ENVIRONMENT AND SPORULATION IN PHYTOPATHOGENIC FUNGI
IV.
THE
EFFECT
OF
LIGHT
ON
THE- FORMATION
OF
CONIDIA
OF
PERONOSPORA
TABACINA ADAM
By I. A. M.
CRUICKSHANK'
[111anuscript received September 21, 1962]
Summary
Using a leaf·disk technique, an analysis of the effect of light on the sporulation
intensity of P. tabacina was carried out. The following points were demonstrated:
(I) Under conditions of continuous light, sporulation of P. tabacina is sensitive
to very low light intensities. ED5!1 for inhibition of sporulation was
16 ftvV cm- 2 for incandescent light (4 f,c.), and 0·58 p.W cm- 2 for a
monochromatic light source (469 mpo) in the region of maximum
effectiveness.
(2) Dark treatments induced sporulation under otherwise continuous light
conditions. The response was directly proportional to the length of the
exposure to darkness over the period 1·5-7 hr.
(3) The time of day at which sporulation occurred could be modified by
adjustment of the time of day at which darkness was initiated.
(4) Within the visible spectrum, the region exerting maximal inhibition on
sporulation occurred at 450-525 mJL.
The results are discussed in relation to the role of light in physiological aspects
of sporulation.
r.
INTRODUCTION
Yarwood (1937) reported that light and the alternation of light and darkness
in the normal day may determine the course of the diurnal cycle of sporulation
in the downy mildews of hop, onion, grape, and lettuce. In preliminary observations
on the effect oflight on sporulation (Cruickshank 1958), Yarwood's general conclusions
were found to apply also to Peronospora tabacina Adam, the causal organism of
do·wny mildew (blue mould) of tobacco. These observations indicated, however,
that the effect of light on sporulation of P. tabacina was more indirect than suggested
by Yarwood. Kajiwara and Iwata (1959) in related studies on the effect of light
on sporulation of Pseudoperonospm"a cubensis (Berk. & Curt.) Rostov. suggested
that alternating light and darkness may be responsible for the cyclic manifestation
of sporulation by an indirect effect through the host. These observations indicated
it might be of interest to examine in detail some aspect of the photosensitive system
which influences sporulation of P. tabacina. This paper is concerned ,,,ith single
cycles of sporulation only. It reports the effects on sporulation of various light
intensities, various photoperiods, and the action spectrum of ,,,hite light on sporulation
of P. tabacina in vivo.
* Division of Plant Industry,
C.S.I.H.O., Canberra.
AV,8t. J. Bioi. Sci., Vol. 16, No.1
ENVIRONMENT AND SPORULATION IN PHYTOPA'l'HOGENIC FUNGI. IV
II.
89
:MATERIALS AND METHODS
Disks cut from tobacco (Nicotiana tabacina cv. Virginia Gold) leaves infected
with P. tabacina were used as the basic biological unit in the experiments to be
described. The tobacco plants were grO\vn under controlled environment conditions
(day temp. 25'0, night temp. IS'O, natural lighting). The leaf disks' were cut on
the 7th day after inoculation of the leaves and were immediately incubated in a
water-water system in a constant-temperature room at 20~C (Cruickshank 1961).
Sporulation intensity per unit area of leaf was measured as described by Cruickshank
and Muller (1957).
A light-tower with an incandescent light source (10-50 W) was used for the
graded light intensity studies. The tower was calibrated "rith an "EEL" photometer
for light incident on the leaf disk surfaces. A light-tight cupboard, in combination
with an area of bench uniformly illuminated (50 f.c.) by a. bank of daylight-type
fluorescent tubes, was used in the photoperiod experiment~.
In the action spectrum experiments, infected .leaf disks were irradiated with
filtered light of wavelengtbs from 402 to 760 m!, under conditions of equal energy
(1· 5 !'W cm- 2 of leaf surface). The light source was a 500-Wtungsten projection
lamp combined with narrow-range Zeiss-Jena interference filters. The light beam
was reflected through 90' and passed through a "Perspex" filter 5 mm thick to
prevent any heat from the original light source passing into the exposure chamber.
A Kipp compensated thermopile and IGntel electronic galvanometer were used
to calibrate the light system for each filter.
