Sporophagy in hymenomycetes

EXPERIMENTAL

MYCOLOGY

9, 74-79 (1985)

BRIEF NOTE Sporophagy

in Hymenomycetes

NILS FRIES AND GUNILLA Institute of Physiological

SWEDJEMARK

Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden Accepted for publication October 17, 1984

FRIES, N., AND SWEDJEMARK, G. 1985. Sporophagy in hymenomycetes. Experimental Mycology 9, 74-79. Sporophagy, a hitherto unknown type of necrotrophic mycoparasitism in the hymenomycetes, is described. Sporophagous species were chiefly found among wood-decomposing Aphyllophorales. The attacks were not directed against hyphal cells but only against living basidiospores, preferably dark-colored ones. After an approach similar to a chemotropically controlled homing reaction the sporophagous hypha contacted the basidiospore and penetrated its cell W&

0 1985 Academic

INDEX

Press, Inc.

sporophagy; hymenomycetes; mycoparasitism; wood-decomposing fungi.

DESCRIPTORS:

penetration;

In an investigation of homing reactions within some species of hymenomycetes a number of interspecific combinations of basidiospores and mycelia were also made. Unexpectedly, in one of these confrontations the mycelium attacked the spores, which were killed and consumed by the myCelia1 hyphae. Further combinations where other species were involved gave similar results in a few cases. To get some insight into the phenomenon a more systematic study was undertaken, the results of which are reported in this communication. The spores to be tested were collected in the autumn of 1983 from basidiocarps of various hymenomycetes and stored in darkness at 4°C. Most of the mycelia came from the stock culture collection at the Institute of Physiological Botany, Uppsala University, and the others were kindly placed at our disposal by Professor Aino Kaarik at the Swedish University of Agricultural Sciences from her large collection of wooddecomposing hymenomycetes. The nomenclature follows Moser (1978), Ryvarden (1976, 1978), and Eriksson and Ryvarden (1973). If not mentioned otherwise the experiments were done in 9-cm-diameter plastic 0147-5975185 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

basidiospores; spore-wall

petri dishes containing approx 12 ml of an autoclaved agar medium, consisting of glucose (4 g * liter-‘), ammonium tartrate (20 mg . liter-‘), agar (1.5 g . liter-‘), mineral salts, and vitamins. The medium differed from the synthetic basic medium of Fries (1981) only by its low content of ammonium tartrate. A suspension of spores (approx 5,000,OOO * ml-‘) in distilled water was plated out on the agar surface, 0.1 ml on each plate. After the water of the suspension had evaporated, inocula of various myCelia of hymenomycetes were tested for activity against the spores by placing the inocula on the agar surface, usually one or two per plate. The interactions, if any, of the developing mycelia with the spores were followed by light microscopy and, for documentation, recorded by photomicrography or electron micrography. For transmission electron microscopy (TEM)’ agar pieces with mycelium interacting with spores were fixed and dehydrated according to Franzen and Sensenbaugh (1983). They were then embedded in t Abbreviations used: TEM, transmission electron microscopy; SEM, scanning electron microscopy.

SPOROPHAGY

IN HYMENOMYCETES

Epon and sectioned with a LKB Ultratome II using a Dupont diamond knife. The sections were stained with 2% aqueous uranyl acetate for 20 min and Reynolds lead acetate for 5 min before viewing in a Jeol JEM 100-B electron microscope equipped with a double condenser operating at 60 kV. For scanning electron microscopy (SEM) the samples were fixed and dehydrated as for TEM, transferred to filtered Freon TF, and critical-point dried from CO,. They were then mounted on stubs, coated with 20 nm of gold in a Jeol JFC-1100 ion sputter, and examined at 15 kV using a Jeol JSM-35 scanning electron microscope. In the first case observed, where basidiospores were attacked by hyphae from a basidiomycete mycelium, the spores came aurantiacum and the myfrom Eeccinum

