Sporulation of Plasmopara viticola: Differentiation and Light ...

Abstract: The development of grape downy mildew �Plasmopara
viticola) was followed histologically during the entire latent
period until the appearance of mature sporangia. Production of
sporangiophores and sporangia was assessed using low-temperature
scanning electron �LTSEM) and fluorescent light microscopy.
Time-course studies using attached leaves of Vitis vinifera
cv. Müller-Thurgau revealed that the production of sporangiophores
and sporangia is a highly coordinated process and is
completed within 7h. As this differentiation is assumed to occur
only in darkness, the influence of light was investigated. For this
purpose, different light regimes were applied to infected leaf
discs of V. vinifera cv. Müller-Thurgau. White light irradiation
prevented formation of sporangia, although the growth of the
mycelium was not affected. Many sporangiophores were observed
that were abnormally shaped, i.e., short hyphae in clusters
or thin, extremely elongated hyphae. For the formation of
mature sporangia, a prolonged dark period was necessary. Light
experiments suggest photosensitivity at the end of the latent
period. A terminal white light irradiation caused an inhibitory effect,
whereas a final phase of darkness promoted sporangium
development. Different light qualities were tested, revealing an
inhibition of sporangium development by blue light whereas
neither red nor far-red light were effective.
Key words: Light regulation, low-temperature scanning electron
microscopy, morphogenesis, oomycetes, sporulation.
Introduction
Plasmopara viticola is an oomycete pathogen causing downy
mildew of grapevine, a severe disease in temperate winegrowing
regions. Oomycetes are fungal-like members of the
kingdom Chromista and are closely related to heterokont algae
forming a distinct group divergent from true fungi, green algae
or plants �Kumar and Rzhetsdy, 1996 [18] ; Van de Peer and De
Wachter, 1997 [29] .
Original Paper
Sporulation of Plasmopara viticola: Differentiation and
Light Regulation
J. Rumbolz 1, 4 , S. Wirtz 1 , H.-H. Kassemeyer 2 , R. Guggenheim 1 , E. Schäfer 3 , and C. Büche 2
1 REM-Labor, Universität Basel, Pharmazentrum, Basel, Switzerland
2 Staatliches Weinbauinstitut, Merzhauser Str.119, 79100 Freiburg i.Br., Germany
3 Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Schänzlestr.1, 79104 Freiburg i.Br., Germany
4 University of California, Department of Plant Pathology, One Shields Ave., Davis, CA 95616, USA
Plant biol. 4 �2002) 413±422
Georg Thieme Verlag Stuttgart ´ New York
ISSN 1435-8603
Received: August 22, 2001; Accepted: April 4, 2002
The asexual part of the life cycle of the pathogen is characterized
by short disease cycles. Under favourable conditions, the
mycelium is able to sporulate 3 days after infection �Dai et al.,
1995 [9] ). A relative humidity of nearly 100% is required for
sporulation �Blaeser and Weltzien, 1978 [2] ). Sporangiophore
development is the process in which mycelium grows out of
stomatal cells forming hyphae that branch in a species-typical
manner. Finally, sporangia develop at the tips of the branches.
Early studies suggested that sporulation of P. viticola occurs at
night �Müller and Sleumer, 1934 [23] ). This was confirmed by
Brook �1979 [3] ), who assessed the sporulation visually after incubation
of infected leaf discs in different light qualities or in
the dark. Sporulation proceeded in darkness, but was inhibited
by both near-UV light and light in the blue-green region of the
spectrum.
In general, only a limited number of detailed studies exist for
light-regulated development in oomycetes. Examples are the
positive phototaxis of Phytophthora cambivora zoospores �Carlile,
1970 [4] ) and the effect of humidity and light on discharge
of sporangia of different oomycete pathogens �Fried and Stuteville,
1977 [13] ; Leach et al., 1982 [20] ; Su et al., 2000 [28] ). The first
reports concerning sporangiophore and sporangium differentiation
are a description of both stages in Peronospora trifoliorum
�Fried and Stuteville, 1977 [13] ) and a study of sporangium
production in Pseudoperonospora cubensis �Cohen and Eyal,
1977 [7] ). The latter reported an inhibition of sporangium production
by blue light which did not prevent emergence of
sporangiophores through stomata.
