Sporulation of Plasmopara Viticola: Differentiation and Light Regulation

Original Paper 413

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

Received: August 22, 2001; Accepted: April 4, 2002

Abstract: The development of grape downy mildew Plasmo- The asexual part of the life cycle of the pathogen is character- para viticola) was followed histologically during the entire latent ized by short disease cycles. Under favourable conditions, the period until the appearance of mature sporangia. Production of mycelium is able to sporulate 3 days after infection Dai et al., sporangiophores and sporangia was assessed using low-tem- 1995[9]). A relative humidity of nearly 100% is required for perature scanning electron LTSEM) and fluorescent light micro- sporulation Blaeser and Weltzien, 1978[2]). Sporangiophore scopy. Time-course studies using attached leaves of Vitis vinifera development is the process in which mycelium grows out of cv. Müller-Thurgau revealed that the production of sporangio- stomatal cells forming hyphae that branch in a species-typical phores and sporangia is a highly coordinated process and is manner. Finally, sporangia develop at the tips of the branches. completed within 7h. As this differentiation is assumed to occur only in darkness, the influence of light was investigated. For this Early studies suggested that sporulation of P. viticola occurs at purpose, different light regimes were applied to infected leaf night Müller and Sleumer, 1934[23]). This was confirmed by discs of V. vinifera cv. Müller-Thurgau. White light irradiation Brook 1979[3]), who assessed the sporulation visually after in- prevented formation of sporangia, although the growth of the cubation of infected leaf discs in different light qualities or in mycelium was not affected. Many sporangiophores were ob- the dark. Sporulation proceeded in darkness, but was inhibited served that were abnormally shaped, i.e., short hyphae in clus- by both near-UV light and light in the blue-green region of the ters or thin, extremely elongated hyphae. For the formation of spectrum. mature sporangia, a prolonged dark period was necessary. Light experiments suggest photosensitivity at the end of the latent In general, only a limited number of detailed studies exist for period. A terminal white light irradiation caused an inhibitory ef- light-regulated development in oomycetes. Examples are the fect, whereas a final phase of darkness promoted sporangium positive phototaxis of Phytophthora cambivora zoospores Car- development. Different light qualities were tested, revealing an lile, 1970[4]) and the effect of humidity and light on discharge inhibition of sporangium development by blue light whereas of sporangia of different oomycete pathogens Fried and Stute- neither red nor far-red light were effective. ville, 1977[13]; Leach et al., 1982[20]; Su et al., 2000[28]). The first reports concerning sporangiophore and sporangium differen- Key words: Light regulation, low-temperature scanning elec- tiation are a description of both stages in Peronospora trifolior- tron microscopy, morphogenesis, oomycetes, sporulation. um 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 Introduction sporangiophores through stomata.

Plasmopara viticola is an oomycete pathogen causing downy The purpose of our studies was to describe in detail the mor- mildew of grapevine, a severe disease in temperate wine- phology of P. viticola during sporulation and to define develop- growing regions. Oomycetes are fungal-like members of the mental stages during this process. Based on these data, we fo- kingdom Chromista and are closely related to heterokont algae cused on the influence of light on sporangiophore and sporan- forming a distinct group divergent from true fungi, green algae gium development. The duration and time point of the light or plants Kumar and Rzhetsdy, 1996[18]; Van de Peer and De treatments leading to a response and the efficiency of different Wachter, 1997[29]. 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 re- spect 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 particu- Plant biol. 4 2002) 413± 422 lar during the late stages of its vegetative infection cycle. Georg Thieme Verlag Stuttgart ´ New York Therefore, these results provide further insights into light reg- ISSN 1435-8603 ulation of an oomycete species. 414 Plant biol. 4 :2002) J. Rumbolz et al.

