Wood rotting by Phallus impudicus

514

Notes and brief articles

of the liquid drop results from the uptake of water vapour from a saturated atmosphere. The possible requirement for water vapour, and the need to avoid contact between basidia and liquid water led to an investigation into the feasibility of using tap water agar as a support for gill sections of an agaric whilst viewing basidia without a coverslip. Hand sections « 1 mm thick) through gills of Laccaria striatula sensu Orton were laid on the surface of filtered tap water agar in a Petri dish and viewed with a x 20 objective without a coverslip. The ripe, z-spored basidia of L. striatula were sought in which both sterigmata were in the same plane offocus, and where the 2 ,urn long protuberant hilar appendices were prominent. Within minutes of mounting such a section, two basidia were seen in which both basidiospores were discharged in turn, and it was possible to photograph the development of Buller's drop in each case (Fig. 1, A-F). As noted by Buller for other agarics, there was a comparatively short interval (less than 1 min) between discharge of successive basidiospores. This may be an adaptation to prevent drops from adjacent basidiospores making contact. There is very little distance between the surface of a fully enlarged drop and the spore immediately opposite it . I t is worth noting that, as in other basidiomycetes

which have been examined, Itersonilia perplexan s Derx and Tilletiopsis washington ensis Nyland, the drop grows in size without appreciable decrease in the dimensions of the spore (W eb ster et al., 1984 ; Webster, Davey & Ingold, 19 84 ). Video-recordings of ballistospore discharge have also been made. Successful discharge appears to be inhibited by high light intensity. We have made recordings in which enlargement of the drop is not followed by spore discharge. In one case, the drop continued to expand until its dimensions eventually exceeded those of the spore. In another recorded case of unsuccessful discharge, we watched a drop first enlarge, then decrease in size, possibly as a result of evaporation. We thank Dr D . A. Reid for identifying the Laccaria, and Mr D . Askew for help with photography. REFERENCES

WEBSTER, J., DAVEY, R. A. , DULLER, G. A. & INGOLD, C. T . (1984) . Ballistospore discharge in Itersonilia perplexans. Transactions of the Brit ish Mycological Soci ety 82, 13-29. WEBSTER, J., DAVEY, R. A. & INGOLD, C . T . ( 1984). Origin of the liquid in Buller's drop . Tra nsactions of the Brit ish Mycological Society 83,524-527.

WOOD ROTTING BY PHALLUS IMPUDICUS BY N. J. DIX AND J. W . G . CAIRNEY* Department of Biological Science, The University, Stirling FK9 4LA, Scotland.'

Wood incubated with Phallus impudicus growing on sawdust-eornmeal agar slowly developed typical macroscopic and microscopic white-rot symptoms. In the absence of an external carbohydrate source, sound wood was decayed slowly or not at all, suggesting that capacity to rot undecayed wood in the field may be rather poor. Several Gasterornycetes are associated with rotting wood, but there have been few published reports of their status as wood-rotting organisms. In Phallus impudicus Pers ., a familiar example, the basidiocarps are attached to an extensive mycelial cord system

* Present address: Department of Botany, The University, P .O . Box 147, Liverpool L69 3BX.

running in the litter or on the soil surface, and this is often traceable to buried wood ~ eneath the soil. Thompson & Rayner (1983) reeo ded weight loss of wood caused by this fungus when colonized wood was placed in soil, but there have been no detailed studies of the dynamics or any other aspects of wood decay by this fungus. It seems likely that P. impudicus produces a type of white-rot in wood. In studies on the course of

Figs 1-5 . Light microscopy of ti ssues of birch wood invaded by P. impudicus, Fig. 1. After 5 weeks' incubat ion ; hyphae in lumens of tracheids and invading parenchyma ray cells (L. S ., x 360). Fig. 2. As in Fig. 1. H yphae in lumens of tracheids (arrows) (T. S., x 902 ) ; Fig. 3. As in Fig. 1. Hyphae in lumen of tracheid branching and pa ssing through wall into adjacent cell (arrow) (L. S ., x 895 ); Fig. 4· At 20 weeks. Hyphae in lumens of tracheids , some cells showing roughening on interior wall s (arrows) (T. S ., x 902 ) ; F ig. 5. As in F ig. 4. Extensive growth in lumens of tracheids without visible damage to walls. (T eased tissues, x 1444).

