Mycofibrillar Cell Wall Extensions in the Hyphal Sheath of Postia Placental

905 Mycofibrillar cell wall extensions in the hyphal sheath of Postia placental

MICHAEL J. LARSEN AND FREDERICK GREEN III U.S. Department of Agriculture, Forest Service, Forest Products Laboratory,2 One Gifford Pinchot-Drive, Madison, WI 53705-2398, U.S.A. Received September 23, 1991 Revision received February 12, 1992 Accepted February 19, 1992

LARSEN, M. J., and GREEN III, F. 1992. Mycofibrillar cell wall extensions in the hyphal sheath of Postia placenta. Can. J. Microbiol. 38: 905-911. Evidence is provided for the existence of linear extracellular fibrillar elements in the brown-rot fungus Postia placenta. These elements appear as structural components of the hyphal sheath and more closely resemble mycofibrils than fungal fimbriae. Mycofibrils are associated with and appear to originate from the hyphal surface when hyphae are grown on wood or inert substrates, such as glass cover slips and polycarbonate filters. These extracellular structures have a nominal diameter of 10-50 nm and are up to 25 tan µmlength. We conclude that mycofibrils are linear structural extensions of the hyphal cell wall. The precise function of mycofibrils in the brown-rot decay process of wood remains to be elucidated. Key words: Postia placenta, mycofibrils, fungal fimbriae, hyphal sheath, electron microscopy.

LARSEN, M. J., et GREEN III, F. 1992. Mycofibrillar cell wall extensions in the hyphal sheath of Postia placenta. Can. J. Microbiol. 38 : 905-911. L'existence d'eld'élémentsrillaires linelinéairesracellulaires chez le Postia placenta, l'agent de la pourriture brune du bois, est raise en evidence. Ces elements apparaissent comme des composants structuraux de la gaine hyphale et ressem- blent davantage aux mycofibrilles qu'aux fibrilles matricielles (fimbriae) des champignons. Les mycofibrilles sont assoasso-ciées surfaces des hyphes et paraissent deridérivéesces derndernièresrsqu'elles sont cultcultivées des substrats ligneux ou mememême substrats inertes, tels des lamelles de verre et des filtres de polycarbonate. Ces structures extracellulaires ont un diamdiamètreinal de 10 is 50 nm et une longueur pouvant atteindre 25 p.m. La conclusion degadégagée que ces mycofibrilles sont des extensions linelinéairesucturales de la paroi cellulaire des hyphes. La fonction precise des mycofibrilles dans le processus evolévolutifla pourriture brune des ligneux reste a elucélucider Mots clesclésostia placenta, mycofibrilles, fimbriae fongiques, gaine hyphale, microscopic elecélectronique [Traduit par la redaction]

Introduction mycofibrils of the hyphal sheath, using gold-labeled The role of the extracellular hyphal sheath in the wood- monoclonal antibodies. decay process by brown- and white-rot fungi has received Other research groups have made significant little attention until the past decade. Numerous reports contributions to the understanding of the extracellular indicate that hyphal sheaths are common ultrastructural hyphal sheath. In a series of papers, Foisner et al. (1985a, features of many groups of fungi (Bonfante-Fasolo et al. 1985b) and Messner et al. (1987) described extracellular 1987; Evans et al. 1981; Nilsson et al. 1989). Ultrastructural trilamellar membranous structures of brown- and white-rot studies on the brown-rot fungus Postia placenta (Fr.) M. wood-decay fungi, using transmission electron microscopy Lars. et Lomb. have revealed an extensive structure that (TEM). Foisner et al. (1985b) isolated these structures from covers and obscures the S3 layer of the wood cell wall during the white-rot fungus Sporotrichum pulverulentum Novobr. decay (Green et al. 1989, 1991b; Highley and Murmanis (Phanerochaete chrysosporium Burds. in Burds. et Eslyn), 1985; Murmanis and Highley 1987; Palmer et al. 1983). The and partial characterization revealed nearly equal amounts structure of the sheath is complex and includes a of carbohydrate, protein, and lipid, covered or embedded in preponderance of cross-linked ß-1,3-1,6-glucan and myco- an excess of nonstructural glucan. The authors conjectured fibrils, a term coined by Leise and Schmid (1962, 1963) for that these structures may contribute