Ultrastructure of Sound and Decayed Birch Woodt ROBERT A

6954 *

Colloidal Gold Cytochemistry of Endo-l,4-f3-Glucanase, l,4-f3-D-Glucan Cellobiohydrolase, and Endo-l,4-f3-Xylanase: Ultrastructure of Sound and Decayed Birch Woodt ROBERT A. BLANCHETTE," ANDRE R. ABAD,' KORY R. CEASE,' REX E. LOVRIEN,2 AND TIMOTHY D. LEATHERS' Department ofPlant Pathologl and Department ofBiochemistry,2 University ofMinnesota, St. Paul, Minnesota' 55108. and Northern Regional Research Center, Agricultural Research Service, U.S. Department ofAgricullllre, Peoria, Illinois 616043 Received 7 April 1989/Accepted 26 June 1989

Colloidal gold coupled to endo-l,4-~-glucanase II (EG II) and 1,4-~-D-glucan cellobiohydrolase 1 (CBH I), isolated- from TricIJodenna reesei (QM9414), and endo~1,4~p,~xylanasefrom Aureobasill11J pllllulans (NRRLY 2311-1) was used successfully to determine the ultrastructural localization ofcellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, PhaneroclJae/e chrysosporillln, Phellinlls pini, and Trametes versicolor, and one brown rot fungus, F011Jitopt's pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the Sf layer ofthe secondary wall, whereas CBH I labeled ~lIlayers ofthe secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH It whereas only CBH I had a specificity for the cut ends ofmicrofibrHs. Labeling with the xylanase~gold complex occurred primarily in the inner regions of the 5z layer, 51' and the middle lameHa. In contrast, little labeling occurred in the middle lameHa with EG II or CBH I. InterceJJular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG 1I~ and xylanase~gold were used. Wood decayed by P. chrysosporium or P. pint' was delignified, and extensive degradation of the middle lameHa was evident. The remaining secondary walls labeled with EG II and CBH 1, but little labeling was fouud with the xylauase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites

of erosion labeled with both EG II and CBH I. Erosion troughs that reached the 8 1 layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Bro\Vn~rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the 5z layer near ceU lumina and corresponded to sites with the most extensive degradation.

Microbial degradation of wood polysaccharides by fungi major hemicellulose is xylan, constituting approximately 20 involves the combined actions ofseveral enzymes. White rot to 25% of the wood (9, 39). Hcmicellulase activity, including fungi produce endo-l,4-~-glucanase (EO) (EC 3.2.1.4) that endo-l,4-[3-xylanase (EC 3.2.1.8), has been found in culture attacks the cellulose chain at random (13, 16, 44) and filtrates of many white and brown rot fungi (23, 27). exo-l,4-~-glucanase (1,4-~-D-glucan cellobiohydrolase [EC Advances in colloidal gold cytochemistry make it possible 3.2.1.91]) (CBH) that attacks the nonreducing end of the to use enzyme-gold complexes to label specific substrates. crystalline cellulose chain, splitting off glucose or cellobiose Since first used to localize nucleic acids in sectioned material (13, 15). It has been postulated that white rot fungi use both (2). the technique has been used with many plant tissues to enzymes synergistically to degrade a cellulosic substrate (15, label polysaccharides (43, 45), pectins (3), and plant and 25, 42). As white rot fungi gradually degrade cellulose, fungal cell walls (4, 10). In the study of Berg et al. (4), crude degradation products are rapidly utilized (34). In contrast, cellulase preparations and purified EO and CBH I were used brown rot fungi cause a rapid depolymerization of cellulose to show specific labeling of fungal and plant cell walls early in the decay process (14, 24). The only cellulase containing cellulose. Colloidal gold labeling of CBH 1 from enzyme reported from brown rot fungi is EO (21, 22), and it Trichoderma reesei also has been used to visualize specific apparently is a multifunctional enzyme, active on both absorption sites on the surface of microcrystalline cellulose polysaccharides and glycosides (46). The exact mechanism produced by Valollia macrophyso (11). Specificity of this responsible for extensive and rapid depolymerization of enzyme for crystal edges, rather than ends, suggested that cellulose is uncertain; however. a nonenzymatic process has CBH I was acting as an endoglucanase-type enzyme. In been strongly suspected during decay by brown rot fungi (14, addition, CBH I alone has been shown to degrade highly 26, 31, 34). crystalline cellulose (12). In addition to cellulose, hemicelluloses also occur in Colloidal gold-xylanase complexes have been used to various amounts within cell walls of wood. In birch, the identify the presence of xylan in cells of Tilia plalyplyllos (45). In this study the secondary wall layers were labeled with the xylanase-gold. but the middle lamella region and * Corresponding author. cell lumina were free from labeling. Ruel and Joseleau (43) t Published as contribution 16.992 of the series of the Minnesota were able to successfully label mannanase in Picea cell Agricultural Experiment Station. walls. Gold particles were distributed over the entire surface

