Resistance of fibre regions in wood of Acer pseudoplatanus degraded by Armillaria mellea

Mycol. Res. 104 (9) : 1126–1132 (September 2000). Printed in the United Kingdom.

1126

Resistance of fibre regions in wood of Acer pseudoplatanus degraded by Armillaria mellea

F. W. M. R. SCHWARZE, S. BAUM and S. FINK Albert-Ludwigs-UniversitaW t Freiburg, Institut fuW r Forstbotanik und Baumphysiologie, Bertoldstr. 17, D-79085 Freiburg i.Br., Germany. E-mail : schwfran!ruf.uni-freiburg.de Received 3 March 1999 ; accepted 18 December 1999.

The white rotting Armillaria mellea induced a spatially very distinctive pattern of degradation in sycamore wood, under both natural and artificial conditions. Degradation began preferentially within groups of fibres containing intercellular spaces, whereas fibre regions lacking such spaces were undegraded and remained largely intact even when decay had become advanced elsewhere. The two types of fibre region differed not only in the presence of intercellular spaces, and hence in the potential for gas exchange, but also in their degree of lignification. This was higher in the more resistant type, as shown by staining of undecayed wood with toluidine blue-O, by microspectrometry after staining for the Ma$ ule colour reaction, and by uv-microscopy. A spatially similar pattern of cellulose degradation was induced by the brown rotting Laetiporus sulphureus, which is known to cause preferential degradation of less strongly lignified cell walls. By contrast, the white-rotting Ganoderma pfeifferi showed a tendency to degrade the stronger lignified cell walls. Thus, in combination with the application of conventional histological methods, the wood degradation modes observed give additional evidence for stronger and weaker lignified fibre regions within the wood of sycamore.

INTRODUCTION Decay fungi are conventionally classified into three main groups, i.e. brown-, white-, and soft-rot fungi (Liese 1970 ; Rayner & Boddy 1988, Eriksson, Blanchette & Ander 1990). Degradation by brown- and soft rot fungi follows fairly consistent patterns, whereas white rot fungi induce a much greater variety of types of degradation. This is partly related to their wide enzymatic capacity, which is possibly reflected by a range of potential modes of degradation. Some of these involve the selective breakdown of different constituents of the secondary cell wall, but many white rot fungi are also capable of degrading all cell wall constituents, even under extreme conditions such as high moisture content (Eriksson et al. 1990, Metzler 1994, Schmidt et al. 1997). The preferential degradation of certain cell wall constituents or of particular regions of the wood is often a feature of the earlier stages of white rot. For example, Phellinus pini causes localised selective delignification in its hosts, inducing a white pocket rot (Blanchette 1980). The white pockets contain little other than crystalline cellulose, due to the fungal breakdown of both lignin and hemicelluloses within these regions. Interestingly, the contents of these pockets cannot be readily decomposed by brown rot fungi, whose cellulolytic ability depends on the presence of hemicelluloses (Blanchette, 1983). Preferential degradation by white rot fungi is often followed by more general utilization of the wood, but some species

show only a limited ability to degrade certain cell types or cell wall constituents. In particular, there are some which leave the vessels of broadleaved trees largely undegraded, even at a relatively advanced stage of decay (Blanchette et al. 1988). This is apparently due to the high lignin : carbohydrate ratio of vessel walls, together with their morphology and the monomeric composition of their lignin (Blanchette et al. 1988). Although some cell types are strongly degraded by fungi, layers within their cell walls may differ in resistance to attack by virtue of the monomeric composition of their lignin content. For example, fibres of beech (Fagus sylvatica) colonised by the ascomycete Ustulina deusta tend to show preservation of the compound middle lamellae, even at an advanced stage of decay, when other cell wall constituents have largely disappeared (Schwarze, Lonsdale & Mattheck 1995). The persistence of this region of the cell wall is probably due to its high percentage of guaiacyl lignin. Members of the Xylariaceae such as U. deusta have a relatively poor lignolytic ability, and this is mainly confined to syringyl lignin (Nilsson et al. 1989). The anatomy of angiospermous wood, in which white rot fungi predominantly occur, is another factor which strongly determines the pattern of degradation. For example, within individual annual increments of beech wood, degradation by Meripilus giganteus shows distinct and very different patterns which correspond to host structure (Schwarze & Fink 1998). In wood of London plane