The abaxial surface of the leaf disks was irradiated unless otherwise stated.
In the light intensity and action spectrum series of experiments, control leaf disks
were used for each intensity level and each filter. They were cut from areas of
infected leaf immediately adjacent to those from which the irradiated disks were
taken, and were placed in micro-environment chambers masked with aluminium
foil, within the exposure chamber. The effect of each light treatment was calculated
from the sporulation intensity counts for each set of paired disks (treated/control X 100).
Leaf disks were randomized and, with the exception of the action spectrum series,
six or eight replications were used in all experiments. For the action spectrum
experiments four replications only were used on account of the restricted area of
uniform illumination available.
The sporulation intensity data were statistically analysed after logarithmic
transformation [y = log (X+l)]. For convenience of comparison, the data within
experiments, were converted to a 0-100 scale and the mean values plotted as sporulation intensity indices.
III.
EXPERIMENTAL AND RESULTS
(a) Effect of Intemity of Continuous Light on Sporulation
Leaf disk samples were cut from plants at 1600 hr and placed in the light-tower.
They were irradiated continuously until 0900 hr the following morning, after which
time they were harvested and sporulation intensity measured.
* Infected leaf disks incubated in this way normally would be expected to sporulate between
0300 and 0600 hr on the day after initiation of incubation (Cruickshank 1958).
90
1. A. M. CRUICKSHANK
In the first experiment in this series, leaf disks were exposed to a series of
light intensities ranging from 0 to 50 f.c. The results of this experiment, presented
in Figure 1, show that, under conditions of continuous light, sporulation of P. tabacina
is sensitive to very low light intensities (ED50 for inhibition of sporulation = 4 f.c.
of incandescent light or approximately 16 p.W cm- 2 ). Almost complete inhibition
100
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" 50
~
Z
c
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040
•
~
30
20
10
o
o
10
W
30
I
!
~
00
INCANDESCENT LIGHT INTENSITY (F.e.)
Fig. I.-Relationship between light intensity for
continuous irradiation of infected tobacco leaf disks
and sporulation of Pero1Wspora tabacina.
occurred at 20 f.c. In the second experiment, the effect of irradiation of the abaxial
versus the adaxial surface of the leaf disks on sporulation was compared. A light
intensity of 50 f.c. was used, and the leaf disks were suitably masked and arranged
to prevent stray light reaching the unexposed leaf surface. Sporulation, which
normally occurs only on the abaxial surface, was completely inhibited, irrespective
of the leaf surface irradiated. In the last experiment in this series, whole plants were
ENVIRONMENT AND SPORULATION IN PHYTOPATHOGENIC FUNGI. IV
91
irradiated (50 f.c.) in clear "Perspex" humidity cabinets. Leaf disks were taken
for sporulation intensity measurements at the end of the irradiation period. The
mean value for the sporulation intensity index of the irradiated plants was 9 as
compared with 100 for the controls in the dark.
(b) Effect of Photoperiod on Sporulation
In this series of experiments the effect of dark treatments given previous to
the time of conidiophore emergence and conidia formation was examined .
WO
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80 ' X
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70
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20
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o
I
I
I
2345678
LENGTH OF DARK PERIOD (HR)
'. .....
J
9CONTINUQUS
DARKNESS
Fig. 2.-Relationship between length of exposure of infected tobacco
leaf disks to darkness and sporulation of PeronDspora tabacina.
(i) Relationship between Length of Dark Treatment and Sp01'Ulation Intewity.-
Leaf disks were cut and placed in the dark at 1200 hr. Sets of replicate disks (8)
were withdrawn from the dark condition after time periods varying from 0 to 9 hr
and transferred to a condition of uniform continuous illumination (50 f.c.) for the
balance of the experimental period, ending at 0900 hr as in the previous experiments.
The results shown in Figure 2 indicate that, under otherwise continuous light conditions, relatively short dark treatments of the leaf disks stimulate sporulation. The
minimum dark treatment to produce some sporulation under these conditions was
between 1 and 2 hr. After 7 hr, sporulation occurred equal in intensity to that
obtained under conditions of continuous darkness.
