Soft rot and multiple T-branching by the ... - Science Direct

Mycol. Res. 96 (1): 49-54 (1992)

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Soft rot and multiple T-branching by the basidiomycete Oudemansiella mucida

G. DANIELl,., J. VOLC 2 AND T. NILSSON l 1 2

Department of Forest Products, Swedish University of Agricultural SCIences, Box 7008, S-750 07, Uppsala, Sweden Institute for Microbiology, Czechoslovakian Academy of Sciences, Prague, Czechoslovakia

A characteristic type of hyphal multiple branching (T-branching) and cavity formation by the basidiomycete fungus Oudemansiella mucida in the secondary walls of pine wood is described. Hyphal branching and cavity formation is reminiscent of that caused by

soft-rot fungi (ascomycetes and fungi imperfecti), but differs in that penetration hyphae tend to branch repeatedly in the wood cell wall and that hyphal alignment with the cellulose microfibrillar axis of fibres is not consistent. Repeated hyphal branching within wood cell walls may aid in a more effective distribution of wood-degrading enzymes, and therefore in the degradation of lignified substrates by this ligninolytic fungus.

The term soft rot was first proposed by Savory (1954) to describe a type of wood decay where fungi produce characteristic cavities within the secondary walls of wood cells. This type of wood decay is normally considered representative of members of the ascomycetes and fungi imperfecti. In addition to cavity formation, hyphae may also cause erosion of wood cell walls at the cell lumen/wall interface. Cavity formation and erosion decay are generally referred to as Type I and Type 2 attacks respectively (Corbett, 1965). By contrast, basidiomycete fungi are normally considered to cause characteristic brown or white rots in the wood, and only a few species have been reported to cause cavity formation (Duncan, 1960; Nilsson & Daniel, 198&). Penetration of wood cell walls by basidiomycete fungi is generally restricted to production of 'borehole hyphae', which pass from one wood cell to another. The present report describes the ability of the basidiomycete fungus Oudemansiella mueida (Schrad. ex Fr.)Hohn. to cause characteristic cavity formation in pine wood following multiple T- or L-branching within secondary cell walls by fine-penetration bore hyphae. Multiple T-branching of penetration hyphae within wood cell walls is unusual, and to the author's knowledge has not been described previously during cavity formation by basidiomycete fungi. Multiple branching has been reported preViously for three microfungi (Hale, 1983), while Schacht (1863) and Bailey & Vestal (1937) also show illustrations which may depict a similar form of attack in material from the field.

MATERIAL AND METHODS Oudemansiella mueida (isolate 428) was obtained from the Czechoslovakian Academy of Science culture collection. The • Corresponding author.

fungus, which was originally isolated from a carpophore produced on beech wood (Fagus sylvatica L.) collected from Voznice, near Dobris, Bohemia, is routinely maintained on 2'5 % malt agar plates. It is acknowledged as an important pathogen of beech trees in Czechoslovakia. For light-microscopic studies and assessment of wooddegrading ability of hard and softwoods, O. mucida was grown together with small pre-weighed wood blocks (3'0 x 1.5 x 0'5 em) of either pine (Pinus sylvestris L.), birch (Betula verrucosa Ehr.) or beech (Fagus sylvatiea L.) placed in garden loam in 125 ml Erlenmeyer flasks. Flasks were sterilized by autoclaving and subsequently inoculated with 2 ml of an aqueous fungal mycelium suspension of O. mucida and incubated at 22°C at a r.h. of 70%. Weight losses were determined after periods ranging from 2 wk to 6 months after drying samples at 40° (Table I). For light microscopy, thin razor blade-cut transverse or longitudinal sections were removed from parallel degraded wood blocks and stained with either 1% w /v safranin (T.S.) or with aniline blue in lactophenol (L.S.). Sections were examined by either bright-field or polarized light illumination using a Leitz Orthoplan microscope. Additional degraded samples were observed using polarized light after prior delignification at 130° in triethylene glycol containing 0'05 % w / v toluenesulphonic-(4) acid (Burkart, 1966). For lignin and carbohydrate analysis, dried wood blocks were ground using a Wiley mill to pass a I mm screen and the dry matter determined. Samples of wood meal (ea 200 mg) were then mixed with 80% aq. ethanol (100 ml) in small tubes and treated in an ultrasonic bath at room temperature (4 x 15 min). Following extraction, the samples were filtered (glass filter No.2) and the solid residues gravimetrically determined and quantified. The residues were thereafter analysed for Klason lignin and neutral polysaccharides MYC 96

Multiple T-branching by Oudemansiella muczda

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G. Daniel,

J. Vole

and T. Nilsson

according to Theander & Westerlund (1986), using I-methylimidazole as the catalyst.

