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Characterization of Plasmopara-Resistance in grapevine using in vitro plants

Posted on: Thursday, 8 January 2004, 06:00 CST

Summary

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in w'/ra-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order: An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection.

The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in w'fro-plants and the resistance of grapevine plants to P. viticola in the field.

Key words: Downy mildew - peroxidase - Plasmopara viticola - Vitis spp.

Abbreviations: dpi = days post infection. - hpi = hours post infection. - POX = peroxidase

Introduction

Although plant disease is the exception rather than the rule, as many plant pathogens are limited to one or a small number of host species, the downy mildews are severe pathogens of many cultured plants, including Plasmopara viticola, the causal agent of grape downy mildew. The exact mechanisms which enable the plant cells to reduce this disease incidence are not fully clarified, and the knowledge of the involved mechanisms remains rudimentary even though this fungus has been known since the beginning of the 20th century (Arens 1929, Muller and Sleumer 1934). Resistance or susceptibility may reflect several features of both organisms, to be able to overcome defenses or virulence, respectively. Often the interaction between a plant and a pathogen results in plant cell death. Plant cell death can occur when the pathogen successfully infects the plant cell. Some pathogens, like many downy mildews, are obligate parasites that depend on living cells to grow and develop, and a susceptible plant cell only dies due to the loss of metabolites or loss of cell integrity. In this case plant cell death is a result of initiated disease and does not interfere with disease resistance. On the other hand plant cell death also occurs rapidly after pathogen attack leading also to the death of the parasite (hypersensitive response, HR). The hypersensitive death of an infected plant cell is an active process triggered by fungal elicitors or endogenous elicitors produced from plant cell walls by the action of fungal hydrolases (Keen and Yoshikawa 1983, De Lorenzo et al. 2001 and refs. therein). Elicitors usually are surface molecules from a plant pathogenic microorganism, which, when applied to host or non-host plants, will induce symptoms of resistance reactions typical for the plant-pathogen-system studied. Many of these reactions rely on rapid transcriptional activation of specific genes which are classified as plant defense or plant defense-related genes. Typical examples include the genes encoding enzymes of phytoalexin biosynthesis (Lamb et al. 1989), lytic enzymes such as chitinases and glucanases (Busam et al. 1997, Xue et al. 1998), cell wall components like hydroxyproline-rich glycoproteins (Showalter et al. 1985) and peroxidases (Mohan and Kolattukudy 1990). Subsequent incorporation of phenolics into the cell walls seems also to be a part of the activated defense response in plants (Beckmann et al. 1974).

Suspension-cultured cells and protoplasts have been widely used to study more closely the rather complex nature of the interaction between plants and pathogens (Davis and Ausubel 1989). Although the defense reactions activated in tissue-cultured cells have been shown to simulate the reactions that occur in plant cells, no information about the reaction in plant cells surrounding the site of pathogen infection in whole plants is available with this test system. Therefore we report in this paper the use of in vitro-plants as a suitable system for studying the plant-fungus interaction, the effect of P. viticola itself, a fungal derived elicitor and some elicitor-like defense activating compounds and other oomycetic pathogens on their HR-inducing capacity and the activation of peroxidase(EC1.11.1.7).

