .. I : -,
‘ '1 r 7 3.4.: I a .
t . ° I
‘ ' . - 5
n . l l . '
1| '

 

28

An additional test was made in order to confirm
whether systemic infection was affected by infection
through leaves of different maturities (Fig. 6, C). Uni-
form 5-week-old plants were inoculated with X5 on 2 upper-
most well-expanded young leaves, approximately 2 weeks old,
and other 5 plants were inoculated on 2 lower leaves
approximately 4 weeks old. Local symptoms developed more
rapidly on the inoculated old leaves than on young leaves,
while systemic symptoms develOped more slowly on plants

inoculated on old leaves.

5. Multiplication of PVX in Stem Epidermis of Epicure

Plants.

Assay of stem epidermis was attempted because this
tissue strips with relative ease and a relatively high per-
centage of inoculated cells were thus available for assay.
About 40 plants uniformly selected were used in eadh of 5
trials. The mature portion of the stem was inoculated by
rubbing carborundum dusted on the surface of the stem with a
cotton ball saturated with virus suSpension. Stems were thorough-
ly rinsed with water after inoculation. All of the plants were
kept in a 22°C glass house under sun light condition in fall

and winter. The stem epidermis of inoculated portions of

29
each 3 plants was stripped off periodically, weighed and

frozen in a small vial. Such strips separated from the stem
between the collendhyma layer and parendhyma layer, and thus
they included epidermis, a layer of chlorendhyma (green
parenchyma), and 2—3 layers of collenchyma tissue. Because
virus infectivity in epidermal strips appeared to be too
high for assay by the 2-dilution method, a 3—dilution system
1-5, 1-50, and 1—500 was used. However, the curves of the
1-50 dilution was satisfactory for the assay (Fig. 7, A).
All of the results in 5 similar trials involving

either X or X8 were essentially similar. For comparing the

5
virus infectivity in epidermal tissue with that in leaf
tissue, data at 1—5 dilution derived from a test with X8 in
leaves in a 22°C glass house under sun light condition in
fall are presented. Infection of PVX in stem epidermal
tissue was established and the virus was recovered on the
second day after inoculation. Infectivity rose considerably
higher on the fourth day and reached a peak about 10 days
after inoculation. Infectivity of virus in stem epidermal
tissues remained high for relatiVely long periods, although
the infectivity of virus in leaves was lost rapidly under the
same environmental conditions. Virus infectivity was still
detected in the strips collected on the thirtieth day. The
infectivity of virus in stem strips at 1-50 dilution showed

the curves similar to those shown by the virus infectivity

in leaves at 1-5 dilution. It seemed that the virus in stem

Fig. 7.

stem surface.

‘-

 

lsolale X5

Lo; rvialivv lnfv«(1v11
p-a
V

[a'llalt‘ Kb

 

‘— Stem thpld-‘l‘lflln

I—°- Stem epiderm-

  

 

 

0

Jr A A
5 10 15
Da)u alter lnnculntlnu

2O

 

U

P

h)
v

Disease index of systeaic nyaptoas

 

 

Fig.7.B

A--- 1nOCUI‘ted on leaf. x

inoculated on stem. X5

 

 

 

A.

10 20
Days after inoculation

Relative infectivity of virus in stem
epidermal strips (1-50 dilution) or in leaves (1-5 dilution),
B, disease indices of plants inoculated either on leaf or on

31
epidermal tissues readhed a concentration at least 10 times

greater than that in the leaves and also that loss of infect-
ivity was not as rapid.

Ten uniformly selected plants were divided into 2
groups and plants of one group were inoculated on stems and
the other plants were inoculated on 2 well-expanded leaves
with X5. Disease development of systemic symptoms was shown
in Fig. 7, B. No apparent difference in systemic disease
develOpment was observed between the 2 groups of plants, al-
though the systemic symptoms deve10ped slightly earlier on

the plants inoculated on leaves.

IV. DISCUSSION

Virus multiplication curves in Epicure plants were
influenced considerably by different environmental conditions.
Two types of curves were obtained in the present investiga-
tion, i.e., type A was‘an upward curve flattened on tOp
without or with a small downward drOp of infectivity, and
the type B curve was characterized by a rapid rise and prompt
decline in infectivity.

