Pro(:. Indian Acad. Sci. (Plant Sci.), Vol. 93, No. 3, July 1984, pp. 205-222 Printed in India.

Spore germination in the higher Basidiomycetes

NILS FRIES Institute ofPhysioiogical Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden

Abstraet. This survey of the spore germination requirements in the Hymenomycetes and the Gasteromycetes shows that saprophytes as xylophiles, which decompose wood and forest iitter, and coprophiles, which live on dun 8, usuaUy germinate easily even on simple nutrient media. Species forminger with trees or living as parasites require asa rule more particular conditions for germination. In the mycorrhiza-formers, chiefly aga¡ and boleti, germination can often be induced by exudates from tree roots or certain yeasts, in species of Leccinum by exudate from self mycelium. The heartrot fungi, chiefly those species of AphyUophorales which ale parasites on trees, germinate preferably when exposed to ah increased CO 2 content in the air or to exudates of certain micro-organisms. In many praticolous fungi, wliich are supposed to parasitize roots of grasses and herbs, germination is stimulated by va¡ yeasts. The possibility of interpreting these particular germination conditions as adaptations to a parasitic or symbiotic life is discussed.

Keywords. Xylophilous fungi; coprophilous fungi; praticolous fungi; ectomycorrhiza- formers; root exudates; activated chareoal.

1. Introduction

In the higher Basidiomycetes, i.e., the Hymenomycetes and the Gasteromycetes, dispersal is essentially based on the sexually produced basidiospores. formation and basidiospore germination represent two critical events in the complete life cycle of these fungi. Both processes are accomplished by a sometimes very delicate interplay between internal and external factors, which is often dit¡ to analyse and understand. In this article I shall only deal with spore germination in the higher Basidiomycetes and try to presenta summary of our present knowledge in this field. Ecological aspects will be particularly considered. Complete coverage of the relevant literature is out of the question in a survey of this rather limited extent and the selection must inevitably be somewhat arbitrary, although focussing on groups where induction of spore germination is still problematic. The germination of spores has always been and is still studied principally with the aid ofa light-microscope. A spore is usually said to be germinating when a germ hypha ora germ vesicle can be observed. For practical reasons many researchers state, arbitrarily, that a spore, tobe scored as germinated, must have produced a germ hypha ofa certain length, for instance as long as it is broad or of a l equal to half the diameter of the spore. As regards germination it should be pointed out that viability is not the same as germinability. A spore may be viable, i.e., living, without being able to germinate, because germination in this case only occurs under very special environmental conditions, which are not present. On the other hand, germination takes place of course only with viable spores. Viability and germinability always gradually disappear with time. Therefore it is often desirable to find out how many spores in a stored

!' --2 205 206 Nils Fries collection are really alive, before a germination experiment is started. This is usually done by testing the enzymatic activity in the spore using suitable fluorescent stains (e.o. Stack et al 1975; Yu and Trione 1983; Sutherland and Cohen 1983). In most of the investigations reported in this review the spores were germinating on nutrient agar plates, sometimes in drops of a liquid medium, under sterile (axenic) conditions. In this survey scientific names of fungi are given in the form they were used by the quoted author. In some tases the name most current at present is added within brackets.

2. The pioneers in the study of homobasidiospore germination

Brefeld, the great pioneer and master in the art of growing fungi under controlled conditions, summarized in one of bis last publications the main results ofhis numerous laboratory experiments on spore germination in different ecological catego¡ of Basidiomycetes (Brefeld 1908). He stated that germination ofbasidiospores from fungi growing on dead wood and litter, which they decompose, generally takes place without difficulty even on very simple substrates, as water and water agar. He had also found that fungi living on animal droppings and manure germinate easily. These two catego¡ the xylophilous and the coprophilous fungi, are typical saprophytes, which utilize dead, organic substrates as sources of carbon and energy. In contrast to these fungŸ the soil-inhabiting Basidiomycetes, nowadays recognized chiefly as ectomycorrhiza formers with trees, had always proved to be very refractory in Brefeld's germination experiments. Evidently he based his conclusion on a large number of unsuccessful experiments with these fungi. Equally negative were the results of earlier and contemporary mycologists. Rarely were any experiments with soil- inhabiting fungi explicitly mentioned, but the negative outcome of such experiments may be inferred from the fact that the publications in this field generaUy deal with xylophilous and coprophilous Basidiomycetes, whereas the well-known mushrooms and toadstools are passed over in silence. Eidarn (1875) is an exception in this respect, since he described his fruitless attempts to germinate spores of Amanita, Lactarius, Russula, and Boletus. New attempts were made during the first three decades of the present century to solve the problem of spore germination in the soil-inhabiting or rather the ectomycorrhiza-forming Basidiomycetes. Again, almost all of these attempts were unsuccessful (Duggar 1901; Ferguson 1902; Cool 1912; Levine 1913; Kniep 1913; Romell 1921; Melin 1922; Hammarlund 1923; Vandendries 1933). Only Fuchs (1911) reported positive results, which seem to be the first reliable ones reported in this field of research. Although most of his experiments failed he managed to get germination of spores from the typical ectomycorrhizal Lactarius delicious and also of spores from Hydnum imbricatum, which in all probability forms ectomycorrhiza with . However, his results with L. deliciosus have been called in question by Oort (1974). As will be reported in the following pages, efforts made by several researchers in this difficult field during the last half-century have led to some, yet still limited, success. As regards the two other ecological groups recognized by Brefeld, the xylophiles and the coprophiles, almost every year during the present century has added new examples of species that conform to his rule of easy germinability. The only noticeable Spore germination in the higher Basidiomycetes 207 exceptions have been found among wood-destroyers, which live as parasites on trees. Finally, it has proved motivated to add a fourth ecological category, the praticolous fungi (Parker-Rhodes 1951), to the three just mentioned. The praticolous species were counted among the soil-inhabiting fungi by Brefeld, and as such they had de- monstrated their unwillingness to germinate in the laboratory. These fungi occur on open fields and are not ectomycorrhiza formers. Their mode of nutrition is still largely unrevealed and so are also their requirements for germination on artificial media in the laboratory.