Material and Methods

Sporangia of P. viticola were collected from leaves of infected grapevine plants grown in the vineyards of the Institute for Grapevine Breeding Geilweilerhof after a over night incubation at room temperature and high relative humidity. In order to obtain sporangia free from contamination, sporangia were propagated on axenic in vitro-culture plants (Blaich 1977). The new produced sporangia were collected and used for the further study. Infection of 8 to 10-week-old in vitro plants was done by an application of 50L drops containing 50,000 sporangia per mL on the adaxial leaf surface. The concentration of sporangia was determined using a hemacytometer. In vitro plants of different cultivars and species (Vitis vinifera cv. , , and , two Vitis riparia selections and the interspecific ) were grown at 27C and a 16h photoperiod (50E/m^sup 2^/s) as decribed in Blaich (1977) and used for reinoculation experiments. Furthermore, plants were elicited by the aseptic addition of autoclaved P. viticola sporangia, viable sporangia of Plasmopara halstedii, Pseudoperonospora cubensis, Phytophthora infestans, Pythium ultimum (each with 50,000 sporangia per mL) and various compounds in 0.5 % aqueous solutions [Laminarin (Sigma, Deisenhofen), Cellulysin (Calbiochem, Schwalbach) and a sporangia-derived elicitor]. In addition, Cellulysin was heat inavtivated (121 C, 15 min) and applicated as described above. Spore suspensions of P. infestans and P. ultimum were prepared by flooding each dish with distilled water and lightly scraping the surface of the colony with a sterile spatula. In case of low spore concentrations spores were collected by a centrifugation step (1000 rpm in a table-top centrifuge) and diluted with sterile water to the appropriate concentration. The protein- and fat-free elioitor of P. viticola sporangia was produced as described by Bach and Seitz (1997). Sporangia of P. halstedii were a kind gift from L. Brahm (University of Gie[beta]en, Germany). Pseudoperonospora cubensis, Phytophthora infestans and Pythium ultimum were taken from the collection of the Institute for Phytomedicine at the University of Hohenheim. Inoculation with sporangia of these different fungal organisms was done as described above. Preparation of samples for light and scanning electron microscopy as well as staining procedure for cell wall peroxidase activity and phenolics using benzidine/ H202 and DMACA or methylred was accomplished following Kortekamp et al. (1998), Nachit and Jakob (1978) or Trump et al. (1961). The production of reactive oxygen species, mainly the Superoxide radical, was visualised with a nitroblue tetrazolium stain as described by Doke (1983).

The relative disease resistance of grape cultivars at different stages of infection was assessed by measuring the cytosolic peroxidase activity. For this purpose ten leaf discs (diameter 5mm) were frozen in liquid nitrogen and ground with mortar and pestle, added to 1.2ml_ 0.1 mol/L sodium acetate buffer (pH 4.0) containing 0.33% H2O2 and 250L of a tetramethylbenzidine (TMB, Serva, Heidelberg) stock solution (10 mg TMB in 5ml_ ethanol), incubated at 25 C for 5 min and centrifugated for 3 min in a table-top centrifuge at maximum speed. The extinction of 1 ml_ of the supernatant was measured at 654 nm.

Results

On both - resistant and susceptible varieties - zoospores of P. viticola could initiate an infection process within one to two hours after application. In the resistant varieties the first reaction that could be seen by light microscopy 4 to 6hpi is the production of Superoxide anions (Figs. 1 a, b) followed by the death of the infected cell (within 6 to 8hpi). At this early stage of infection the host cell had turned brown (Figs. 1 c, d). The brown pigment in the plant cell undergoing sudden cell death appeared to be localized in the cytoplasm due to the disruption of compartments. The hypersensitive cell death resulted later during the infection process (1 to 2 dpi) in restricted lesions delimited from surrounding healthy tissue (not shown).

Soon after the fungus encountered the host, POX activity increased (10 to 12 hpi). This increase was only observed in host cells surrounding an infection area (Fig. 1e) and was followed by the deposition of oxidized (poly-) phenolics which could be seen 12 to 15hpi after treatment with suitable stains (Figs. 1 f, g). Whereas induction of POX activity was only observed in the adjacent non-infected cells (Figs. 1e and 2a), deposition of phenolic compounds occurred as well in the infection area itself as in the flanking cells (Figs. 1 e, g and 2 b). POX activity \was most prominent in parenchyma cells and vascular bundles (Fig. 2c) and appeared to be concentrated at or close to the cell walls. The described histochemical processes were not noted in the susceptible cultivars and in water treated controls.

Figure 1. Cytological reactions of the resistent variety after an infection with P. viticola: Detection of Superoxide using NBT 4-6 hpi (a, b; bar = 10 m), HR of the infected cell 6-8hpi (c, d; bar= 10m), enhanced POX activity 10-12hpi (e; bar= 100m), deposition of phenolics 12-15hpi (f, DMACA stain; g, methyl red stain; bars = 100 M).

The early infection events were also investigated with the aid of a scanning electron microscope. In the susceptible variety (Fig. 3a) the germ tubes generated from encysted zoospores penetrated the stomata and produced first appressoria, then typical substomatal vesicles and secondary hyphae within 12 to 15 hpi indicating a successful transfer of nutrients from the plant into the fungal cell. In the case of an infection of fungal development was restricted. This refers primarily to the substomatal vesicles and the secondary hyphae - which were always absent and to the slight and small appressoria (Fig. 3 b). The infected cells appeared collapsed and shrunken due to the loss of cell integrity.

Since histochemical investigation revealed a clear difference between the resistant variety and the susceptible variety regarding POX activity, we decided to extend our experiments to a test with extractable POX.

