Ultrastructural Cytochemistry of the Host-Fungus Interfaces in the Endornycorrhizal Association Glomus mosseae / Allium cepa

Originalarbeiten . Original Papers

Laboratoire de Botanique II, Universitt~ de Nancy I, France, and Station de Physiopathologie Vegthale, INRA, Dijon, France

Ultrastructural Cytochemistry of the Host-Fungus Interfaces in the Endomycorrhizal Association Glomus mosseae / Allium cepa

JEAN DEXHEIMER, SILVIO GIANINAZZI and VIVIENNE GIANINAZZI-PEARSON

With 14 figures

Received June 2,1978' Accepted October 30, 1978

Summary

The structure of the host-fungus interface in the VA mycorrhizal aSSOCiation Glomus mosseael Allium cepa has been investigated cytochemically (Thiery test; differential DMSO and EDTA extraction; phosphotungstic acid staining) and using different fixation procedures (glutaraldehyde and OS04 post-fixation; simultaneous glutaraldehyde - OS04 fixation).

The continous, highly invaginated host plasmalemma surrounding the intracellular fungal structures retains its affinity for phosphotungstic acid and continues to produce polysaccharide fibrils, but in the presence of the endophyte it progressively loses the capacity to organise these into well-structured wall material.

At the point of cell penetration and in the primary arbuscule branches the fungal wall, which is stained by the Thiery reaction and phosphotungstic acid but unaffected by DMSO or EDTA, is surrounded by a collar of host wall material which has the same cytochemical properties as the primary walls of the plant cell. The fine arbuscule branches are surrounded by a layer of condensed matrical fibrils. When the arbuscule branches senesce and collapse, they become encased by the polysaccharide fibrillar material derived from the host.

The appearance of the interfacial matrix separating the host plasmalemma from the fungal wall is always the same, no matter which fixation procedure is used. It contains membranous vesicles and scattered polysaccharide fibrils, both of which are elaborated by the host plasmalemma.

The importance of the host and endophyte plasmalemma formations is emphasised. The vesicles arising from the host plasmalemma are often in contact with the walls of the endophytic hyphae. In the fungus, these bulbous or tubular configurations of the plasmalemma can become very numerous and can occupy the whole hyphal width. Although these structures could be involved in host-fungus exchanges, their eventual participation in wall synthesis is also proposed.

Key words: Ultrastructure, host-fungus interface, mycorrhiza, Glomus mosseae, Allium cepa.

Z. Pflanzenphysiol. Bd. 92. S. 191-206. 1979.

192 JEAN DEXHEIMER, SILVIO GIANINAZZI and VIVIENNE GIANINAZZI-PEARSON

Introduction

Vesicular-arbuscular (VA) endomycorrhizas represent the most widespread association between fungi and higher plants (NICOLSON, 1967). The role of the fungus in the mineral nutrition of the host plant is now well established (HAYMAN and MOSSE, 1972; HATTINGH et aI., 1973; SANDERS and TINKER, 1973; PEARSON and TINKER, 1975; TINKER, 1975; RHODES and GERDEMANN, 1975).

It is therefore not surprising that during the last few years there have been several ultrastructural studies on VA mycorrhizas and in particular on the organisation of the interface between the cytoplasm of the host cells and the intracellular hyphae of the endophyte (SCANNERINI and BELLANDO, 1968; SCANNERINI, 1972; Cox and SANDERS, 1974; KASPARI, 1975; CARLING et aI., 1977). CARLING et aI., using a new fixation method (glutaraldehyde and OS04 simultaneously), reported that the interfacial matrix contains a dense granular material that had never been observed before. According to these authors, the structure of the matrix as described in previous papers represents an artefact, its true structure being conserved only by use of the simultaneous fixation procedure. All these previous ultrastructural studies, however, have been purely descriptive. The aim of the present investigation was to analyse more closely the structure of the host-fungus interfaces in VA mycorrhizas, using cytochemical techniques combined with different fixation procedures, in order to gain insight into the nature and possible functions of the different components of this interfacial complex.