3. Spore germination in different ecological groups

3.1 Xylophilousfungi Spore germination of a considerable number of xylophilous Basidiomycetes had been observed and studied even early in the 19th century. There is not space here to enumerate all those workers who about a hundred years ago cont¡ to ah increased insight into the life cycle of wood-decomposing fungi. Only a few outstanding names are H Hoffmann, E Eidam, Ph. van Tieghem, and O Brefeld. More detailed information on this period can be obtained elsewhere (see Bavendamm 1936; Fries 1943 for more details). After the turn of the century the interest in these generally easily culfivated fungi increased, partly because of their economic importance as wood-destroyers. Later, when Bensaude (1918) and Kniep (1920) discovered the sexuality in Basidiomycetes, ah interest in the genetics of these fungi arose. Monosporous cultures were a necessary prerequisite for such studies and therefore the xylophilous---together with the coprophilous--fungi became objects ofchoice because of their readiness to germinate. Furthermore, since the 1940's taxonomists have increasingly made use ofcrossing tests between monosporous mycelia to elucidate taxonomic relationships. Most of these studies were made with species belonging to Aphyllophorales, the dominating group among the xylophilous fungi. As an example may be mentioned Boidin's (1958) comprehensive, biotaxonomic investigation of 147 species and subspecies of Hydnaceae and Corticiacae. Spore germination occurred in 125 species and mono- karyons could be isolated in 87. It is perhaps signi¡ that in the Gasteromycetes, where the spores are usually difficult to germinate, the rather few easily germinating species are all found in the xylophilous and coprophilous families Nidulariaceae and Sphaerobolaceae (Hoffmann 1859; Hesse 1876; Eidam 1877; Brodie 1975; and others). The number of xylophilous species which have been brought to germination in vitro is now immense. It may suffice to refer to some relevant overviews (Whitehouse 1949; Kneebone 1950; Merrill 1970). It must be remembered that xylophilous fungi not only attack cellulose and/or lignin in such materials as trees, timber, and wooden houses, but also, although less conspicuously, the litter on the forest soils. Spore germination in these litter- decomposers, many ofwhich are small aga¡ also seems to be rather unproblematic. Some of the rather few cases where germination in vitro has failed have led to efforts to elucidate the reason. In Merulius (Serpula) lacrymans some authors reported successful results (e.g., Hartig (1885)), whereas others failed. The reason for the failure was evidently that the pH of the media used had been too high (M611er 1903; Falck 1912). Germination occurred on most media if a suitable amount of an organic or 208 Nils Fries inorganic acid was added, malic acid being recommended as particularly efficient (Findlay 1932; Harmsen 1960). The only group of xylophilic Hymenomycetes which still presents serious difficulties for workers trying to induce in vitro spore germination, comprises some species of Polyporaceae, which live as parasites on trees. White (1920) found that Polyporus applanatus, which causes heartrot in various trees, never germinated at more than 1 and sometimes not at all. Later workers found that up to 78 % germination could be obtained ir the spores were situated close to growing mycelia of Ceratocystis sp. or colonies of yeasts and bacteria (Brown and Merrill 1973). The effect was caused by volatile substances, still unidentified, which were produced by the adjacent activator organisms. In Polyporus dryophilus and related spr the germination-inducing mechanism seems to be somewhat more complicated. Bailey (1941) reported that germination occurred only on a malt extract medium and only under the condition that a mycelium of the fungus had previously been grown on the medium. Spores also germinated in water drops to which the same medium, filtered and sterilized, had been added. However, Mog and Morton (1970) induced up to 92 ~ germination in this fungus by increasing the CO2 content to 65 ~o of the air phase. Later, Morton and French (1974) showed that the effect of other microorganisms, especially Ceratocystis fagacearum, could not be due only to their CO 2 production but also to volatile organic emanations of unknown identity. In Fomes rimosus, another heartrot fungus, a maximum germination of 41 ~ could be obtained by keeping the spores in an atm0sphere of I00 ~ CO z. Tests with x4CO2 showed that the carbon dioxide was fixed by the spores (Mog and Morton 1970). According to Hintikka (1970) the germination-promoting effect of ah enhanced CO2 content in the air is most pronounced in those wood- decomposing fungi which in nature inhabit living trees as parasites. Among heartrot fungi, which have never germinated on artificial media, Merrill (1970) mentions Fomes everhartii, Polyporus hispidus, P. lucidus, and P. tsuoae. There are indications that microorganisms of various sorts influence the germi- nation of xylophilous fungŸnot only under laboratory conditions but also in nature. Paine (1968) reported that spore germination in Polyporus betulinus, Fomes pinicola, and F. subroseus was higher on bluestained (i.e., infected with blue-stain fungi) than on unstained branch stubs from all tested species of trees. Evidently some microorganisms inhabiting dead coniferous branch stub wood do not retard basidiospore germination but rather stimulate it. From this point of view it seems suggestive that bark extracts from diseased (infected) roots of spruce support better spore germinations in Polyporus tomentosus than do bark extracts from healthy roots (Whitney and Bohaychuk 1971). The positive effect of wood saprophytes on germination may not necessarily depend on production of stimulatory substances but on a removal of toxic compounds, as was shown by Carey and Savory (unpublished)in Trichoderma viride and blue stain fungi. Similarly, water extracts from dead branches of aspen, the host species of Fomes igniarius var. populinus, stimulated spore germination of this parasite, whereas water extracts from living branches had no positive effect (Wall and Kuntz 1964). Whether the stimulatory substances in the dead branches had been produced by the tree or by microorganisms in the decaying wood could not be decided. In certain truly saprophytic xylophiles germination requires special conditions as well. Boidin (1958) found that in a few species of AphyUophorales gerrainating spores Spore oermination in the higher Basidiomycetes 209 were observed only together with colonies of bacte¡ belonging to the Bacillus brevis group. Flammula (Pholiota) alnicola is one of the rather few xylophilous agarics which are difficult to germinate. It is a saprophyte on wood but lives sometimes also asa parasite on trees, which are infected through the root system. Denyer (1960) reported that germination on malt agar is slow and never exceeds 1%. The germination percentage could be doubled if the spores were stored at -7~ for 10 weeks to 13 months. Lower temperatures were inefficient. The closely related F. conissans reacted similarly, the effect of the cold treatment in this case being even more conspicuous. It would be interesting to test whether the spores also in these cases are susceptible to the influence of CO 2 and adjacent microorganism colonies. As mentioned earlier, spore germination in the litter-decomposing xylophilous fungi generally takes place easily and without any particular pretentions on the medium. As examples may be mentioned two genera specialized on this mode of living: Marasmius (Lindeberg 1944) and Mycena (Fries 1949). Most tested species in both genera germinated even in distilled water and on water agar, the germination in some species starting within a few hours. Still, some authors have reported difficulties with species of Mycena (Quintanilha 1944; Quintanilha et al 1941), one reason possibly being that the concentration of ammonium ions in the nutrient medium used had been too high, since germination in some Mycena species is totaUy inhibited in this way (Fries 1949).

