Mechanisms of reaction zone penetration by decay fungi in wood of beech (Fagus sylvatica)

Transcription

1 RESEARCH New Phytol. (2000), 146, Mechanisms of reaction zone penetration by decay fungi in wood of beech (Fagus sylvatica) F. W. M. R. SCHWARZE* AND S. BAUM Albert-Ludwigs-Universita t Freiburg, Institut fu r Forstbotanik, Bertoldstr. 17, D Freiburg i.br., Germany Received 5 August 1999; accepted 26 November 1999 Dedicated to R. B. Pearce SUMMARY Fungal growth within reaction zones of beech (Fagus sylvatica) challenged by three basidiomycetes, Inonotus hispidus, Ganoderma adspersum, Fomitopsis pinicola, and one ascomycete, Ustulina deusta, was studied in naturally colonized and artificially inoculated wood. All the fungi, except F. pinicola, breached reaction zones, but the mechanisms involved were all somewhat different. Both I. hispidus and U. deusta bypassed blocked cell lumina by tunnelling through cell walls (soft-rot mode), but the latter caused far more decomposition of cell walls. Degradation of polyphenols was slight with I. hispidus and absent with U. deusta. By contrast, G. adspersum preferentially degraded the polyphenolic occlusions in the cell lumina. The failure of F. pinicola to invade reaction zones was typical of a brown rot fungus having limited enzymatic potential and a uniform growth pattern. Mechanisms of lesion expansion, illustrated and summarized in schematic diagrams, are consistent with earlier observations that reaction zones in beech sapwood are static boundaries, which may be successfully breached by white and soft-rot fungi. Keywords: reaction zones, polyphenols, soft-rot, selective delignification, degradation modes. INTRODUCTION Current concepts of the delimitation of fungal colonization in xylem of trees are largely based on descriptions of patterns of discoloration and decay (Shain, 1967, 1971, 1979; Shigo & Marx, 1977). On the basis of such observations, the CODIT-model (Compartmentalization Of Decay In Trees) was proposed and developed by Shigo & Marx (1977). They proposed that there are distinct boundaries between a decay column and the surrounding sound wood, whereby the decay column is confined within a defined compartment. They recognised four different types of boundary, corresponding to various anatomical interfaces within the xylem, and termed these walls 1, 2, 3 and 4. Walls 1 to 3 are formed in the wood extant at the time of wounding and represent modified anatomical features. Wall 4, the barrier zone, corresponds to pre-existing anatomical features which may be modified by the deposition of materials laid down by the host as defensive barriers. Wall 4 also differs from walls 1 to 3 in that it is *Author for correspondence (fax 49 (0) ; schwfran ruf.uni-freiburg.de). formed in the plane of the cambium in response to wounding. Shain (1967, 1971, 1979) proposed the term reaction zone to describe the discoloured region between colonized and uncolonized xylem, which corresponds to walls 1 to 3 of the CODIT model, and distinguish it from the structurally and functionally distinct barrier zone. Another term, the column boundary layer has more recently been applied to the altered tissue around a zone of colonized or dysfunctional xylem (Shortle & Smith, 1990). Unlike barrier zones, reaction zones are not structurally homogeneous and do not form a continuous morphological barrier to ingress of air and or fungal attack (Pearce & Rutherford, 1981; Boddy & Rayner, 1983; Pearce, 1990; Blanchette, 1992). In cases where a fungus grows beyond a reaction zone, it has been envisaged that the reaction zone migrates progressively into the previously functional xylem, being preceded by a margin of drying wood (the transition zone), and followed by incipient and eventually actual decay (Shain, 1967, 1971, 1979). More recently, there has been evidence

2 130 RESEARCH F. W. M. R. Schwarze and S. Baum Decayed wood Sound wood Reaction zone (a) Paraffin-covered No paraffin (c) Inoculum (b) Melted paraffin Fig. 1. Artificial inoculation of wood blocks. (a) Small blocks containing reaction zones and healthy wood from a naturally infected beech tree. (b) After sterilization, blocks inserted into melted paraffin, the side of the reaction zone remaining unsealed. (c) For colonization, test blocks placed separately onto pure cultures of different fungal isolates. that various fungal species are able to extend into functional sapwood some way beyond the reaction zone without stimulating the simultaneous migration of the zone. Instead, a new reaction zone forms later at the new colonization front. This type of discontinuous host response may be recognized by the