to the formation and the fibrillar structures associated with hyphal walls of transport of extracellular wood-decay enzymes. Chaetomium globosum Kunze, Polyporus versicolor L. : Fr., Poon and Day (1974, 1975) and Gardiner and Day (1985, and Trichoderma viride Pers. : Fr. Ruel et al. (1990), 1988) described fungal fimbriae of hyphal cell walls of Dickerson and Baker (1979), and Eriksson (1990) noted the numerous ascomycete and basidiomycete yeasts, including potential importance that extracellular glucan may have in the smut fungus Ustilago violacea Fukl., using negative- the decay process, because the sheath bridges the space staining and shadow-casting techniques with TEM. Gardiner between fungal hyphae and substrate and surrounds the and Day (1988), using a fimbrial antibody derived from U. hyphae. Green et al. (1991a) recently provided evidence for violacea and fluorescence techniques, also described a an association of extracellular polysaccharidases with partial cross-antigenic reaction to numerous filamentous fungi, including the white-rot fungus Trametes versicolor (L. : Fr.) Pil. They suggested that fimbriae permit fungi to The use of trade or firm names in this article does not imply 30 µm endorsement by the U.S. Department of Agriculture of any product or contact and interact with substrates as far away as service. from the hyphae. Poon and Day (1975) also showed that 2The Forest Products Laboratory is maintained in cooperation with individual fungal fimbriae are generally too small to be the University of Wisconsin. resolved clearly by TEM unless negatively stained, but Pnnted in Canada / Imprime au Canada 906 CAN. J. MICROBIOL. VOL. 38, 1992

FIG. 1. Transmission electron micrograph of cross section of southern yellow pine (Pinus sp.) decayed by the brown-rot fungus P. placenta. The fibrillar sheath (arrowheads) appears to extend from the hypha (H) and accumulate at the surface of the S3 (x 5000; scale bar represents 2 µm; glutaraldehyde - osmium tetroxide fixation). ML, middle lamella. FIGS. 2-6. Scanning electron micrographs of hyphae and fibrillar sheath structures of P. placenta on inert substrates. Fig. 2. Membranous-like sheath structure associated with LARSEN AND GREEN 111 907

thicker fibrils emanating from the surface of the cell wall of U. in a 6.5- to 7.5-nm-thick Au layer. Specimens were examined with violacea, which appear to be cabled fimbriae, are frequently either a Hitachi S-530 scanning electron microscope at an acceler- observed. ating voltage of 25 kV and working distances varying between 5 Benhamou and Ouellette (1987) demonstrated by TEM an and 10 mm or a Hitachi S-4000 field emission scanning electron extracellular fibrillar sheath of Asocalyx abietina (Lagerb.) microscope at an accelerating voltage of 5 kV and working distance of 5 mm. Schl.-Bern., bordered sometimes by a pellicle-like structure. Also, Benhamou et al. (1986) confirmed antigenic cross- Transmission electron microscopy reactivity between the polyclonal antifimbrial antiserum, Western hemlock (Tsuga heterophylla (Raf.) Sarg.) sawdust derived from U. violacea by Gardiner and Day (1985), and was degraded by P. placenta (isolate Mad-698), using the soil- the fibrillar sheath of A. abietina. block procedure (six replications) (ASTM 1971). After 4 weeks, Our observations on Postia placenta growing on inert the degraded sawdust was removed from bottles and fixed in a formaldehydeglutaraldehyde mixture in 0.1 M phosphate buffer, substrates or wood provide further evidence for the existence pH 7.2, postfixed in 1.33% 0504 in sym-collidine buffer, pH 7.2, of extracellular fibrillar sheath structures and further clarify and stained with 1.5% uranyl acetate. Samples were dehydrated in the spatial relationships of these structures to hyphal cell a graduated acetone series, followed by propylene oxide, and walls and associated substrates. Based on available evidence, embedded in Araldite - Epon - dodecenyl succinic anhydride we prefer to use the term mycofibril for these structures (1:1:3). Polymerization was carried out at 80°C, and micrographs rather than fungal fimbriae. were taken with a RCA EMU 3G electron microscope at an accelerating voltage of 50 kV.

