Basidiospore germination in some mycorrhiza-forming hymenomycetes

June 1978

Vol. 70, Part 3 Trans. Br. mycol. Soc. 70 (3) 319-324 (1978)

Printed in Great Britain

BASIDIOSPORE GERMINATION IN SOME MYCORRHIZAFORMING HYMENOMYCETES By NILS FRIES Institute of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, S weden Basidiospores from five randomly selected species of mycorrhiza-forming Hymenomycetes, viz. Laccaria laccata, Amanita muscaria, Lactarius helous, Paxillus inooiutus, and Leccinum scabrum, did not germinate on any agar media tested (except for very few and irregular germinations in A. muscaria), nor did an addition of activated charcoal or a living Rhodotorula glutinis colony induce spore germination. However, with these two supplements together on the same agar plate slight germination occurred in all five species. In P. inoolutus percentage germination was further increased by a volatile factor produced by the mycelium of P. inuolutus. In L. scabrum a non-volatile substance exuded from its own mycelium strongly promoted germ vesicle and germ tube formation in the presence of activated charcoal. In both of these mycelial germination-inducing factors a certain specificity was indicated. under the lid of a sterilized glass Petri dish, so that the spores were cast onto the bottom of the dish . After some hours the lid was replaced by another, sterile one and itself transferred to anew, sterilized bottom part of a Petri dish. Thus several spore prints from the same fruit-body could be collected in separate Petri dishes. The Petri dishes with the spore prints were kept in darkness at 4 "C. The spores retained their germinability for three months in the case of L laccata, A . muscaria and L. helous, for 5 months with P. inoolutus, and for at least 10 months with L. scabrum. This limited length oflife of the spores made it impossible to conduct more than a few germination experiments with spores from the same collection. Agar medium The germination tests were always performed in 9 em diam plastic Petri dishes containing 15 ml of medium. The medium contained glucose 4 g, ammonium tartrate 1 g, KH ZP0 4 0'2 g, MgS04.7HzO 0'1 g, NaCl 20 mg, CaCl z.2HzO 26 mg, ZnSO•. 7H zO MATERIAL AND METHODS 0'88 mg, MnS04.4HzO 0·81 mg, FeCl a .6H zO Basidiospore samples 0 '8 mg, malt extract (Difco) 1 g, agar (Difco) 15 g, Species were selected which were known to be a vitamin mixture composed of thiamin 100 J.lg, capable of growing on agar media as tissue pyridoxine 100 J.lg, riboflavin 100 J.lg, biotin 25 J.lg, cultures. They were : Laccaria laccata (Scop . ex nicotinamide 100 J.lg, p-amino-benzoic acid 100 pg, Fr.) Berk. & Br., Amanita muscaria (L. ex Fr.) pantothenic acid 100 J.lg and inositol 10000 pg, Hooker , Lactarius helous Fr., Paxillus imiolutus and distilled water to 1 1. The medium was (Bau ch) Fr ., and Leccinum scabrum (Bull. ex Fr.) sterilized by autoclaving at 1200 for 10 min. S. F. Gray. Fruit-bodies of these fungi were Techniques collected during the autumn of 1976 in the forests around Uppsala, Sweden. Spores were harvested The spores to be tested for germination were by fastening a fruit-body cap, or pieces of a cap, removed from one of the stored spore prints by

Basidiospore germination in the soil-inhabiting Basidiomycetes has long been a problem in experimental mycology. In most of the mycorrhiza-forming species within Amanita, Entoloma, Cortinarius, Gomphidius, Paxillus, Lactarius and Russula spore germination has never, or only occasionally, been observed under laboratory conditions. This has been interpreted in various ways, e.g. as an expression of dormancy, as a need for a germination-inducing factor, or as the effect of an endo- or exogenous inhibitor. Some evidence in favour of these latter possibilities has been obtained in experiments with Boletus (Suillus) species, where amino acids stimulate and ammonium ions inhibit germination (Fries, 1976). In the present paper the possibility of such interactions between induction and inhibition of basidiospore germination has been further investigated in some soil-inhabiting and mycorrhizaforming Hymenomycetes,