presence of reaction zone relics within decayed wood (Pearce, 1991; Schwarze & Fink, 1997). In such interactions, the reaction zone is essentially acting as a static boundary to decay, and not as the product of a dynamic host response. The mechanisms by which decay fungi may overcome host barriers have so far been the subject of only a few studies (Pearce, 1996). Elucidation of the means by which hyphae penetrate the barriers and thus bring about lesion expansion requires direct microscopical observation. Recently, light microscopy studies by Schwarze & Fink (1997) showed that the white-rot fungus Inonotus hispidus was able to penetrate reaction zones in London plane (Platanus hispanica) by tunnelling through the cell walls like a soft-rot fungus, thus apparently circumventing blockages within the cell lumina. A similar mode of action was also described for Meripilus giganteus in false heartwood of beech (Fagus sylvatica) (Schwarze & Fink, 1998). It was postulated that the ability of these and other white-rot basidiomycetes to switch to a soft-rot mode of growth may be a feature of a wide range of decay fungi which have the ability to extend beyond host barriers (Schwarze & Fink, 1997, 1998). The objective of the present study was to investigate mechanisms of reaction zone penetration by various decay fungi in living beech trees. By making comparative observations on four fungi inoculated into excised, autoclaved reaction zone material, the role of the active host response could be assessed. Three basidiomycetes, Inonotus hispidus, Ganoderma adspersum and Fomitopsis pinicola, and one ascomycete, Ustulina deusta, selected. With the exception of F. pinicola, all the decay fungi used to investigate reaction zone penetration were chosen as being capable of degrading both lignin and cellulose, and having the ability to invade living host sapwood (Wilkins, 1943; Pearce & Woodward, 1986; Pearce, 1991; Schwarze & Fink, 1997). MATERIALS AND METHODS Inoculation of wood containing previously formed reaction zones The fungal isolates used for artificial inoculation of wood containing the interfaces of reaction zones and healthy sapwood, were: Inonotus hispidus (Bull.:Fr.) (Freiburg collection: ATCC-No ), isolated in 1992 from a basidiome on an ash tree (Fraxinus excelsior L.) at Worpleson, Surrey, UK; Fomitopsis pinicola (Fr.) Karst.(Isolate No ) isolated from a basidiome from a beech tree at Schauinsland, Baden-Wu rttemberg, Germany; Ustulina deusta (Fr.) Petrak (Isolate No ) from Norway maple (Acer platanoides L.) at Freiburg, Baden-Wu rttemberg, Germany; Ganoderma adspersum (Schulz.) (Isolate No ) was supplied by the ETH-Zu rich, Switzerland and, like the other basidiomycete cultures, was dikaryotic.

3 RESEARCH Reaction zone penetration by decay fungi 131 L 1 L L L 3 L 3 Fig. 2. Schematic diagram showing the diffuse porous structure of beech wood. Longitudinally aligned fibretracheids, vessels and axial parenchyma and transversely aligned cell lumina of xylem ray parenchyma correspond with walls 1 and 3, the latewood with wall 2 of the CODIT model. Numbers and arrows within the diagram indicate the alignment of walls 1 3, which enhance or suppress hyphal growth within the wood structure. L, cell lumen. Fig. 3. Schematic diagram showing the modified wood structure within a reaction zone of beech. Tylose formation is apparent within vessels. The inner cell wall of axial parenchyma cells is encrusted with a polyphenolic layer, whereas cell lumina of fibre-tracheids are occluded with abundant polyphenolic deposits. For the basidiomycetes, identity of the pure cultures was confirmed using mycelial characters as observed on plates of Stalpers (1978) malt extract agar. The test wood blocks incorporating the interfaces of reaction zones and healthy sapwood were obtained from a living 100-yr-old beech tree at Mooswald, Freiburg, Baden-Wu rttemberg, Germany, naturally infected with U. deusta. The stem was felled and a number of discs containing reaction zones and living sapwood were sawn from it. From these discs, 50 blocks each measuring mm were cut (Fig. 1). The blocks were autoclaved twice within 48 h at 121 C for 30 min. After cooling, wood blocks were inserted for 1 min into paraffin wax (Merck- Darmstadt 7155, Merck, Darmstadt, Germany) heated at 55 C (melting point 50 C) under aseptic conditions in order to seal all faces of each block except for one exposing the reaction zone (Fig. 1). Each test block was placed onto a pure 14 d-old culture of one of the fungi, on a 20 ml plate of 2%

4 132 RESEARCH F. W. M. R. Schwarze and S. Baum (a) (b) (c) (d) (e) (f) Fig. 4. For legend see opposite.

