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ACTA FACULTATIS FORESTALIS XXXVI
ZVOLEN – SLOVAKIA 1994
THE EFFECT OF MEDIUM AND AGAR CONTENT ON
GROWTH OF PURE CULTURES OF
ECTOMYCORRHIZAL FUNGI AMANITA MUSCARIA
(L.: FR.) HOOK. AND SUILLUS GRANULATUS (L.:
FR.) O. KUNTZE.
Ivan R e p á č
Repáč, I.: The effect of medium and agar content on growth of pure cultures of
ectomycorrhizal fungi Amanita muscaria (L.:FR.) HOOK. and Suillus granulatus (L.:FR.)
O. KUNTZE. Acta Facultatis Forestalis Zvolen, XXXVI, 1994, s. 85-92.
The effect of medium and agar content (cultivation method) on growth of pure
cultures of ectomycorrhizal fungi Amanita muscaria (isolate 109 TUZ) and Suillus
granulatus (isolate 140 TUZ)was investigated in flask scale experiment under laboratory
conditions. MMN, KHO and MPM (maltpepton medium) media with three different
contents of agar (1.5%, 0.3% and no agar) were tested.
The intensity of growth of both fungi was significantly influenced by the
composition of the nutrient medium as well as method of cultivation. MMN solid medium
was the most suitable for mycelial growth of both fungi A. muscaria and S. granulatus. The
mycelial yields of A. muscaria in MMN medium were enhanced 32% and 26%, S.
granulatus 43% and 32% as compared to growth in KHO and MPM media, respectively.
As compared to mycelial growth in liquid media, growth of A. muscaria was enhanced
114% and S.granulatus 94% on solid media, whereas in semi-liqiud media was practically
the same.
Key words: Amanita muscaria, Suillus granulatus, mycelial growth, nutrient media
1. INTRODUCTION
Isolation and experimental manipulation of ectomycorrhizal fungus
cultures have been critical in developing all phases of ectomycorrhizal research
(MOLINA – PALMER 1982). Isolation and comparative study of diverse fungal
species and isolates within species provide a basis for selecting specific isolates for
artificial inoculation of nursery stock. The important criterion for selection of the
fungi is the ability of the selected fungus to grow in pure culture and withstand
manipulation. Ideally, the fungus should be able to grow rapidly.
Unfortunately, ectomycorrhizal fungi are difficult to grow in the laboratory.
Many species have never been isolated and grown in pure culture. However, most
species of Suillus and Rhizopogon are easily isolated and grow well in culture. In the
genus Amanita, species in subgenus Amanita are more easy to isolate and grow in
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culture than species of subgenus Amanitopsis (MOLINA – PALMER 1982).
The cultivation of pure cultures of ectomycorrhizal fungi is unavoidable for
preparation of vegetative fungal inoculum, which has been repeatedly recommended
as the most biologically sound method of inoculation (MARX – KENNY 1982). A
variety of culture media and methods of cultivation can be used for propagation,
inoculum preparation, storage on agar, fruiting, and other experimental procedures
(CUDLÍN et al. 1980, HUTCHISON 1990, HARVEY 1991).
Ectomycorrhizal fungi often grow very slowly in ordinary liquid culture, or not
at all (MARX – KENNY 1982). Agar-solidified nutrient media may be used to
produce small amounts of mycelium (CHAPMAN et al. 1990). The cultivation of
fungi in semi-liquid medium (agar added at low concentration) has several
advantages (CHAPMAN et al. 1990). Faster growth is sustained without frequent
shaking or addition of glass fragments. In nearly every case, the initial growth lag
phase is shorter compared to standard liquid culture (MOLINA – PALMER 1982) and a
separate "fuzzing out" procedure is not necessary.
Amanita muscaria and Suillus granulatus are typical symbionts with Picea abies
(L.) KARST. and Pinus sylvestris L., respectively, the most spread and significant
coniferous tree species in Slovakia. The objective of this experiment was to
determine the effects of medium composition and agar content on growth of
mycelial cultures of ectomycorrhizal fungi Amanita muscaria (isolate 109 TUZ) and
Suillus granulatus (isolate 140 TUZ).
2. MATERIALS AND METHODS
2.1. Cultures of mycorrhizal fungi
Cultures of fungi under investigation were obtained by the explantation of
tissues from the fruitbodies (boundary of cap and stipe). Media KHO, MMN, BAF
and agar with malt extract (IMUNA Šarišské Michaľany) were employed for
maintenance of cultures in the recultivation cycles.