dark period of 16 h Schäfer, 1975[26]). Continuous white light Materials and Methods cWL, 80 mmol m±2 s±1) and continuous monochromatic irra- Plant and fungal material diation 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, 14mmol Two-node cuttings of grapevine Vitis vinifera L. cv. Müller- m±2 s±1) was applied as previously described in Schäfer Thurgau) were rooted in pots and grown in the greenhouse un- 1977[27]). der a natural photoperiod until shoots reached a length of 40 cm. Plants were kept free from powdery mildew infections To determine the time course of sporangium differentiation in by vaporization of sulphur for 5 h per day. For all experiments, leaf disc experiments under short-day, constant darkness and the fifth or sixth expanded leaf of each plant, counted from the white light conditions, each sample consisted of 5 leaf discs apex, was used. Leaf discs were prepared after surface sterili- per petri dish. Discs were harvested at various time points be- zation with a tissue wipe soaked with 75% ethanol and subse- tween 68 and 98 h post-inoculation p.i.) and either transfer- quent rinsing in distilled water. Discs were excised with a cork red into 50 ml of 1M KOH for light microscopy or cryofixed in borer diameter 16 mm) and placed onto water agar 0.8 %) in liquid nitrogen for LTSEM. Assessment of pathogen develop- petri dishes. In additional experiments, leaf discs were also ment was made with light and fluorescence microscopy, as de- placed on water agar containing 2% sucrose. scribed below, as well as with scanning electron microscopy. Experiments were repeated in triplicate. Plasmopara viticola Berk. and Curt.) Berl. and De Toni was obtained from a natural field infection and maintained on In light experiments, where inoculated leaf discs 5 discs per cuttings of V. vinifera cv. Müller-Thurgau see above) in the experiment, petri dish and variant) were exposed to white greenhouse. Propagation of the pathogen was carried out by light and kept in darkness for different time intervals see misting abaxial leaf sides of cuttings with a spore suspension Figs. 4,6±8 for irradiation schedules), incubation was termi- 20 000 sporangia ml±1 distilled water) until run-off, and cov- nated 88 h p.i. Experiments were repeated in triplicate. Assess- ering of entire plants with wet polyethylene bags overnight. ment of sporangiophore and sporangium development was Bags were removed on the following day and plants were kept done by light and epifluorescence microscopy, as described in the greenhouse for 5±6 days, corresponding to the latent below. period of P. viticola,at20±268C. Another overnight incubation of inoculated plants under the wet plastic bag yielded new Light and epifluorescence microscopy sporangia. Fresh inoculum was harvested with a paintbrush into centrifuge tubes. To prepare spore suspensions used in Leaf discs transferred to KOH after various incubation periods the experiments, sporangia were counted using a Fuchs±Ro- were cleared by autoclaving the samples for 15 min. After cool- senthal counting chamber and diluted to a final concentration ing down, KOH was decanted and samples were washed three of 20 000 sporangia ml±1. times in 50 ml distilled water. 0.05% Aniline blue Merck,

Darmstadt, Germany) was dissolved in 0.067 M K2HPO4 pH Sporulation experiments 9.0 and used to stain intercellular hyphae, as well as fungal structures of P. viticola which protruded from stomata of the To observe the morphogenesis of P. viticola during sporulation, host epidermis Williamson et al., 1995[30]). attached leaves of cuttings of V. vinifera cv. Müller-Thurgau) were inoculated by misting the abaxial leaf surface with a Cleared and stained leaf discs were examined by light bright spore suspension until run-off. Plants were covered overnight field) and epifluorescence microscopy using an Axiophot with wet polyethylene bags. Bags were removed on the follow- Zeiss, Oberkochen, Germany) microscope equipped with an ing day and plants were kept in the greenhouse under the nat- epifluorescence device excitation at 365 nm; LP 397 nm). For ural photoperiod. Sporulation was induced after 6 days by cov- microscopic analyses, a leaf area encircled by leaf veinlets was ering the entire plants with wet polyethylene bags and trans- defined as a ªleaf unitº Fig.1A). 20±25 leaf units per leaf disc ferring them to darkness at dusk 8 p.m.). To monitor the time exhibiting tissue colonized by P. viticola were selected ran- course of sporulation, infected leaves were detached and areas domly and used to determine the degree of differentiation. with downy mildew lesions were cryofixed in liquid nitrogen The three stages occurring during sporulation of P. viticola at hourly intervals until the following morning 6 a.m.). Sam- were defined as follows: 1. hyphal cluster short or extremely ples were further prepared for low-temperature scanning elongated hyphae in clusters, no branching), 2. sporangio- electron microscopy LTSEM) studies, as described below. All phores elongated hyphae, unbranched and branched, without experiments were carried out between April and June and re- sporangia) and 3. mature sporangia branched hyphae with peated in triplicate. spherical or drop-shaped sporangia). By assessing 20 ±25 leaf units, the average number of sporangiophores with sporangia Light experiments stage 3) per leaf and the number of sporangiophores without sporangia stages 1and 2) per leaf unit was determined, indi- Leaf discs placed on water agar were inoculated abaxially with cating the degree of differentiation of P. viticola on the respec- a100-ml droplet of a spore suspension. Petri dishes were sealed tive leaf disc. Three leaf discs per variant and experiment were in order to avoid desiccation of the inoculation droplet and in- randomly selected and all experiments were repeated in tripli- cubated at 228C ambient temperature. To apply short-day con- cate. Since data for each replicate were almost identical, we ditions, samples were incubated in growth chambers, immedi- combined them in the final analysis. Data were compared ately after inoculation, with an irradiation of WL 300 mmol using the unpaired Students t-test p £ 0.05). 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 Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 :2002) 415