5 RESEARCH Reaction zone penetration by decay fungi 133 MEA, in a 90 mm Petri dish. Ten such units were set up for each fungal species, together with 10 controls, each containing a single non-inoculated wood block. Incubation of the units was carried out in a random array in an incubator at 25 C and with 50 70% rh, either for 6 or 14 wk. After incubation, the blocks were tested to confirm whether the decay fungi were the only micro-organisms present. Before the incubated wood blocks were cleaned they were sampled at random points by removing chips of negligible weight. These were plated on to MEA for checking the presence of the causal decay agent, which was confirmed in all cases. To check whether the decay fungi had penetrated the reaction zone, isolations were made from different regions of the wood blocks. Preparation of naturally infected material Naturally infected material was obtained from two c. 100 yr-old beech trees (Fagus sylvatica L.), one from the city of Freiburg (Baden Wu rttemberg, Germany) infected with G. adspersum and the other from a site near Mo hringen (Baden Wu rttemberg, Germany) infected with U. deusta. The trees were felled on 22 Feb 1998 and 24 Mar 1999, respectively, and samples containing the host-fungus interface extracted. Isolations were made from all these samples to verify the identity of the decay fungi and to identify any other micro-organisms present. Light microscopy For light microscopy of inoculated beech wood (see above), the test blocks were sawn into smaller samples of c mm following incubation. Similar samples were also removed from the naturally infected beech wood. The samples, with transverse, radial, and tangential faces exposed for examination, were fixed in 2% glutaraldehyde buffered at ph , dehydrated with acetone and embedded in a methacrylate medium. The embedded samples were sectioned at approx. 2 and 4 µm using a rotary microtome (Leica 2040 Supercut, Leica, Baden-Wu rttemberg, Germany) fitted with a diamond knife. For general observation of cell wall degradation and hyphal growth, sections containing reaction zones were stained for 12 h in safranine and then counter-stained for 3 min in methylene blue and for 30 min in auramin. The initial stages of selective delignification were detected in replicate sections by staining with safranine and astra blue (Srebotnik & Messner, 1994). Early stages of brown-rot (i.e. loss of birefringence due to cellulolysis) were detected by viewing sections between crossed Nicols (Wilcox, 1993). Micrographs were taken, using a colour film (Kodak EPY 64T), with a Leitz Orthoplan microscope (Leitz, Baden-Wu rttemberg, Germany) fitted with a Leitz-Vario-Orthomat camera system. Further replicate sections were treated as follows for histochemical examination. For detection of polyphenolic deposits, an ethanolic solution of 2% ferric chloride, which produces a greenish colour (Gahan, 1984), was used. For detection of suberin, sections were treated for 30 min with trichlorethylene so as to remove mainly soluble lipids (Schmitt & Liese, 1993) before being washed in distilled water and then stained with a 0 5% ethanolic solution of Sudan IV. For detection of various active defence materials rich in aromatic residues (including suberin and phytoalexin-like compounds), unstained sections were mounted in glycerine and examined for autofluorescence with a Zeiss fluorescence microscope, using incident illumination from a mercury vapour lamp with a filter combination (BP 365, FT 395 and LP 397). Fig. 4. (a) Transverse section (T.S.) of a reaction zone in beech showing tyloses (T) and accumulation of polyphenols (pointers) in the lumina of parenchyma and fibre-tracheid cells. Bar, 50 µm. (b) T.S. of a reaction zone in beech wood artificially inoculated with Inonotus hispidus showing soft-rot and cavity formation (arrows) within the secondary walls of fibre-tracheids. Darkening of the cell wall is visible immediately around each cavity. Note the occlusion (pointers) of cell lumina with reddish-brownish polyphenolic deposits. T, tyloses. Bar, 10 µm. (c) Tangential longitudinal section (T.L.S.) of a reaction zone in beech wood artificially inoculated with Inonotus hispidus. Hyphae (arrows) within tunnel-like cavities follow the alignment of microfibrils within the secondary wall of occluded fibre-tracheids. Abundant polyphenolic deposits (pointers) with