Trans. Br, my col. Soc. 8S (3), (1985)

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5

5 16

Notes and brief articles

Figs. 6-8. SEM freeze fracture preparations of interior tissues of birch wood blocks after 15 months' incubation with P. impudicus. Fig. 6. T .S . oftracheids showing signs of wall thinning and cavities in cell walls, notably in the middle lamella region ( x 2250 ).

invasion of felled tree stumps by basidiomycetes, Rayner (1977) observed white-rot associated with the presence of the mycelium ; however, the capacity of the fungus to produce white-rot has not been tested by exclusive experiments. The decay of wood colonized by P . impudicus was studied microscopically using small autoclaved blocks of birch wood (Betula sp. ) that had been incubated with an established mycelium of the fungus growing on sawdust-cornmeal agar (5 g Acer pseudoplatanus L. (sycamore) fine sawdust in 80 em" of cornmeal agar (Oxoid)) in 500 ern" medicine flat bottles. The isolate of P . impudicus was kindly supplied by Dr A. D. M. Rayner. Wood blocks were recovered after 5, 20 and 52 weeks and prepared for examination. Radial and tangential longitudinal sections and tranverse sections were cut on a Reichert OME sledge microtome with a freezing stage. If necessary, blocks were softened prior to sectioning by boiling in a 5 : 1 water-glycerol mixture and left to soak in water overnight. Sections were stained with safranin and picroaniline blue (Cartwright, 1929) and inspected by Trans. Br, mycol. Soc . 85 (3), (1985)

light microscopy. Stained preparations were also made of tissue teased from the softer, more decayed parts of the blocks. Wood incubated with the fungus for 15 months was used to make freeze fracture preparations for examination by scanning electron microscopy (SEM). After 5 weeks' incubation, extensive invasion of tracheids and the parenchyma cells of some of the medullary rays by hyphae could be seen by light microscopy in sections of surface tissues (Figs 1, 2). In tracheids hyphae were mainly confined to the cell lumen, although rare instances of hyphae passing through cell walls to the lumens of adjacent cells were also seen (F ig. 3). As time passed, tissues became progressively more invaded in deeper positions, with ray tissues becoming particularly heavily colonized. After 20 weeks of incubation the outer tissues of the wood had become noticeably softer. Internally some roughening on the inner walls could be seen in some tracheids (Fig. 4), but many well-colonized tracheids showed no obvious wall damage (Fig. 5).

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Notes and brief articles

Fig. 7. Destruction of parenchyma ray tissue ( x 2000); Fig. 8. Severe damage to parenchyma ray tissues (cross walls destroyed) and in the pit fields of tracheids below ( x 300).

Trans. Br. mycol. Soc. 85 (3), (1985)