3.2 Coprophilous fungŸ From a nut¡ point of view the coprophilous fungi do not differ from the litter- decomposing xylophiles. Like those they are--as far as we know--capable of decomposing cellulose and/or lignin, although they avoid wood and prefer the less compact substrate offered by dung and manure, which is also richer in nitrogenous compounds. Typical coprophilous species are those of Coprinus, Panaeolus, Psilocybe and Bolbitius. Since many of them forra fruit-bodies in culture and grow rapidly they have been frequently utilized for genetic studies. Almost all of them germinate within a few hours or days and at a high pr This can be concluded from the fact that the geneticists working with these fungi very rarely have reported any difficulties with spore germination (Vandendries 1923; Brunswik 1924; Quintanilha 1944; Lange 1952; Kemp 1975). There is one coprophilous fungus, however, whose spore germination has intrigued and has been studied by more mycologists than any other, namely the edible mushroom, Agaricus bisporus. The reason for this interest is its great practical importance asa commercially produced and appreciated vegetable and the difficulties early researchers met with in growing it from germinated spores. These difficulties are nowadays difficult to understand, since germination occurs easily on most common, slightly acidic, agar media ifthe spores have been incubated upon them for one to three weeks (Hoffmann 1860; Ferguson 1902; Falck and Falck 1924). Some failures might have been due to the occurrence of strains or morphologically slightly divergent species with poor germination capacity. Furthermore, the spores cast from one and the same fruit-body often differ considerably in germinability depending on the age of the fruit- body (Cayley 1936). During the course of these early studies Ferguson (1902) observed that germination 210 Nils Fries started earlier if a piece of a growing mycelium was present close to the spores. This simple measure has now become a routine among workers in this field (e.g. Elliott and Wood 1978) who want to secure a satisfactory outcome of germinated spores. Further studies by Hutchinson and coUaborators (Hutchinson 1971; McTeague et al 1959; LSsel 1964) revealed that the germination-stimulating substance exuded from the Agaricus mycelium was also produced by yeasts and other fungi. Being volatile it diffused through air to the spores in the vicinity. It could finally be identified as isovaleric acid. Its mode of action was skiUfully elucidated by Rast and St~uble (1970). Briefly, the isovaleric acid triggers germination of the spores by overcoming the self- inhibition caused by metabolically produced, internal carbon dioxide. By a carboxy- lation reaction this carbon dioxide is removed by being bound to B-methylcrotonyl- CoA, formed from isovaleric acid. As yet this is the only case where the biochemical mechanism behind the germination-inducing effect of a specific compound has become fully understood. Volvariella volvacea is another coprophilous fungus which ought to be mentioned because of its particular spore germination requirements. This is, like Agaricus bisporus, an edible fungus, extensively grown in the tropics. Thriving well on decaying plant material, especially paddy straw, it has a less coprophilic character than Agaricus and may justas well be placed among the litter-decomposers. Chang and Chu (1969) found that spores placed directly on agar plates germinated relatively well, but that presoaking with water increased the percentage germination from ca 40 ~o-85 ~. The soaking effect was explained as either a breakage of permeability barriers or a washing off of inhibitory substances in the spores. The optimum germination temperature was as high as 40~ This could be interpreted asa mild heat shock, stimulatory to spore germination in this species.

