APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1984, p. 670-676 Vol. 47, No. 4 0099-2240/84/040670-07$02.00/0 Copyright ©) 1984, American Society for Microbiology frustulatus Decay of : Patterns of Selective Delignification and Subsequent Cellulose Removalt LEWIS OTJEN* AND ROBERT A. BLANCHETTE Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108 Received 14 November 1983/Accepted 19 January 1984

Xylobolusfrustulatus caused a distinct pocket rot in decorticated oak. Polymerization products appeared to accumulate in advance of delignified wood to form barriers to decay. Medullary ray parenchyma and earlywood vessels were not readily degraded and remained between pockets of decay. Chemical analyses indicated that 97% lignin, 96% xylose, and 69% mannose were removed from pockets of wood during incipient decay. Although 53% of the cellulose was removed from these areas, the remaining white tissues were composed of relatively pure cellulose. Hyphae became abundant as the released cellulose was subsequently removed. In the most advanced stages of decay, hyphae were absent from pockets, and only a sparse lining of crystals, found to contain a high concentration of calcium, remained.

Many microorganisms can be implicated in the process of the United States it is more commonly known as honeycomb wood decay, but fungi are responsible for most lignin and rot due to the complete removal of wood from localized cellulose degradation. Fungi are fundamental in recycling pockets within the log (7, 22). To obtain a better understand- most of the world's carbon source (18). Although lignin is ing of the decay process caused by X. frustulatus, it is considered to be the cell wall component degraded slowest important to determine the factors that restrict delignifica- within the forest ecosystem (25), white rot basidiomycetes tion to localized areas or pockets and to provide a better are particularly well adapted for lignin degradation. Usually, understanding of the mechanism responsible for selective white rot fungi remove lignin, cellulose, and hemicellulose in delignification. The intent of this research was to (i) examine equal proportions (9). Decay by white rot fungi is character- the capacity of X. frustulatus to selectively remove lignin ized microscopically by a uniform removal of all cell wall from wood, (ii) elucidate the micromorphological patterns of constituents resulting in a shot-hole appearance of cell walls. decay, and (iii) compare decay caused by X. frustlulatus with Some white rot fungi, however, show a marked variation in previously studied white pocket rots (e.g., I. dryophilus and the pattern of attack on cell wall components by selectively P. pini). removing lignin (3, 4, 23; R. A. Blanchette, L. Otjen, M. J. Effland, and W. E. Eslyn, Wood Sci. Technol., in press). MATERIALS AND METHODS These fungi cause many localized areas or pockets of degradation within wood which are composed of cellulosic Bolts approximately 30 cm long containing various stages white tissues and are virtually free of lignin. Phellinus pini of decay by X. frustulatus were cut from 12 fallen red and Inonotus dryophilus cause a selective delignification of (Quercus rubra L.) and 5 fallen white oaks (Quercus alba L.) the heartwood of living trees which results in a white pocket in Ramsey and Winona Counties, Minn. The sapwood of all rot (3, 23). The microscopic characteristics of selectively trees sampled was completely degraded, indicating that they delignified wood are different from those of wood with had been down for many years. Sporophores of X. frustula- typical white rot. Lignified middle lamellae are completely tus were present on the outer surface of these decorticated removed, resulting in a separation of cells. The remaining logs. Bolts were cut from trees displaying all stages of decay cell walls have a fibrillar structure and consist primarily of and aseptically split; isolations were made with a sterile cellulose (23). Recently, several basidiomycetes not causing scalpel from all representative areas of the longitudinal a white pocket rot have also been shown to selectively surface. These areas included white tissues from pockets remove lignin from wood (Blanchette et al., in press). The during incipient stages of decay, hyphae occupying pockets specificity of these fungi for lignin degradation demonstrates during later stages of decay, wood present between pockets their potential for industrial applications such as biological of decay, and sound wood present in advance of decay. pulping or bleaching of wood, removing waste lignin from Sections of wood were cultured on three different media: pulp mill effluent, and releasing wood sugars for subsequent malt yeast agar (15.0 g of Difco malt extract, 15.0 g of Difco fermentation in ethanol production or for use as a carbohy- agar, 2.0 g of Difco yeast extract per 1,000 ml of water), acid drate source for ruminant animals (18, 21). malt yeast agar (4 ml of lactic acid added to malt yeast agar ( frustulosum) is a basidio- after autoclaving), and a semiselective medium for basidio- mycete (order Aphyllophorales; family ) that is mycetes (5). Cultures were incubated at room temperature capable of causing a white pocket rot of dead oaks through- and observed after 10 days. out Europe (8, 14) and North America (6, 7, 22, 26). Hartig Radial, tangential, and transverse sections of wood were (14) described the decayed wood as partridge wood, but in cut from all representative areas of affected wood. Unfixed specimens were dried in a desiccator and coated with 40% gold-60% palladium or carbon in a Kinney KSE-2A-M vacuum evaporator. Energy dispersive X-ray microanalyses * Corresponding author. were made with an Edax-711 X-ray analyzer in conjunction t Paper no. 13,675, Scientific Journal Series, Minnesota Agricul- with a Philips scanning electron microscope to determine the tural Experiment Station, St. Paul, MN 55108. elemental constitution of crystals left after degradation. An 670 VOL. 47, 1984 X. FRUSTULATUS DECAY OF OAK 671

