FACTORS ENFLUEN‘CENG LQCAL LESION
FORMATEQN 0F POTATO WRUS X
EN LEAVES QF GOMPHRENA GLOBOSA L.

Thai. for fine Dam of Ph. D.
MICHIGAN STATE UNIVERSITY

Joseph E. Huguelet
19:64

 

 

was

LIBRARY

Michigan State
University  

 

This is to certify that the

thesis entitled

Factors Influencing Local Lesion Formation

Of Potato Virus X In Leaves Of
Gomphrena glgogsa L.

presente

Joseph Edward Huguelet

has been accepted towards fulfillment
of the requirements for

Ph. D. dqpeein Botany and Plant Pathology

W/M/

flajor professor

Date Sept. 29, 1961+

0-169

 

ROOM USE ONLY

ROOM USE ONLY

ABSTRACT
FACTORS INFLUENCING LOCAL LESION FORMATION OF POTATO

VIRUS X IN LEAVES OF GOMPHRENA.GLOBOSA L.

by Joseph E. Huguelet

The reproducibility of results of the biceassay of
potato virus X (PVX) using Gomphrena globosa L. is governed
in part by several environmental factors. Among these,
light and temperature are of importance in symptom reSponse:
accordingly their influence was examined critically and the
relation of host carbohydrate level.determined.

Plants used for inoculation were selected for uni-
formity, and.inoculations were made using clarified PVX.
Bio-assays were made by grinding.infected.leaves,.expressing
the sap, preparing suitable dilutions, and determining
infectivity.on.healthy Q. globosa. Each.inoculation was
replicated 6,or more times using a random arrangement.

One leaf of each inoculated leaf pair was covered
with aluminum foil to exclude all light. At the end of a
suitable incubation period, covers were removed and lesion
counts made.

Decreasing numbers of.lesions were obtained as the
length of the dark period was increased. Aftercl4 days of
darkness few, if any, lesions were apparent at the time of
uncovering. In addition, the appearance of the first lesions

was delayed by the length of the dark period.

Joseph E. Huguelet

No lesion reduction effect occurred if the dark

treatment was delayed for 1—4 days after inoculation. The
same number of lesions formed on leaves which were not
covered until 3 days after inoculation as were formed on the
controls (not covered).

Similar experiments Were carried out using excised
leaves. No lesions were ever apparent at the time of uncover-
ing. Virus infectivity titre in darkened leaves as deter-
mined by bio-assay, was found to exceed by as much as.3 fold,
the titre in illuminated leaves. Thus, although symptom
reaponse was latent, virus multiplication occurred in
darkened leaves. In addition, limited movement of virus was
demonstrated from inoculated to non-inoculated regions of
the darkened leaf.

The influence of the diurnal fluctuation in suscept-
ibility was determined by inoculating leaves over 24 hour
periods at 4 hour intervals. Susceptibility was correlated
with light conditions. Resistance was greatest at 8 a.m.
and susceptibility continued to increase from inoculations
made up to 8 p.m.

The influence of different temperatures before
inoculation on symptom eXpression varied as the light condi-
tions varied. When natural illumination was not supplemented
with artificial light, results were inconsistent: with
continuous illumination the minimum number of lesions
develOped at 280C and the maximum at 1707 with 1 leaf

covered with foil the minimum number occurred at 22°C.

Joseph E. Huguelet

Using excised leaves, both sucrose and glucose
nutrient solutions reversed the influence.of darkness.
Lesion numbers increased in darkened leaves as carbohydrate
concentration was increased.up to .5 M, and concurrently,
virus infectivity titre decreased.

In inoculated leaves virus titre was consistently
greater in darkened leaves than in illuminated leaves.
Inhibition was apparent by an initial increase followed by a
decrease in virus titre as leaf juice from leaves grown in
the dark was diluted and assayed on g. globosa. In addition,
juice from.non-inoculated leaves grown in the.dark and.mixed
with clarified PVX, inhibited the virus more than did juice

from leaves grown in the light.

FACTORS INFLUENCING LOCAL LESION FORMATION OF POTATO

VIRUS X IN LEAVES OF GOMEHRENA GLOBOSA L.

BY

Joseph E: Huguelet

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

1964

 

ACKNOWLEDGEMENT

I am greatly indebted to a number of persons who
have guided and encouraged me throughout the research and
writing of this paper. To Dr. W. J. Hooker an expression
of sincere gratitude seems to be an inadequate return for his
thoughtful efforts and help.

Special thanks are due to Dr. D. J. deZeeuwcand
Dr. H..H. Murakishi for their genuine interest, assistance,
and encouragement. I also wish to extend my thanks to
Dr. J. E. Cantlon and Dr. W. B. Drew for their help in the
evaluation of the manuscript.

To Sue, my wife, for her patience-and understanding,
and to Joey, Jeffy, and Jamie for their ability to envoke

smiles, I am deeply grateful.

ii

mats or CONTENTS

Page
INTRODUCTION

0.00.0...OOOOOOOOOOOOOCOOOOOOOOOOOOOOOOQO 1

REVIEW OF LITERATURE

O'OOOOOOOOOCOOOOOOCOOOOOOOOOOOOOOO 2

MATERIALS AND METHODS

COCOOOOOOOOIO0.000000000000000CO 8

EXPERIMENTAL! RESULTS COCO00......OOOOOOOOOOOOOOOOOOOO lo

Lesion formation in intact leaves as

influenced by a dark period ................. lO
Lesion formation in excised leaves ............ 14
Influence of diurnal fluctuation .............. 25
Influence of pre— and posteinoculation

environments ..........-..................... 27
Influence of carbohydrate nutrition in

virus localization .......................... 31
Virus inhibition in illuminated and in

darkened leaves ............................. 35

DISCUSSION 000.00....OOOOOOOOOOOOOOOOOOO0.00.00.00.00 39

SWARY0.0.0....000000.0.00.0...COOOOQOO°OOOOQOOOOOOCO 50

LITERATURE CITED.0000.0.0.000OOOOOOOOOOOOOOOOOOOOOOO 52

iii

TABLE

1.

LIST OF TABLES

Page

.Lesion formation in intact Gomphrena

globosa plants as influenced by darkening
1 leaf of a pair after inoculation ........... 13

Latent infection and multiplication of
PVX in inoculated, excised, and darkened
leaves of Gomphrena globosa .................. 22

PVX infectivity in inoculated and non-

inoculated regions of excised leaves of

Gomphrena globosa incubated in the light

or in the dark ............................... 24

Influence of light and temperature pre-
inoculation conditions on the lesion
response of Gomphrena globosa to PVX ......... 28

Influence of prea and post-inoculation
temperatures on the lesion reSponse of
Gomphrena globosa to PVX ..................... 30

Influence of exposing entire plant to l2,
l7, 22,0r 28°C and to light or to dark,
4 days before inoculation .................... 32

Influence of glucose nutrition on
localization and multiplication of PVX
in excised leaves ............................ 34

Inhibition of PVX by juice from non-

inoculated leaves of Gomphrena globosa
incubated in light or darkness for 7 days .... 38

iv

LIST OF FIGURES

FIGURE Page

1. Apparatus for maintaining air movement
in covered leaves of Gomphrena globosa
after inoculation with PVX ..................... ll

2. Delay and reduction in lesion appearance
in intact, inoculated, and darkened

Gomphrena globosa leaves ....................... 15

3. Local lesions in intact leaves of
Gomphrena globosa inoculated with PVX
27 days previously. TOp row, illuminated
control leaves of each pair. Bottom row,
Opposite leaf of the pair darkened for
(A-E) 7, 6, 5, 4, and 3 days after
inoculation respectively ....................... l6

4. Number of lesions on PVX inoculated
leaves illuminated for varying periods
and darkened for the remainder of a 14—day
period ......................................... 17

5. Method for incubating excised leaves
of Gomphrena globosa inoculated with PVX.
Note Opening at tip of each tube to allow
nutrient solution to rise to the level of
the solution in the tray ....................... l9

6. Daily variation in susceptibility of
Gomphrena globosa to PVX (A: initial
inoculation made on one-half leaf of all plants,
and B: initial inoculation and subsequent
periodic inoculations.made on separate
groups of plants) .............................. 26

7. Virus.infectivity in PVX inoculated
leaves incubated under illuminated and
darkened conditions ............................ 36

INTRODUCTION

In recent years quantitative assay methods for
determining infectivity have become increasingly important.
Many investigations rely on bio-assay.as the most.accurate
'method of quantitative virus determination. The.elucidation
of the exact mechanisms of viral multiplication and of host
symptom eXpression depend in.part on the development of more
accurate and precise methods of assay.