RESUL TS AND DISCUSSION Initial fungal colonization and decay of pine wood blocks occurred in ray parenchyma cells from where vegetative hyphae (ca 2·01..lm diam.) began to penetrate and ramify throughout the cell lumina of both early and latewood tracheids. The first sign of tracheid cell wall attack was the development of small penetration hyphae (Le. borehole hyphae) into wood secondary walls. These fine hyphae (ca O'3-D's I..Im diam.) either passed directly through the wood cell walls of both colonized and adjacent fibres to produce fine boreholes or branched within the secondary cell walls (S2 or Sl layers) themselves (Figs 1-4). In advanced stages of decay, these boreholes widened considerably and showed features typical of those produced by basidiomycete fungi (ef. Figs 3-4 and 8-9). Within secondary wood cell walls, the fine-penetration borehole hyphae frequently branched in a characteristic manner. Various stages of hyphal branching were observed in individual tracheids (Figs 1-4). Often branching resulted in both the penetration hyphae and newly formed branch turning sharply and growing perpendicularly in opposite directions, thus forming a characteristic hyphal T-branch reminiscent of those of soft-rot fungi (Corbett, 1965; Levi, 1965) (Fig. 1). More frequently, the main penetration hyphae branched repeatedly to produce multiple T-branches which then grew in opposite directions (Figs 1-3). In addition to producing T-branches, single branching from the main hypha in one direction (L-branching) was also regularly observed (Figs 2-4). The number of branches produced by penetration hyphae and the length to which branches developed varied considerably. Branches as long as ca 5'0 I..Im were noted, which may represent a final preset length. Occasionally T-branches branched further, producing their own characteristic secondary side branches. In this manner, abundant ramification of finepenetration hyphae within the wood cell walls occurred. Hyphal penetration and T- or L-branching was not restricted to the S2 layer of tracheids but was also abundant in the 51 layer of both early and latewood tracheids. Branching within the S3 layer was however not recorded. T- and L-branches produced from penetration hyphae generally followed the

51 microfibrillar axis of individual rracheid layers (i.e. showed alignment with cellulose microfibrils) in much the same manner as that reported for soft rot fungi (Figs 2-5). However, this was not consistent, and side branches often developed in a more irregular fashion within the secondary tracheid walls (Fig. 8). The parallel alignment of soft-rot Tbranch hyphae with the cellulose microfibrils of the 52 of wood cell walls has been considered a prerequisite for degradation and cavity formation (Nilsson, 1976). Studies have further shown that many soft-rot fungi causing erosion and cavity formation in hardwoods produce only cavities in S2 during the decay of softwoods (Nilsson et al., 1989). This is thought to relate to the important influence of lignin in forming a barrier around cellulose microfibrils, especially in some of the wall layers of softwoods. Unlike most soft-rot fungi attacking softwoods, 0. mucida showed considerable ligninolytic activity on pine wood (Table 2). Therefore, while alignment of T-branch hyphae with cellulose microfibrils may not be a requirement for their degradation, it may provide a means for a more effective decay of lignified substrates, particularly those containing softwood lignin residues. During initial phases of degradation of pine, decay was restricted to the wood directly surrounding the penetration and T-branch hyphae. Early cavities had serrated edges and were typically cylindrical in shape with pointed ends (Figs 5, 6). Series of very small biconical and diamond-shaped cavities were also occasionally recorded. With subsequent decay these cavities united to form typical cylindrical cavities. In crosssections early cavities were seen as characteristic small round holes within the secondary cell wall layers. Evidence for branching of penetration hyphae was also apparent (Fig. 10). During later stages of decay large numbers of cavities were produced and a general dissolution of wood cell wall layers, particularly the S2 occurred (Fig. 11). Continued dissolution of wood materials around penetration hyphae and side branches led to their coalescence and the development of large cavities. This was most apparent in L.S. of delignified material observed with polarized light (Fig. 9). Dissolution of wood materials around penetration hyphae resulted in enlarged boreholes, a feature characteristic of white-rot fungi. Enlargement and coalescence of cavities often proceeded in an outward direction from the main penetration hyphae along Tand L-branches (Fig. 7). Unlike soft-rot fungi, penetration and side-branch hyphae often remained thin (ca 0'3-D's I..Im) and