Materials and methods Results Culture parameters The results of our investigations, which were designed to Postia placenta (Fr.) M. Lars. et Lomb. (isolate MAD-698) was inoculated onto 90-mm polycarbonate plastic Petri plates contain- demonstrate the presence and distribution of extracellular ing 2% malt and 1.5% agar (w/v) and incubated at 24°C. Four fibrillar structures, are presented in Figs. 1 to 11. sterile cover slips (No. 2) were then placed 10 mm from the point Variability associated with visualization of mycofibrils of inoculation. In one series of experiments, microgram quantities appeared to be minimal, and that which was observed of xylan were placed on four separate cover slips. After the fungus appeared as a result of the differing ages of various parts of had overgrown approximately two-thirds of the cover slips, the the mycelial mats or differing amounts of extracellular fungus was removed and processed through various protocols of glucan throughout mycelial mats. Mycofibrils were visible fixation and dehydration (described later). Four sterile poly- using various preparative protocols. The structural carbonate filters (pore size 0.2 µm, Nucleopore Inc.) were also relationships among hyphae of P. placenta, mycofibrillar used as inert substrates in some experiments. structures within the tracheid lumen of T. heterophylla, and On woody substrates, P. placenta isolate MAD-698 was S3 surface of the tracheid cell wall are presented in Fig. 1. inoculated onto 12 southern yellow pine (Pinus sp.) wood blocks (8 x 8 x 4 mm), using the ASTM soil-block procedure (ASTM The fibrillar elements appear to provide a continuum 1971), and allowed to incubate at 24°C for 10 days. between the hypha and wood cell wall and to aggregate in larger numbers on the surface of the S3. The hyphal cell Scanning electron microscopy (SEM) wall is electron transparent and possesses a barely visible Individual specimens were removed and processed through various fringe of fibrillar material. protocols of fixation and dehydration. Cryofixed and lyophilized specimens on both inert Fixation substrates (Fig. 2, glass cover slip) and southern yellow pine Half of the prepared samples was exposed to each of two fixa- (Figs. 9 and 10) are comparable. Membranous structures are tion schedules: (i) cryofixation by rapid quenching in precooled present in all three images. In Fig. 2, these membranous (l.4 kPa, -210°C) liquid nitrogen, followed by lyophilization elements cover and extend among numerous hyphae. The without chemical fixation, and (ii) l% ruthenium red in a solution effects of dehydration are evidenced by tears, holes, and con- of 1% glutaraldehyde - 1 % CaCl2 (w/v) for 12 h. In a preliminary labeling experiment using glass cover slips, samples tractions. Close examination reveals the presence of linear were exposed to 30-nm colloidal gold labeled rabbit antixylanase fibrillar elements intimately associated with these mem- polyclonal antibodies (PAbs), rinsed with 1% sodium acetate, branous structures. In Fig. 9, mycofibrillar structures are not and air dried. readily seen embedded in membranous elements extending from the hypha to the surface of the S3, but the surface of the Dehydration hypha is notably striated and ridged, suggesting mycofibrillar Samples were either freeze dried or air dried. For freeze drying, structures are present. Figure 10 depicts the interface between cryofixed decayed pine blocks or hyphae on glass cover slips were transferred to a precooled cryovessel and lyophilized overnight at - a membranous element and S3 surface, and entangled linear 55°C for 12 h. fibrillar elements are clearly visible at this interface. The Specimens (wood and glass cover slips) were coated with gold identity of these elements away from this interface cannot be (Au) in a Polaron sputter coater for approximately 22 s, resulting resolved, or these elements are absent. hyphae (H) on glass with xylan. Note fibrillar and (or) long rod-like components (arrowheads) embedded