The purpose of our studies was to describe in detail the morphology
of P. viticola during sporulation and to define developmental
stages during this process. Based on these data, we focused
on the influence of light on sporangiophore and sporangium
development. The duration and time point of the light
treatments leading to a response and the efficiency of different
light treatments were of major interest. The experiments in
which infected host tissue is exposed to different light regimes
display the plasticity of the host±pathogen system with respect
to differentiation of the obligate biotrophic pathogen.
Our data document a strong impact of different light conditions
on the development of grape downy mildew, in particular
during the late stages of its vegetative infection cycle.
Therefore, these results provide further insights into light regulation
of an oomycete species.
413

414
Plant biol. 4 �2002) J. Rumbolz et al.
Materials and Methods
Plant and fungal material
Two-node cuttings of grapevine �Vitis vinifera L. cv. Müller-
Thurgau) were rooted in pots and grown in the greenhouse under
a natural photoperiod until shoots reached a length of
40 cm. Plants were kept free from powdery mildew infections
by vaporization of sulphur for 5 h per day. For all experiments,
the fifth or sixth expanded leaf of each plant, counted from the
apex, was used. Leaf discs were prepared after surface sterilization
with a tissue wipe soaked with 75 % ethanol and subsequent
rinsing in distilled water. Discs were excised with a cork
borer �diameter 16 mm) and placed onto water agar �0.8 %) in
petri dishes. In additional experiments, leaf discs were also
placed on water agar containing 2 % sucrose.
Plasmopara viticola �Berk. and Curt.) Berl. and De Toni was
obtained from a natural field infection and maintained on
cuttings of V. vinifera cv. Müller-Thurgau �see above) in the
greenhouse. Propagation of the pathogen was carried out by
misting abaxial leaf sides of cuttings with a spore suspension
�20 000 sporangia ml ±1 distilled water) until run-off, and covering
of entire plants with wet polyethylene bags overnight.
Bags were removed on the following day and plants were kept
in the greenhouse for 5 ± 6 days, corresponding to the latent
period of P. viticola,at20±268C. Another overnight incubation
of inoculated plants under the wet plastic bag yielded new
sporangia. Fresh inoculum was harvested with a paintbrush
into centrifuge tubes. To prepare spore suspensions used in
the experiments, sporangia were counted using a Fuchs±Rosenthal
counting chamber and diluted to a final concentration
of 20 000 sporangia ml ±1 .
Sporulation experiments
To observe the morphogenesis of P. viticola during sporulation,
attached leaves of cuttings of V. vinifera �cv. Müller-Thurgau)
were inoculated by misting the abaxial leaf surface with a
spore suspension until run-off. Plants were covered overnight
with wet polyethylene bags. Bags were removed on the following
day and plants were kept in the greenhouse under the natural
photoperiod. Sporulation was induced after 6 days by covering
the entire plants with wet polyethylene bags and transferring
them to darkness at dusk �8 p.m.). To monitor the time
course of sporulation, infected leaves were detached and areas
with downy mildew lesions were cryofixed in liquid nitrogen
at hourly intervals until the following morning �6 a.m.). Samples
were further prepared for low-temperature scanning
electron microscopy �LTSEM) studies, as described below. All
experiments were carried out between April and June and repeated
in triplicate.
Light experiments
Leaf discs placed on water agar were inoculated abaxially with
a100-ml droplet of a spore suspension. Petri dishes were sealed
in order to avoid desiccation of the inoculation droplet and incubated
at 22 8C ambient temperature. To apply short-day conditions,
samples were incubated in growth chambers, immediately
after inoculation, with an irradiation of WL �300 mmol
m ±2 s ±1 , 10 Osram HQIL 400 W-lamps plus four Osram L40/
W60 fluorescent bulbs; Osram, Berlin, Germany) for 8 h and a
dark period of 16 h �Schäfer, 1975 [26] ). Continuous white light
�cWL, 80 mmol m ±2 s ±1 ) and continuous monochromatic irradiation
of blue light �436 nm, 38.5 mmol m ±2 s ±1 ), red light
�656 nm, 3 mmol m ±2 s ±1 ) and far-red light �730 nm, 14 mmol
m ±2 s ±1 ) was applied as previously described in Schäfer
�1977 [27] ).
