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Stimulation of germination of spores of some ectomycorrhizal fungi by other micro-organisms
Mycal. Res. 93 (2): 182-186 (1989)
182
Prinled in Greal Brilain
Stimulation of germination of spores of some ectomycorrhizal fungi by other micro-organisms
NAHIA A. ALI'" AND RICHARD M. JACKSON Deparlmenl of Microbiology, Universily of Surrey, Gui/dford, Surrey, GU2 5XH, UK
Stimulation of germination of spores of some ectomycorrhizal fungi by other micro-organisms. Mycological Research 93 (2): 182-186 (1989). Seven bacterial isolates out of 42 obtained from sporophores of ectomycorrhizal fungi, two out of 36 obtained from mycorrhizas and two out of 40 obtained from soil stimulated germination of certain mycorrhizal fungi. None of the fungal isolates obtained from the same sources stimulated germination of any of the test fungi. One fungus (Trilirachium roseum) and one bacterium (Micrococcus roseus), which appeared among the spores of Hebe/oma crustuliniforme on the surface of agar plates as natural contaminants, also stimulated germination of spores of that fungus. The most active isolate was that of Pseudomonas stuturi which was obtained from a sporophore of H. crustuliniforme and induced ca 21 % of spores of the fungus to germinate within 1 em of its colonies. Isolates of CO'llnebacterium spp. obtained from sporophores of H. crusluliniforme and roots of willow (Salix spp.), mycorrhizal with H. crusluliniforme, stimulated spores of that fungus and of Paxillus involulus. Spores of Laccaria larcala were stimulated by only one isolate (S19 an unidentified soil bacterium). This isolate also stimulated spores of P. involutus and H. crusluliniforme. Spores of the remaining test fungi did not respond to any of the bacterial and fungal isolates tested. Key words: Ectomycorrhizas, Spore, Germination, Stimulation, Bacteria.
Spore germination of the ectomycorrhizal hymenomycetes is very difficult to achieve under laboratory conditions (Brefeld, 1908; Fries, 1978, 1985; Marx & Ross, 1970; Rommell, 1921; Stack, Sinclair & Larsen, 1975). It is oHen dependent upon, or stimulated by, the presence of activating factors, usually of biological origin. Fries (1943) was the first to succeed in germinating spores of species of Boletus, Amanita and Tricholoma, using a living yeast, i.e. Torulopsis sanguinea or Rhodotorula glutinis, as a stimulator. Lamb & Richards (1974) induced germination of basidiospores of Pisolifhus tinetorius, Rhiwpogon roseolus and Suillus granulatus only in the presence of the yeast Rhodotorula glutinis. Spores of Laetarius species were stimulated to germinate by Ceratocystis fagacearum (Oart, 1974). Living mycelia of some ectomycorrhizal fungi were reported to induce germination of spores of the same species (Fries, 1978, 1981, 1983; Straatsma, Konings & Griensven, 1985). The routine germination of spores of ectomycorrhizal fungi in the laboratory is still a problem, and the role of micro-organisms as stimulators merits further examination. In this study different isolates of bacteria and fungi obtained from soil, sporophores and mycorrhizal sheaths of the test fungi, or occurring as natural spore contaminants, were tested for their ability to stimulate germination of spores of ectomycorrhizal fungi. Attempts were made to determine • Present address: Agriculture and Water Resources Research Center, Council of Scientific Research, P.O. Box 2416, Baghdad, Iraq.
the nature of the stimulatory factors produced by the active organisms.
MATERIALS AND METHODS Mycorrhizal fungi Basidiocarps of Paxillus involufus (Batsch: Fr.) Fr., Laccaria laccata (Scop.: Fr.) Cooke, Amanifa fulva (Schaeff.) Seer., Amanifa rubescens (Pers.: Fr.) S. F. Gray, Lacfarius turpis (Weinm.) Fr. and Russula nitida (Pers.: Fr.) Fr. were collected beneath birch (Betula spp.) at Whitmoor Common, Surrey. Those of Hebeloma crustuliniforme (Bull.) Que!' were collected beneath willow (Salix spp.) at the University of Surrey, GUildford, Surrey. Basidiospores of these fungi were collected from clean and mature sporocarps in sterile plastic Petri dishes under humid conditions (Ali & Jackson, 1988) and used within 24 h.
Media The four media used were: Fries agar (Fries, 1978); modified Melin-Norkrans agar (MMN) (Marx, 1969); nutrient agar CM3 (Oxoid Ltd); potato-dextrose agar (PDA) CM139 (Oxoid Ltd).
Stimulating organisms Bacteria and fungi obtained from birch forest soil; bacteria and
Nahia A. Ali and R. M. Jackson
183
Table 1. Numbers and sources of isolates of bacteria and fungi tested for germination stimulation
Paxillus involutus Laeearia laeeata Hebeloma crustuliniforme Amanita fulva Amanita rubescens Lactarius turpis Russula nitida
Soil isolates ...