Fig.1 Intercellular growth of Plasmopara viticola within the spongy graphs of freeze-fractured spongy mesophyll colonized by P. viticola mesophyll of host leaves :Vitis vinifera cv. Müller-Thurgau). :A) Epi- :side view). :B) Note granular structure of hyphae :h) compared with fluorescence light micrograph of unseptate hyphae colonizing the mesophyll cells :m), which contain chloroplasts :cp). :C,D) Bulb- leaf areas between leaf veinlets, :top view). The square area encir- shaped structures :arrows) are situated underneath guard cells :g), cled by the leaf veinlets :arrowheads) was defined as a ªleaf unitº. giving rise to sporangiophores :sph). Scale bar: 50 mm. :B±D) Low-temperature scanning electron micro-

Scanning electron microscopy Results

Hyphal structures of P. viticola were examined with LTSEM Morphogenesis of P. viticola according to Rumbolz et al. 2000[25]). Following incubation, fresh plant material attached leaves or leaf discs from cut- The development of the pathogen was followed during the tings) was taken, cut into pieces of 0.8±1cm 2 and mounted entire latent period until the appearance of mature sporangia. on a Balzer specimen holder using low-temperature mounting Colonization of the leaf mesophyll by P. viticola prior to sporu- medium. Samples were rapidly frozen by plunging them into lation was assessed using both FLM fluorescence light micro- liquid nitrogen. After cryofixation, samples were transferred scopy) and LTSEM. Under the fluorescence microscope, inter- under nitrogen gas to a Balzers cryopreparation unit SCU 020 cellular unseptate hyphae were easily distinguishable from attached to a JEOL JSM 6300 scanning electron microscope. the background by their greenish fluorescence Fig.1A). Com- Ice crystals on the surface were allowed to sublime from the parative studies on freeze-fractured specimens revealed inter- specimens by raising the temperature to ± 808C for 5±8 min. cellular hyphae of P. viticola inside the spongy mesophyll, Sputter coating with 20 nm gold was carried out in an argon identifiable by their granular structure, in comparison to the gas atmosphere Müller et al., 1991[24]). For the examination surrounding host cells that contained large organelles, i.e., of fungal growth inside the leaf mesophyll, samples were frac- chloroplasts Fig.1B). The granular structures were also pres- tured at ± 1208C with a precooled knife prior to immediate ent inside bulb-shaped fungal cells that were situated under- sputter coating. Coated specimens were transferred online neath guard cells during sporulation Figs.1C,D). Hyphal into the SEM under high-vacuum conditions. The samples growth within the leaf was mostly restricted to the intercellu- were observed and photographed at a stage temperature of lar spaces of the spongy mesophyll. Colonization of interveinal ±1658C using an accelerating voltage between 5 and 10 kV. 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 developing external hyphae :3 h). :D) Branches of the first, second and third mental stages of Plasmopara viticola during sporulation on leaves of order formed on sporangiophores :5 h). :E) Development of spherical Vitis vinifera cv. Müller-Thurgau. Sporulation was induced by transfer sporangia at the distal end of sporangiophores :6 h). :F) Drop- of plants to darkness and exposure to high humidity. :A) Stoma of shaped, mature sporangia :7 h). :G) Distal end of a sporangiophore host leaf that is already filled with sporangiophore initials :0 h after bearing mature sporangia, which appear shrunken :9 h). Some spor- darkening). :B) Hyphal tips protruding from stoma :2h). :C) Elongat- angia are already released :arrows).

