Intemational Journal of Medicinal Mushrooms, Vol. 6, pp.

347-360 (2004)

Morphological, Physiological, and Growth

Characteristics of Mycelia of Several Wood-Decaying
Medicinal Mushrooms (Aphyllophoromycetideae)
. . i- .

Suzanna M. Badalyan and Carmen Z. Sakeyan

Yerevan State University, Dept. of Botany, 1 Aleg Manoogian St., 375025, Yerevan, Armenia
Address all correspondence to S.M. Badalyan, Yerevan State University, Department of Botany, 1 Aleg Manoogian St., 375025,
Yerevan, Armenia; suzannab@yahoo.com

ABSTRACT: In this article, we describe the morphological, physiological, and cultural characteristics
(growth rate and coefficient, macro- and micromorphologlcal peculiarities, and extracellular laccase
activity tests) of vegetative mycelia of wood-decaying medicinal mushrooms Ganoderma lucidum (W.
Curt.:Fr.) P. Karst., G. applanatum (Pers.) Pat., Fomes fomentarius (L.) Fr., Laetiporus sulphureus (Bull.:
Fr.) P. Karst., Piptoporus betulinus (Bull.) P. Karst., and Daedalea quercina (L.) Pers. and estimated their
taxonomic significance. The screening was carried out on different nutrient media: malt-extract agar
(MEIA), potato-dextrose agar (PDA), and glucose-peptone agar (GPA). Mycelia of white-rot mushrooms
(G. lucidum, G. applanatum, and E fomentarius} preferred MEA medium and revealed strong laccase
activity. Laccase tests were negative in brown-rot species {L. sulphureus^ P. betulinus, D. quercina) which
grew relatively well on PDA medium.
I

KEYWORDS: medicinal mushrooms, Aphyllophoromycetidae, growth characteristics, macro- and

micromorphology, laccase activity

INTRODUCTION and nerve tome, thrombolytic, fibrinolytic, etc.) of

Preventive and curing properties of mushrooms have
been known since the ancient times. They are widely
used in the folk medicine of various countries: North
and Central America, Africa, Asia, and Europe (Yang
and Jong, 1987; Vaidya and Rabba, 1993; Hobbs,
1995; Denisova, 1998; Gr2ywnowicz,2001).
A large spectrum of therapeutic action (immune-
modulating, antifiingal, antibacterial, anti-inflam-
matory, antiparasitic, hepatoprotective, kidney
many mushroom metabolites (glucans, terpenoids,
steroids, etc.) and enzymes (e.g. proteolytic) has been
mentioned in several reviews (Wasser and Weis,
1999; Badalyan, 2(XX); Wasser, 2000; Smith et al.,
2002; Gao et al., 2003). There are also numerous
publications concerning fungal bioactive metabolites
and their physiological activity (Ikekawa et al, 1985;
Sterner, 1995; Sugiyama et al., 1995; Miura et al.,
1996; Badalyan and Serrano, 1999; Badalyan et al.,
2(X)0; Badalyan,2003a).These publications indicate

ABBREVATIONS

FB: fruiting body; GR: growth rate; GC: growth coefficient; GPA: glucose-peptone agar; MM: medicmal mushrooms;
MEA: malt-extract agar; PDA: potato-dextrose agar