Isolates associated with spores or sporophores
Isolates associated with mycorrhizal roots'
Bacteria
Fungi
Bacteria
5 6 10 5 5 5 6
3 2 3 5 4 4
6 4 6 7 7 4 2
2
Bacteria 40
Fungi 3 4 2 3 3 4 5 Fungi 30
Totals 17 16 21 20 19 17 15 70
• Roots of birch (&tula spp.) close to sporophores of the named fungus, except in the case of H. CTustuliniforme where the roots were willow (Salix spp.).
fungi associated with spores or sporophores of the test ectomycorrhizal fungi; bacteria and fungi associated with the mycorrhizal sheath of the test ectomycorrhizal fungi; bacteria and fungi appearing among the spores of the test fungi as natural contaminants on laboratory media. The number and sources of the isolates of the first three groups are shown in Table 1.
Isolation of stimulating organisms Soil bacteria and fungi were obtained by the dilution plating method. Fresh soil samples were collected from the top 30 cm of the soil profile in July, 1985 from 12 different locations beneath birch trees at Whitmoor Common, Surrey. They were passed through a 3 mm sieve and four composite samples were made. From each, 10 g of soil was suspended in 90 ml sterile distilled water, shaken in a Gallenkamp wrist-action shaker for 15 min and allowed to stand for 5 min. From this suspension serial dilutions (up to 10- 4 ) were made, and 1 ml of each dilution was plated on nutrient agar for bacteria and PDA for fungi. Three plates were prepared for each dilution and incubated at 20°C for 7 d. Forty bacterial colonies and 30 fungal isolates were selected and screened for their effect on the germination of the spores of the test ectomycorrhizal fungi. As with isolates obtained from other sources, the bacteria were chosen at random and the fungi were isolates with macroscopically distinct colonies, assumed to be either different species or different strains of the same species. Identification was attempted only of those organisms showing stimulatory activity. Isolates associated with spores or sporophores of the ectomycorrhizal fungi were obtained from caps of clean and healthy sporophores. They were surface-sterilized by wiping with tissue soaked in ethanol, peeled and broken up under aseptic conditions; small fragments of the inner tissue were placed on MMN agar (five pieces per plate). Several plates were made for each fungus and incubated at 20° for 7 d. Forty two bacterial and 23 fungal isolates were selected for study. 13
For isolates associated with mycorrhizal sheaths of the test fungi, mycorrhizal roots of birch (Betula spp.) or willow (Salix spp.) were collected beneath sporophores of the test mycorrhizal fungi. Segments of roots, 5-10 mm long and bearing mycorrhizal tips were agitated in 0'003 % Tween 80 in distilled water for 15 min to remove soil debris, followed by agitation in three changes of distilled water. The roots were then surface-sterilized by immersion in 30 % (v/ v) hydrogen peroxide for 15 s and washed again by agitation for 10 min in sterile distilled water. Individual mycorrhizal tips (ea 40 for both birch and willow) were then plated on MMN agar, five to eight per plate, and incubated at 20°. Thirty-six bacterial isolates and 24 fungal isolates were selected for study.
Germination tests Basidiospores to be tested for germination were removed from the plastic Petri dishes (within 24 h of collection) by dispersing them in sterile distilled water (mixtures of spores from 3-5 fruit bodies of the same collection were used). Fries agar plates were inoculated with 0'1 ml of spore suspension 5 (ea 5 x 10 spores per plate) spread over the agar surface, and allowed to stand overnight for absorption of surplus water. Test plates were then inoculated with the appropriate microorganism using a 6 mm diam disc of fungal mycelium from the margin of an actively-growing colony or, for bacteria, a streak ea 4 em long. Uninoculated plates and the areas of inoculated plates distant from the inoculum served as controls. Half of the surface of each plate was sprinkled with a thin layer (ea 7 mg per plate) of autoclaved activated charcoal (Darco G60, BDH Chemicals Ltd, Poole, England) that covered the basidiospores. The plates were incubated in darkness at 20° and examined daily by light microscopy for spore germination.
RESULTS AND DISCUSSION A minority of the bacterial and fungal isolates were found to MYC 93
Germination of ectomycorrhizal fungi
184
Table 2. Identity, source, and stimulatory activity of the test bacterial and fungal isolates
Isolate number
2 3 4 5
6 7
8 9 10 II
12 13
Identity of the active isolates Pseudomonas stutzeri Lehmann & Neumann Pseudomonas sp. no. I Pseudomonas sp. no. 2 Pseudomonas sp. no. 3 Pseudomonas sp. no. 4 Corynebacterium sp. no. I Enterobacteriaceae Micrococcus roseus Flugg Tritirachium yoseum van Beyma Arthrobacter sp. Corynebacterium sp. no.6a
Source
Spores stimulated
H. cyustu/iniforme
Sporophores of H. cyustu/iniforme H. cyustu/iniforme
and P. inDo/utus
} l
J
Pseudomonas sp. } no. S30 Unidentified bacterium no. S19
Spores of H. cyustu/iniforme Mycorrhizas of Salix spp. with H. crustu/iniforme Birch forest soil
stimulate germination of spores of ectomycorrhizal fungi. Of 42 bacterial isolates associated with sporophores, seven stimulated spore germination. All the active isolates came from sporophores of H. crustuliniforme and stimulated germination of spores of that fungus. One isolate only (Corynebacterium sp.) stimulated spore germination of P. involutus as well as H. crustuliniforme. Two of the 36 bacterial isolates associated with the mycorrhizal sheath stimulated spore germination. These isolates were obtained from mycorrhizas of Salix collected beneath sporophores of H. crustuliniforme and stimulated spores of the same fungus and of P. involutus (Table 2). Two bacterial isolates out of 40 obtained from birch forest soil stimulated spore germination; one of them (Pseudomonas sp. 530) stimulated germination of H. crustuliniforme spores, and the other (an unidentified Gram-positive coccus 519) stimulated spore germination of H. crustuliniforme, P. involutus and L. laccata (Table 2). One fungus (Tritirachium roseum) and one bacterium (Micrococcus roseus), which appeared on the surface of agar plates among spores of H. crustuliniforme as natural spore contaminants (Table 2), stimulated spores of H. crustuliniforme around their colonies. None of the fungal isolates obtained from sporophores, mycorrhizal sheaths or from soil, stimulated spore germination of any of the test ectomycorrhizal fungi after up to 15 d incubation; observation for longer was not possible, as the fungi covered the whole surface of the plate within 15 d. Pseudomonas stutzeri was the most active bacterium in stimulating germination of H. crustuliniforme spores. Germination began within 3 d and increased until it was ca 21 % (Table 3) up to I cm from the bacterial colony after 10 d incubation (spores were counted in ten microscope fields, chosen randomly in each of three replicate plates). Germination was ca 1'7% at a distance of 1-2 cm from the bacterial colony.