To define developmental stages of P. viticola during the sporu- of darkening 0 h), stomata were already filled with hyphal tips lation process, time-course studies using attached leaves were protruding from the interior of the leaf Fig. 2A). Two hours la- carried out. Observations were started immediately after ter, hyphal tips were clearly visible Fig. 2B). External hyphae transfer of sporulation-induced plants to darkness. Differen- further elongated 3 h, Fig. 2C) until 4 h. Five hours after trans- tiation proceeded in a highly coordinated manner. At the time fer to darkness, hyphae with branches of the second and third Light-Regulated Sporulation of Plasmopara viticola Plant biol. 4 :2002) 417

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 Meth- odsº) 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 clus- tersº stage 1) due to their spatial organization, were charac- teristically 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 de- creased 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 dif- Fig. 3 Epifluorescence light micrographs of sporangiophore and ferentiate further than stages 1and 2. Vegetative growth was sporangium development of Plasmopara viticola on leaf discs of Vitis not affected, as the leaf mesophyll was densely interspersed vinifera cv. Müller-Thurgau. :A) Typical, branched sporangiophores with intercellular mycelium Figs. 3B,C). Instead of mature :sph) most abundantly found during sporulation. Some sporangio- sporangia or regularly formed sporangiophores, hyphal clus- phores bear mature sporangia :s). :B,C) Sporangiophores with abnor- ters stage 1) covered the diseased leaf areas. mal 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 Since full differentiation to mature sporangia was reached at sporangiophores were found :C). Scale bars: 50 mm. 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. order were observed Fig. 2D). By 6 h, spherical sporangia de- Sucrose effects veloped Fig. 2E), followed by drop-shaped, mature sporangia 7 h, Fig. 2F). At 9 h in the dark, sporangiophores exclusively As shown in Fig. 4, short day and continuous darkness yielded bore shrunken sporangia, which appeared desiccated after an excess of the stage ªmature sporangiaº p<0.05). In contrast, cryofixation Fig. 2G). under continuous white light the developmental progress ceased after the elongation of external hyphae. To check for Effects of light on differentiation of sporangia an impairment of photosynthesis and thus of fungal growth in continuous white light or darkness in comparison to short- As sporulation occurred as a coordinated developmental pro- day conditions, inoculated leaf discs were also incubated on cess, we carried out more detailed studies on its regulation by agar containing sucrose Fig. 5). While development in short external factors. We focused on the effects of different light day and white light was not altered, an increased number of treatments on sporulation. These experiments were done hyphal clusters was observed in darkness compared to incuba- using leaf discs to obtain a sufficient data base. tion without the sugar supplement. However, there was no impact of sucrose on the observed light effects. 418 Plant biol. 4 :2002) J. Rumbolz et al.