F. W. M. R. Schwarze, S. Baum and S. Fink (Platanusihispanica) naturally degraded by Inonotus hispidus, xylem rays persist within the wood at an advanced stage of decay. Degradation of xylem rays is markedly delayed and apparently associated with the infiltration of intercellular spaces by polyphenolic deposits (Schwarze & Fink 1997). The present studies involved the basidiomycete Armillaria mellea which occurs as an important pathogen in forest and park trees, causing a white rot of the butt and roots. The classification of A. mellea as a white rot fungus is based on its lignolytic ability, although chemical analysis has shown this to be rather low at early stages of degradation when compared with many other white rot fungi (Campbell 1931, 1932). Preliminary studies of inoculated wood blocks of sycamore (Acer pseudoplatanus) showed a preferential attack of certain cell wall regions by A. mellea (Engels 1997). In order to verify and investigate this phenomenon, a series of microscopical observations was carried out, using naturally colonised and artificially inoculated wood of sycamore. For comparison, wood blocks of sycamore were also inoculated with the brown rot fungus Laetiporus sulphureus and the white rot fungus Ganoderma pfeifferi.

MATERIALS AND METHODS Inoculation of wood blocks For the inoculation of wood blocks, dikaryotic isolates of Armillaria mellea, Laetiporus sulphureus, and Ganoderma pfeifferi were used. Armillaria mellea was isolated from decayed wood of a sycamore (Acer pseudoplatanus) in Umkirch, BadenWu$ rttemberg, Germany (isolate 220197.3) in 1997. A dikaryotic isolate of Ganoderma pfeifferi (isolate 250592.1) was obtained from a basidiome on a beech tree (Fagus sylvatica) at Windsor Great Park, Berkshire, UK. Laetiporus sulphureus (isolate 940904.1) was supplied by the ETH-Zu$ rich. Armillaria mellea and G. pfeifferi were cultured by extracting wood samples which were plated onto 20 ml plates of water agar amended with 4 mg l−" methyl benzimidazole-2yl carbamate (MBC). Potential contamination by bacteria was eliminated by the use of Raper’s rings in the isolation plates (Raper 1937). Cultures growing out from the rings were transferred to peptone-yeast-glucose-agar (PYGA) as defined by Lelliott & Stead (1987), with ingredients from Unipath, (Basingstoke). Cultures were maintained on 3 % malt extract agar (MEA), 20 ml per Petri dish. All cultures were incubated in the dark at 25 mC. The inoculum for the test blocks was prepared by growing A. mellea, G. pfeifferi and L. sulphureus on cylindrical ‘ feeder ’ blocks of approx. 10i10 mm, cut from 5–6 y-old debarked stems of hazel (Corylus avellana). The blocks were autoclaved at 121 m for 20 min in 250 ml glass Erlenmeyer flasks, 20 to a flask, together with 20 g sharp sand, 0n6 g maizemeal and 15 ml distilled water. Each flask was inoculated with five pieces of mycelial inoculum taken from 14 d-old pure cultures of the isolates and then incubated at 25 m for 14 d. Three such flasks were set up, one for each of the isolates. The test wood blocks were obtained from the sapwood of a living 70–80 yr-old sycamore (A. pseudoplatanus) located at St Peter, Baden-Wu$ rttemberg.

1127 The test blocks were prepared using the procedures of Schwarze & Fink (1998). Ten replicate jars were set up for each species, together with eight control jars which contained noninoculated feeder blocks. Incubation of the jars was carried out in a random array in an incubator at 25 m and with relative humidity between 50 and 70 %, either for 6, 12 or 18 wk. Before the incubated test blocks were dried for measurement of weight loss, they were cleaned and sampled at random points by removing small chips of negligible weight. These were plated on to MEA to check whether A. mellea, G. pfeifferi or L. sulphureus were the only microorganisms present, and this was confirmed in all cases.

Light microscopical observation of wood from inoculated blocks and naturally infected sycamore For light microscopy of inoculated wood, samples of approx. 20i5i5 mm were sawn from the inoculated blocks. The samples, with transverse, radial, and tangential faces exposed for examination, were dehydrated with acetone and then infiltrated with a methacrylate medium which was subsequently polymerized at 50 m. The embedded samples were sectioned at approx. 2 and 3 µm, using a rotary microtome (Leica 2040 Supercut) fitted with a diamond knife. The sections were finally stained according to our standard procedure for 12 h in safranine and then counter-stained for 30 min in auramin and for 3 min in methylene blue. For observation of wood naturally infected with A. mellea, samples were obtained from a 90–100 yr old sycamore at a site in Umkirch, Baden-Wu$ rttemberg. The samples were taken exclusively from decayed regions within the lower stem. Isolations were made to verify the presence of A. mellea. The samples were then treated, embedded and stained as above. Micrographs were taken using black and white (Agfapan) APX 25 film with a Leitz-Orthoplan microscope fitted with a Leitz-Vario-Orthomat camera system.