Materials and Methods Studies were made on onion roots (Allium cepa var. Topaze F1 hybrid) infected with

Glomus mosseae (NICOL. & GERD.) and cultured as described previously (GIANINAZZI-PEARSON and GIANINAZZI, 1976). Roots of 6-week old onions were rapidly washed in ice-cold distilled water and the infected zones, identifiable by their yellow colour, removed and fixed at 4°C for 31/ 2 h in either 2.5 % glutaraldehyde in cacodylate buffer (pH 7.2), or a mixture of 2.5 % glutaraldehyde plus 1 % OS04 buffered to pH 7.2 with phosphate. Specimens were washed overnight in the corresponding fixation buffer containing 10 % saccharose, followed by 30 min in 10 Ufo saccharose in H 20 and post-fixed 1 h at 4°C in 1 % OS04 in phosphate buffer (pH 7.2). After fixation, specimens were dehydrated through an acetone series and embedded in Epon (Epikote) 812.

Sections, cut with a diamond knife ultramicrotome, were examined either poststained in uranyl acetate and lead nitrate, or unstained and submitted to the Thiery test for polysaccharides (DEXHEIMER, 1976).

Some specimens were also stained 2 min in 1 % phosphotungstic acid in 10 % chromic acid, the former being a specific stain for the plasmalemma (RAMBOURG, 1967; ROLAND et aI., 1972; VIAN and ROLAND, 1972). Preferential extraction of wall material was carried out on specimens prefixed in glutaraldehyde. After a rapid rinse, they were incubated 7 h in either pure DMSO (dimethyl sulfoxide) to remove xylose-rich compounds, or in an aqueous solution of 0.03 M EDTA (ethylenedinitrilotetraacetic acid, sodium salt) to dissolve galacturonic acid-containing compounds. Specimens were washed thoroughly, postfixed and embedded as described above, and sections were examined unstained after being submitted to the Thiery test.

Z. Pjlanzenphysiol. Ed. 92. S. 191-206. 1979.

Ultrastructure of host-fungus interfaces 193

Results

Vesicular-arbuscular mycorrhizas are characterised by the formation of highly branched haustoria, the arbuscules, within the host cells. Arbuscules arise from a network of intercellular hyphae which spread through the root cortex by destruction of the host middle lamella (Fig. 1). A branch from an intercellular hypha penetrates the host wall and enters the cell. This penetration hypha which gives rise to the arbuscule trunk differs little in aspect and dimension from the intercellular hyphae. Once inside the cell the arbuscule trunk bifurcates repeatedly to give finer and finer branches. The terminal branches, which have a diameter (c. 0.5,um) equal to that of the host mitochondria (Fig. 6), are filled with dense cytoplasm and contain few vacuoles (Fig. 3, 6 and 11). This is also typical of the very young intercellular hyphae (Fig. 1), the older hyphae becoming more vacuolated (Fig. 1, 7 and 8 c). After a certain time the arbuscule begins to degenerate; the branches empty and collapse, and their walls aggregate into clumps (Fig. 9 and 10) which were originally, and misleadingly, termed «sporangioles» (jANSE, 1897).

The penetrating hypha and later the entire arbuscule are surrounded by the continuous plasmalemma of the host cell (Fig. 1). The endophytic hyphae and the host cytoplasm are therefore never in direct contact with each other. The structure of the interfacial zone, or interface, between the fungus and the host cytoplasm is relatively complex. It consists of the invaginated host plasmalemma, a matrix which according to Cox and SANDERS (1974) is continuous with the host periplasm, wall structures of varying thickness and complexity, and the fungal plasmalemma. These components of the host-fungus interface have been examined in detail.

Host plasmalemma

Since membranes are capable of developmental change and differentiation (BRACKER and LITTLEFIELD, 1973), the possibility that the host plasmalemma may become modified during its invagination by the endophyte has been investigated by phosphotungstic acid staining and the Thiery test. Both the host plasmalemma and the membrane surrounding the fungal hyphae are strongly stained by phosphotungstic acid (Fig. 1 and 2), a property that distingushes them from other cytoplasmic membranes (ROLAND et aI., 1972). In addition, the invaginated host membrane has a very clear 3 layered structure (Fig. 6) and it gives a positive reaction to the Thiery test. It has therefore remained identical, with respect to both its structure and biochemical properties, to the plasmalemma from which it is derived.