FIG. 1. (a) Sporophores ofX. frustulatus on the surface of decorticated oak. (b) Pockets, relatively uniform in size and shape, are present in tangential view. (c) A cross section of infected oak with honeycomb appearance of advanced decayed wood. accelerating voltage of 12 kV and a spot diameter of 64.0 nm ance of decayed wood was evident in a cross section of were used. Both small roster and spot analyses of crystals advanced decayed wood (Fig. lc). were performed. Incipient decay was characterized by the formation of Lignin and wood sugar determinations were made by M. J. white pockets (Fig. 2a). White pockets were observed with Effland, Forest Products Laboratory, Madison, Wis., by the scanning electron microscope to consist of delignified previously described techniques (Blanchette et al., in press). cells (Fig. 2c and e). Distinct borders were apparent between Percent loss of wood components was determined by using the pockets (Fig. 2c). Separated tissues lacked middle lamel- the specific gravity based on green volume by previously lae, and the fibrillar arrangement of cellulosic macrofibrils described techniques (2, 16). within cell walls was apparent (Fig. 2e). Advanced decay was characterized by the absence of white tissues within pockets (Fig. 2b). Micromorphological examination of the RESULTS degraded area showed them to be void of any delignified Logs infected with X. frustulatus were identified by the cells (Fig. 2d), but areas of apparently sound wood still presence of small, white, resupinate fruiting bodies (Fig. la) existed between pockets of decay (Fig. 2d). The walls of the on the surface of decorticated logs. The decayed wood empty pockets were sparsely lined with crystals (Fig. 2f) and consisted of empty, spindle-shaped pockets separated by found to contain high amounts of calcium by using X-ray apparently sound wood (Fig. lb). The honeycomb appear- analysis techniques. FIG. 2. (a) Incipient decay showing delignified white tissues within pockets. (b) Pockets in advanced decayed wood void of delignified tissues. (c) Separation of tissues in pockets of incipient decay, and borders of apparently unaltered wood between pockets. (d) Empty pockets in advanced stage of decay, but borders still exist between pockets. (e) Lignified middle lamellae absent from separated tissues and exposed cellulosic macrofibrils. (f) A sparse lining of crystals containing calcium were present on the walls of empty pockets. 672 VOL. 47, 1984 X. FRUSTULATUS DECAY OF OAK 673

