World Journal of Microbiology and Biotechnology (2018) 34:166

https://doi.org/10.1007/s11274-018-2550-4

ORIGINAL PAPER

Preservation affects the vegetative growth and fruiting body

production of Cordyceps militaris
Henan Sun1 · Ting Hu2 · Yanbin Guo2 · Yue Liang1 

Received: 16 July 2018 / Accepted: 26 October 2018 / Published online: 30 October 2018
© Springer Nature B.V. 2018

Abstract
Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fun-
gus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as
healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield
and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal
strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated
that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated
subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and
protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The devel-
opment of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed
to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fun-
gal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal
inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris.
The results may shed light on preservation for other Cordyceps species and other edible fungi.

Keywords  Cordyceps militaris · Fruiting body · Inoculum · Preservation · Subculture

Introduction cordycepic acid, polysaccharides and pentostatin, which have

Cordyceps militaris is widely used as a traditional medicinal
material and an edible mushroom in China, Korea and some
other Asian regions (Lee et al. 2015). This fungus belongs to
Hypocreales in Ascomycota Division (Sung et al. 2007). In
natural conditions, C. militaris often parasitizes on the pupa
or larva of insects (e.g., Lepidoptera) and forms fruiting bodies
or stroma (Clarkson and Charnley 1996). The fruiting body of
C. militaris is rich in bioactive metabolites such as cordycepin,
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s1127​4-018-2550-4) contains
supplementary material, which is available to authorized users.
* Yue Liang
liangyuet@126.com
1
College of Plant Protection, Shenyang Agricultural
University, Shenyang 110866, China
2
College of Resources and Environmental Sciences, China
Agricultural University, Beijing 100193, China
healthy effects to human being including anticancer, anti-
bacteria and immunity improvement (Xia et al. 2017). The
pharmacologically active ingredients of C. militaris is similar
to or more efficient than those of the traditionally medicinal
mushroom Ophiocordyceps sinensis, which made C. militaris
a substitute of O. sinensis for medicinal and nutritional use
(Shrestha et al. 2012). As a result, market demand of wild
resources and artificial Cordyceps products (e.g., fruiting
body) increased (Xia et al. 2017). However, wild C. milita-
ris became scarce due to its requirement of specific growing
conditions and excessive excavation, resulting in a shortage
of the natural supply and stagnation in the Cordyceps indus-
try (Dong et al. 2016). Although artificial cultivation of C.
militaris was available, the yield and quality of fruiting bodies
were normally affected by the degeneration or mutagenesis of
the fungal strains (Shrestha et al. 2012). The fungal degen-
eration or mutagenesis generally showed as slow growth of
mycelia, irregular shape of colonies, excessive aerial hypha,
sector mutation, and poor development of stroma and fruit-
ing bodies (Sun et al. 2017). For example, an entomogenous