To determine the time course of sporangium differentiation in
leaf disc experiments under short-day, constant darkness and
white light conditions, each sample consisted of 5 leaf discs
per petri dish. Discs were harvested at various time points between
68 and 98 h post-inoculation �p.i.) and either transferred
into 50 ml of 1M KOH for light microscopy or cryofixed in
liquid nitrogen for LTSEM. Assessment of pathogen development
was made with light and fluorescence microscopy, as described
below, as well as with scanning electron microscopy.
Experiments were repeated in triplicate.
In light experiments, where inoculated leaf discs �5 discs per
experiment, petri dish and variant) were exposed to white
light and kept in darkness for different time intervals �see
Figs. 4,6±8 for irradiation schedules), incubation was terminated
88 h p.i. Experiments were repeated in triplicate. Assessment
of sporangiophore and sporangium development was
done by light and epifluorescence microscopy, as described
below.
Light and epifluorescence microscopy
Leaf discs transferred to KOH after various incubation periods
were cleared by autoclaving the samples for 15 min. After cooling
down, KOH was decanted and samples were washed three
times in 50 ml distilled water. 0.05% Aniline blue �Merck,
Darmstadt, Germany) was dissolved in 0.067 M K 2HPO 4 pH
9.0 and used to stain intercellular hyphae, as well as fungal
structures of P. viticola which protruded from stomata of the
host epidermis �Williamson et al., 1995 [30] ).
Cleared and stained leaf discs were examined by light �bright
field) and epifluorescence microscopy using an Axiophot
�Zeiss, Oberkochen, Germany) microscope equipped with an
epifluorescence device �excitation at 365 nm; LP 397 nm). For
microscopic analyses, a leaf area encircled by leaf veinlets was
defined as a ªleaf unitº �Fig.1A). 20±25 leaf units per leaf disc
exhibiting tissue colonized by P. viticola were selected randomly
and used to determine the degree of differentiation.
The three stages occurring during sporulation of P. viticola
were defined as follows: 1. hyphal cluster �short or extremely
elongated hyphae in clusters, no branching), 2. sporangiophores
�elongated hyphae, unbranched and branched, without
sporangia) and 3. mature sporangia �branched hyphae with
spherical or drop-shaped sporangia). By assessing 20 ±25 leaf
units, the average number of sporangiophores with sporangia
�stage 3) per leaf and the number of sporangiophores without
sporangia �stages 1and 2) per leaf unit was determined, indicating
the degree of differentiation of P. viticola on the respective
leaf disc. Three leaf discs per variant and experiment were
randomly selected and all experiments were repeated in triplicate.
Since data for each replicate were almost identical, we
combined them in the final analysis. Data were compared
using the unpaired Student s t-test �p £ 0.05).

Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 �2002) 415
Fig.1 Intercellular growth of Plasmopara viticola within the spongy
mesophyll of host leaves �Vitis vinifera cv. Müller-Thurgau). �A) Epifluorescence
light micrograph of unseptate hyphae colonizing the
leaf areas between leaf veinlets, �top view). The square area encircled
by the leaf veinlets �arrowheads) was defined as a ªleaf unitº.
Scale bar: 50 mm. �B±D) Low-temperature scanning electron micro-
Scanning electron microscopy
Hyphal structures of P. viticola were examined with LTSEM
according to Rumbolz et al. �2000 [25] ). Following incubation,
fresh plant material �attached leaves or leaf discs from cuttings)
was taken, cut into pieces of 0.8 ± 1cm 2 and mounted
on a Balzer specimen holder using low-temperature mounting
medium. Samples were rapidly frozen by plunging them into
liquid nitrogen. After cryofixation, samples were transferred
under nitrogen gas to a Balzers cryopreparation unit SCU 020
attached to a JEOL JSM 6300 scanning electron microscope.
Ice crystals on the surface were allowed to sublime from the
specimens by raising the temperature to ± 808C for 5 ± 8 min.