The literature indicates that.many.factors appear to
be.correlated with symptom expression and therefore may
affect the reliability of the.assay of virus infectivity.
Thus the initial objective was to determine what influence
light, temperature, and the diurnal fluctuationchad on the
reSponse of g. globosa to PVX.

Symptom expression of‘go globosa to PVX after periods
of darkness was found to be much different from the response
following illumination: thus the investigation was.broadened
to include an examination of the requirement of carbohydrate
in lesion formation.

This research was carried out to improve assay
methods, and to enlarge upon our.knowledge of the physio—
logical factors which are involved in virus multiplication

and host reSponse.

REVIEW OF LITERATURE

The influence of environment on host symptom

expression to viral infection has been the tOpiC of concern

in many investigations since Johnson's (1922) early work.

It has been known for some time that light (Bawden and
Roberts, 1947, 1948: Best, 1936a, b: Takahashi, 1941, 1947)
and air temperature (Goss and Peltier, 1925: Johnson, 1922:
Samuel, 1931: Tompkins, 1926) affect symptom reSponse.

Recent literature reviews on the subject of the influence of
light and temperature have been made (Bawden and_Pirie, 1952:
Bawden, 1959: and Kassanis, 1957).

The specific influence of light on symptom response
has been found to vary with the virus-host combination
examined and with the time of introduction of light with
reSpect to inoculation. The severity of some symptoms.seems
to be entirely a function of light intensity (Kassanis, 1957).
Bawden and Roberts (1947, 1948) have shown that condutions
to which plants are eXposed before inoculation, are more
important than conditions after inoculation: tobacco mosaic
virus (TMV), tobacco necrosis virus (TMV), or tomato bushy
stunt virus in Nicotiana glutinosa and tomato aucuba mosaic
virus in tobacco were examined and short periods of darkness
before inoculation increased susceptibility.

Lindner gt El. (1959) found that a dark period before

inoculation was necessary for the maximum number of lesions

3
to form in cucumber inoculated with TMV. Wiltshire (1956a, b)
described the same effect using bean and TMV.

On the other hand, Cheo and.Pound (1952) found that
a long day and high light intensity prior.to.inoculation, as
well as after inoculation, enhanced virus multiplication of
cucumber virus I in Spinach.

Post-inoculation light conditions have been shown to
have less effect than pre-inoculation.light conditions
(Bawden.and Roberts 1948) and again the effect seems to vary
with the virus-host combination in question.

Lesions were reduced by 60 percent following a 1 day
dark period after TMV inoculation of.cucumber (Lindner 25 31.,
1959). Infection was reduced by darkening Pinto bean after
inoculation with TMV (Nienhaus.and Yarwood, 1963). .Lesion
numbers were reduced in Gomphrena globosa inoculated with
PVX and shaded for 48 hours after inoculation (Wilkinson and
Blodgett,1948). ”Bauden and Roberts (1948) showed that post
inoculation dark conditions most often decreased susceptibil-
ity in the virus-host combinations they examined.

On the other hand,-and.in addition to the report of
Cheo anleound (1952), Takahashi (1941, 1947) found that
more virus was produced in light culture than in dark
culture of TMV infected tobacco. A similar report was made
by Pound and Bancroft (1956).

Another influence on symptom reSponse related to the
influence of_light is that of diurnal fluctuation. Matthews

(1953a) inoculated Sydney Wonder been with TMV and found

4
that the.number of local.lesions reached a.maximum.from
inoculations madein the afternoon and fell off .later -in the
evening: this fluctuation correSponded with the accumulation
of sugars produced by photosynthesis. Light was.more
important than temperature in controlling the.diurnal
fluctuation in susceptibility (Matthews, 1953b), but inter-
acting changes.in light and temperature.have an affect
(Bawden, 1959).and may.be additive (Kassanis, 1957).
Yarwood (1956) found more lesions developed on bean.inoculated
with TMV from 11 a.m. to 5 p.m. than from.inoculations made
from 6 a.m. to 8 a.m. and a similar, but not as great an
effect, was observed.using E, glutinosaas the host plant.

Seasonal variations in symptom response and virus
multiplication have been demonstrated and these may be due
to photoperiod (Pound and Helms, 1955).

Pound.and walker (1945) found that high air
temperatures.increased the concentration of cabbage virus A
(CVA) and decreased concentration of cabbage virus B (CVB) in
cabbage. Systemic movement and multiplication of OVA in g.
glutinosa were low at 28°C while in the inoculated leaves
themselves the_rate of multiplication was.increased with
temperature (Round and Weathers, 1953). In §,.mu1tivalvis
both cabbage virus strains_multiplied in inoculated leaves
at 28°C while their concentration in systemically invaded
leaves varied little with temperature (Pound and Weathers,

1953).

5

The concentration Of PVX varied with temperature and
host (Pound and Helms, 1955). Symptoms of turnip virus I
were masked in horseradish at 28°C and severe at 16°C
(Pound,l949).

,Bancroft and Pound (1954) found that.mu1tiplication
of TMV increased in N. tabacum (H-38) with increased
temperature.up to 28°C and that the increase was correlated
with symptom severity.

In observations Of TNV inoculated Erench.bean,
Harrison (1956) found that increasing the temperature.above
100C increased virus accumulation in inoculated.leaves with
the Optimum at 22°C.

cabbage virus A in both E, glutinosa-and N, tabacum
(H-38) was enhanced by.pre-inocu1ation temperatures of 28°C
(Gonzalez and Pound, 1963). In general.high preeinoculation
temperatures greatly increase.host susceptibility to virus
infection (Kassanis, 1957).

Goodchild (1960) showed that cell to cell movement
Of TMV in E. glutinosa increased in the dark, While the
amount Of virus development per unit area was similar in the
light or in the dark.

The mechanism of the.influence.of darkness and high
temperature on susceptibility may be similar, since both
reduce carbohydrate content Of leaves (Kassanis,.lQSZ). The
relationship carbohydrates play in symptom reSponse has been

examined and a recent review is available (Diener, 1963).

6

Yarwood (1952) examined the relation of.carbohydrate
to.lesion.response using several virusehost.combinations.

NO symptoms develOped-from TMV inoculated tobacco.incubated
in the dark.in-sucrose or in water, but virus titre was
higher from leaves in sucrose culture. Similarlresults
occurred.using.tobacco ring spot virus (TRSV) in tobacco.
Smaller lesions.and a-decreased virus titre were.obtained in
sucrose culture of TMV in bean than without sucrose. In E.
glutinosa,inoculated with TMV.no difference was obtained in
virus_infectivity titre between water and sucrose culture
in the.dark. White clover mosaic virus (WCMV) inoculation
of bean produced no symptoms in water or in sucrose culture
in the dark, but a higher virus infectivity titre develOped
with sucrose culture than with water culture.