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fungus Metarhizium anisopliae might suffer from a spontane-
ous degeneration, resulting in sterile sectors and decline on
sporulation (Wang et al. 2005). Producing of abnormal fruiting
bodies by C. militaris strains has been attributed to the degen-
eration and mutagenesis of the fungus (Lin et al. 2010). Fungal
age also affects the mycelial growth rate, hyphal extension and
fungal biomass (Taniwaki et al. 2006). Repeated subculturing
is a simple and effective method for fungal propagation (Sid-
diqui and Kataoka 2011). The biological characters and the
fruiting body biomass were reported to be affected by subcul-
turing of C. militaris (Liu et al. 2006). Therefore, the stabil-
ity and viability of mycelial growth as well as fruiting body
formation initiated by a fungal strain stocked under optimized
preservation conditions were considered important for aca-
demic research and industrial production (Dong et al. 2016).
Fungal preservation is an effective approach to maintain
the fungal vitality and further ensure the normal growth and
genetic stability during the subculturing (Ayala-Zermeno
et al. 2017; Bainard et al. 2010). An appropriate fungal
preservation method generally relies on a certain suspend-
ing metabolism to avoid fungal mutation, contamination
and death (Zhang et al. 2016). The process of preservation
might include dehydration, cryopreservation, and immersion
in sterilized water, mineral oil or anti-crystallization agents
(Homolka 2014). For example, dehydration such as dry-
ing or freezing-drying (lyophilization) is a method to keep
the fungi from air and suspends metabolism (Prakash et al.
2013). The lyophilization was an efficient drying technology
to frozen fungal cultures (Homolka 2014). Liquid nitrogen
or freezers were widely used to freeze fungi for preserva-
tion (Jong et al. 1991). Protective agents, such as glycerol,
skim milk and peptone, were applied in the preservation
to protect the microorganisms from crystallization damage
in low temperatures (Dahmen et al. 1983; Homolka 2014).
In this study, preservation conditions for C. militaris pres-
ervation were investigated and optimized by assessing fungal
growth and artificial inoculum features essentially relevant
to fruiting body production. Thereafter, the development of
stroma or fruiting body and the period of preservation time
were further assessed under the optimized preservation con-
ditions to improve the stability and quality of the fruiting
body production and development. This study standardized
an economic and efficient preservation method for C. mili-
taris and provided reference information on preservation for
other Cordyceps species and edible fungi.
Materials and methods
Microorganism and cultural conditions
Strains of C. militaris were collected from Qipan Moun-
tain (Shenyang, Liaoning, China) and maintained on the
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World Journal of Microbiology and Biotechnology (2018) 34:166
modified potato dextrose agar (MPDA; 20% (w/v) potato,
2% dextrose, 1.6% agar, 0.3% peptone, 0.1% M ­ gSO4·7H2O
and 0.2% K ­ H2PO4). Representative strains of C. militaris
were cultured on MPDA at 23 °C in the dark for 3 days and
then under continuous light for 18 days. Thereafter, mycelial
plugs were excised from the activate edges of the growing
colonies as replicates used in the following experiments.
Particularly, for fruiting body induction, the fungal plugs
were inoculated in 50 mL modified potato dextrose broth
medium (MPDB; MPDA without agar) shaking at 150 rpm
at 23 °C for three days. Subsequently, the liquid cultures
were inoculated on the cultivation medium (20 g rice mixed
with 30 mL MPDB) and the resultant cultures were incu-
bated at 23 °C under the cycle of 8 h light and 16 h dark
until the formation of fruiting body was visualized (Zhang
and Liang 2013).
Radial growth and repeated subculturing
One mycelial plug excised from each of five strains was
incubated on MPDA in a 90 mm Petri plate until the grow-
ing hyphae reached the edge of the plate. From each plate,
three mycelial plugs at 35 mm away from the colony center
were excised and transferred to a new MPDA plate. The
diameters of the colony growing on the new MPDA plate
were measured every second day from the fifth day after
inoculation (dai) to 19 dai.
Cordycep militaris grows slow on MPDA media and
generally needs 21 days to cover up a 90 mm Petri plate.
Variation of fungal age would exist among mycelial plugs
from different parts of the plate if such a plate is being used
as source of inocula. Therefore, one mycelial plug from the
center (3 mm), the middle (20 mm) and the edge (40 mm) of
a fungal colony on each of three radial lines was excised and
inoculated on a new MPDA plate. The diameter of the colo-
nies was measured every second day from the 5 to 19 dai.