in a membranous or pellicle-like structure. ( x 10 000; scale bar represents 1 µm; slushed liquid N2, lyophilized). Fig. 3. Hyphae (H) on polycarbonate filter. Note dense fibrillar (arrowhead) nature of surface of hyphal cell wall. Many fibrils appear to branch and coalesce, caused by cabling of individual mycofibrils ( x 10 000; scale bar represents 1 µm; air dried only). Fig. 4. Hypha (H) on glass and associated mycofibrillar structures appear to be fragmented (arrowheads) both on glass and hypha; 30-nm gold bead PAbs to hemicellulase are also present ( x 8000; scale bar represents 1 µm; acetate buffer, air dried). Fig. 5. Field emission SEM of mycofibrillar structures (arrowheads) on polycarbonate filter. Note that the dimensions of fibrillar structures vary from about 10 to 50 nm, caused by roping and cabling of individual mycofibrils ( x 15 000; scale bar represents 1 µm; acetate buffer, air dried). Fig. 6. Hyphae (H) on glass treated with the detergent Triton X-100 ( x 3000; scale bar represents 5 µm; air dried). 908 CAN. I. MICROBIOL. VOL. 38 1992

nos. 7-11. Scanning electron micrographs of P. placenta on glass and wood of southern yellow pine (Pinus sp.). Fig. 7. Hyphae (H) on glass with mycofibrils (arrowheads) associated with hyphal cell wall. Note branching and coalescing of cabled mycofibrils ( x 20 000; scale bar represents 1µm; acetate buffer, air dried). Fig. 8. Hypha (H) on S3 layer of wood cell wall. Mycofibrils (arrowheads) are seen to extend from striated hyphal surface to S3 surface ( x 23 000; scale bar represents 0.5 µm; ruthenium red - glutaraldehyde CaCl2; air dried). Fig. 9. Fibrillar and membranous sheath structure on hyphal (H) surface and extending from hypha to S3. Note similarity of this sheath structure on wood to that depicted in Fig. 2 on glass slide ( x 15 0&3; scale bar represents 1 µm; slushed liquid N2, lyophilized). Fig. 10. Visible edge of membranous sheath structure (seen in Fig. 9) showing cabled mycofibrils (arrow) on S3 (x 30 000; scale bar represents 0.5 µm; slushed liquid N2, lyophilized). Fig. 11. Hypha (H) on S3 surface of Pinus sp. Mycofibrillar structures (arrowheads) emerge systematically from the surface of hyphal cell wall onto the S3. The identity of mycofibrils is not readily apparent on surface of S3 (x 40 000; scale bar represents 0.5 pm; ruthenium red - glutaraldehyde - CaCl2, air dried).

The surface of the S3 appears occluded by the hyphal sion scanning electron micrograph (Fig. 5) clearly resolves sheath. linear structures (mycofibrils), 20-25 µm long, that are Mycofibrils were clearly resolved following acetate buffer noticeably of smaller diameter than the associated 30-nm washing of hyphae grown on inert substrates (Figs. 4 and 7, gold beads. Although partial removal of the sheath matrix glass cover slips; Fig. 5, polycarbonate filters) with only with buffers is demonstrated in Figs. 4 and 7, complete minor differences apparent. Treatment with phosphate (con- removal of sheath from both hyphae and glass cover slips taining gold-labeled PAbs) and acetate buffers removed was accomplished with the detergent Triton-X 100 (Fig. 6). excess hyphal sheath matrix (water-soluble glucan), leaving Mycofibrils were readily demonstrated on the surfaces of residual and more clearly visible fragmented mycofibrillar hyphae on polycarbonate filters through simple air drying elements that would normally be obscured. The field emis- (Fig. 3). Dimensional variability of mycofibrils is readily LARSEN AND GREEN III 909 apparent in our micrographs, with diameters ranging from proteases was Botrytis cinerea Pers. (teleomorph: Sclerotinia 10 to 50 nm, and is probably due to the fusion and separa- fuckeliana (deBary) Fckl.). Furthermore, Gardiner and Day tion of individual mycofibrils and associated glucan. In (1988) documented the presence of fimbriae on outer cell Figs. 8 and 11, the edges of hyphae juxtaposed to the S3 wall surfaces of Trametes versicolor, using PAbs. This was surface appear