Silicon in cell walls and papillae of Cucumis sativus during infection by Sphaerotheca fuliginea

P~~ssiological arrd Molecular

Silicon during A.

L.

PIaM Pathologv

in cell walls and papillae infection by Sphaerotheca A. D. M.

SAMUELS*$,

GLASS*,

J. G.

for publication

Januay

of Cucumis fuliginea

MENZIES~

* .!JGwrsip of British Columbia, Vancouuer, B.C., Canada, P.O. Box 1000, Agassiz, B.C., Canada, VOM IA0 (Amped

237

( 1994) 44, 237-242

l/67

1<4;

and D. L.

sativus

EHRET~

f Research Sfation, Agricullure

Canada,

1994)

The role of silicon in the enhancement of cucumber defence reactions against powdery mildew fungi was examined. Scanning transmission electron microscopy coupled with energy dispersive X-ray analysis and image analysis were used to map iu situ distribution ofsilicon in cucumber Icaves infected with Sphaero!heco fuliginea. Silicon was found in the papillae, in host cell walls, around the haustorial neck and in deposits between the host cell wall and plasma membrane. The deposition orsilicon in these sites could be a rapid mechanism for modifying the cell wall during pathogen attack.

INTRODUCTION Cucumber plants grown in hydroponic systemshave increased resistanceto Sphaerotheca fkliginea when the nutrient media is supplemented with soluble silicon in the form of

silicate [I, 13, 1.51. In scanning electron microscopy studies of powdery mildew infection on the surface of cucumber leaves, sites enriched in silicon were localized surrounding germinating conidia and along fungal hyphae [17]. Basedon similar work on monocotyledonous species[3, II, 161, these sitesof high silicon were interpreted as areas where fungal haustoria,penetrated the host epidermis. In this study, sections of cucumber leaves infected with powdery mildew were examined using light microscopy (LM), transmission electron microscopy (TEM), and scanning TEM (STEM) linked to an energy dispersive X-ray analyser (EDX)/’ rmage analysis system. The objective was to verify that the sites of high silicon associated with powdery mildew *infection were sites of fungal penetration to form haustoria. EXPERIMENTS

AND RESULTS

Cucumis sa&ivus cv. Corona plants were grown hydroponically, as previously described [17]. This cultivar of cucumber is susceptible to Sphaerotheca fuliginea; the appearance

ofdiseasesymptoms, and their reduction in silicate treated.plants, have been previously described [14,17]. The hydroponic medium was based on the cucumber nutrient :To whom correspondence should be addressed at. Abbrc..&tions used in text: EDX, energy dispersive X-ray analyser; LM, treated; -Si, untreated; STEM, scanning transmission electron microscopy; microscopy.

light microscopy; +Si, silicon TEM, transmission electron

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FIG. I. Transmission electron micrographs of cucumber leaves, 96 h after inoculation with .S/~/~oero&ca J;~~&%Yz. (a) Epidermal cell (e) from the leaf of untreated control plant, which contained three haustoria complexes (arrows) consisting of extrahaustorial membranes, estrahaustorial matrices, haustoria with multiple lobes. This section passed through the lobed region of the haustorial complex. The cell walls were darkly stained; there was a cross-section through the fungal mycelium (m) outside the leafsurface. (Bar = 5 pm). (b) Epidermal cell (e) containing haustorial complex (h) from silicon treated leaf. Cell wall darkly stained, with electron-dense deposits on inner surface of wall in the papilla (arrow). (Bar = 1 pm). (c) Outer host cell wall penetrated by haustorial neck (n). Silica bodies aggregated in the papilla, adjacent to the neck (arrow). (Bar = I urn).

formula usedin commercial greenhouseoperations in British Columbia [4]. For silicon supplemented cultures, 1.7 mM (100 ppm) potassium silicate was added (National Silicates Kasil #6, Toronto, Canada). Six weeks after germination, the adaxial surfaces of mature cucumber leaves were inoculated with conidia. Twenty-four hours prior to inoculation, leaves of heavily infected cucumber were shaken briskly to dislodge old conidia; this provided fresh conidia with high viability for inoculation. Colonies were excised from the infected leaves and pressedgently on leaves from silicon treated (+Si) or untreated (-Si) plants. At 72 and 96 h after inoculation, leaf sampleswere cut out of the inoculated area with a razor blade and plunged into 2.5% glutaraldehyde in 0.1 y0 sodium phosphate buffer, pH 7.2. The samples were dissected into 0.2 mm” pieces while immersed in fixative then transferred to fresh fixative for 2 h. After dehydration in ethanol, sampleswere infiltrated with Epon-Araldite resin with propylene oxide as a transition solvent. Sections (0.5 pm) were cut on glassknives for LM and stained with 1y. toluidine