H. cyustu/iniforme
H. cyustu/iniforme,
P. inDo/utus and L. /accata
Numerous spores also germinated underneath the bacterial growth. The effect of P. stutzeri was tested in the presence of activated charcoal and growing mycelium of H. crustuliniforme either separately or together (Table 3); charcoal slightly increased germination in the presence of mycelium but not the bacterium. Mycelium had no effect on germination alone or in combination with the bacterium. Spores of H. crustuliniforme were stimulated to germinate by the two isolates of Corynebacterium after two wk incubation, and after five wk, by Micrococcus roseus and Tritirachium roseum. The exact percentage germination could not be counted with these isolates, but it was < I % very close to the activator colonies, in the presence or absence of charcoal. Slight stimulation « 0'1 %) germination) occurred in the presence of other bacteria (Pseudomonas nos 1,2,3,4; Arthrobacter sp. and an isolate belonging to the Enterobacteriaceae). Spores of P. involutus were stimulated by the two isolates of Corynebacterium, Arthrobacter and the unidentified soil bacterium (519) (Table 2), but only in the presence of activated charcoal. Percentage germination « 0'1 %) was not accurately determined, as most of the spores germinated either very close to or underneath the bacterial growth. Spores of Laccaria laccata were stimulated by the unidentified soil bacterium (519) in the presence of activated charcoal but percentage of germination was very low « 0'01 %). In all cases germinating spores produced mycelial colonies which were then transferred to MMN agar. These findings thus indicate that there is some specificity in the phenomenon of germination stimulation; only certain species of micro-organisms can stimulate spore germination of certain species of ectomycorrhizal fungi. Published data about the effect of other micro-organisms on
Nahia A. Ali and R. M. Jackson
185
Table 3. Spore germination of H. crustuliniforme on Fries agar with different supplements (after 10 d incubation)
Treatment Control (no addition) + Charcoal + Bacteriat + Mycelium + Bacteriat + Charcoal + Bacteriat + Mycelium + Bacteriat + Mycelium + Charcoal + Mycelium
Numbers of spores observed
Germinated
Germination
3'2 x 10 6
0
(%)"
0
106 10 3
0 2'0
X
10 3
0 21'3
106 10 3
0 1'5
X
10 3
20'7
6'6 x 10 3
1'4
X
10 3
21'3
6'1 x 103
1'3
X
10 3
21-6
3'2 x 106
9
3'2 9'4 3'2 7'1
x x x x
0
0'0002
• Germination values at sites 0-1 cm from bacterial colony edge. t Pseudomonas stutz.eri.
spore germination of ectomycorrhizal fungi are very limited and most studies have concerned the yeast Rhodotorula where germination was typically reported as being slow and sparse, and seldom exceeded 1'0% (Fries, 1943, 1966; Lamb & Richards, 1974). More recently Fries (unpub!., cited by Fries, 1984) claimed that no new germination inducers more powerful than those of the rather inefficient Rhodotorula were discovered during his extensive testing of a variety of microorganisms isolated from forest soil. Stimulation of spore germination of ectomycorrhizal fungi by named bacteria has not previously been reported. However, there are recent reports of the stimulation of germination of spores of VA mycorrhizal fungi and sometimes of subsequent growth by various soil micro-organisms. Azcon-Aguilar et al. (1986) tested the effects of several micro-organisms on the spore germination and development of Glomus mosseae. They found that the germination rate of surface-sterilized chlamydospores was significantly increased and the subsequent growth of hyphae was promoted by the unnamed free-living fungi tested. Mayo, Davis & Motta (1986), working with Glomus versiforme, observed that increased spore germination was caused by several genera of bacteria, including Corynebacterium and Pseudomonas; the hyphae produced following germination were longer and smoother than in the absence of bacteria. Mugnier & Mosse (1987) reported that various contaminants developing on agar plates stimulated the germination of Glomus mosseae spores. These contaminants included various filamentous fungi and in one instance Streptomyces orientalis. This actinomycete was strongly stimulatory and could induce germination at a distance by what appeared to be a volatile metabolite. Bacteria have been shown to influence the colonization of roots by ectomycorrhizal fungi (Bowen & Theodorou, 1979). One bacterium (Bacillus sp. WRl), isolated from washed Pinus radiata roots mycorrhizal with Rhizopogon luteolus significantly increased colonization of pine roots by four of the mycorrhizal fungi tested, and also stimulated growth of R. luteolus on agar media. Effects on spore germination were not examined in this study.