Figs 1-9. Micrographs showing various stages of O. muclda T-/L-hypha! branching within pine tracheid cell walls. Figs 1-4, 7-9 are of decayed and subsequently delignified tracheids and Figs 5-7 decayed tracheids without delignification. All micrographs are from observations using polarized light and lactophenol blue staining. Samples are taken from wood blocks exposed to the fungus for 16 weeks. Figs 1, 2. Early stages showing T- (Tb) and L-branching (Lb) patterns produced by penetration hyphae. After branching, cavities show orientation along the cellulose microfibrillar axis of the 52 layer. Bars (1) 5'0 !Jm; (2) 10 1Jffi. Figs 3. 4. Individual fibres showing ramification of fine cavities within the cell walls and various patterns of previous hyphal T- and L-branching. Cellulose microfibrillar axis of the tracheids is indicated by arrows. Bars (3, 4) 10'0 !Jm. Fig. 5. Higher magnification showing multiple T-branching of penetration hyphae and the cylindrical shape and pointed ends (arrow) of cavities. Bar 5'0 !Jm. Fig. 6. Micrograph showing the typical serrated edge (arrows) of biconical-like cavities and uneven dissolution of wood substances surrounding the cavity hyphae (H). Note the very fine nature of the hypha. Bar 2'0 !Jm. Fig. 7. Delignified tracheids showing the outward coalescence of fine cavities formed from previous multiple T-branching and cavity formation. Bar 5'0 !Jm. Fig. 8. Hyphal alignment and non-alignment after T-branching with the microfibrillar axis (arrows) of the tracheid wall. Bar 2'0 !Jm. Fig. 9. Advanced cavity formation and typical coalescence of cavities to form voids (V) within the cell walls of pine tracheids. Many of the voids appear orientated along the microfibrillar axis (arrows) of the tracheids and are continuous across the middle lamella (MI) region. Bar 5'0 !Jm. 4·2

Multiple T-branching by Oudemansiella mucida

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Figs 10-13. Micrographs showing various aspects of soft-rot cavity formation and erosion within pine tracheids and a birch vessel. Fig. 10. Cross-section of pine showing early stages (8 weeks) of cavity (C) formation, erosion attack and presence of fine-penetration hyphae (Ph) (inset) within tracheids. Note increased safranin stainmg of cell wall regions adjacent to the tracheid lumma. Bar 5'0 I.lm; inset 2'0 I.lm. Fig. 11. Late stages (12 weeks) of cavity (C) formation within the S2 layer of pine. Several tracheids (arrows) show advanced decay and coalescence of cavities to form a concentric ring of cavities within the S2' Cell wall thinning and cavity formation in the same tracheids is also apparent. Bar 10'0 I.lm. Fig. 12. Advanced cell wall thinning of pine trachelds WIth rupture of the radial cell walls (arrows) leaving the more resistant middle lamella cell comer (Mlc) regions. Bar 10'0 ;.lm. Fig. 13. Birch vessel showing typical decay patterns of penetration hyphae (Ph) and T-branching (Tb) (inset) withm the S2 wall. Sample stained WIth laetophenol blue and observed with polarized light. Bar 10'0 I.lm; inset 1'0 I.lffi. Abbreviations used in micrographs:

C soft rot cavity; Ph, penetration hypha; Bh, bore hole; Tb, T-branch; Lb, L-branch; H, hypha; Ml, middle lamella; Mlc, middle lamella cell comer, V, cavity voids.