The assays were performed using five strains as biological
replicates.
To investigate the effect of subculturing on the fungal
biomass development, a mycelial plug from the edge of
actively growing colony was excised and inoculated on a
MPDA plate until the hyphae grew close to the edge of the
plate. Then a new mycelial plug excised from the edge of
the growing colony was transferred on a MPDA plate as one
generation of the repeated subculturing. Such procedure was
repeated eight more times. The colonial morphologies and
the radial growth were recorded in each generation. The five
strains were treated as biological replicates and each biologi-
cal replicate consisted of a technical triplicates.
World Journal of Microbiology and Biotechnology (2018) 34:166 Page 3 of 9  166
Evaluation of preservation conditions
Mycelial plugs bearing conidia from C. militaris cultures are
used as initial inocula in/on artificial media in the large-scale
production (Shrestha et al. 2005). The ability of the initial
inoculum to produce mycelia and conidia of was important
and considered as a criterion for the inoculation potential
on fruiting body production (Lomberh et al. 2000). There-
fore, mycelial plugs or spore suspension of five strains were
treated under 4 °C, room temperature or − 20 °C with/with-
out lyophilization treatment for 24 h, and then were mixed
with 500 µL sterile water to inoculate into MPDB at 150 rpm
agitation for 3  days. The resulting liquid cultures were
referred as artificial inoculum because it was normally used
to initiate large-scale culturing in industrial production. The
features of the artificial inoculum were categorized based on
the medium features, mycelial formation and conidia abun-
dance by counting the mycelial balls using gradient dilution
and the number of conidia using a hemocytometer.
Optimization of preservation conditions
Two stock cultures of C. militaris, mycelial plugs (5 mm in
diameter) and liquid cultures at 3 dai, were selected for opti-
mizing the preservation. In a 1.5 mL microcentrifuge tube,
a mycelial plug or 200 µL liquid culture was mixed with
300 µL protective agents including 6% peptone, pasteurized
skim milk, or sterilized skim milk. Subsequently, each of
the fungal samples was equaled into two portions, one por-
tion was lyophilized overnight and the other was processed
without lyophilizing treatment. Thereafter, fungal samples
were stored at 4 °C, room temperature, − 20 °C, or − 80 °C
for 24 h. Each of preservation conditions was performed
with three technical replicates.
Effect of preservation on fruiting body induction
Fruiting body production under the optimized preservation
conditions was further investigated to determine the optimal
preservation period. The fungal samples generated from the
optimized conditions were individually stored at 4 °C or
room temperature for several preservation periods (i.e., 0.5,
1, 3, 6 or 12 months). At the end of each period, the fungal
samples were used for the induction of fruiting body and the
fresh biomass with morphological characterization was fur-
ther assessed (Zhang and Liang 2013). The experiment was
performed duplicate for each of the preservation periods.
Data analysis
Linear regression analysis (LRA) of mycelial growth in C.
militaris was performed and the correlation was clarified
between the colony diameter and culture days based on
the theoretically hypothesized intercepts in the previous
procedure (Hendricks et al. 2017). Principal component
analysis (PCA) was further conducted based on the results
analyzed by LRA of each biological replicate using PAST
software (version 3.01; Hammer et al. 2001).
Results
Radial growth and repeated subculturing
Radial growth of C. militaris was determined on the basis
of the colony diameters measured by a two-day interval
for the successive 14 days before the hyphae overgrew
a 90 mm Petri plate. LRA was performed and the result-
ing growth rate was further analyzed by PCA. Among the
tested strains, mycelial growth was similar to each other
and expressed as a linear model with a positive correlation
(y = 4.7814x − 13.418 and ­R2 = 0.9938, in which x is the
culture day after inoculation and y is the colony diameter
at the corresponding culture day) (Fig. 1a). The growth
rate of each biological replicate by LRA was further ana-
lyzed within 95% confidence interval and indicated that
the first principal component is 95.708% at the accumula-
tion contribution rate by PCA (Supplementary Fig. 1). The
results of LRA and PCA suggested that the growth status
of C. militaris was independent with fungal strains and
predominantly determined by the radial rate of mycelial
growth.
The potential effect of mycelial ages on the fungal
growth was also investigated. The mycelial growth of three
representative fungal ages was evaluated using LRA and