(ii) Effect of High Light Intensities on Sporulation Subsequent to Dark Treatment.Leaf disks, after 6 hr continuous dark treatment (from 1200 to 1800 hr), were exposed
to light intensities (fluorescent) ranging from 50 to 800 f.c. from 1800 hr to 0900 hr.
92
I. A. M. CRUICKSHANK
The results of this test (Table 1) suggested that a slight depressive effect on sporulation
occurred at the maximum light intensity used. However, the effect was not significant
(P>0·05). This result confirmed that even at light intensities greatly in excess of
saturation (25 f.e.) there was no inhibitive effect on the conidiophore emergence or
the conidia formation phases of sporulation.
(iii) Effect of Multiple Short Dark Treatments.-Leaf disks were cut from infected
plants and immediately placed in darkness as described in Section III(b)(i). The
total treatment period for all treatments was 6 hr of darkness, and exposures in the
different treatments in this experiment ,vere given intermittently with exposures
to light (50 f.c.). The disks were exposed to multiple dark periods of 0'75, 1·50,
and 3·0 hr and a single continuous period of 6 hr. The length of the corresponding
intermittent light periods was 0·25, 0·50, 2, and 0 hr respectively. The experiment
TABLE I
EFFECT OF HIGH LIGHT INTENSITY ON SPORULATION FOLLOWING A 6·HR
DARK PERIOD
Light
Intensity
(f.c.)
50
100
200
Mean Sporulation
Intensity Index
82·6
98·0
72·6
Light
Intensity
(f.c.)
400
800
Continuous dark
Mean Sporulation
Intensity Index
80·7
75·7
100
was arranged so that the total of 6 hr dark treatment was given over approximately
the same period of time irrespective of the length of the individual multiple dark
treatments. Mter the full 6 hr dark treatment was complete, all leaf disks were
transferred to uniform light conditions (50 f.c.) until the termination of the experimental period (0900 hr). The results are presented in Figure 3. Two periods of 3 hr
darkness were as effective as one period of 6 hr or continuous darkness. Multiple
dark treatments of the two shorter durations were less effective than the 3~hr
treatment. Their effect was, however, highly significant and greater than would
have been the effect of single dark treatments of length equivalent to one exposure
of 0·75 or 1·5 hr.
(iv) Relationship between Time of Onset of Darkness and Time of Initiation of
Conidial Formation.~As pointed out earlier in this paper the time of day at which
sporulation, that is, conidiophore emergence and conidia formation occurs under
conditions of natural lighting is between 0300 and 0600 hr on the day subsequent
to the initiation of favourable conditions of relative humidity and temperature.
This is' not influenced by the time of initiation of humidity provided the period of
humidity coincides with the normal time of sporulation (Cruickshank 1958). To
determine if the time of initiation of darkness affects the time of initiation of the
formation of conidia, infected leaves of tobacco plants were sampled at 1200 and
1600 hr. The leaf disks were immediately placed in darkness in the micro~environment
ENvmONMENT AND SPORULATION IN PHYTOPATHOGENIC FUNGI. IV
93
chambers with the covers removed to prevent the occurrence of high humidity. All
covers were replaced at 2000 hr and the leaf disks harvested sequentially until
0900 hr. The results shown in Figure 4 indicate that the time of day at which sporulation occurred could be influenced by the time of day at which dark treatments
.00
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3'0
6·0
CONTINUOUS
DARKNESS
OURATION OF DARK PERIODS (HR)
o
8
4
2
NO. OF DARK PERIODS
Fig. 3.-Relationship between duration X number of periods
of exposure to darkness of infected tobacco leaf disks and
intensity of sporulation of Peronospora tabacina.
were initiated within a 24-hr sporulation cycle. Additional sampling of infected
plants, at shorter intervals through the day, to determine if the final intensity of
sporulation response was affected by the time of day leaf disks were cut from plants,
was also carried out. No effect was demonstrated when plant material at the optimal
stage of incubation (Cruickshank 1961) was used.