Leben and Fulton (1951) showed that typical lesions
develOped in cowpeas inoculated with TNV or TRSV in the dark
only if glucose was supplied.in the culture media. Iarwood
(1951,.1952) reported that lesions formed in Pinto-bean
inoculated with TMV were larger if the leaves were cultured
on water than on 8 percent sucrose solution. Watson (1955)
has shown that sugar beet yellows virus titre was decreased
and symptoms increased by spraying sugar beet leaves with
sugar.

Environmental affects on symptom response and multi-
plication of the virus may result from production of inhibi-
tors varying both.qualitatively_and.quantitatively in respect

to environmental conditions. Literature reviews of virus

7
inhibition are available (Bawden, 1954:.Hooker and.Kim, 1962:
and Blaszczak, §t_al.,l959). In their study, Blaszczak gt
al..found that dilution of g. globosa juice.to.1elo
increased infectivitypby.143 percent when the diluted juice
was mixed with PVX and a bio—assay made. Bawden and Roberts
(1948) found that sap from been or tobacco_plants, grown in
the dark or light,.and used as diluent of TMV or INV had the

same inhibitory effect.

MATERIALS AND.METHODS

Gomphrena globosa L. plants were grown in sterilized

U.C. soil (Baker,_l957).and transplanted to 4—inch pots.

The soil was a.mixture of equal parts.sand.and peat with

4 oz. potassium.nitrate, 4 oz. potassium sulfate, 2.5.lb.
single superphosphate, 7.5 lb. dolomite.lime.and 2.5.lb.
calcium carbonate lime added per-cubic yard of the sand-peat
mixture. Plants were watered daily, and twice a week were
supplemented with nutrient solution (Plant.Marvelz.Plant
Marvel Laboratories, Chicago 28, Illinois: 12, 31, and 14
percent.re3pectively N, P, and K, at the rate of 1 table-
Spoon per gallon of water). .Light was supplemented during
the winter with a combination of fluorescent and incadescent
lamps which gave.an intensity of 450-700 foot candles at
mean.plant height. .This_light was supplied continuously.for
l6_hours each day and the temperature held.between 220 and
28°C.

Plants used for inoculation were selected for
uniformity Of age, height, and size Of leaf pair. One day
prior to inoculation, all leaves below the fully expanded
apical pair were.removed from the.plants. On the.following
day the fully expanded pair was inoculated with clarified
PVX.

Stock inoculum was prepared from Datura tatula (L.)
Torr. plants inoculated with PVX-5 (Timian st 31., 1955).
Systemically infected leaves were.removed.10e14.days after

8

9
inoculation and immediately frozen. The frozen leaves were
ground.and the juice extracted through a double_layer Of
cheesecloth. The sap was 1) clarified by.heating in a water
bath at 54°C for .15 minutes, 2) centrifuged at -l2,ooo x o
for 15 minutes and 3) the supernatant frozen, thawed,and
centrifuged 2 more times. After.the final centrifugation,
the clear, lightly colored virus preparation was stored at
~200C. .Fresh.inoculum was;prepared_every 4-6 months and the
virulence periodically determined on g. globosa.

Inoculation of the Opposite leaves of Q. globosa
plants with PVX was made using.a dilution.of stock inoculum
which produced 40-80 lesions per half leaf. Carborundum,
400.mesh, as an abrasive was dusted on the leaves with a
powder.blower, a polished glass spatula was used for inocul-
ation and a folded paper towel for leaf support.

.Bio-assay of virus infectivity.was made.by grinding
infected leaves.and.diluting the juice with distilled water.
In some.cases juice was extracted through cheese cloth and
serially diluted. Initially.inoculatedcleaves were divided
into 2 or more groups and the assay made on healthy _G_.
globosa.using 6 or more half.1eaves per dilution, and with
each dilution and treatment randomly arranged on the assay
plants. Often, a standard PVX inoculation was made on one—
luxlf leaf Of each assay plant and the results calculated as

percent Of standard.

v‘-‘

EXPERIMENTAL.RESULTS

.Lesion formation in.intact-leaves.as.influenced€py_g
dark period.4Leaves of g. globosa wereeselected.and.inoculated
as previously.outlined. As.soon as the inoculum had dried
on the leaf, usually within.oneehalf.hour,.l leaf of each
inoculated pair was covered with aluminum foil. In.later
trials, an apparatus was designed which permitted.covering
the leaves and atthe same .time provided ventilation (Fig. .1).
Small.woodenifremes.were constructed and.covered.with foil
to form a.box. One.end-of eachcbox:was;Open to provide for
the.entrance Of the 1eaf.and was then closed by folding the
overlapping.foi1 around the petiole of the-inserted.leaf,
thus.excluding light. At the.other end of the.box, an air-
hose connected to a.manifold was inserted. The.manifold
was supplied with forced air which was.regulated to provide
aircmovement over each leaf. The Opposite.leaf of the
pair, which served as a control, was not covered but remained
illuminated under normal greenhouse conditions. Temperatures
under the foil covers, in either ventilated or non—
ventilated tests, dud.not differ more than O.5°C.between the
inside and outside of the covers.

In some trials, leaves were covered immediately after
inoculation and at daily intervals groups of leaves were un—
covered. In other trials, leaves were covered after certain

periods in the_light following inoculation.

10

_- _.-.

ll

 

Fig. 1. Apparatus for maintaining air movement in covered
leaves Of Gomphrena globosa after inoculation with

PVX.

12

In preliminary tests, fewer lesions formed in leaves
which were covered immediately after inoculation than in
illuminated leaves. Plants maintained in the dark for
several hours prior to inoculation, or plants which were
inoculated in the early morning, and then covered, produced
even fewer lesions than those maintained in the light.

Thus, in subsequent trials, inoculations were made after
the plants had been previously held in the dark.

Results of 3 tests in which darkened leaves were un-
covered at periodic intervals after inoculation are shown
(Table 1). When intact leaves were grown in the dark, and
then covered once again after inoculation, only a few and
sometimes no lesions had developed at the time of uncovering.
Then, after eXposure to light, lesions began to form, and
more formed in leaves covered for short periods than in
leaves covered for longer periods. Leaves which were
covered for periods up to 13 days or less, produced lesions
after exposure to light although they were always fewer in
number than in illuminated leaves (Table 1). Leaves covered
for as long a period as 14 days were somewhat chlorotic
tmhen uncovered, but for the most part they appeared healthy
land.usually without lesions. Frequently they became dried
amid collapsed within a few days following uncovering.

Lesion appearance was delayed by approximately the
14mmgth of the dark period, with lesions appearing 3-4 days
aftJer eXposure to light (Fig. 2). A single typical

repfl_ication of g. qlobosa leaves (Fig. 3) is illustrative of

13

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14
the reduction in lesion numbers by increasing the
length of the dark period.

In order to determine the length of the period Of
light required for lesion formation, leaf pairs were
inoculated with PVX and 1 leaf of each pair of 6 plants was
darkened at daily intervals and maintained in the dark for
the balance of the experiment (14 days). A low number of
lesions was formed in leaves darkened immediately after
inoculation (Fig. 4). The number of lesions increased in
leaves darkened at later periods with the maximum being
reached on leaves darkened 3 days after inoculation. This
suggests that approximately 24—48 hours of light after
inoculation are required to initiate lesion development.