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5 18

Notes and brief articles

By 52 weeks a very obvious wh ite-rot condition had developed in the outer tissues. These had become conspicuously bleached, very soft, wet and easily separated. The wood was clearly delignified and gave no positive red reaction for lignin when tested with phloroglucinol /H'Cl. Even with the very thin blocks that were used, examination of inner tissues at 52 weeks showed many invaded tracheids that were still without much visible sign of damage that could be seen by light microscopy. SEM examination of freeze fracture preparations of inner tissues (with no obvious external symptom of white-rot after 15 months' incubation) showed signs of possible thinning on interior walls of tracheids, damage to pit fields and the development of small cavities in the middle lamella region (Figs 6, 8). The latter are probably caused by the fungus and may be similar to those observed by Eriksson et al. (1980) in the walls of spruce tracheids in wood rotted by the white-rot Sporotrichum puloerulentum. In other SEM preparations of the same tissue, extensive damage to parenchyma ray tissues was seen (F igs 7, 8). Thus it was confirmed that P. impudicus produces a type of white-rot in wood analogous to that produced by Hymenomycetes. The bleaching of the wood by the removal of lignin is the characteristic feature of advanced decay produced by this type of rot. At the microscopic level, the thinning of the secondary walls of tracheids, the enlargement of pits and damage to pit fields, the primary location of infection in parenchyma rays and the spread to the lumens of tracheids, the progressive destruction ofray tissues, the formation of small cavities in SEM preparations in the lignin-rich middle lamella regions of tracheids with little or no shrinkage, collapse or separation of tissues until advanced decay, are all symptoms associated with various white-rots (W ilcox, 1970). The ability of the fungus to decay various kinds of wood was followed using thin blocks ca l' 5 x r -5 x o· 4 em weighing about 2 g . These were cut from felled large undecayed branches of Betula sp . (birch), Fagus sylvatica L. (beech), Picea abies (L.) Karst. (spruce), Fraxinus excelsior L. (ash), Quercus robur L. (oak) and Acer pseudoplatanus L. (sycamore) after seasoning for 1 year. Individual air-dry weights of wood blocks were recorded for each tree species. Moisture content in the wood was adjusted by plunging briefly into water to wet the surface and humidifying on damp filter paper in dishes held over water in an enclosed tank for 2 weeks. At the start of the experiment the mean moisture content of the wood exceeded 55 % of the wet weight. Moistened wood was sterilized by exposure to ethylene oxide for 18 h in order to avoid Trans . Br. mycol, Soc. 85 (3), (1985)

some of the changes that could affect the results if autoclaved wood had been used. The swelling of cellulose fibres by steaming or subjecting materials to high temperatures, for example, is known to increase hydrolysis (Millet, Baker & Satter, 1976) and would increase decay potential. For similar reasons the wood was not oven-dried prior to weighing, because this could have led to the loss of inhibitory volatiles and to an increase in stimulatory compounds. In Pinus sylvestris for example the stimulatory auto-oxidation products of unsaturated fatty acids are increased by drying (Flodin & Fries, 1978 ). Thirty to forty sterilized blocks of each wood species were transferred to Petri dishes containing Abram's medium (NH 4NO a, 3 g ; K 2H P 0 4 , 2 g; KH 2P0 4l 2'5 g; MgS04 .7H20, 2 g; agar, 20 g; I- I). Dishes were inoculated with 1 cm diam disks of P. impudicus cut from cultures growing on malt agar. A similar number were placed on Abram's medium but were not inoculated and served as controls. All Petri dishes were partially sealed with autoclave tape and incubated at 25-27° over water in almost completely closed deep tanks for 10 weeks for birch, beech and spruce, and 15 weeks for the other woods. After harvest, the wood blocks were soaked in daily changes of distilled water for 1 week to remove soluble materials (preliminary experiments indicated that this was the least time required for blocks to reach a minimum constant weight). Wood was subsequently dried to a constant weight at 55° and weight losses recorded as a percentage of the original air-dry weight. Calculations of the original water content of the air-dried wood based on ten wood blocks of each species dried to a constant weight at 55° showed a maximum difference between species of about 2 % (Table 1). Small differences of this order are unimportant when comparing final weight losses in different wood species, and valid comparisons can be made without the need to apply a correction factor to bring all the woods to oven-dry weight. The fungus made conspicuous growth over all wood and appeared to colonize it on the Abram's medium. At the end of the experiment some of the wood had softened, but apart from this there was very little obvious sign of rot. Only the angiosperm woods of lower density (birch, sycamore and ash) lost significantly greater weight when incubated with the fungus compared with the controls (T able 1). Mean percentage weight losses were small and rate of loss was slow, at 0 '229 %, 0 '225 % and 0'065 % week"! respectively . Maximum weight loss for individual samples compared with mean values for controls were 15 '05 % for birch, 6'92 % for sycamore and 5'73 % for ash. The results suggest that birch and sycamore have

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Notes and brief articles Table Mean

1.

wt

Beech

Controls 7'85 ±o'15

Birch

7'9 2±o '19

Spruce

8'80 ±O'13

Sycamore

12'11 ±O'17

Ash

13-11±o'27

Oak

13'9 6±0 '15

Decay of wood by Phallus impudicus loss (%) with standard errors Expt 8'43±O '30

Numbers in brackets are values for wt

nur.