1521-9437/04 $20.00
2004 by BegeU House, Inc. 347
 S. M. BADALYAN & C. Z. SAKEYAN
that medicinal mushrooms (MM) are promising
sources of bioactive compounds that require further
biotechnological and mycopharmacological research
for obtaining novel biotech products and functional
food additives with medicinal importance {Wasser
et al., 2000; Badalyan, 2001).
Data concerning the medicinal properties of in-
vestigated Aphyliophoromycetideae mushrooms is
also available. However, the majority of published
material concerns Ganoderma species, particularly
G. lucidum (W. Curt.; Fr.) P. Karst. (Reishi or Ling
Zhi), widely used in the traditional medicine of
Eastern countries (Hobbs, 1995, 2001; Smith et
al., 2002; Krasnopolskaya et al., 2003).
Tlie biopreparations and food additives in different
formulations (tablets, powders, teas, etc.) obtained
from fruiting bodies (FB) and/or mycelium of G.
lucidum are widely consumed in the world market
(Wasser and Weis, 1999; Smith et al., 2002).
Polysaccharides and triterpenoid compounds
are biologically active substances isolated from
this mushroom (Mizuno, 1995; Badalyan, 2000;
Wasser, 2002; Smith et al., 2003; Gao et al., 2003).
They possess strong immune-modulating activity,
which is revealed through their antitumor, anti-
inflammatory, antibacterial, and antifungal effects
(Ooi et al.,2001; Zhou and Gao, 2002; Mau et al.,
2002). In addition, the analgesic, tonic, antifever,
adaptogenic,and sedative actions of G. lucidum have
also been reported (Smith et al., 2002). Regular
consumption of this mushroom stimulates defense
mechanisms and maintains homeostasis in the
human organism (Gavrilova and Yakovleva,
2003; Shah and Shah, 2004). The FB extract of
G. lucidum possesses not only hep atop rotective
and hypocholesterolemic effects but also inhibits
the production of 5a-reductase enzyme, which
plays a crucial role in the development of benign
prostate hyperplasia and cancer (Liu et al., 2004).
It is successfully used in the combined therapy of
hypertension, neurasthenia, and arthritis (Niksic
et al., 2001). Tlie ability of G. lucidum to prevent
oxidative damage of DNA has been also reported
(Shi et al., 2001; Gao et al., 2003).
There are bibliographic data about the immune-
modulating, antibacterial, and antiviral effects of
348
polysaccharides and steroid compounds isolated
from other Ganoderma speciesG. applanatum
(Pers.) Pat. (Artist conk) (Smania et al., 1999).
Information concerning the medicinal properties
of other investigated species Fomesfomentarius (L.)
Fr., Laetiporus sulphureus (Bull.; Fr.) P. Karst., Pipto-
porus betulinus (Bull.) P. Karst., and Daedalea quer-
cina (L.) Pers.) is relatively rare. However, some data
concerning their immune-modulating, antifungal,
antitumor, antiviral, and antibacterial activities is
now available (Smith et al., 2002; Stamets, 2002).
The strong antibacterial activity of white crystals
(preliminary calcium oxalate acid) isolated from La-
etiporus sulphureus (Chicken ofthe woods or Sulphur
tuft) against Bacillus subtilis^ B. mycoides, B.pumilis,
Micrococcus luteus, Staphyllococcm aureus, Escherichia
coli, Comamonas terrigena, and Pseudomonas aeru-
ginosa has been revealed (Tichonova et al., 2003).
This author also describes the hallucinogenic effect
of L. sulphureus.
Recent investigation of Z-. sulphureus^ pigments
show that this mushroom produces three different
fractions of carro tinoids with antioxidant, radiopro-
tective, and antiviral activities (Kapich et al., 2004;
Gvozdkova et al., 2004). About 10 mg of carroti-
noids are contained in 1 g of dry mycelial biomass.
Recently, the antibacterial 2s:xi>nVf oi Fomesfomen-
tarius (Tinder fungus) and Piptoporus betulinus (Birch
polypore) against P. aeruginosa and Serratia marc-
escens has been reported by Stamets (2002). He also
described bacteriostatic effect of i? betulinus against
S. aureus, B. subtilis and Mycobacterium smegmatis
(closely related to Mycobacterium tuberculosis).
Daedalea quercina have been found to possess
immune-modulating and insecticide activities (Koch
et ai., 2001; Grzywnowicz, 2001).
The screening results of G. lucidum, G. applana-
tum, Ffomentarius^ L. sulphureus, P. betulinus, and D.
quercina mycelia for their antifungal activity prove
to significantly inhibit the growth of several phy-
topathogenic filamentous fungi, such as Fusarium
culmorum, Rhizoctonia cerealis, Rhizoctonia solani,
Bipolaris sorokiniana, Gaeumannomycesgraminis vas.
triticii, Verticillium dahliae (Badalyan and Sakeyan,
unpublished data).
It is known that both FB and mycelia of mush-
/nternat/ona/ Journal of Medicinal Mushrooms
 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS

rooms contain active metabolites. Investigation of MATERIALS AND METHODS

bio-ecological and morphological characteristics
of vegetative mycelium {definition of growth
parameters, screening of macro- and micromor-
phological peculiarities, biochemical and genetic
properties, etc.) will allow us to control the culti-
vation processes for obtaining high-yield biomass
and desired mushroom-based bioteeh products
from this group of fungi (Badalyan et al., 2000;
Grzywnowicz, 2001; Badalyan, 2003a; Gavrilova
and Vakovleva, 2003).
On the other hand, estimation of taxonomic
significance of mycelial characteristics is an important
step for i?7 vitro identification protocol of mushrooms.
The further investigation of taxonomic features of
both vegetative and generative stages will greatly assist
in Basidiomycetes systematics (Nobles, 1965; Stalpers,
1978; Buchalo, 1988; Badalyan, 2003b; Badalyan and
Garibyan, 2003; Garibova et al., 2003).
This paper deals with the screening of morpho-
logical, ecological, and physiological characteristics
of mycelia of wood-decaying Aphyllophoromyce-
tideae G. lucidum, G. applanatum., F.fomentarius^L.
sulphureus^ P. betulinus, and D. quercina on different
agar media. - "
Fungal Material
Six species and 12 strains of white-rot and brown-rot
Aphyllophoromycetideae G. lucidum (strains Gl-1-1,
Gl-l-2,Gl-l-3,Gl-4,Gl-5), G. applanatum (Ga-1);
F. fomentarius (Ff-1); L. sulphureus (Ls-1); P betu-
linus (Pb-2; Pb-4) and D. quercina (Dq-1, Dq-2)
were investigated (Table 1).
The cultures were separated by the tissue plug
method from fresh FB collected in Armenia and
France. All strains were easily isolated in pure ailture
over 2-3 days.They are preserved under the catalog
numbers of culture collection ofthe Fungal Biology
and Biotechnology Group (FBBG), Department of
Botany, Yerevan State University (Armenia).
Culture Conditions
The investigated strains were inoculated in Petri
dishes with ventilation using different agar media
(pH 6): 1.5% MEA (Serva), PDA (Difco), and GPA
(Difco). Incubation lasted 15 days at 25 "C. Macro-