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 posi- tioned 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. 4 Time-course of differentiation of Plasmopara viticola on leaf Fig. 7). Significantly more sporangiophores with sporangia discs of Vitis vinifera cv. Müller-Thurgau in short day :SD, A), continu- were found if terminal dark periods were 48 h or longer ous darkness :cD, B) and continuous white light :cWL, C) after the p < 0.05). Shorter dark intervals led to an increase of sporan- end of the latent period :l sporangiophores with sporangia; ~ spor- giophores without sporangia concomitantly with a reduction angiophores without sporangia). Leaf discs were constantly exposed of sporangial development p < 0.05). to high humidity on water agar. Horizontal bars on top of figures show the irradiation design with the period of data assessment indi- Evidence for the requirement of a dark period before the com- cated by the bracket. Data points represent means of three experiments with three leaf discs each, and error bars correspond to the pletion of the vegetative life cycle was also supported by an- standard error of the means. other 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. 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 Fig. 6 Sporangiophore and sporangium development of Plasmopara the infection cycle have been the subject of several micro- viticola on leaf discs of Vitis vinifera cv. Müller-Thurgau. White light scopical studies Locci, 1969[21]; Langcake and Lovell, 1980[19]; was applied for 88 h, interrupted by dark periods of different number [17] and position. One night :16 h darkness) :B) or two nights :A) during Kortekamp et al., 1998 ). The main focus was to describe the the light/dark cycle were replaced by cWL. Horizontal bars above developmental stages of the pathogen from infection until each figure show the irradiation regimes. Values represent means of sporulation. There is little information on the terminal phase three experiments with three leaf discs each, and error bars corre- of the life cycle, in particular, on the dynamics of sporangial spond to the standard error of the means. 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 hu- short-time periods of 10 or 16 h of white light irradiation a sig- midity reach 75±100%. In the present study, the single devel- nificant number of sporangiophores with sporangia was still opmental stages occurring during sporulation were docu- observed, exposure times to WL of 24 h and longer led to a sur- mented by a detailed microscopical analysis. From the time- plus of sporangiophores without sporangia p < 0.05). course data, it is evident that sporulation is a highly coordinated process. The good temporal resolution might be the result Effect of blue light of a synchronicity in development of fungal structures, which has been described for other species of the Peronosporales Since exposure of P. viticola to constant WL revealed a drastic Dick, 1990[10]; Falloon and Sutherland, 1996[12]). effect on differentiation, inoculated leaf discs were subjected to light of different wavelengths. Red, as well as far-red light An important feature of the mature sporangia is their ability to yielded sporangia Fig. 9). Far-red light caused similar differen- shrink Fig. 4G). The collapse of mature sporangia visualized in tiation as cD. In contrast, blue light incubation gave rise to a the LTSEM was also described for cryofixed Peronospora viciae surplus of sporangiophores without sporangia, as observed in Falloon and Sutherland, 1996[12]), as well as for P. viticola after 420 Plant biol. 4 :2002) J. Rumbolz et al.

