Ultrastructure of wilt syndrome caused by ...

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fibrillar coating, and bubbly coating stained differentially. .... similar structures which stain intensely blue. ..... similar to that described in hops infected by Ver-.
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Ultrastructure of wilt syndrome caused by Verticillium dahliae. VII. Correlated light and transmission electron microscope identification of vessel coatings and tyloses J A N EROBB,'J . D. BRISSON ,' LLOYDBUSCH,A N D B. C. LU Dep(rrt~nentof'En~irot~tnentol Biology rrnd D e p ~ r t ~ nOf'Botrrny e~~r 011d Generics, Ut~ioer~iry oj'Grielph, G~relph,Out., Cot~arloN l G 2Wl Received August 17, 1978 L. BUSCH,and B. C. Lu. 1979. Ultrastructureofwilt syndromecaused ROBB,J., J . D. BRISSON, by Verricillilrtn rlrrhlioe. VII. Correlated light and transmission electron microscope identification of vessel coatings and tyloses. Can. J . Bot. 57: 822-834. Thin cross sections of petioles from wilted leaves of chrysanthemums infected with Verticilli~rmrlrrhlirre were fixed in glutaraldehyde f FeCI, and embedded for electron microscopy. Alternate thick (LM) and thin (TEM) sections were cut. The thick sections were stained with ( l ) the Prussian blue reaction, (2) Sudan black B, (3) toluidine blue 0 , or (4) Schiffs reagent. Correlated LM and TEM of exactly the same vessels showed that tylosis walls, smooth coating, fibrillar coating, and bubbly coating stained differentially. The tyloses are more abundant than formerly anticipated but are restricted to the primary vessels; fungal cells and coating on vessel walls are confined to smaller secondary vessels. ROBB,J., J . D. BRISSON, L. BUSCHet B. C. Lu. 1979. Ultrastructure ofwilt syndrome caused by Verricilli~rtrrdohline. VII. Correlated light and transmission electron microscope identification of vessel coatings and tyloses. Can. J . Bot. 57: 822-834. L'etude a porte sur de minces tranches transversales de pktioles provenant de feuilles flCtries de chrysanthkmes infectes par le Verticillirrtn rlnhlirre. Les tranches, fixees dans un melange de glutaraldehyde et FeCI,, furent enrobees pour la microscopie electronique et decoupees en sections alternees semi-mince pour la microscopie photonique (MP) et ultrafines pour la electronique B transmission (MET). Les sections semi-mince furent colorCes par ( l ) une reaction au bleu de Prusse, (2) le noir Sudan B, (3) la toluidine bleue 0 , ou (4) une solution de Schiff. L'analyse correlee par MP et MET d'exactement les m h e s vaisseaux a montlt que les palpis des thylles, le materiel lisse de recouvrement, le matkriel fibrillaire et le recouvrement bulbeux se colorent differentiellement. Les resultats de cette analyse ont montre que le nombre de thylles presentes fut beaucoup plus grand qu'anticipe anterieurement. Ces thylles ne se foment que dans les vaisseaux du xylkme primaire; le tapissement sur les parois de vaisseaux et les cellules du pathogkne ne se trouvent que dans les plus petits vaisseaux du xylkme secondaire.

Introduction which is capable of resolving some of the interpretational problems. In this technique, the tissue is Previous studies in chrysanthemums and sunflowers infected with Verticilliu~n dahliue prepared for electron microscopy; alternate thick showed that many interpretational problems may and thin sections are cut for light microscopy (LM) be encountered in the cytological analysis of vessel- and transmission electron microscopy (TEM), reoccluding structures (Robb et al. 1979). In the spectively. Standard histochemical stains (LM) are petioles of these hosts, the main occluding ele- applied in order to differentiate the structures: ( l ) ments are fungus, three or possibly four types of Prussian blue for phenolic compounds, (2) Sudan vessel coating material (i.e., smooth, fibrillar, bub- black B for lipids, (3) Schiff s reagent for carbohybly, and irregular), tyloses, a vessel filling material drates, and (4) toluidine blue 0 , a general metawhich may be produced by the tyloses, and vessel chromatic stain. Chrysanthemums were used plugs (Robb et al. 1979). The problems are most throughout this study. acute in infected chrysanthemums since almost Materials and Methods every vessel in a petiolar cross section of a wilted Chrysanthemum cuttings (Chrysrrnthetnutn tnorifoli~rm leaf is occupied by one or more types of these Ramat. CV. Brilliant Anne) were grown and inoculated with structures. Verticilli~rm (lrrl~lirre Kleb. as previously described (Robb, The following paper demonstrates one technique Busch, and Lu 1975). When foliar flaccidity was well advanced, 'Mailing address: Department of Botany and Genetics, University ofGuelph, Guelph, Ont., Canada NIG 2W1. ZPresent address: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA 0R6.