TABLE 1. Characteristics of Tested Aphyliophoromycetideae Mushroom Strains

Natural
Date of culture Physiological substrate
N Species Strain Origination separation group (host-wood)
1. G. lucidum Gl-1-1 France 2002 White-rot Oak
2. GI-1-2 France 2002 White-rot Oak
3. GI-1-3 France 2002 White-rot Oak
4. Gl-4 Armenia 2002 White-rot Oak
5. Gi-5 Armenia 2002 White-rot Oak
6. G. applanatum Ga-1 Armenia 2003 White-rot Ash
7. F. fomentarius Ff-1 Armenia 2003 White-rot Oak
8. L. sulphureus U-1 Armenia 2002 Brown-rot Stump of
deciduous tree
9. P. betulinus Pb-2 France 2002 Brown-rot Oak
10. Pb-4 France 2002 Brown-rot Oak
11. D. quercina Dq-1 France 2002 Brown-rot Oak stump
12. Dq-2 France 2002 Brown-rot Oak stump

Volume 6, Issue 4, 2004 349

 S. M. BADALYAN & C. Z. SAKEYAN
and micromorphological observations of mycelial
colonies and vegetative structures were carried out
over 30 days of growth.
Macro morphological Investigation
Macromorphological and growth characteristics of
mycelial colonies were described on tested nutrient
media. Mycelial growth rate (GR) and growth
coefficient (GC) were determined according
to Buchalo (1988) formulas: GR = AD/Ar and
VK = DGH/T; whert D ~ diameter of colony, AD =
diiference between colony diameters during A 7"time;
G = density of colony,//= height of colony; and T =
time of colony growth. Morphology and texture of
colonies were described by Stalpers' (1978) scale.
Micromorphological Investigation
The light microscopy (Microscope PZO 38357) was
used for micromorphological studies of vegetative
mycelial structures. Preparations were obtained by
the slide culture method using a MEA medium at
25 "C. Mycelium was inoculated at the margin of
the Petri plate, and three cover slips were placed at
different distances from the inoculum. When the
mycelium was partially or completely overgrown by
cover slips, they were carefiiUy taken off and stained
for further microscopic observation.
Clamp connections, vegetative structures, hyphal
loops, microtilaments, crystals, and types of asexual
sporulation were thoroughly described within the
investigated species/strains. Preparations were pho-
tographed using the Sony DSC-P71 digital camera
under x40 objective lenses.
Laccase Activity Tests
Extracellular phenoloxidase (laccases) activity was
tested after the growth of mycelial colonies on the
MEA medium (pH 5) at 25 "C by Bavendamm
tests, using syringaldazin, a-naphtol, and guaia-
col as reagents (Saponsatien, 1998). Spots were
streaked well beyond the mycelial margin. Re-
3S0
actions were recorded after 15 minutes and the
next hour. The intensity of reactions was recorded
by a four-point scale.
RESULTS AND DISCUSSION
Mycelial Growth Characteristics:
GR and GC
The following groups of mushroom strains were
separated based on mycelial GR indicators obtained
on tested nutrient media: I, slowly growing (GR < 7.2
mm/day); II, growing with average rate (GR = 7.2-
12.0 mm/day); and III, fast growing (GR>12.0
mm/day) strains (Table 2).
On MEA 58.4% of strains (GI-1-2, Gl-1-3,
G M , Gl-5, Dq-1, Dq-2, and Ls-1) belong to the
second group, 25% were slowly growing (strains
of P. betulinus, G. applanatum)., and only 16.7%
of strains (Ff-1 and Gl-1-1) made up the fast
growing group.
The numbers of slowly (41.7%) and average (50%)
growing strains of tested mushrooms were almost
equal on the PDA medium, whereas high GR (14.4
mm/day) was only detected within the Pb-4 strain
oi P. betulinus.
The GPA was not favourable for mycelial growth
of all tested species/strains. Slow GR was revealed in
about 91.6% ofstrains. Only one strain Gl-1-1 of G.
lucidum possessed average GR value (Table 2).
On the seventh day of mycelial growth, all inves-
tigated species/strains were divided into three groups
based on their GC vaiue: I, GC < 40; II, GC = 40-60
and III, GC > 60 (Fig. 1, Table 3).
On MEA the D. quercina's strains and four strains
of G. lucidum (50%) belonged to the second group,
whereas F. fomentarius's strains and Gl-1-1 strain
(16.7%) belonged to the third one. Four ofthe 12
tested strains (33.4%), including G. applanatum, L.
sulphureus, and P. betulinus, had low GCs on MEA
medium (Table 3).
The strains with average GC were not observed
on the PDA medium. The tested strains of Z,. sul-
phureuSy D. quercina, and P betulinus (41.7%) had
high GCs, whereas almost 60% revealed GC < 40
(Fig. 1).
International Journal of Medicinal Mushrooms
 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS

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Volume 6, Issue 4, 2004 351

 S. M. BADALYAN & C. Z. SAKEYAN
GCs
Gl-1-1 Gl-1-2 Gl-1-3 GU Gl-5
FIGURE 1. Growth coefficients of several Aphyllophoromycetideae mushrooms on the seventh day of mycelial cultiva-
tion on different media: MEA, PDA, and GPA.
On the GPA medium, 75% belonged to the first
group with relatively low GC indicators. The sec-
ond group (25%) included only L. sulphureus and D.
quercina strains. None ofthe strains possessed high
GC on GPA (Fig. 1; Table 3).
The most favorable nutrient medium for mycelial
growth of white-rot mushrooms G. lucidum, G.
applanatum, and F. fomentarius was found to be
MEA, while for brown-rot species P betulinus, L.
sulphureus, and D. quercina it was PDA, then GPA
(Figs. l,2a-b).
It is worth mentioning that on PDA medium
Gl-1-1 and Gl-1-2 strains formed scarce, large dia-
meter colonies.The same phenomenon was observed
within P. betulinusy Z. sulphureus, and D. quercina
strains on MEA (Figs. 2 and 3).
Extracellular Laccase Activity
Three white-rot speciesG. lucidum, G. applana-
tum, and F.fomentariuspossessed strong laccase
activity. In contrast, brown-rot species L. sulphureus,
P. betulinus, and >. quercina didn't react to applied
tests (Table 4). The obtained results confirm that
brown-rot species lack extracellular oxidases, while
white-rot species produce them (Nobles, 1965).The
352
Ga-1 FM Ls-1 Pb-2 Pb-4 Dq-1 Dq-2
Strains
laccase activity was found within the aforementioned
species by Nobles (1965), Stalpers (1978), and
Buchalo (1988), except i?^i'rt//;n5, in which positive
reaction by Buchalo was mentioned.
The laccase activity tests could be included in spe-
cies culture description and he useful as an express
test in culture identification protocol during mycelial
cultivation processes.
Macromorphological/Micromorphoiogical
Characteristics of Mycelial Colonies
Investigated species/strains have constant
morphological descriptions on all tested nutrient
media (Figs. 2-3). Differences mentioned within
G. lucidum\ strains (Gl-1-1, Gl-1-2, and Gl-4) on
MEA were not remarkable.
The clamp connections formed at the hyphal
septa are characteristic features of dikaryotic mycelia
of many Homobasidiomycetes fungi. The presence
or absence of clamps, their shapes and sizes (single,
pairs, oval, round, giant, small) are ako considered
species-specific criteria (Fig. 4a-i).
Hyphal loops and springs often described
in Deuteromycetes fiingi (e.g. Dactylaria forms
mycelial loops to destroy the nematodes, etc.) were
rarely observed in Basidiomycetes mushrooms
Internationa/ Journal of Medicinal Mushrooms
 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS

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Volume 6, Issue 4, 2004 353

 S. M. BADALYAN & C. Z. SAKEYAN

FIGURE 2. Morphology of mycelial colonies of Laetiporus sulphureus (a) and Ganoderma lucidum (b) on MEA, PDA,
and GPA.