Vol. 70, Part 2, was issued 3 April 1978 1 :2

MY C 70

N. Fries

320

dispersing them in 0'5 ml sterile distilled water. By dilution a spore suspension was obtained which contained 0'5 x 106 to 2 X 106 spores per ml. From this suspension 50 pI was pipetted and spread evenly over the surface of each plate. The plates were left overnight to permit the thin water film to dry out. Three experimental measures were tested, singly and in combination, for their possible capacity to induce germination of the spores: (a) Activated charcoal was tested since it is known to trigger various developmental processes in plants, including fungi, an effect which usually is interpreted as being due to the capacity of charcoal to adsorb and thus remove inhibitory compounds from the culture medium. The charcoal powder (Aktivkohl, pro analysi, Merck) was dusted out over one half of the agar plate, c. 3 mg per plate in the first experiment (Fig. 1) and then c. 7 mg per plate in the following experiments. The charcoal particles covered less than 10 % of the powdered surface. (b) Rhodotorula glutinis (Fres.) Harrison was used as a germination activator since it, as well as some other Rhodotorula species, had proved useful in earlier studies of a similar kind (Fries, 1941; Bulmer & Beneke, 1964). To that purpose it was inoculated on the agar close to the periphery of the plate (Fig. 1). The diameter of the developing colony after one month was c. 1 ern and after two months c. 2 em. (c) Growing mycelium of the same species as the spores has sometimes been found to induce germination (Cayley, 1936; Losel, 1964) and was therefore tested also in the present investigation. The inoculum was placed in the centre of the plate. Table

1.

Petri dishes were sealed with 'Parafilm' and incubated in darkness at 25°. Each experimental series comprised 4 or 5 Petri dishes. At least once or twice a week the plates were inspected in daylight and where germination led to the production of a mycelium visible to the naked eye it was marked. The final number of mycelia, i.e. germinated spores, was scored according to the scale given in Table 1. RESULTS

Nutrient agar medium without any further supplements

The inhibitory effect of the ammonium ion found in germination experiments with Boletus (Fries, 1976) was reduced or eliminated with the more dilute nutrient medium used in the present investigation. However, only A. muscaria germinated on this medium. At the most five mycelia were scored per plate after 2 months, the average number being about two per plate, which means c. 0'001 % germination. With none of the other media commonly used in mycological work did germination occur, neither in A. muscaria nor in the four other species tested. Effects oj activated charcoal and Rhodotorula Activated charcoal alone did not affect germination in any of the five species tested in the present investigation, neither did Rhodotorula, known to induce spore germination in e.g. Suillus (Fries, 1941). However, when combined on the same agar plate these two supplements together induced germination in all five species (Table 1). Laccaria laccata was the first species found to

In vitro germination of five Hymenomycete species on agar plates with different supplements Germination rare] with different supplements

,-----------------"-------No. of experiments

No supplement

Charcoal + Rhodotorula

Charcoal Rhodotorula Species* o o o Laccaria 3 laccata 2 2 2 2 Amanita muscaria o o o 2 Lactarius helvus 6 Paxillus o inoolutus o o Leccinum 5 scabrum * In each species the same spore print was used in all experiments. t Germination: 0 = none; 1 = < 0'0001 %i 2 = 0'0001 to 0'01 %i 3 = 0'01 to tested. Germ vesicle or germ-tube formation but no (or rarely) further development.

*

Charcoal + Rhodotorula + own mycelium

4

4

3

3

1-2

3

4

2

1

%; 4 = >

1

%; - = not

Spore germination in Hymenomycetes

32 1

Fig. 1. Agar plate seeded with 200000 basidiospores from Amanita muscaria, incubated for 3 months. Monosporous mycelia are developing within the area strewn with activated charcoal. R. = colony of Rhodotorula glutinis,

germinate under these conditions, with up to 20 % germination in some plates. Experimental details have been published elsewhere (Fries, 1977)· Amanita muscaria. Despite the occurrence of up to fifty per million 'spontaneous' germinations on the unsupplemented agar plates it was quite obvious that an increased rate of germination was obtained on the plates with Rhodotorula in combination with charcoal (Fig. 1). It is possible that the presence of living mycelium of A. muscaria further improved germination, but the considerable variation in germination rate even among the plates belonging to the same series made a conclusion uncertain. The highest germination percentage attained was about 1) '1 %. The first germinating spores were observed after 2 weeks. Only one germ tube was produced, which soon started forming branches (Fig . 2). The hyphae developed in a tortuous way, which gave the young mycelium a

curly appearance. Sizeable mycelia could easily be transferred to stock culture tubes with a synthetic agar medium. Lactariu s helvus. Germinating spores were observed after a month's incubation but the germination rate was below 0'001 % (Fig . 2). All germinations took place within a distance of 1 em from the Rhodotorula colony and within the area dusted with charcoal. Efforts to transfer the young, developed mycelia to unsupplemented agar media failed. Paxillus inuolutus. Germination started after about two weeks and always among the charcoal fragments but at any distance from the Rhodotorula colony. After 6 weeks a germination percentage of 0'01 to 0'1 % was scored . Occasionally solitary germinations occurred also with charcoal or Rhodotorula alone. As soon as the germ tube had started to branch the mycelium developed rapidly so that closely situated spores germinating later were easily overlooked (F ig. 2). It could 12- 2

322

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F Fig. 2. Germinating spores and young mycelia of three species. A and B: Amanita muscaria, incubati on time 38 days, x 280; C and D : Lactarius helous, incubation time 38 days, x 520; E and F: Paxil/us inuolutus, incubation time 78 days, x 280.