Type A curves are typical of virus titre in tolerant
potato plants grown at 16°~24°C (Hooker and.Kim, unpubliShed
data) and in Epicure plants as listed below. Type B curves
were obtained in Epicure plants kept at 20°C, in long days
after inoculation, in leaves predisposed at high temperature
(28°C) or prediSposed by short days before inoculation, in
old leaves, and with certain other growing conditions. Type
B curves were obtained when environmental or physiological
factors were favorable for very early, rapid virus multipli—
cation. This type of curve was demonstrated by Hutton and
Wark (1952), with Epicure plants. Since the Epicure plants
used by Hutton and Wark were inoculated under summer condi—
tions, the type of curve they obtained is logical and.
results in their trial and mine are in agreement. Hutton
and Wark did not test virus titre under varied environmental

conditions. Hutton (1948) stated that sudh a hypersensitive

reaction as localization could be complete under midsummer

condition but was incomplete in mid winter.

32

33

Virus multiplication type A curves were usually
obtained in inoculated leaves of Epicure plants kept at 16°C
under Short days after inoculation, in plants pred13posed at
16°C, or in young leaves or stem epidermal tissues. These
curves (type A) were distinct from those of Hutton and Wark
and more nearly resemble virus titre curves of tolerant
plants (Hooker and Kim, unpublished data).

thler (1958) accepted the theory of Hutton and Wark
(1952) in that a common virus-inactivation system determined
the immunity of S. 41956 and the localized resistance of
Epicure. Both types of curves were obtained with Epicure in
the present test. It is difficult to accept without reserva-
tion the theory that a common virus inactivating system is
present in both hypersensitive and in immune types of potato
since virus titres from inoculated leaves of Epicure more
nearly resemble those of tolerant varieties than of immune
types. Data obtained to date from inoculation of immune
types such as S. 41956 or its derivatives (Hutton and Wark,
1952: Kohler, 1958: Benson and.Hooker, 1960: Bagnall, 1961)
suggest neither the A nor the B type of curve.

Mature plant resistance in tolerant potato plants to
PVX was demonstrated by Bercks (1951) and by Beemster (1957)
in tolerant potato varieties. They found that the older the
potato plants were at the time of inoculation with PVX, the
fewer were the tubers that became infected. At present, the

cause of the mature plant resistance is unknown. In the

34

present trials, rapid virus multiplication followed by rapid
decline in infectivity took place in the old leaves of
Epicure plants. In young leaves, the tissue maintained high
levels of infectivity for relatively long periods.

Bercks (1954, 1956) found that PVX concentration as
determined by serological and electronmicrosc0pic methods
was very low in the oldest leaves of Samsun tobacco plants
and relatively high in the middle leaves. The older foliage
contained mostly fractions with particles at most half the
length of the Whole virus particles and so he supposed that
PVX tended to disintegrate in the old leaves. Possibly a
slightly different situation is present in old Epicure
leaves since virus multiplied very quickly, readhed a high
titre, and declined rapidly. Inoculated old leaves were
killed quickly. Few of the Epicure plants inoculated on old
leaves became systemically infected. These plants further-
more did not generally show top necrosis and were usually
free of PVX as determined by the Gomphrena test. The fact
that PVX.multiplied more rapidly in old leaves followed by
early defoliation probably played a role in mature plant
resistance in this hypersensitive potato variety. The rapid
disease develOpment and quick death of the inoculated old
leaves might be attributed to rapid increase of virus content
in old leaves associated with the early attainment of an in-

fectivity level whidh was high enough to cause death of old
leaf tissue due to hypersensitivity. Probably some of these

35

plants escaped further systemic invasion because the in-
oculated leaves were quickly killed and drOpped. The tissue
of young leaves was more tolerant to high virus concentration,
and virus infectivity remained high in sudh leaves even
though relatively non-necrotic. At least some evidence is
presented to suggest that within tissues of a hypersensitive
potato plant, degrees of susceptibility exist suggesting
tolerance (young tissue) as Opposed to hypersensitivity (old
tissue).

PVX in stem epidermal tissue maintained high levels
of infectivity over a relatively long period. It seems
probable that epidermal stem tissues were more tolerant to
PVX infection or were not as hypersensitive as the leaves.
Thus, different degrees of sensitivity to PVX may exist with—
in different tissues of the host.

Timian gt‘al (1955) found that local symptoms in
potato plants of susceptible progenies developed equally well
at 18° and at 24°C, and that systemic symptoms were more
pronounced at 24° than at 18°C. At 28°C, no systemic symptoms
develOped and local disease develOped slower on inoculated
leaves. In the present tests with Epicure plants, the
Optimum temperature for disease develOpment was within more
narrow limits and more definitely inhibited by high temperature.

Disease develOped more rapidly in hypersensitive

plants in continuous light at temperatures below 24°C While

at 28°C higher disease indices were obtained in the 8 hour

36

day than in continuous light. In contrast, increased symp-
tom severity in susceptible plants subjected to reduced
light has been reported (Timian gt §_J_._, 1955b).