Sputter coating with 20 nm gold was carried out in an argon
gas atmosphere �Müller et al., 1991 [24] ). For the examination
of fungal growth inside the leaf mesophyll, samples were fractured
at ± 1208C with a precooled knife prior to immediate
sputter coating. Coated specimens were transferred online
into the SEM under high-vacuum conditions. The samples
were observed and photographed at a stage temperature of
±1658C using an accelerating voltage between 5 and 10 kV.
graphs of freeze-fractured spongy mesophyll colonized by P. viticola
�side view). �B) Note granular structure of hyphae �h) compared with
mesophyll cells �m), which contain chloroplasts �cp). �C,D) Bulbshaped
structures �arrows) are situated underneath guard cells �g),
giving rise to sporangiophores �sph).
Results
Morphogenesis of P. viticola
The development of the pathogen was followed during the
entire latent period until the appearance of mature sporangia.
Colonization of the leaf mesophyll by P. viticola prior to sporulation
was assessed using both FLM �fluorescence light microscopy)
and LTSEM. Under the fluorescence microscope, intercellular
unseptate hyphae were easily distinguishable from
the background by their greenish fluorescence �Fig.1A). Comparative
studies on freeze-fractured specimens revealed intercellular
hyphae of P. viticola inside the spongy mesophyll,
identifiable by their granular structure, in comparison to the
surrounding host cells that contained large organelles, i.e.,
chloroplasts �Fig.1B). The granular structures were also present
inside bulb-shaped fungal cells that were situated underneath
guard cells during sporulation �Figs.1C,D). Hyphal
growth within the leaf was mostly restricted to the intercellular
spaces of the spongy mesophyll. Colonization of interveinal
tissue by P. viticola was further limited by the anatomy of leaf
veins, which appeared impassable for hyphae due to the dense
package of cells in the vascular tissue.

416
Plant biol. 4 �2002) J. Rumbolz et al.
Fig. 2 Low-temperature scanning electron micrographs of developmental
stages of Plasmopara viticola during sporulation on leaves of
Vitis vinifera cv. Müller-Thurgau. Sporulation was induced by transfer
of plants to darkness and exposure to high humidity. �A) Stoma of
host leaf that is already filled with sporangiophore initials �0 h after
darkening). �B) Hyphal tips protruding from stoma �2h). �C) Elongat-
To define developmental stages of P. viticola during the sporulation
process, time-course studies using attached leaves were
carried out. Observations were started immediately after
transfer of sporulation-induced plants to darkness. Differentiation
proceeded in a highly coordinated manner. At the time
ing external hyphae �3 h). �D) Branches of the first, second and third
order formed on sporangiophores �5 h). �E) Development of spherical
sporangia at the distal end of sporangiophores �6 h). �F) Dropshaped,
mature sporangia �7 h). �G) Distal end of a sporangiophore
bearing mature sporangia, which appear shrunken �9 h). Some sporangia
are already released �arrows).
of darkening �0 h), stomata were already filled with hyphal tips
protruding from the interior of the leaf �Fig. 2A). Two hours later,
hyphal tips were clearly visible �Fig. 2B). External hyphae
further elongated �3 h, Fig. 2C) until 4 h. Five hours after transfer
to darkness, hyphae with branches of the second and third

Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 �2002) 417
Fig. 3 Epifluorescence light micrographs of sporangiophore and
sporangium development of Plasmopara viticola on leaf discs of Vitis
vinifera cv. Müller-Thurgau. �A) Typical, branched sporangiophores
�sph) most abundantly found during sporulation. Some sporangiophores
bear mature sporangia �s). �B,C) Sporangiophores with abnormal
morphology observed after extended white light irradiations. On
some leaf discs, ªhyphal clustersº �hb) of unbranched hyphae were
dominating �B), whereas on other discs thin, extremely elongated
sporangiophores were found �C). Scale bars: 50 mm.
order were observed �Fig. 2D). By 6 h, spherical sporangia developed
�Fig. 2E), followed by drop-shaped, mature sporangia
�7 h, Fig. 2F). At 9 h in the dark, sporangiophores exclusively
bore shrunken sporangia, which appeared desiccated after
cryofixation �Fig. 2G).