PCA, which confirmed that the growth rate of mycelia was
predominant (98.624%) within 95% confidence interval for
biological replicates (Fig. 1b). The results also indicated
that the fungal growth followed a linear model of mycelial
growth, but was not affected by fungal ages.
The repeated subculturing on the fungal biomass accu-
mulation was investigated because potentially degenera-
tion or mutagenesis might occur in repeated subcultures.
The morphologies of fungal colonies in each generation
were found to be identical to one another, typically apri-
cot orange in color, with few aerial hyphae and abundant
conidia, and no sector mutation (Fig. 2a). Moreover, myce-
lia showed stable in growth rate and were not significantly
altered between any two of subculture generation (Fig. 2b).
The results indicated that the individual cultures of C. mil-
itaris could maintain consistent fungal growth between
generations. The above results should be a significant
consideration to establish industrial propagation and for
subsequent further studies.
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Fig. 1  Mycelial growth of Cordyceps militaris. a Linear regression
analysis of radial growth and culturing days. b Principal component
analysis of three representative fungal ages from an individual fungal
colony. Colors indicate the biological replicates with shapes as tech-
nical replicates within a circle at 95% confidence interval
Fig. 2  Morphology and growth of Cordyceps militaris on repeated
subcultures. a Colonies of a strain after subculturing. From left
to right, the parental strain, the fifth and the ninth repeated subcul-
tures. b Box plot of mycelial growth for each of repeated subcultures.
Colors indicate five of biological replicates with technical triplicates
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World Journal of Microbiology and Biotechnology (2018) 34:166
Evaluation of preservation treatments
Fruiting body was generally induced from the inoculum on
the artificial media in the industrial production of C. milita-
ris. The quality of the inoculum and the capacity of media
for mycelia and conidia production were critical for fruiting
body induction in C. militaris (Liu et al. 2006). Therefore,
the characteristic levels (termed as cultural index) of the
inoculum for artificial production according to the features
of mycelial formation and media were categorized. Six fea-
ture groups could be classified: G0, transparent and clear
cultural medium without visible mycelia by the unaided
eyes; G1, limpid cultural medium with the visible and lower
abundance of mycelia [about 10–100 mycelial balls per flask
(50 mL)]; G2, limpid cultural medium with the low abun-
dance of mycelia [about 100–300 mycelial balls per flask];
G3, turbid cultural medium with the moderate abundance
of mycelia [about 300–1000 mycelial balls per flask]; G4,
turbid cultural medium with the high abundance of mycelia
[about 1000–3000 mycelial balls per flask]; G5, turbid cul-
tural medium as the congee with the higher abundance of
mycelia [more than 3000 per flask] (Fig. 3). Additionally,
the production of conidia was also investigated for further
assessment of inoculation culture treated with different pres-
ervation conditions.
Optimization of preservation conditions
The effects of fungal preservation with the protective agents
under different temperatures by lyophilization were evalu-
ated by assessing the ability of mycelial formation and
conidia production of the artificial inoculum cultures in C.
militaris. The preservation of mycelial plugs was optimized
base on the cultural index coupled with conidia abundance.
In the lyophilization treatment, the cultural index of the arti-
ficial inocula was lower than that of the normal conditions
(i.e., without lyophilization) (Fig. 4a). The artificial inocu-
lum culture at the G3 or higher levels was generally used to
induce fruiting body in industrial production (Zhang and
Liang 2013). Under the normal condition (without lyophi-
lization), 43.8% of the samples were at the G3 or beyond
while the proportion in the lyophilized samples was only
6.3% (Fig.  4a). The ability on conidia production was
much higher in samples without lyophilization than those
with lyophilization (Fig. 4b). Furthermore, the influence
of preservation temperatures on fungal growth was esti-
mated. The percentage (75%) of samples preserved at 4 °C
or room temperature without lyophilization were at the G3
or beyond with the productive conidia while the features of
samples preserved at − 20 °C and − 80 °C were not higher
than G3 level (Fig. 4). Similar observation of the tempera-
ture effect was consistently observed under the lyophiliza-
tion treatment. The protective agents were further evaluated
World Journal of Microbiology and Biotechnology (2018) 34:166 Page 5 of 9  166