systematically striated, with each stria giv- presumably done knowing that washed mycelia were free of ing rise to an individual mycofibril. carbohydrate or that their PAbs were not produced in response to carbohydrate moieties in the original fimbrial antigen. However, Xu and Day (1990) reported the presence Discussion of carbohydrate in fimbrial structure of two Ustilago species. Fibrillar extracellular structures associated with cell walls Thus, the positive immunofluorescence test reported by and hyphal sheath of fungi have been observed by numerous Gardiner and Day (1988) for Trametes versicolor may be a investigators. Liese and Schmid (1962) and Gardiner and function of cross-reactivity between a fimbrial carbohydrate Day (1988) reported linear structures for several filamentous antibody and carbohydrate moiety in the extra-cellular fungi. Poon and Day (1974) reported long, fine hairs hyphal sheath of Trametes versicolor. associated with the walls of yeast-like sporidial cells of U. An alternative interpretation of linear sheath structures is violaceae and compared the hairs to bacterial fimbriae or that they are ordered ß-1,3-glucan and not proteinaceous, as pili. They characterized these hairs as proteinaceous proposed by Gardiner and Day (1988). Wang and Bartnicki- filaments of polymerized 74-kDa protein subunits, 6-7 nm in Barcia (1976) synthesized ß-1,3-glucan mycofibrils (about diameter, up to 10 µm long, and apparently originating on 25-50 nm in diameter, our measurements) in vitro utilizing the surface of the plasma membrane and traversing the cell ß-glucan synthase in cell-free extracts of Phytopthora wall, and they called them fungal fimbriae. Similar struc- cinnamomi Rands. Their micrographs appear strikingly tures were observed on yeast cells in such genera as similar to our Figs. 4 and 5 and to many micrographs of Lipomyces, Nadsonia, and Saccharomyces (Poon and Day Green et al. (1991a). Therefore, if the fibrillar structures 1974). The function of these structures was also the subject demonstrated here prove to be glucan, then the data of speculation and included processes associated with published by Ruel et al. (1991), Eriksson (1990), and conjugation, substrate recognition, and other roles in Dickerson and Baker (1979) take on added significance; pathogenesis, adhesion, self- or nonself-recognition phe- they implicate the ß-1,3-1,6-d-glucan of hyphal sheaths of nomena, and nutrition (Gardiner et al. 1982). wood-decay fungi as the medium to which enzymes are Gardiner et al. (1982) observed that most ascomycetous bound and as the junction or interface between hyphal walls and deuteromycetous yeasts investigated possessed shorter and substrates. fimbriae than did most basidiomycetous yeast forms. The Artifactual considerations were foremost in our attempt at fimbriae of most filamentous fungi investigated were also defining the presence and developmental extent of usually longer than ascomycete and deuteromycete yeast mycofibrils. As Read et al. (1983) stated regarding their forms (Gardiner and Day 1988). However, fimbriae were study on Sordaria humicola (Fckl.) Wint., all methods that not fully characterized for this fungus but were presumed they used in specimen preparation produced artifacts. In pre- to be present based on immunofluorescence and light liminary experiments, Green et al. (1991b) provided data on microscopy tests using antifimbrial antiserum (PAbs). the effects of varied preparative procedures for SEM on Gardiner and Day (1985) provided an extensive list of several wood-rotting fungi. Those methods, which detailed chemical treatments of fimbriae of U. violacea and, by partial or selective fixation and preservaton, yielded agglutination tests and electron microscopy, determined the informative images that provided additional insight into the presence or absence of fimbriae. Our investigations on the physical nature of extrahyphal sheath structures. We present chemical nature of the mycofibrils of P. placenta