Wood ScienceWood Sci. Technol. 19:313-321 (1985) andTechnology

© Springer-Verlag 1985

Electron-dense particles in wood decayed byGanoderma applanatum L. Murmanis, J. G. Palmer and T. L. Highley, Madison, USA

Summary. Hemlock sawdust samples degraded by Ganoderma applanatum showed no elec­tron-dense particles either in hyphae or in wood cell walls after aldehyde/OsO4 fixation. After KMnO4 fixation at early stage of attack. particles were in hyphae, hyphal sheath and wood cell walls. In samples prepared by a cytochemical technique which localizes cellulase activity at the ultrastructural level, particles were in hyphae. hyphal sheath and wood cell walls. The smallest diameter range of the particles lay between 3 and 7 nm which corresponds to the size of cellulases. Larger diameter particles were present which are probably aggregates of the smaller units. We believe that particles present in hyphal cytoplasm and hyphal sheath are cellulolytic enzymes. Whether particles present in attacked wood cell walls are enzymes or degradation products cannot be determined by this study. Nevertheless. the particles reveal the decay pattern in wood by the white-rot fungus G. applanatum.

Introduction

Electron-dense particles having diameters from 3 to 7 nm have been found to be associated with fungi and decayed wood. Wilcox et al. (1974), in a study of pentachlorophenol-treated wood decayed by the brown-rot fungi Lenzites trabea and Poria monticola, interpreted the particles as reaction products of pentachloro­phenol and KMnO4. Ruel et al. (1981), investigating wood degradation by the white-rot fungus Sporotrichum pulverulentum in JMnO4-fixed samples, saw 7nm granules coming from disintegrated lignin lamellae. Messner and Stachelberger (1983), studying aldehyde/OsO4-fixed samples, observed particles in wood degraded by white- and brown-rot fungi and provided elaborate explanations for the particles being either enzymes or degradation products. Murmanis et al. (1984), studying KMnO4-fixed samples decayed by the white-rot fungus Ganoderma applanatum, speculated that particles present within hyphae and hyphal sheath could be enzymes. whereas particles present in wood cell walls could be either enzymes or lignin granules, both of which have the same diameter. Messner and Stachelberger (1983) called the particles “osmiophilic” because they strongly react with OsO4. However, because particles were also present in KMnO4-fixed samples, we thought that the general term, “electron-dense particles.” would be more appro-

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In the present study our purpose was to elaborate on the location of electron-dense particles in wood decayed by G. applanatum. From the location of the particles, we hoped to speculate on their nature and role in the degradation process ofwood.

Materials and methods

Western hemlock (Tsuga heterophylla (Raf.) Sarg.) sawdust was degraded by the white-rot fungus G. applanatum (Pers.: S. F. Gray) Pat. (MAD 708) using ASTM soil-block procedure. After 4 weeks. the degraded sawdust was removed from bottles and prepared for transmission electron microscopy as previously described (Highley et al. 1983. Murmanis et al. 1984). The localization of cellulase activity was tested in the present study by using the cytochemical technique of Bal et al. (1976) and Lieberman et al. (1982). This test includes aldehyde fixation. carboxymethyl­cellulose. Benedict‘s solution treatment, and OsO4 postfixation. Control samples of undecayed western hemlock sawdust were fixed in a conventional way and had no carboxymethylcellulose orBenedict’s solution.

Results

Hemlock sawdust samples degraded by G. upplanatum showed no electron-dense particles when fixed in paraformaldehyde-glutaraldehyde mixture and postfixed in OsO4 (Fig. 1). The particles were not found in hyphal cytoplasm. hyphal sheath or wood cell walls, although the walls were severely degraded. At late stages of fungal attack, when the wood cell walls consisted entirely of a fine reticulum. the particles were not present either.

The picture was entirely different when samples were fixed in KMnO4 . In early stages ofattack (when wood cell walls showed no damage), electron-dense particles were present only in hyphal cytoplasm (Fig. 2). In more advanced stages, the particles were present in hyphal cytoplasm (Fig. 3. arrows), hyphal sheath (Fig. 4) and wood cell walls (Figs. 3, 4). Figure 4 shows the particles amassed in the sheath around the hyphae; from the sheath they also penetrated the cell wall. A remnant ofdecayed wall (at right) was surrounded by the particles.