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 pro- Fig. 8 Differentiation of Plasmopara viticola on leaf discs of Vitis vinifera cv. Müller-Thurgau after incubation in darkness followed by a duced at night. Knowledge of the effects of light on differentia- white light irradiation :total incubation time: 88 h). The length of a tion of P. viticola exclusively relies on the studies of Arens terminal light interval was progressively reduced :inverse to Fig. 7). 1929[1]), Yarwood 1937[31]) and Brook 1979[3]). While Arens Horizontal bars above figure show the irradiation regimes. Values re- 1929[1]) and Yarwood 1937[31]) both found an inhibition of present means of three experiments with three leaf discs each, and sporangiophore formation on detached leaves after irradiation error bars correspond to the standard error of the means. 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 chemical fixation Kortekamp et al., 1998[17]). From compara- of the incubation period. In our experiments, we exposed in- tive LM studies, which revealed a recovery of turgescence of fected leaf discs to continuous white light during the entire in- the propagules, it was concluded that the observed phenom- cubation period, or at least for an extended time during the life enon is not an artefact but an adaptation of the pathogen to cycle. Light inhibited the differentiation of sporangia, resulting changing humidity conditions in the field. Studies on sporula- in a reduction in the amount of sporangia in favour of sporan- tion of Peronospora trifoliorum showed that lowering relative giophore formation. Interestingly, continuous white light did humidity caused rapid shrivelling of the sporangia and their not affect intercellular colonization by P. viticola. Thus, release from sporangiophores Fried and Stuteville, 1977[13]). restricted growth was not responsible for this effect. Moreover, Reports that sporangia of P. viticola are still viable after storage the mycelium was able to produce many sporangiophores for several days under dry air conditions Müller and Sleumer, emerging from nearly all stomata. The microscopical observa- 1934[23]) support the thesis of reversible shrinkage. tion of abnormal sporangiophores formed in light aligns with findings on Pseudoperonospora cubensis, the downy mildew Observations on freeze-fractured specimens of P. viticola dur- pathogen of cucurbits Cohen and Eyal, 1977[7]). In addition to ing intercellular growth prior to sporulation Fig.1) corro- the abnormally shortened sporangiophores described for this borate earlier TEM studies Langcake and Lovell, 1980[19])in pathogen, P. viticola sometimes showed extremely elongated, which the bulb-shaped fungal structures were described as thin sporangiophores bearing no sporangia. Taking into con- swellings of hyphae within a substomatal cavity that give rise sideration the SEM observation that late stages of sporangia to sporangiophores. This is in line with our own observations development are completed over short periods Fig. 4), it is im- of hyphal aggregations underneath stomata prior to the out- pressive that the described inhibitory influence of light be- break of sporangiophores data not shown). These structures comes apparent long after the potential for differentiation is may be necessary to build up pressure for penetration through established. closed stomata. The granular structure characteristic of cryofixed intercellular hyphae of P. viticola might be due to the ex- The strongest impact of light on morphogenesis was observed tensive vacuolization, as revealed by transmission electron mi- when it was applied towards the end of the incubation period. croscopy. 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 Indications for involvement of light in regulation of the sporu- is much longer than is necessary for differentiation 7 h). Thus, lation process were provided by the observation of several au- the competence to fulfil this inhibitory effect must be estab- thors Cuboni, 1885[8]; Istvanffi and Palinkas, 1913[16]; Gregory, lished. 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 de- sporangia may emerge. In this case, the potential for successful termined 48 h). This light-dependent reaction is very differ- sporulation the next night might be increased. The extent of a ent compared to the light-induced conidiation in several true sporulation event may be influenced by such preceded events. fungi, such as Aspergillus nidulans Mooney and Yager, This should be considered in epidemiological models. Taking 1990[22]). Here, only 15 or 30 min of exposure is necessary for into account the protective characteristics of most oomycete a light response. However, in this organism, a light-sensitive fungicides, a decision as to whether a control measure is nec- critical period also exists, from initiation of conidiation until essary immediately after conditions favourable for the patho- formation of differentiated structures. gen, but not fully sufficient for an outbreak, should be carefully evaluated. Although the competence to respond to the light signal is greatest at the end of the incubation period, there is also an Acknowledgements influence of light at the beginning of the infection cycle. One dark phase of 16 h during an irradiation with cWL had no Thanks to M. Düggelin and D. Mathys, REM-Labor Universität effect, no matter if applied at the beginning or at the end of Basel, for technical assistance in part of the experiments and the incubation period. However, synergistic effects could be to N. Papadopoulos, Davis, for advice on statistical analysis. observed, if two dark phases were applied interrupted by an The authors are further grateful to C. Körner, Botanisches Insti- irradiation of cWL of 8 h. The dark phases at the end or in the tut, Universität Basel, for providing facilities for light experi- middle of the incubation period led to a significant amount of ments and to L. Hennig, Zürich, and P. McCabe, Davis, for criti- sporangia. cally reading the manuscript.