paralleled similar modifications of structure, and mycofibrils were those of Gardiner and Day (1985). In preliminary experi- revealed under a variety of protocols. Thus, we conclude ments, we were unable to depolymerize mycofibrils of P. that the presence of mycofibrils is predictable and their placenta, using commercial pronase or laminarinase (M.J. visualization is a function of partial removal of glucan and Larsen and F. Green III, unpublished results). We cannot fixation of remaining material. Close examination reveals exclude the possibility that a complex extracellular glucan linear structures that frequently branch and coalesce and are matrix may block solubilization of fimbrial protein or 10-50 nm in diameter and up to 20-25 µm long when com- mycofibrillar glucan (carbohydrate). Leise and Schmid pared with companion 30-nm gold beads. The cabling of (1962, 1963) did not provide any data on the chemical nature fimbriae of fungi has been noted by Poon and Day (1975) in of mycofibrils, but did provide illustrations and dimensions U. violacea and of pili in Neissaria gonorrhea by Todd of 20-50 nm in diameter and 20 µm long. We also utilized (1986) and Todd et al. (1984). Todd (1986) and Todd et al. the nonionic detergent Triton-X 100, which disrupts hydro- (1984) noted that linear surface structures of N. gonorrhea phobic bonds, to completely remove mycofibrils. Although appear quite differently depending on the method of speci- this is coincident with the observation that large numbers of men processing. Cabling of pili was evident from branching hydrophobic interactions are known to occur in bacterial pili and rejoining when critical point dried, but the pili main- (Folkhard et al. 1979), which appear ultrastructurally similar tained a recognizable level of integrity with less cabling on to fungal fimbriae and mycofibrils, we cannot conclude that the surface of the cell under conditions prescribed for freeze mycofibrils are proteinaceous. fracturing and etching. Existing evidence in the literature suggests that fibrillar We have enhanced visualization of mycofibrils by allow- extensions of fungal cell walls, inclusive of truly filamentous ing P. placenta hyphae to encroach from an agar surface fungi, are proteinaceous. However, the only filamentous onto the surface of inert substrates. However, the arrange- fungus that Gardiner and Day (1988) "defimbriated" with ment and form of mycofibrils may be altered by the 910 CAN. J. MICROBIOL. VOL. 38, 1992 chemically inert nature of such substrates. In addition, since Foisner, R., Messner, K., and Roehr, M. 1985a. Wood decay by there is no S3 fiber angle, some dissimilarity should be basidiomycetes: extracellular tripartite membranous structures. expected. The mycofibrils appear to be individually shorter Trans. Br. Mycol. Soc. 85: 257-266. on inert substrates and perhaps "confused," as expressed by Foisner, R., Messner, K., Stachelberger, H., and Roehr, M. 1985b. Isolation and characterization of extracellular three-lamellar shorter lengths and lack of spatial organization. structures of Sporotrichum pulverulentum. J. Ultrastruct. Res. We obtained no physical evidence in this study that pro- 92: 36-46. vides a precise explanation for the ontogeny of these struc- Folkhard, W., Leonard, K.R., Malsey, S., and Marvin, D.A. 1979. tures. The conceptualization of cell wall ontogeny and struc- X-ray diffraction and electron microscope studies on the structure provided by Bartnicki-Garcia (1987), Farkas (1985), ture of bacterial F pili. J. Mol. Biol. 130: 145-160. Garraway and Evans (1984), and Wessels and Sietsma Gardiner, R.B., and Day, A.W. 1985. Fungal fimbriae. IV. Com- (1981) does not account for these structures. Whether they position and properties of fimbriae from Ustilago violacea. are produced basipetally, e.g., synthesized in the cytoplasm Exp. Mycol. 