(Carlile et al., 2001). They were often observed
within D. quercina, P. betulinus, and G. lucidum
species (Fig. 4j, k).
TKick-walled vegetative resting sporesapi-
cally and intercalary formed chlamydospores were
seen in G. lucidum and L. sulphureus (Fig. 4n, o, r).
The chlamydospores were numerous, particularly
in L. sulphureus. They were rarely described in G.
lucidum, which was also confirmed by Garibova et
al. (2003). According to Clemen^on (1997), the
chlamydospores of L. sulpbureus belong to aleuric
type of conidiophores.
Previously, the asexual blastic type of conidial
sporulation (anamorpha) described within L. sul-
phureus (Ls-1) and P betulinus (Pb-1) strains (Fig.
4p,q) was mentioned by Nobles (1965) and Buchalo
(1988) as weil.
Production of calcium oxalic crystals with vari-
able shapes and sizes (tetrahedral, rectangle) at-
tached to the cell wall or free in the medium is a

3S4 /nternationa/ Journal of Medic/na/ Mushrooms

 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS

FIGURE 3. Morphology of mycelial colonies Daedalea quercina (a) and Piptoporus betutinus (b) on PDA, and Fomes
fomentarius (c) and Ganoderma applanatum (d) on MEA.

characteristic feature of Aphyllophoromycetideae Reverse agar is slightly yellowish. No strain-specific

mushrooms (Larrson, 1994) (Fig. 4m). The crystals
were revealed in almost all cultures.
Mycelial growth characteristics and macro- and
micromorphological descriptions of investigated
MM species/strains are presented below.
Daedalea quercinacolony is white, woolly/felty
with slightly raised reticulations. The colony edge is
vrell expressed. Usually grows with concentric circles-
morphological differences were observed (Fig. 3a).
Dikaryotic hyphae are strongly vacuolized.The nu-
merous chaotic, giant, and small clamp-connections
are present (Fig. 4a-c). Hyphal loops and crystals
were present (Fig. 4k).
The laccase test was negative on MEA, mycelial
GR = 9.6 mm/day, GC = 43.7-^7.1.
Fomes fomentariuscolony is white at first, cottony,

Volume 6, Issue 4, 2004 355

 S.M, BADALYAN & C. Z. SAKEYAN

TABLE 4. Extracellular Laccase Tests in Several Aphytlophoromycetideae Mushrooms

Reagents
Species Strains a-Naphtol Guaiacol Syringaldazin
White-rot species
GM-1
GI-1-2
G. lucidum GI-1-3
GI-4
GI-5
G. app(anatum Ga-1
F. fomentarius
Brown-rot species
L. sulphureus Ls-1
Pb-2
P. betulinus
Pb-4
Dq-1
D. quercina
Dq-2

Notes; (-) - no reaction, (+) - {++++) - intensity of reaction

becomes creamy to brown, felty to chamois-like,
with some raised white, woolly plateaus and compact
lumps on the surface (Fig. 3c). The hyphae are not
vacuolized.Semiround clamps, mostly without pores,
are typical for dikaryotic mycelium (Fig. 4g).
Tlie laccase test was positive: mycelial GR= 16.8
mm/day and GC = 77.1.
Ganoderma applanatumcolony texture is white,
cottony, or farinaceous, in part opaque, felty, or pel-
licular,yellow to buff". Usually grows with concentric
circles. Colony's edge is well expressed (Fig. 3d).
The typically branched hyphae and oval clamps were
often described in this species.
The iaccase test was positive: the colony GR = 7.2
mm/day and GC = 18.4.
Ganoderma lucidumcolony is white, later becomes
brown, felty to chamois-like, buff or olive-bufF, pel-
licular with yellow exudate, and grows \vith concen-
tric circles. Only in strain Gl-1-1 do the mats not be-
come buff". Armenian strains (Gl-4, Gl-5) are more
pellicular than strains from France (Gl-1-1, Gl-1-2,
and Gl-l-3).Tlie colony's edge is well expressed.The
reverse of agar is partly brown (Fig. 2b). Dikaryotic
hyphae are large and strongly vacuolated. The "coral
shaped" branched hyphae previously described by
Garibova and coauthors (2003) were also present.
Tlie clamps are oval with narrow central pore (Fig.
4h and i). TKe hyphal anastomosis and rectangular
crystals, apical and intercallar chlamydospores have
been seen (Fig. 4n).
The laccase activity test was positive. The growth
characteristics of colonies on MEA were the follow-
ing: GR=9.6 mm/day, GC=40.3-48.9.
Laetiporus sulphureuscolony mats are pale orange,
cottony and farinaceous, and form pellets on the
surface with slightly orange on the reverse side ofthe
agar (Fig. 2a). Clamps are without pores, rare, small,
and not well expressed. Chlamydospores are numer-
ous, apical and intercallar (Fig. 4o and r). Asexual
blastic sporulation was also present (Fig. 4q).
The laccase test was negative: GR=9.6 mm/day
and GC = 6.7.
Piptoporus betulinuscolony is white, slightly
raised at first, cottony to floccose, sub-felty and

356 /nternat/onal Journal of Medicinal Mushrooms

 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS

FIGURE 4. Micromorphology of vegetative structures of several Aphyllophoromycetideae mushrooms. Different

types of clamp-connections in Daedaiea quercina (a, b, c), Fomes fomentarius (g), Ganoderma lucidum (h, i), and
Piptoporus betulfnus {d, e, f); mycelial loops in R betutinus (j) and Daedalea quercirja (k); anastomosis (I) and crystals
(m) in Piptoporus betulinus; chlamydospores in Ganoderma luddum (n) and Laetiporus sulphureus (o, r); blastospores
in Piptoporus betulinus (p), and Laetiporus sulphureus (q).