3.3 Praticolous fungi The ecology of the praticolous fungi is insufficiently known. They are characterized by growing outside the forests, on meadows and lawns, and probably living on dead and dying roots of grasses and herbs. Their nutritional capacities have been studied chiefly on species close to the coprophilic group, e.g., Agaricus species. Typical representatives of the praticoles are the Lycoperdaceae (the ) and many species of Hygrophorus among the agarics. Although the borderline towards the other ecological categories, the xylophilous and coprophilous fungi, is diffuse, the praticoles distinguish themselves by being exceptionally adverse to germination in vitro. The numerous and common species of Lycoperdaceae with their enormous basidiospore production resisted all attempts to induce spore germination for almost a century. Some claims of success proved to be false alarms and could not be repeated (Hoffmann 1859, 1860; Swartz 1928). Constantly negative results were obtained by, among others, Brefeld (1877), De Bary (1884), Ferguson (1902), Cool (1912), and Kaufmann (1934). The first fruitful experiment was the result ofa happy coincidence (Fries 1941). On a malt extract agar plate sown with spores of umbrinum some contami- nations appeared, probably originating together with the spores from the fruit-body. One of them was an unidentified yeast. Close to this deveioping yeast colony a few germinating Lycoperdon spores could be seen. Repeated experiments gave the same Spore germination in the higher Basidiomycetes 211 result and also demonstrated that germination started after five days. Some other yeasts, especially species of Torulopsis, also proved capable of inducing germination. Thus, four more Lycoperdon species were germinated. Moreover, ir was shown that malt extract agar plates on which Torulopsis, or some other red yeast, had grown permitted germination of Lycoperdon spores even after removal of the yeast from the surface and after a renewed autoclaving of the substrate. From experiments with synthetic nutfient media it could be concluded that the activator yeast not only produced a germination-inducing substance (or substances) but also eliminated a germination-inhibiting compound, which otherwise could be removed only by a thorough ¡ of the agar before autoclaving. Similar effects, but less striking, were produced by some filamentous fungi. Some twenty years later Bulmer and Beneke started a more comprehensive investigation of the spore germination conditions in Gasteromycetes. Through their careful and elaborate expe¡ much new and valuable information was obtained (Bulmer and Beneke 1961, 1964; Bulmer 1964). With red yeast, mainly Rhodotorula raucilaoinosa, germination was induced in about 50 species of Gasteromycetes, the majofity being praticoles of the genera Calvatia, Bovista, and Lycoperdon. In all cases the percentage germination was extremely low, rarely above 0-1%. Interestingly enough, germination was observed in Calvatia oig~ntea even without Rhodotorula if the spores were incubated in shake culture itasks with barley extract medium or Calvatia extract medium. After such treatments approximately 1 spore germinated out of 20 millions. In agar plate tests malt extract was the most efficient component, and with Rhodotorula up to 3 spores per million germinated in Calvatia oioantea. The spores proved capable of retaining their germinability for several years in the dried sporophores. Later experiments by Wilson and Beneke (1966) corroborated some of these results, notably the importance of malt extract, and gave new information on the role of carbon and nitrogen sources in the germination process. Several species of Rhodotorula were tested for activity. Not only red strains, but also yeUow ones, promoted germination in Calvatia goantea. Generally, however, the species were the more active the redder they were. When turning to the praticolous agarics very little can be reported of their modes of basidiospore germination. Among the dominating genera within this ecological category the following may be mentioned: Clitocybe, Entoloma, Nolanea, Galera, and Hygrophorus (Parker-Rhodes 1951). According to my own (unpublished) experiences, the Hyorophorus species differ widely from each other as to their ability to germinate in vitro. In H. (Caraarophyllus) niveus-viroineus group as well as in H. (Hyorocybe) psittacinus, spores often germinate on synthetic nut¡ agar without any activator organism. The germinability of the spores seems to differ considerably from one basidiocarp to another. In Clitocybe and Hyorophorus ir is often hard to judge which species are really true praticoles; some may be facultative ectomycorrhiza formers.