9: 344-350. or periplasmic space (Ottow 1975) and extruded through the Gardiner, R.B., and Day, A.W. 1988. Surface proteinaceous fibrils cell wall, or self-assembled outside the cell wall in an (fimbriae) on filamentous fungi. Can. J. Bot. 66: 2474-2484. acropetal process (see Kushner 1969) is unknown. Never- Gardiner, R.B., Podgorski, C., and Day, A.W. 1982. Serological studies on the fimbriae of yeasts and yeast-like species. Bot. theless, the presence of fibrillar structures and their small Gaz. (Chicago), 143: 534-541. size raise numerous hypotheses for their role in the decay Garraway, M.O., and Evans, R.C. 1984. Fungal nutrition and process. physiology. John Wiley and Sons, New York. We have provided evidence for the existence of fibrillar Green, F., Larsen, M.J., Murmanis, L.L., and Highley, T.L. 1989. extensions of the cell wall of P. placenta and have shown Proposed model for the penetration and decay of wood by the that these structures are ultrastructurally similar to other hyphal sheath of the brown-rot fungus Postia placenta. Inter- extracellular hyphal structures, i.e., fungal fimbriae. How- national Research Group on Wood Preservation, Document ever, fimbriae are characterized as proteinaceous, with a very IRG/WP/1391. small carbohydrate component. Furthermore, the extensive Green, F., Clausen, C.A., Larsen, M.J., and Highley, T.L. 1991a. Immunoscanning electron microscopic localization of extrahyphal sheath of the brown-rot fungus P. placenta is com- cellular polysaccharidases within the fibrillar sheath of the posed primarily of extracellular polymeric glucose that is brown-rot fungus Postia placenta. International Research readily observed by both TEM and SEM. Existing evidence Group on Wood Preservation, Document IRG/WP/1497. suggests that the extracellular fibrillar structures described Green, F., Larsen, M.J., and Highley, T.L. 1991b. Ultrastructural here are mycofibrils and are composed of linear glucan. morphology of the hyphal sheath of wood-decay fungi modified However, we have not excluded the possibility that a prote- by preparation for scanning electron microscopy. In inaceous component may be present. Biodeterioration research. 3. General biodeterioration, degradation, mycotoxins, biotoxins, and wood decay. Proceedings of the 3rd Pan American Biodeterioration Society Meeting, 1989, Washington, D.C. Edited by C.E. O'Rear and ASTM. 1971. Standard method for accelerated laboratory test of G.C. Llewellyn. Plenum Press, New York. natural decay resistance of woods. ASTM D 2017. American Highley, T.L., and Murmanis, L.L. 1985. Micromorphology of Society for Testing and Materials, Philadelphia, Pa. degradation in western hemlock and sweetgum by the brown- Bartnicki-Garcia, S. 1987. The cell wall: a crucial structure in rot fungus Poria placenta. Holzforschung, 39: 73-74. fungal evolution. In Evolutionary biology of the fungi. Edited Kushner, D.J. 1969. Self assembly in biological studies. by D.D.M. Rayner, C.M. Brasier, and D. Moore. Cambridge Bacteriol. Rev. 33: 302-345. University Press, Cambridge. pp. 387-403. Leise, W., and Schmid, R. 1962. Elektronmicroscopische Unter- Benhamou, N., and Ouellette, G.B. 1987. Ultrastructural charac- suchungen fiber den Abbau des Holzes durch Pilze. Angew. terization of an extracellular fibrillar sheath on cells of Bot. 36: 291-298. Ascocalyx abietina, the scleroderris canker agent of conifers. Leise, W., and Schmid, R. 1963. Fibrillare Strukturen an dem Can. J. Bot. 65: 154-167. Hyphen Holz zerstorender Pilze. Naturwissenschaften, 50: Benhamou, N., Ouellette, G.B., Gardiner, R.B., and Day, A.W. 102-103. 1986. Immunocytochemical localization of antigen binding Messner, K., Srebotnik, E., Erther, G., et al. 1987. Cell wall systems, sites in the cell surface of two ascomycete fungi using extracellular membranous structures and ligninase of wood-rotting antibodies produced against fimbriae from Ustilago violacea fungi. In Lignin Enzymic and Microbial Degradation, and Rhodotorula rubra. Can. J. Microbiol. 