Light and uv microscopical observation of sound wood of sycamore For determination of lignin and its distribution within cell walls of sound wood, the following methods were applied. For qualitative analysis of lignin distribution, semi-thin sections (2–3 µm) were stained for 5 min in 0n05 % aqueous toluidine blue-O and thereafter rinsed for 20 min in 20 % aqueous CaCl # (Herr 1992). Additional sections 15 µm thick were treated as follows for induction of the Ma$ ule colour reaction (Nakano & Meshituska 1978, Iiyama & Pant 1988). The sections were stained for 5 min in 1 % aqueous KMnO , then rinsed in % distilled water, immersed in 18  HCl for 2 min and finally for a few seconds in 10 % aqueous NH . Semi-quantitative $ assessment of syringyl lignin monomer units was carried out using microspectrometry (Takabe et al. 1992, Wu, Fukazawa & Ohtani 1990). The absorbance of visible spectra in the wavelength range 435–570 nm was measured at intervals of 5 nm. Measurements were made within 30 min of staining, as the Ma$ ule colour reaction is not permanent. Additionally, uv microscopy was carried out using semi-

Armillaria mellea and degradation in sycamore wood

1128

Figs 1–4. Sycamore wood (TS) inoculated with Armillaria mellea. Fig. 1. At an early stage of decay, fibre regions inbetween vessels (arrows) are preferentially degraded, whereas fibre regions surrounding vessels are resistant to decay. Bar l 100 µm. Fig. 2. Within preferentially degraded fibre regions, intercellular spaces are apparent (arrowheads). Within fibre regions surrounding vessels intercellular spaces are absent. Bar l 25 µm. Fig. 3. Fibres with intercellular spaces (arrowheads) are strongly degraded. Although hyphae (arrows) are present within the lumina of fibres without intercellular spaces, these show inherent resistance to degradation by A. mellea. Bar l 10 µm. Fig. 4. TS of sycamore wood naturally infected with A. mellea at an advanced stage of decay. Fibres within low-density fibre regions (arrows) are completely degraded. High-density fibre regions and vessels persist within the degraded wood. Bar l 50 µm.

thin sections (0n5 µm). Each section was mounted on a quartz slide, immersed in glycerine and covered with a quartz coverslip for observation under ultraviolet light using a Xenon-ArcLamp source and a Leitz-Monochromator. Images were taken with a cooled CCD-camera (Sensys 400 Photometrics) and compiled with the computer program V for Windows (Photometrics). For every investigated cell type and\or cell area, seven uv-images were taken at a wavelength of 280 nm, five readings made and the mean value calculated. These data were converted into relative uv-absorbance as described by Scott et al. (1969). To obtain information on the lignin monomer composition, the uv-absorbance spectrum was measured in the wavelength range 250–300 nm at intervals of 2 nm. To avoid photolysis of lignin, each area investigated

was exposed to uv light for no more than 10 min (Scott & Goring 1970).

RESULTS Anatomy of sycamore wood Sycamore wood, which is diffuse-porous, contains groups of fibres which appear to be of alternating high and low-density when viewed in transverse section. The less dense groups lie between vessels and consist of dead fibres with abundant intercellular spaces (Braun 1970). The denser groups are associated with the vessels, and each of them forms a complete paratracheal sheath of living fibres with no intercellular

F. W. M. R. Schwarze, S. Baum and S. Fink

1129

Figs 5–7. Fig. 5. TS of sycamore wood naturally infected with Ganoderma pfeifferi. During initial stages of degradation preferential lignin degradation within high-density regions is apparent. Selective delignification of the middle lamellae between fibres results in cell wall separation (arrows). Cell wall corner regions (arrowheads) of middle lamellae persist within delignified fibres. Bar l 15 µm. Figs 6–7. Sound sycamore fibres : uv micrograph at 280 nm (bars l 10 µm). Fig. 6. The relatively low uv-absorption of the fibre secondary walls within areas containing intercellular spaces (arrows) is apparent from their light colour (cf. Fig. 7). Fig. 7. The fibre secondary walls within areas without intercellular spaces appear dark, indicating a higher uv-absorption compared with the fibres in Fig. 6.

