The analysis of 12 grapevine cultivars differing in susceptibility to powdery mildew revealed a correlation between the thickness of cuticle plus cell wall of young leaves and resistance to mildew. Neither in mature leaves or in leaves of in vitro plants or in berries such correlations could be established. Coating of the leaf surface with a polymetacrylate antitranspirant prevented sporulation in all cases, although infection structures were formed

Blaich, R.

Heintz, Cathérine

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 28, 153-160 (1989) Bundesforschungsansta lt für Rebenzüchtung Geilweilerhof, Siebeldingen, BR Deutschland Structural characters of epidermal cell walls and resistance to powdery mildew of different grapevine cultivars by CATHERINE HEJNTZ and R. BLAICH Strukturelle Eigenschaften von Epidermiszellwänden und Oidiumresistenz bei verschiedenen Rebsorten Zusammenfassung : Untersuchungen an 12 unterschiedlich oidiumanfälligen Rebsor-ten ergaben bei jungen Blättern eine Korrelation zwischen Dicke von Cuticula plus Zellwand und Oidiumresistenz. Bei älteren Blättern, solchen von in vitro-Pflanzen und bei Beerenhäuten konnte keine Korrelation gefunden werden. Beschichten der Blattoberfläche mit einem Antitranspirant auf Polymetacrylat-Basis verhinderte in allen Fällen eine Sporulation, obwohl Infektionsstruktu-ren gebildet wurden. K e y wo r d s : oidium, resistance, variety of vine, leaf, berry, epidermis, cell wall, cuticle. Introduction In previous reports, it was shown that powdery mildew (Uncinula necator, Oidium tuckeri) may penetrate artificial membranes by mechanical force (HEINTZ 1986) and that a very important active defence mechanism of the epidermal cells of Vitis against Oidium is the formation of mechanical barriers in or near cell walls - e:g. papillae (HEINTZ and BLAICH 1989) and silica deposits (BLAICH and WI ND 1989). Hence the thick-ness of the epidermal cell walls and cuticles might play a role as a passive character of resistance to powdery mildew in Vitis cultivars. Materials and methods Leaves and berries of 12 grapevine varieties (see Table 1) with different suscepti-bility to Oidium were taken from plants grown in the field, in the glasshouse or under in vitro conditions at different times during the growing season. Two types of leaves were used : 5th (young, just fully expanded) leaves and 8th (mature) leaves, counted from the top of the shoot. As the thickness of leaf cell walls was less than 1 µm, measurements with the light microscope proved very difficult and required semithin sections of resin embedded leaves. Therefore, margi ns of broken leaf segments freeze dried at - 60 ° C were ana-lyzed using a Zeiss DSM 950 stereoscan electron microscope (SEM). This method was more exact and far simpler than might be expected (for details of preparation see BLAICH et ae. 1984). Each value given in Tables 1 and 2 and Figs. 1 and 3 is based on 150 single measurements u sing 5 fragments of different leaves of one variety. Culture of Oidium and in vitro grapevines as well as leaf disc tests for susceptibil-ity to Oidium (indexed as intensity of sporulation) are described by STEIN et al. (1985). Surface treatment with Protec (polymetacrylate normally used as an antitranspi-rant): leaf discs (mature and young leaves from field, in vitro and glasshouse plants, 154 c. HEINTZ and R. BLAICH 10 discs/variant in triplicate) of all varieties were either dipped into the product or sprayed to obtain a thin but complete film, allowed to dry and then kept in petri dishes on damp filter paper. The surfaces were then inoculated with oidiospores as described previously (STEIN et al. 1985) and assessed after 12 d. Results and discussion L e a f c u t i c 1 e : In the SEM it is rarely possible to differentiate between cuti-cle and cell wall (Fig . 