VISUALIZATION OF HYPHAL SHEATH IN WOOD-DECAY HYMENOMYCETES. I. BROWN-ROTTERS

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1 Mycologia, 75(6), 1983, pp , by The New York Botanical Garden, Bronx, NY VISUALIZATION OF HYPHAL SHEATH IN WOOD-DECAY HYMENOMYCETES. I. BROWN-ROTTERS J. G. PALMER Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia L. MURMANIS AND T. L. HIGHLEY ForestProductsLaboratory, 1 P.O. Box 5130, Madison, Wisconsin ABSTRACT Several brown-rot fungi were studied with respect to formation of hyphal sheaths in axenic culture. The 12 fungi examined showed hyphal sheaths when grown on each type of solid substrate; in isolates grown in liquid media, sheaths were poorly demonstrated. Sheath structure vaned among and within species, but a fine fibrillar texture was common to all. Sheath of Poria carbonica might also be granular or composed of electron dense granules and rods. A sheath often contained cellular contents released from hyphae autolyzing within the sheath. Occasionally protoplasm could be seen escaping through openings in cell walls. Necrotic hyphae lacked sheaths. Key Words: hyphal sheath, Hymenomycetes, wood-decay fungi, brown-rotters, autolysis. An extracellular matrix or hyphal sheath is thought to be the site and the pool of fungal enzymes, and sheaths have been reported in many recent studies (Bullock et al., 1980; Dowsett, 1981; Evans et al., 1981; Pechak and Crang, 1977; Van der Valk et al., 1977). Some fungi may have a hyphal sheath while others do not. For example, Green et al. (1980) reported the work of Crossley who found sheaths in white-rot and soft-rot fungi but not in a brown-rot fungus. However, Highley et al. (1983) and others, e.g., Jutte and Sachs (1976), have demonstrated a sheath in the brown-rot fungus Poria placenta. Consequently, we decided to examine a number of brown-rot fungi that are important in decay of trees and wood products for development of hyphal sheath in axenic culture. Occurrences, forms, and inclusions of hyphal sheaths are reported for brown-rot fungi. We hope that the results will support speculation on the function of hyphal sheath in the wood decaying mechanism. White rotters will be considered in a separate report. MATERIALS AND METHODS Cultures of 12 North American brown-rot fungi were obtained from the culture collections of the Center for Forest Mycology Research at the Forest Products Laboratory. Six were isolated from wood in use: Coniophora arida (Fr.) Karst., MS-42-R; C. puteana (Schum. : Fr.) Karst., MAD-5 15; Gloeophyllum trabeum (Pers. : Fr.) Murr., MAD-6 17; Lentinus lepideus Fr., MAD-534; Poria placenta (Fr.) Cke., MAD-698; and Serpula incrassata (Berk. et Curt.) Donk, MAD-563. Six were isolated from forest trees, logs, or slash: Fomitopsis meliae (Underw.) Gilbn., FP Sp; Laetiporus sulphureus (Bull. : Fr.) Bond. et Sing., Sh-27- R; Leucogyrophana arizonica Ginns, RLG-9902-Sp.; L. olivascens (Berk. et Curt.) 1 Maintained at Madison, Wisconsin, in cooperation with the University of Wisconsin. 995 Purchased by USDA, Forest Products Laboratory, for Official Use