spaces. Living wood fibres are also concentrated at the borders of the annual increments, where they are associated with apotracheal terminal parenchyma (Braun 1970). Measurements obtained in this study show that secondary walls of the paratracheal fibres have a mean thickness of 2n3 µm, whereas the walls of fibres in the ‘ low-density ’ groups are thinner, with a mean thickness of 1n6 µm. Wood colonization and cell wall degradation modes After 6, 12 and 18 wk, mean weight losses of inoculated wood blocks were 1n4 %, 3n0 % and 6n1 % respectively. The overall pattern of colonisation by Armillaria mellea was best observed after an incubation period of 18 wk. Cell wall degradation by A. mellea in sycamore wood did not differ greatly between artificially incubated and naturally infected material. The fungus was observed within the cell lumina of fibres, vessels and parenchyma cells of xylem rays. The hyphae were 1–2 µm wide, septate and rarely showed clamp connections. Hyphal growth was most abundant in the cell lumina of fibres with intercellular spaces, which occurred in regions between the vessels. Single hyphae were often also observed in the intercellular spaces of these fibre regions. The preferential degradation of the low-density regions produced a distinctive pattern when sections of the wood were viewed at low magnification (Figs 1–2). The general

erosion of the cell walls caused by hyphae lying within the lumina of fibres was shown to involve delignification, which resulted in a distinct colour change of the inner secondary wall (Fig. 3). This occurred initially in the immediate vicinity of individual hyphae. In the early stages of decay, a general dissolution of the cell walls typical of a simultaneous rot was apparent, so that hyphae lying in the cell lumina induced a general thinning of the walls. At this time, wall thinning was not observed within the fibre regions without intercellular spaces, even though hyphae were present within their cell lumina (Fig. 3). When the decay of the low-density fibre regions reached an advanced stage, the walls of fibres and of vessels within the high-density regions persisted within the otherwise strongly decomposed wood (Fig. 4). By this stage there was, however, some structural alteration within these less decayed cells, which resulted in a distinct colour change of the inner secondary wall. The initial mode of degradation in sycamore wood artificially inoculated with the brown rotting Laetiporus sulphureus, was similar to that induced by A. mellea. In transverse sections, when viewed between crossed Nicols, preferential degradation and loss of birefringence were apparent in fibre regions without intercellular spaces. By contrast, the mode of degradation in sycamore wood, inoculated with the whiterotting Ganoderma pfeifferi, showed typical features of preferential selective delignification after 6 wk of incubation (Fig. 5).

Armillaria mellea and degradation in sycamore wood

1130 0·3

0·25 0·25 0·20 Relative uv-absorbance

Relative uv-absorbance

0·30

0·15 0·10 0·05 0·00 (a)

(b)

0·2

0·15 0·1

(c) 0·05

Fig. 8. Mean relative uv-absorbance at a wavelength of 280 nm in secondary walls of sound sycamore cells. (a) l vessel ; (b) l fibres without intercellular spaces ; and (c) l fibres with intercellular spaces.

The middle lamella in the walls of fibres and of vessels within the high-density regions was degraded, and the cells separated from one another (Fig. 5).

0 250

260

270 280 Wavelength (nm)

290

300

Figu. 9. uv-absorbance spectra in the secondary walls of sound sycamore cells showing a peak at 280 nm for vessels (#) and peaks at 276 nm for fibres both with intercellular spaces (=) and without intercellular spaces ( ). 0·8