Volume 6, Issue 4, 2004 357

 S. M. BADALYAN &C. Z. SAKEYAN
'7'-
tratislucent at edges, except for scattered small
areas of more compact mycelium in tbe middle
and small, compact balls of mycelium, usually
grown against Petri disb walls, bearing poroid
fruiting areas (Ftg. 3b). Dikaryotic mycelium is
vacuolated. The clamps are numerous, chaotic, oval
or round, giant and small (Ftg. 4d-f). Hyphal loops,
anastomosis, and tetrahedral or/and oval shaped
crystals were typical for P. betulinus strains (Ftg.
4j, 1, m). Asexual blastospores were also described
(Fig.4p).
The laccase test was negative. The colony's GR =
4.8-7.2 mm/day; GC= 19.7-28.3.
Thus, macro- and micromorphological descriptions
of vegetative mycelia, as well as tbe growth charac-
teristics of screened medicinal Aphyllophoro-
mycetideae species, are species specific and could
be used tor their in vitro identiiication processes.
CONCLUSIONS
The taxonomic significance of several growth and
macro- and micromorphological characteristics of
vegetative mycelia was revealed as a resitlt of the
screening of six MM mushroom cultures (Aphyl-
lophoromycetideae). The texture and pigmentation
of colonies, mycelial growth rate and coefficient,
shape and size of clamp connections, type of asexual
sporulation are species-specific features.
The brown-rot mushrooms can be distinguished
from white-rot ones by extracellular laccase
activity tests. The white-rot mushrooms {G.
lucidum, G. applanatum, and F. fomentarius) prefer
MEA, whereas brown-rot species {P. betulinus,
L. sulphureus, and D. quercina) prefer PDA, then
GPA media.
The above-mentioned characteristics of vegetative
mycelium could be useful in culture in vitro
identification protocol of screened species during
their biotechnological cultivation processes.
Further studies directed tovrard revealing opti-
mal in vitro grovrth parameters (pH, temperature,
and aeration), medicinal properties of investigated
tnuslirooms, and estimation of their biotechnologi-
cal potential are in progress.
358
ACKNOWLEDGMENTS
This work was supported by the Ministry of Science
and Education of Armenia (#0104). SMB gratefully
acknowledge the DAAD (#548.104401.174),
NATO (#FEL.RIG. 980764), and ANSEF (#04-
NS-biotech-814-73) for financial support.
REFERENCES
Badalyan S.M. 2000. Immunomodulating and antitumor
acti\ity of macroscopic fungi {Basidiomycofina).
Review. ProM Med Mycology. 2, 22-30,
Badalyan S.M. 2001. Higher Basidiomycetes as perspec-
tive objects for myco-pbarmacological research. Int
JMecf Musbr, 3, IQS.
Badalyan S.M. 2003a. Edible and medicinal higher
Basidiomycetes mushrooms as a source of natural
antioxidants. IntJ Med Musbr, 5,153-162.
Badalyan S.M. 2003b. Taxonomic significance of tbe
biological features of vegetative structures within
Basidiomycete mushrooms. In: Bot. Res. Asian
Russia, Proc. XI Congr. Russ. Bot. Soc, August
18-22, Novosibirsk-Barnaoul, pp. 7-8.
Badalyan S.M. and Serrano J,-J. 1999. Hypoglycemic
activity oiHypbolomafasciculare (Fr.) Kumm. IntJ
Med Musbr, 1,245-50.
Badalyan S.M. and Garibyan N.G. 2003. Cultural char-
acteristics of several medicinal musbrooms. In: Bot.
Res. Asian Russia. Proc, XI Congr. Russ. Bot. Soc.
August 18-22, Novosibirsk-Barnaoul, pp. 10-11.
Badalyan S.M., Serrano J.-J., Rapior S., and Andary
C. 2000. Pharmacological activity of macroscopic
fi.ingi: Flammulina velutipes (Curt. : Fr.) Sing.,
Paxillus involutus (Batsch.: Fr.) Fr. and Tricboloma
tigrinum SchaefF. (Basidiomycotina). Int J Med
Buchaio A.S. 1988. The Higher Edible Basidiomycetes