a granular appearance within the cell lumina of fibre-tracheids. Note occluded inner pit apertures (large arrows) Bar, 10 µm. (d) T.L.S. of a reaction zone in beech wood naturally infected with Ganoderma adspersum. Brownish polyphenols (pointers) are apparent within the lumina of xylem ray parenchyma. Note hypha (H) penetrating polyphenols. Bar, 10 µm. (e) T.L.S. of a reaction zone in beech wood artificially inoculated with Ganoderma adspersum showing localized growth of hypha (small arrow) and cavities appearing as tunnels (pointers) within secondary walls of fibre-tracheids. Note hyphal growth through a formerly occluded pit chamber (large arrow; cf. occluded inner pit apertures in Fig. 6). Bar, 10µm. (f) T.S. of beech wood adjacent to a reaction zone artificially inoculated with Ganoderma adspersum showing initial features of delignification. Delignification within fibre-tracheids is induced by hyphae (small arrows) growing within the cell lumina and results in a distinct colour change of the inner secondary wall (large arrows). Note widening of simple pits (large pointers) within parenchyma cells. Fragments of degraded polyphenols, small pointers. Bar, 10 µm.

6 134 RESEARCH F. W. M. R. Schwarze and S. Baum (a) (b) (c) (d) (e) (f) Fig. 5. For legend see opposite.

7 RESEARCH Reaction zone penetration by decay fungi 135 RESULTS Histology of reaction zones and healthy sapwood in beech The anatomical features of sound wood of beech and the alterations recorded within the reaction zone are presented in Figs 2 and 3. The schematic diagram of the reaction zone (Fig. 3) will be used to summarize and illustrate the different mechanisms of fungal penetration by decay fungi. In all cases, reaction zones appeared macroscopically as dark mm-wide bands adjacent to sound wood. In these zones, tyloses were found blocking lumina of vessels. The axial parenchyma cells were also obstructed either by a complete occlusion of the cell lumina by deposits or by an encrustation upon the inner cell wall. Also, c % of fibre-tracheids were fully or partly occluded with polyphenols, recognizable by their natural reddish-brown colour (Fig. 4a). Pit apertures and chambers between parenchyma cells and fibre-tracheids, the lumina of xylem ray parenchyma and intercellular spaces were also heavily occluded with polyphenols. Within the cells, these deposits stained green with ferric chloride. Within sections treated with trichlorethylene and Sudan IV for detection of suberin, a positive reaction was found in tyloses within vessels and also in polyphenol-rich deposits within the cell lumina of fibre-tracheids and parenchyma cells. However, autofluorescence was found only in the walls of the tyloses. Within healthy wood, neither tyloses nor polyphenolic deposits in the lumina of fibre-tracheids or parenchyma cells were ever observed. Fungal spread and wood degradation patterns within reaction zones It was necessary to establish that hyphae in the inoculated wood blocks belonged exclusively to the test fungi, and not to the fungus (Ustulina deusta) which had induced reaction zone formation before the removal of the blocks from the naturally infected tree. This was confirmed by the total absence of hyphae from the reaction zones of the control wood blocks (i.e. blocks which had been autoclaved but not inoculated). The test blocks, which had been placed on fungal culture plates, became colonized only via the unsealed reaction zone surfaces. The paraffin wax seals on the other sides of all wood blocks remained completely intact throughout incubation. Although colonization by all decay fungi took place rapidly, the behaviour and modes of degradation of the different species within reaction zones and adjacent healthy sapwood varied greatly. Therefore a separate account is given for each decay fungus. Inonotus hispidus. This fungus, which was observed only within artificially inoculated wood blocks, showed two distinct modes of colonization and degradation, as previously described (Schwarze & Fink, 1997), i.e. a typical white-rot mode, involving hyphal growth along cell lumina, rather than hyphal tunnelling within cell walls. The whiterot mode, which involved cell wall erosion mainly in the fibre-tracheids, was present only in the previously functional wood outside the reaction zone. Close to the edge of the reaction zone, fungal development in cell lumina