We also found electron-dense particles in samples prepared by the cytochemical technique, which localizes cellulase activity at the ultrastructural level. A copper precipitate (which results when Benedict’s solution reacts with cellulose sugars) marks the sites ofcellulase activity. In electron micrographs, copper precipitate was depicted as electron-dense particles with their smallest diameters between 3 and 7 nm. Larger size particles were present, and they probably are aggregates of the smaller units (Fig. 5). The particles were present in hyphal cytoplasm, where they showed a tendency to align into the rows (Fig. 6). They were also present in hyphal sheath (Fig. 7, arrow), wood cell walls (Figs. 5, 7, 8) and cell comers (Fig. 9). Often the S2 layer was greatly swollen and uniformly covered by particles (Fig. 7), whereas the portion of wall toward the lumen showed no particles or showed localized removal of the wall material (Figs. 7, 8; arrows). Patchy distribution of

Electron-dense particles in decayed Ganoderma applanatum 317

Fig. 5. Particles not uniformly distributed are present in the cell wall (CW). Cellulase activity test. (×15,000)

Fig. 6. Particles (arrow) in cytoplasm of hypha (H). Cellulase activity test. (× 22,100)

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Electron-dense particles in decayed Ganoderma applanatum 319

Fig. 9. Hypha (H) and particles within cell corner. Cellulase activity test. ( x 19 270)

particles within the S2 layer was also encountered (Fig. 8). Figure 9 shows that the particles permeated the attacked cell comer areas and surrounded the hypha. The wall layers around the cell comer showed no particles. Control samples showed no particles.

Thus, after the cytochemical technique the particles were not evenly distributed over the cell walls; some areas were massively covered: whereas others were entirely free of particles (Figs. 5, 7, 8, 9). The particles outlined the areas where the wall material was gone (Figs. 7, 8); they penetrated through the inner wall portion (Fig. 8); and they aligned the wood cell lumen when the lumen was covered by the hyphal sheath (Figs. 7,8).

Discussion

We cannot explain why we do not find electron-dense particles in degraded wood after aldehyde/OsO4 fixation in the present or previous studies (Murmanis et al. 1984, Highley et al. (in preparation)) as did Messner and Stachelberger (1983).

Fig. 7. Particles in hyphal sheath aligning cell lumen (long arrow) and in the swollen S2 layer. The inner portion of the cell wall shows localized attack (short arrow). Cellulase activity test. (× 25,500)

Fig. 8. Particles unevenly distributed in the cell wall (CW). Part of the inner portion of the wall has been removed allowing particles to move through (arrow). Cellulase activity test. (× 24 000)

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However, we do find particles when we use the cytochemical test of Bal et al. (1976) and Lieberman et al. (1982). In these samples, we find particles in hyphal cyto­plasm. hyphal sheath, wood cell walls. and cell corners. The particles have diameters from 3 to 7 nm, but can also aggregate in larger size units. The control samples show no electron-dense particles. Bal et al. (1976) demonstrate two types of cellulases by this test: the two cellulases have been purified and reprecipitated by an immunocytochemical technique. Lieberman et al. (1982) show a high level of copper precipitate in the cell wall where breakdown occurred, and they conclude that cellulase-type enzymes are present.

We also find electron-dense particles in decayed wood when samples are fixed in KMnO4, as do Ruel et al. (1981). In KMnO4-fixed samples, we find particles in hyphal cytoplasm, hyphal sheath, wood cell walls, and cell comers. In every case when particles are encountered, their diameters range between 3 and 7 nm. This is also the size range for cellulases demonstrated by White and Brown (1981) in Trichoderma viride by negative staining.

KMnO4 stains lignin, and 7-nm particles may correspond to degraded lignin (Ruel et al. 1981). However, when particles are in hyphal cytoplasm and hyphal sheath, they could not be lignin and probably are enzymes (Murmanis et al. 1984). If the particles are enzymes, it remains unclear why we do not see them in aldehyde/OsO4-fixed samples when the wood is greatly degraded. One explanation could be that the particles are enzymes which carry chemical groups which stain only with KMnO4 and not with OsO4. In that case, particles in OsO4-fixed samples could remain invisible. Of course. this does not explain why Messner and Stachel­berger (1983) did see the particles.

In the cytochemical test for cellulase activity, the samples reveal the site of cellulase activity by copper precipitate. We speculate that in our study when the copper precipitate occurs, cellulases must be present. The identical size range (3-7 nm) of diameters of copper precipitate and negatively stained cellulases in Trichoderma viride (White, Brown 1981) is interesting, and it may be that the copper precipitate is incorporated in the enzyme molecule. Additional support for this statement comes from the work by Bal et al. (1976), who demonstrate two cellulases by the same technique, and from the work by Lieberman et al. (1982), who conclude that copper precipitate accumulations in wall breakdown areas demonstrate cellulase-like enzymes. In our opinion, the 3- to 7-nm-diameter particles demonstrated by KMnO4 staining and cytochemical test for cellulase activity are cellulolytic enzymes. This study does not distinguish enzymes and degradation products when present in the cell wall. However, the particles reveal the decay pattern in wood by the white-rot G. applanatum.

References

Electron-dense particles in decayed Ganoderma applanatum 32 1

(Received May 29, 1984)

L.Murmanis, J. G. Palmer, T.L.Highley, Forest Products Laboratory. P.O.Box5130, Madison,WI53705,USA

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