Fungus decomposition of beech cupules

[ 437 ] Trans. Brit. mycol. Soc. 47 (3), 437-444 (1964) Printed in Great Britain

FUNGUS DECOMPOSITION OF BEECH CUPULES By C. G. CARRE

Bryanston School, Blandford, Dorset (With

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The decay of the cupules of Fagus sylvatica L. in the natural environment is described. The appearance and abundance of fungus fruit bodies produced on weekly samples of cupules, over a z-year period, is recorded. A characteristic flora occurs, certain species fruiting in spring, others in autumn and winter, and some throughout the year. It is suggested that during the first 2 years of decay the incidence of fruit bodies shows the influence of weather rather than accumulation and availability of food materials. The course of mycelial invasion ofintemal tissues is described and also the gradual change in pH from 3'5 to 7, and a considerable decrease in hardness of the cupule; the fall in total nitrogen content is initially rapid, whereas the loss of total organic matter is very slow.

Ecological successions of fungi on naturally occurring plant debris, decomposing on the surface of the soil have been subjects of intensive study by various workers (e.g. Webster, 1956; Hudson & Webster, 1958; Kendrick, 1958), and the importance of the microhabitat has frequently been stressed (Garrett, 1951). The microbial flora of the surface litter ofa beech wood has been examined by Saito (1956) and Caldwell (1963) has investigated the fungus flora of decomposing beech stems and roots in soil. The present study concerns the beech cupule decomposing under natural conditions. MATERIAL AND METHODS

The sampling area was in semi-natural woodland at Bryanston School, Dorset (National Grid Ref. 31/870075), with Fagus sylvatica and Aesculus hippocastanum L. as dominant trees. The field layer was poor and in some areas absent; the co-dominant plants were Mercurialis perennis L. and Hedera helix L. Collections were made at least once a week. All cupules were taken from the surface of the soil to the laboratory and examined immediately after collection; twenty to thirty taken at random gave a picture of the relative frequency of any particular fungus fruiting on the cupule. In addition to the compilation of a complete list of species, an attempt was made to record the frequency of the fruit bodies on the cupules in the natural environment. In estimating numbers of fruit bodies it was unrealistic to count accurately the hundreds of fruit bodies of a particular fungus which sometimes occurred on a single cupule. Although dominant on one cupule of a sample, its fruit bodies might be absent from all other cupules in the same sample. Thus symbols of abundance were used to denote frequency of a particular fungus in the area studied, rather than on 28-2

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a particular cupule. The following arbitrary symbols were used to denote frequency on the cupules examined: + + + + practically all with the fungus + + + approximately half with the fungus + + only occasionally encountered (on less than one third of the cupules) + rare, seen once or twice only. In addition, the condition of the cupules and particulars of habitat and weather conditions were recorded for each sample. OBSERVATIONS

An analysis of the records of fruit bodies common on the cupules is given in Fig. I. Periodicity varied with the species which were classifiable in three groups on this basis: Species fruiting throughout the year: Xylosphaera carpophila (Pers.) Dumort., Lophiotrema praemorsum (Lasch) Sacc., Hyaloscypha leuconica (Cooke) Nannf., Helotium ?granulosellum (Karst.) Karst., Unguicularia sp. It is quite likely that some specimens of Lophiotrema were L. oagabundum Sacco since they agreed with the measurements given by Dennis (1960) for this species. No marked difference was noticed in the abundance and incidence of imperfect fungi on the cupules throughout the year. The commonest species recorded were: Trichothecium roseum (Pers.) Link ex Fr., Bactrodesmium arnaudii Hughes, Volutella ciliata (Alb. & Schwein.) Fr., Cylindrocladium sp., Cylindrium sp., Asterosporium sp. The incidence of this group of species was independent of weather conditions and showed little correlation with rainfall, air temperature or temperature of leaf litter. Species fruiting in late autumn and winter: Inocybe ?patoullardii Bres., Mycena mirata (Peck) Sacc., Marasmius recubans Quel., Helotiumfagineum (Pers.) Fr., Calycellina ochracea Hohnel, Calycella sulfurina (Quel.) Boud., Niesslia pusilla (Fr.) Schroet. Fruiting occurred mainly late September-March. It was interesting to note that even in mid-winter, below snow and ice-encrusted leaflitter, delicate species such as Calycellina ochracea were found. N. pusilla, rare, was found as late as March in 1963 after many weeks of snow. (The dimensions of the ascospores of this species were consistently smaller than those given by Dennis (1960), 6.4-7.36 x 1.5-1.81-', but other characters were less variable.) Apothecia of Calycella sulfurina are usually associated with members of the Sphaeriales, but were not so when found on the cupule. Mycena mirata and Marasmius recubans were both limited to the autumn and the latter has only rarely been found in this country. (Private communication from Mrs F. L. Balfour Browne.) Although Clavaria cristata Pers., Melanoleuca exscissa (Fr.) Sing., Lepiota castanea Qu el. and Peeiza varia (Hedwig) Fr. have been found growing from the inside of the cupule, they were all probably associated with soil packed inside and did not penetrate the tissues of the cupule. Inocybe ?patouillardii, however, was found growing out of the side of the cupule, which it used as the sole source of nutriment. Abundance of Mycena mirata, Marasmius recubans, and Helotium fagineum was greatest during October 1963, after a very wet August-September and