2c), whereas in light microscopy the cuticle (about 0.4 ~tm) is at the limit of resolution. Therefore, the values in Tables 1 and 2 give the thickness of cuticle plus cell wall. In yo ung leaves from field-grown plants a correlation between thickness of cell walls and susceptibility to Oidium could be demonstrated (Fig. 1). All Tl. vinifera cvs (susceptibility > 7) lie around 0.85 ~lm and all resistant varieties ( < 2) above 1.1 µm. Mature field leaves, however, showed no significant correlation: They are generally more resistant in all varieties (see also DosTER and ScHNATHORST 1985) and differences in thickness between susceptible and resistant varieties h ave diminished (Table 1). In vitro leaves uniformly have very thin walls (0 .6 µm and less) and are much more sensitive to powdery mildew (see STEIN et al. 1985). Table 1 Thickness of epidermal cell walls including cuticle of different grapevine varieties · Young leaf: 5th, mature leaf: 8th, counted from the top of the shoot Dicke der Epidermiszellwand einschließlich der Cuticula ve rschiedener Rebsorten · Junge Blätter in Position 5, ältere in Position 8, von der Triebspitze aus gezählt Variety Riesling1) Kerner1) V 31251) Pollux Phoenix Gf. Ga-50-34 Sirius Gf. Ga-54-14 Orion Gf. 67-198-3 Marechal Fach Vidal 256 Mean 1) Varieties of Vitis vinifera. Young leaves 0.85 0.85 0.85 0.86 1.11 1.14 1.05 1.23 1.03 1.10 1.16 1.46 1.10 Thickness of cell wall + cuticle (µm) Field plants Mature leaves 1.20 0.90 1.07 1.02 1.18 1.16 1.12 1.26 1.06 1.06 1.24 1.47 1.15 In vitro plants 0.62 0.52 0.40 0.37 0.62 0.63 0.52 0.51 0.40 0.55 0.44 0.44 0.50 LSD(5%) 0.13 0.04 Epidermal cell walls and resistance to powdery mildew 155 Co a t i n g o f 1 e a v e s : The treatment of leaf surfaces with polymetacrylate (Protec) to produce an artificial thickening of the cuticle was successful for all varieties and all leaf types: The mean (all varieties) sporulation notes for untreated young leaves were 5.3 (for field plants) and 2.3 (for glasshouse plants), for mature leaves < 1. Pro-tected leaves never showed sporulations. Cytological observations revealed that the fungus formed appressoria and that some penetration attempts were successful as indicated by the accumulation of callose around· the penetration peg (fluorescence after staining with aniline blue; see HEINTZ and BLAICH 1989). This was, however, never suffi-cient to produce visible symptoms of sporulation on the leaf. SEM showed that the coat was very uneven and had thin areas and even holes (Fig. 2 e) . The capability of the parasite to penetrate polymetacrylate membranes of < 1 µm is well known (HEI NTZ 1986). These membranes were, however, formed using true solutions whereas the poly-metacrylate layers on leaves were produced by drying an aqueous dispersion of small polymetacrylate beads which fuse due to capillary forces. Be r r y c u t i c 1 e : Peripheral cell walls and cuticle of berries are much thicker than in leaves (for more details see CoNSIDINE and K NOX 1979). The cuticle con-sists of 3 layers which may be differentiated under the fluorescence microscope (Fig. 2 f) and - in rare cases - by SEM (Fig. 2d). The changing thickness during maturation is demonstrated for a resistant and a susceptible variety (cv. Vidal 56 and cv. Kerner) in Fig. 3. lt reached a maximum during the 2nd growth phase and decrea-sed slightly until the end of maturation. The time when maximal thickness was reached differed between both varieties (Fig. 3); it seems also to depend on climatic conditions Table 2 Thickness of the epidermal cell walls of berries of different grapevine varieties at one sample time (4th October 1984) · If possible, mildew infected berries were used for the analysis · These values are only intended to give hints for discussion; they are not suitable for further comparisions s ince varieties are different in maturity and wall thickness might have been measured before or after maximum (compare Fig. 3) · Dicke der Epidermiszellwand der Beeren verschiedener Rebsorten zu einem bestimmten Zeitpunkt (4 . Oktober 1984) · Nach Möglichkeit wurden oidiumbefallene Beeren ausgewertet · Die Werte sol-len lediglich als Diskuss ionsgrundlage dienen; für weitergehende Vergleiche sind sie nicht geeig-net, da die einzelnen Sorten zu verschiedenen Zeiten reif werden und die Wanddicke deshalb vor oder nach dem Maximum gemessen worden sein könnte (vgl. Fig. 3) Variety Thickness of Sporulation Sugar content wall + cuticle (µm) intensity (g/l) Riesling 5.0 2.3 57.7 Kerner 4.7 7.2 93 .5 V3125 4.3 3.8 40.0 Pollux 4.8 0.1 139.5 Phoenix 3.9 0.3 117.7 Gf. Ga-50-34 3.1 0 140.5 Sirius 4.6 2.1 101.2 Gf. Ga-54-14 4.7 0.2 84.5 Orion 4.4 2.7 101.2 Marechal Foch 4.9 0 181.5 Vidal 256 6.6 0 93 .5 156 Sporulation 10 5 0 0.8 c. HEINTZ and R. BLAlCH r= - 0 .803** Y= -12.7 X +18.4 •7 •9 1.0 •8 .10 Thickness of cuticle + cell wall ( 1-'m ) 1. Riesling 2„ Kerner 3. V 3125 4. Pollux 5. Phoenix 6. Ga-50-34 7. Sirius 8. Ga-54 - 14 9. Orion 10. 