was much more sparse and mainly involved the growth of hyphae 1 µm wide in the fibre-tracheids. Cell wall degradation in this boundary region consisted only of a helical pattern of hyphal tunnelling (soft-rot mode) in the walls of fibre-tracheids and ray parenchyma (Fig. 4b, c). The reaction zone itself had been breached by hyphae tunnelling transversely through the fibretracheid walls. Fungal growth was prevented by the deposits in the cell lumina, with the conspicuous diversion of penetration hyphae around such deposits. Ganoderma adspersum. The breaching of reaction zones by this fungus, whether in artificially inoculated or naturally infected material, involved the degradation of the tyloses and polyphenolic deposits in the lumina of the axial parenchyma and fibretracheids (Fig. 4d). Degraded remnants of tyloses, Fig. 5. (a) T.S. of beech wood adjacent to a reaction zone artificially inoculated with Ganoderma adspersum, showing advanced stages of delignification. Strongly delignified fibre-tracheids appear light blue (arrows) and are separated from one another. Cell wall corners (pointers) persist between delignified fibre-tracheids. H, hypha. Bar, 10 µm. (b) T.S. of a reaction zone in beech wood artificially inoculated with Ustulina deusta showing soft-rot and dark hyphae (arrows) within cavities of fibre-tracheids. Note polyphenolic deposits (pointers) within the cell lumina of fibre-tracheids and axial parenchyma. Bar, 10 µm. (c) T.L.S. of a reaction zone in beech wood artificially inoculated with Ustulina deusta showing cavities (large arrows) with pointed ends following the alignment of microfibrils within the secondary walls of fibre-tracheids. Note dark hyphae within cavities (H). Bar, 10 µm. (d) T.S. of a reaction zone in beech wood naturally infected with Ustulina deusta showing abundant tyloses (arrows) and polyphenols (pointers) within the lumina of vessels and fibre-tracheids in the otherwise heavily degraded wood. Bar, 50 µm. (e) T. S. showing details of (d). Note: the persistence of brownish polyphenolic deposits (pointers) and compound middle lamellae (arrows) in the otherwise strongly degraded wood; polyphenols derived from an axial parenchyma cell (P). H, hypha. Bar, 10 µm. (f) Unstained section of (d) treated with trichlorethylene and viewed under the fluorescence microscope. Tyloses within vessels appear light blue, indicating suberization (large pointers). Polyphenols (small pointers) within the cell lumina of parenchyma cells and fibre-tracheids do not show a typical blue fluorescence. Cavities are apparent within secondary walls of xylem ray parenchyma (small arrows). Bar, 20 µm.

8 136 RESEARCH F. W. M. R. Schwarze and S. Baum unlike intact ones, did not show autofluorescence. Hyphae, 1 2 µm in diameter, could be found singly in most of the lumina after partial degradation of the blocking materials, and there was free hyphal penetration of the formerly occluded pit chambers (Fig. 4e). The degradation of blockages in the cell lumina preceded any degradation of the secondary cell walls, but at a later stage there was some localized cavity formation by hyphal tunnelling within the walls of the fibre-tracheids. The cavities were branched tunnels, 1 4 µm in diameter, surrounded by a zone of discoloration and deviating somewhat from the orientation of the cellulose microfibrils. Only after the degradation of most of the tyloses and polyphenolic deposits, which was almost complete after 14 wk, did the fungus enter its typical white-rot mode. This resulted in heavy degradation of axial parenchyma, with the widening of the simple pits and the formation of bore-holes (Fig. 4f). The secondary cell walls of the fibre-tracheids showed erosion, starting around hyphae lying in the cell lumina, and their inner layers showed a blue staining reaction with safranine and astra blue, indicating delignification. By this time, white-rot was advanced in the previously sound sapwood beyond the reaction zone, with some separation of adjacent fibretracheids due to delignification of the middle lamellae (Fig. 5a). Selective delignification also commenced from within the middle lamellae of ray parenchyma cells. Ustulina deusta. Two different modes of degradation were observed in beech wood colonized by U. deusta. At the host-fungus-interface in the naturally infected tree from Freiburg, neither hyphal growth nor typical