Activity increases in extractable peroxidase were associated with fungal infection in all cultivars tested (Fig. 4). Nevertheless great differences were seen in regard to the value of POX activity. The resistant varieties are characterised by either a higher basal activity (V. riparia selections) compared to the other cultivars tested and/or a fast increase in enzyme activity (two- to threefold more than basal activity) during the first 24hpi, which could be exceptionally seen for the cultivar reaching the highest peak of activity after an infection. At 5 dpi these three cultivars reached the same level.

Figure 2. Localization of peroxidase (arrow heads) and oxidized phenolics (arrows) at infection sites 15 hpi (a, b; bars = 10m) and in cells of a vascular bundle 12 hpi (c; bar = 50 m) in leaves of Gloire de Montpellier>.

Figures 3. SEM micrographs of infection events in the susceptible variety () and the resistant variety () 12 hpi. Note substomatal vesicle and secondary hypha in (a) indicating a successful infection of the leaf, and the lack of these structures in (b) as well as the collapse of the infected cell in ; Ph = primary hypha, Sh = secondary hypha, A = appressorium, iM = infected mesophyll cell, nM = non-infected mesophyll cell, V = vesicle.

Figure 4. Relative differences in increase of peroxidase activity of several grapevine varieties at different times (days) after an inoculation with P. viticola (time-course studies). Data represent means SE of n = 5-10 experiments.

In the two tolerant/field-resistant varieties <0rion> and we observed a substantial and transient increase during the first 2 dpi () or 3 dpi () and a slight decrease in both cases after the 4th dpi. in the susceptible varieties and no significant or only small changes in POX avtivity were observed throughout the first 24 hpi. in activity slowly increased after inoculation to a maximum of 3 dpi followed by a decline at 4 and 5 dpi, additionally a similar trend of increasing POX activity occurred in . At 1 dpi there was little difference between activity in control and inoculated leaves. After 2 days, a weak but distinct activity was detectable which increased on day 3 after an infection. The highest increase in activity occurred at 5 dpi and exceeded the activity of the tolerant varieties at the same time point. This increase occurred in the subsequent phase of infection, only after the colonisation of additional cells.

Figures 5. Localization of POX activity (arrow heads) in leaves 12 hpi with sporangia of Plasmopara halstedii; (a) , (b) ; bars = 1 cm.

Figure 6. Induction of HR and POX activity in (a) and (b) in response to inoculation with Pythium ultimum. The HR occurs in the centre of the infection site whereas POX activity is located in the border area; bars = 100 m.

Table 1. Peroxidase activity of in vitro-plants 12 hpi (- = no activity, + = weak activity, ++ = moderate activity, +++ = strong activity) and occurrence of an HR.

In addition the infection experiments with viable sporangia of P. viticola, the two cultivars and were treated with different compounds and several oomycetic fungi. Although the cultivar showed no visible histochemical reaction after treatment with autoclaved sporangia of P. viticoia, after application of the sporangia derived elicitor, the cell wall fragment mimic laminarin or cellulolytic enzymes (Cellulysin), inoculation with other Oomycetes resulted in a clearly to strong observable POX activity (stains, Figs. 5, 6 and Table 1). Interestingly inoculation with members of the family Pythiaceae led to a higher POX activity in than after treatment with the downy mildews. Nevertheless, all oomycetic pathogens had a HR- inducing capacity in both, and . Whereas did also express a HR after treatment with P. viticola and the cell wall derived elicitor, this was not the case for .