The apparent difference in cell wall density between both types of fibre regions described above was initially observed in sections of undecayed wood that had been prepared using our standard staining procedure. This produced darker staining within the walls of fibres with no intercellular spaces. This difference was also shown in additional sections stained with toluidine blue-O, in which the darker staining regions appeared turquoise, whilst the remaining areas stained light blue. For confirmation of these qualitative results, semiquantitative analysis with uv-microscopy was undertaken. This showed a correlation between uv-absorbance and the visually assessed depth of staining within the secondary walls of the fibres (Figs 6–7). For the regions with and without intercellular spaces, the mean uv absorbance values were 0n093 and 0n141 units respectively (Fig. 8). The greatest uv-absorbance was detected in the secondary walls of vessels, where the mean value was 0n283 units (Fig. 8). In addition to the above microscopical observations, which indicated differences in the degree of lignification in different cell types, microspectroscopy was used for the assessment of the lignin monomer composition. The uv-absorbance of vessel secondary walls showed a peak at 280 nm (Fig. 9). A peak absorbance of 276 nm was measured within the secondary walls of fibres, irrespective of whether they occurred in regions with or without intercellular spaces (Fig. 9). Similarly, the cell walls in both types of fibre regions showed a peak absorbance at 520 nm in the visible spectrum, as measured in sections which had been stained for the Ma$ ule colour reaction (Fig. 10). Walls of fibres in the regions with intercellular spaces showed, however, a lower mean absorbance than the other category of fibres over the whole investigated spectrum. Vessel cell walls showed a slight peak at 515 nm, but their overall absorbance was clearly lower (Fig. 10).

0·7 Relative light absorbance

Lignin distribution and monomer composition in sound wood of sycamore

0·6 0·5 0·4 0·3 0·2 0·1 0 435

460

485 510 Wavelength (nm)

535

560

Fig. 10. Visible light absorbance spectra of sound sycamore cells after staining for the Ma$ ule colour reaction. The secondary walls of fibres without intercellular spaces ( ) show the highest mean light absorbance over the whole spectra. The absorbance of secondary walls of fibres with intercellular spaces (=) and vessels (#) are distinctly lower.

DISCUSSION In sycamore wood inoculated or naturally infected with Armillaria mellea a distinctive mode of degradation was observed. This was characterized by the preferential degradation of fibres with intercellular spaces, whereas fibres without such spaces persisted even at an advanced stage of decay. These differences in decay resistance corresponded to the degree of lignification within these two types of fibre, as shown by the density of staining with toluidine blue-O and the absorbance of uv light. These observations indicated that the fibres without intercellular spaces were more strongly lignified. The absorbance of uv light is not necessarily an indication of total lignin content per se, as the two main forms of lignin differ considerably in their absorbance within the entire uv spectrum. Compared with syringyl lignin, equivalent amounts of guaiacyl lignin absorb approximately four times more uv

F. W. M. R. Schwarze, S. Baum and S. Fink radiation (Aulin-Erdtman 1957). By contrast, the absorbance of visible light following staining for the Ma$ ule reaction appears to be positive mainly for syringyl lignin (Nakano & Meshituska 1978, Iiyama & Pant 1988, Wu et al. 1990). Thus, the slightly higher absorbance of visible light that was recorded after staining in the walls of fibres without intercellular spaces was not necessarily a measure of total lignin content. The additional use of microspectroscopy indicated, however, that the difference between the two types of fibre was indeed in their total lignin content, rather than in the monomeric composition of their lignin. Both types showed a peak absorbance at 276 nm, typical of lignin with a high proportion of syringyl units (Fergus & Goring 1970 a, b). The vessels, unlike both types of fibre, appear to have a high proportion of guaiacyl lignin within their secondary walls, as shown by their peak uv absorbance at 280 nm. This interpretation is supported by the low absorbance of visible light at 520 nm after staining for Ma$ ule colour reaction, as this appears to be characteristic of a low syringyl monomer content (Wu et al. 1990). There is also good agreement with observations made by Fergus & Goring (1970 b), who showed a high proportion of guaiacyl lignin in the vessel cell walls of a range of angiosperms. Resistance of hardwood vessels to degradation by white rot fungi has been previously described and among other factors also appears to be related to the lignin monomer composition (Blanchette et al. 1988). Furthermore, studies on tropical timbers showed that hardwoods containing guaiacyl-rich lignin are more resistant to decay than those containing syringyl-rich lignin (Syafii & Yoshimoto 1991). Although the monomeric composition of lignin may play a part in the decay resistance of hardwood vessels, the two types of fibre regions observed in the present study appeared to have similar forms of lignin in their secondary walls. It thus seems apparent that lignin concentration is more likely than lignin composition to play a role in the decay resistance of the fibres without intercellular spaces. A further explanation for the prolonged decay resistance of discrete regions of sapwood may be the involvement of host responses (Rayner & Boddy 1988, Pearce 1997). Paratracheal fibres within the sapwood of sycamore remain living for a long time, unlike the groups of fibres that lie between the vessels (Braun 1970). They may, therefore, retain the ability to lay down defensive compounds within their cell lumina in response to the ingress of air and\or fungal colonization. Deposits of such materials were, however, rarely observed within sections of naturally infected wood. In any case, resistance to naturally occurring decay was observed not only within the living fibres of the paratracheal sheath, but also within dead fibres adjacent to them. Moreover, the same pattern of decay resistance occurred within inoculated wood. Although locally high lignin concentrations probably have a role in determining the pattern of decay induced by A. mellea in sycamore wood, it is important also to consider the possible influence of accompanying differences in aeration of the tissues. A high water content and a low availability of oxygen restrict wood degradation processes and therefore act as a passive microenvironmental form of defence within intact sapwood (Boddy & Rayner 1983, Pearce 1997). In this