The nature of substances produced by micro-organisms that stimulate the germination of spores of mycorrhizal fungi is not known. However, Fries (1966, 1976) reported that yeast could stimulate germination by production of a diffusible substance(s), e.g. amino-acids, and volatile compounds (unknown) or by removing inhibitory compounds, e.g. NH 4 , from the medium. In the case of P. stutzeri volatile compounds, cell-free culture filtrates and cell extracts were tested in a further study (Ali, 1986), but none stimulated germination of the test ectomycorrhizal fungus (H. crustuliniforme). Also, germination did not occur on agar on which P. stutzeri had been allowed to grow for some time and then removed before being autoclaved and re-used for germination tests without any living bacteria. Germination occurred only in the presence of active bacteria close to the spores. This suggests that the stimulatory compound(s) produced by P stutzeri may be chemically labile and inactivated after exudation into the medium. The very high percentage of P. involutus spores germinating near roots of birch in soil, previously reported (Ali & Jackson, 1988) may have been due to the effects of both root exudates and micro-organisms similar to those reported here. N. A. Ali wishes to thank the Iraqi Government for the financial support for this work. REFERENCES ALI, N. A. (1986). Spore germination and the pre-infection phase in ectomycorrhizal fungi. Ph.D. Thesis, University of Surrey. ALI, N. A. & JACKSON, R. M. (1988). Effects of plant roots and their exudates on the germination of spores of ectomycorrhizal fungi. Transactions of the British Mycological Society 91, 253-260.
AZCON-AGUILAR, c.. ROSA, M., RODRIGUES, D. & BAREA, J. M. (1986). Effect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Transactions of the British Mycological Society 86, 337-340.
BOWEN, G. D. & THEODOROU, C. (1979). Interactions between bacteria and ectomycorrhizal fungi. Soil Biology and Biochemistry 2, 119-126. 13-2
Germination of ectomycorrhizal fungi BREFELD, O. (1908). Untersuchungen aus dem Gesamtgebiet der Mykologie. 14. Die Kultur der Pilze. Munster, I. W. Heinrich Schoningh. FRIES, N. (1943). Ober die Sporenkeimung bei Gasteromyceten und Mykorrhiza bildenden Hymenomycetin. Archiv fur Mikrobiologie 12, 266-284.
FRIES, N. (I966). Chemical factors in the germination of spores of Basidiomycetes. In The Fungus Spore (ed. M. F. Madelin), pp. 189-199. London, u.K.: BuUerworths Scientific Publications. FRIES, N. (1976). Spore germination in Boletus induced by aminoacids. Proceedings Koninklijke Nederlandse Akademie van WeHenschappen Series C79, 142-324. FRIES, N. (1978). Basidiospore germination in some mycorrhizaforming hymenomycetes. Transactions of the British Mycological Society 70, 319-324.
FRIES, N. (1981). Effects of plant roots and growing mycelia on basidiospore germination in mycorrhiza-forming fungi. In Arctic and Alpine Mycology (ed. G. A. Lauson & J. F. Ammirati), pp. 493-508. Seattle, U.S.A.: University of Washington Press. FRIES, N. (1983). Spore germination, homing reaction, and intersterility groups in Laccaria laccata (Agaricales). Mycologia 75, 221-227.
FRIES, N. (1984). Spore germination in the higher Basidiomycetes. Proceedings of the Indian Academy of Sciences (Plant Sciences) 93, 205-222. FRIES, N. (1985). Spore germination in ectomycorrhizal fungi. In Proceedings of the 6th North American Conference on Mycorrhizae, June, 1984 (ed. R. Molina). Corvallis, USA.: Forest Research
Laboratory. LAMB, R.]. & RICHARDS, B. N. (1974). Survival potential of sexual
186 and asexual spores of ectomycorrhizal fungi. Transactions of the British Mycological Society 62, 182-191.
MARX, D. H. (1969). The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163. MARX, D. H. & ROSS, D. W. (1970). Aseptic synthesis of ectomycorrhizae on Pinus taeda by basidiospores of Thelephora terrestris. Canadian Journal of Botany 48, 197-198.
MAYO, K., DAVIS, R. E. & MOTTA, J. (1986). Stimulation of germination of spores of Glomus versiforme by spore-associated bacteria. Mycologia 78, 426-431. MUGNIER, ]. & MOSSE, B. (1987). Spore germination and viability of a vesicular arbuscular fungus, Glomus mosseae. Transactions of the British Mycological Society 88, 411-413.