in Pure Culture, Naukova Dumka Press, Kiev 143
pp. (in Russian).
CarUle M.J., Watkinson S.C., and Gooday G.W. 2001.
Tbe Fungi. Academic Press, London, UK. 588 pp.
Clemen^on H. 1997. Anatomie der Hymenomyceten.
Univ. Lausanne, Switzerland, 990 S.
DenisovaN,P. 1998. Medicinal properties of musbrooms.
Etbno-mycological sketch. Saint-Petersburg, 59
pp. (in Russian).
Gao Y., Lin Z.-B., Huang M., and Zhou S. 2003.
/nternati'ona/ Journal of Medicinal Mushrooms
 CULTURAL CHARACTERISTICS OF SEVERAL MEDICINAL MUSHROOMS
Hepatoprotective activity and tbe mecbanisms of
action of Ganoderma lucidum (Curt.:Fr.) P. Karst.
(Ling Zbi, Reishi Mushroom) (Apbyllopboromy-
cetideae) (R^tvf).IntJMedMusbr,5, Ul-131.
Garibo\'a L.V., Antimonova A. V., Zavyalova L.A., and
Krosnopolskaya L.M. 2003. Growth and morpbo-
logical features of Ganoderma lucidum mycelium
depending on cxilture conditions. Mykol i Fitopatol^
37,14-19.
Gavrilova V.P. and Yakovleva N.S, 2003. Biotecbno-
logical using of Basidiomycete Ganoderma lucidum
(Curt.: Fr.) P. Karst. In: Adv. Med. Mycology. Proc.
I Int. Congr. Med. Mycology, Moscow, Nat. Myc.
Acad. Press, 1,261.
Grzywnowicz K. 2001. Medicinal mushrooms in PoUsb
folk medicine. Int J Med Musbr, 3,154.
G^'ozdkova T.S., Mishin L.T, ChemokTV., Plenina
L.V., and Kapich A.N. 2004. Submerged mycelium
of xantbophyll-containing mushroom L. sulpburous
basis for a novel bioactive supplements. In: Adv.
Med. Mycology. Proc. II Int. Congr. Med, Mycology,
Moscow, Nat. Myc. Acad. Press, 3,218.
Hobbs Ch.R. 1995. Medicinal Mushrooms: an Explor-
ation ofTradition, Healing and Culture. Botanica
Press, Santa Cruz, 251 pp.
Hobbs Cb.R. 2001. Medicinal mushrooms: modem clin-
ical uses. Overview. Int J Med Mushr, 3, 86.
Ikekawa T , Maruyama H., Miyano T et al. 1985.
Proflamin, a new antitumor agent: preparation,
pbysicocbemical properties and antitumor activity.
JapJ Cancer Res, 76,142-148.
Kapich A.N., Gvozdkova TS., Kvacheva Z,B., Niko-
lae\'a S.N., Shisbkina L.N., Gaikin S., Kha-
takka N., Konoplya E.F., Verescbako G.G.,
Khodosovskaya A.M., and Rutkovskaya J.A.
2004. Antioxidant, radioprotective and antiviral
properties of mycelium extracts of Laetiporus sul-
pburem mushroom. In: Adv. Med. Mycology. Proc.
II Int. Congr. Med, Mycology, Moscow, Nat. Myc.
Acad. Press, 3,146.
Koch J., Witt S., and Lindequist U. 2001. Screening of
several Basidiomycetes extracts for tbeir immuno-
modulating activity. IntJ Med Musbr, 3,170.
Krasnopolskaya L.M., Antimonova A. V., Belickii I. V.,
SobolevaN.J., and Garibova L.V. 2003. Obtaining
biomass from medicinal musbrooms Ganoderma
lucidum (Curt. : Fr.)P. Karst. and Lentinus edodes
(Berk.) Sing, in submerged culture. In: Adv Med.
Mycology. Proc. I Int. Congr. Med. Mycology,
Moscow, Nat. Myc. Acad. Press, 1,281.
Volume 6, Issue 4, 2004
Larrson K.-H. 1994. Poroid species in Trecbispora and
the use of calcium oxalate cr)'stals for species iden-
fication. Mycol Res, 98,1153-1172.
Liu J., Shimizu K., Konisbi F., Noda K., Kumamoto
S., Ueda C , Tajiri H., Kaneko S., Stiimi Y., and
Kondo R. 2004. The anti-androgenic effect of
: Ganoderma lucidum. In: Scientific Cultivation of
Edible Medicinal Fungi. Proc. XVI Int. Congr.,
14-17 March, Miami, Florida, 645-652.
Mau J.-L., Lin H . - C , and Cben C.-C. 2002. Antioxi-
dant properties of several medicinal mushrooms,/
Agric Food Cbem, 50,6072-6077.
MitiraN., Ohno N.,AketagawaJ., Tamura H., Tanaka
S., and Yadomae T. 1996. Blood clearance of (1-
3)-beta-D-giucan in MRL LPR/LPR mice. FEMS
Immunol Med Microbiol, 13,1, 51-57.