Characteristics of studied fungal pure cultures:
Amanita muscaria (L.: Fr.) HOOK.: isolate 109 TUZ, isolated on KHO medium, the
fruitbody collection in 1990 under Norway spruce, altitude approximately 500 m,
near the town of Banská Bystrica, Slovakia.
Suillus granulatus (L.: Fr.) O. KUNTZE.: isolate 140 TUZ, isolated on MMN
medium, the fruitbody collection in 1991 under Norway spruce, altitude
approximately 450 m, near the town of Zvolen, Slovakia.
2.2. Nutrient media
The growth of pure cultures was investigated in three nutrient media:
MMN, KHO and MPM - maltpepton medium, each with three different contents of
agar: 1,5% (solid media), 0.3% (semi-liquid) and no agar added (liquid).
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The media employed were of the following composition (g/1000 ml of distilled
water):
MMN (modification of MELIN-NORKRANS medium, MARX 1969): malt extract 3.0;
glucose 10.0; KH2PO4 0.5; MgSO4.7H2O 0.15; FeCl3.6H2O 0.012; NaCl 0.025;
CaCl2 0.05; (NH4)2HPO4 0.25; Thiamine 100µg, pH 5.5-5.7.
KHO (modification of ODDOUX medium 1953, in ŠAŠEK – MUSÍLEK 1967): malt
extract 5.0; glucose 5.0; casein hydrolysate 1.0; NH4Cl 0.5; KH2PO4 0.5;
MgSO4.7H2O 0.5; Fe2(SO4)3 0.05, pH 5.5 - 5.7.
MPM (maltpepton medium): beer malt 50 ml; pepton 5.0, pH 5.5 - 5.7.
2.3. Inoculation and cultivation conditions
The media were placed in 100 ml Erlenmeyer flasks at a rate of 15 ml per
flask. All flasks were autoclaved 20 min at 120 °C and allowed to cool. Twelve
flasks were used for each combination of medium and agar content, 3x3x12=108
flasks altogether. Mycelium grown on MMN agar medium was used for inoculation.
Inoculation was carried out in air-filtered box. Each inoculation consisted of the
addition of a 4 mm agar plug from an agar-plate colony to Erlenmeyer flask.
The inoculating plug was placed gently in the semi-liquid agar flask so that it
would float on top of the agar. The cultures were then allowed to rest undisturbed
until growth had begun on the top of the medium, then shaken by hand vigorously
once, and allowed to rest until evaluated. The liquid cultures grew stationary,
shaking vigorously for a few seconds two times per week on a small laboratory
shaker. The cultures were grown at 21 °C in darkness for five weeks.
To evaluate growth, cultures were heated to boiling and drained on filter paper
and rinsed with boiling water (CHAPMAN et al. 1990). Mycelium was air-dried for
24 hours and dry mass was determined.
Data were analyzed by two-way analysis of variance (ANOVA) singly for each
fungi and differences among means were evaluated with TUCKEY's test.
3. RESULTS
The ANOVA indicated the influence of medium and cultivation method on
growth of mycelial cultures of A. muscaria and S. granulatus was highly significant
(p = 0.01). Better dry weight growth of S. granulatus in pure culture in comparison
with A. muscaria was observed (Table 1, Figs. 1, 2).
Dry weights of pure cultures of both fungi in MMN medium were significantly
higher than in KHO and MPM media (Table 1, Fig. 1). No significant differences
between KHO and MPM media were detected. The maximal mycelial yield of A.
muscaria (2.50 mg.ml-1) was found in MMN medium, as compared to 1.98 and 1.90
mg.ml-1 in MPM and KHO media, respectively. Similar results were obtained with
mycelial cultures of S. granulatus, when the maximal growth was 3.33 mg.ml-1 in
MMN medium and the minimal one in KHO medium (2.33 mg.ml-1).