94
I. A. M. CRmCKSHANK
(0) Effect of Filtered Light on Sporulation Intensity
The variability of sporulation exhibited as a difference in response at various
wavelengths, under conditions of equal energy (1·5 /kW cm- 2 ), was measured as
described in' Section II. The results are presented in Figure 5. Inhibition was in
excess of 50% at all wavelengths from 432 to 671 mf'. No significant inhibition
occurred at 402 mf' or in the spectral region 715-760 mf'. Within the effective
range, an inhibition maximum occurred at 469-524 mtt; a shoulder occurred in
o
'00
•
o
90
TREATMENT
INITIATING TIMES
eOr
x
W
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0 1200 HR
•
1600 HR
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•
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~ 50
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o
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40
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•
•
30
zo
10 ' -
o
zooo
2.4~~
0400
0700
TIME OF DAY (HR)
Fig. 4.-Relationship between time of initiation of darkness
and time of sporulation.
the 600-625 mfL region. Thus, within the visible spectrum, the blue-green region was
primarily responsible for sporulation inhibition under continuous irradiation condi~
tions. The effect of graded energy levels of a monochromatic light source (469 ffif')
on sporulation intensity is shown in Figure 6. For the narrow spectral region
represented by this filter, the ED50 value for inhibition in terms of energy required
for the reaction was 0·58 fL W cm- 2 of leaf surface.
IV.
D,SCUSSION
In this discussion, sporulation is considered to be a multi~step process of
change from vegetative to reproductive growth culminating in the formation of
conidia. This study has revealed that sporulation of P. tabacina in leaf tissues is
inhibited by continuous incident light of very low intensity on either surface (Fig. 1).
It has also shown, however, that light is ineffective as an inhibiting agent if the
ENVIRONMENT AND SPORULATION IN PHYTOPATHOGENIC FUNGI. IV
95
light treatment is preceded by relatively short exposures to darkness (Fig. 2). This
applies even when light intensities given are many times in exces~ of light saturation
levels under continuous irradiation.
The relationship between length of dark treatment given either as a single
continuous period, or as multiple intermittent periods, suggests that a biochemical
step or steps essential to sporulation takes place in a dark inductive period which
may be experienced many hours prior to the external appearance of conidiophores
90
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00
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[
400
,00
---
---
---
[!
600
650
I
700
-750
WAVELENGTH OF INC1DENT LIGHT (M,u.)
Fig. 5.-Action spectrum of sporulation of Peronospora
tabacina in infected tobacco leaf disks. Energy level constant
and equal to 1·5 p.W per square centimetre of leaf.
and conidia. The time at which darkness is initiated appears to exert some measure
of control over the time at which conidiophores emerge and conidia are formed.
Darkness thus appears to play a dominant and irreversible role in sporulation of
P. tabacina. Sporulation of P. tabacina appears, in fact, to be a dark induction
phenomenon.
A more detailed consideration of the results presented above indicates that
the biochemical changes which occur during the dark period are quantitative ones.
Evidence in support of this is firstly that intensity of sporulation is dependent on the
length of the dark period, and secondly that sporulation intensity is related to
96
1. A. 1\1. CRUICKSHANK
the incident light intensity and the spectral region of light. The changes which occur
are also cumulative and not nullified by intermittent light periods. The simplest
biochemical situation which would explain most facets of the phenomenon described
would be the formation or destruction over the dark period of a metabolite, which
was required for or was inhibiting to the subsequent biochemical steps which result
in the formation of conidia. Once this dark-dependent step or steps is complete
the subsequent steps occur independently of light.
'00
90
BO
70
•0
X
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>
60
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"?: 50
W
Z
0
C
<
J
o
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40
0
"
ill
'0
20
•
>0
0
0
0-5
ENERGY LEVEL
1'0
(A
\·5
469 MIL)
Fig. 6.-Relationship between energy level of monochromatic
light (>t = 469 mIL) at surface of infected tobacco leaf disks and
intensity of sporulation of Peronospora faba.cina.
Although continuous light is not a normal condition in nature, it is of interest,
from the point of view of the physiology of reproduction in fungi, to consider this
aspect of the studies reported. The first point which emerges is that sporulation
inhibition by light involves a very Imv energy reaction. From this it is concluded
ENVIRONMENT AND SPORULATION IN PHYTOPATHOGENIC FUNGI. IV
97
that major metabolic cyoles are not involved and that the dark induction phenomenon
is probably linked with hormonal ohanges whioh are inhibited by a photosensitive
mechanism.