features of soft rot were observed within the reaction zone. By contrast, reaction zones within the naturally infected beech tree from Mo hringen were readily breached by hyphal tunnelling through secondary cell walls (Fig. 5b, c) without any obvious degradation of the tyloses or polyphenolic deposits which blocked the cell lumina. This mode of degradation was also observed within artificially inoculated wood blocks by U. deusta (Isolate No ). Development of this mode took place largely as previously described (Schwarze et al., 1995a), with chains of helical cavities forming in the fibre-tracheids along the orientation of the cellulose microfibrils (Fig. 5c), both in artificially inoculated blocks and in naturally infected material. The cavities were often found to contain dark hyphae, µm in diameter. Unlike the cavities formed by I. hispidus or G. adspersum, they were not surrounded by discoloration in the surrounding cell wall material. In the case of the artificially inoculated material and within the beech tree at Mo hringen, hyphal tunnelling also occurred in the ray parenchyma. The resulting cavities were helical and of indefinite length (Fig. 5f). Cell wall degradation continued to an advanced stage by the widening of the cell wall cavities, up to 2 6 µm in diameter in the fibre-tracheids, so that the whole cell wall was breached in places (Fig. 5b). Cavity widening in the ray parenchyma of naturally infected wood progressed to a diameter of 1 3 µm. At an advanced stage of decay, a skeleton remained, consisting of the largely intact compound middle lamellae of the fibre-tracheids, parenchyma and vessels, together with the largely unaltered polyphenols (Fig. 5d f). Auto-fluorescence, indicating the presence of suberin, was still present in the tyloses within the vessels of the breached reaction zone (Fig. 5f). At this stage polyphenols formerly infiltrating bordered pits were visible as casts between degraded fibre-tracheids and axial parenchyma cells (Fig. 5e). Fomitopsis pinicola. Although the surfaces of all artificially inoculated wood blocks were colonized by F. pinicola, the fungus was not isolated from wood regions beyond the reaction zone. Also, the wood showed no loss of birefringence under crossed Nicols, indicating the absence of any brown-rot decay, even at an early stage. DISCUSSION Three of the four fungi observed in the present study were able to defeat reaction zones in beech, and did so in different ways. Both I. hispidus and U. deusta bypassed obstructed cell lumina by hyphal tunnelling within secondary cell walls without substantially degrading the obstructing materials, but the latter caused much more cell wall degradation in the process. In contrast to both these fungi, G. adspersum overcame obstructions in the cell lumina by degrading them before causing any significant cell wall degradation. For a better understanding, illustrations of these different modes of reaction zone penetration are presented in schematic diagrams (Fig. 6). The remaining species, F. pinicola, did not defeat reaction zones in artificially inoculated beech wood blocks, and this finding was consistent with the field observation that it does not invade living sapwood. Having observed that I. hispidus could defeat reaction zones by hyphal tunnelling and by the transverse growth of penetration hyphae between adjacent cell walls, Schwarze & Fink (1997) discussed these modes of growth as a possible explanation of the invasive abilities of other decay fungi in standing trees. The particular pattern of hyphal tunnelling by I. hispidus resembled that described for certain soft-rot fungi which have been assigned to form-group 11 by Courtois (1963). Although not generally recognized in the past, softrot is a common feature which has been described for a range of wood decay basidiomycetes (Nilsson &

9 RESEARCH Reaction zone penetration by decay fungi 137 (a) (b) (c) Fig. 6. Schematic diagram of the different mechanisms of reaction-zone penetration in beech by decay fungi. (a) Inonotus hispidus defeats reaction zones by penetration hyphae and a soft-rot mode within secondary walls of xylem ray parenchyma. (b). Ganoderma adspersum initially degrades polyphenols allowing subsequent hyphal growth through the cell lumina. (c) Ustulina deusta defeats reaction zones by a soft-rot mode without significant degradation of polyphenols.