OORT. A. J. P. (1974). Activation of spore germination in Lactarius species by volatile compounds of Ceratocystis fagacearum. Proceedings Koninklijke Nederlandse Akademie van Wetenschappen Series C77, 301-307. ROMMELL, L. G. (1921). Parallelvorkimmen gewisser Boleten und Nadelbaume. Svensk Botanisk Tidskrift 15, 204-213. STACK, R. W., SINCLAIR, W. A. & LARSEN, A. O. (1975). Preservation of basidiospores of Laccaria laccata for use as mycorrhizal inoculum. Mycologia 67, 167-170. STRAATSMA, G.. KONINGS, R. N. H. & GRIENSVEN, L. J. L. D. (1985). A strain collection of the mycorrhizal mushroom Cantharellus cibarius obtained by germination of spores and culture of fruit body tissue. Transactions of the British Mycology Society 85, 689-697.
(Received for publication 5 October 1987 and in revised form 6 September 1988)
182
Prinled in Greal Brilain
Stimulation of germination of spores of some ectomycorrhizal fungi by other micro-organisms
NAHIA A. ALI'" AND RICHARD M. JACKSON Deparlmenl of Microbiology, Universily of Surrey, Gui/dford, Surrey, GU2 5XH, UK
Stimulation of germination of spores of some ectomycorrhizal fungi by other micro-organisms. Mycological Research 93 (2): 182-186 (1989). Seven bacterial isolates out of 42 obtained from sporophores of ectomycorrhizal fungi, two out of 36 obtained from mycorrhizas and two out of 40 obtained from soil stimulated germination of certain mycorrhizal fungi. None of the fungal isolates obtained from the same sources stimulated germination of any of the test fungi. One fungus (Trilirachium roseum) and one bacterium (Micrococcus roseus), which appeared among the spores of Hebe/oma crustuliniforme on the surface of agar plates as natural contaminants, also stimulated germination of spores of that fungus. The most active isolate was that of Pseudomonas stuturi which was obtained from a sporophore of H. crustuliniforme and induced ca 21 % of spores of the fungus to germinate within 1 em of its colonies. Isolates of CO'llnebacterium spp. obtained from sporophores of H. crusluliniforme and roots of willow (Salix spp.), mycorrhizal with H. crusluliniforme, stimulated spores of that fungus and of Paxillus involulus. Spores of Laccaria larcala were stimulated by only one isolate (S19 an unidentified soil bacterium). This isolate also stimulated spores of P. involutus and H. crusluliniforme. Spores of the remaining test fungi did not respond to any of the bacterial and fungal isolates tested. Key words: Ectomycorrhizas, Spore, Germination, Stimulation, Bacteria.
Spore germination of the ectomycorrhizal hymenomycetes is very difficult to achieve under laboratory conditions (Brefeld, 1908; Fries, 1978, 1985; Marx & Ross, 1970; Rommell, 1921; Stack, Sinclair & Larsen, 1975). It is oHen dependent upon, or stimulated by, the presence of activating factors, usually of biological origin. Fries (1943) was the first to succeed in germinating spores of species of Boletus, Amanita and Tricholoma, using a living yeast, i.e. Torulopsis sanguinea or Rhodotorula glutinis, as a stimulator. Lamb & Richards (1974) induced germination of basidiospores of Pisolifhus tinetorius, Rhiwpogon roseolus and Suillus granulatus only in the presence of the yeast Rhodotorula glutinis. Spores of Laetarius species were stimulated to germinate by Ceratocystis fagacearum (Oart, 1974). Living mycelia of some ectomycorrhizal fungi were reported to induce germination of spores of the same species (Fries, 1978, 1981, 1983; Straatsma, Konings & Griensven, 1985). The routine germination of spores of ectomycorrhizal fungi in the laboratory is still a problem, and the role of micro-organisms as stimulators merits further examination. In this study different isolates of bacteria and fungi obtained from soil, sporophores and mycorrhizal sheaths of the test fungi, or occurring as natural spore contaminants, were tested for their ability to stimulate germination of spores of ectomycorrhizal fungi. Attempts were made to determine • Present address: Agriculture and Water Resources Research Center, Council of Scientific Research, P.O. Box 2416, Baghdad, Iraq.
the nature of the stimulatory factors produced by the active organisms.
MATERIALS AND METHODS Mycorrhizal fungi Basidiocarps of Paxillus involufus (Batsch: Fr.) Fr., Laccaria laccata (Scop.: Fr.) Cooke, Amanifa fulva (Schaeff.) Seer., Amanifa rubescens (Pers.: Fr.) S. F. Gray, Lacfarius turpis (Weinm.) Fr. and Russula nitida (Pers.: Fr.) Fr. were collected beneath birch (Betula spp.) at Whitmoor Common, Surrey. Those of Hebeloma crustuliniforme (Bull.) Que!' were collected beneath willow (Salix spp.) at the University of Surrey, GUildford, Surrey. Basidiospores of these fungi were collected from clean and mature sporocarps in sterile plastic Petri dishes under humid conditions (Ali & Jackson, 1988) and used within 24 h.
Media The four media used were: Fries agar (Fries, 1978); modified Melin-Norkrans agar (MMN) (Marx, 1969); nutrient agar CM3 (Oxoid Ltd); potato-dextrose agar (PDA) CM139 (Oxoid Ltd).