32: 871-883. International Symposium, Paris. Edited by E. Odier. Institut Bonfante-Fasolo, P., Perotto, S., Testa, B., and Faccio, A. 1987. national de la recherche agronomique, Paris. pp. 243-249. Ultrastructural localization of cell surface sugar residues in Murmanis, L.L., and Highley, T.L. 1987. Cytochemical localiza- ericoid mycorrhizal fungi by gold-labeled lectins. Protoplasma, tion of cellulases in decayed and nondecayed wood. Wood Sci. 139: 25-35. Technol. 21: 101-109. Dickerson, A.G., and Baker, R.C.F. 1979. The binding of Nilsson, T., Daniel, G., Kirk, T.K., and Obst, J.R. 1989. Chemistry enzymes to fungal ß-glucans. J. Gen. Microbiol. 112: 67-75. and microscopy of wood decay by some higher ascomycetes. Eriksson, K.-E. 1990. Microbial delignification-basics, potentials, Holzforschung, 48: 11-18. and applications. In Biochemistry and genetics of cellulose Ottow, J.G.C. 1975. Ecology, physiology, and genetics of degradation. Edited by J.-P. Aubert, P. Bequin, and J. Millet. fimbriae and pili. Annu. Rev. Microbiol. 29: 79-108. Academic Press, London. pp. 285-302. Palmer, J.G., Murmanis, L.L., and Highley, T.L. 1983. Visualiza- Evans, R.C., Stempen, H., and Stewart, S.J. 1981. Development tion of hyphal sheath in wood-decay hymenomycetes. II. of hyphal sheaths in Bipolaris maydis race T. Can. J. Bot. 59: White rotters. Mycologia, 75: 1005-1010. 453-459. Poon, N.H., and Day, A.W. 1974. "Fimbriae" in the fungus Farkas, V. 1985. The fungal cell wall. In Fungal protoplasts. Ustilago violacea. Nature (London), 250: 648-649. Edited by J.F. Peberdy and L. Ferenzy. Marcel Dekker, New Poon, N.H., and Day, A.W. 1975. Fungal fimbriae. I. Structure, York. pp. 3-29. origin, and synthesis. Can. J. Microbiol. 21: 537-546. LARSEN AND GREEN III 911 Read, N.D., Porter, R., and Beckett, A. 1983. A comparison of niques for microorganisms. Edited by H.C. Aldrich and W.J. preparative techniques for the examination of the external mor- Todd. Plenum Press, New York. pp. 87-100. phology of fungal material with the scanning electron Todd, W.J., Wray, G.P., and Hitchcock, P.J. 1984. Arrangement microscope. Can. J. Bot. 61: 2059-2078. of pili in colonies of Neisseria gonorrhea. J. Bacteriol. 159: Ruel, K., Odier, E., and Joseleau, J.-P. 1990. Immunocytochemical 312-320. observations of fungal enzymes during degradation of wood cells Wang, M.C., and Bartnicki-Garcia, S. 1976. Synthesis of ß-1,3- by Phanerochaete chrysosporium (strain K-3). In Biotechnology glucan mycofibrils by a cell-free extract from Phytophthora in pulp and paper manufacturing. Edited by T.K. Kirk and H.M. cinnamomi. Arch. Biochem. Biophys. 175: 351-354. Chang. pp. 83-97. Wessels, J.G.H., and Sietsma, J.H. 1981. Fungal cell walls: a Ruel, K., Odier, E., and Joseleau, J.-P. 1991. Involvement of an survey. Edited by W. Tanner and F. Loewus. Springer-Verlag, extracellular glucan sheath during degradation of Populus wood Heidelberg. pp. 352-394. by Phanerochaete chrysosporium. Appl. Environ. Microbiol. Xu, J., and Day, A.W. 1990. A comparison of fimbrial proteins 57: 374-384. in Ustilago violacea and Ustilago maydis. Abstracts of the 4th Todd, W.J. 1986. Effects of specimen preparation on the apparent International Mycology Congress. Botanical Institute, Uni- ultrastructure of microorganisms. In Ultrastructure tech- versity of Regensburg, Regensburg, Germany. p. 101.

Errata

Page 909, column 1, under Disscussion, paragraph 2 should read:

Gardiner et al. (1982) observed that most ascomycetous and deuteromycetous yeasts investigated possessed shorter fimbriae than did most basidiomycetous yeast forms. The fimbriae of most filamentous fungi investigated were also usually longer than ascomycete and deuteromycete yeast forms (Gardiner and Day 1988). Of interest here is that Gardiner and Day (1988) extended their observations to the filamentous white-rot fungus Trametes versicolor. However, fimbriae were not fully characterized for this fungus but were presumed to be present based on immunofluorescence and light microscopy tests using antifimbrial antiserum (PAbs).

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