Discussion

The observation that appressoria did not develop to a normal size and subsequent fungal structures were lacking in the resistant variety - a few hours after an infection - indicates that early defense reactions inhibit the uptake of nutrients which are required for further fungal growth. As the production of Superoxide radicals and not the production of peroxide is the first observable reaction of resistant grapevine plants, their production seems to play a fundamental role in blocking microbial ingress. The production of ROS such as Superoxide and H^sub 2^O^sub 2^ has been noted when plants are challenged with particular viral, bacterial of fungal pathogens (Low and Merida 1996, Mehdy 1994, Wojtaszek 1997). It is generally considered as a component of the hypersensitive response and is often highly specific to particular pathogen-host-interactions. The oxidative burst may be related to subsequent events in the challenged cells, although the exact nature of the relationships is unclear. Free radical oxidation of plasma membrane lipids, proteins or nucleic acids may kill cells directly. Alternatively Superoxide (Jabs et al. 1996) or H^sub 2^O^sub 2^ (Levine et al. 1994) may serve as signals leading indirectly to mortality. This HR - occurring subsequent to the appearance of ROS - may act as a final shot down for P. viticola and perhaps for most of the obligate biotrophic microorganisms, especially for other oomycetes (Kamoun et al. 1999). Previous data with the field-resistant V. vinifera-cultivars and showed that there is a (limited) growth of the pathogen that stopped 4 dpi. An HR, as shown for V. riparia , does not seem to be part of the resistance response and indicates that rather biochemical reactions are involved in the defence response in these cultivars. The single cell HR may be specific to V. riparia and other wild species. Since V. riparia was used to breed new resistant cultivars like (classified as V. vinifera), it has to be recognised that the HR type of resistance could not be introduced into these cultivars.

In further inducible barriers beside the HR are formed at the infection site to prevent the fungus from intruding into neighbouring tissues. Several cell wall-bound enzymes, like peroxidases, have been shown to contribute to the reinforcement of the cell wall through enhanced synthesis or incorporation of phenolic compounds. These peroxidases catalyze the conversion of cinnamyl alcohols into their corresponding free-radicals, which spontaneously polymerize into lignin. Several other biochemical processes which have been shown to occur during plant disease resistance also require (extracellular) peroxidase. These are covalent insolubilisation of hydroxyprolin-rich glycoproteins within the cell wall, cross-linking of wall-esterified p-coumaric or ferulic acids, membrane lipid peroxidation and generation of H^sub 2^O^sub 2^ (from molecular oxygen and NADPH) required for these events. Both a higher basal level, or fast enhancement of peroxidase activity, as seen in the two resistant V. riparia selections and , and the activation of this enzyme in the vicinity of HR- undergoing cells seem to be very important for the protection of the non-infected tissue. In addition, POX activity in cells of the vascular bundles could take part in the restriction of the pathogen in case of multiple infections near the bundles. The observation of age-related resistance - which means that older leaves from the bottoms of the shoots were more resistant to natural or to artificial inoculation with P. viticola than were younger leaves from the tops of the shoots - appears also to be a consequence of an increase in enzyme activity, especially in POX activity (Reuveni 1998). As indicated in this paper and in the cited paper above it can be concluded that there is a good correlation between POX activity and the resistance to P. viticola. Plants displaying a high inducible POX activity in our test system also show resistance or at least tolerance in the field.

Another common response of plant cells to stress such as infection, elicitation or wounding, is the induced incorporation of phenolic material into the cell wall (\Matern and Grimmig 1994), and the activation of phenylpropanoid metabolism is one of the initial disease resistance reactions in many plants (Dixon and Paira 1995). High POX levels can cause an accumulation and wide dispersal of oxidized phenolic products in grapevine species and the resistance to P. viticola seems to correlate with such compounds (Dai et al. 1995, Kortekamp et al. 1998). In addition to phenolics modified by POX, quinonoid products produced by the oxidation via polyphenol oxidases leads to the covalent modification and cross-linking of (a variety of) nucleophilic substituents of amino acids and proteins which are also thought to possess a direct antibiotic and cytotoxic activity to pathogens (Li and Steffens 2002).