Yarwood (1956, 1958) reported that susceptibility of
bean leaves to certain viruses was increased by pre- and
post-inoculation hot water treatment. In my tests, no in-
creased susceptibility was obtained in inoculated leaves
heated to 50°C in hot water for 60 seconds before inoculation.
In contrast, heating leaves within 30 minutes before or after
inoculation reduced infection. Infection was decreased to
approximately the same extent by either pre- or post-inocula-
tion treatment. Conceivably susceptible sites may have been
destroyed either before or after infection or some process
in virus synthesis within the cell may‘have been impaired.
Whatever the underlying reason, the treatment temperature
was well below the thermal inactivation point of PVX, and
within 15 hours of inoculation treated tissue had resumed a
level of susceptibility similar to that of nontreated tissue.

Other types of pre-inoculation treatment such as
variation in growing temperature and photOperiOds were factors
in susceptibility of Epicure plants to PVX. PrediSposition
by growing plants for 2 weeks at 28°C increased susceptibil-
ity, whereas at 16°C susceptibility was decreased. These
results are difficult to interpret since 28°C was generally

a very poor temperature for disease develOpment and 16°C was

very favorable. A relatively short exposure to 36°C

\
a
. .
O
a
. ; .
.
. .
.
.

 

 

37

(2 days) was not as effective as 2 weeks at 28°C in enhancing
susceptibility. Kassanis (1952) increased susceptibility of
local lesion hosts to 5 mechanically transmitted viruses by
keeping healthy plants at 36°C for 1-2 days before inoculation.
It is recognized that age of plants and conditions
under which they have been grown affect virus susceptibility
(Kassanis, 1957: Yarwood, 1959). In my trials, symptom sever-
ity and virus multiplication in inoculated leaves were en-
hanced by growing plants in short photOperiOds eSpecially at
20° or 28°C, and was retarded by pre-inoculation incubation
in continuous light eSpecially at 16°C for 2 weeks. Many
viruses produce more severe lesions both local and systemic
in plants grown under low than under high light intensities
(Bawden and Roberts, 1947: Ross, 1953). The effect of en-
vironment on virus disease develOpment was demonstrated by
subjecting plants to various periods of darkness and reducing
illumination before and after inoculation (Bawden and
Roberts, 1948). Shaded leaves are much more fragile than
those grown in bright light.. Thus they may be more readily
injured during inoculation and provide a greater number of
entry points for virus. Leaves of plants grown in continuous
light were tougher than those grown in Short days. Mechanical
resistance may at least play a part in this methanism. How-
ever, other more subtle changes in physiology may be of
greater importance in this reSponse. Takahashi (1947) re-

ported that TMV multiplied more rapidly in leaves in the

38

light than in the dark. According to Pound and Bancroft
(1956), TMV concentration in tobacco plants kept under long
photOperiods (12-16 hour day) was higher 4 days after inocu-
lation than that of plants kept in short day (4—8 hour days)
although these differences were reversed after 18-32 days.
Direct correlation between virus multiplication and
symptom severity has been demonstrated in Nicotiana spp.
with PVX (Pound and Helms, 1955) and other host-virus combina-
tions (Harrison, 1956: Bancroft and Pound, 1956). In my
trials, infectivity of virus in inoculated leaves was positiv-
ely related to local symptom severity as mentioned above.
High levels of virus infectivity were demonstrated in the
inoculated leaves of plants which showed high disease indices.
Generally disease develOpment in the plants inoculated

with isolate X of PVX was slightly more rapid than that in

8
plants inoculated with isolate X5, although isolate X5 pro-
duced severe symptoms and isolate X8 either no symptoms or
mild symptoms on susceptible potato plants (Timian gg‘al,
1955a). Cockerham (verbal communication) has indicated that
for infection of necrotically reacting potato varieties, non-

necrotic strains of PVX are relatively more effective than

necrotic isolates.

 

Illnlllllll . II |i

V. SUMMARY

Effects of certain environmental factors and certain
factors within host plant on disease develOpment and multi-
plication of PVX in Epicure plants hypersensitive to PVX
were studied. Most rapid diseaSe development occurred in
the plants at post-inoculation temperature of approximately
20°C, and plants at 16°C showed almost as severe symptoms as
plants at 20°C. At 24°C, there was considerable reduction
in severity of systemic symptoms, while local symptoms develOped
somewhat slower than on plants at 16°C. No systemic symptoms
develOped on plants at 28°C, and local symptom development
was slower than that at lower temperatures.