The availability of nutrients does not seem to influence the References light responses. As the pathogen lives in an environment where the light and dark cycles determine the productivity of 1 Arens, K. 1929) Physiologische Untersuchungen an Plasmopara vi- the host plant, a reaction to these changes could, hypothetical- ticola, unter besonderer Berücksichtigung der Infektionsbedin- ly, be present. Also, an interaction of sucrose- and light-depen- gungen. Jahrb. f. wiss. Bot. 70, 93±157. dent pathways could be possible, as observed in certain plant 2 Blaeser, M. and Weltzien, H. C. 1978) The importance of sporula- species Dijkwel et al., 1997[11]; Harter et al., 1993[15]). As an ad- tion, dispersal, and germination of sporangia of Plasmopara vitico- dition of sucrose to the medium led only to an increase of pro- la. J. Plant Dis. Prot. 3, 155±161. 3 ductivity of the mycelium and not to a change in the light re- Brook, P. J. 1979) Effect of light on sporulation of Plasmopara viti- sponse, we can exclude any impact of nutrient availability. cola. New Zeal. J. Bot. 17, 135±138. 4 Carlile, M. J. 1970) The photoresponses of fungi. In Photobiology of microorganisms Halladal, P., ed.), Wiley-Interscience, pp. 309± P. viticola is an obligate parasite which makes it impossible to 344. investigate the light responses singly in one of the two organ- 5 Cohen, Y., Eyal, H., and Sadon, T. 1975) Light-induced inhibition of isms. Thus, both organisms can theoretically contribute to the sporangial formation of Phytophthora infestans on tomato leaves. observed light effects. The ecological benefit is for the parasite, Can. J. Bot. 53, 2680±2686. as the temporal regulation of sporulation promotes successful 6 Cohen, Y. 1976) Interacting effects of light and temperature on development following infection. Under natural conditions, sporulation of Peronospora tabacina on tobacco leaves. Austral. J. sporulation occurs in the dampness of the early morning Biol. Sci. 29, 281±289. hours, when humidity conditions are suitable for infection. 7 Cohen, Y. and Eyal, H. 1977) Growth and differentiation of spor- From this point of view, it seems to be most likely that the angia and sporangiophores of Pseudoperonospora cubensis on cu- pathogen plays an important role in mediating the light re- cumber cotyledons under various combinations of light and tem- sponse. perature. Physiol. Plant Pathol. 10, 93±103. 8 Cuboni, G. 1885) Gli effeti dell idrato di calce nella cura delle viti As light in the green to blue-green region of the spectrum was contro la Peronospora. Lab. Bot. R. Scuola Vit. Conegliano, 10 p. 9 described to inhibit sporangium formation Brook, 1979[3]), we Dai, G. H., Andary, C., Mondolet-Cosson, L., and Boubals, D. 1995) tested the influence of BL on the differentiation of sporangio- Histochemical responses of leaves of in vitro plantlets of Vitis spp. to infection with Plasmopara viticola. Phytopathology 85, 149± phores and sporangia. We found strong reduction of sporangia 154. in continuous blue light, similar to that observed under cWL. 10 Dick, M. W. 1990) Phylum Oomycota. In Handbook of protoctista Thus, blue light of high photon fluence rate, as used in our ex- Margulis, L., Corliss, J. O., Melkonian, M., Chapman D. J., and periments, might have been sufficient to cause inhibition. A McKhann H. I., eds.), Boston: Jones and Bartlett Publ., pp. 661± continuous irradiation with R or FR did not lead to an inhibi- 685. tory effect. The inhibition of sporangia formation as a blue 11 Dijkwel, P. P., Huijser, C., Weisbeek, P. J., Chua, N.-H., and Smeekens, light response seems to be common for other members of the S. C. M. 1997) Sucrose control of phytochrome A signaling in Ara- Peronosporales Cohen et al., 1975[5]; Cohen, 1976[6]; Cohen bidopsis. Plant Cell 9, 583±595. and Eyal, 1977[7]). To obtain more insight into the photorecep- 12 Falloon, R. E. and Sutherland, P. W. 1996) Peronospora viciae on Pi- tor action, further information, such as action spectra, will be sum sativum: Morphology of asexual and sexual reproductive required. structures. Mycologia 88, 473±483. 13 Fried, P. M. and Stuteville, D. L. 1977) Peronospora trifoliorum The inhibition of sporangial differentiation by light might be of sporangium development and effects of humidity and light on dis- particular interest when estimating the potency of a sporula- charge and germination. Phytopathology 67, 890±894. tion event under marginal conditions. Assuming that humidity 14 Gregory, C. T. 1912) Spore germination and infection withPlasmo- conditions favourable for an outbreak are not achieved until para viticola. Phytopathol. 2, 235±249. the early morning hours, sporangiophores bearing almost no 422 Plant biol. 4 :2002) J. Rumbolz et al.