Lesion formation in excised leaves.-Darkened intact
leaves occasionally develOped a few lesions prior to ex—
posure to light. Possibly a substance or substances of
photosynthetic origin, essential to lesion development, and
translocated from illuminated to darkened leaves in
sufficient quantity, might account for the develOpment of
the few lesions which were formed. Therefore, factors
influencing lesion formation in excised leaves were examined.
52. globosa leaves were inoculated with PVX, excised, and
incubated by floating on Vickery solution (Vickery 33 El-
1&337) in petri plates or in Pyrex glass tubes. Vickery
solirtion was supplemented with 0.2 ppm ethylmercurithiosali-

CYliate to suppress mold growth (deZeeuw 1962), and the

soliition changed frequently. Light was excluded by wrapping

15

 

mo>eOH «mocon.ocoscoeoo
cocoesoc can .coeaasooce .poopcw OH mooosoooce conOH cw coflpospos one amaoo
coHunasoocH scams m>oo
mm mm me me HH 5

 

.m .mee

 

 

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mean we coso>oo

memo u cono>oo

 

  

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Ow 110T1‘ L11. 11-71»
when m coso>oo

 

ma

on

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ow

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3

(onluoo JO lueoaed) AirArioalul

l6

 

Fig. 3. Local lesions in intact leaves of Gomphrena globosa
inoculated with PVX 27 days previously. Top row,
illuminated control leaves of each pair. Bottom
row, opposite leaf of the pair darkened for (A-E)
7, 6, 5, 4, and 3 days after inoculation reSpect—

ively.

1.7

100 .

80 .

60 .

 

Trial 1: O

Infectivity (percent of control)

 

 

40 Trial 2: 0
Trial 3: A
20
o n .1 e .
o 1 2 3 4

Days of illumination
Fig. 4. Number of lesions on PVX inoculated leaves
illuminated for varying periods and dark-

ened for the remainder of a 14 day period

18
the plates or tubes in aluminum foil. The tubes, which

measured 10.5 by 2.5 cm, were pierced.providing an air out—
let and were usedAinverted so that they held the leaves in
an upright position (Fig. 5). Leaves were held in the tubes
by corks through which holes had been drilled. Leaf
petioles extended through the,holes into the continously
stirred Vickery solution. .Light was.maintained at 750 foot
candles for 16 hours/day. Air temperature in the growth
chamber ranged between 22-24°c but the temperature within
the tubes was.not determined.

For some reason fewer lesions develOped in excised
leaves than in intact.leaves with the same concentration of
virus: thus a-higher.concentration of virus was.nsed for
excised leaves. Leaves floated.on.nutrient.solution in
petri.p1ates develOped fewer lesions than did_leaves
incubated with only the petioles in the solution.

These tests established that, as with intact dark-
ened leaves, lesion develOpment was delayed by the length of
the dark period. Furthermore continuous darkness consist-
ently precluded lesion formation in inoculated-and excised
leaves when examined at the time Of exposure tollight.
Difference in results between intact and excised leaves
suggested movement of some substance, possibly.of photo-
synthetic origin, from illuminated to darkened_leaves permit-
ting formation Of a few lesions in the latter.

Tests were designed to determine the length of the

light period after inoculation required for initiation of

l9

 
 

 

Fig. 5. Method for incubating excised leaves of Gomphrena

globosa inoculated with PVX. Note opening at tip
of each tube to allow nutrient solution to rise to

the level of the solution in the tray.

20

local lesions. In the first of.these tests, leaves were
inoculated, excised, and floated on Vickery solution. One
leaf of each pair was darkened with foil. Light periods of
.25, .75, 1.5, 3, 6, 12, and 24 hours were given 3, 5, and 7
days respectively following inoculation and placing leaves
in the dark. Since only a few lesions develOped on any Of
the darkened leaves, it was apparent that either illumination
periods of longer duration than 24 hours were required, or
that the period.of illumination must be initiated prior to
the 3 day period. In a second test, illumination periods of
6, 12, 24, and 48 hours duration were.begun.12, 24, 36, and
48 hours after inoculation. Again no lesions develOped on
darkened leaves illuminated for less than 48 hours regard—
less of when illumination was initiated. The possibility
existed that shorter periods of light, although not enough
to produce visible.lesions, may well have been sufficient to
cause limited infections. Therefore.infectivity of symptom~
less leaves from several experiments was assayed
separately on healthy 9. globosa. Although no lesions had
formed on any of these leaves, all leaves incubated in the
dark contained from 2—3 times as much infective virus as
did leaves which had been incubated in the light and in
Which typical local lesions had develOped.

The extent Of virus multiplication was.determined in
additional trials using similar methods of excised leaf
culture. After an 8 day.incubation period in the light or

dark, leaves were weighed, ground in a mortar and pestle,

21
and diluted by adding twice their weight of distilled water.

The resulting juice served as inoculum for subsequent bio-
assay. Light and.dark treatments were assayed.on Opposite
half-leaves of both the same anddifferent plants. In these
trials (Table 2), leaves incubated in the dark never develOped
lesions, but always produced a higher virus infectivity
titre than did illuminated leaves. These differences were
significant at the 1 percent level (analysis of variance).
Thus PVX was.latent.in darkened leaves and was more.infective
than in illuminated leaves with local lesions.

Since the virus was latent in darkened leaves, it
may also have been systemic, thus accounting for the high
titre. g, globosa leaves were marked with India ink to
differentiate inoculated regions from non-inoculated regions.
Each leaf was divided into 3 or 4 regions of approximately
equal size and these regions designated.apical, intercalary,
and basal. In leaves divided into 4 areas, the intercalary
region was further subdivided into an upper (near apical)
and-a lower (near basal) region. Other leaves were divided
into 2 regions using the midrib as the boundary.line.

After inoculation of 1 or more specific regions on each
leaf, leaves were excised and incubated as described. After
7 days, leaves were cut above and below the India.ink lines.
An intermediate section of 1-2 mm Of leaf tissue,.on which

the ink line had been made, was discarded. Individual

regions.of the.leaves were.ground.separately.in a mortar and

pestle and assayed on healthy g. glObosa leaves.

22

Table 2. Latent infection and multiplication of PVX in
inoculated, excised, and darkened leaves of

Gomphrena globosa.

 

 

Number of lesions
formed in the
inoculated leaf1

Average number of
lesions obtained in
bio-assay of the
inoculated leaf2

 

 

Trial Light Dark Light Dark
1 34 0 35.5 65.7
18 0 30.2 109.2

34 0 30.5 66.7

36 O 41.0 76.7

2 45 O 51.7 131.5
38 0 45.5 75.0

60 0 82.0 117.0

64 O 52.2 91.0

37 0 57.0 75.2

63 0 48.7 97.7

92 0 56.7 76.5

56 0 -32.5 62.7

51 0 60.2 _113.2

53 0 21.0 49.0

 

lEach figure represents the number of lesions developing on
opposite leaves of a pair.

2Each figure represents infectivity assay of the paired
leaves in columns 1 and 2. Each assay was~made on 4 half
leaves of g. globosa . The data were significant at the 1%
level (analysis of variance).

23

In leaves divided longitudinally into 2 regions by
the midrib, the midrib was removed and discarded. No virus
movement was demonstrated between opposite halves of leaves
separated by the midrib.

No virus movement could be detected_in illuminated
leaves from inoculated into non-inoculated regions except in
2 cases in which the titre was very low (Table 3). In
darkened leaves, limited virus movement was consistently
demonstrated. Virus movement took place from both the
apical and the basal positions, but was not consistently
greater in either direction. The.bi-directional.movement
and the low titre, suggest very limited cell to cell move—
ment, although limited vascular movement is a possibility.

Since limited movement of the virus from inoculated
regions in darkened leaves was very slight, a more refined
technique was applied. A tiny glass Spatula, with an
inoculating surface measuring 1 mm in diameter was used to
inoculate leaves within the circumference of several 4 mm
diameter circles drawn on leaves with India ink. After in-
cdbation in the usual manner, 3 sterile cork.borers with
diameters of 4, 5, and 8 mm were used to obtain, first discs,
and then rings of leaf tissue from regions previously marked
with ink and inoculated with PVX. Borers 5 and 8 cut-rings
larger than the diameter of the inked, inoculated area.
Discs and rings were ground separately and assayed on
healthy g. globosa. Again the highest virus infectivity titre

was obtained from darkened leaves, and limited movement of

 

2.4

 

 

 

Table 3. PVX infectivity'in inoculated and non-inoculated regions
of excised leaves of Gomphrena globosa incubated in the
light or in the dark.