Mizuno T. 1995. Bioactive biomolecules of musbrooms:
food function and medicinal effect of musbroom
fungi. Food Rev Int, 11, 7-21.
Niksic M., Nikicevic N., Tesevic V., and Klaus A. 2001.
Evaluation of alcoholic beverages based on Gano-
derma lucidum (Curt. : Fr.) P. Karst. extract. InfJ
Med Musbr, 3,192.
Nobles K.M. 1965. Identification of cultures of wood-
inhabiting Hymenomycetes, Can J Botany, 43,
1097-1139.
Ooi S.M.L., Ooi V.E.C., and Fung M.C. 2001. Induc-
tion of immunomodulatory cytokine gene expres-
sion by Ganoderma lucidum (Curt. : Fr.) P. Karst.
poiysaccharide in tbe mouse, Int J Med Musbr, 3,
195.
Saponsatien S. 1998. Some characteristics of ammonia
iunglj Gen ApplMicrobiol, 44,337-345.
Shab N.R. and Sbab B.N. 2004. Ganoderma lucidum: a
novel addition to modem medicine. In: Scientific
Cultivation of Edible Medicinal Fungi. Proc.
XVI Int. Congr. 14-17 March, Miami, Florida.
653-658.
Shi Y.-L., James A.E., Benzie F.F., and J.A. Buswell.
2001. Prevention of oxidative damage to cellular
DNA by mushroom-derived components. IntJ
Med Musbr, 3,221.
Smania J.A., Monache F.D., Smania E,F.A., and Cu-
neo R.S. 1999. Antibacterial activity of steroidal
compounds isolated from Ganoderma applanatum
(Pers.) Pat. (Apbyllopboromycetideae) fruit body.
IntJ Med Musbr, 1, 325-330.
Smith E.J., Rowan N., and Sullivan R. 2002. Medici-
nal musbrooms: tbeir tberapeutic properties and
current medicinal usage with special emphasis
359
 S. M. BADALYAN & C. Z. SAKEYAN
on cancer treatments. Cancer Research, UK,
250 pp.
Smith EJ., Sullivan R., and Rowan N. 2003. The role
of polysaccharides derived trom mediciinal mush-
rooms in cancer treatment programs current per-
spectives (Review). Int J Med Mushr., 5,217-234.
Stalpers J.A. 1978. Identification of wood-inhabiting
Aphyllophorales in pure culture. Stud Mycol, 16,
248 pp.
Stamets P. 2002. Novel antimicrobials from mushrooms.
Herbal Gram, 54, 28-33.
Sterner 0.1995. Toxic terpenoids from higher fungi and
their possible role in chemical defence systems.
Cryptogam Mycol, 16,1,47-57.
Sugiyama K., Akachi T , and Yamakawa A. 1995. The
hypocholesterolemic action of eritadenine is medi-
ated by a modification of hepatic phospholipid
metabolism in lats.JNutrif, 125, 2134-2144.
Tichonova O.V., Ersho\'a E.J., Lurye L.M., Kulyayeva
V.V., Katrukha G.S., KamzoUdna O.V., Efi^men-
kova O. V., and Dudnik Yu.V. 2003. Antimicrobial
properties oiLaetiponts mlphurem (Fr.) Bond et Sing.
liK Adv, Med. Mycology. Proc. 1 Int. Congr. Med.
360
Mycology, Moscow, Nat. Myc. Acad. Press, 1,313.
Vaidya J. and Rabba A. 1993. Fungi in folk medicine.
Mvra/o^w/', 3,131-133.
Wasser S.P. 2002. Medicinal mushrooms as a source of
antitumor and immunomodulating polysaccha-
rides. .f^/>/'/Mirro^w/5ioA?c,^wo/, 60,258-274.
Wasser S.P. and Weis A.L. 1999. Medicinal properties
of substances occurring in liigher basidiomycetes
mushrooms; Current perspectives (Review). Int]
Med Musbr, 1,31-62.
Wasser S.P., Nevo E., Sokolov D., Reshetnikov S., and
Timor-Tismenetski M. 2000. Dietary supple-
ments from medicinal mushrooms: diversity of
types and variety of regulations. Int J Med Mushr,
2,1-19.
Yang Y.Q. and Jong C.S. 1987. Medicinal mushrooms
in China. In: Scientific Culti\^tion of Edible Medi-
cinal Fungi. Proc. XII Int. Congr. Braunschweig,
Germany, pp. 631-643.
Zhou S. and Gao Y. 2002. The immunomodulating ef-
fects of Ganoderma lucidum (Curt.: Fr) P. Karst.
(Ling Zhi, Reishi Mushroom) (Aph}'Uophoromye-
ddeae). hit J Med Mushr, 4,1-11.
/nternationa/ Journal of Medicinal Mushrooms