Distribution

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blue for 30 s then examined for cells containing haustoria. The + Si treated leaves have been shown to contain significantly fewer haustoria than -Si leaves (at 96 h after inoculation, +Si leaves had mean haustoria per colony of 3, whereas -Si leaves had 35) [la], thus extensive screening of leaf sectionsat the LM level was necessaryto locate haustoria. Sections (O-1-0*25 pm) were cut on glassknives and mounted on Formvar coated beryllium grids for X-ray microanalyses. Thin sections (70-90 nm) were cut on a diamond knife for TEM and stained with uranyl acetate and lead citrate for ultrastructural analyses. For EDX analysis, the electron microscope was operated in scanning transmission mode with a spot size of 15 nm, interactively with a Kontron Image Analysis computer and the LINK AN 10000 X-ray analyser. After collecting a sample spectrum, windows of X-ray energy levels characteristic for the elements of interest were designated. Each window had a width of seven channels of 0.02 keV channel-’ for four-channel maps. X-ray maps of 256 x 256 pixels were collected using a dwell time of 100 or 200 ms pixel -l. The chemical fixation procedure results in the leaching of soluble elements from the sample, so the silicon detected in these experiments was the insoluble, polymerized form. The characteristic Sphaerotheca haustorial morphology of an oval central body, flanked by lobesand surrounded by an extrahaustorial membrane, was observed in the epidermal cells. There were no apparent differences in the ultrastructure of haustoria from + Si or - Si leaves. The cell walls of infected cells from - Si plants were modified in that they were more electron dense than uninfected cells, but they did not show a tendency to shatter upon sectioning, and the density was confined to the wall proper [Fig. 1(a)]. No silicon was detected in leaves from -Si plants in X-ray spectra or maps. There were obvious changes to the cell wall in +Si leaves: in infected cells and the cells surrounding the trichome bases, the cell walls were extremely electron dense [Fig. l(b)] and often shattered upon sectioning, a common problem with silicified material [Fig. 2(a)]. Electron-dense deposits were observed within the host cell walls, as well as in the papillae region between the host cell wall and the plasma membrane [Fig. 1(c)]. These small electron-dense bodies have a similar appearance to the silica bodies reported previously [la, 161. Using point mode excitation, as well as in maps (not shown), these bodies were found to contain silicon. Using X-ray analysis, the areascontaining the electron-dense depositswere tested for the presenceof silicon. For each spot scanned on the section, the presenceof silicon is reported by a bright dot on the computer screen; when a whole infected cell is scanned, this creates a map of the silicon distribution [compare Fig. 2(a) (scanning transmission image of an infected cell) with Fig. 2(b) (map of the silicon distribution of the same area)]. The presence of silicon was confirmed in the cell walls of infected cells, in the deposits between the plasma membrane and cell wall, in the papillae, and around the haustorial neck. No silicon was detected in the extrahaustorial membranes, extrahaustorial matrices, haustoria, fungal mycelium, or uninfected epidermal cells with the exception of the trichome bases.The map of silicon distribution can be electronically overlaid on an EM image to correlate silicon-rich areas with ultrastructure. When the silicon X-ray map is superimposed on the scanning transmission image, the electrondense deposits of the host cell wall and the collar region surrounding the haustorial neck showed high silicon contents [Fig. 2(c)]. IG-2