2293 2294 BLANCHETTE ET AL. ArrL. ENVIRON. MICROBIOL. ofthe wall, with the greatest concentration in the S21ayer of the secondary wall. The present study was done to demonstrate the specificity of EG II, CBH I, and xylanase for cell walls of sound birch (Betula papyri/era) wood and wood decayed by brown or white rot fungi and to determine' the ultrastructurallocaliza tion of these enzymes wi.thin woody cell walls. One brown rot fungus, Fomitopis pinicola, and three white rot fungi, Pizanerocizaete chrysosporiufl'l, Phellillus pini, and Trametes versicolor, were used in this study. The white rot fungi represented species responsible for two different forms of decay (5, 38): selective degradation of lignin (P. clzrysas porium and P. pinf) and nonselective degradation of all cell wall components (T. versicolor).

MATERIALS AND METHODS Wood blocks of sound, freshly cut birch, B. papyri/era, were decayed in the laboratory for 12 weeks as previously described (39). The wood blocks were incubated in the dark at 27'C and a relative humidity of approximately 90%. With these methods a combination of intermediate and advanced stages of decay could be expected within the wood blocks (8). Three white rot fungi, T. (Coriaills) versicolor (MAD 697-R), P. clzrysospori1lln (BKM-F-1767), and P. pilli (RAB FlO. 1. Transmission electron micrograph of sound birch wood 83-19), and one brown rot fungus, F. pillicola (RAB-32), fixed in KMnO.;, showing cell wall layers in fibers (F) and vessels were used to inoculate the wood blocks. Noninol;ulated (V). ML, Middle lamella (arrows indicate intercellular region of cell wood blocks served as controls. corner); $1> Sz, and S), layers of secondary walls. No S) is apparent Sound and decayed wood blocks were cut into small in fibers. Bar = 2 j.Lm. segments (approximately 1 by I by 0.4 mm) and fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 after ultracentrifugation with citric phosphate buffer, pH 4.5. for 2 h. Samples were rinsed in buffer three times for 20 min containing 0.6% NaCI and 0.05% Tween 80. The EG II and each and dehydrated through a graded acetone series. Em eBB I probes were suspended after ultracentrifugation with bedding procedures with Quetol 651 resin (32) were as citric phosphate buffer, pH 5.4, containing 0.06% NaCI and previously described (1). Samples in resin were cured at 0.05% Tween 80. Sections on grids were floated in citric 74'C for 8 h and cut with a diamond knife. Sections (100 to phosphate buffer, at pH 4.5 or 5.4, for 5 min and then placed 120 nm) were collected on nickel grids. Additional samples in the respective enzyme-gold complexes for 15 min. To were cut from the wood blocks and fixed with 2% KMn04 in determine the effects ofa longer incubation time on labeling. distilled water, followed by dehydration in Quetol 651 and additional sections ofsound wood were also incubated for 30 embedment (1) to observe the extent of lignification within min in EG II- or CBH I-gOld complexes. Grids were rinsed cell walls. with a jet spray of distilled H20 and air dried. The sections Colloidal gold solutions with a gold particle size of13 to 15 were observed and examined with a Hitachi 600 transmis nm were produced using the methods of Frens (18). Highly sion electron microscope without additional staining. Cy purified EG !l and CBH I were obtained from T. re.esci tochemical controls included labeling with heat-treated EG (QM9414), using procedures described previously (37), and II, CBH I, or xylanase and labeling with nonenzymatic endo-1,4-fj-xylanase was obtained from Aureobasidium pul proteins (4). Iliialls (NRRLY-2311-1) by methods of Leathers (33; T. D. Leathers, J. Ind. Microbiol., in press). Each enzyme was combined with the colloidal gold solutions at pH 7.4 by RESULTS methods of Geoghegan and Ackerman (19) and Goodman et Cell walls of fibers and vessels in birch wood. fixed in

al. (20) to form the enzyme-gold complex. The amount of KMn04 and prepared for transmission electron microscopy, enzyme used to stabilize 0.5 ml ofcolloidal gold solution was show the ultrastructure of various cell wall layers (Fig. 1). 0.01 to 0.02 mg. The concentration ofall gold complexes was Transverse thin sections of wood incubated with the gold determined using a spectrophotometer at 560 nm and an EG 11 complex. using a 30-rnin incubation, showed that the absorbance of 0.27. Xylanase-gold probes were suspended enzyme had a preference for the Sl layer of secondary walls