3.4 Ectomycorrhiza formers Most of Brefeld's soil-inhabiting fungi are nowadays recognized as ectomycorrhiza formers with forest trees. They represent the great majority of the species in certain genera of (e.g., Tricholoma, Amanita, Inocybe, Hebeloma and Cortinarius), of Russulales (Russula and Lactarius), and almost all genera of Boletales (e.g., Boletus, 212 Nils Fries

Suillus, Leccinum, Paxillus, and Gomphidius). Some representatives are found in the Gasteromycetes (e.g., Scleroderma and Rhizopoffon) and the Aphyllophorales (e.g., Thelephora). As mentioned earlier, Fuchs (1911) was the first to see germinating spores of a typical ectomycorrhiza-forming fungus, namely Lactarius deliciosus. The germination evidently occurred without any pretreatments of the spores or any particular supplements to the medium. Nobody seemed to have been able to repeat it, until Kneebone (1950) induced Lactarius luteolus to germinate after having stored the spores at -6~ for 135 days. Hammarlund (1923) spent several years in attempts to germinate Boletus (Suill~) grevillei by trying innumerable combinations of nutrient media and variations in other environmental conditions. Germinating spores were found in three experiments, but apparently without any demonstrable correlation to the prevailing conditions. The results could not be repeated. Asa consequence of the experiments with praticolous Gasteromycetes already desc¡ the effect of yeast colonies on germination was tested by Fries (1941) also with mycorrhizal species, like Suillus luteus. These experiments met with success and their simple technique proved applicable to other mycorrhizal species within several genera of Agaricales and Boletaceae. Thus, thanks to the germination-inducing power of Torulopsis sanguŸ (and later Rhodotorula glutinis), germinating spores and monosporous mycelia could be obtained from several species belonging to the genera Amanita, Tricholoma, Clitopilus, and Suillus (F¡ 1943). However, the germination induced in this way was usually characterized by being slow and sparse, the percentage seldom exceeding 1 There were still many mycorrhizal fungi which did not respond at all to the influence of Rhodotorula. Since some circumstances indicated the presence of inhibitory factors in the nut¡ agar media employed, steps were taken to identify and remove these inhibitors. First it was found in experiments with species of Suillus that germination is very sensitive to the content of ammonium ions in the substrate. A radical reduction of the ammonium concentration considerably improved both tate and percentage of germination in Suillus (Fries 1976) and, as was later found, also in other genera. Later it was discovered that germination in various ectomycorrhizal fungi was prevented by an inhibitor in agar which is formed from agarose during the autoclaving of the agar medium. The inhibitor could be removed by activated charcoal (F¡ 1978) and has been identified as a weak organic acid chiefly active on mycorrhiza-forming Hymenomycetes (Bjurman 1984). This inhibitor is not identical with the one earlier mentioned, which is not formed from agar by autoclaving and affects germination in Lycoperdon (F¡ 1941). By relieving the nutrient agar media from these inhibitors and using Rhodotorula glutinis as an inductor organism, germination could be induced in a further number of ectomycorrhizal species, e.g., Laccaria laccata (F¡ 1977), Lactarius helvus, Paxillus involutus, Leccinum scabrum (Fries 1978), and Cantharellus cibarius (Fries 1979b), and also improved the rate of germination in many others. The practising of these principles proved to be particularly fruitful in the Boletaceae genera Boletus, Leccinum, Tylopilus, and Suillus, where 23 of 25 tested species could be brought to germination (F¡ 1983 c) (table 1). Ir is evident that there are great differences among the many genera of ectomycor- rhizal fungi as regards their response to both inhibitory and stimulatory factors. Oort Spore germination in the higher Basidiomycetes 213

T,,bir 1. Effect of plant roots, red yeasts (Rhodotorula) and sr mycr on the germination of basidiospores of ectomycorrhizalfungi. (Revised from Fd 1981b).

No. Pine or bit'eh Rhodotorula Sr addition seedling roots glutinis mycelium Spr a b a b a b a b

Leccinum spp. x ..... (+) _ + Thelephora terrestris ~ - - + + (+) (+) (+) (+) Paxillus involutus - - - + - + - + Cantharellus cibarius ..... + - (+) Lactarius spp. 2 ..... + - - Hebeloma spp. 3 (+) - + + .... Suillu$ spp. 4 (-t-) (+) + + - (+) Laccaria laccata - - - + -- + Tricholoma spp. ~ + + Russu/a spp. z ......

+ : gr (+): sparse or slow gerrnination; - : no germination; .: not tested, a: agar medium not treated with charcoal, b: agar medium treated with charcoal, t: Leccinum $cabrum, L. hoiopus, L. aurantiacum, L. ver$ipelle, L. variicolor, L. vulpinum. 2: Lactariu~ helvu~, L. torminosus, L. deterrimu& 3: Hebeloma mesphaeum, H. crustuliniforme. 4: Suillus luteus, S. granulatus, S. variegatus. 5: Tricholoma equestre, T. imbricatum, T. pessundatum, 7". terreum. ~ : Russula emetica, R. decolorans. 7: Birraux and F¡ (1981).