Fig. 3  Characteristic levels
of the inoculation culture of
Cordyceps militaris. Cultural
features of liquid artificial
inoculum to initiate artificial
cultivation of fruiting body were
categorized as the culture index
from G0 to G5 on the basis of
the mycelia and medium

Fig. 4  Culture index (a) and

sporulation (b) of the artificial
inoculum initiated from from
the fungal strains preserved for
24 h under different condi-
tions. These conditions were
individual and combinational
treatments of lyophilization,
non-lyophilization, 4 °C, room
temperature (RT), − 20 °C,
− 80 °C, with or without protec-
tive agents (6% peptone, pas-
teurized skim milk or sterilized
skim milk). Culture index of the
inoculation cultures incubated
for 3 days from the fungal
strains preserved for 24 h under
different conditions. These
conditions were combinations
of lyophilization, non- lyophili-
zation, 4 °C, room temperature
(RT), − 20 °C, − 80 °C, with or
without protective agents (6%
peptone, pasteurized skim milk
or sterilized skim milk)

and did not significantly improve the feature of inoculation
culture (Fig. 4). Moreover, the optimization of preserva-
tion conditions by liquid culture showed similar results as
by mycelial plugs. Under normal conditions, 37.5% of the
samples were at the G3 or beyond while the proportion of
samples with lyophilization was only 6.3% (Supplementary
Fig. 2a). Meanwhile, the lyophilized samples produced much
less conidia than in the normal conditions (Supplementary
Fig. 2b). The cultural features of fungal strains preserved
at 4 °C or room temperature were 50% at the G3 or beyond

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Page 6 of 9 World Journal of Microbiology and Biotechnology (2018) 34:166

with productive conidia while the sample proportion at

− 20 °C and − 80 °C were not over than G3 level (Supple-
mentary Fig. 2). Overall, the optimal preservation condi-
tions of C. militaris were found to be storing C. militaris
using mycelial plugs without lyophilization at 4 °C or room
temperature without or with peptone as a protective agent.

Fruiting body induction affected by preservation

Fruiting body induction was further investigated to assess

the periods of preservation time under the optimized condi-
tions. Mycelial plugs were preserved at 4 °C or room tem-
perature with/without a protective agent (e.g., peptone) for
various periods of time. Morphologies and biomass of fruit-
ing body induced on the artificial medium were recorded and
measured, respectively (Fig. 5). Abnormal fruiting bodies
or stroma with fewer perithecia were developed for a half or
one month of the preservation time (Fig. 5a). Additionally,
the strains preserved at room temperature for over a month
were more prone to contamination compared to strains pre-
served at 4 °C. Such contamination to fungal preservation
caused the failure of the artificial inoculation and fruiting
body production. Therefore, the subsequent investigation
was continuously assigned at 4 °C alone. On the cultures
with three-month preservation, the fruiting body or stroma
was induced and developed, but the perithecia that were
produced by this fungus preserved with the protective agent
Fig. 5  Fruiting body production and preservation term under the opti-
showed lower abundance and undeveloped (Fig. 5b). The
mized conditions. a Biomass of fruiting bodies induced by strains
fruiting bodies induced by the strains preserved for six or treated by the optimized preservation conditions. b Development of
twelve months at 4 °C without protective agents consistently fruiting body (top panel) or stroma with abnormally developed (left),
showed productive in biomass as well as abundantly fully poor and low density (middle), developed and abundant (right) peri-
thecia (bottom panel)
developed perithecia (Fig. 5).