Percentage loss, week"!

Percentage H 2o of air-dried wood 8'85

0'57 P = 0'11 (N,S,) 10'21±O '62 2'29 [2'47] 0'229 7'64 P = 0'0009 0-08 8'69 8'88±o'28 P = 0'79 (N,S.) 3,83 [4'22] 0'255 15'94±O '42 9'3 P = 0-0001 0'97 [1'07) 0'065 9'6 14'08±o'33 P = 0'029 9'0 14'29±O '25 0'33 P = 0'25 (N.S.) loss recalculated as percentage of oven-dried wt of wood.

the greatest potential for decay by P. impudicus. No significant loss of weight occurred in the time span of the experiments with beech, spruce or oak . The result for beech contrasts with that obtained for this wood by Thompson & Rayner ( 1983) who found significant dry weight losses using autoclaved beech wood (equivalent to a rate of 1 '64 % week?") when incubated with P . impudicus. Mean weight losses in beech and spruce can be seen to be entirely covered by the loss of water from air dried wood on oven drying. Higher weight losses in oak controls than can be accounted for by loss of water are most probably due to loss of soluble matter by the water soaking given at the end of the experiment. Sycamore and ash also apparently contain appreciable amounts of soluble material. The percentages of wood blocks where weight loss in individual samples exceeded mean value for controls were 73'3 % for beech, 59'1 % for spruce and 48'0 % for oak. These calculations may indicate that some slight decay has taken place in these woods but that losses are so slight compared with the range of values in the controls that these cannot be shown to be statistically significant. In white-rot decay by Hymenomycetes there is simultaneous oxidation of lignin and metabolism of carbohydrate (Cowling, 1961) and white-rot fungi apparently require an available source of carbohydrate to act as a growth substrate before lignin decomposition can take place (K irk, Connors & Zeikus, 1976). For C. versicolor a brief exposure of wood for 4 days to the fungus seems all that is necessary for growth to continue at the expense of carbohydrates in the wood after it is removed from the culture. Weight losses of 25 % resulted following this treatment after 9 weeks' incubaTrans. Br. mycol, Soc. 8S (3), (1985)

519

tion (Cowling, 1961). The poor performance of P. impudicus under similar circumstances in our experiments could have been caused by an inability of the fungus to gain access to the carbohydrates of our woods. The results of Thompson & Rayner (1983) suggest that by contrast with C. versicolor, P. impudicus requires access to readily available carbohydrates before it can rot wood. In their experiments this was supplied in large amounts by the malt agar of the fungus culture on which the wood was incubated for 5 weeks prior to transferring it to soil, and it is possible that most of the weight loss that they recorded occurred while the wood was in contact with the culture. These results may indicate that P. impudicus is a late colonizer of wood and has little potential to rot newly available undecayed wood. Certain field experiments lend support to this view. Rayner (1977 ) found that it was slow to invade freshly exposed wood and that it could not be detected by the destructive sampling of tree stumps in the first 12 months following felling and exposure, but was present in samples taken 14 months later. In the field the fungus is most often associated with certain sorts of rotting angiosperm wood. In such wood, disrupted cell walls, broken-down cellulose fibrils and the partial depolymerization of cellulose caused by the growth ofother wood-rotting basidiomycetes may ease access to the wood carbohydrates needed for growth. Migrating mycelium cord systems will undoubtedly carry sufficient inoculum potential to bring about establishment on rotted wood, but colonization from spores may depend upon the build-up of an inoculum by growth in the leaf litter first .

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Notes and brief articles

52 0 REFERENCES

CARTWRIGHT, K. ST G . ( 1929) . A satisfactory method of staining fungal mycelium in wood sections. Annals of B otany 43, 412-4 13. CoWLING,E. B. ( 196 1). Comparative biochemistry of the decay of sweetgum sapwood by white-rot and brown-rot fungi. Technical Bulletin 1258, Forest Service, U .S . Dept of Agriculture, Washington D .C . FLODIN, K. & FRIES, N. (1978). Studies on volatile compounds from Pinus silvestris and their effect on wood decomposing fungi. II. Effects of some volatile compounds on fungal growth. Eu ropean Journal of Forest Pathology 8, 300-310. ERIKSSON, K . E ., GRUNEWALD, A., NILSSON, I. & VALLANDER, L. (1980). A scanning electron microscopy study of the growth and attack on wood by three wh ite-rot fungi and their cellulase-less mutants. Holzforschung 34, 20 7-213 . KIRK, T. K ., CONNORS, W. J. & ZEIKUS, J. G . (1976).