Disease indices of plants kept in continuous light
at 16°C were higher than those of plants eXposed to 8 hour
illumination in 24 hours. Difference between photOperiOds
were small at 20°C. At 24°C, early disease symptoms were
more severe in plants with continuous light, Whereas 12 to 14
days after inoculation symptoms were more severe in plants
grown in an 8 hour day. At 28°C, disease develOped in 8
hour day plants earlier than in plants grown in continuous
light.

Virus infectivity in inoculated leaves was positively
correlated to symptom severity. Higher relative infectivity
of virus was demonstrated in inoculated leaves at Optimum

temperatures for disease development, and little infective

39

 

4O

virus content was present in leaves at 24° and 28°C. Higher
infectivity was usually demonstrated during the initial rise
in long photOperiod than that in short photOperiod. Loss of
virus infectivity occurred after 8 days in leaves under all
conditions tested except at 16°C in short day. Differences
in virus concentration were small between photOperiOds at
other temperatures.

At each post-inoculation temperature, local symptom
develOpment was increased by a pre-inoculation high tempera-
ture of 28°C and decreased by pre-inoculation incubation at
17° for 2 weeks. Plants grown at 22°C were prediSposed
intermediately. The virus multiplied more rapidly in the
leaves of plants prediSposed at 28°C, and the virus infectiv-
ity lost more rapidly. While the virus multiplied slower in
leaves of plants grown at 17°C, and virus infectivity con-
tinued to increase up to the end of Observation period.

Virus multiplication in leaves predisposed at 22°C was inter-
mediate.

Continuous light at all temperatures before inocula-
tion retarded symptom develOpment and virus multiplication
in inoculated leaves, while symptom develOpment and early
virus multiplication were enhanced by predisposition in short
day before inoculation. Loss of infectivity was most rapid
When plants were prediSposed in short day at 20° or 28°C. and
was delayed by 16°C prediSposition at either day length as
well as by long day at 20°C.

41

Increased susceptibility to PVX was slightly increased
by pre-inoculation temperatures of 36°C 2 days before inocula-
tion.

Disease indices of inoculated plants were reduced by
hot water leaf treatment (50°C), within 30 minutes before or
after inoculation. After 15 hours tissues had recovered and
disease indices were essentially similar to plants with un-
treated leaves.

Symptoms on inoculated old leaves develOped very
quidkly. The virus multiplied very rapidly and infectivity was
lost suddenly after reaChing a high peak. Disease develOped
slower and virus multiplication was slightly retarded but in-
fectivity remained high over a 20 day period in inoculated
young leaves.

Infection of the virus in stem epidermal tissues was
established. The virus titer in stem strips remained high
for relatively long period, although in leaves it drOpped
rapidly under the same conditions. Infective virus content
in stem epidermal tissues was at least 10 times greater than
that in leaves. About the same degree of systemic infection
was noticed among plants inoculated on leaf or stem tissue.

In general relatively low systemic invasion develOped
on the plants Which showed higher local symptoms under the
conditions favorable for virus multiplication. The X8 isolate
of the virus caused slightly more rapid disease development in

Epicure plants in comparison with X5 isolate, although X8 was a

mild isolate on some other potato varieties susceptible to PVX.

V I . L ITERATURE CITED

Bagnall, R. H. 1961. Hypersensitivity to virus‘A and X in

Canadian and American potato varieties. .Amer. Potato
Jour. 38: 192-202.

Bancroft, J. B., and G. 8. Pound. 1956. Cumulative concen-
tration of TMV in tobacco and tomato at different
temperatures. Virology 2:29-43.

Bawden, F. C. 1936. The viruses causing tOp necrosis
(acronecrosis) of the potato. Ann. Appl. Bio. 23:
487-497.

Bawden, F. C. and F. M. Roberts. 1947. The influence of

light intensity on the susceptibility of plants to
certain viruses. Ann. Appl. Biol. 34:286-296.
Bawden, F. C., and F. M. Roberts. 1948. Photosynthesis and

predisposition of plants to infection with certain
viruses. Ann. Appl. Biol. 35:418-428.

Beemster, A. B. R. 1957. Some aspects Of mature plant
resistance to viruses in the potato. Proceedings of
the Third Conference on potato virus Diseases. 212-
217.

Benson, A. P., and W. J. Hooker. 1960. Isolation Of virus
X from "immune“ varieties of potato, Solanum
tuberosum. PhytOpathology 50:231-234.

Bercks, R. 1951. Waiter Untersudhungen zur Frags der
Altersresistanz der Kartoffelpflanzen gegen das X-

virus. Phytopath. Z. 18:249-269.

42

43

Bercks, R. 1954. Untersuchungen fiber Anderungen dss
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