(1974) found that some species of Lactarius germinated when exposed to volatile exudations from Ceratocystis fagacearum, a fungus which already has been mentioned as an inducer of germination in two xylophilous Polyporus species. Other Lactarius species responded positively to Rhodotorula and required charcoal-treated agar for a substrate (Fries 1981 b). At the end of the 1950's Melin caUed attention to the influence of tree root exudates on ectomycorrhiza-forming fungi. These exudates promoted the mycelial growth rate in several species (the "M factor"), but also sometimes affected spore germination. He reported observations of germinating spores from species of Suillus, Amanita, Lactarius and Paxillus, when exposed to exudates from pine and tomato roots. When he used the same technique with spores of Cortinarius and Russula species his positive results were most impressive. Some Russula species responded to both pine and tomato roots, others only to pine roots (Melin 1959, 1962). Many attempts have been made to repeat Melin's experiments, but always without success as regards the latter genera. However, there are other, later observations which also point to a germination-inducing effect of tree roots. The only existing picture of germinating Russula spores was taken from a flask culture of a birch plant, where the spores were situated close to the root surface (Heinemann and Gaie 1979). In fact, germination-inducing effects of roots have been observed or presumed by several authors. Germination in Hebeloma spp. was strongly stimulated by pine roots (Fries and Birraux 1980) and germination in Thelephora terrestris by pine and birch roots (Birraux and Fries 1981). These experiments were performed on agar plates. Roots of herbs were inactive (table 1). By adding spore suspensions to axenically grown tree seedlings ectomycorrhizae 214 Nils Fries were formed, which demonstrated that the spores---from species of Scleroderma, Thelephora, Rhizopogon, Laccaria, and Pisolithus--actually had germinated and formed mycelia, although the germination process could not be observed (Thapar et al 1967; Marx and Ross 1970; Theodorou and Bowen 1973; Stack et al 1975; Marx 1976). The experiments made by Orlos and Twarowska (1965) are more dit¡ to interpret. They found that if spores ofBoletus edulis and Suillus luteus were placed on glass slides and buried in forest soil, germination often took place after some time. For unexplained reasons it occurred only in certain experiments, however. These germinations could have been due to volatile or diffusible exudates from roots of the forest trees or from soil microorganisms. The most striking example of an exceedingly specialized, but also very efficient, system for germination induction was found in the Boletaceae genus Leccinum (F¡ 1979 a, 1981 a, 1973 b). Like many other ectomycorrhizal fungi the Leccinum species can be brought to germination, although as usual a very poor one, under the influence of Rhodotorula glutinis. In contrast, a Leccinum mycelium growing among the Leccinum spores on an inhibitor-free nut¡ agar plate induces germination of the spores within a few days at a percentage nearly always above 10 9£ and often above 50 ~. However, a prerequisite is that the spores belong to the same species group as that of the mycelium: either to the L. aurantiacum (6 species) or to the L. scabrum (2 species) group. Crosswise combinations always give negative results. Further investigations in this peculiar system have demonstrated that the germi- nation induction is due to a non-volatile, diffusible pheromone produced by the mycelium and recognised by the closely related spores, which respond by developing a germination vesicle (Fries 1981 a). The chemical constitution of this pheromone has not yet been identi¡ In a search for analogous modes of germination induction in other genera than Leccinum I have studied the possible effect of self mycelium on spore germination in various mycorrhizal fungi. In some cases, e.g. in Laccaria spp., a positive influence on germination was observed, although unspeci¡ and relatively weak, more like that of Rhodotorula (F¡ 1983a). Less spectacular but apparently without any equivalent among other mycorrhiza formers is the effect ofamino acids on the spore germination in Suillus. In S. luteus, S. granulatus, and S. variegatus germination is strongly stimulated by casein hydrolysate and by particular mixtures of certain amino acids, glutamic acid being ah especially active component (Fries 1976). How the amino acids affect germination is stiU an open question. Only few ectomycorrhiza-forming hymenomycetes have been found which are capable ofgerminating on synthetic nutrient media without any activator organistas or without any special organic supplements. To these fungi belong some Tricholoma (Fries 1943) and Hebeloma species, in the latter case chiefly members of the section Denudata, subsection C (Bruchet 1973). After long incubation "spontaneous" germinations sometimes occur also in species of the genera Suillus and Thelephora. Finally it should be mentioned that according to Voglino (1895) spores of Inocybe, Russula and Lactarius species eaten and digested by slugs germinate after passage through the digestive tract. The validity of his results has been called in question, however, and the experiments have never been successfully repeated (Ferguson 1902; Buller 1909). Spore germination in the higher Basidiomycetes 215

4. Comments on some of the factors controlling spore germination and associated phenomena

4.1 Duration of germinability The duration of germinability varŸ from one species to another and sometimes also within the species. A few examples may suflice to illustrate this. Preservation at a sub-zero temperature is known to prolong the viability of the spores, at least in many species. Spores of Lycoperdon pusillum germinated even after 4 yr if stored at -18~ (Bulmer and Beneke 1964). In this species the percentage germination slowly increased du¡ storage up to an age of at least two and halfyears irrespective of the temperature. This occurred at all three storage temperatures tested: 26~ 10~ and - 18~ In L. curtis¡ on the other hand, almost no spores germinated after two anda half yr, not even those which had been kept at -18~ In contrast, the spores of all tested Laccaria species lose their germination capacity within only three months, even if preserved at - 18~ (Fries 1983a). Species of Suillus remain germinable for about half ayear at low temperature storage (Fries 1943) and species of Leccinum up to ten months (Fries 1978). In all these cases the germinability decreases faster if the spore prints are kept at a higher temperature, i.e., at 4~ or 25 ~C. In Fomes igniarius var. populinus studied by Good and Spanis (1958), spores from different differed considerably as to durability of germination power. Collections from some basidiocarps lost their germinability within 10 days, others after 40 days, whereas the spores from one of the basidiocarps (out of seven tested) still remained germinable after 80 days. In a very comprehensive and careful study, Whitney (1966) demonstrated that spores of Polyporus tomentosus, when kept at -18~ generally ceased to germinate after six to eight months, but before that they exhibited a percentage germination after three months which was higher than the original one. These results were based on average values from 332 tested spore collections. The variabilitv in the collections could be extraordinary: in some of them no spores germinated, in others germinability was seemingly lost but returned after some months, etc. Still, Whitney's results suggest the existente of an after-ripening process in P. tomentosus, analogous to that noticed by Bulmer and Beneke (1964) in Lycoperdon pusillum. However, real and total dormancy, which requires after-ripening or intervention of external factors to be overcome, has never been asserted in Basidiomycetes. It may be that Flammula alnicola and F. conissans represent exceptions to this rule, since they were found to germinate only after treatment at -7~ (Denyer 1960). It should be kept in mind that spores during storage may acquire an increased sensitivity to the conditions under which their germinability is tested. Watling (1963) demonstrated that herbarium collections of spores from some species of Bolbitiaceae only germinated if they had been incubated foi" several hours in a water-saturated atmosphere before they were plated out on nut¡ agar.