Stimulating organisms Bacteria and fungi obtained from birch forest soil; bacteria and
Nahia A. Ali and R. M. Jackson
183
Table 1. Numbers and sources of isolates of bacteria and fungi tested for germination stimulation
Paxillus involutus Laeearia laeeata Hebeloma crustuliniforme Amanita fulva Amanita rubescens Lactarius turpis Russula nitida
Soil isolates ...
Isolates associated with spores or sporophores
Isolates associated with mycorrhizal roots'
Bacteria
Fungi
Bacteria
5 6 10 5 5 5 6
3 2 3 5 4 4
6 4 6 7 7 4 2
2
Bacteria 40
Fungi 3 4 2 3 3 4 5 Fungi 30
Totals 17 16 21 20 19 17 15 70
• Roots of birch (&tula spp.) close to sporophores of the named fungus, except in the case of H. CTustuliniforme where the roots were willow (Salix spp.).
fungi associated with spores or sporophores of the test ectomycorrhizal fungi; bacteria and fungi associated with the mycorrhizal sheath of the test ectomycorrhizal fungi; bacteria and fungi appearing among the spores of the test fungi as natural contaminants on laboratory media. The number and sources of the isolates of the first three groups are shown in Table 1.
Isolation of stimulating organisms Soil bacteria and fungi were obtained by the dilution plating method. Fresh soil samples were collected from the top 30 cm of the soil profile in July, 1985 from 12 different locations beneath birch trees at Whitmoor Common, Surrey. They were passed through a 3 mm sieve and four composite samples were made. From each, 10 g of soil was suspended in 90 ml sterile distilled water, shaken in a Gallenkamp wrist-action shaker for 15 min and allowed to stand for 5 min. From this suspension serial dilutions (up to 10- 4 ) were made, and 1 ml of each dilution was plated on nutrient agar for bacteria and PDA for fungi. Three plates were prepared for each dilution and incubated at 20°C for 7 d. Forty bacterial colonies and 30 fungal isolates were selected and screened for their effect on the germination of the spores of the test ectomycorrhizal fungi. As with isolates obtained from other sources, the bacteria were chosen at random and the fungi were isolates with macroscopically distinct colonies, assumed to be either different species or different strains of the same species. Identification was attempted only of those organisms showing stimulatory activity. Isolates associated with spores or sporophores of the ectomycorrhizal fungi were obtained from caps of clean and healthy sporophores. They were surface-sterilized by wiping with tissue soaked in ethanol, peeled and broken up under aseptic conditions; small fragments of the inner tissue were placed on MMN agar (five pieces per plate). Several plates were made for each fungus and incubated at 20° for 7 d. Forty two bacterial and 23 fungal isolates were selected for study. 13
For isolates associated with mycorrhizal sheaths of the test fungi, mycorrhizal roots of birch (Betula spp.) or willow (Salix spp.) were collected beneath sporophores of the test mycorrhizal fungi. Segments of roots, 5-10 mm long and bearing mycorrhizal tips were agitated in 0'003 % Tween 80 in distilled water for 15 min to remove soil debris, followed by agitation in three changes of distilled water. The roots were then surface-sterilized by immersion in 30 % (v/ v) hydrogen peroxide for 15 s and washed again by agitation for 10 min in sterile distilled water. Individual mycorrhizal tips (ea 40 for both birch and willow) were then plated on MMN agar, five to eight per plate, and incubated at 20°. Thirty-six bacterial isolates and 24 fungal isolates were selected for study.
Germination tests Basidiospores to be tested for germination were removed from the plastic Petri dishes (within 24 h of collection) by dispersing them in sterile distilled water (mixtures of spores from 3-5 fruit bodies of the same collection were used). Fries agar plates were inoculated with 0'1 ml of spore suspension 5 (ea 5 x 10 spores per plate) spread over the agar surface, and allowed to stand overnight for absorption of surplus water. Test plates were then inoculated with the appropriate microorganism using a 6 mm diam disc of fungal mycelium from the margin of an actively-growing colony or, for bacteria, a streak ea 4 em long. Uninoculated plates and the areas of inoculated plates distant from the inoculum served as controls. Half of the surface of each plate was sprinkled with a thin layer (ea 7 mg per plate) of autoclaved activated charcoal (Darco G60, BDH Chemicals Ltd, Poole, England) that covered the basidiospores. The plates were incubated in darkness at 20° and examined daily by light microscopy for spore germination.
