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Instability in in vitro fruiting of Cordyceps militaris

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Journal of Mushroom Science and Production Vol. 2, No 3, p140-144 Sep. 2004
Copyright ⓒ 2004 by The Korean Society of Mushroom Science Printed in S. KOREA

Instability in in vitro fruiting of Cordyceps militaris

Bhushan Shrestha, Young-Jin Park, Sang-Kuk Han, Sung-Keun Choi and Jae-Mo Sung*
Department of Applied Biology, Division of Bio-Resources Engineering and Entomopathogenic
Fungal Culture Collection, Kangwon National University, Chuncheon 200-701, Korea

강원대학교 농업생명과학대학 생물자원공학부 응용생물학과

ABSTRACT : Traditionally, Cordyceps species have been used as a part of herbal medicine in Oriental countries,
including Korea for internal health, vigor and to cure different diseases related to heart, lung etc. In recent years, research
on artificial fruiting of some species of the genus Cordyceps including C. militaris has been carried out extensively because
of their medicinal value. Instability observed in the in vitro fruiting of C. militaris is reported in the present study.

KEYWORDS : Cordyceps militaris, Artificial fruiting

Cordyceps is a highly esteemed herb in oriental medicine
(Hobbs, 1995; McKenna, 2002). Recent studies have
shown different pharmacological effects of C. militaris
such as immunomodulatory, immunoenhancing and
antitumor activities (Zhao et al, 2000; Liu et al, 1997).
Antioxidant, anti-inflammatory, antiangiogenic and
antinociceptive effects have also been reported from
both cultured mycelium and fruiting bodies of C.
militaris (Won and Park, 2005). Yoo et al (2004) have
shown that C. militaris has high potential for
antiangiogenesis and anti-tumor effects both in vitro
and in vivo. Polysaccharides isolated from C. militaris
have been identified as possessing anti-inflammatory
and suppressive activities on humoral immunity (Yu et
al, 2004). Cordyceps militaris is especially well known
for the production of cordycepin, which is effective in
inhibiting the growth of tumor cells (Kodama et al,
1999).
Among Cordyceps species, C. militaris has been
most successfully grown in complex artificial media
such as rice medium (Kobayasi, 1941; Liang, 1990;
Pen, 1995) as well as in defined medium (Basith and
Madelin, 1968). Similarly, cultural characteristics and
in vitro fruiting have been studied using Korean
isolates of C. militaris (Choi et al, 1999; Sung 1996;
Sung et al 1993, 1999, 2002). But regular artificial
*Corresponding author: <jmsung@kangwon.ac.kr>
fruiting-body production has been found difficult (Choi
et al, 1999; Sung, 1996). Recently, bipolar heterothallism
has been shown in C. militaris from an in vitro fruiting
study (Sung and Shrestha, 2002; Shrestha et al,
2004). Besides cultural studies, molecular techniques
have also been applied to understand mating systems
and phylogeny of different Cordyceps species (Yokoyama
et al 2003, 2004). Single spore isolations are necessary
to study mating type and fruiting-body production,
and this requires some basic knowledge of fungal
genetics as well as basic laboratory facilities such as a
microscope, and good facilities for preservation of pure
isolates. However, for mass fruiting at the farmer’ s
level, multispore isolates are suitable sources because
no specific techniques of isolation nor preservation
facilities for opposite mating type isolates are required.
Hence, in this study, multispore isolates of C. militaris
were used to induce fruiting bodies in order to observe
the stability of fruiting from both original cultures and
their subcultures.
Multispore isolates of C. militaris preserved in the
Entomopathogenic Fungal Culture Collection (EFCC),
Kangwon National University were used in the present
experiment. The isolates were prepared directly from
 In vitro에서 큰번데기 동충하초 자실체형성의 불안정성 141