FIG. 2. Transmission electron micrographs oftransverse (a, b, e, and f) and oblique (c and d) sections ofsound birch wood incubated with EO lI·gold (a to c) and CBH I-gold (d to f). (a) Section incubated with EG ll-gold for 30 min, showing intense labeling of the Sllayerand some labeling of other fiber cell wall layers. In vessels, S1 and S) have high concentrations of gold. (b) Incubation of EO II·gold for 15 min, with reduced concentrations of gold throughout the cell wall. The preference of EO II for the S1 layer is evident. Low levels of gold labeling are seen in S2' and no labeling is evident in the electron-dense middle lamella. Intercellular areas of cell corner regions, however, were labeled (arrows). (c) Extensive labeling ofall cell wall layers of fibers and vessels·is observed in an oblique section. (d) CBH I·gold labeling ofall cell wall layers in an oblique section, with the greatest concentrations in the $1 layer. (e) Incubation of CBH I for 30 min, showing labeling of all secondary wall layers in· fibers and vessels. <0 Fifteen·minute incubation with CBH 1, exhibiting moderate labeling of secondary wall layers

but not in boundaries between 8 1·S2 in fibers and Sl-$2 or 82.5) in vessels or in the middle lamella. No labeling was apparent in the intercellular regions of cell corners (arrows). Bar == 2 !J.m. VOL 55, 1989 ULTRASTRUCTURE OF SOUND AND DECAYED BIRCH WOOD 2295