Effects of light on differentiation of sporangia
As sporulation occurred as a coordinated developmental process,
we carried out more detailed studies on its regulation by
external factors. We focused on the effects of different light
treatments on sporulation. These experiments were done
using leaf discs to obtain a sufficient data base.
Using FLM, structures equal to those described in our LTSEM
studies were found. The most frequent stages identified were
branched sporangiophores �stage 2; see ªMaterials and Methodsº)
and sporangiophores bearing mature sporangia �stage 3,
Fig. 3A). A variation in the morphology of sporangiophores
was only present after extended white light irradiation
�Figs. 3B,C). These external hyphae, defined as ªhyphal clustersº
�stage 1) due to their spatial organization, were characteristically
unbranched. The latter were sometimes abnormally
shortened �Fig. 3B) or extremely elongated, with a reduced
diameter �Fig. 3C) compared with standard sporangiophores.
Since the formation of sporangia marks the borderline between
successful and unsuccessful asexual reproduction of
the pathogen, the number of both types of sporangiophores
without sporangia �stage 1and 2) was compared with the
number of sporangiophores yielding sporangia �stage 3) in
the following experiments.
Sporulation in short days, continuous white light
and continuous darkness
The time-course of differentiation under continuous light after
the end of the latent period is shown in Fig. 4. In short days
�Fig. 4A), sporangiophores appeared by the end of the third
day. Later on in darkness, the number of hyphal clusters/sporangiophores
without sporangia �stages 1and 2) slightly decreased
in favour of sporangiophores with sporangia �stage 3),
which were first found 73 h after inoculation. After 87 h, the
majority of sporangiophores possessed sporangia �p < 0.05). In
continuous darkness �cD) �Fig. 4B), sporangiophores and sporangia
were both present after 3 days, but were less frequent
than under short-day conditions. During the entire observation
period, levels of both stages remained constant. In contrast,
a strong inhibition of sporangium formation was found
under continuous white light �Fig. 4C). Many leaf areas with
external hyphae of P. viticola were observed which did not differentiate
further than stages 1and 2. Vegetative growth was
not affected, as the leaf mesophyll was densely interspersed
with intercellular mycelium �Figs. 3B,C). Instead of mature
sporangia or regularly formed sporangiophores, hyphal clusters
�stage 1) covered the diseased leaf areas.
Since full differentiation to mature sporangia was reached at
88 h p.i., this time point was used for the following experiments
to assess the impact of variable light and dark periods
on differentiation.
Sucrose effects
As shown in Fig. 4, short day and continuous darkness yielded
an excess of the stage ªmature sporangiaº �p

418
Plant biol. 4 �2002) J. Rumbolz et al.
Fig. 4 Time-course of differentiation of Plasmopara viticola on leaf
discs of Vitis vinifera cv. Müller-Thurgau in short day �SD, A), continuous
darkness �cD, B) and continuous white light �cWL, C) after the
end of the latent period �l sporangiophores with sporangia; ~ sporangiophores
without sporangia). Leaf discs were constantly exposed
to high humidity on water agar. Horizontal bars on top of figures
show the irradiation design with the period of data assessment indicated
by the bracket. Data points represent means of three experiments
with three leaf discs each, and error bars correspond to the
standard error of the means.
Fig. 5 Sporangiophore and sporangium development of Plasmopara
viticola on leaf discs of Vitis vinifera cv. Müller-Thurgau in short day
�SD), continuous darkness �cD) and continuous white light �cWL).
Leaf discs were incubated for 88 h on agar containing no or 2% �w/
v) sucrose. Values represent means of three experiments with three
leaf discs each, and error bars correspond to the standard error of
the means.
Effects of a dark period on the differentiation to sporangia
As the formation of sporangia requires a dark period, the time
and length of the dark phase necessary for this process was determined.
In the first set of experiments, one �B1± B3) or two
nights �A1±A3) during the light/dark cycle were replaced by
cWL. Fig. 6A shows that one night �16 h) was insufficient to
yield sporangia at all when placed at the beginning �A1) or
after 24 h in cWL �A2). However, shifting the dark period towards
the end of the incubation period resulted in the production
of a few sporangiophores with sporangia �A3). When irradiation
with cWL was interrupted by two intervals of darkness,
sporangia developed, but only if the dark periods were positioned
towards the end of the incubation period �B2 and B3;
Fig. 6B). A dark period at the beginning plus a dark period
24 h p.i. was not sufficient to allow sporulation �B1).