-‘ Lesions in Infectivity Lesions in Infectivity

designated assay of designated assay of
Leaf Region regionl designated region1 designated
region Inoculated region2 region3

Light Dark Light Dark Light Dark Light Dark
Apical (Inoculated) 18.2 0.0 10.6 43.1 50.9 0.0 54.3 55.1
Basal 0.0 0.0 0.1 0.6 0.0 0.0 0.0 1.8
Apical 0.0 0.0 0.0 3.2 0.0 0.0 0.0 0.8
Basal (Inoculated) 20.7 0.0 19.0 39.2 62.8 0.0 37.0 78.8
Apical (Inoculated) 4.5 0.0 27.1 89.7
Intercalary 0.0 0.0 0.04 23.7
Basal (Inoculated) 12.7 0.0 63.7 84.3
Apical 0.0 0.0 0.0 3.5
Inter-
calary (Inoculated) 14.7 . 63.3 83.7
Basal 0.0 0.0 0.0 0.0
.Apical (Inoculated) 11.7 0. 8.1 34.1
Upper Int. 0.0 0. 0.0 0.37
Lower Int. 0.0 .0 0.0 0.18
Basal 0.0 0.0 0.0 0.25
Apical 0.0 . 0.0 0.0
Upper Int. 0.0 0.0 0.0 0.25
Lower Int. 0.0 . 0.0 0.0
JBasal. (Inoculated) 5.2 . 5.5 32.4

 

lEacfli figure represents the average of 4 and 10 leaf regions in tests
1 arui 2 respectively, leaves were incubated for 8 days and then assayed.

Earl: figure represents the average infectivity assay of similar
regirans from 4 leaves ground together and assayed on 6 half leaves.

Back: figure represents the average infectivity assay of 10 regions
sack: assayed separately on 4 half leaves.

25

the virus.was_demonstrated outside of the inoculation site in
darkened.leaves.

Influence of diurnal fluctuation.-To determine the
influence of diurnal fluctuation in.susceptibility of Q.
glgbgsg to PVX, plants were inoculated over 24 hour periods
at 4_hour.intervals during April and May. Post inoculation
temperatures were held constant at 22°C or 28°C and with
normal non-supplemented light conditions.

In 2 trials, plants were more susceptible in the
evening and least susceptible in the morning. ,Lesioni numbers
were.slight1y.1ower at the end of these tests than at the
beginning. Thus the possible influence of inhibitory sub-
stances originating from the initial base inoculation was
evaluated by dividing a final and more extensive testrinto 2
parts: A) with an initial standard inoculation at the
initiation of the trial and made on.one-half-leaf of each of
the 28.plants.included_in this part of the test, Fig. 6 (A):
and B) with the initial inoculation and each subsequent
periodic inoculation made on all 16 half-leaves of 4 plants,
Fig. 6 (B).

No significant difference using the analysis of vari-
ance was obtained between plants inoculated by design A or by
design B.

A significant difference at the 1 percent level was
1established between the intervals at whiCh inoculations were
rnade in design A as well as in design B. .The Sheffe method
cof contrasts (Sheffe, 1953) was.applied to both design A and.B.

 

26

 

90
1; 80 B
a I
o
"-4
U)
O)
H
‘H A
0 7O
5..
(D
.D
S
c
2:. 6°-
:6
$4
0)
>
53
>» 50
.p
"-4
>
-H
4.)
U \I
O)
‘H
.5 40,
30 I l n I n L
4 pom. 8 pom. 12 4 a.m. 8 a.m. 12 4 pom.
midnight noon
Time of inoculation
Fig. 6. Daily variation in susceptibility of Gomphrena globosa

 

to PVX (A: initial inoculation made on one-half leaf of
all plants, and B: initial inoculation and subsequent
periodic inoculations made on separate groups of plants).

27

At the 1 percent level, significant decreases were dis-
tinguished between the means from inoculations made from

8 p.m. to 8 a.m., and the means were significantly increased
in those made from 8 a.m. to 4 p.m. Thus Q. globosa was
more resistant to PVX inoculations made at 8 a.m. and be-
came increasingly susceptible until 8 p.m., after which
resistance increased.

Influence of pr — and post-inoculation.environments.-
To determine the influence of prediSposition on symptom
response of Q. globosa to PVX, opposite leaves of intact
plants were prediSposed in either light or darkness (leaf
covered with aluminum foil) at temperatures ofl7, 22, and
28°C. After predisposition periods of l, 2, 3, 6, and 9
days, leaves were uncovered, both leaves of each pair in-
oculated with PVX, and the plants held at 22°C. Each test
consisted of 6 replications per treatment.

More lesions developed on leaves prediSposed by
light than on leaves predisposed in the dark in 27 of 29
comparisons (Table 4). Of the 2 reversals, 1 occurred in 1
trial at the 170-1 day period and the other in 1 trial at
the 220-3 day period.

To determine the -influenceof temperature alone,
pre— and post—inoculation temperatures were.held at 17, 22,
or 28°C under.either continuous illumination (800 foot
candles) in a growth chamber during February and March or

non-supplemental greenhouse illumination during December,

January,and March. No definite relationship existed between

28

 

 

 

 

Table 4. Influence of light and temperature pre-inoculation
conditions on the lesion response of Gomphrena
glgbggg to val.
I Average number
ofiesions2
Number Length of pre- PrediSposition Predégposed
of trials diSposition period temperature light dark
2 I“ 1 day 17°C 159.1 165.6
22°C 89.6 54.6
28°C 167.0 155.7
2 2 days 17°C 164.3 141.4
22°C 79.7 49.8
28°C 154.9 105.6
3 3 days 17°C 98.5 63.5
22°C 38.8 23.9
28°C 81.8 34.2
1 6 days 12°C 41.3 19.1
17°C 28.3 25.1
22°C 25.5 22.1
28°C 66.6 14.8
1 9 days 12°C 1.5 0.16
17°C 25.6 12.0
22°C 14.6 7.1
28°C .64.0 13.6
1

2

Post-inoculation temperature was held at 22°C.

Each figure represents the average number of lesions from

6 replications per trial per treatment.

29
prediSposition temperature and the number of lesions formed.
In each test, differences were obtained but they were in-
consistent between tests (Table 5). More lesions formed
when plants were predisposed at 28° than at lower temperatures
When no supplemental light was given but this trend was not
consistent in each replication (Table 5). On the other hand
with continuous illumination, fewer lesions consistently
formed when plants were prediSposed at 28°C (Table 5). In
plants pred18posed with 1 leaf covered with foil, in both
the illuminated and the darkened leaves and in 33 of 36
comparisons, fewer lesions were produced at 22°C than at 170
or at 28°C (Table 4).

A relationship did exist between the number of
lesions formed and post-inoculation temperature using con-
tinuous illumination (Table 5): more lesions develOped when
the post inoculation temperature was 22° than at 28° or 17°C.
Without the supplemental light there was no such clear
correlation with temperature.

Lesions were better defined and were more easily
counted, that is the red border around the lesions was
clearer, when post-inoculation temperatures in both supple—
mented and non-supplemented light conditions were held at
28°C than at lower temperatures. At 12° and 17° mechanical
damage seemed to be greater and more necrosis evident on the
entire inoculated leaf, making lesion counts difficult.

The reaction was not observed on leaves similarly rubbed

with water and carborundum.

30

Table 5. Influence of pre- and post-inoculation:temperatures
on lesion response of Gomphrena globosa to PVX.