The mechanisms of resistance may involve preformed biochemical, and morphological barriers (Kortekamp and Zyprian 1999, Kortekamp et al. 1999), as well as active defense reactions (as shown in this paper). However the signal transduction and activation of plant defense genes in the nucleus requires some type of recognition of the pathogen, probably at the plant cell surface. We are currently investigating the molecular nature of the differential regulation of these cellular responses. Since the Plasmopara-derived elicitor used in this study was only partially purified and is certainly biochemically heterogenous, containing protein or amino acid residues and oligosaccharides of different fragment sizes, it remains to be determined which of the molecules has eliciting properties. Several oligomeric sugar residues may serve as signalling molecules as the application of the pure cell wall mimic laminarin or the cellulase enzyme (Cellulysin) promoted POX activation. Interestingly, an increase in POX activity and an accumulation of phenolic compounds were also induced by exogenously applied cellulase or pectinase on fruit peel or apple suspension cultures (Eshel et al. 2002, Lux-Endrich et al. 2002). Both, degradation of the host cell wall leading to a reduced rigidity and the release of cell wall fragments seems to activate the cell wall- bound or -associated POX. Furthermore, the degradation of chitin, which is a cell wall component of P. viticola (Werner et al. 2002), by chitinases may lead to a recognition of the fungus via the release of (chitin-) oligomers even though no direct antifungal effect was found after an enhanced expression of a class III chitinase (Busam et al. 1997). Although an induction of POX activity could be provoked by Cellulysin or laminarin (but not with heat inactivated Cellulysin), these compounds did not initiate an HR in or and did not act in a synergistic manner. Therefore degradation of the host cell wall and the subsequent release of oligomers (alone) and/or application of a fungal cell wall mimic is not sufficient to induce the plant apoptotic pathway. The direct contact between the plant cell membrane and the invading haustorium as well as the separation of cell wall constituents (e.g. proteins) as assumed during an infection may be necessary for an appropriate receptor-ligand binding and thus to a signal transduction as autoclaved sporangia were not able to induce a HR and caused only a weak induction of POX. Since an HR occurred in after an application of the P. viticola derived elicitor the concentration of low-weight molecules enabled with eliciting features was high enough to cause a reaction after diffusion into the leaf through stomata even though they were denatured during the preparation process. An infiltration or injection of the putative elicitor was avoided, because the reaction provoked by this treatment (control) was stronger than the effect caused by the elicitor.

The detection of a putative oomycetic non-host pathogen and the initiation of a defense response in plants seems to depend on the phylogenetic distance of the pathogenic organism to P. viticola as members of the family Peronosporaceae caused only a moderate increase in POX activity whereas inoculation with other Oomycetes belonging to the family Pythiaceae led to a higher induction of POX activity.

Each kind of plant is a host to only a small number of pathogens and thus a non-host to the vast majority of known plant pathogens. In general, non-hosts are completely resistant to all pathogens of other plants (Agrios 1988). This nonhost resistance is most robust but its specificity is unclear. Resistance to non-host pathogens may occur when the plant can specifically recognise the pathogen. An alteration in the cell wall composition as supposed for the members of the Pythiaceae may help grapevine plants that are rather susceptible to become aware of an infection of a putative pathogen and to rapidly induce a variety of potential defenses that limit - accompanied by preformed morphological barriers - its growth and development.

As the cytological defense response of in vitro plants corresponds to that of grapevine plants in the field and based on the easy handling and also the absence of other organisms in axenic in vitro cultures, the challenge of in vitro-grown plants with P. viticola and elicitors offers a good experimental system for the elucidation/investigation of the physiological and molecular interaction including the elicitor recognition and expression of genes involved in plant defense response.

Acknowledgements. The authors wish to thank R. Wind for technical assistance. We are also indebted to O, Bachmann for fruitful and helpful discussions.

References

Agrios GN (1988) Plant pathology. Academic Press, San Diego

Arens K (1929) Physiologische Untersuchungen an Plasmopara viticola unter besonderer Berucksichtigung der Infektionsbedingungen. Jb wiss Bot 70: 98-157

Bach M, Seitz HU (1997) Elicitor-induced defense response of a suspension-cultured woody plant and possible mechanisms of signal transduotion. Can J Bot 75: 1243-1251

Beckmann CH, Mueller WC, Mace ME (1974) The stabilization of artificial and natural cell wall membranes by phenolic infusion and its relation to wilt disease resistance. Phytopathol 64: 1214-1220

Blaich R (1977) Versuche zur kunstlichen Mykorrhizabildung bei Vitis riparia. Vitis 16: 32-37

Busam G, Kassemeyer HH, Matern U (1997) Differential expression of chitinases in Vitis vinifera L. responding to systemic acquired resistance activators or fungal challenge. Plant Physiol 115: 10291038

Dai GH, Andary C, Mondolot-Cosson L, Boubals D (1995) Involvement of phenolic compounds in the resistance of grapevine callus to downy mildew (Plasmopara viticola). Eur J Plant Pathol 101: 541547

Davis KR, Ausubel FM (1989) Characterization of elicitor-induced defense responses in suspension-cultured cells of Arabidopsis. Mol Plant Microbe Inter 2: 363-368

De Lorenzo G, D'Ovidio R, Cervone F (2001) The role of polygalacturonase-inhibiting proteins (PGIPS) in defense against pathogenic fungi. Annu Rev Phytopathol 39: 313-335

Dixon RA, Paira NL (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097

Doke N (1983) Involvement of Superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to hyphal components. Physiol Plant Pathol 23: 345-357