10 138 RESEARCH F. W. M. R. Schwarze and S. Baum Daniel, 1988; Daniel et al., 1992; Schwarze et al., 1995a; Schwarze & Fink, 1997, 1998; Worrall et al., 1997). The ability of U. deusta to defeat reaction zones was not necessarily predictable from previous observations. It had generally not been isolated from reaction zones and it appeared to be absent from the reaction zone material which was excised in the present study for artificial inoculation. Indeed, it was classified as a weakly invasive decay fungus (Pearce et al., 1994), partly because of its inability to degrade deposited polyphenolic materials. Despite its capacity for hyphal tunnelling, which had been considered as a possible means of bypassing occluded cell lumina (Schwarze, 1995), it had been found unable to pass laterally between cells of various host species except via pits (Wilkins, 1936, 1943; Schwarze et al., 1995b). Its inability to form boreholes through the compound middle lamella seems consistent with its tendency to leave this layer as an intact skeleton even at quite an advanced stage of decay (Nilsson et al., 1989; Schwarze et al., 1995b). In explaining the behaviour of the three isolates of U. deusta in the present study, comparison with earlier findings seems to suggest that the isolate used in the inoculated wood blocks was perhaps atypical in being able to cause localized rupturing of the middle lamella. Interestingly, the isolate within one of the natural infected beech trees also caused localized rupturing of the middle lamella. It is conceivable that this might have involved mechanical puncturing by hyphae, as bore holes were not observed. Another feature of these two isolates, which had not been previously reported for U. deusta, was the presence of abundant hyphal growth and soft-rot within the secondary walls of xylem ray parenchyma. This growth pattern at the hostpathogen interface may account for reaction zone penetration and hence lesion expansion. Although studies with a greater number of isolates are necessary, it seems conceivable that hyphal growth within secondary walls of xylem ray parenchyma may be due to intra-specific variation and may distinguish strongly invasive from mildly invasive isolates of U. deusta. Despite the localized rupturing of the middle lamella by U. deusta in the present study, the absence of any significant breakdown of the cell occlusions present in the reaction zone indicates a limited ability to degrade polyphenolic materials such as the lignin-rich middle lamella. This, combined with the ability of the fungus to traverse the reaction zone, could be expected to allow the persistence of reaction zone relics within otherwise heavily degraded wood, as occurs in the case of I. hispidus (Pearce, 1991; Schwarze & Fink, 1997). Such relics are, however, not typical of beech wood degraded by U. deusta, and a possible explanation for this is that the polyphenolics are modified or degraded by other micro-organisms gaining access to them due to the substantial degradation of the cell wall by U. deusta. In contrast with both I. hispidus and U. deusta, G. adspersum degraded the occlusions in the lumina of axial parenchyma and fibre-tracheids in order to defeat reaction zones, both in the standing tree and in artificially inoculated wood blocks. This ability may in part account for its reported role as a strongly invasive decay fungus (Pearce et al., 1994). Its radial spread through the reaction zone was further assisted by a preferential degradation of the middle lamella within the walls of xylem ray parenchyma. The fungus did not traverse reaction zones by hyphal tunnelling but, interestingly, it nevertheless showed localized tunnelling in the secondary walls of formerly occluded fibre-tracheids, resulting in the formation of cavities. Hyphal tunnelling and sequential degradation of cellulose as a rich carbon source within the secondary walls by G. adspersum may aid in the modification and degradation of polyphenolic deposits, which was almost complete after 14 wk incubation. Substantial degradation of polyphenols by G. adspersum and Ganoderma resinaceum Boud. in Pat. had already been observed in a range of other broadleaved hosts (F. W. M. R. Schwarze, unpublished). The extraordinary preferential development of G. adspersum within an environment rich in polyphenols, observed in the present study, can perhaps be related to an ability to respond to certain chemical stimuli. Rayner & Boddy (1988) have observed that some volatiles can stimulate not only the overall growth of Ganoderma spp., but also their direction of growth towards the source. Fries (1961) observed preferential growth of Ganoderma applanatum (Pers.:Wallr.) Pat. towards a vessel containing the volatile nonanal. Selective delignification, which was observed in the present study within areas of wood white-rotted by G. adspersum, is well documented for Ganoderma spp. (Blanchette, 1984) and corresponds with their ability preferentially to degrade polyphenols, as these are chemically similar to lignin. An identical mode of degradation was observed at the host-pathogeninterface within samples of sycamore (Acer pseudoplatanus), artificially infected by G. adspersum, kindly supplied by R. B. Pearce. Reaction zones were breached after degradation of polyphenols had commenced and selective delignification and hyphal tunnelling within cell walls were apparent. The brown-rot fungus Fomitopsis pinicola was not able to invade and defeat reaction zones in artificially inoculated wood blocks of beech after 14 wk of incubation. This was of interest in relation to its reported inability to invade living sapwood, which might be related to its limited enzymatic capacity as a brown rot fungus to degrade polyphenols, such as those observed forming abundant occlusions of cell