The invaginated host plasmalemma is characteristically elaborated into paramural bodies or lomasomes which react positively to the Thiery test (Fig. 7). Round or elongated vesicles bud off from the plasmalemma into the matrix (Fig. 12). The size of these vesicles is very variable and the largest can contain cytoplasm (Fig. 6). They come into direct contact with the hyphae of both the arbuscule trunk and branches (Fig. 3, 11 and 12).

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Endophyte plasmalemma

The fungal plasmalemma is stained, though to a lesser degree than that of the host, by phosphotungstic acid and is also characterised by the formation of multi vesicular

2 Z. Pjlanzenphysiol. Bd. 92. S. 191-206. 1979.


paramural bodies (Fig. 6 and 13 a). These are made up of small round vesicles (Fig. 13 a, b), and membranous elements which can sometimes be very long (Fig. 14). All these structures react very strongly to the Thiery test and can thus be distinguished from other multivesicular bodies in the cytoplasm (Fig. 3 and 7) which do not react to this cytochemical test. The paramural bodies or lomasomes are present in both the intercellular hyphae (Fig. 13 a, b) and the intracellular arbuscule trunk and branches ,(Fig. 13 b). They are particularly well-developed and frequent in the finest arbuscule branches where they can occupy the whole width of the hypha (Fig. 7 and 13 b). Their very clear 3 layered structure (Fig. 13 b) shows that they originate from the plasmalemma.

No link has ever been observed between these vesicles and those of the host plasmalemma.

Interfacial matrix

The matrix surrounding the arbuscule branches is clearly an extension of the host periplasm (Fig. 1, 3 and 4). No significant differences in its structure have been observed between specimens fixed using the conventional method and those prepared using the simultaneous fixation procedure of CARLING et al. (1977). As most authors have reported, the matrix is nearly empty and contains only the vesicles arising from the host plasmalemma together with scattered fibrils. The latter are of a polysaccharide nature since they react positively to the Thiery test (Fig. 5, 6 and 7). They are sometimes in contact with the host plasmalemma (Fig. 5) and they can form a more or less diffuse layer around the arbuscule branches (Fig. 5, 6 and 7). The dense, granular matrical material of CARLING et al. has never been observed.

Walls and wall appositions

The various cytochemical tests employed distinguish clearly the host wall and appositions from those of the endophyte. The differences in the cytochemical properties of the endophyte and host walls can be summarised as follows:

Fig. 1: Phosphotungstic acid stained section of two intercellular hyphae. The upper mature hypha is highly vacuolised whilst the young hypha penetrating into the host cell (H) contains few vacuoles and dense cytoplasm. The latter is rich in organelles and unidentified, dark-staining, membrane bound inclusions (i) are frequent (see also Fig. 3). The fungal wall (fw) and the invaginated host plasmalemma (hp) are specifically stained by phosphotungstic acid whilst the host cell wall (hw) is unaffected. The host middle lamella is destroyed by the fungus leaving only a few fibrils (arrow). X 15,000. Key to lettering: c, collar; E, endophyte; fi, fibrils; fp, fungal plasmalemma; fw, fungal wall; H, host; hp, host plasmalemma; hw, host cell wall; i, inclusions; 1, lomasomes; m, matrix; pe, periplasm; pf, plasmalemma formations; t, host tonoplast; v, host vacuole; wa, wall apposition layer.

Fig. 2: Phosphotungstic acid stained section through a primary arbuscule branch (E). The fungal plasmalemma (fp) and the host tonoplast (t) are more lightly stained than the host plasmalemma (hp). The dark-staining fungal wall is surrounded by a thin layer of fibrils (fi). X 46,000.