G. Daniel, J. Vole and T. Nilsson

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Table 1. Weight loss (%) obtamed with O. mucida on birch, pine and beech blocks Weight loss' (%) Time (months)

Birch

Pine

Beech

0'5 0'4 0'7 0'3 4'7 1'2 3'2 I 2 17'1 2'0 16'6 3 35'0 2'3 27'1 43'3 15'1 38'0 4 6 54' I 29' I 63'0 9 78'9 52'4 69'7 • Weight loss values represent the mean of SIX replicates.

Table 2. Weight. lignin and carbohydrates lost (%) from birch. pine and beech blocks after 4 months decay by O. mUClda Loss (%) Weight

Xylose

Mannose

Glucose

Lignin

Birch 43'3 46'9 40'4 44'2 62'9 Pine 15'1 36'8 19'6 22'1 15'9 Beech 38'0 30'9 59'5 42'6 42'4 Undegraded wood: Birch: xyl. 24'6%, Man. 2'5%, Glu, 44'9%. Lig. 16'4%. pIne. Xyl. 6'8%. Man 12'4%, Glu. 44'0%. Llg. 28'1 %; beech' Xyl. 19'0, Man. 2'1%. Glu. 46'0%. Lig. 22'0%.

relatively constant in size throughout all stages of cavity formation. In advanced stages of decay of pine wood the enlargement and unification of cavities around both penetration hyphae and respective side branches resulted in an extensive decay network. Under polarized light, such cavities appeared as large cell-wall voids, indicating considerable substrate removal and localized enzyme attack. Like T-branch cavities, these voids were often aligned with the tracheid microfibrillar axis (Fig. 9). At this time, wood blocks were typically soft and easily broken when wet (i.e. a macro-decay characteristic of soft rotted wood) and showed slight bleaching similar to white rotted wood. In comparison, wood blocks of beech and birch were highly bleached. In addition to producing cavities, localized erosion of secondary cell walls by hyphae lying on the wood cell lumen wall interface of pine tracheids was also apparent. This was most obvious in early stages of decay and resulted in increased safranin staining of secondary walls adjacent to the cell lumen (Fig. 10). Cell wall erosion and cavity formation frequently occurred in the same fibres. In advanced stages of decay, the coalescence of cavities and progressive erosion of secondary cell walls resulted in a pattern of cell-wall thinning typical of that reported for white-rot fungi, with the cell comers of the middle lamella resisting decay the longest (Fig. 12).

Results from the wood block decay test (Table I) showed O. mucida to have a greater capacity to degrade the hardwoods birch and beech than pine. Microscopic examination of these hardwoods showed a similar degradative pattern, although erosion decay and cell-wall thinning tended to be more prominent than cavity formation. Birch vessels were more

poorly attacked than birch tracheids, but nevertheless showed degradative evidence of penetration hyphae and T-branching (Fig. 13). By contrast, beech vessels were highly degraded in advanced stages of decay. Birch vessels are known to contain guaiacyl lignin (Saka & Goring, 1985), and their greater resistance to decay may therefore reflect the type of lignin present. Gross chemical analyses of wood blocks degraded by O. mucida similarly showed the fungus to have a greater capacity to remove hardwood (syringyl-guaiacyl lignin) than softwood (guaiacyl lignin) residues (Table 2). In comparison with other wood decay fungi, 0. mucida shows decay characteristics of both soft- and white-rot fungi. Production of fine-penetration hyphae and boreholes, wood cell-wall thinning, middle lamella degradation, localized wood cell-wall decay beneath lumina hyphae, and wood bleaching are all features typical of white-rot fungi. Middle lamellae regions in hard and softwoods are resistant to degradation by soft-rot fungi (Nilsson et ai., 1989). Although cavity formation has been reported previously for basidiomycete fungi (Nilsson & Daniel, 1988), it is a wood decay feature generally associated with soft-rot fungi. However, unlike 0. mucida, soft-rot fungi produce cavities which are normally aligned with the cellulose microfibrils. Chemical analyses of degraded wood further showed removal of all cell-wall components including lignin in the hard and softwoods tested, a further characteristic of white-rot fungi (Table 2). The ability of O. mucida to produce repeated T- and Lbranching of fine-penetration hyphae within wood cell walls and the manner in which the cavities produced coalesce during wood degradation appears unique compared with that presently known for other cavity-forming ascomycetes and basidiomycetes. Development of fine-penetration hyphae is a common feature of fungi attacking and penetrating hard and insoluble substrates (e.g. keratinized substrates, English, 1965; and even metal foils, Liese & Schmid, 1964). Multiple branching by penetration hyphae of 0. mucida may therefore represent a further development of this characteristic decay pattern for attacking more resistant substrates. Interestingly, our recent studies on Oudemansiella radicata (Relh. ex Fr.)Sing. ( = Collybia radicata (Relh. ex Fr.) Quel.) have shown a similar unique type of hyphal T-jL-branching and cavity formation in both pine and birch wood. This fungus is also known to produce a range of ligninolytic enzymes (i.e. lignin peroxidase, Mn(n)-dependent peroxidase and laccase) consistent with that known for white-rot fungi (Huttermann et al., 1988). Further studies are in progress to understand the ultrastructural and physiological characteristics of T-branching by O. mucida in wood. The authors would like to thank Dr M. SemerdZieva for making cultures available from the Czechoslovakian Academy of Science culture collection. This work was supported partly by the Swedish Council for Forestry and Agricultural Research and by the Czechoslovakian and Swedish Academies of Sciences.