ascospores discharged from natural fresh stromata of

C. militaris and hence designated as original cultures.
The original cultures were preserved in SDAY medium
in test-tubes. Liquid spawn of original cultures of the
isolates were prepared by inoculating five small pieces Isolates of C. militaris which produced a high grade
of mycelial agar from test tubes into 60 ml of SDAY of fruiting-bodies are described in Table 1. Out of
broth and incubating in rotary shaker (120 rpm) for 5 seventeen isolates, only seven isolates produced high
days at 25℃. Fruiting medium was prepared by mixing grade fruiting-bodies from second inoculations. Five
60 g of brown rice, 10 g of silkworm pupae and 90 ml isolates produced medium grade fruiting-bodies, and
of distilled water in 1 litre Polypropylene (PP) fruiting the rests produced low grade fruiting-bodies (Table
bottles which were then autoclaved at 121℃ for 20 1). It can be shown from the present experiment that
min. Liquid spawn of each isolate was inoculated into isolates vary in fruiting ability when they are used
the fruiting bottles in triplicate (20 ml in each bottle). repeatedly. This character of variation in fruiting-
This inoculation was referred to as first inoculation. body production from the same isolate is termed
The inoculated bottles were incubated at 20±1℃ instability. This instability has caused problems in the
under high humidity (70~90%) and continuous light mass fruiting-body production at the farmers’level.
conditions for 60 days. The number of stromata in The cause of this instability, however, could not be
each fruiting bottle was counted after 60 days of determined in the present study.
cultivation. The fruiting-body production of each Sung and Shrestha (2002) and Shrestha et al (2004)
isolate was graded as high, medium and low depending showed that two single spore isolates with opposite
upon the average number of stromata per fruiting mating types are required for fruiting-body production
bottle. Fruiting-body production was graded as high if in artificial conditions. In this study, it was assumed
the average number of stromata was 100 or more; that multispore isolates of C. militaris would contain
medium when the average number was 50~99, or low both mating types, so that fruiting-bodies could be
when less than 50 per fruiting bottle. The isolates
which produced high grade fruiting-bodies were again Table 1. Fruiting-body production of C. militaris from first and
inoculated into brown rice medium, in triplicate, for second inoculations of original cultures of different isolates
fruiting-body production following the method Isolate No. 1st inoculation 2nd inoculation
mentioned above. This was the second inoculation. EFCC C-3551 High* High
The numbers of stromata produced from second EFCC C-3559 High Medium
inoculations were counted and graded again into high, EFCC C-3561 High High
medium and low types. EFCC C-3577 High Low
EFCC C-3578 High High
EFCC C-3710 High Low
Original cultures of C. militaris isolates which EFCC C-3713 High Medium
produced high grade fruiting-bodies were subcultured EFCC C-3741 High Low
to fresh SDAY media in test tubes and were referred EFCC C-3746 High Low
to as first subcultures. Liquid spawns of first EFCC C-3747 High Medium
subcultures were also inoculated into brown rice EFCC C-3751 High High
medium to produce artificial fruiting-bodies following
EFCC C-3927 High High
the method mentioned above. First subcultures were
EFCC C-3931 High Medium
again transferred to fresh SDAY agar in test-tubes
EFCC C-3945 High High
and grown and these were referred to as second
EFCC C-3966 High High
subcultures. Second subcultures were also inoculated,
EFCC C-3970 High Medium
in triplicate, into brown rice medium, to produce
EFCC C-3973 High Low
fruiting-bodies. These fruiting-bodies in turn, were
* Fruiting-body production was graded as high if the average number
observed and graded high, medium or low according to of stromata was 100 or more; medium when the average number
the average number of stromata per fruiting bottle. was 50~99, or low when less than 50 per fruiting bottle.
142 한국버섯학회지 2(3) 2004

produced repeatedly. However, such continuous

production was not observed in this study. Sato and
Shimazu (2002) showed that C. militaris is a
homothallic fungus. In that case, fruiting-bodies of C.
militaris can be produced for a number of times from
the same culture. This was not observed in the
present study. Further studies are required to
understand the reason for instable fruiting-body
production of C. militaris. Fig. 1. Artificial fruiting-bodies produced from original culture
(left) and its subculture (right) of C. militaris isolate
EFCC C-3578 (high quality subculture).
Again, variation could be observed in artificial
fruiting-body production in the subcultures from
original cultures of C. militaris isolates (Table 2). Eight
isolates out of seventeen showed high grade fruiting-
bodies both from original cultures and their
subcultures (Table 2, Fig. 1). Subcultures of three
isolates produced medium grade fruiting-bodies
(Table 2), while two subcultures produced low grade
fruiting-bodies (Table 2, Fig. 2). Four subcultures Fig. 2. Artificial fruiting-bodies produced from original culture
produced only mycelium over brown rice medium (left) and its subculture (right) of C. militaris isolate
(Table 2, Fig. 3). EFCC C-3553 (low quality subculture).
Subculture is necessary to preserve the vitality of
fungal isolates for a long time. It is easy and does not