75

comatus. The hycelium from Coprinus phae grew toward the spores in a way which strongly suggested a chemotropic reaction (Fig. la). After having reached one of the spores the hypha wound itself round it, ramified, and sometimes almost totally enclosed the spore by dense branches (Figs. lb, d; Figs. 2a, b). Finally the spore wall was penetrated by a hypha and the spore was killed (Figs. 2c, d). Processes like this are known as in intrageneric hypha-basidiospore interactions (Morton and French, 1940; Fries, 1981). Similar, although intergeneric, reactions, usually called tropisms, have also been observed in mycoparasitism as interachypha-hypha and hypha-spore tions (e.g., Barnett and Sly, 1958). In the type of interaction now described the at-

FIG. 1. Light micrographs showing hyphae of Coprims comatus attacking basidiospores of kccinum arrrantiacum on an agar plate surface. (a) Homing-like approach; (b) and (c) contact established between hypha and spore; (d) five spores entwined by lateral hyphae from the same main hypha. x 60(1.

76

FRIES AND SWEDJEMARK

Frc. 2. SEM (a-c) and TEM (d) of Cop&us comatus. (a) Lateral entwining a spore; (c) two hyphae the spore wall; (d) cross section of (c) x3800; (d) x 10,000.

micrographs of Leccinum auruntiacum spores attacked by hyphae hypha winding around a spore; (b) several branches of hyphae coming up through the agar surface, one of which is penetrating a spore showing the penetration of a hypha. (a) x 4000; (b) x 3200;

tacking hypha and the killed basidiospore always represented species of different genera. Therefore we judged the phenomenon as being an example of necrotrophic mycoparasitism (Barnett, 1964). When it turned out that, in contrast to other types of necrotrophic mycoparasitism, only basidiospores and no hyphae were attacked by the aggressive counterparty, we called this sort of interaction sporophagy. With basidiospores of L. aurantiacum as “bait” a large number of species from various groups of Homobasidiomycetes, chiefly agarics and Aphyllophorales, were

tested for sporophagous activity. The species were grouped in ecological categories (Table 1). Among the 39 species classified as ectomycorrhiza formers only one, Clitocybe sinopica, attacked, albeit weakly, the Lecchum spores. However, it is doubtful whether C. sinopica is mycorrhizal. It seems always to occur in the vicinity of pine (Harmaja, 1969), but ectomycorrhiza formation has never been demonstrated. None of the four praticoles (ParkerRhodes, 19.51) were sporophagous. Of the 11 coprophiles tested only one, Coprinus

SPOROPHAGY

IN HYMENOMYCETES

77

TABLE 1 Numbers of Sporophagous Species Found in Different Ecological Groups of Hymenomycetes Sporophagous species

Ecological categories

No. of species tested

Strongly active

Weakly active

Inactive species

Ectomycorrhiza formers Praticoles Coprophiles Xylophiles Total number of species

39 4 11 82 136

0 0 1 9 10

1 0 0 14 is

38 4 10 59 III

coma&s, which is not very typical of its group and difficult to classify ecologically, was sporophagous. By far the largest number of sporophagous species was found in the xylophilous, i.e., wood and litter decomposing, species, most of which belonged to the Aphyllophorales. Among the 82 tested species 9 were clearly and strongly active: Lentinellus omphalodes, Gloeophyllum saepiarium, Pluteus cervinus, Lentinus lepideus, Climacocystis borealis, Crepidotus mollis, Heteroporus biennis, Anthrodia heteromorpha, and Byssomerulius corium; a further 14 species showed a weaker reaction by attacking only a few spores or by possessing many hyphae without homing activity. Hn a subsequent series of experiments, basidiospores from different hymenomycete species were exposed to mycelia from three sporophagous fungi, viz., Coprinus comatus, Lentinellus omphalodes, and Pluteus cervinus. These experiments comprised spore collections from 22 ectomycorrhizal and xylophilous species. The white spores from species of the genera Amanita, Lepiota, Luccaria, Mycena, Lactar&, and Russula were left alone by the sporophagous mycelia, whereas the darkcolored spores from the genera Cortinarius, Paxillus, Gomphidius, Suillus, Leccinum, Bole&s, and Thelephora were attacked and destroyed. In a few cases more then one isolate of a sporophagous species was available. Out of