1131 context, one prominent feature of fibre regions in sycamore wood preferentially degraded by A. mellea is the presence of abundant intercellular spaces. It is conceivable that the gaseous microenvironment in these regions is more favourable for degradation by A. mellea than in the regions without intercellular spaces. It should be noted that numerous studies have shown that some decay fungi, such as A. mellea s.lat., have the capacity to degrade wood with a high water and a low oxygen content (Metzler 1994, Schmidt et al. 1997). Thus, even watersaturated wood of Norway spruce (Picea abies), stored under water sprinklers is degraded by this fungus. Its ability to overcome poor aeration is not, however, due simply to a tolerance of a low availability of oxygen. Instead, it is able to induce the ingress of air into channels within the wood via airfilled rhizomorphs which lie on the surface (Metzler 1994). This indicates that aeration enhances decay by this fungus, despite its apparent tolerance of microaerophilic conditions. It therefore seems possible that aeration via intercellular spaces may have been a factor in determining the spatial decay pattern observed in the present study. These spaces may also have enhanced decay by allowing the penetration of hyphae between as well as within the fibres. By contrast, the spatial degradation pattern of Ganoderma pfeifferi in inoculated wood of sycamore shows a preferential attack of the stronger lignified fibre regions. Low-density regions, preferentially degraded by A. mellea and Laetiporus sulphureus, persisted during initial stages of degradation by G. pfeifferi. Ganoderma species are known to cause a selective delignification (Blanchette 1984 a, b, Adaskaveg, Gilbertson & Blanchette 1990, Schwarze 1995). Thus selective delignification by G. pfeifferi apparently provides further evidence that lignin distribution rather than oxygen conditions affect the mode of degradation in sycamore wood. Although A. mellea is a white rot fungus and the enzymatic and non-enzymatic processes produced by this fungus are completely different from any brown rot, its spatial pattern of degradation within inoculated wood blocks of sycamore was very similar to that of the brown rotting L. sulphureus. The first evidence of cell wall degradation by brown rot fungi is the loss of birefringence (Schulze & Theden 1937, Wilcox 1993, Schwarze 1995). Previous studies have shown that the walls of certain cell types such as latewood fibres and vessels show an inherent resistance against brown rot fungi (Schulze & Theden 1937, Schwarze 1995). This appears to be related to a higher degree of cell wall lignification which hampers diffusion of cellulolytic enzymes into the cell wall, thus resulting in a greater resistance towards degradation (Schwarze 1995). It is therefore not surprising that L. sulphureus preferentially degrades fibre regions within sycamore wood which are less strongly lignified. Interestingly, these observations are in good agreement with chemical analysis by Campbell (1931, 1932), which shows that the lignolytic ability of A. mellea s. lat. is low at early stages of degradation, compared to that of many other white rot fungi. Thus, although A. mellea has the capacity to degrade all cell wall constituents, the present study confirms that it preferentially degrades cell walls with a relatively low lignin content.

Armillaria mellea and degradation in sycamore wood The present study shows that in combination with conventional histological methods, microorganisms can be of great value as selective tools for anatomical investigations. The specificity of their enzymes and the mild conditions under which degradation proceeds, make them potentially suitable agents for more precise studies of the distribution of cell wall constituents. A C K N O W L E D G E M E N TS The authors are indebted to Prof. Dr Ottmar Holdenrieder for providing the isolate of Armillaria mellea and to Dr Julia Engels for providing samples of naturally and artificially infected wood. We also appreciate the assistance of Karin Waldmann and Astrid Fischer in preparing sections for light microscopy. Finally, the authors thank Dr David Lonsdale for comments on the manuscript.

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