TABLE 1. Chemical composition of sound and decayed wood lamellae. Since lignin degradation has been noted by several Chemical composition (%)f investigators to occur at a distance from fungal hyphae (11), Wood type it has been postulated that a diffusible process may be Lignin Glucose Xylose Mannose responsible. The chemical analyses presented in this study Sound red oak 24.5 41.6 23.5 3.0 suggest that extensive amounts of lignin are removed from Decorticated oak 24.0 40.9 20.2 1.2 the entire cell wall. Since a large amount of lignin can be White pocket 2.5 (97) 76.9 (53) 3.7 (96) 1.5 (69) found in the secondary wall (24), it may be possible that tissues minute avenues of lignin and hemicellulose are removed so a Values in parentheses represent percent loss. access to the middle lamellae by enzymes or oxidizing agents (12, 19, 20) could be realized. X. frustulatus was found to cause a distinctive pocket rot in decorticated oak. Other studies have suggested that fungi Chemical analyses of sound oak heartwood, sound decor- causing white pocket rots may have a preference for various ticated oak, and white pocket tissues are presented in Table cell types within wood (3, 23). Cell types not preferred by 1. Very little difference was apparent between sound oak, those fungi were left as borders between decayed areas. X. which had a 25% lignin and 42% glucose content, as com- frustulatus, however, degraded all cell types, with the ex- pared with sound decorticated oak, which had a 24% lignin ception of medullary rays which only partly composed wood and 41% glucose content. White pocket tissues, however, between pockets. A clear demarcation between delignified were found to contain 3% lignin and 77% glucose, represent- and unaltered wood was evident during the decay process ing a 97% loss of lignin and only a 53% loss of cellulose. The (Fig. 3c). Occluding substances, presumably decomposition hemicelluloses were removed in relatively large amounts, products, appeared to be primarily responsible for delimit- with a 96% loss of xylose and 69% loss of mannose. ing pockets of degradation. Since hyphae were present in the The progressive stages of decay were observed and com- occluded wood between pockets and no decay was evident, pared with sound unaltered wood. Intact cells composing occluding substances appeared to prevent cell wall degrada- sound decorticated oak are displayed in Fig. 3a. Decay of tion. oak by X. frustulatus began with the removal of lignin from After delignification occurred in isolated pockets, X. frus- cell walls resulting in a loss of middle lamellae and a tulatus removed the exposed cellulose. Other white pocket separation of cells (Fig. 3b). Substances that accumulated in rot fungi do not remove the cellulose that remains after advance of delignification appeared to limit the size of the selective delignification (3, 23). Decay by X. frustulatus also pockets as well as to form borders between selectively differed from decay produced by fungi causing a typical delignified areas (Fig. 3c). After delignification, the remain- white rot. Instead of cellulose and lignin being simultaneous- ing cellulose was degraded. Mycelia became abundant ly removed in equal proportion, X. frustulatus selectively during this stage of decomposition (Fig. 3d). After cellulose removed lignin first and in a separate but successive process was completely removed, only a mass of mycelia remained removed the remaining intact cellulose. Since lignin protects within the pockets (Fig. 3e). Orange-colored mycelia, abun- cellulose from enzymatic attack (18), large amounts of dant in pockets during cellulose removal, were cultured on cellulose were made available after delignification. Degrada- nutrient media and identified as X. frustulatus. In the most tion of cellulose in the absence of lignin appeared to differ advanced stage of decay, mycelial masses were absent, from that during lignin removal. Only after lignin was leaving pockets void (Fig. 3f). removed did micromorphologically discernible cellulase ac- X. frustulatus was capable of attacking axial parenchyma, tivity occur. There was abundant hyphal growth in response latewood fibers, and uniseriate rays in oak (Fig. 4a; p, f, and to the degradation of cellulose remaining after delignifica- r, respectively). Medullary ray parenchyma were rarely tion. The reason for this increase in fungal growth may be degraded and comprised a significant portion of the wood due to the increased utilization of available carbohydrate. after degradation (Fig. 4b; m). Earlywood vessels appeared Alternatively, if the cellulose system produced after deligni- to be more difficult to degrade and were common as borders fication were not diffusible, close hyphal contact would be (Fig. 4c). Substances produced during the delignification necessary for degradation. Thus, stimulation in growth may process surrounded the pockets and appeared to create be a genetically controlled response to provide hyphal boundaries (Fig. 4d, arrowheads). contact with substrate. The decay caused by X. frustulatus differs from that DISCUSSION caused by other white rots and illustrates the different decay Chemical analyses indicated that a selective removal of capacities that exist among white rot fungi. One major lignin and hemicellulose occurred during the incipient (white difference between X. frustulatus decay and other white rots pocket) stage of decay. Since a carbohydrate source appears is the poor production of laccase and the negative Baven- necessary for delignification (18), X. frustulatus may utilize damm reaction (10). Since laccase has been implicated in xylose preferentially during lignin degradation. Alternative- lignin degradation (1), studies to elucidate the process of ly, hemicellulose may be selectively removed with lignin selective lignin removal by X. frustulatus deserve additional because of the close spatial association that exists within the attention (13). Decay by white rot fungi appears to vary cell wall (17, 24). Other fungi that selectively degrade lignin tremendously, resulting in white pocket (3, 22), white mot- have also been found to degrade hemicellulose in preference tled (Blanchette et al., in press), and simultaneous rots (4). to cellulose (15; Blanchette et al., in press). X. frustulatus is unique because the micromorphological Bore holes and erosion troughs, indicative of the nondif- patterns of degradation are different from these other white fusible lignin degrading systems of white rot fungi (18), were rot fungi. More information is needed to differentiate the not observed. Instead, lignin degradation occurred at a degradative capacities of white rot fungi so that less ambigu- distance from fungal hyphae located within cell lumens. ous terms can be used to describe them and so that more Selective delignification resulted in exposure of the cellulose adequate descriptions can be made of a group of fungi that is macrofibrils of the cell wall and complete removal of middle of great ecological importance and potential use for humans. FIG. 3. Progressive stages of decay by X. frustulatus. (a) Sound decorticated oak heartwood. (b) Selective lignin removal resulted in a loss of middle lamellae and a separation of cells. (c) Occluding substances (arrowhead) accumulated in advance of the delignified wood delimiting the decay to pockets. (d) Delignified cellulosic tissues (arrowhead) colonized after delignification and pocket formation. (e) Pockets filled with mycelia after cellulose removal. (f) Mycelia gradually disappeared and pockets were empty. 674 VOL. 47, 1984 X. FRUSTULATUS DECAY OF OAK 675

FIG. 4. (a) Cross section of wood with advanced decay caused by X. frustulatus; note degraded fibers (f), axial parenchyma (p), and uniseriate rays (r). (b) Medullary ray parenchyma (m) were not degraded and occurred along borders of pockets. (c) Earlywood vessels not readily degraded and bordered pockets. (d) Occluding substances accumulated in advance of decay responsible for restricting degradation to localized areas.

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