67-198-3 11. M. Foch 12. Vidal 256 .12 1.5 Fig. 1: Correlation between the thickness of epidermal cell wall (including cuticle) of leaves of some grapevine cvs (see Table 1) and their susceptibility to powdery mildew. Korrelation zwischen der Dicke der Zellwand (einschließlich Cuticula) de r Blattepidermiszellen ve rschiedener Rebsorten (siehe Tabelle 1) und ihrer Oidiumanfälligkeit. Fig. 2: a- e) SEM images, f) light microscopic image; A = appressorium, C = cuticle, W = peri-clinal wall of epidermal cell. - a) Tvw hyphae of Oidium on the leaf surface with typical s ingle appressoria; note some wrinkles on the leaf surface. - b) Hyphae with a typical cluster of appres-soria on berry surface. - c) Edge of a broken epidermal cell wall near a leaf ve in where the epider-mal surface forms numerous wrinkles; in this particular case, cell wall and cuticle can be differen-tiated, which is rare . - d) Edge of a broken epidermis cell of a berry (cv. Vidai°256) where cell wa ll and layers of cu ticle can be differentiated. - e) Surface of a leaf coated with Protec; the polyacry-late layer is incomplete, leaving some holes and some areas where the space between wrinkles is not completely filled up. - f) UV microscopic image (405 nm) of 1 µm section of berry epidermis stained with thioflavin; the shrinkage of the underlying cells during preparation was stronger than that of the cuticle which (near an infection site) forms a ply separated from the cell wall; 2- 3 layers can be differentiated. a- e ) REM-Bilder, f) Lichtmikroskopisches Bild; A = Appressorium, C = Cuticula, W = perikline Zellwand der Epidermiszelle. - a) Zwei Oidium-Hyphen auf e iner Blattoberfl äche mit typischen Einzelappressorien; man beachte einige Runzeln der Blattoberfläche. - b) Hyphe mit typischer Anhäufung von Appressorien auf Beerenhau.t. - c) Kante an e iner abgebrochenen Epidermiszell-wand in der Nähe einer Blattader, wo die Epidermisoberfläche zahlreiche Leisten aufweist; aus-nahmsweise lassen sich Zellwand und Cuticula differenzieren. - d) Bruchkante einer Beerenepi-dermiszelle (cv. Vidal 256); auch hier können Zellwand und geschichtete Cuticula ausnahmsweise differenziert werden. - e) Oberfläche e ines Blattes, das mit Protec behandelt wurde; di e Polyacryl-schicht hat einige Löcher und dünne Stellen , an denen die Zwischenräume zwischen den Epider-misleisten nicht ganz ausgefü llt sind. - f) UV-Fluoreszenzbild (405 nm) eines kunstharzeingebette-ten 1 µm dicken Schnittes einer Beerenepidermis; die Epidermiszellen sind während der Präpara-tion stärker geschrumpft a ls die Cuticula, die sich (neben einer Oidium-Infektion) fa ltenar tig von de r pe ripheren Zellwand abgehoben hat: 2-3 Schichten können unterschieden werden. Epidermal cell walls and resistance to powdery mildew. 157 158 c. HEINTZ and R. BLAICH since in Australia the maximum for V 1rinifera cv. Muscat of Alexandria occurred during the lst growth phase (CüNSIDINE and K NOX 1979). CHRISTIN- BOURGUIGNON et al. (1986) found an analogous behaviour of the whole berry skin. The other varieties were assessed only at one time. Reproducible inoculation of berries under laboratory conditions being extremely difficult, infected berries were collected in the fie ld to obtain some additional hints on susceptibility. Because this, of course, was not possible for the highly resistant varieties and both Oidiwn susceptibil-ity and wall thiclmess of berries are influenced by the state of maturity (Fig. 3), the data must be interpreted cautiously, although considerable varietal differences of the wall thickness of ripe berries were found (from 3.1 to 8.6 µm) which could be correlated (r = 0.63, significant at P = 5 % ) to the wall thickness of mature field leaves. Thickness of cuticle + cell wall ( JJm ) 8 ·- -· ·--· 7 6 Vidal Kerner / • I / • „"' ' ."' ' I ' I ' I •-Sugar content of juice (g/I) 0 ---- 0 o--o -. 0 200 5 4 3 / / / / / / / / • /. __ ;·y. /. //" • 100 / /1,--','o . , ,' ,' 0 ,' 0 °'::"'- ,o------ 0 "/ 0 ...... ~ ,.- ../" o ... ._ o'..=:.:.__ o /' . 