and PCA. Cordyceps militaris, similar to other Cordyceps

Discussion
Cordyceps militaris is an edible and medicinal mushroom
with diverse bioactive metabolites and properties (Lee
et al. 2015). The yield and quality of fruiting bodies in the
large-scale production are affected by the limitation of wild
resources and the mutagenesis and degeneration of fungal
strains in the artificial cultivation of C. militaris (Lin et al.
2010). Therefore, the fungal preservation method was opti-
mized by assessing the ability of the preserved strains on
fruiting body production.
Fungal growth generally includes that of vegetative and
reproductive, which is influenced by various factors such as
ambient temperature and humidity (Pietikäinen et al. 2005;
Pasanen et al. 1991). Due to the special biological characters
(e.g., frequent degeneration coupled with less primordium,
decreased spore abundance, and abnormal development of
stroma after cultivation) of C. militaris (Sun et al. 2017), the
mycelial growth was investigated and determined by LRA
species, has a slow growth (Dong 2013), which was proved
to follow a linear model with the radial growth positive cor-
relating to culture time. The slow growth of C. militaris and
other fungi was supposed to cause the potential difference
on the hyphal ages among different parts of an individual
culture, which might lead to the degeneration or mutagenesis
(Shrestha et al. 2012; Wang et al. 2005). However, myce-
lial plugs excised from an individual culture with different
ages produced cultures with identical colonial morpholo-
gies and mycelial growth rate. Repeated subculturing is a
common strategy for fungal propagation and preservation,
which might affect the fungal growth, mutation or degenera-
tion (Homolka 2014). For example, the ability of mycelial
growth and conidia production of Beauveria bassiana was
decreased after multiple subcultures (Santoro et al. 2014). In
this study, the repeated subculturing of C. militaris was car-
ried out for ten transferred generations through six months.
The results indicated that repeated subculturing did not
affect propagation of C. militaris.