2 996 MYCOLOGIA

3 PALMER ET AL.: HYPHAL SHEATH I 997 Ginns et Weresub, FP Sp.; Poria carbonica Overh., FP sp.; and Serpula himantioides (Fr.) Karst., FP R. Mycelia were grown on two per cent malt agar in glass Petri plates on some surfaces of which had been placed sterile cellophane disks, glass cover slips, and stainless steel squares; on sterile cellulose (cotton, Hercules Type A-600), and in liquid culture with 1% glucose (Highley, 1973). Procedures for growth on cotton fibers were those described for P. placenta (Highley et al., 1983). Cellophane disks and cover slips supporting mycelia were trimmed along edges with a razor blade, peeled from the agar surface, and placed on glass slides for reflectance light microscopy (RLM). Samples were negatively stained with 40% phosphotungstic acid (PTA) for 10 min, rinsed in distilled water, washed in 50% ethanol, air-dried, and examined in RLM. Samples for transmission electron microscopy (TEM) and OsO 4 vapor-fixed samples for scanning electron microscopy (SEM) were prepared as previously described (Highley et al., 1983). RESULTS Every isolate of brown-rot fungi examined had a hyphal sheath on each type of solid substrate including agar. The sheath appeared to be fragile and was not always well preserved but was always evident. Its texture differed among species and within isolates. The fine, fibrillar texture of sheath was common in all species studied. Sometimes the sheath contained vesicles and other protoplasmic inclusions that had come from cells autolyzing within the sheath. This phenomenon was observed in all the brown-rot isolates and was reported previously in P. placenta (Highley et al., 1983). For RLM, negative staining with PTA was the easiest technique and provided satisfactory results. Poria placenta (FIG. 1) and P. carbonica (FIG. 2) clearly showed distinct sheaths around the hyphal tips in contact with a solid substrate. In addition, the sheath extended along the hyphae, and was especially noticeable where they touched the solid substrate. In our samples, hyphae in the age range from 2 to 8 wk invariably had a mixed population of young, mature, and necrotic cells. Young cells, mature cells, conidia, and chlamydospores had sheaths; necrotic cells did not. The sheath was also easily demonstrated by conventional TEM techniques. Poria placenta, grown on cotton (FIG. 3), exhibited a characteristic fibrillar sheath that showed a well delineated border. The same sheath texture was seen in F. meliae grown on cotton (FIG. 4). Coniophora puteana illustrated the aforementioned variability in sheath texture within a species (FIG. 5-fine, fibrillar; FIG. 6 -fibrillar with included vesicles (arrow); FIG. 7 -fine, granular). Coniophora puteana had been reported to have no hyphal sheath by Crossley (reported by Green et al., 1980), but in our study it had a sheath on whatever solid substrate it was grown. Poria carbonica had a sheath that contained granules or short rods of extreme electron density (FIGS. 8, 9). A hypha shown at the top of FIG. 9 was autolyzing (arrow), and its protoplasmic contents were dispersed into the sheath. This type of sheath resembles a mucilaginous zone described by Olah and Reisinger (1974) in the imperfect fungus, Phialophora richardsiae, which is known to contain mel FIGS. 1, 2. Hyphal sheaths surrounding growing points and rest of hyphae. RLM. 1. Poria placenta, Poria carbonica, 220. FIGS. 3, 4. Fine fibrillar sheaths (Sh). TEM. 3. Poria placenta, 10, Fomitopsis meliae, 11,070.