' .. e , f . 2296 BLANCHETTE ET AL. ApPL ENVIRON. MICROBIOL. in fibers and vessels (Fig. 2a). The boundary zones between cell wall immediately adjacent to the decayed areas had large S, and the compound middle lamella and S,-S, layers were concentrations of gold particles. labeled intensely. The S, layer of fiber walls contained small Wood decayed by P. pilli or P. cll1ysosporium showed amounts of label near the lumen, but concentrations in~ evidence of selective delignification (Fig. 3c and d). In cells creased toward the Sl layer. Ves'sel cell walls were labeled with advanced decay. the middle lamella regions were throughout all layers, with greatest concentrations in Sl and completely degraded while the S1 and S2 layers remained. S3' while concentrations of gold particles within the middle These cell wall layers appeared slightly swollen, but no cell lamella remained low. Usually, some labeling occurred in wall erosion was evident. EG II-treated sections of deligniM the cell corner regions but very slight amounts in the middle fied cells showed labeling within the S, layer (Fig. 3c). lamella between cells (Fig. 2a).· Sections with CBH I had labeling throughout the secondary Incubation times used for labeling sections with enzyme wall (Fig. 3d). Even in cells where lignin was removed from gold complexes affected the intensity ofgold concentrations the secondary wall and the entire middle lamella was re observed. Longer incubation times of 30 min fully saturated moved, the secondary wall maintained high concentrations all available sites for enzyme activity and resulted in. an of gold particles. increased amount of background labeling that was not spe Wood decayed by the brown rot fungus F. pinkola was cific or representative of sites for enzyme activity. Sections extensively degraded, and secondary wall layers were swolM incubated for a shorter time, 15 min, had considerably less len and appeared porous. Both secondary wall layers and the labeling, but the location of the labeling and the patterns of middle lamella remained discernible after the 12 weeks of gold particle deposition were similar to those of the longer decay. Sections of brown-rotted wood incubated with EG II incubation time (Fig. 2b). A preference of EG II for the S, had very low levels of labeling (Fig. 3e). Only a thin outline layer was evident. In sections of wood, spaces were often of the S, layer was evident by the gold particles. The CBH observed that were less electron dense in cell corners. Gold I-gold treatment displayed high concentrations ofgold in the particles were always present in these areas (Fig. 2b, ar secondary walls, but in many· cells considerably less gold rows). The middle lamella, however, remained relatively was present in the S2 layer near cell lumina (Fig. 3D. No free from gold particles. When sections of birch wood were labeling ofthe middle lamella was observed for CBH I or EG cut at oblique angles instead of in a transverse section, II. moderate amounts of gold labeling were observed through A xylanaseMgold probe used on transverse sections of out the secondary cell wall (Fig. 2c). Results were similar sound wood showed some labeling in all cell wall layers, but using the 15~ and 30Mmin-incubation procedures. Other the greatest concentrations were found within the middle oblique sections incubated with CBH I also showed labeling lamella and adjacent secondary wall regions (Fig. 4a and b). throughout the secondary wall, with the greatest concentra~ The distribution of gold particles within cell walls varied tions in S, and the least within the middle lamella (Fig. 2d). among different samples ofsound wood observed, with some Transverse sections of wood incubated with CBH I for 30 cells exhibiting more labeling of secondary wall layers than min (Fig. 2e) or 15 min (Fig. 2f) exhibited labeling in all others (Fig. 4a and b). Sections from wood decayed by T. secondary wall layers of fibers and vessels. The distribution versicolor and incubated with the xylanase-gold complex of gold appeared to be relatively uniform throughout the showed gold labeling that was localized primarily in S1 and cells. The boundary zones of S1-S2 and S2~S3 in vessels and the middle lamella. Few gold particles were observed in SCS2 in fibers were distinct and labeled to a lesser extent areas where erosion troughs, produced by the fungus, than other areas. The middle lamella and the less electron reached S, Or the middle lamella (Fig. 4c, arrows). Gold dense spaces within the cell corner regions were not labeled concentrations in other parts of the cell were similar to those to any appreciable extent. observed in sections of sound wood. Spaces appearing less All sections of wood decay by brown ·or white rot fungi electron dense within the cell corner regions of the middle were treated with EG II or CBH I, using the 15-min lamella were labeled with the xylanase-gold probe. Wood incubation. Wood decayed by T. versicolor showed an delignified by P. pilli or P. chrysosporillm had gold labeling attack on all cell wall layers (Fig. 3a and b). The secondary only in cell corner regions (Fig. 4d and e). The middle wall was eroded from cell lumina, where hyphae were lamella between cells was in various stages of decomposi located toward the middle lamella. Sections incubated with tion. In these areas where the middle lamella was removed, EG II were labeled primarily in the S, layer (Fig. 3a). In no gold particles were evident. reflecting the lack of xylan. areas of the cell where the secondary wall was eroded to Electron·dense areas of the middle lamella which apparently expose the Sl layer, a reduced amount of labeling was were not delignified were sites for gold labeling. Very few evident (Fig. 3a, arrow). Sections incubated with CBH I had gold particles were found in sections ofwood decayed by F. gold particles distributed throughout the residual secondary pillicola, the brown rot fungus (Fig. 4f). Only scattered wall that remained after degradation (Fig. 3b). Although particles of gold occurred randomly over the various cell extensive cell wall erosion had taken place in some cells, the wall layers, indicating the absence of xylan in the wood.

FIG. 3. Transmission electron micrographs of transverse sections of wood decayed for 12 weeks by T. versicolor (a and b), P. pill" (c), P. chrysosporium (d), and F. pinicoltl (e and O. (a) Erosion of cell wall layers removed portions of the cell wall. Labeling with EG II was restricted to the Sl layer. Intercellular regions ofcell corners are labeled (arrow). Hyphae (H) are located in cell lumina. (b) Eroded cell walls, showing gold labeling of remaining wall layers with CBH 1. Intercellular regions are not labeled (arrow). (c) Delignified fibers after extensive degmdation of the middle lamella show gold labeling by EO primarily in the SI layer. (d) Partially delignified fibers exhibit areas with a completely degraded middle lamella (arrowheads) and areas with an intact middle lamella. CBH I labeling occurs in all secondary wall layers. (e) Sections of brown·rotted wood. with cells having a porous, swollen appearance. Little gold labeling of the SI layer is evident with EO II. mSection labeled with CBH 1. showing moderate to low levels ofgold in the secondary wall. Concentrations ofgold particles were extremely low in extensively degraded areas of cell walls. A fiber is seen with small quantities of gold near cell lumina (arrowheads), which is the most severely degraded part of the cell. Bar = 2 IJ-m. VOL. 55, 1989 ULTRASTRUCTURE OF SOUND AND DECAYED BIRCH WOOD 2297 2298 BLANCHETTE ET AL. ApPL. ENVIRON. MICROBIOL.