Average number offllesions‘l

  
 

 

post Pre-inoculation
Number Light Inoculation o tempergture o
of trials Conditions Temperature 17 C 22 C 28 C
3 Without 17°C 78.7 65.3 87.3
supplemental
light2 0
" 22 C 70.7 71.9 71.4
" 28°C 48.2 57.7 72.3
2 Supplemented 17°C 47.2 46.1 16.9
with 800 F.C.
continuous o
" 22 C 63.2 52.7 27.7
" 28°C 40.4 33.9 12.0

 

1Each figure represents the average number of lesions from
8 replications per trial per treatment.

2The 3 trials were made in the greenhouse during December,

January and March reSpectively.

3The 2 trials were made in a growth chamber during February
and.March respectively.

31

Lesions on leaves prediSposed in the dark develOped
more slowly than on leaves prediSposed in the.light.

Lesions were first apparent 3 days after inoculation in
light predisposed leaves and at least 12-24 hours later on
dark predisposed.1eaves. The length of the dark pre—
disposition period had little.influence on the length of the
delay; lesions appeared at about the same time after ex-
posure.to light whether prediSposed to the dark for 1, 2, or
3 days.

Plants prediSposed in the dark by enclosing the
entire plant within a foil covered box for 4 days, develOped
fewer lesions than illuminated plants accompanied by a 12-
24 hour delay in lesion appearance. At 28°c, a SCQICh type
symptom developed on the leaves predisposed by darkness,
lesions were poorly defined and difficult to count (Table 6).
The appearance of such leaves was similar to that often
present in systemic virus infections, however, using ground
non-inoculated plant parts as inoculum, no systemic movement
was demonstrated. Control plants in this test were not pre-
disposed to darkness nor supplemented with light, however
they produced more lesions as the predisposition temperature
was increased from 17° (Table 6).

Influence of carbohydrate_nutrition in virus_local-
ization.-Since virus was localized in inoculated leaves
eXposed to light, the possible role of carbohydrates in

lesion formation was explored. For this study, inoculated

excised leaves were maintained in the light or dark in

32

Table 6. Influence of expos.ing entire plant to 12,17, 22,
or 28° C and to light or to dark, 4 days before

 

 

 

inoculation.
Average number of lesions2
Preinoculation light
Number Preinoculation ' condition
of trials temperature light dark
0
2 12 C 70.4 50.1
2 17°C 72.8 61.4
2 22°C 129.1 73.5
2 28°C 178.8 —-3

 

lPost inoculation temperature was 28°C, without supplemental

light.

2Eachfigure represents the average number of lesions from

6 replications per trial per treatment.

It was not possible to make lesion counts on the 280 dark
prediSposed leaves due to extensive necrosis.

33
inverted tubes. In preliminary tests,.local.lesions formed
in excised.leaves incubated in the dark and supplied with
either glucose or sucrose. In later, more extensive tests
using only glucose (Table 7), 4 leaves were inoculated for
each glucose concentration and.incubated in the light and
an equal number incubated in the dark. After 7 days of
incubation, lesion counts were made, the leaves ground
separately,di1uted with 2 and 4 times their weight in dis—
tilled water, and assayed on 6 half—leaves of healthy Q;
globosa.plants.per dilution. A standard inoculation was
made on the.other.half-leaf of each.assay plant and the
assay eXpressed as percent of control.

As the concentration of glucose was increased, the
number of lesions in leaves incubated in.the-dark progress-
ively increased (Table 7). With an increase in glucose con—
centration, virus infectivity titre decreased.in darkened
leaves (Table 7). Virus infectivity titre in illuminated
leaves was not correlated with glucose concentration. Thus
leaves in the dark and supplemented with glucose developed
local lesions after inoculation with PVX similar to
illuminated.leaves without supplemental glucose.

In addition, the red—bordered PVX lesions of
inoculated g. globosa leaves.incubated with low concentra-
tions of glucose (.1 M), were indistinct, while those on

leaves incubated in higher concentrations of glucose were

distinct and well defined borders had develOped.

34

 

 

 

 

Table 7. Influence of glucose nutrition on localization
and multiplication of PVX in excised leaves.
Number of
lesions in Infectivity assay of inoculated
inoculated leaves.(percent of control)
leaves . '
light 3 dark
Glucose in in 1-2 1-4 1-2 1-4
Trial molarity' light dark ”dilution dilution dilution dilution
1 0.000 92.7 0.0 56.0 55.1 74.0 84.2
0.001 97.5 0.0 50.3 54.4 65.7 69.6
0.01 73.2 0.0 40.0 36.7 52.0 62.8
0.1 80.7 19.0 .13.1 22.9 27.5 33.1
0.2 84.5 41.5 30.5 43.2 27.8 31.8
0.5 84.5 65.5 60.8 56.8 30.1 35.5
2 0.000 97.7 0.0 76.9 90.5 123.8 145.8
0.1 139.2 97.0 46.6 78.8 51.0 89.9
0.2 124.2 111.5 79.8 69.7 70.6 73.8
0.5 143.2 140.2 97.3 61.8 88.9 59.8

 

35

Leaves incubated in the dark had slightly higher
virus infectivity titres at a dilution of 1-4 than at 1-2
regardless of glucose concentration. Infectivity titres of
leaves in the light on the other hand, were.not as con-
sistently increased by dilution. This suggested that pos-
sibly virus inhibitors are formed to.a greater extent or
that inhibitors are more active, in darkened leaves than in
illuminated leaves.

Virus inhibition in illuminated and in darkened
leaves.-Differences in infectivity of virus from illuminated
and.non-illuminated leaves may.have been associated with
differences in either virus concentration and/ordiffer-
ences in concentration of virus inhibitors in such leaves.
Tests were designed to determine biologically the extent
of inhibition in leaves incubated in the light or in the
dark.

Leaves were inoculated,.excised, and incubated with
or without light in inverted tubes. Virus titre was
assayed using g. globosa and a 2-fold dilution series in
distilled water. In similar trials, 16 plants were
initially inoculated and each assayed in 2 replications
with 6 half-leaves per replication per dilution. A
standard inoculation was made on one—half leaf of each of
the assay plants and infectivity data eXpressed as percent
of standard. Averages of both replications of a typical

trial are shown (Fig. 7). Virus infectivity titre was

higher in dark incubated leaves at all dilutions than in
illuminated leaves. The steep rise in infectivity with

36

90;

80

Bio-assay of

darkened leaves

70+.

60_.

50.

40

30.,

Infectivity (percent of control)

  
  
 

20 Bio-assay of

illuminated leaves

 

 

10.
O A 1 A 1 J n
undiluted 1-2.5 1-5 1-10 1-20 1-40 1-30

Dilution of leaf juice
Fig. 7. Virus infectivity in PVX inoculated leaves incu-

bated under illuminated and darkened conditions

37
increased dilution in darkened leaves and the lack of such a
steep rise in illuminated leaves suggest that an inhibitor
was formed in leaves incubated in the dark.

In another approaCh, non-inoculated leaves were
excised and incubated for 7 days in inverted tubes under
illuminated or dark conditions. Using 2 replications of 20
leaves per replication per treatment, each group of 20
leaves was ground separately and their juice extracted
through cheesecloth. Clarified PVX was diluted with the
respective healthy leaf juice and a standard inoculum was pre-
pared with water in the usual manner. Assays were made on
healthy'g. globosa and the results expressed as percent of
standard (Table 8). The number of lesions develOping in juice
from illuminated and darkened leaves is significantly different!
(1 percent level using the analysis of variance). As in the
previous test, more inhibition was demonstrated in juice from
leaves incubated in the dark than from leaves incubated in

the light.

38

Table 8. Inhibition of PVX by juice from non-inoculated

leaves of Gomphrena globosa incubated in light
or darkness for 7 days.