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Table 1 Effects of media and agar contents on the mycelial growth of ectomycorrhizal fungi Amanita muscaria and
Suillus granulatus
Source of
variation
Amanita muscaria
Suillus granulatus
Mycelial dry mass
(mg.ml-1)
Standard deviation
(mg.ml-1)
Mycelial dry mass
(mg.ml-1)
Standard deviation
(mg.ml-1)
Medium
MMN
KHO
MPM
2.50b
1.90a
1.98a
±0.22
±0.16
±0.19
3.33b
2.33a
2.52a
±0.17
±0.13
±0.30
Agar
0.0 %
0.3 %
1.5 %
1.54a
1.70a
3.30b
±0.14
±0.14
±0.17
2.29a
2.35a
3.65b
±0.17
±0.13
±0.16
Within a given source of variation and fungal species, means not followed by the same letter are significantly
different at the p = 0 .01 level.
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Fig. 1 Growth intensity of pure cultures
of Amanita muscaria and Suillus
granulatus in different media
The dry weight growth of both fungi in pure cultures on solid media was
significantly greater than in semi-liquid and liquid media (Table 1, Fig. 2). As
compared to mycelial growth in liquid media, growth of A. muscaria was enhanced
114% and S. granulatus 94% on solid media, whereas in semi-liqiud media was
practically the same. The results suggest that in this experiment MMN solid medium
is the most suitable for cultivation of studied isolates of ectomycorrhizal fungi
Amanita muscaria and Suillus granulatus.
4. DISCUSSION
Investigations into the effect of media and cultivation methods on in vitro
growth of ectomycorrhizal fungi have been stimulated by the desire to select those
which are best suited to use in inoculation procedures. In the present study MMN
medium and solid media proved to be the most efficient on mycelial growth of
Amanita muscaria and Suillus granulatus.
ŠAŠEK - MUSÍLEK (1967) found the influence of composition of the media
on the ability and intensity of growth of pure cultures of 17 ectomycorrhizal species
(35 strains). The best growing were all studied strains of the genus Suillus while the
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Fig. 2 Growth intensity of pure cultures
of Amanita muscaria and Suillus
granulatus in media with different
agar content
representatives of the genus Amanita (including A. muscaria) belonged to the worst
cultivable fungi. The pure cultures of Suillus granulatus and Paxillus involutus
exhibited better growth than Leccinum scabrum on KHO medium without and with
addition of NaCl (GÁPER 1992).
It appears the s pecies of genus Suillus really belong to the best growing
ectomycorrhizal fungi in pure cultures. Among 6 species of ectomycorrhizal fungi S.
granulatus achieved the largest diameter of pure culture colonies in the majority of
tested media, with the exception of two media including MMN medium (TORRES -
HONRUBIA 1991). In general, A. muscaria grew more weakly than S. granulatus,
however similar results for these fungi were recorded in MMN medium. S. collinitus
also belonged to well growing fungi. The reports of the authors mentioned above
tends to be very similar with our results.
HYUNG-WON et al. (1991) also confirmed the significant influence of the
composition of medium on mycelial growth, when at optimum cultivation
conditions in MMN broth containing 10 g.l-1 D-glucose supplemented with 800
mg.l-1 lysine the mycelial yield of Pisolithus tinctorius was enhanced 120 % as
compared to the yield in standard MMN broth.
In experiment of CHAPMAN et al. (1990) eleven (including isolate A46 of
Suillus sp.) of the 12 fungal cultures grew sig nificantly faster in semi-liquid (0.3 %
content of agar) than in the liquid medium. In our experiment the fungal cultures
grew significantly better on agar media than in both semi-liquid and liquid media,
where the mycelial growth was practically the same. The results can be influenced
by procedure of growth evaluation of mycelial cultures and also probably by
cultivation methods, although ŠAŠEK - MUSÍLEK (1967) did not find differences in
the ability and rapidity of growth between the stationary and submerged cultivation.
HARVEY (1991) reported, that for liquid cultivation in static flask culture the plugs
should float on the surface to allow maximum aeration. Ectomycorrhizal fungi do
not grow well in static culture unless they are growing on the surface.
In spite of our effort consistently separate the mycelium by boiling of agar
medium and washing of fungal cultures with boiling water the remnants of agar in
mycelial dry mass are not excluded. However, if the growth of in vitro fungal
cultures growing on agar medium is evaluated by measuring of colony area (ŠAŠEK -
MUSÍLEK 1967) or diameter (DANNEL - FRIES 1990, HUTCHISON 1990, TORRES -
HONRUBIA 1991) the results are not entirely reliable because possible differences in
mycelial density (ŠAŠEK - MUSÍLEK 1967).