4 998 MYCOLOGIA

5 PALMER ET AL.: HYPHAL SHEATH I 999 anin granules. We did not determine the chemical nature of the sheath or its contents in P. carbonica. FIGURE 10 showed many hyphae of Lentinus lepideus within a sheath delimited by a border (at left, arrows). The sheath also included released protoplasmic contents. Released protoplasmic contents often were seen without a connection to a sheath (FIGS. 11, 12). However, the many samples examined indicated that released cell contents generally lay within sheaths (FIGS. 9, 13). In FIG. 13 the cell organelle (MB) breaking apart within the sheath had structural features characteristic of microbodies. Microbodies were observed in all brown-rot isolates examined and often were seen to be released from cells through the cell wall (FIG. 14, arrow). The sheath of hyphae was either poorly preserved or formed from liquid culture (FIG. 15). Only its delimiting border (FIG. 15, arrow), enclosing hyphae with walls and cell remnants of autolysis, was retained. Here sheaths also were not found surrounding the cell contents released from autolyzing cells (FIGS. 16, 17). Scanning electron microscopy micrographs supported observations made with RLM and TEM. They showed sheath (arrows) around growing points and nonnecrotic hyphae, especially where the hyphae were appressed to a solid substrate (FIGS ). They showed no sheath around necrotic cells but often showed apparent release of protoplasmic contents, well organized or unorganized. DISCUSSION This study supported results of other investigators indicating that a hyphal sheath is a morphological feature of many fungi. Its presence in all 12 brown-rot fungi examined suggested that it may be a universal structural entity in this group. Sheath was found around the growing points and along the length of hyphae; it encircled conidia and chlamydospores, but was not present on necrotic hyphae. Evans et al. (1981) reported that in Bipolaris maydis race T sheath was present only on rapidly growing hyphae, such as tips and germ tubes. This further suggested that among species differences may exist with respect to the distribution patterns of sheath as well. The denser limiting border of sheath in our samples was not a true biological membrane but only a condensation of the sheath material. We disagreed with Crossley s contention that the brown-rot fungus, Coniophora puteana, lacks a sheath (see Green et al., 1980). Green et al. (1980) showed that wood degrading white-rot and soft-rot fungi bind cellulases while brown-rot fungi do not. The binding site is a 1,3-beta glucan that could be either a cell wall component or the extracellular mucilage, which, as shown by Crossley, was present in the white-rot fungus, Coriolus versicolor, and the soft-rot fungus, Chaetomium globosum, but not in the brown-rot fungus, Coniophora puteana. In any case, the lack of sheath cannot explain how the attack of brown-rot fungi upon lignocellulosic material differs from that of white-rot and soft-rot fungi. In the brown-rot fungi examined, the hyphal sheath enclosing released cell contents surrounded the hyphae or the cellulose fiber away from hyphae. This suggested that the sheath is a structure that houses and provides translocation of cellular contents, which in turn may contain a depolymerizing agent necessary for the initial attack on cellulose. The cell organelle directly responsible for necessary FIGS Sheaths (Sh) of Coniophora puteana. TEM. 5. Fine fibrillar sheath, 10, Sheath including vesicles (arrow), 12, Fine granular sheath, 18,000. FIGS. 8, 9. Sheaths (Sh) of Poria carbonica. TEM. 8. Densely fine-granulate, Densely fine-granulate with rods; autolyzing hypha at top (arrows), 18,000.

6 1000 MYCOLOGIA FIGS. 10, 11. Sheath (Sh) enclosing released protoplasmic contents. TEM. 10. Lentinus lepideus. Arrows point to delimiting border of sheath, 12, Coniophora arida. Fine fibrillar sheath at bottom and with released protoplasmic contents at top, 5600.

PALMER ET AL.: HYPHAL SHEATH I 1001 FIGS. 12-14. Protoplasmic contents released from hyphal cells. TEM. 12. Coniophora puteana, 12,000. 13.

7 PALMER ET AL.: HYPHAL SHEATH I 1001 FIGS Protoplasmic contents released from hyphal cells. TEM. 12. Coniophora puteana, 12, Poria placenta. Microbody (MB) breaking apart within sheath (Sh), 15, Laetiporus sulphureus Microbody (arrow) breaking through cell wall, 8200.

1002 MYCOLOGIA FIGS. 15-17. Poria placenta grown in liquid culture. TEM. 15. Delimiting border of sheath (arrow) enclosing wall-less cell contents and cells with walls, 15,600. 16.

8 1002 MYCOLOGIA FIGS Poria placenta grown in liquid culture. TEM. 15. Delimiting border of sheath (arrow) enclosing wall-less cell contents and cells with walls, 15, Autolyzing hypha with dispersal of protoplasmic contents in early stage, 14, Autolyzing hypha with dispersal of protoplasmic contents in advanced state, 10,800. hydrolyzing or oxidizing agents may be the microbody. This organelle is known to be associated with oxidative degradations, i.e., catalase and H 2 O 2 -producing oxidases (Choinski and Mullins, 1977; Fukui et al., 1975; Maxwell et al., 1977, and others). This would be in line with the cellulose degrading mechanism of brown-rot fungi involving a H 2 O 2 /Fe system as suggested by Koenigs (1974). Rounded or oval cell organelles with characteristics of microbodies were present in the fungi examined in this study. They were released from hyphae through cell walls and released contents, granular or membranous, into the sheath.