RESULTS AND DISCUSSION A minority of the bacterial and fungal isolates were found to MYC 93
Germination of ectomycorrhizal fungi
184
Table 2. Identity, source, and stimulatory activity of the test bacterial and fungal isolates
Isolate number
2 3 4 5
6 7
8 9 10 II
12 13
Identity of the active isolates Pseudomonas stutzeri Lehmann & Neumann Pseudomonas sp. no. I Pseudomonas sp. no. 2 Pseudomonas sp. no. 3 Pseudomonas sp. no. 4 Corynebacterium sp. no. I Enterobacteriaceae Micrococcus roseus Flugg Tritirachium yoseum van Beyma Arthrobacter sp. Corynebacterium sp. no.6a
Source
Spores stimulated
H. cyustu/iniforme
Sporophores of H. cyustu/iniforme H. cyustu/iniforme
and P. inDo/utus
} l
J
Pseudomonas sp. } no. S30 Unidentified bacterium no. S19
Spores of H. cyustu/iniforme Mycorrhizas of Salix spp. with H. crustu/iniforme Birch forest soil
stimulate germination of spores of ectomycorrhizal fungi. Of 42 bacterial isolates associated with sporophores, seven stimulated spore germination. All the active isolates came from sporophores of H. crustuliniforme and stimulated germination of spores of that fungus. One isolate only (Corynebacterium sp.) stimulated spore germination of P. involutus as well as H. crustuliniforme. Two of the 36 bacterial isolates associated with the mycorrhizal sheath stimulated spore germination. These isolates were obtained from mycorrhizas of Salix collected beneath sporophores of H. crustuliniforme and stimulated spores of the same fungus and of P. involutus (Table 2). Two bacterial isolates out of 40 obtained from birch forest soil stimulated spore germination; one of them (Pseudomonas sp. 530) stimulated germination of H. crustuliniforme spores, and the other (an unidentified Gram-positive coccus 519) stimulated spore germination of H. crustuliniforme, P. involutus and L. laccata (Table 2). One fungus (Tritirachium roseum) and one bacterium (Micrococcus roseus), which appeared on the surface of agar plates among spores of H. crustuliniforme as natural spore contaminants (Table 2), stimulated spores of H. crustuliniforme around their colonies. None of the fungal isolates obtained from sporophores, mycorrhizal sheaths or from soil, stimulated spore germination of any of the test ectomycorrhizal fungi after up to 15 d incubation; observation for longer was not possible, as the fungi covered the whole surface of the plate within 15 d. Pseudomonas stutzeri was the most active bacterium in stimulating germination of H. crustuliniforme spores. Germination began within 3 d and increased until it was ca 21 % (Table 3) up to I cm from the bacterial colony after 10 d incubation (spores were counted in ten microscope fields, chosen randomly in each of three replicate plates). Germination was ca 1'7% at a distance of 1-2 cm from the bacterial colony.
H. cyustu/iniforme
H. cyustu/iniforme,
P. inDo/utus and L. /accata
Numerous spores also germinated underneath the bacterial growth. The effect of P. stutzeri was tested in the presence of activated charcoal and growing mycelium of H. crustuliniforme either separately or together (Table 3); charcoal slightly increased germination in the presence of mycelium but not the bacterium. Mycelium had no effect on germination alone or in combination with the bacterium. Spores of H. crustuliniforme were stimulated to germinate by the two isolates of Corynebacterium after two wk incubation, and after five wk, by Micrococcus roseus and Tritirachium roseum. The exact percentage germination could not be counted with these isolates, but it was < I % very close to the activator colonies, in the presence or absence of charcoal. Slight stimulation « 0'1 %) germination) occurred in the presence of other bacteria (Pseudomonas nos 1,2,3,4; Arthrobacter sp. and an isolate belonging to the Enterobacteriaceae). Spores of P. involutus were stimulated by the two isolates of Corynebacterium, Arthrobacter and the unidentified soil bacterium (519) (Table 2), but only in the presence of activated charcoal. Percentage germination « 0'1 %) was not accurately determined, as most of the spores germinated either very close to or underneath the bacterial growth. Spores of Laccaria laccata were stimulated by the unidentified soil bacterium (519) in the presence of activated charcoal but percentage of germination was very low « 0'01 %). In all cases germinating spores produced mycelial colonies which were then transferred to MMN agar. These findings thus indicate that there is some specificity in the phenomenon of germination stimulation; only certain species of micro-organisms can stimulate spore germination of certain species of ectomycorrhizal fungi. Published data about the effect of other micro-organisms on
Nahia A. Ali and R. M. Jackson
185
Table 3. Spore germination of H. crustuliniforme on Fries agar with different supplements (after 10 d incubation)
Treatment Control (no addition) + Charcoal + Bacteriat + Mycelium + Bacteriat + Charcoal + Bacteriat + Mycelium + Bacteriat + Mycelium + Charcoal + Mycelium
Numbers of spores observed
Germinated
Germination
3'2 x 10 6
0
(%)"
0
106 10 3
0 2'0
X
10 3
0 21'3
106 10 3
0 1'5
X
10 3
20'7
6'6 x 10 3
1'4
X
10 3
21'3
6'1 x 103
1'3
X
10 3
21-6
3'2 x 106
9
3'2 9'4 3'2 7'1
x x x x
0
0'0002
• Germination values at sites 0-1 cm from bacterial colony edge. t Pseudomonas stutz.eri.