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Fig. 3: Section through an arbuscule trunk giving rise to a large branch, and showing that the matrix (m) is an extension of the host periplasm (pe). The Thiery test for polysaccharides distinguishes clearly the densely stained fungal wall from the surrounding less reactive, more or less layered, coating of fibrils. This wall apposition layer or collar (c) gently tapers along the arbuscule branch and disappears in the fine branches (arrow). The host cell wall (hw) also reacts positively. Numerous vesicles in the matrix are in contact with the arbuscule trunk and branch. X 15,000.

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0,5 H 5 • t

Fig. 4: Detail of penetration point of a hypha into a host cell: Thiery test. The collar (c) of apposition material surrounding the endophyte (E) is continuous with the host cell wall (hw). The matrix (m) which separates the host plasmalemma from the collar is an extension of the host periplasm (pe). Polysaccharide fibrils in the matrix are attached to the host plasmalemma (hp) (arrow). X 72,000. Fig. 5: Detail of the interface in the finer arbuscule branches (E): Thiery test. Within the matrix (m) both the fibrils and the membrane of the vesicles are stained by the reaction. Some fibrils are attached to the host plasmalemma (hp) (arrow) as well as the fungal wall. X 60,000.


Fig. 6: Lead citrate/uranyl acetate poststained section through an arbuscule. The diameter of the fine arbuscule branches (E) is similar to that of the host mitochondria. Both the membrane surrounding the endophyte and that of the vesicles in the matrix (m) have, compared to the host tonoplast (t), a clear 3 layered structure (arrows). Cytoplasm is enclosed within the largest of these matrical vesicles. The fibrils contained within the matrix also form a loose network around the fungal hyphae. X 45,000.

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treatment wall

Thiery test

host +

endophyte + +


EDTA DMSO phosphotungstic acid

middle + middle -lamella lamella matnx - matrix +

+ +, positive reaction; + +, strongly positive reaction; -, unaffected

The host cell walls react positively to the Thiery test (Fig. 4 and 8). After treatment with EDTA most of the middle lamella disappears, the primary wall thins out and its fibrillar architecture becomes clearly visible. With DMSO the primary wall matrix is nearly completely destroyed, leaving only a few fibrils.

The endophyte walls react more strongly to the Thiery test than the host walls, thus appearing much darker (Fig. 3 and 4). They are, on the contrary however, unaffected by DMSO or EDTA. The fungal walls, furthermore, have an unusually marked affinity for phosphotungstic acid (Fig. 1), the reason for which is not known.

The appearance of the wall appositions varies according to their position in the arbuscule. At the point of penetration of a hyphal branch into the host cell a thick outer wall, which is continuous with the host cell wall (Fig. 4), surrounds the inner densely stained fungal wall. This outer wall layer is termed the )collar<. The cytochemical properties of these wall layers confirm their origin. The fungal wall reacts much more strongly to the Thiery test (Fig. 3 and 4) and stain more densely with phosphotungstic acid (Fig. 1) than the collar; it is not modified by DMSO treatment whilst the outer collar layer is almost completely destroyed except for a few fibrils (Fig. 8 c); neither of the wall layers are significantly affected by EDTA (Fig. 8 b). Thus, the collar is definitely of host origin and it corresponds to an extension of the primary cell wall. The fact that EDTA has no effect on the two wall layers shows that they are in direct contact with each other and are not separated by a wall formation analogous to the middle lamella. Around the larger arbuscule branches there is a thinner coating of fibrils which sometimes present a layered arrangement (Fig. 2), and which possess all the cytochemical properties of the collar (positive reaction to the Thiery test; extraction by DMSO). The collar does not therefore stop abruptly around the penetration point but it extends along the arbuscule branches as a gently tapering wall apposition. In the finest arbuscule branches (Fig. 5, 6 and 7) the outer part of the fungal wall is never distinct as it has a coating of disorganised polysaccharide frbrils (Fig. 5 and 7), which are obvious even after double staining with

Fig. 7: Hyphal branches (E) in the region of an arbuscule similar to that shown in Fig. 6: Thiery test. The fungal wall and plasmalemma formations in the lomasomes (1) are darkly stained; the host-produced vesicles in the matrix (m) react less strongly. The contents of the matrix and the host vacuole (v) are very different. X 30,000.