PALMER ET AL.: HYPHAL SHEATH I 1003 FIGS. 18-20. Sheaths (arrows) surrounding hyphae. SEM. 18. Sheath surrounding growing point of Poria placenta, 5000. 19.

9 PALMER ET AL.: HYPHAL SHEATH I 1003 FIGS Sheaths (arrows) surrounding hyphae. SEM. 18. Sheath surrounding growing point of Poria placenta, Distinct sheath around hyphae of Gloeophyllum trabeum appressed along glass surface becoming less recognizable around elevated cells, Sheath containing rods in Poria carbonica, Release of cellular contents from hyphae into sheaths may provide food supplies for growing hyphae. Sheaths are known to contain proteinaceous materials and polysaccharides (Bullock et al., 1980; Olah and Reisinger, 1974; Saito, 1974; Van der Valk et al., 1977), which could be used immediately or stored. Saito (1974) showed that the sheath (which he calls the outer fibrillar layer) rather than glycogen is utilized during germination of sclerotia of Sclerotinia sclerotium. For wood decaying fungi, the sheath s proteinaceous compounds could supply nitrogen, the quantity of which is very low in wood substrates. LITERATURE CITED Bullock, S., A. E. Ashford, and H. J. Willetts The structure and histochemistry of sclerotia of Sclerotinia minor Jagger. II. Histochemistry of extracellular substances and cytoplasmic reserves. Protoplasma 104: Choinski, J. S., and J. T. Mullins Ultrastructural and enzymatic evidence for the presence of microbodies in the fungus Achlya. Amer. J. Bot. 64: Dowsett, J. A Extracellular hyphal sheaths of Dactylaria brochophaga. Mycologia 73: Evans, R. C., H. Stempen, and S. J. Stewart Development of hyphal sheaths in Bipolaris maydis race T. Canad. J. Bot. 59: Fukui, S., S. Kawamoto, S. Yasuhara, and A. Tanaka Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody. Eur. J. Biochem. 59: Green, N. B., D. J. Dickinson, and J. F. Levy A biochemical explanation for the observed patterns of fungal decay in timber. The International Research Group of Wood Preservation. Working Group I. Document No. IRG/WP/1111.

10 1004 MYCOLOGIA Highley, T. L Influence of carbon source on cellulase activity of white-rot and brown-rot fungi. Wood and Fiber 5: , J. G. Palmer, and L. Murmanis Decomposition of cellulose by Poria placenta: light and electron microscopy study. Holzforschung 37: Jutte, S. M., and I. B. Sachs SEM observations of brown-rot fungus Poria placenta in normal and compression wood of Picea abies. Proceedings of the Workshop on Plant Science Application, Scanning Electron Microscopy, Part IV, pp Koenigs, J. W Hydrogen peroxide and iron: a proposed system for decomposition of wood by brown-rot Basidiomycetes. Wood and Fiber 6: Maxwell, D. P., V. N. Armentrout, and L. B. Graves, Jr Microbodies in plant pathogenic fungi. Annual Rev. Phytopathol. 15: Olah, G. M., and 0.Reisinger Etude ultrastructurale et cytochimique de l appareil sporifère chez Phialophora richardsiae. Canad. J. Bot. 52: Pechak, D. G., and R. E. Crang An analysis of Aureobasidium pullulans developmental stages by means of scanning electron microscopy. Mycologia 69: Saito, I Utilization of ß-glucans in germinating sclerotia of Sclerotinia sclerotium (Lib.) de Bary. Ann. Phytopathol. Soc. Japan 40: Van der Valk, P., R. Marchant, and J. G. H. Wessels Ultrastructural localization of polysaccharides in the wall and septum of the basidiomycete Schizophyllum commune. Exp. Mycol. 1: Accepted for publication May 22, 1983

Micromorphology of Degradation in Western Hemlock and Sweetgum by the White-Rot Fungus Coriolus versicolor

Vol. 41 (1987) No. 2 Micromorphology of Degradation in Western Hemlock and Sweetgum 67 Holzforschung 41 (1987) 67-71 Micromorphology of Degradation in Western Hemlock and Sweetgum by the White-Rot Fungus