 

Infectivity (percent of control)1

 

 

Dilution of virus with sap from “with sap from
and leaf juice * illuminated leaves darkened leaves
1/2 111.0 94.8
1/10 102.1 89.9
1/20 107.3 73.8
1/40 69.2 35.2
1/80 38.3 11.6

1

Each figure represents the average percent of control
from 2 replications of 6 half leaves per replication per
dilution: significant at the 1% level (analysis of
variance).

DISCUSSION

Often the reSponse of the.host plant to infection
depends.partially on carbohydrate level (Scheffer,-l959).
In the.necrotic reaction, virus multiplication is greater
in leaves at low carbohydrate level (Yarwood, 1952). The
altered response ofdarkened é. globosa leaves to inocu-
lation with PVX compared to illuminated leaves, points to
the need for a product of photosynthesis required for
lesion.formation. Emphasis must be,placed on the fact
that the.requirement for such a product is on.host_res-
ponse and not on virus synthesis as virusmultiplication
took place in darkened leaves (Table 2).

Virus multiplication may have been enhanced because
necrosis was absent under-darkened conditions. Possibly an
increase in enzyme activity resulting in the formation of
substances toxic to both host and virus (Solymosy, Farkas,
and Kiraly, 1959: Farkas, Kiraly, and Solymosy, 1960:
Solymosy and Farkas, 1963) may take place in illuminated
but not in darkened leaves. Whether this is the case or
not is not known, but it is clear that the host—virus
relationship exists as a latent condition in darkened
leaves in the system studied. If toxic substances such as
quinones are.involved in the necrotic reaction in the
light, they are apparently produced by a process of host

metabolism which is altered under darkened conditions.

The latent reSponse of dark incubated Q. globosa

leaves to PVX may be overcome by supplemental sugar.

39

 

40
Giucose.or sucrose as the.nutrient.solution.of excised
leaves, stimulated the formation of local lesions under
darkened condutions: this.is.in accord with_a.report of
Leben and Fulton (1951).using TNV or TRSV. .By what
mechanism this occurs is not clear, but probably the carbo-
hydrate is only indirectly reSponsible. It may be that
carbohydrate reSpiration reactions provide energy and/or
by products necessary either for the formation or activa-
tion of toxic substances.

When either intact or excised leaves were inoculated
with PVX and incubated in the dark,.a few lesions had
fonmed in intact leaves while no lesions had formed in
excised leaves at the time of uncovering. This suggested
that with the intact leaves, the photosynthetic product
responsible for lesion formation was translocated from
illuminated leaVes to darkened leaves. Delay in lesion
formation due to short periods of darkness after inocula-
tion strengthens the case for the.requirement of a carbo—
hydrate source in local lesion response (Fig.-2). These
results are in agreement with the observation of Watson
(1955) on the change in symptoms of beet yellows.in
relation to sugar content.

If we consider polyphenol oxidation products, e.g.
quinones, as being at least partially responsible for the
necrotic hypersensitive reaction, then natural occurring

reducing agents such as ascorbic acid could limit the

necrotic reaction by reducing the toxic quinones back to

 

41
polyphenols (Farkas e5 $1., 1960). It is of interest to
note that Wiltshire (1956b) reported ascorbic.acid concen-
tration decreased in.French bean and tobacco plants in-
cubated in the dark. If this also occurs ipfig;dglobosa
inoculated with PVX, it would follow that thenecrbtic re—
action would be rampant in.proportion to the increased
virus titre which.was_shown to occur by bio-assay. .Howe
ever, in the system studied,necrosis did not occur in
leaves incubated in the dark. Therefore, it would follow
that ascorbic acid concentration is either not decreased,
or the polyphenol oxidase system is not operating in the
necrotic reaction of é.globosa. If it were, it would seem
that the lack of ascorbic.acid occurring under darkened con—
ditions would tend to increase oxidation of phenolic sub—
stances to toxic compounds, and theoretically more necrosis
would result.

Increased virus.infectivity titre in darkened leaves
has implications beyond that of the lack of necrosis. Un-
less darkness triggers activation of additional multiplica~
tion sites, presumably there would be the same.number of
sites per unit.area in illuminated as in darkened leaves.
What then, accounts for the increased virus;multiplication?

Virus multiplication is limited in a local lesion
host possibly.because of_necrosis due to oxidation of poly-
phenols. It may be possible that the increased titre is
associated.with the limited movement of PVX in Q. glgpggg

which occurs in leaves incubated in the dark. Weintraub 25 21.

 

 

42
(1961, 1963) has suggested that a continuous supply of
virus as from a systemically.invadedlhost grafted to a
hypersensitive host, or by an increase in multiplication
by changing environmental conditions may result in a
systemic invasion. Zaitlin (1962), on the other hand,
believes a systemic invasion of a hypersensitive host re-
quires the introduction of the virus into the vascular
elements. Whether we call the darkened and inoculated Q.
globosa leaf systemically invaded or not is of little con-
cern, but what is of concern is the apparent limited move—
ment of virus to uninoculated regions due to an environmental
condition, 1.8. darkness. In this system then, an in-
.creased multiplication associated with an environmental
change stimulated limited invasion of uninoculated regions.
This in turn provided more multiplication sites at the
eXpense of lodal lesion formation. Furthermore, it is
doubtful that systemic Spread occurred through the vascular
tissue.

No correlation seems to exist in the system studied
between host.local.1esion response and virus titre. If a
correlation did exist it would seem that more local
lesions would haveresulted from short P°Fi°d° of darkness
rather than a decreased number simply because multiplica—
tion is enhanced. This view supports the statement of
Bawden and Harrison (1955) in a paper concerned with TNV
that cell to cell.spread may require only 1 virus—particle,

but a single lesion requires about 105 particles. It is

 

43
also in support of the work of Goodchild (1960) in which it

was shown that symptoms occurring in N, glutinosa and 2.
stramonium from TMV infection are less severe in plants
incubated in the dark than in the light and at the same time
cell to cell movement increased in the dark. However, in
this case the amount of virus per unit area was similar in
plants incubated in the light or in the dark.

In discussing the limited movement of the virus
under darkened.conditions, it may be appropriate to include
a discussion of pigment fonmation. The formation or
accumulation of pigments, probably anthocyanins, was not
apparent in leaves incubated under darkened conditions. It
may be that the anthocyanins responsible for the colored
halo around the lesion in illuminated leaves exist as agly-
cones in darkened leaves or when sugar is not available.
Oxidation reactions of the glucoside may be more-extensive
than that of the aglycone, thus resulting in toxic sub-
stances and then necrosis. This is rather doubtful because
of the apparent.stability of the glucoside. Furthermore,
the pigmented border seems more to reSult.from an accumula-
tion of red pigment, a degradation of masking pigments-al-
ready present around the lesion, or a change in color of
existing pigments via pH changes. At any rate the lag in
the time of appearance of the pigmented border suggests
that the pigment is a result, rather than a cause of the

necrotic reaction.

 

 

44
Kerling (1962) has shown that re—inoculation of Q.

globosa leaves already showing PVX local lesions, resulted
in new lesions touching the old ones and that inhibitory
activity of some component in the Cells may counteract virus
multiplication and expansion. He also showed that the
virus disappears from the center of the lesion first and
appears in the periphery of the lesion last, and with none
occurring outside of the lesion.

Kohler (1959) also was unable to show virus movement
outside of tissue obtained from a local lesion using a 48 mm
cork borer. This work as well as that of Kerling (1962)
was done on Q. globosa under illuminated conditions.

These observations, in addition to those.showing that the
virus does move and that no pigment is apparent under dark-
ened conditions, circumstantially points to the pigment as
being intimately associated with the necrotic reaction and
may be the mechanism involved in limitation of viral trans-
location. More work along these lines is required for a
definite conclusion.