4.2 Intra-species and intra-basidiocarp differences As mentioned previously, spore collections from different basidiocarps within the same species often differ markedly in germination frequency and environmental requirements. Therefore, the characterization of a species from the point of view of 216 Nils Fries germination behaviour should not be based on experiments with a single spore collection only, but on several collections from different basidiocarps. As examples of this intraspecific diversity, references may be made to the studies by White (1920) in Polyporus applanatus, by Bulmer and Beneke (1961) in Calvatia gigantea, Bulmer and Beneke (1964) in species of Lycoperdon, Fries (1943, 1976, and unpublished) in Amanita rubescens, Boletus (Suillus) luteus, and Hygrophorus species, and Whitney (1966) in Polyporus tomentosus. The rate of germination may vary not only among different basidiocarps from the same species, but also among different parts of the hymenium in one and the same basidiocarp. Cayley (1936) observed that in Psalliota (Agaricus) spp. the germinability of spores depends on the age of the basidiocarp when the spores were cast. Whitney (1966) found the same in Polyporus tomentosus, where the best spore production was from 4-week-old basidiocarps and the best germination was in spores from 3-week-old basidiocarps. In Lycoperdon pusillum, on the other hand, Bulmer and Beneke (1964) could not ¡ any significant differences in germinability among spores from different areas (top, side, and bottom) of the basidiocarps studied.

4.3 The role of some physical environmental factors In the higher Basidiomycetes the optimum temperature for spore germination asa rule seems to be about the same as the optimum temperature for mycelial growth (e.g., Merril11970; McCracken 1982). An exception is the coprophilous Volvariella volvacea, which has an optimum germination temperature of40~ (Chang and Chu 1969). The spores of the similarly coprophilous Cyathus stercoreus (Gasteromycetes) require two days incubation at 40~ for good germination (Brodie 1975). The importance of a low storage temperature for the maintenance of germinability has already been discussed. The influence of light on spore germination has been very little studied in these higher fungi. Apart from some old, occasional observations on the harmful consequences of direct sunlight, the only methodical investigation of light effects on spore germination seems to have been made by McCracken (1982) with Pleurotus sapidus. He showed that in this fungus germination is strongly inhibited by white and blue, but not by yellow and red light. Thus, wavelengths below about 500 nm inhibit germination.

4.4 Interactions between spores and hyphae In the foregoing several cases have been mentioned where germination of spores has been induced by substances produced from mycelia, yeast colonies or bacteria in the vicinity of the spores. It. ought to be added that chemically transmitted influences sometimes also go the other way: germinating or germinable spores produce substances which entice hyphae nearby to grow towards the spore, evidently by a positive chemotropic reaction. This mode of reaction was ¡ observed by Morton and French (1970) in Polyporus dryophilus and by Bistis (1970) in Clitocybe truncicola. It was later found also in Schizophyllum commune (Voorhees and Peterson 1983). The change of growth direction of the attracted hypha (or hyphae) leads to a conjunction between the hyphal tip cell and the spore, probably ending up with plasmogamy. The phenomenon (with oidia as attractants) was further studied in many species of Spore germination in the higher Basidiomycetes 217

Coprinus by Kemp (1975, 1977), who described the reaction as "homing". It also occurs among ectomycorrhizal Hymenomycetes, as Leccinum (Ffies 198 la), Laccaria (Fries 1983a), and Thelephora (Birraux, unpublished). In the present context there is no reason to go into greater detail, but reference may be made to the interesting correlations found between homing reactions on one hand and and genetics on the other (Kemp 1977; Fries 1983b). So far it seems that homing occurs only with living spores, and in Leccinum spp. only with spores which have already germinated. Another case of interplay between spores and hyphae is the recently discovered reaction which is provisionaUy called "sporophagy" (Fries, unpublished). As yet it has only been observed and studied in the higher Basidiomycetes, but might well occur also in other groups of fungi. Sporophagy implies that hyphae of one species attack basidiospores ofanother species, twist around them, and to all appearances penetrate the spore wall. About 20 sporophagous species have as yet been found, most of them xylophilous. Dead spores are not attacked, nor hyphae, whether alive or dead. It appears that relatively large and coloured basidiospores are most attractive. Group-size effects among densely sown spores are well-known in some Phycomycetes and Fungi imperfecti. They manifest themselves as mutual germination stimulations or inhibitions (for a review see Robinson 1973). In the higher Basidiomycetes such effects seem to be less common. However, according to Kª (1938) spores of certain species of Mycena germinate rapidly only when they were sown out densely on the agar surface. More isolated spores may germinate but the germ hypha soon stops growing and dies. Boidin (1958) made similar observations with some xylophilous species of Aphyllophorales. This need of density for germination made the isolation of monosporous mycelial dilficult. In Calvatia gigantea both very low and very high spore densities were unfavourable for germination, the optimum being between 1 and 5 million spores per agar plate (Bulmer and Beneke 1962).