2 1 0 17.7 31.7 14.8 31.8 19.9 4.10 24.10 Date Fig. 3: Thickness of the epidermal cell wall (including cuticle) and sugar content of the berries of grapevine cvs Vidal 256 (res istant to mildew) and Kerner (susceptible) during maturation. Dicke von Epidermiszellwand (einschließlich Cuticula) und Zuckergehalt von Beeren der Rebsorten Vida l 256 (oidiumresisten t) und Kerner (anfällig) während der Reife. Epidermal cell walls and resistance to powdery mildew 159 Conclusions Although the existing correlation between cuticular thickness and resistance to powdery mildew of young grapevine leaves does not prove the role of thickness as a preformed resistance character, this fact corroborated earlier assumptions from exper-irnents with artificial membranes (HEINTZ 1986) which became unpenetrable at a thick-ness corresponding to the dirnensiori of the cuticle of resistant varieties. Of course, we are aware that the mechanical properties of polyacrylate membranes and cutin struc-tures may be quite different an<l even between the cuticle of the leaf and that of the berry there must be considerable differences because berries with cuticles of serval ~tm thickness may be susceptible. Such differences are recognized by Oidium because the density of appressoria is much higher on berries (Fig. 2 b) than on leaves (Fig. 2 a) - a phenomenon that has been already described for appressoria on leaves of resistant cul-tivars by DOSTER and SCHNATHORST (1985). Thus it is certainly not mere thickness but also other properties which determine resistance to mildew. lt has been shown by BLAicH et al. (1984) that in the surface of berries natural perforations are formed during maturation which start from preformed weak zones. The high density of appressoria might simply reflect numerous penetration attempts to find such weak zones. On the other band, it is weil known that resistance is determined by a number of overlapping characters which may replace each other in different cultivars (HEINTZ and BLAICH 1989 and in preparation). As AusT and HOYNINGEN-HUENE (1986) point out, every barrier which slows down the penetration process may provide the time necessary for other mechanisms (formation of papillae, deposition of cell wall incrusts). Summary The analysis of 12 grapevine cultivars differing in susceptibility to powdery mildew revealed a correlation between the thickness of cuticle plus cell wall of :roung leaves and resistance to mildew. Neither in mature leaves or in leaves of in vitro plants or in berries such correlations could be established. Coating of the leaf surface with a poly-metacrylate antitranspirant prevented sporulation in all cases, although infection structures were formed . This work was supported by the Deutsche Forschungsgemeinschaft References AusT, H. J .; HoYNINGEN-HUENE, J.; 1986: Microclimate in relation to epidemics of powdery mildew. Ann. Rev. Phytopathol. 24, 491-510. B LA!CH, R.; STEIN, U.; WI ND, R.; 1984: Perforationen in der Cuticula von Weinbeeren als morpholo-gischer Faktor der Botrytisresistenz. Viti s 23, 242-256. BLA!CH, R.; WI ND, R.; 1989: Inducible silica incrusts in cell walls of, Vitis leaves. Vitis 28, 73-80. CHRISTIN-BOURGUIGNON, F.; MAUJEAN, A.; AUDRAN, J. C.; 1986: Pellicule et resistance de Ja baie de rai-sin a Ja pourriture grise chez deux cepages champenois. Vigneron Champenois 3, 143-148. CoNSIDI NE, J. A.; KNox, R. B.; 1979: Development and histochemistry of the cells, cell walls, and cuti-cle of the dermal system of fruit of the grape, Vitis l'inifera L. Protoplasma 99, 347-365. DosTER, M. A.; ScHNATHORST, W. C.; 1985: Effects of leaf maturity and cultivar resistance on develop-ment of the powdery mildew fungus on grapevines. Phytopathology 75, 318-321. 160 c. HEINTZ and R. BLAICH HEINTZ, CATHERINE; 1986: Infection mechanisms of grapevine powdery mildew (Oidium tuckeri): Comparative studies on the penetration process on artificial membranes and leaf epidermis. Vitis 25, 215-225. HEINTZ, CATHERINE; BLAJCH, R.; 1989: Ultrastructural and histochemical studies on host-parasite rela-tions between Vitis spp. and Uncinula necator. (Subrnitted). STEIN, U.; HEINTZ, CATHERINE; BLAICH, R.; 1985: Die in-vitro-Prüfung von Rebsorten auf Oidium- und Plasmopararesistenz. Z. Pflanzenkrankh. Pflanzensch. 4, 355-369. Eingegangen am 20. 6. 1989 Prof. Dr. R. BLAICH BFA für Rebenzüchtung Geilweilerhof D 6741 Siebeldingen 

Structural characters of epidermal cell walls and resistance to powdery mildew of different grapevine cultivars

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Structural characters of epidermal cell walls and resistance to powdery mildew of different grapevine cultivars

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