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All cytochemical controls used in this study for each and do not appear lignified are frequently observed (Fig. 1). enzyme were negative, and no labeling was observed. These areas, often referred to as intercellular spaces (17), are sites where EO II-gold and xylanase-gold probes had posi DISCUSSION tive labeling. No labeling, however, occurred with the CBH I-gold probe. These results demonstrate that these areas are Colloidal gold cytochemistry, using EO II, CBH I, and not voids but contain hemicellulose and probably noncrys xylanase, successfully demonstrated the ultrastructural 10 talline forms of cellulose. The presence of polysaccharides calization of cellulose and xylan substrates within cell walls within nonlignified zones of the middle lamella in hardwood of birch wood. The microfibrillar orientation of cellulose species is an interesting phenomenon that has not been varies among different layers ofthe secondary wall (40), and reported previously. these differences affect the sites of endo~ and exo-type Colloidal gold labeling of sections from wood blocks enzyme activity. In transverse sections afwood, EG'II was decayed by P. chrysosporium or P. pini with EO II or CBH specific for the S, layer rather than the S2 layer and orien I indicated that cellulose was not substantially removed after tations of the cut microfibrils were not sites for EO .11 extensive delignification. In sections where the middle la~ binding. However, in oblique sections, where the rni mella had been completely degraded and secondary walls crofibrils are cut differently, exposing their sides, EO II was were free from lignin, patterns of gold labeling by EO II or able to label intensely Ihe 8, layer. These results confirm the CBH I were not different from those observed in cell walls of affinity of this enzyme for lateral faces ofmicrofibrils and not sound wood. Chemical analyses from previous investiga~ their cut edges. Labeling ofthe SI layer in transverse section tions of wood decayed by P. chrysosporium and P. phd, also suggests an affinity for sides of the crystalline cellulose using the same time period of degradation, demonstrated structure since the direction of rnicrofibtils is different in this that after a weight loss of 17 to 39%, 54 to 73% of the lignin, layer than in the S2 layer. Differences in the amount of 5 to 15% of the cellulose, and 30 to 55% of the xylan were crystalline and amorphous cellulose also may vary between removed (7,39). The small percentage of cellulose degraded these two layers (40). Most crystalline cellulose is located in by these fungi apparently was not sufficient to detect a the S2 region. Results with the CBH I-gold complex indicated that large change in the labeling of EO 11 or CBH 1. Although the concentrations of gold particles were present in transverse secondary wall was swollen and chemically altered, acces . and oblique sections. Since specificity for CBH I is consid sibility to EO II was not enhanced. The loss of xylan from ered to take ·place at the nonreducing ends of the cellulose the wood, however, was detected when the xylanase-gold chains (13, 15), positive labeling of transverse sections was probe was used. These results support the view that when expected since large quantities of end chains would occur at lignin is degraded, hemicellulose is also removed. The this cut surface. The intense labeling ofthe oblique sections, selective loss of lignin and hemicellulose and repression of where few end chains would be expected, raises the possi~ cellulase by some white rot fungi make these particular bility that this enzyme has some characteristics of an en species ideally suited for industrial applications where lignin doglucanase. Chanzy et al. (11) also demonstrated that the or various phenolic compounds must be altered or removed sides ofmicrocrystals produced by V. macrophysa were well (29,36). labeled when treated with colloidal gold-CBH I. In another The degradation of wood by T. versicolor was different study (12) it was demonstrated that CBH I alone was able to from that caused by white rot fungi that preferentially degrade highly crystalline Valonia cellulose. The results of degrade lignin. A nonselective attack removed all layers of our investigations also show affinity of CBH I for all spatial the cell wall. The remaining cell wall showed no differences aspects of cellulose chains within woody cell walls and from sound wood in gold labeling after EO 11- or CBH I-gold support the possibility that this enzyme recognizes more treatment. These results demonstrate that cellulose is not than just the nonreducing ends of cellulose. altered beyond the eroded cell wall region. Evidence from Micrographs of the xylanase probe demonstrated that the xylanase-gold-treated sections suggests, however, that the enzyme had endo~type activity and that gold particles were xylan content of cell walls near eroded areas is reduced. distributed primarily in the 8, layer and the middle lamella. Hemicellulases apparently move into the cell wall and de~ Although some xylan is undoubtedly located throughout the grade hemicellulose before cellulases. Lignin removal also secondary wall (6), the greatest concentration appears in has been postulated to precede cellulose degradation (8). inner regions of woody cell wall. Other investigations have Hemicellulases most likely accompany or follow immedi suggested that lignin and hemicellulose are closely associ~ ately lignin~degrading enzymes or possibly nonenzymatic ated within cell walls (28, 40). Areas of the cell with higher processes. It is also possible that the lignin degradative concentrations of lignin, such as the middle lamella (8), processes alone may affect hemicellulose (41). would also be expected to have a higher hemicellulose Brown rot fungi remove cellulose and hemicellulose from content. The information presented here, using a xylanase wood without degrading large amounts of lignin (30). The probe, supports these previous findings. highly lignified cell wall that remained after decay by F. Within cell corner regions of middle lamellae, less~elec pinkola observed in our study had little gold labeling after