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a dry, warm October. In September 1964 the abundance of these species was greatly reduced after much drier weather. Presumably the fall in temperature during late November and/or the consequential drop in availability of water is associated with the decrease or absence offungi in this group. Species fruiting in spring and early summer (April-June): Dasyscyphusfuscescens (Pers.) S. F. Gray, D. virgineus S. F. Gray, Chaetomium bostrychodes Zopf, Mollisia ligni (Desm.) Karst., Solenia sp. M. ligni and D. virgineus both occurred sporadically at other times of the year, but they had obvious maxima in spring. Only in April 1962 was Masseea quisquilarum (Berk. & Cooke) Sacco recorded. May, June and July were months of low rainfall and rising temperature, and with the preceding wet month of April, gave suitable environmental condition for the abundant production of fruit bodies of these species. A very poor flora was recorded in September 1962 and 1963 (no records were made in August of either year). THE COURSE OF INVASION BY FUNGI

It was obvious that incidence of fruit bodies did not give a measure of the relative abundance of species present as mycelium. Accordingly, the density and distribution of hyphae in the tissues of the cupules of different ages was studied. It was not possible to determine the species of the invading hyphae. The images of the hyphae were traced on to graph paper, and the total length of hypha per unit area was recorded by means ofa map measurer. Hand sections of the infected cupule wall were stained by the modified periodic acid-Schiff technique (Dring, 1955). During growth of the cupule on the tree there was no internal infection, though spores and external hyphae were visible. Three months after fall a few hyphae were found in the outer tissues of the cupule wall, and intracellular hyphae in the cortex. This cortical tissue was further attacked and after 12 months there was considerable invasion of the parenchymatous cells, the walls of which were frequently destroyed and the cells distorted. However, parts of the same tissue showed no infection or decay. At this stage the spines of the cupule were broken, and the hairs on the outer surfaces had decayed. After 18 months the whole cortical region was permeated by intra- and extracellular hyphae and the fibrous zones just below the outer and inner surfaces of the cupule had slight intercellular infection. Sections ofcupules more than 2 years old were cut with a freezing microtome. The outer fibrous zones were beginning to break down and the cortical layers were reduced in parts to a pulp without cellular detail. An interesting feature of the decay of the cupule was the apparently random way in which various parts of the cupule were invaded by hyphae. Destruction was not usually the result of a simultaneous invasion of all the parenchymatous parts, nor were the various food materials used in strict succession. At any stage a particular cupule exhibited a mosaic of activity, different parts with different degrees of decay. Thus it was impossible to give a precise estimate of the stage of decay of any particular specimen.

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Decomposition of beech cupules. C. G. Carre THE CHANGING NATURE OF THE CUP ULE

H-ion concentration Measurements were made on a sample ofeach weekly collection throughout the period of study. There was a gradual change in reaction from an initial pH 3'5-4 to 6'5-7 after the cupule had been lying on the soil for 2 years. Soil reaction was markedly alkaline (7'2-7' 7) and varied little throughout the year. Hardness As decay proceeds the texture or hardness of the cupule declines. An apparatus was constructed which measured the pressure required to drive a needle through the valve of the cupule at a point as near the centre of the valve as possible. The sample taken from the field was tested before it dried; variations in recordings were due largely to water content of the tissues. However, the significant result was the considerable (86' 2 %) reduction in hardness over a period of 2 years, 100

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Nitrogen and carbon content Records were kept throughout of the disappearance of the total nitrogen and total carbon content as decay progressed. There is no way ofseparating synthesized fungus material from the unchanged host residues, and therefore the results include some fungus material. Also, it is difficult to relate the results of gross chemical analysis with the presence of a particular fungus. Fig. 2 shows that the rate ofloss of total nitrogen was not constant. After an initial rapid decrease in the first 6 months, the following 12 months were characterized by a stable period, which did not indicate a static state but reflected the conversion of cupule protein to fungus protein. The cupule just before falling was considerably lignified and being such a compl ex substratum for colonization its rate of decomposition was, not surprisingly, slow, After 2 years only 6 % of the total organic matter was

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lost (Fig. 2). It was interesting to compare this lossoftotal organic material with the disappearance of the lignin component of Casuarina needles (fig. 5 in Burges, 1958).