Because darkness was most effective at the end of the incubation
period to promote full sporangial differentiation of P. viticola,
terminal dark intervals were progressively reduced
�Fig. 7). Significantly more sporangiophores with sporangia
were found if terminal dark periods were 48 h or longer
�p < 0.05). Shorter dark intervals led to an increase of sporangiophores
without sporangia concomitantly with a reduction
of sporangial development �p < 0.05).
Evidence for the requirement of a dark period before the completion
of the vegetative life cycle was also supported by another
set of experiments. To determine the minimum WL period
required for inhibition of sporangium formation following
incubation in darkness, we performed an inverse irradiation
schedule to that shown in Fig. 7. Irrespective of irradiation
duration, WL applied at the end of the latent period generally
reduced the number of mature sporangia �Fig. 8). While in

Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 �2002) 419
Fig. 6 Sporangiophore and sporangium development of Plasmopara
viticola on leaf discs of Vitis vinifera cv. Müller-Thurgau. White light
was applied for 88 h, interrupted by dark periods of different number
and position. One night �16 h darkness) �B) or two nights �A) during
the light/dark cycle were replaced by cWL. Horizontal bars above
each figure show the irradiation regimes. Values represent means of
three experiments with three leaf discs each, and error bars correspond
to the standard error of the means.
short-time periods of 10 or 16 h of white light irradiation a significant
number of sporangiophores with sporangia was still
observed, exposure times to WL of 24 h and longer led to a surplus
of sporangiophores without sporangia �p < 0.05).
Effect of blue light
Since exposure of P. viticola to constant WL revealed a drastic
effect on differentiation, inoculated leaf discs were subjected
to light of different wavelengths. Red, as well as far-red light
yielded sporangia �Fig. 9). Far-red light caused similar differentiation
as cD. In contrast, blue light incubation gave rise to a
surplus of sporangiophores without sporangia, as observed in
Fig. 7 Differentiation of Plasmopara viticola on leaf discs of Vitis vinifera
cv. Müller-Thurgau after a white light irradiation followed by an
incubation in darkness �total incubation time: 88 h). The length of a
terminal dark interval was progressively reduced. Horizontal bars
above figure show the irradiation regimes. Values represent means
of three experiments with three leaf discs each, and error bars correspond
to the standard error of the means.
continuous white light �p < 0.05). Supplementing the agar with
sucrose had no effect on the light-dependent differentiation in
red, far-red and blue light �data not shown).
Discussion
The early steps of morphogenesis of Plasmopara viticola during
the infection cycle have been the subject of several microscopical
studies �Locci, 1969 [21] ; Langcake and Lovell, 1980 [19] ;
Kortekamp et al., 1998 [17] ). The main focus was to describe the
developmental stages of the pathogen from infection until
sporulation. There is little information on the terminal phase
of the life cycle, in particular, on the dynamics of sporangial
development. However, Arens �1929 [1] ), as well as Müller and
Sleumer �1934 [23] ), pointed out that sporangia appear between
6 to 10 h after incubation in the dark, if values for relative humidity
reach 75 ± 100%. In the present study, the single developmental
stages occurring during sporulation were documented
by a detailed microscopical analysis. From the timecourse
data, it is evident that sporulation is a highly coordinated
process. The good temporal resolution might be the result
of a synchronicity in development of fungal structures, which
has been described for other species of the Peronosporales
�Dick, 1990 [10] ; Falloon and Sutherland, 1996 [12] ).
An important feature of the mature sporangia is their ability to
shrink �Fig. 4G). The collapse of mature sporangia visualized in
the LTSEM was also described for cryofixed Peronospora viciae
�Falloon and Sutherland, 1996 [12] ), as well as for P. viticola after

420
Plant biol. 4 �2002) J. Rumbolz et al.
Fig. 8 Differentiation of Plasmopara viticola on leaf discs of Vitis vinifera
cv. Müller-Thurgau after incubation in darkness followed by a
white light irradiation �total incubation time: 88 h). The length of a
terminal light interval was progressively reduced �inverse to Fig. 7).