tron-dense areas that apparently do not stain with KMnOol sections were incubated with EG II- or xylanase-gold

FIG. 4. Transmission electron micrographs of tnmsverse sections from sound wood (a and b) and wood decayed for 12 weeks by T. \'crs;co!or (c), P. pini (d), P. chrysosporiuftl (e), and F. pinicoltl (0 after incubation with a xylanase-gold complex. (a) Gold labeling occurring within the inner regions of the secondary wall, 51' and the middle lamella. {bl Some sections. such as this one. exhibit labeling only in parts of the 51 layer and the middle InmeJla. Labeling can also be seen in the intercellular regions ofcell corners (arrow). (c) Gold labeling is evident in some areas of 51 and the middle lamella. but absent in the cell. wall near eroded areas (arrowheads). (d and e) Fiber cells that were only partially delignified show some of the middle lamella still present. Gold labeling is seen only in cell corner regions. (0 Cells from brown~rotted wood show very low levels of gold within the ceil walls. Bar = 2 }.lm. :2.300 BLANCHETTE ET AL. App!.. ENVIRON. MICRQDlOL. probes, demonstrating the reduced amount of substrate Hollaender (ed.), Trends in the biology of fermentations: for available for these enzymes. Some cellulose remained, how fuels and chemicals. Plenum Publishing Corp., New York. ever, within secondary walls and gold labeling by the CBH I 16. Eriksson, K. E., and n. Pcttersson. 1975. Extracellular enzyme probe was evident. The parts of the cell wall with the most system utilized by the fungus Sporotrichum puh'erulellwm advanced decay, exhibiting a swollen appearance, had the (Chrysosporium ligllorum) for the breakdown of cellulose. 1. separation, purification and physio·chemical characterization of lowest levels ofgold labeling. These regions apparently have five endo~1,4-I3~glucanases. Eur. J. Biochem. 51:193-206. extremely small amounts of cellulose present. Although 17. E.>;au, K. 1977. Anatomy of seed plants. 10hn Wiley & Sons, initial processes of cellulose de polymerization by brown rot Inc., New York. fungi are diffusible and occur extensively throughout the 18. Frens, G. 1973. Controlled nucleation for regulation of the wood (14, 25), removal of cellulose by enzyme action particle size in mono dispersed gold suspensions. Nature Phys. appears to take place in cell wall layers near the lumen. As Sci. 241:20-22. the brown rot degradation progresses, cellulose is subse~ 19. Geoghegan, W. D., and G. A. Ackerman. 1977. Absorption of quently removed from the inner portions ofthe cell wall. The horseradish peroxidase, ovomucoid and antiimmunoglobulin to colloidal gold for the indirect detection of concanavalin A, movement of enzymes into the wood apparently follows the wheat germ agglutinin and goat anti-human immunoglobulin G precelluloytic degradation system (35), on cell surfaces at the electron microscopic level: a new Colloidal gold cytochemistry can provide a unique way to method, theory and application. J. Histochem. Cytochem. 25: visualize the precise location of sites for enzyme activity. In 1187-1200. sound wood, this provides a mechanism to determine the 20. Goodman, S. L., G. M. Hodges, and D. C. Livingston. 1980. A ultrastructural arrangement of cell wall components. It also review of the colloidal gold marker system, p. 133-146./11 R. P. has been shown here to provide important new information Becker and O. Johari (ed.), Scanning electron microscopy, part about how white and brown rot fungi degrade woody sub 8 II. SEM Inc. AMF O'Hare, Chicago. strates. 21. 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