spore germination of ectomycorrhizal fungi are very limited and most studies have concerned the yeast Rhodotorula where germination was typically reported as being slow and sparse, and seldom exceeded 1'0% (Fries, 1943, 1966; Lamb & Richards, 1974). More recently Fries (unpub!., cited by Fries, 1984) claimed that no new germination inducers more powerful than those of the rather inefficient Rhodotorula were discovered during his extensive testing of a variety of microorganisms isolated from forest soil. Stimulation of spore germination of ectomycorrhizal fungi by named bacteria has not previously been reported. However, there are recent reports of the stimulation of germination of spores of VA mycorrhizal fungi and sometimes of subsequent growth by various soil micro-organisms. Azcon-Aguilar et al. (1986) tested the effects of several micro-organisms on the spore germination and development of Glomus mosseae. They found that the germination rate of surface-sterilized chlamydospores was significantly increased and the subsequent growth of hyphae was promoted by the unnamed free-living fungi tested. Mayo, Davis & Motta (1986), working with Glomus versiforme, observed that increased spore germination was caused by several genera of bacteria, including Corynebacterium and Pseudomonas; the hyphae produced following germination were longer and smoother than in the absence of bacteria. Mugnier & Mosse (1987) reported that various contaminants developing on agar plates stimulated the germination of Glomus mosseae spores. These contaminants included various filamentous fungi and in one instance Streptomyces orientalis. This actinomycete was strongly stimulatory and could induce germination at a distance by what appeared to be a volatile metabolite. Bacteria have been shown to influence the colonization of roots by ectomycorrhizal fungi (Bowen & Theodorou, 1979). One bacterium (Bacillus sp. WRl), isolated from washed Pinus radiata roots mycorrhizal with Rhizopogon luteolus significantly increased colonization of pine roots by four of the mycorrhizal fungi tested, and also stimulated growth of R. luteolus on agar media. Effects on spore germination were not examined in this study.
The nature of substances produced by micro-organisms that stimulate the germination of spores of mycorrhizal fungi is not known. However, Fries (1966, 1976) reported that yeast could stimulate germination by production of a diffusible substance(s), e.g. amino-acids, and volatile compounds (unknown) or by removing inhibitory compounds, e.g. NH 4 , from the medium. In the case of P. stutzeri volatile compounds, cell-free culture filtrates and cell extracts were tested in a further study (Ali, 1986), but none stimulated germination of the test ectomycorrhizal fungus (H. crustuliniforme). Also, germination did not occur on agar on which P. stutzeri had been allowed to grow for some time and then removed before being autoclaved and re-used for germination tests without any living bacteria. Germination occurred only in the presence of active bacteria close to the spores. This suggests that the stimulatory compound(s) produced by P stutzeri may be chemically labile and inactivated after exudation into the medium. The very high percentage of P. involutus spores germinating near roots of birch in soil, previously reported (Ali & Jackson, 1988) may have been due to the effects of both root exudates and micro-organisms similar to those reported here. N. A. Ali wishes to thank the Iraqi Government for the financial support for this work. REFERENCES ALI, N. A. (1986). Spore germination and the pre-infection phase in ectomycorrhizal fungi. Ph.D. Thesis, University of Surrey. ALI, N. A. & JACKSON, R. M. (1988). Effects of plant roots and their exudates on the germination of spores of ectomycorrhizal fungi. Transactions of the British Mycological Society 91, 253-260.
AZCON-AGUILAR, c.. ROSA, M., RODRIGUES, D. & BAREA, J. M. (1986). Effect of soil micro-organisms on spore germination and growth of the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Transactions of the British Mycological Society 86, 337-340.
BOWEN, G. D. & THEODOROU, C. (1979). Interactions between bacteria and ectomycorrhizal fungi. Soil Biology and Biochemistry 2, 119-126. 13-2
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FRIES, N. (I966). Chemical factors in the germination of spores of Basidiomycetes. In The Fungus Spore (ed. M. F. Madelin), pp. 189-199. London, u.K.: BuUerworths Scientific Publications. FRIES, N. (1976). Spore germination in Boletus induced by aminoacids. Proceedings Koninklijke Nederlandse Akademie van WeHenschappen Series C79, 142-324. FRIES, N. (1978). Basidiospore germination in some mycorrhizaforming hymenomycetes. Transactions of the British Mycological Society 70, 319-324.
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FRIES, N. (1984). Spore germination in the higher Basidiomycetes. Proceedings of the Indian Academy of Sciences (Plant Sciences) 93, 205-222. FRIES, N. (1985). Spore germination in ectomycorrhizal fungi. In Proceedings of the 6th North American Conference on Mycorrhizae, June, 1984 (ed. R. Molina). Corvallis, USA.: Forest Research
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MAYO, K., DAVIS, R. E. & MOTTA, J. (1986). Stimulation of germination of spores of Glomus versiforme by spore-associated bacteria. Mycologia 78, 426-431. MUGNIER, ]. & MOSSE, B. (1987). Spore germination and viability of a vesicular arbuscular fungus, Glomus mosseae. Transactions of the British Mycological Society 88, 411-413.
OORT. A. J. P. (1974). Activation of spore germination in Lactarius species by volatile compounds of Ceratocystis fagacearum. Proceedings Koninklijke Nederlandse Akademie van Wetenschappen Series C77, 301-307. ROMMELL, L. G. (1921). Parallelvorkimmen gewisser Boleten und Nadelbaume. Svensk Botanisk Tidskrift 15, 204-213. STACK, R. W., SINCLAIR, W. A. & LARSEN, A. O. (1975). Preservation of basidiospores of Laccaria laccata for use as mycorrhizal inoculum. Mycologia 67, 167-170. STRAATSMA, G.. KONINGS, R. N. H. & GRIENSVEN, L. J. L. D. (1985). A strain collection of the mycorrhizal mushroom Cantharellus cibarius obtained by germination of spores and culture of fruit body tissue. Transactions of the British Mycology Society 85, 689-697.
(Received for publication 5 October 1987 and in revised form 6 September 1988)