5. CONCLUSION
The pure cultures of Suillus granulatus grew better than Amanita muscaria
and MMN solid medium, of those tested, proved to be the most efficient on mycelial
growth of both fungi. With respect to symbiotic relationships of A. muscaria and S.
granulatus with Picea abies and Pinus sylvestris, recpectively, the benefit of these
results is obvios.
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Acknowledgement
I am grateful to Mrs. Kate Argay for technical assistance.
References
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on dilute agar. Mycologia, 82, 1990, pp. 526-527.
CUDLÍN, P., MEJSTŘÍK, V., ŠAŠEK, V.: The effect of the fungicide Dithane M-45 and the
herbicide Gramoxone on the growth of mycorrhizal fungi in vitro. Česká Mykologie, 34,
1980, pp. 191-198.
DANELL, E., FRIES, N.: Methods for isolation of Cantharellus species, and the synthesis of
ectomycorrhizae with Picea abies. Mycotaxon, 38, 1990, pp. 141-148.
GÁPER, J.: Mycorrhizas in the urbanized landscape of Slovakia (in Slovak). In: P. HRUBÍK
(ed.): International symposium at the occasion of the 100th anniversary of the arboretum
Mlyňany foundation 1982-1992. Bratislava, Veda 1992, pp. 124-129.
HARVEY, L. M.: Cultivation techniques for the production of ectomycorrhizal fungi.
Biotechnology Advances, 9, 1991, pp. 13-29.
HUTCHISON, L. J.: Studies on the systematics of ectomycorrhizal fungi in axenic culture. V.
Linear growth response to standard extreme temperatures used as a taxonomic character.
Canadian Journal of Botany, 68, 1990, pp. 2179-2184.
HYUNG-WON, S., CRAWFORD, D. L., KORUS, R. A.: Effects of varying nutritional and
cultural conditions on growth of the ectomycorrhizal fungus Pisolithus tinctorius SMF.
Journal of Microbiology and Biotechnology, 1, 1991, pp. 121-125.
MARX, D. H.: The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots
to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi
and soil bacteria. Phytopathology, 59, 1969, pp. 153-163.
MARX, D. H., KENNEY, D. S.: Production of ectomycorrhizal fungus inoculum. In:
N.C.SCHENCK (ed.): Methods and principles of mycorrhizal research. St. Paul, The
American Phytopathological Society, 1982, pp.131-146.
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VPLYV MÉDIA A OBSAHU AGARU NA RAST MYCÉLIOVÝCH KULTÚR
EKTOMYKORHÍZNYCH HÚB AMANITA MUSCARIA (L. EX FR.) HOOK. A
SUILLUS GRANULATUS (L. EX FR.) O. KUNTZE.
SÚHRN
Rast mycéliových kultúr ektomykoríznych húb Amanita muscaria (izolát 109TUZ
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a Suillus granulatus (izolát 140 TUZ) bol sledovaný v laborat¢rnych podmienkach na
živných médiách MMN, KHO a MPM, s rôznym obsahom agaru: 1.5% (tuhé médiá), 0.3%
(polotekuté) a bez agaru (tekuté). Čisté kultúry rástli v tme pri 21°C v Erlenmeyerových
bankách a po 5 týždňoch sa intenzita rastu vyjadrila hmotnosťou mycélia v sušine.
Podľa dvojfaktorovej analýzy variancie vplyv druhu média a obsahu agaru na rast
mycélia obidvoch húb bol štatisticky vysoko významný. Všeobecne, čisté kultúry S. bovinus
rástli intenzívnejšie než kultúry A. muscaria. Najvyššiu priemernú hmotnosť mycélia v
sušine (3.33 mg.ml-1) dosiahla huba S. bovinus na MMN médiu, čo je o 43% viac než na
KHO a o 32% viac než na MPM. Rovnako Amanita. muscaria rástla najintenzívnejšie (2.50
mg.ml-1) na MMN, o 32% a 26% intenzívnejšie než na médiách KHO a MPM. Mycéliové
kultúry obidvoch húb rástli štatisticky významne lepšie na tuhých médiách v porovnaní s
polotekutými a tekutými médiami, kde bol rast prakticky vyrovnaný.
Vzhľadom na to, že Amanita muscaria je typický symbiont s Picea abies a Suillus
granulatus s Pinus sylvestris, využitie výsledkov je aktuálne pri kultivácii väčšieho množstva
mycéliových kultúr v procese umelej mykorizácie semenáčikov týchto drevín.
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