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.).

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