13
World Journal of Microbiology and Biotechnology (2018) 34:166 Page 7 of 9  166
Fungal preservation generally involves dehydration, tem-
perature, protective agents, and storage period for maintain-
ing fungal variability and reducing contamination (Ayala-
Zermeno et al. 2017; Prakash et al. 2012). Mycelial growth
of O. sinensis was a critical standard for determination of
different preservation methods (Fang et al. 2011). Addition-
ally, the ability of mycelial formation and sporulation in the
artificial inoculum were critical factors affecting the fruiting
body production of C. militaris (Lin et al. 2006). Therefore,
the cultural categories were established on the basis of the
mycelial formation and sporulation of the artificial inocu-
lums, which could be beneficial to industrial fruiting body
production (Shrestha et al. 2012). Meanwhile, the preserva-
tion conditions of C. militaris were preliminarily screened
and optimized mainly based on the feature of the artificial
inoculum for artificial cultivation of fruiting bodies.
Mycelial plugs and liquid culture were widely used for
fungal preservation (Homolka 2013; Xu and Feng 2001).
Use of mycelial plugs was simple and inexpensive for fast
preservation of many fungi (Baskarathevan et al. 2009). Liq-
uid culture was also used for industrial production of the
edible mushroom Hypsizygus marmorens (Zhang and Shi
2010). Therefore, the choices of fungal stock cultures (e.g.,
mycelial plugs and liquid culture) were compared and the
results indicated that mycelial plug was more suitable for C.
militaris preservation. In the preservation of Botryosphaeria
species, mycelial plugs were used and found to be suitable to
maintain fungal viability (Baskarathevan et al. 2009).
Lyophilization is an efficient method to reduce the mois-
ture in microorganism preservation (Prakash et al. 2013).
However, many factors such as growing stage, protective
agents and spore abundance needed to be optimized before
freeze-drying treatment. On the other hand, the requirement
of special and expensive equipment also limited the use of
lyophilization (Homolka 2014). In this study, the ability of
mycelial formation and sporulation in the artificial inocu-
lum initiated by the lyophilized strains were lower than
that without freeze-drying, which is possibly related to the
nature behavior and ecological characteristics of C. militaris
(Zhong et al. 2006).
Many fungal species are routinely stored in − 20 °C or
− 80  °C (Homolka 2014). For example, Nmuraea rileyi
maintained vitality while Verticillium lecanii processed
stable growth and sporulation after stored at − 80  °C
(López Lastra et al. 2002). The coral tooth fungus Heri-
cium coralloides was preserved at − 80 °C for 15 years and
still remained the stability of normal growth, which further
indicated the cryopreservation was an economic and effec-
tive preservation method for basidiomycotous fungi (Ito and
Nakagiri 1996). Moreover, fungal cultures were also usu-
ally stored at 4 °C (Kitamoto et al. 2002). For example, a
filamentous fungus Cryptococcus laurentii showed higher
viability at 4 °C than room temperature (Li and Tian 2007).
In this study, preservation at 4 °C was found to be feasible
to C. militaris as a preferred treatment.
Protective agents were used in fungal preservation to
reduce cell damage, such as skim milk, peptone or glycerol
(Dahmen et al. 1983; Homolka 2014; Ayala-Zermeno et al.
2017). For example, skim milk was applied as a protective
agent for preserving a plant pathogenic fungus Monilinia
fructigena and offered protection against the storage damage
of freezing or thawing (Dahmen et al. 1983). Alternatively,
peptone as a protective agent can maintain the virulence of
Alfalfa mosaic virus (Fukumoto 2008). In this study, the
preservation efficiency of C. militaris was more productive
without protective agents than those of the usually protec-
tive agent skim milk. The major function of most protective
agents is to help the fungal materials resist crystallization
when the temperature is below the freezing point (Homolka
2014). When preserved at 4 °C, crystallization is not a threat
to C. militaris and the presence of protective agents might
slightly compromise the fungal development (Kitamoto et al.
2002).
Fruiting bodies of C. militaris were widely recognized as
an edible and medicinal supply, which resulted in a flour-
ishing industrial production (Shrestha et  al. 2012). The
biomass and quality of the fruiting bodies are the critical
factors affecting the industrial production and yield (Dong
et al. 2016). Moreover, the formation and development of
perithecia was a feature to determine the quality of fruit-
ing bodies in C. militaris (Zhang and Liang 2013). There-
fore, the preservation conditions and periods were evaluated
based on the growth stability and viability as well as the
fruiting body induction. The results indicated that the fun-
gal strains stocked under the optimized preservation condi-
tions remained the viability for the routine production of
qualified fruiting bodies for at least 12 months. However,
different fungi have different optimal preservation periods
under their special conditions. For example, Verticillium
lecanii was preserved for 12 months and its viability was
still remained (López Lastra et al. 2002), meanwhile a mush-
room Schizophyllum commune was also preserved for 12
months with the stable mycelial growth and biomass (Kara-
duman 2012). Nonetheless, N. rileyi and M. anisopliae only
remained growth stability for 3 and 6 months (Freitas et al.
2014) while the growth ability and sporulation of Hirsutella
citriformis were found to weaken and even lost after a six
month preservation (Ayala-Zermeno et al. 2017).
In this study, the preservation conditions for C. militaris
were optimized based on assessments of mycelial growth,
sporulation, fruiting body production and perithecium devel-
opment. The distinguished features of the initial inoculum
for the artificial cultivation of fruiting bodies were deter-
mined. Particularly, C. militaris is ideally preserved using
mycelial plugs at 4 °C for not less than one year without
lyophilization. Moreover, cultural criteria of the artificial
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Page 8 of 9
inoculum used to initiate fungal strains were suggested for
industrial fruiting bodies induction. This study may shed
light on the fungal preservation for the large-scale produc-
tion of the fruiting bodies in C. militaris and Cordycepes
species, even other medicinal and edible fungi.
Acknowledgements  This study was funded by Program for Liaon-
ing Excellent Talents in University (LR2015058) and the Scientific
Research Foundation for the Introduced Talents of Shenyang Agricul-
tural University (20153040).
Compliance with ethical standards  Appl Microbiol 44:437–442
Conflict of interest  Authors declare that they have no conflict of inter-
est.
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