Horizontal bars above figure show the irradiation regimes. Values represent
means of three experiments with three leaf discs each, and
error bars correspond to the standard error of the means.
chemical fixation �Kortekamp et al., 1998 [17] ). From comparative
LM studies, which revealed a recovery of turgescence of
the propagules, it was concluded that the observed phenomenon
is not an artefact but an adaptation of the pathogen to
changing humidity conditions in the field. Studies on sporulation
of Peronospora trifoliorum showed that lowering relative
humidity caused rapid shrivelling of the sporangia and their
release from sporangiophores �Fried and Stuteville, 1977 [13] ).
Reports that sporangia of P. viticola are still viable after storage
for several days under dry air conditions �Müller and Sleumer,
1934 [23] ) support the thesis of reversible shrinkage.
Observations on freeze-fractured specimens of P. viticola during
intercellular growth prior to sporulation �Fig.1) corroborate
earlier TEM studies �Langcake and Lovell, 1980 [19] )in
which the bulb-shaped fungal structures were described as
swellings of hyphae within a substomatal cavity that give rise
to sporangiophores. This is in line with our own observations
of hyphal aggregations underneath stomata prior to the outbreak
of sporangiophores �data not shown). These structures
may be necessary to build up pressure for penetration through
closed stomata. The granular structure characteristic of cryofixed
intercellular hyphae of P. viticola might be due to the extensive
vacuolization, as revealed by transmission electron microscopy.
Indications for involvement of light in regulation of the sporulation
process were provided by the observation of several authors
�Cuboni, 1885 [8] ; Istvanffi and Palinkas, 1913 [16] ; Gregory,
Fig. 9 Differentiation of Plasmopara viticola on leaf discs of Vitis vinifera
cv. Müller-Thurgau after irradiation for 88 h with continuous blue
light �cBL), continuous red light �cR) and continuous far-red light
�cFR). Values represent means of three experiments with three leaf
discs each, and error bars correspond to the standard error of the
means.
1913 [14] ; Arens, 1929 [1] ) that sporangia are exclusively produced
at night. Knowledge of the effects of light on differentiation
of P. viticola exclusively relies on the studies of Arens
�1929 [1] ), Yarwood �1937 [31] ) and Brook �1979 [3] ). While Arens
�1929 [1] ) and Yarwood �1937 [31] ) both found an inhibition of
sporangiophore formation on detached leaves after irradiation
with light of high photon fluences, Brook �1979 [3] ) qualitatively
described a shift in differentiation from mature sporangia towards
sporangiophores after a treatment with light at the end
of the incubation period. In our experiments, we exposed infected
leaf discs to continuous white light during the entire incubation
period, or at least for an extended time during the life
cycle. Light inhibited the differentiation of sporangia, resulting
in a reduction in the amount of sporangia in favour of sporangiophore
formation. Interestingly, continuous white light did
not affect intercellular colonization by P. viticola. Thus,
restricted growth was not responsible for this effect. Moreover,
the mycelium was able to produce many sporangiophores
emerging from nearly all stomata. The microscopical observation
of abnormal sporangiophores formed in light aligns with
findings on Pseudoperonospora cubensis, the downy mildew
pathogen of cucurbits �Cohen and Eyal, 1977 [7] ). In addition to
the abnormally shortened sporangiophores described for this
pathogen, P. viticola sometimes showed extremely elongated,
thin sporangiophores bearing no sporangia. Taking into consideration
the SEM observation that late stages of sporangia
development are completed over short periods �Fig. 4), it is impressive
that the described inhibitory influence of light becomes
apparent long after the potential for differentiation is
established.
The strongest impact of light on morphogenesis was observed
when it was applied towards the end of the incubation period.
The length of an irradiation with WL required for an inhibitory
effect at a sensitive time was at least 24 h. This period of time
is much longer than is necessary for differentiation �7 h). Thus,
the competence to fulfil this inhibitory effect must be established.
A similar phenomenon was observed when the mini-

Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 �2002) 421
mum dark phase, necessary for sporangia formation, was determined
�48 h). This light-dependent reaction is very different
compared to the light-induced conidiation in several true
fungi, such as Aspergillus nidulans �Mooney and Yager,
1990 [22] ). Here, only 15 or 30 min of exposure is necessary for
a light response. However, in this organism, a light-sensitive
critical period also exists, from initiation of conidiation until
formation of differentiated structures.