5. Conclusions

The present, accumulated knowledge of spore germination in the Homobasidio- mycetes gives a picture of st¡ diversity as far as germination conditions are concemr This diversity is most pronounced among those fungi which in nature live as parasites on, or as symbionts with, higher plants, as is the case with the heartrot fungŸ and the ectomycorrhiza formers. Many of the praticolous mushrooms and puffballs, whose ecology is still little understood, have also proved to require very special conditions for germination and could therefore, per analogiam, be supposed to live as parasites on herb or grass roots. Would it therefore be justified to interpret these special requirements for germi- nation as adaptations to a parasitic or symbiotic mode of life? In several cases such interpretations have br suggested. The spores of Flammula alnicola and F. conissans attain their optimal germination if stored at -7~ for 10 weeks up to one year, which fits in with their mission of spending a winter in the soil and then attacking a tree-root in the spring or the summer (Denyer 1960). In Polyporus dryophilus the spores require an increased CO z content in the ambient atmosphere, which can be found in their normal infection court: the decaying wood in branch stubs of living trees (Jensen 1969; Morton and French 1974). This seems to be the case with tree parasites in general (Hintikka 1970). In Fomes igniarius var. populinus the germination of the spores is 218 Nils Fries stimulated by substances present in the dead branches of the aspen tree, the host ofthis fungus (Wall and Kuntz 1964). Other va¡ of F. igniarius, which are parasites on other trees, are inhibited in their germination by the same substances from aspen wood. Selectively stimulatory or antifungal substances may thus be a factor favou¡ or preventing the establishment ofan infection via dead branches or stubs ofa tree. When leaving the basidiocarp the spores of Fomes applanatus carrY with them numerous infections, some of which are known to stimulate spore germination (Brown and Mer¡ 1973). It is also attractive to consider the cases ofspore germination induced by root exudates, which occurs in some ectomycorrhizal fungi, as an adaptation to symbiotic life. More examples can be found in the literature. However, it is of course next to impossible to prove or disprove the correctness of such ecological interpretations. A conspicuous trait in those species which can be germinated only with more or less speci¡ and often complicated measures, is their very low percentage germination. AII tested Lycoperdaceae and almost all Boletaceae species show this. Does this reluctant response reflect an int¡ low germination power or does it simply show that the methods worked out by the mycologists are still poor substitutes for the true, natural mechanism--picklocks instead of the real key, which still remains to be found? The case of Leccinum is perhaps a warning against running into premature conclusions: when the effect of self mycelium was ¡ discovered, the attainable germination percentage rose to 50 9£ and higher, from the scarcely 0.1 Y/o earlier produced with Rhodotorula species as inducers (Fries 1979a). To elucidate aU these speci¡ mechanisms for spore germination one should probably try to look more into the situation in nature (Fries 1981 b). Leccinum may also from this point ofview be taken as an example. To function as an efficient distributor of the species to new habitats, other inducers than the own mycelium must be in action. However, so far extensive testings of various sorts of microorganisms isolated from forest soil have not led to the discovery of any new germination inducers more powerful than the rather inefficient Rhodotorula (Fries unpublished). It is hard to imagine that such more efficient factors do not exist, which would induce germination of Leccinum spores in the absence of self mycelium. Occasional observations of mass germination in Russula species (Melin 1959, 1962; Heinemann and Gaie 1979) and in Boletus edulis (Orlos and Twarowska 1965) indicate that very efficient germination-inducing mechanisms may be found operating in the soil. Their nature is still unknown, however. It may be volatile exudates from roots or from micro- organisms, which as yet have escaped isolation or are difficult to cultivate in vitro for activity tests. On the other hand it may be significant that many fungi which are difficult to germinate are also difficult to cultivate as vegetative mycelia derived from basidiocarp .tissues. Several species of Lactarius can be germinated as well as cultivated, even ifwith difficulty (Oort 1974; Fries 1981b), whereas the Russula species are extremely refractory in both respects. A profitable mode of attacking the spore germination problem in Russula, Cortinarius, Inocybe, Gomphidius and other recalcitrant genera would perhaps be to improve the medium for vegetative growth. When satisfactory nutrient media have been composed, they might form a better basis also for spore germination experiments with species of tbese genera. The germination of the spores does not necessarily depend only on the presence of more or less specific substances of biological origin but also on delicately adjusted combinations of chemical and physical Spore germination in the higher Basidiomycetes 219 environmental factors, conditions which may be difficult to detect and reconstruct in vitro.

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