15 Harter, K., Talke-Messerer, C., Barz, W., and Schäfer, E. 1993) Light- Erratum and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape Brassica napus L.). Plant Biology vol. 4, no. 2, 2002 Plant J. 4, 507±516. 16 Istvanffi, G. and Palinkas, G. 1913) Etudes sur le mildiou de la vigne. Ann. Inst. Ampelogr. Hongorois 4, 1±125. Due to an unfortunate mistake which occurred after the then 17 Kortekamp, A., Wind, R., and Zyprian, E. 1998) Investigation of the still correct page proofs of Plant Biology vol. 4, no. 2, 2002, interaction of Plasmopara viticola with susceptible and resistant had been checked by the editor Ulrich Lüttge) responsible for grapevine cultivars. Z. PflKrankh. PflSchutz 105, 475±488. the contributions covered by the guest editorial of Matyssek et 18 Kumar, S. and Rzhetsky, A. 1996) Evolutionary relationships of eu- al. p.133, the contribution by Reitmayer et al. p. 228 got mis- karyotic kingdoms. J. Mol. Evol. 42, 183±193. placed. It belongs to the set of 7 contributions covered in the 19 Langcake, P. and Lovell, P. A. 1980) Light and electron microscopi- guest editorial and was supposed to be placed after Grams et cal studies of the infection of Vitis spp. by Plasmopara viticola, the al. p.153 and before Pretzsch p.159. downy mildew pathogen. Vitis 19, 321 ±337. 20 Leach, C. M., Hildebrand, P. D., and Sutton, J. C. 1982) Sporangium discharge by Peronospora destructor: Influence of humidity, red- infrared radiation, and vibration. Phytopathology 72, 1052 ±1056. 21 Locci, R. 1969) Direct observations by scanning electron microscopy of the invasion of grapevine leaf tissues by Plasmopara viticola. Riv. Patol. Veg. Ital. Serie IV 5, 199±212. 22 Mooney, J. L. and Yager, L. N. 1990) Light is required for conidation in Aspergillus nidulans. Genes Dev. 4, 1473±1482. 23 Müller, K. and Sleumer, H. 1934) Biologische Untersuchungen über die Peronosporakrankheit des Weinstocks. Landw. Jahrb. Ber- lin 79, 509±576. 24 Müller, T., Guggenheim, R., Düggelin, M., and Scheidegger, C. 1991) Freeze fracturing for conventional and field emission low- temperature scanning electron microscopy: The cryo scanning unit SCU 020. J. Microscopy 161, 73±83. 25 Rumbolz, J., Kassemeyer, H.-H., Steinmetz, V., Deising, H. B., Mend- gen, K., Mathys, D., Wirtz, S., and Guggenheim, R. 2000) Differen- tiation of infection structures of the powdery mildew fungus Unci- nula necator and adhesion to the host cuticle. Can. J. Bot. 78, 409± 421. 26 Schäfer, E. 1975) A new approach to explain the ªhigh irradiance responsesº of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41±56. 27 Schäfer, E. 1977) Kunstlicht und Pflanzenzucht. In Optische Strah- lungsquellen Albrecht, H., ed.), Grafenau: Lexika-Verlag, pp. 249± 266. 28 Su, H., van Bruggen, A. H.C., and Subbarao, K. V. 2000) Spore release of Bremia lactucae on lettuce is affected by timing of light initiation and decrease in relative humidity. Phytopathology 90, 67±71. 29 Van De Peer, Y. and De Wachter, R. 1997) Evolutionary relationships among the eukaryotic crown taxa taking into account site- to-site rate variation in 18S rRNA. J. Mol. Evol. 45, 619±630. 30 Williamson, B., Breese, W. A., and Shattock, R. C. 1995) A histo- logical study of downy mildew Peronospora rubi) infection of leaves, flowers and developing fruits of Tummelberry and other Rubus spp. Mycol. Res. 99, 1311 ±1316. 31 Yarwood, C. E. 1937) The relation of light to the diurnal cycle of sporulation of certain downy mildews. J. Agr. Res. 54, 365 ±373.

J. Rumbolz University of California Department of Plant Pathology One Shields Ave. Davis, CA 95616 USA E-mail: [email protected]

Section Editor: J. Draper