11 RESEARCH Reaction zone penetration by decay fungi 139 lumina in beech reaction zones. Under conditions of controlled incubation, heat-killed beech wood blocks which are free from reaction zones are readily colonized by the fungus (Schwarze, 1995). Polyphenolics are not present in significant quantities in the types of wood in which F. pinicola commonly occurs in nature, i.e. the sapwood of dying or dead trees and in trees with heartwood of low extractive content. Its various host species include old or dying beech trees and it is also an important member of the coniferous forest ecosystem, because it decays dead trees and logging slash (Sinclair et al., 1987). In North America it causes some loss of timber value in old growth western conifers, but it acts slowly and perhaps for this reason is not among the major decay pathogens of second-growth forests (Sinclair et al., 1987). The present observations directly demonstrate the ability of the hyphae of some decay fungi to grow through reaction zones in the xylem of beech and, presumably, other broadleaved trees. Some of the mechanisms whereby this can happen were apparent both in artificially inoculated reaction zone material and in samples taken from standing trees. In the latter case, hyphae penetrated beyond the reaction zones into previously sound wood despite the presence of putatively active defences. It could therefore be concluded that dynamic maintenance of the reaction zones was either absent or inadequate. Absence of such a defensive mechanism seems the more likely of these possibilities, because a dynamically supported but failing reaction zone could be expected to migrate progressively ahead of the advancing hyphae instead of being penetrated. Penetration of a reaction zone does not represent total failure of active defence, provided that a new reaction zone subsequently forms beyond the new invasion front. If fungal invasion and host response are alternating events, it seems likely that the phases of this interaction are seasonal. Seasonal differences in the susceptibility of various temperate broadleaved hosts to invasion by fungi such as Chondrostereum purpureum have been demonstrated (Brooks & Moore, 1926; Spiers et al., 1998). Cyclic changes in carbohydrate reserves, which are the feedstock of defensive materials such as polyphenolics, may account largely for these temporal patterns, but accompanying changes in wood moisture content, which limit fungal development at or above normal sapwood concentrations (Boddy & Rayner, 1983), may also be a key factor. Indeed, the active enhancement of water content at the lesion margin by the replacement of gases within fibre lumina seems to be a feature of reaction zone formation (Kemp & Burden, 1986; Pearce et al., 1994). The long term maintenance of such a regime might help to explain why some reaction zones seem to be very stable and persistent. Although reaction zones did not migrate ahead of the fungi in the two trees examined in the present study, it remains unclear whether reaction zones can nevertheless migrate under other circumstances such as different host-pathogen combinations. Such migration could be regarded as evidence of a dynamic host response, but would not be identical with the dynamic maintenance of the reaction zone in situ. Indeed it could be considered not to be fundamentally very different from the stepwise alternation of fungal advance and renewed host response which we have envisaged above. In any case, evidence of migration would probably require the use of sequential non-destructive methods of sampling, such as magnetic resonance imaging (Pearce et al., 1994). The comparative study of challenged reaction zones in artificially inoculated wood and in standing trees, as used in the present study, seems to provide a means of elucidating the potential mechanisms whereby fungi may overcome host defences, while also determining whether or not these mechanisms operate in nature. In parallel with other methods, direct microscopical observations are essential if real value is to be attached to the primary observations of discoloration and decay that have been made at the macroscopic level. ACKNOWLEDGEMENTS This work was partially funded by the Deutsche Forschungsgesellschaft (DFG) as a part of the project SFB 433 Buchendominierte Laubwa lder unter dem Einfluß von Klima und Bewirtschaftung. The authors are indebted to the late Dr R. B. 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