Multiple T-branching by Oudemansiella muclda REFERENCES Balley, I. W. & Vestal, M. R. (1937). The significance of certain wood destroying fungi in the study of the enzymatic hydrolysis of cellulose. Journal of the Arnold Arboretum 18, 196-205. Burkart. L. F. (1966). New technique for maceration of woody tissue Forest Produds Journal 16, 52. Corbett, N H. (1965). MICromorphology studies on the degradation of lignified cell walls of ascomycetes and fungI imperfectI. Journal Inshtute of Wood SClmce, 14, 18-29. Duncan, C. G. (1960) Wood-altackmg Capab,IJiles and PhYSIOlogy of Soft Rot FungI Report No. 2173, Forest Products Laboratory, u.s. Department of Agriculture: MadIson, Wisconsm, U.S.A 55 pp English, M. P (1965). The saprophytic growth of non-keratinophilic fungi on keratinized substrata, and a companson WIth keratinophIlIC fungI. Transactions of the British MycologIcal SOCIety 48, 219-235. Hale, M. D. C. (1983). The mechanisms of soft rot cavIty formatIon m wood Ph.D. TheSIS, C.N.AA, Portsmouth Polytechruc, England. Huttermarm, A, Milstein, 0., Nicklas, B., Trojanowski, J.. Haars, A & Kharazlpour, A (1988). Enzymatic modificatIon of lignin for technical use. strategies and results. In Llgnm' Properhes and Matenals (ed. W. G Glasser & S. Sarkanen), pp. 361-370. Amencan Chemical Society symposIUm senes 397: Washmgton D.C., USA

(Accepted 8 August 1991)

54 LeVI, M. P. (1965) Decay patterns produced by ChaetomlUm globosum m beechwood fibres. Matenals und Orgamsmm Belheft 1, 119-126. Liese, W. & Schmid, R. (1964). Uber das wachs tum von blauepilzen durch verholzte zellwande. Phytopathologlsche Zeltschnft 51, 385-393. NIlsson, T (1976) Soft-rot fungi: decay patterns and enzyme production. Material und Organismm Beiheft 3, 307-318. Nilsson, T. & Daniel, G (1988). MICromorphology of the Decay Caused by Chondrostereum purpureum (Pees.. Fr.) Pouzar and Flammuhna velutipes (Curl.: Fr.) Smger International Research Group on Wood Preservation Document No. IRG/WP/1358. [Can be received &om the IRG Secretanat. Box 5607, Drottnmg Knstinas Vag 67B, S-114 86 Stockholm, Sweden J Nilsson, T, Daniel, G" Kirk. T K. & Obst, j. R. (1989). Chemistry and microscopy of wood decay by some higher ascomycetes. Holzforschung 43, 11-18. Schacht, H. (1863). Ueber die Ver