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Fig . 3. Agar plate seeded with 175000 basidiospores of Leccinum scabrum, incubated for 40 days. The effect of charcoal, Rhodotorula and Leccinum scabrum mycelium on the germination of the spores on different parts of the agar plate shown in photomicrographs. (A) Localization of the photomicrographs (B- E) in relation to the charcoal-dusted area, Rhodotorula (R .) colony and Leccinum (L .) mycelium . (B) Commencing vesicle formation; (C) No germination ; (D) Vesicle formation completed in most spores; (E) Germ hypha formation in some spores, with or without a preceding vesicle formation . (A) Half size; (D-E ) x 410.

Spore germination in Hymenomycetes clearly be seen that even a small, developing Paxil/us mycelium increased the germination rate among the surrounding spores. Leccinum scabrum, The first spores to germinate were observed after 3-4 weeks close to the Rhodotorula colony and within the charcoal-sown area . After 2 months up to about 0'01 % of the spores had germinated. The germ-tube always developed from one of the end points of the spore (Fig. 3E). Finally, richly branched mycelia were formed, which could be transferred to other media in culture tubes. As soon as such a mycelium had attained a diam of c. 2 mm the still ungerminated spores around it started producing spherical germ vesicles. Since this occurred in at least half of the surrounding spores it was interpreted as an expression of a much higher potential germinability than had earlier been presumed. This observation led to the following experiments with living mycelia of the same species.

Effects of a mycelium representing the same species as the spores Paxillus inuolutus. On plates with Rhodotorula and activated charcoal a growing Paxillus mycelium accelerated the germination of the spores near the progressing mycelial frontier. This influence was manifest even across a fissure in the agar plate. A further experiment using theinverted dish method of Brown & Merrill (1973) gave the same result. This indicates the presence of a volatile, germination-stimulating substance produced by the mycelium of P. inuolutus. Volatile compounds inducing spore germinations have been observed in a few other Hymenomycetes (Brown & Merrill, 1973; Oort, 1974; and others). In Agaricus bisporus the active compound is isovaleric acid (Losel, 1964; Rast & Stauble, 1970). Therefore, isovaleric acid at various concentrations was tested on the spores of P. inuolutus incubated either with charcoal and Rhodotorula or without these supplements. No effect on germination was observed, however. Leccinum scabrum. As already indicated the spores of L. scabrum responded differently to Rhodotorula and to its own mycelium. The very few spores - about one out of ten thousandgerminating under the influence of Rhodotorula (plus charcoal) directly produced a germ tube, which soon started branching and gradually formed a dense mycelium (Fig . 3E). In contrast, at least every second spore around the slowly growing L. scabrum mycelium reacted by forming a spherical germ vesicle or (Fig . 3 D), more rarely, a short germ tube at one end of the spore. Only exceptionally did these vesicles and germ tubes undergo any further development, and therefore

only very few or no mycelia were formed on agar plates with L. scabrum mycelium without a coexistant Rhodotorula colony. There was also a difference in germination induction rate: with Rhodotorula the first germinations started after about 3 weeks, with L. scabrum mycelium the first vesicles became visible as soon as after 1 week. In the former case, germinations occurred only rather close to the yeast colony, in the latter an induction of vesicle formation spread centrifugally rather rapidly among the spores over the whole agar plate. If these effects are considered to be the result of stimulatory substances only, diffusing out of the Rhodotorula colony and the L. scabrum mycelium, respectively, then the former seem to diffuse more slowly than the latter, to be less stable or to exert an effect on the spores more slowly. Using the simple agar plate fissure technique it was conclusively shown that neither Rhodotorula nor L . scabrum produced any effect on the spores via the gas phase. DISCUSSION

Earlier reports of germinabiliry of basidiospores of the fungi studied in the present paper are rather summary and do not include any descriptions of the germination process (Brefeld, 1889; Fries, 1943; Kneebone, 1950; Melin, 1962; Stack et al., 1975). However, with the aid of activated charcoal in combination with living colonies of Rhodotorula glutinis it has now proved possible to induce and to observe under the microscope the basidiospore germination in these species and to establish reproducible conditions for the process. The reasoning was that the charcoal should remove inhibitory substances from the medium, as had been supposed to be its function in other fungal systems (Day & Anagnostakis, 1971; Butler & Bolkan, 1973), and Rhodotorula should provide the spores with germination-stimulating substances as was supposed to be the case in Boletus (Suil/us) luteus (Fries, 1941, 1966) and Calvatia (Bulmer & Beneke, 1961). The reason why the combination of charcoal and Rhodotorula failed to induce germination in some other species tested could either be that certain inhibitors were not removed by the charcoal and/or that the spores required germination stimulators other than those present in the medium or produced by Rhodotorula. It must also be remembered that the possible influence of many other environmental factors like temperature, carbon dioxide and light, as well as pretreatments of the spores, have not yet been methodically investigated. However, even our very limited knowledge