Activities of the microfauna The effect of these was not only to cause reduction in weight and comminution ofthe cupule, but also to bring about the dissemination offungus spores. Nematodes and mites encrusted with spores and hyphae were often present, and spores were found in animal excreta, especially inside the cupule valves. LABORATORY ISOLATION STUDIES

It was likely that many fungi might be present in the cupules, but because of antagonism or competition failed to produce fruit bodies or spores on the outer surfaces. An attempt was made to isolate these, two methods being employed; (i) a modification of the serial washing technique of Harley & Waid (1955) for isolating surface hyphae and spores; and (ii) a surface sterilization technique for isolation of hyphae from inside the tissues of the cupule. A large number of imperfect fungi were isolated, including many common soil saprophytes, but it was not possible to show what part they played in the decomposition of the cupule. Numerous perithecia of Gelasinospora cerealis Dowding developed from surface washings on potato agar, yet perithecia of this species were never observed on cupules taken from the natural environment. Chaetomium bostrychodes Zopf was also isolated from surface washings from cupules during the first 18 months after fall; presumably it is an active cellulose-splitting fungus which entered the succession early. In the natural environment this species produced fruit bodies on the cupules only in spring. CONCLUSIONS

The beech cupule when dropped from the tree was a reasonably sterile discrete unit of organic debris. A characteristic fungus flora developed on the fallen cupule. The visible manifestation of this, fruit bodies and spores, varied with time and season during the slow decomposition of the extensively lignified tissues. The effect of the environment was obviously complex, probably the result of the interaction of many factors. The succession of the majority of fruit bodies on the cupule during the first 2 years suggested the influence of weather rather than the accumulation and availability of food substrates. The autumn crop of basidiomycetes was most pronounced after a few weeks of moist warm conditions, and a typical spring flora developed on first- and second-year cupules. Periods of abnormal cold were correlated with the lateness of species appearing in spring 1963, and periods of drought were associated with the nonappearance of species on cupules lying on exposed beechwood slopes. Fruit bodies which did not show seasonal periodicity were protected from weather changes by their tough perithecial covering or by development

Decomposition of beech cupules. C. G. Carre

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within the host tissues. In species such as Niesslia pusilla, which had a restricted distribution, the controlling factor could have been the lack of competition from other fungi, and not necessarily climatic conditions. After 2 years the cupule changed from a distinctly acid to a neutral condition and there was a reduction in hardness. It was becoming a part of a more stabilized micro-environment in the compacted mass of the humus layer, and would obviously be less influenced by weather changes. Saito (1957) found that when beech litter was being decomposed under natural conditions almost half of the lignin had gone when only onequarter ofthe cellulose had been decomposed. This fact is interesting in the light of the seasonal appearance of fruit bodies on the cupule. The lignicolous basidiomycetes, such as Mycena mirata and Marasmius recubans, together with lignicolous members of Helotiaceae, appeared as early colonizers of the cupule, and were members of the autumn group. After 2 years only 6 % of the total organic matter was lost, reflecting the slow rate of decomposition of the substrate. Internally the concentration of hyphae in the cortex reached a maximum after 18 months, after which the more resistant fibrous zones were attacked. Caldwell (1963) has drawn attention to the differences found in adjacent fragments of rotting material and Burges (1963) has emphasized this pattern of different fungal units found on cellulose added to the soil. The beech cupule shows a similar pattern. After 2 years parts of the cupule were firm and contained little internal mycelium, while adjacent parts had been reduced to an amorphous residue. This mosaic of activity explains the observed seasonal crop of fruit bodies on cupules of different ages. A different flora in the second year would be expected if the usage and availability of food materials throughout the cupule had been uniform. This work formed part of a thesis submitted for the degree of M.Sc. in the University of Bristol. I am extremely grateful to Dr L. E. Hawker under whose supervision this study was carried out, for her interest and advice, and to Dr R. W. G. Dennis, W. D. Graddon and Mrs F. L. Balfour-Browne for their help with identification of specimens. The Royal Society and Bryanston School kindly provided grants towards the cost of equipment. REFERENCES

BURGES, A. (1958). Micro-organisms in the soil. London: Hutchinson. BURGES, A. (1963)' Presidential address. Trans. Brit. mycol. Soc. 46, 1-14. CALDWELL, R. (1963). Observations on the fungal flora of decomposing beech litter in soil. Trans. Brit. mycol. Soc. 46, 249-261. DENNIS, R. W. G. (1960). British cup fungi. Ray Society, London. DRING, D. M. (1955). A periodic acid-Schiff technique for staining fungi in higher plants. New Phytol. 54, 277-279. GARRETT, S. D. (1951). Ecological groups of soil fungi: a survey of substrate relationships. New Phytol. 50, 149-166. HARLEY,j. L. & WAlD,j. S. (1955). A method of studying active mycelia on living roots and other surfaces in the soil. Trans. Brit. mycol, Soc. 38, 104-118. HUDSON, H, ]. & WEBsTER,j. (1958). Succession offungi on decaying stems of Agropyron repens. Trans. Brit. mycol. Soc. 41, 165-177. KENDRICK, W. B. (1958). Micro-fungi in pine litter. Nature, Lond., 181,432.

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SAITO, T. (1956). Microbiological decomposition of beech litter. Ecol. Rev. 14, 141-147. SAlTO, T. (1957). Chemical changes in beech litter under microbiological decomposition. Ecol. Rev. 14,209-216. WEBSTER, J. (1956). Succession of fungi on decaying cocksfoot culms. I. ]. Ecol. 44, 5 17-544.

(Acceptedfor publication

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March 1964)