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Fig. 8: Cytochemical analyses of the host wall apposition layer. a) Thiery test, clearly distinguishing the fungal wall (fw) from the wall apposition layer (wa); b) EDTA treatment, not affecting the fungal wall and with little modification in the wall apposition layer; c) DMSO treatment, almost completely destroying the wall apposition layer but leaving the fungal wall intact. X 64,000.

Fig. 9: Aggregated, collapsed hyphae in a senescing arbuscule: Thiery test. The dead hyphae (E) are encased by a polysaccharide fibrillar material which also contains fragments of the host plasmalemma. X 76,000.

Fig. 10: Aggregated; collapsed arbuscule hyphae subjected to the Thiery test after treatment with DMSO. Most of the polysaccharide encasement material has disappeared. X 50,000.


0,5 . 12

" . f .. '.

Fig, 11: Lead citrate/uranyl acetate post stained section through an arbuscule branch showing host plasmalemma formations in the matrix (m), Several vesicles are in contact with the wall of the endophyte (E). X 65,000.

Fig. 12: Phosphotungstic acid post stained section showing vesicles budding off the host (H) plasmalemma and becoming attached to the endophyte (E) wall. X 80,000.

Fig. 13: Lead citrate/uranyl acetate post stained paramural bodies of the endophyte (E) showing the 3 layered structure of the enclosed plasmalemma formations. a) invaginated plasmalemma surrounds vesicles in an intracellular hypha; b) plasmalemma formations occupy nearly the whole width of a fine arbuscule branch. X 76,000.


Fig. 14: Diagrammatical representation of an arbuscule reconstructed from the various figures. The sections A, B, C, and D correspond to Figures 1, 4, 3 and 9 respectively. Lettering as in Figures.

uranyl acetate and lead citrate (Fig. 6). These fibrils form a loose network which varies greatly in thickness and which seems to result from condensation of the polysaccharide fibrillar contents of the matrix.

As the arbuscule degenerates the empty, collapsed hyphae aggregate in clumps and become encased by polysaccharide fibrillar material which also contains fragments of the host plasmalemma (Fig. 9). The fibrils have a relatively disorganised arrangement and cytochemical studies have shown that they are produced by the host cell (positive reaction to the Thiery test (Fig. 9); extracted by DMSO (Fig. 10).

Discussion and Conclusions

The interpretations given by different authors on the structure and nature of the VA mycorrhiza interfaces can now be examined in the light of the present cytochemical observations made on the VA association C. mosseael A. cepa.

It should first of all be pointed out that the appearance of the interfacial matrix has always been the same, no matter which fixation procedure is used. Only scattered polysaccharide fibrils and vesicles arising from the host plasmalemma have been observed within the matrix and never the dense, granular material described by CARLING et al. (1977). Although good morphological fixation can be obtained with

Z. P/lanzenphysiol. Bd. 92. S. 191-206. 1979.


the procedure employed by the latter authors, it is no better than that obtained by the more conventional method. The different resultats of CARLING et al. could perhaps arise from the fact that they studied a different V A association (G. caledoniusl Glycine max). Since the interfacial matrix is an extension of the plant periplasm, it would not be surprising if it varied from host to host. There is no evidence whatsoever that, as the above authors have concluded, the apparently empty matrices are fixation artefacts.

Cytochemical analyses of the walls and wall appositions in the arbuscule confirm previous hypotheses that the apposition layer or collar around the penetration point and arbuscule trunk is of host origin, and they show clearly that it corresponds to

an extension of the primary cell wall. Its structure is comparable to the host wall appositions induced by parasitic infections in a number of plant species (TEMMINK et aI., 1968; EDWARDS et aI., 1970; CHOU, 1970; DELON, 1972). In the small arbuscule branches, the fungal wall is surrounded by a zone, varying in thickness, of disorganised polysaccharide fibrils and which is formed by condensation of the fibrils in the matrix. It is highly probable that this fibrillar zone represents the last of the host wall apposition which persists along the whole length of the arbuscule branches. This constant feature of the interface, present independent of the fixation method, has not been noted by previous authors. Finally, the cytochemical analysis confirm that with degeneration of the arbuscule the collapsed hyphae become encased by a fibrillar deposit produced by the host, as though there is a reaction by the latter to the senescence of the fungus.