N. Fries shows how much the conditions required for basidiospore germination differ from one specie s to another among the soil-inhabiting Hymenomycetes . In contrast to Boletus (Sui/Ius) luteu s, for example, the germination of which is stimulated by Rhodotorula alone or by amino acids (Fries, 1976), the present five species did not germinate unless charcoal plus Rhodotorula were present on the agar plate. In two of them the germination process was further stimulated by mycelium belonging to the same species as the spores. The activity of both of these germinationst imulating mycelial products, the volatile one in P. imiolutus and the non-volatile one in L. scabrum, seems at least to some degree to be specific. With P. inuolutus five and with L. scabrum seventeen different mycelia of other fungi were tested for germination induction but without any success. Occasional airborne contaminations were also inactive. This supports the conclusion that the effect is not produced by a trivial and commonly occurring catabolite, like CO 2 , and cannot simply be due to a change in pH of the med ium either. A chemical characterization of these germination stimulators is under way. The author wishes to thank Mrs Harriette Cedervall for patient and skilful technical assistance. REFERENCES O. (1889). Untersuchungen aus dem Gesammtgebiete der Mykologie. VIII. Basidiomyc etes: Ill. Le ipzig. BROWN, T. S. & MERRILL, W. (1973). Germination of basidiospores of Fomes applanatu s. Phytopathology 63, 547-550. BULMER, G. S. & BENEKE, E. S. (1961). Studies on Caluatia gigantea. I. Germination of the basidiospores. Mycologia 53, 123-136. BULMER, G. S. & BENEKE, E. S. (1964). Germination of basidiospores of Lycoperdon species and Scleroderma lycoperdoides. Mycologia 56, 70-76. BUTLER, E. E. & BOLKAN, H. (1973). A medium for heterokaryon formation in Rhizoctonia solani. Phytopathology 63, 542-543. BREFELD,

CAYLEY, D. M . (1936). Spores and spore germination in wild and cultivated mushrooms (Psalliota spp.). Tran sactions of the Br itish Mycological S ociety 20, 225-241. DAY, P. R. & ANAGNOSTAKIS, S. L. (1971). Corn smut dikar yon in culture. Nature New Biology 231 ,19-20. FRIES, N. (1941). Uber die Sporenkeimung bei einigen Gast erom yceten und mykorrhiza-bildenden Hymenom yceten . Archiv fur Mikrobiologi e 12, 266-284. FRIES, N. (1943). Untersuchungen tiber Sporenkei mung und M ycelentwicklung bodenbewohnender H ymenomyceten . Symb olae Botanicae Upsalienses 6, 1-81. FRIES, N . (1966). Chemical factors in the germination of spores of basidiomycetes. The fungus spore (ed. M. F. Madelin), pp . 189-200. London: Butterworths Scientific Publications. FRIES, N. (1976). Spore germination in Boletu s induced by amino acids. Proceedings Koninklijke Nederlands e Akademie van Wetenschappen C 79, 142-146. FRIES, N. (1977). Germination of Laccaria laccata spores in vitro . Mycologia 59, 848-850. KNEEBONE, L. R. (1950). An investigation of basidiospore germination in the hymenomycetes, especially in the Agaricaceae. Ph.D. Thesis, Pennsylvania State College. L6sEL, D . M . (1964). The stimulation of spore germ ination in Ag aricus bisporus by living mycelium . Annals of Botany, N.S. 28, 541-554. MELIN, E. (1962). Physiological aspects of mycorrhizae of forest trees. Tree growth (ed. Th.T. Kozlowski), pp. 247-263. Ronald Press Co. OORT, A. J. P. (1974). Activation of spore germination in Lactarius species by volatile compounds of Ceratocystis f agacearum. Proceedings K oninklijke Nederlandse Akademie van Weten schappen, 77, 301-307. RAsT, D. & STAUBLE, E. J. (1970). On the mode of action of isovaleric acid in stimulating the germination of Agaricus bisporus spores. New Phytologist 69, 557-566. STACK, R. W. , SINCLAIR, W. A. & LARSEN, A. O. (1975). Preservation of basidiospores of Laccaria laccata for use as mycorrhizal inoculum. Mycologia 67, 167-17°.

(Accepted for publication 10 November 1977)