If we examine the curve of daily variation in sus—
ceptibility (Fig. 6) and that for apparent photosynthesis
(Thomas and Hill, 1937, 1949) we note that susceptibility
lags behind photosynthesis. That is, in photosynthesis
more carbohydrate is produced during high light intensity
with a peak at around noon and a low from 6 p.m. to 6 a.m.

while the susceptibility peak occurs at about—4:00 p.m.

and with a low at about 8:00 a.m. This lag may be due to

 

45
the ttme required for solubility changes in carbohydrates or
the time required for energy transfer. At any rate, light
conditions during inoculation and immediately prior to
inoculation seem to be intimately related to the local
lesion.response.

The diurnal effect could bedue to other factors such
as humidity and the state of turgidity of the epidermal
cells (.Humphries .and Kassanis, 1955). For example, it may
be that the more flacid an epidermal cell is, the more
easily-damaged by rubbing it would be. This might be re-
lated to both soil moisture and humidity. In the diurnal
eXperiments reported in this paper however, the soil moisture
was maintained by watering twice-during the 24 hour period.
Humidity fluctuated to some degree due to cloud movement
and.watering in the same house. The influence of relative
humidity could be reSponsible for variations which would
occurseveral times in any 24 hour period, while the over-
all diurnal effect may be indirectly due to light conditions
at the time of inoculation, and more directly, to carbo-
hydrate levels within the leaf. Little published data are
available on the subject.

Results with Q. globosa—and PVX reported in this
paper are in agreement-with the diurnal studies of Matthews
(1953a) using bean-and TNV and those of Yarwood (1956)

using bean and TMV.

 

46

In considering the influence of pre- and post-
inoculation environment on the number of local lesions,
illumination is related to the.number of lesions formed
(Table 4). Bawden and Roberts (1947, 1948) indicated that
predisposition factors are.more important than conditions
after inoculation, this is not the case with illumination
of Q. globosa inoculated with PVX. Although this may be
true if the consideration is given only to susceptibility
because predisposition may well affect susceptibility while
post—inoculation treatments affect more directly virus
multiplication.

The role temperatureplays provides information for
further Speculation. The influence of temperature before
inoculation varied as light conditions were modified.

That is, if no additional light were supplemented, the
Imaxtmum number of lesions developed at 28°C while with con-
tinuous illumination the minimum number develOped at 28°C.
With no supplemental light but with 1 leaf of each plant
covered with foil the minimum number was formed at 22°C on
both the foil covered and uncovered leaves. If a predis-
position temperature of 22°C is considered.as the Optimum
for the production of an antiviral substance or a virus in—
hibitor,.and.if predisposition in the dark enhances form-
ation and systemic movement of such a substance, then the
decrease in lesion number at 22°C and under dark predis-

position would be explained. If such a substance is formed,

then it seems that temperatures above and below 22° are not

47

favorable for its formation. .Naturally many other reasons
may be given for such a reduction in lesion number,.among
these is the susceptibility change whidh occurs with pre-
inoculation environmental changes suggested by Wiltshire
(1956a).

Carbohydrate.production probably is not the single
reason for increases or decreases at any one temperature,
because photosynthesis and respiration occur over a wide
range of temperatures, It seems unlikely that photosynthesis
or respiration at 220C would be much slower than at 17°C.
However it is postulated that the carbohydrate pool
established by the photosynthesis/reSpiration ratio as well
as inhibitor production, play important roles in local
lesion formation. The prOper combination of these two
materials may well govern local lesion formation. It may
not be out of line to hypothesize that the darkened leaf of
each plant may serve as a reservoir for anti-viral (or
better, anti-lesion) substances and as a drain for carbo—
hydrates synthesized in the Opposite and illuminated leaf.

When continuous illumination was maintained, the
maximum.number of lesions develOped at a post inoculation
temperature of 22°c. It is thought however, that the
clearly delimited lesions which are produced at 28°C pro-

vide a mudh better assay than at the lower temperatures.

Results of-the tests concerned.with pre- and post—

inoculation environments varied as the light conditions

were varied. Lesions were consistently reduced in number

48

When plants were predisposed by darkness. The results
suggest that light played a more direct ,..role in lesion
formation than did temperature. These results are similar
to those reported for other virus—host combinations
(Lindner gt 3;. 1959).

PVX infectivity is reduced by Q. globosa leaf sap
obtained from inoculated or non-inoculated leaves, and the
greatest inhibition was obtained with sap from leaves
incubated in the dark (Fig. 7; Table 8). On the other
hand, it has been shown by Bawden and Roberts (1948), that
bean or tobacco sap inhibit TMV or TNV to the same degree
regardless of the illumination.conditions of the plants.
Their hypothesis was that labile products of photosynthesis
combine with virus particles and render them non-infective.
They could not support this View with a-decreased inhibitor
titre from darkened leaves. Later, Matthews (1953a)
showed that the time of maximum sugar accumulation
corresponded to the time of maximum susceptibility, and
suggested that the accumulation of inhibitory photosynthetic
products is unlikely. Increased inhibition reported in the
present paper, is in accord with the view of Matthews.

The action of inhibitors from inoculated and non-
inoculated leaves seems to be quantitatively different.
Differences_may simply be due to quantitative differences

in formation, but they may also be due to the production
of virus pre-cursors i.e. interferon (Isaacs—and Lindemmann

1957: Loebenstein, 1963) in inoculated leaves. It is of

49
interest to note that virus multiplication continues and
in fact increases, regardless of the formation of hypo—
thesized inhibitors under darkened conditions. An inter-
feron system could be responsible, and appear as an
inhibitor in bio—assay. Such precursors, if blocked,
might result in inhibition as they pile up. If such pre—
cursors are present and non-infective, presumably
dilution would be required before the maximum virus titre
was realized by bio—assay.

Virus infectivity of light and dark incubated
leaves and plotted against dilution (Fig. 7) show curves
which are not parallel. This indicates that inhibitors
in light and dark incubated leaves may not be the same, or
if they are the same, the activity or concentration of
each may be different. Inhibition which results after
leaves have been ground up, is no evidence that such in-
hibition occurs when'the leaves are intact. The exact
cellular loci of inhibitor formation may be far removed
from that of viral synthesis. The evidence at hand seems
to suggest that this is the case; although inhibition was
demonstrated in leaves in the dark, multiplication of the

virus proceeds at an accelerated rate.

 

 

SUMMARY

The influence of light and temperature onthost
symptom response and virus multiplication in Gomphrena
globosa L. inoculated with PVX was studied. Of the 2
factors, light exerted the greater and more consistant
influence.

The number of local lesions was associated with the
length of the dark period. Periods of 14 days in the dark
all but excluded the formation of lesions. The appearance
of the first lesions on leaves darkened for less than 13
days was delayed by the length of the dark period. The
effect of the dark period was lost if leaves were not
darkened until 1-4 days after inoculation.

Symptom response was latent in darkened and excised
leaves but virus infectivity titre increased by as much as
3 fold. In addition, slight limited movement of the virus
was demonstrated from inoculated to non—inoculated regions
in darkened leaves and may have accounted for the increased
infectivity titre, Susceptibility was correlated with
light conditions, resistance being greatest from morning
inoculations and decreased from afternoon and evening
inoculations.

The influence of darkness was reversed when sucrose
or glucose were used as nutrient solutions. Lesion

numbers were increased and infectivity titre decreased in

50

 

51
darkened leaves as the molarity of the carbohydrate was

increased up to 0.5 M.

More virus inhibition was demonstrated in juice
from leaves incubated in the dark than from leaves in-
cubated in the light. The virus infectivity curve from
bio—assay of inoculated leaves plotted against dilution,
was more steep from leaves incubated in the dark than from
leaves incubated in the light. This was thought to indi-
cate the presence of substances which had an inhibitory

influence.

 

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