FIG. 2. Scanning transmission electron micrograph and energy dispersive X-ray maps of silicon treated (+Si) cucumber leaves, 96 h after inoculation with Sphaerotheca fuliginen. (a) Scanning transmission image ofunstained semi-thick section of +Si leaf epidermal cell (e), cut tangentially through the epidermal layer. Neck of an haustorium was seen in the center of the cell (arrow) ; section had shattered along cell walls connecting epidermis and mesophyll cells (white areas), a common problem with sections of silicified material (Bar = 10 l.tm). (b) X-ray map of epidermal cell (e) shown in Fig. 2(a). Silicon was found in the collar region surrounding the haustorial neck (arrow) and in the cell wall of the infected epidermal cell, as well as extending into adjacent cells. (Bar = 10 urn). (c) Scanning transmission image of cross-section of epidermal cell (e) containing a haustorial complex (h). The distribution of silicon in this section has been displayed by superimposing the silicon X-ray map on the image. Areas of high silicon (white dots) were found in the papilla and neck. Some beam damage occurred on this section during the mapping process: light scan lines across the top and light points can be seen in the adjoining mesophyll cell where the electron beam dwelt on each pixel of the raster as the image analysis computer collected the X-ray map. (Bar = 5 pm).

There was a wide range in the extent of silicification of the cell walls of infected leaf cells from +Si plants: some cells showed little or no silicon deposition. For example, X-ray analysis of the cell shown in Fig. 1 (b) revealed silicon restricted to the papilla and cell wall immediately adjacent to the papilla. Other cells were completely encased in silicon, with the silicification extending to adjacent epidermal and mesophyll cells, as illustrated in Fig. 2(a and b). The plane of section through the leaf could have a major impact on whether silicon is perceived as being present. For example, in a cell with silicon deposited in a small papilla, a section that missed that ‘papilla region would appear not to contain silicon.

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CONCLUSIONS

Silicon was found only in trichome base cells and infection sites surrounding fungal haustoria. This supports the interpretation of earlier studies [24] where regions of high silicon on the leaf surface were postulated to be the sitesof fungal penetration into the cucumber leaf. The appressoria of Sphaero~heca are indistinct, so in previous studies it was not possible to tell where a haustorium would arise based on the presence of a distinct appressorium, as is the case for El;l~@he graminis [2U]. The X-ray mapping system is useful for identifying the nature of electron-dense deposits seen using TEM. The cell walls of all infected cells were electron dense; but how much of that density was due to silicon could be distinguished. The increased electron density observed in cell walls of -Si leaves probably represents phenolic depositions, as no silicon was detected using EDX. Light microscopy studies of this system have demonstrated the presence of phenolics in the cell walls surrounding infected epidermal cells [M] ; the presenceof phenolics have been widely documented in other systems [reviewed in 51. In contrast, cell walls from +Si leaf cells had more extensive dark deposits and these could be identified as silicon-containing sites. The presenceof silicon has been reported in the papillae incited by powdery mildew fungi [3, II, 16,201, but the distribution around the infected cell has not been mapped. Sections cut through infected leaves allow the haustorial complex, aswell as the papilla and collar to be examined. The haustoria observed in the +Si plants appeared to be healthy despite the presence of the silicon in the adjacent cell wall and papilla. This suggeststhat silicon per seis not directly toxic to the fungus. Several studies of enhanced resistance with silicate supplementation have shown +Si plants to have more rapid and extensive phenolic depositions around sites of fungal penetration [5, 131. Silicon supplemented cucumber plants infected with PJdium spp. have shown enhanced levels of several defence-related enzymes and the appearance of fungistatic phenolic compounds (M. Cherif, A. Asselin, R. Btlanger, pers. comm.). Thus the enhanced resistanceof cucumbers supplemented with silicon appears to be more complex than simple alterations in the apoplastic architecture, as shown in this study, but instead is the result of a multicomponent change in the plants’ defences. This work was supported by the Science Council of British Columbia. We thank Dr R. Belanger for reviewing the manuscript. REFERENCES MH, Besford RT. 1986. The effectsof siliconon cucumber plantsgrownin recirculating nutrientsolution.,&~nals of Bofany 58: 343-351. Aist JR, Israel HW. 1977. Papillaformation:timingand significance auringpenetrationof barley coleoptiles by Erysiphe gmninis hordei. Ptylopathology 67: 455-461. Akutsu K, Doi Y, Yora K. 1980.Elementaryanalysis of papillacand cytoplasmic vesicles formed at

I. Adatia 2. 3.

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