Requirement for a growth substrate during lignin decomposition by two wood-rotting fungi. Applied Environmental Microbiology 32, 192-194. MILLET, M. A., BAKER, A. J. & SATTER, L. D . ( 1976). Physical and chemical pretreatment for enhancing cellulose saccharification. Biotechnology and Bio engineering 17, 1199-1210. RAYNER, A. D. M. ( 1977) . Fungal colonization of hardwood stumps from natural sources. II. Basidiomycetes. Transactions of the British Mycological Society 69, 30 3- 3 12 . THOMPSON, W . & RAYNER, A. D . M. "(1983). Extent, development and function of mycelial cord systems in soil. Transactions of the British Mycological Society 81, 333-345· WILCOX, W . W . (1970) . Anatomical changes in wood cell walls attacked by fungi and bacteria. Botanical Reviews 36,1-28 .

COMPARISON OF SPATIAL PATTERNS OF SEXUAL AND VEGETATIVE STATES OF BOLETINELLUS MERULIOIDES BY H. VAN T. COTTER AND G. F . BILLS

Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A. The spatial pattern of Boletinellus merulioides sclerotia in a forest was compared with basidiocarp frequency recorded over 4 years. Both estimates of the spatial pattern coincided, but year-to-year basidiocarp frequency varied greatly. Basidiocarp and sclerotial densities were centred around and declined outward from Fra xinus americana trees . Quantitative studies of macrofungal communities (Lange, 1948; Hering, 1966; Petersen, 1977 ; Arnolds, 1981; Fogel, 1982) have often relied on basidiocarp numbers or basidiocarp biomass in sample areas of fixed dimensions. A fundamental assumption of these studies is that the relative production ofbasidiocarps among fungal species in some way reflects their dominance or mycelial biomass . Only some limited evidence indicates this is true (Laiho, 1970; Newell, 1984 ). A second assumption - that the absence of basidiocarps at a location over several years reliably indicates that the vegetative state is absent - has rarely been directly tested. Boletinellus merulioides (Schwein.) Murrill produces abundant, persistent sclerotia in nature which can be easily found and recognized in the field (Cott er & Miller, 1985 ). Mapping sclerotia and comparing their spatial pattern with that of basidiocarps offered a unique opportunity to test the hypothesis that repeated observations of basidiocarps over several years can accurately estimate the spatial pattern ofa fungus . In addition, the question of whether density of basidiocarp Trans . Br, mycol. Soc. 85 (3), (1985)

occurrence is related to the density of the occurrence of vegetative mycelium as indicated by sclerotia is addressed. Finally, the spatial pattern of B . merulioides is described in relation to its associate Fraxinus americana L. The study area was a stand of 66-year-old hardwood trees on the crest of Kennison Mt (elev. 1207 m ) in the Monongahela National Forest, Pocahontas Co., WV, U.S.A. (380 11' lat., 80 0 17 ' W long.). The soil at the site was a shallow, rocky, well-drained clay-loam with a pH of 3"8±0'3 S.D. (n = 8) and organic matter content of 8·5 ± 3"7 % S.D . (n = 8). The canopy was dominated by Prunus serotina Ehrh., Acer saccharum Marsh., Fagus grandifolia Ehrh., Fraxinus americana and Betula al/eghaniensis Britt. Other Acer spp. were abundant in the understory. Two permanent plots, 5 and 6, were established as representative samples of a mixed hardwood forest. Each plot was 16 x 16 m in size and was further su bd ivided into 64 2 x 2 m subplots for sampling, each identified by a column letter and row number (Fig. 2). Three Fraxinus trees were located within plot 6 (F ig. 2), one in subplot d 7 (49"5 em DBH (diam at breast

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