In the in vivo system used, the variability of sporulation exhibited as a
difference in response at various wavelengths (Fig. 5) represents the net action spec.trum for sporulation of P. tabacina. Ingold (1959) pointed out that, in the fungi
generally, when light exerts an effect, as in phototropism, stimulation of spore
disoharge, or in carotinoid pigment production, it is the blue rays ("max. 450 m,u)
which are effective. The studies referred to were carried out in vitro. The direct
effect of light on the fungus was not in doubt. In the studies reported here, the
fungus was initially growing vegetatively within the leaf tissues. Where inhibition
of sporulation was complete, fungal development remained within the leaf in the
vegetative condition. The effect of light on the fungus, in this case, could only be
assumed because of the correlation between the fungal responses which could be
measured and the light treatments applied. Support for the ilirect effect of light
on the microorganism is given by the "max. for sporulation inhibition at 450-525 m,u;
however, it must be pointed out that, when the full action spectrum for sporulation
of P. tabacina over the visible light range is compared with the action spectrum
for phototropism of Phycomyces (Curry and Gruen 1959), it is seen that they are
different.
Figure 5 represents the action spectrum due to incident light at the leaf surface
under constant energy conditions. However, as pointed out above, P. tabacina,
in its vegetative stage, grows within the tissues of the tobacco leaf. The photosensitive system which is associated with sporulation inhibition must thus be related
to the light absorbed by the leaf or by the fungus withln the leaf. If the former
. situation is considered together with the data of Moss and Loomis (1952) for the
absorption spectrum of tobacco leaf and the ratio of sporulation to percentage
light absorption calculated, the derived curve for sporulation inhibition does not
vary qualitatively from that in Figure 5. The quantitative level of the inhibition
maximum in the 450-525 m,u remained unchanged. The inhibition of sporulation
at 611 mp. was more pronounced than in Figure 5, but owing to the uncertainty of the
position of the curve due to the experimental data in this spectral region, 110
significant difference between the ratios at 542 and 611 mp. could be sh0'vn. The second
situation, namely the absorption of light by the fungus in the leaf, does not readily
lend itself to experimental testing as colour changes occur in an infected leaf
associated with infection and development of the fungus, but not directly due to
the fungus per se. In vitro studies are not possible as P. tabacina is an obligate
pathogen. The experimental data obtained in the action spectrum series reported
thus could not be adjusted with due allowance for light absorption by the fungus.
Owing to the complications of the photosensitive system involved in these studies,
the action spectrum can only be regarded as an empirical one. No biochemical
implications can be drawn from it in regard to the nature of the photoreceptor
involved.
The measurements on the sporulation responses to phototreatments reported_
here show that under natural light conditions sporulation of P. tabacina is a dark-
98
I. A. M. ORUICKSHANK
induction phenomenon. Under experimental conditions light was shown to exert an
effective control over sporulation; the most effective spectral region of white light
being 450-525 m,u. These results are of interest as observations on which may be
based biochemical investigations of the photosensitive system involved in the
physiology of asexual reproduction of tills fungal pathogen.
V.
ACKNOWLEDGMENTS
The author wishes to thank Mr. S. W. Thorne for the design and calibration
of the light system used in the action spectrum experiments and Dr. E. J. Williams,
Division of Mathematical Statistics, C.S.I.R.O., for his examination and analysis
of the data.
VI.
REFERENCES
CRUICKSHANK, I. A. M. (1958).-Aust. J. Biol. Sci. 11: 162-70.
CRUICKSHANK, I. A. M. (1961).-Aust. J. Biol. Sci. 14: 19S---207.
CRUICKSHANK, I. A. M., and MfuLER, K. O. (1957).-Nature 180: 44--5.
CURRY, G. M., and GRUEN, H. E. (1959).-Proc. Nat. Acad. Sci., Wash. 45: 797-804.
INGOLD, C. T. (1959).-In "Vistas in Botany". pp. 348--86. (Ed. W. B. Turrell.) (Pergamon
Press: London.)
KAJIWARA, J., and IWATA, Y. (1959).-Ann. Phytopath. Soc. Japan 24: 109-13.
Moss, A. R., and Looms, W. E. (1952).-Plant Physiol. 27: 370-9l.
YARWOOD, C. E. (1937).-J. Agric. Res. 54, 365-73.