Although the competence to respond to the light signal is
greatest at the end of the incubation period, there is also an
influence of light at the beginning of the infection cycle. One
dark phase of 16 h during an irradiation with cWL had no
effect, no matter if applied at the beginning or at the end of
the incubation period. However, synergistic effects could be
observed, if two dark phases were applied interrupted by an
irradiation of cWL of 8 h. The dark phases at the end or in the
middle of the incubation period led to a significant amount of
sporangia.
The availability of nutrients does not seem to influence the
light responses. As the pathogen lives in an environment
where the light and dark cycles determine the productivity of
the host plant, a reaction to these changes could, hypothetically,
be present. Also, an interaction of sucrose- and light-dependent
pathways could be possible, as observed in certain plant
species �Dijkwel et al., 1997 [11] ; Harter et al., 1993 [15] ). As an addition
of sucrose to the medium led only to an increase of productivity
of the mycelium and not to a change in the light response,
we can exclude any impact of nutrient availability.
P. viticola is an obligate parasite which makes it impossible to
investigate the light responses singly in one of the two organisms.
Thus, both organisms can theoretically contribute to the
observed light effects. The ecological benefit is for the parasite,
as the temporal regulation of sporulation promotes successful
development following infection. Under natural conditions,
sporulation occurs in the dampness of the early morning
hours, when humidity conditions are suitable for infection.
From this point of view, it seems to be most likely that the
pathogen plays an important role in mediating the light response.
As light in the green to blue-green region of the spectrum was
described to inhibit sporangium formation �Brook, 1979 [3] ), we
tested the influence of BL on the differentiation of sporangiophores
and sporangia. We found strong reduction of sporangia
in continuous blue light, similar to that observed under cWL.
Thus, blue light of high photon fluence rate, as used in our experiments,
might have been sufficient to cause inhibition. A
continuous irradiation with R or FR did not lead to an inhibitory
effect. The inhibition of sporangia formation as a blue
light response seems to be common for other members of the
Peronosporales �Cohen et al., 1975 [5] ; Cohen, 1976 [6] ; Cohen
and Eyal, 1977 [7] ). To obtain more insight into the photoreceptor
action, further information, such as action spectra, will be
required.
The inhibition of sporangial differentiation by light might be of
particular interest when estimating the potency of a sporulation
event under marginal conditions. Assuming that humidity
conditions favourable for an outbreak are not achieved until
the early morning hours, sporangiophores bearing almost no
sporangia may emerge. In this case, the potential for successful
sporulation the next night might be increased. The extent of a
sporulation event may be influenced by such preceded events.
This should be considered in epidemiological models. Taking
into account the protective characteristics of most oomycete
fungicides, a decision as to whether a control measure is necessary
immediately after conditions favourable for the pathogen,
but not fully sufficient for an outbreak, should be carefully
evaluated.
Acknowledgements
Thanks to M. Düggelin and D. Mathys, REM-Labor Universität
Basel, for technical assistance in part of the experiments and
to N. Papadopoulos, Davis, for advice on statistical analysis.
The authors are further grateful to C. Körner, Botanisches Institut,
Universität Basel, for providing facilities for light experiments
and to L. Hennig, Zürich, and P. McCabe, Davis, for critically
reading the manuscript.
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J. Rumbolz
University of California
Department of Plant Pathology
One Shields Ave.
Davis, CA 95616
USA
E-mail: jrumbolz@ucdavis.edu
Section Editor: J. Draper
Erratum
Plant Biology vol. 4, no. 2, 2002
Due to an unfortunate mistake which occurred after the then
still correct page proofs of Plant Biology vol. 4, no. 2, 2002,
had been checked by the editor �Ulrich Lüttge) responsible for
the contributions covered by the guest editorial of Matyssek et
al. p.133, the contribution by Reitmayer et al. p. 228 got misplaced.
It belongs to the set of 7 contributions covered in the
guest editorial and was supposed to be placed after Grams et
al. p.153 and before Pretzsch p.159.