Eshel D, Beno-Moualem D, Lorang JM, Dinoor A, Prusky D (2002) Induction of peroxidase during infection of unripe persimmon fruit by Alternaria alternate: A possible quiescence mechanism. J Phytopathol 150: 357-362

Jabs T, Dietrich RA, Dangl JL (1996) Initiation of runaway cell death In an Arabidopsis mutant by extracellular Superoxide. Science 273: 1853-1856

Kamoun S, Huitema E, Vleeshouwers VGAA (1999) Resistance to oomycetes: A general role for the hypersensitive response? Trends Plant Sci 4: 196-200

Keen NT, Yoshikawa M (1983) [beta]-1,3-Endoglucanase from soybean releases elicitor-active carbohydrates from fungus cell walls. Plant Physiol 71: 460-465

Kortekamp A, Zyprian E (1999) Leaf hairs as a basic protective barrier against downy mildew of grape. J Phytopathol 147: 453-459

Kortekamp A, Wind R, Zyprian E (1998) Investigation of the interaction of Plasmopara viticola with susceptible and resistant grapevine cultivars. J Plant Dis Protec 105: 475-488

Kortekamp A, Wind R, Zyprian E (1999) The role of hairs on the wettability of grapevine (Vitis spp.) leaves. Vitis 38: 101-105

Lamb CJ, Lawton MA, Dron M, Dixon RA (1989) Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56: 215-224

Levine A, Tenhaken R, Dixon R, Lamb C (1994) H^sub 2^O^sub 2^ from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79: 583-593

Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215: 239-247

Low PS, Merida JR (1996) The oxidative burst in plant defense: function and signal transduction. Physiol Plant 96: 533-542

Lux-Endrich A, Treutter D, Feucht W (2002) Response of apple (Malus domestica) cell suspension cultures cv. Alkmene on elicitation with biotic elicitors. J Appl Bot 76: 121-126

Matern U, Grimmig B (1994) Natural phenols as stress metabolites Acta Hort 381: 448-462

Mehdy MC (1994) Active oxygen species in plant defense against pathogens. Plant Physiol 105: 467-472

Mohan R, Kolattukudy PE (1990) Differential activation of expression of a subenzation-associated anionic peroxidase gene in near-isogenic resistant and susceptible tomato lines by elicitors of Verticillium albo-atrum. Plant Physiol 921: 276-280

Muller K, Sleumer H (1934) Biologische Untersuchungen uber die Peronosporakrankheit des Weinstockes. Landwirtsch Jb 79: 1-68

Nachit MM, Jakob H (1978) Flavans in leaves of Ribes nigrum infected with Black Currant Reversion Virus. Phytopathol Z 91: 280- 283

Reuveni M (1998) Relationships between leaf age, peroxidase and 1,3-glucanase activity, and resistance to downy mild\ew in grapevines. J Phytopathol 146: 525-530

Showalter AM, Bell JN, Cramer CL, Bailey JA, Varner JE, Lamb CJ (1985) Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection. Proc Natl Acad Sci USA 82: 6551-6555

Trump BF, Smuckler EA, Benditt EP (1961) A method for staining epoxy sections for light microscopy. Ultrastruct Res 5: 343-348

Werner S, Steiner U, Becher R, Kortekamp A, Zyprian E, Deising HB (2002) Chitin synthesis during in planta growth and asexual propagation of the cellulosic oomycete and obligate biotrophic grapevine pathogen Plasmopara viticola. FEMS Microbiol Letters 208: 169-173

Wojtaszek P (1997) Oxidative burst: an early plant response to pathogen infection. Biochem J 322: 681-692

Xue L, Charest PM, Jubaji-Hare SH (1998) Systemic induction of peroxidases, 1,3-[beta]-glucanases, chitinases, and resistance in bean plants by binucleate Rhizoctonia species. Phytopathol 88: 359365

Andreas Kortekamp(a),*, Eva Zyprian

Federal Centre for Breeding Research on Cultivated Plants, Institute for Grapevine Breeding Geilweilerhof, D-76833 Siebeldingen, Germany

Received December 9, 2002 Accepted March 28, 2003

* E-mail corresponding author: kortekam@uni-hohenheim.de

a Present address: Institute for Phytomedicine, University of Hohenheim, D-70593 Stuttgart, Germany

Copyright Urban & Fischer Verlag Nov 2003

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