Along the length of the arbuscular hyphae, different host-fungus interfaces can be defined according to the scheme of BRACKER and LITTLEFIELD (1973). As Cox and SANDERS (1974) and SCANNERINI et al. (1975) have previously reported, the interface around the penetration point and the arbuscule trunk is type IT 21, that is, it is made up of host plasmalemma, matrix (often very reduced), host wall apposition, endophyte wall and plasmalemma. In the original interface type IT 21, however, the omnipresent matrix between host plasmalemma and collar is not included.

The interface in the finer arbuscule branches is comprised of host plasmalemma, matrix, endophyte wall and plasmalemma. This is considered by the above authors as type IT 24 or IT 18, but it probably in fact an intermediate between IT 21 and IT 24 since there is always a layer of polysaccharide fibrils surrounding the fungal wall. As Cox and SANDERS (1974) have indicated, the interface in the encased senescent arbuscule is similar to that of the trunk (IT 21) except that the endophytic hyphae are empty and collapsed.

Although the host membrane surrounding the arbuscule shows, after phosphotungstic acid and the Thiery test, the typical 3 layered structure of non-invaginated host plasmalemma, its activity appears to be slightly modified with invagination. Around the penetration point and arbuscule trunk it retains the same functions described by ROLAND (1973) for a normal cell, that is, the synthesis of polysaccharide fibrils and their organisation into well-stuctured wall material identical to host primary

Z. P/lanzenphysiol. Bd. 92. S. 191-206. 1979.


wall. With formation of the finer arbuscule branches, fibril synthesis continues but the host plasmalemma progressively loses its capacity to organise them so that the fungus becomes surrounded by a loose network of disorganised fibrils. In the senescent arbuscule, this fibrillar network remains disorganised but it develops and condenses, encasing the collapsed hyphae (Fig. 14). Host plasmalemma activity thus seems to be partially inhibited whilst the arbuscular hyphae are active. The resulting decrease in fibril synthesis and deposit may be an important determinant in the exchange processes between the symbionts.

Membranes are capable of developmental change and in V A mycorrhiza a specialisation of the invaginated host plasmalemma is the formation of vesicles or membranous tubules in the matrix (see also Cox and SANDERS, 1974). These are particularly abundant and well-developed along the finer arbuscule branches where the endophyte wall is thin, and they frequently come into contact with the fungal hyphae. Such observations suggest that they could be involved in the exchange processes between the host and the endophyte. However, similar vesicle formation by the host plasmalemma also occurs around the penetration point and arbuscule trunk where wall structures are thick and unfavourable for any exchange between the symbionts. In addition, vesicles are found around or within the fibrillar material encasing senescent, collapsed hyphae. All these distinctive features and configurations of the invaginated host plasmalemma no doubt signify specialisation of the membrane where it is associated with the arbuscule. Although their specific significance cannot be ascertained from the observations of the present study, it is probable that certain are involved in exchange processes between the symbionts whilst others may playa role in the deposition of wall material by the host cell.

The endophyte plasmalemma similarly invaginates and produces multi vesicular paramural bodies (see also SCANNERINI and BELLANDO, 1968; Cox and SANDERS, 1974; KASPARI, 1973, 1975) which are particularly ahundant in the arbuscule branches. These are generally regarded as sites where active exchanges occur. The paramural bodies greatly increase the surface area of the fungal plasmalemma and, according to the Thiery test, they are particularly rich in polysaccharides. However, further studies are necessary to determine their precise function. The importance of these configurations, especially in the arbuscule branches, together with their differences in structure and probably function, has led us to propose a new type of interface in the VA mycorrhizal association: host plasmalemma/matrix/endophyte wall! endophyte paramural bodies.

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Documents

Ultrastructural Cytochemistry of the Host-Fungus Interfaces in the Endornycorrhizal Association Glomus mosseae / Allium cepa