Table 2. Fruiting-body production of C. militaris from original

cultures and their subcultures
Isolate No. Original culture Subculture
EFCC C-3264 High* Medium
EFCC C-3533 High High
EFCC C-3551 High High Fig. 3. Artificial fruiting-bodies produced from original culture
EFCC C-3553 High Low (left) and its subculture (right) of C. militaris isolate
EFCC C-3561 High Only white mycelium EFCC C-3954 (yellow mycelium subculture).
EFCC C-3563 High High
EFCC C-3568 High High require sophisticated equipment and conditions. Hence,
EFCC C-3578 High High it is preferred by most of the mushroom growers. But,
EFCC C-3746 High High the present study showed that subculturing may affect
EFCC C-3747 High Yellow cotton like mycelium the quality of fruiting-body production of C. militaris.
EFCC C-3921 High Light yellow mycelium The extent of degeneration was felt to be too drastic
EFCC C-3937 High High as no fruiting-bodies at all were produced from four of
EFCC C-3954 High Yellow mycelium the seventeen subcultures (Table 2). It is concluded
that subculture may result in severe physiological or
EFCC C-3956 High Medium
morphological degeneration of the isolates.
EFCC C-3970 High Low
Table 3 shows that out of seven isolates producing
EFCC C-3973 High Medium
high grade fruiting-bodies, only two isolates EFCC C-
EFCC C-4013 High High
3551 and EFCC C-3578 showed consistency in
* Fruiting-body production was graded as high if the average number
of stromata was 100 or more; medium when the average number fruiting-body production from their original as well as
was 50~99, or low when less than 50 per fruiting bottle. their subcultures. Original cultures of isolates EFCC
 In vitro에서 큰번데기 동충하초 자실체형성의 불안정성 143

Table 3. Stability of fruiting-body production of C. militaris

Original culture
Isolate No. 1st subculture
1 inoculation
st
2nd inoculation
EFCC C-3551 High* High High
EFCC C-3561 High High Only white mycelium
EFCC C-3578 High High High
EFCC C-3746 High Low High
EFCC C-3747 High Medium Yellow cotton like mycelium
EFCC C-3970 High Medium Low
EFCC C-3973 High Low Medium
* Fruiting-body production was graded as high if the average number of stromata was 100 or more; medium when the average number
was 50~99, or low when less than 50 per fruiting bottle.

Fig. 4. Fruiting-bodies produced from first inoculation (left), second inoculation (middle) and subculture (right) of C. militaris
isolate EFCC C-3561.

C-3561 (Fig. 4) and EFCC C-3747 produced high
grades of fruiting-bodies from both first and second
inoculations, but their subcultures produced no
fruiting-bodies at all. Two isolates EFCC C-3746 and
EFCC C-3973 produced high grade fruiting-bodies
from first inoculation, but produced low fruiting-
bodies from second inoculation. Surprisingly, their
subcultures again produced high and medium grade
fruiting-bodies, respectively. Isolate EFCC C-3970
showed continuous degeneration in fruiting-bodies
production (Table 3).
Fruiting-body production of C. militaris was found
to be more unstable when further subcultures were
used (Table 4). First subculture of all three isolates
EFCC C-5252, EFCC C-5253 and EFCC C-5254
produced low grade of fruiting-bodies. It was expected
that second subculture would produce low or no fruiting-
bodies at all. The second subculture of only EFCC C-
5252 produced low fruiting-bodies, but those of EFCC
C-5253 and EFCC C-5254 surprisingly produced
medium grade fruiting-bodies. Fruiting-body production
from further subcultures is required to study the effect
of subculturing on fruiting ability of C. militaris.
Table 4. Effect of subculture on artificial fruiting
Isolate No. Original culture 1st subculture 2nd subculture
EFCC C-5252 High* Low Low
EFCC C-5253 High Low Medium
EFCC C-5254 High Low Medium
* Fruiting-body production was graded as high if the average number
of stromata was 100 or more; medium when the average number
was 50~99, or low when less than 50 per fruiting bottle.
Fruiting of Cordyceps militaris varied among
different isolates. The multispore isolation method is
relatively simple and provides the gross morphological,
cultural and fruiting characters of C. militaris. Mature
stromata were profusely produced from multiple
ascospore isolates in brown rice medium. But
continuous fruiting-body production from their
subcultures produced instable fruiting, ranging from
profuse stromata formation to no stromata formation.
 144 한국버섯학회지 2(3) 2004
This causes a big problem in mass cultivation of C.
militaris. Even high fruiting isolates did not produce the
same quality of fruiting either from their repeated
inoculation or from their subcultures. Further
phenotypic and genotypic studies are required to
understand the character of fruiting instability of C.
militaris in artificial conditions.
전통적으로 Cordyceps종은 한국을 포함한 동양에서 건
강증진과 심장과 폐에 관련된 질환을 치료하는 약용식물
의 하나로 사용되어왔다. 최근에 동충하초의 약리적 가치
때문에 큰번데기 동충하초를 포함한 몇가지 Cordyceps
종의 인공재배에 관한 연구가 활발하게 이루어지고 있다.
본 연구에서는 큰번데기 동충하초를 in vitro에서 인공재
배한 결과 자실체 형성이 불안정한 것을 관찰하였기에 보
고하고자 한다.
This work was supported by a grant from the
Strategic National R&D Program through the Genetic
Resources and Information Network Center funded by
the Korean Ministry of Science and Technology and
Rural Development Administration
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