very thin freehand cross sections were cut from petioles of infected plants and uninoculated controls (Robb et al. 1978). The petiolar sections were fixed for 5 h at 4°C in 3% glutaraldehyde made up in 0.07 M phosphate buffer (pH 6.8) containing 5% (wlv) ferric chloride. The sections were monitored with the

0008-4026179lO70822-l3$O 1.OO/O 01979 National Research Council of CanadaIConseil national de recherches du Canada

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ROBB ET AL.

light microscope in the buffer wash; they were dehydrated, embedded in Spun's plastic, and sectioned for LM and TEM as previously described (Robb et r r l . 1978). Each piece of tissue (i.e., one freehand section) was serial sectioned in sets consisting of approximately 20 gold sections (i.e., 80-90 nm thick) followed by 8 red-green sections (i.e., 0.25 pm thick). Gold sections (TEM) were viewed without additional staining using a Phillips 200A electron microscope operating at 80 kV. Red-green sections (LM) were stained in one of four. ways (i.e., two sections each): (1) 2% potassium ferricyanide in 1% HCI in distilled water (i.e., Prussian blue reaction, Pearse (1972), Robb et r r l . (1978)); (2) Sudan black B in ethylene glycol; (3) 0.5% toluidine blue 0 in 1% sodium borate (TBO); and (4) Schiff s reagent following oxidation by H,O, (PAS reaction, Pool (1973)). All thick sections were photographed using a Leitz orthoplan microscope.

Results and Discussion Figure 1 is a light micrograph of a cross section through a petiole of a wilted leaf; the section is stained by the Prussian blue reaction. The section of vein bounded by Fig. l is the region subjected to detailed cytological analysis. The monitor photograph of the original freehand section was previously illustrated (Robb et al. 1978, Fig. 5). The region of vein illustrated in Fig. 1 is located approximately in the centre of the xylem as visualized in the monitor section. The region of vein studied contains 86 vessel cross sections. An estimate was made of the number of coated vessels and tyloses based on TEM analysis of the first set of gold sections. This estimate yielded the following counts: tyloses, l l ; coated vessels, 34 (one also contained a tylose); nonoccluded, 35; vessels under grid bars, 6. Figures 2 to 5 illustrate the vessels in the upper part of region A (indicated on Fig. 1) as they appear with LM after staining. The section illustrated in Fig. 2 is stained with the Prussian blue reaction. The vessels labelled t , and t2 are 'coated' with similar structures which stain intensely blue. TEM visualization of vessels t, and t, showed that the blue structures which are ferric positive are tylosis walls (Figs. 10 and 100; Robb et al. 1979). As indicated in Table l , tylosis walls are unstained with Sudan black B (Fig. 3), purple or blue with toluidine blue (Fig. 4), and deep red with PAS (Fig. 5). As a further check on the colour reactions of the tylosis walls, each staining procedure was observed in at least one case in which the tylose parenchyma cell connection was visible (Figs. 12, 13). As indicated in Figs. 12 and 13, the tylosis wall stains identically with the parenchyma cell wall or, more specifically, with the protective layer which is also strongly ferric positive (Robb et al. 1978). Figure 14 is a TEM view through the protective layer - tylosis wall transition zone indicated in Figs. 12

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and 13; compare the structure of the tylosis wall with that of the wall of the parenchyma cell adjacent to the tylosed vessel. The similarity in the staining reactions of the protection layer and the tylosis wall is compatible with the theory that the former gives rise to the latter (Foster 1967; Meyer and C6te 1968). Figures 6 to 9 illustrate the vessels in region B (indicated on Fig. 1) as they appear with LM after staining. According to the corresponding TEM observations, all seven vessels shown in the light micrographs are truly coated (Fig. l l , c,