four different strains of Coprinus comaeus three exhibited sporophagy, while one was inactive. Similarly, one of three strains of Lentinellus omphalodes was inactive; the two others were sporophagous. these inactive strains had been k ture for several years, which c that after many transfers on art they had lost their sporophago Comparisons between freshly isolate strains will make dear whether or not this interpretation of the inactivity is correct. Hence, it cannot yet be decided whether sporophagy is a species c~~ra~t~r~st~~ or not. On the other hand, different spore collections from one and the same species sometimes differed in attractivity. In contrast to all other spore prints of Eeccinurn aurantiacum collecled in the aut~rn~ 1983, one, No. 501, was not attacked Coprinus comatus mycelium. however, that the spores fr No. 501 could not be ger usual way (by self mycehum; Fries, 1979); for some unknown reason they were not alive. When tests were made of severalyears-old spore collections which had host their germinability and viability through senescence, they did not show any attractivity to sporophagous myceha. The same held true for spores that had been heating. This indicates that only living spores are attacked by the s~or~~h~g~~~~ fungi. The response of a sporophagous myce-

78

FRIES

AND

lium also depends on the composition of the nutrient agar medium. Although the results could not be quantified, experiments clearly demonstrated that the frequency of sporophagy was highest if the concentration of ammonium tartrate in the medium was kept low, i.e., at 20 to 100 mg * liter-’ and the concentration of glucose kept between 100 and 500 mg * liter-‘. This agrees with the situation in other types of mycoparasitism as shown by Griffith and Barnett (1967). Some sporophagous species responded differently in this respect, however. On a basic medium with 100 mg * liter-’ of glucose both Coprinus comatus and Pluteus cervinus attacked the Leccinum spores, whereas only Coprinus comatus attacked these spores on a medium without glucose. No sporophagy was observed on water agar. The possibility was investigated whether sporophagous mycelia also attack hyphae of species whose spores they parasitized. Four mycelia, representing species with attractive spores (Pluteus cervinus, Paxillus involutus, Leccinum aurantiacum, and Thelephora terrestris), were combined on agar plates with mycelia from five vigorously sporophagous species (Coprinus comatus, Pluteus cervinus, Lentinellus omphalodes, Climacocystis borealis, and Byssomerulis corium). The inocula were placed both on nutrient agar plates with a naked surface and on plates covered with cellophane. In all combinations the two mycelia intermingled without showing any tendency to mycoparasitism or hyphal interference (Ikediugwu et al., 1970). The hyphae appeared to lack the chemical attractiveness possessed by the spores and necessary for triggering an attack from a sporophagous mycelium . In conclusion it can be said that sporophagy is a type of necrotrophic mycoparasitic interaction, where hyphae of one species attack and kill spores, but not hyphal cells, of another species. As yet it has been found only within the hymenomycetes. The

SWEDJEMARK

spores apparently exude a substance which acts as an attractant to sporophagous hyphae of another species nearby. The approach of the hyphae has all the characteristics of a positive chemotropical reaction, and proceeds in the same way as an intrageneric homing reaction in Polyporus dryophilus (Morton and French, 1970) or Leccinum aurantiacum (Fries, 1981). After contacting the spore a sporophagous hypha penetrates the spore wall and apparently assimilates the contents inside. The thinning out of the spore cell wall around the hole made by the sporophagous hypha suggests an enzymatic mechanism for the penetration (Fig. 2d). Like intrageneric basidiospore homing, the process of sporophagy requires living spores. However, unlike the situation in intrageneric basidiospore homing, a sporophagous species does not respond chemotropically to spores of self but to spores from very distantly related species. Furthermore, sporophagy does not lead to plasmogamy with its possible genetic implications, since the attacking hypha retains its cell wall after having penetrated the wall of the assailed basidiospore. Therefore, the sporophagous hypha is not killed by the spore contact, as is often the case after basidiospore (and oidium) homing followed by plasmogamy (Kemp, 1977; Fries, 1981). It is still an open question whether the attacked spore is killed before or after penetration by the sporophagous hypha. It is also still unexplained why only dark-colored, mostly brown, spores are attractive. It might indicate a difference in metabolism between colorless and colored spores resulting in different sorts of exudates. The fact that sporophagy is most conspicuous on media poor in carbon and nitrogen sources suggests that the attacking mycelium obtains nutrients from the spores. Since the phenomenon of sporophagy has been studied only as interactions between hymenomycetes, it is premature to

SPOROPHAGY

IN HYMENOMYCETES

speculate on its possible ecological implications. The basidiospores are the only means of dispersal known in most hymenomycetes, particularly in the ectomycorrhiza-forming species. The fate of the airborne basidiospores after having landed in the soil is still little known. If in addition to some xylophilous hymenomycetes, common saprophytic soil fungi of other taxonomic groups are also sporophagous, the role of the basidiospores as propagules may be less important than is usually assumed. ACKNOWLEDGMENTS The authors thank Anna Aspan for valuable cooperation during an early phase of the work. We are also indebted to Ms. Anette Axen and Mr. Terry Sensenbaugh for skillful technical assistance in producing the electron micrographs. The study was supported by a grant from the Swedish Natural Science Research Council. REFERENCES H. L. 1964. Mycoparasitism.

Mycologin 56: l-19. BARNETT, I-l. L., AND LILLY, V. G. 1958. Parasitism of Calcarisporium parasiticurn on species of Pkysalospora and related fungi. Bull. W. Va. Agric. Exp. Sm. 420 T. ERIKSSON, I., AND RYVARDEN, L. 1973. The Corticiaceae of North Europe, Vol. 2. Fungiflora, Oslo. FRAN&N, A., AND SENSENBAUGH, T. 1983. Fine structure of the apical plate in the larva of the freshwater BARNETT,

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bryozoan Plumatella fungosa (Pallas) (Bryozoa: Phylactolaemata). ZoomorphoIogy 102: U-98. FRIES, N. 1979. The taxon-specific germination reaction in Leccinam. Trans. Brit. Mycol. Sot. 73: 337341. FRIES, N. 1981. Recognition reactions between basidiospores and hyphae in Leccinum. Trans. Brit. Mycol. sot. 77: 9-14. GRIFFITH, N. T., AND BARNETT, H. L. 1967. Mycoparasitism by basidiomycetes in culture. JJ:~coiopin 59: 149- 154. HARMAJA, H. 1969.

The genus Clitocybe (Agaricales) in Fennoscandia. Karstenia 10: 5-f21. TKEDIUGWU, E E. G., DENNIS, C.. AND WEBSTER. .I. 1970. Hyphal interference by Penio.ohoru gigantea against Heterobasidion annosum. Traw. Brir. Mycol. Sot. 54: 307-309. KEMP, R. E 0. 1977. Oidial homing and the taxonomy and speciation of basidiomycetes with special reference to the genus Coprinus. In The Species Concept in Hymenomycetcs (H. Clemencon, Ed.;, pp. 259-273. Cramer, Vaduz. MORTON, H. L., AND FRENCH, D. W. 1970. Attraction toward and penetration of Po/yporus dtyophill4s va:. vzrlpinus basidiospores by hyphae of the same species. Mycolopia 62: 714-720. MOSER, M. 1978. Kleine Ktyptogamenjlora, Basidiomycetes 2 Teil. Die Rohrlinge und Bititterpilze. Fischer, Stuttgart. PARKER-RHODES, A. F. 1951. Tne Basidiomycetes of Skokholm Island. VII. Some floristic distributions. New Phytologist SO: 221-243. RYVARDEN, L. 1976. The Polyporaceae of North EMrope, Vol. 1. Fungiflora, Oslo. RYVARDEN. L. 1978. The Polyporaceae ofNorth Europe, Vol. 2. Fungiflora, Oslo.