Nutritional and environmental requirements for the morphogenesis of Ophiocordyceps sobolifera

8/12/2019 Nutritional and environmental requirements for the morphogenesis of Ophiocordyceps sobolifera

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1MORPHOGENESISOFOPHIOCORDYCEPS

MICOL. APL. INT., 26(1), 2014, PP. 1-8

MICOLOGIAAPLICADAINTERNATIONAL, 26(1), 2014, pp. 1-8 2014, BERKELEY, CA, U.S.A.

www.micaplint.com

NUTRITIONALANDENVIRONMENTALREQUIREMENTS

FORTHEMORPHOGENESISOFOPHIOCORDYCEPS

SOBOLIFERA

A. IMTIAJ1,2ANDS. OHGA1*

1 Division of Forest Environmental Sciences, Department of Agro-environmental Sciences, Faculty of

Agriculture, Kyushu University, Fukuoka 811-2415, Japan.2 Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh.

Accepted for publication December 30, 2013

ABSTRACT

Nutritional and environmental requirements for spore germination, mycelial growth

and sporocarp development ofOphiocordyceps sobolifera were evaluated. There was no

spore germination after 5 days of incubation at 25 C. However, pre-incubation at 4 C

for 7 days stimulated the spore germination. Incubation with the tissues of sporocarp

of O. soboliferaon PDA and yeast-malt agar resulted in mycelial growth and callus-

like structure (sporocarp) development. The callus-like structure was examined under

a microscope, revealing many sporocarps. Among 12 media, PDA-yeast malt extract,

PDA-yeast extract and yeast malt extract, showed more sporocarps and branch

formation than other media. The greatest growth was supported by PDA-yeast malt

extract, which also contained more ingredients than any other media. Sporocarp

formation was poorly supported by either PDA or yeast malt extract. However, the

combined media accelerated mycelial growth and development of the sporocarp. Plant

growth hormones were also found to stimulate the formation of ascocarps and could be

considered as an enhancer of sporocarp formation of O.sobolifera.

Key words: Activators, ascocarp, germination, growth hormones, morphogenesis.

* Corresponding address: Tel.: +81-929483118; Mobile: +81-80-5283-2259; Fax: +81-929483116.

E-mail: [email protected]


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2 A. IMTIAJANDS. OHGA

MICOL. APL. INT., 26(1), 2014, PP. 1-8

INTRODUCTION

Ophiocordyceps sobolifera (Hill ex Wat-

son) G.H. Sung, J.M. Sung, Hywel-Jones

& Spatafora is an insect parasitic fungusbelonging to Ophiocordycipitaceae, As-

comycota. This fungus is found in China,

Cuba, Japan, Korea, Madagascar, Mexico

and Sri Lanka; it is also synonymously

known as Cordycepssobolifera, Clavaria

sobolifera, Sphaeria soboliferaand Torru-

bia sobolifera.Beauveria soboliferais the

anamorph (asexual reproductive stage) of

O. sobolifera9. It is assumed that the syn-

anamorphic fungus O.soboliferahas ana-

morphic, teleomorphic and holomorphicphases. Most of the members of the family

are known worldwide as herbal folk me-

dicinal mushroom, due to the presence of

important bioactive compounds, such as

adenosine, cordycepin and exopolysaccha-

rides7. The chemical constituents for most

species include cordycepin (3-de-oxy-

adenosine), ergosterol, polysaccharides,

a glycoprotein and peptides containing

-aminoisobutyric acid. Polysaccharides

account for anti-inammatory, antioxi-dant, anti-tumour, anti-metastatic, immu-

nomodulatory, hypoglycemic, steroido-

genic and hypolipidaemic effects, whereas

cordycepin contributes to the anti-tumour,

insecticidal and antibacterial activities6.

Liquid culture ltrate of Cordyceps spe-

cies can inhibit the growth of bacteria4. It is

also reported that this fungal species may

boost energy and ameliorate nephrotoxici-

ty-induced renal dysfunction in rats via an-

tioxidant, anti-apoptosis, and anti-autoph-agy mechanisms10. In general, the morpho-

logical biomass of Cordycepsspecies has

been regarded as preferred resources with

perceived health benets. Thus, there is a

great demand for the mycelial biomass to

harvest desired ingredients. However, nat-

ural slow growth pattern of wild O. sobo-

liferacan not supply the industrial require-

ment and purposes. Moreover, its presence

in nature is restricted to specic areas and

the size of sporocarps is extremely small.It is essential to develop methods for com-

mercial production of biomass. Some at-

tempts have been made to obtain biomass

by submerged culture. Submerged culture

gives potential advantages of higher my-

celial production in a compact space and

shorter time with less chance of contami-

nation1.

There are differing synonyms for O. so-

bolifera including different species, mor-

phic status, and growth types leading toconfusion about its taxonomic identica-

tion. Therefore, DNA sequences were ob-

tained for authentication of the fungal spe-

cies. Thereafter, a phenotypic study includ-

ing key requirements for mycelial growth,

spore germination and ascocarp formation

was carried out, which can be useful for in-

dustrial applications ofO. sobolifera.

MATERIALS AND METHODS

Collection and identication of O.

sobolifera. An ascocarp of this species

growing on a cicada nymph was collected

in the forest and brought to the Laboratory,

Department of Agro-Environmental

Sciences, Kyushu University, Japan. To

identify the fungal species, morphological

characteristics were noted, and a strain

was isolated, cultured on potato dextrose

agar (PDA) medium, and incubated at25 C for further study. Identication was

conrmed by a polymerase chain reaction

(PCR) using universal primers ITS1F

(5-GTAACAAGGT(T/C)TCCGT-3) and

ITS1R (5-CGTTCTTCATCGATG-3),

and sequencing was done by Genenet Co.


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Ltd., Tokyo, Japan (www.genenet.co.jp).

DNA extraction, PCR, gel electrophoresis,

sequencing and analysis ofDNA sequences

was carried out following the methods of

our previous work5

. The identied mycelialpure culture was deposited at Kyushu

University, Japan, and an accession number

was acquired from the Kyushu University

Mushroom Bank (KUMB122).

Preparation of media. Twelve culture

media [PDA, carrot dextrose agar (CDA),

yeast-malt agar (YMA), cow-milk agar

(CMA), coconut-milk agar (CtMA),

prawn agar (PA), egg agar (EA), residue of

ribosomal nucleic acid and agar (RNAA),

nutrient agar (NA), PDA-malt extract(PDAM), PDA-yeast extract (PDAY), and

PDA-yeast malt extract (PDA-YM)] were

tested in this study. PDA and NA powder

(Difco) were used at the concentration

of 3.9% and 2.3% (g/100 ml). CDA was

prepared using water from boiling 30 g of

peeled carrots, 2 g glucose, and 1.5 g agar to

make 100 ml, in the same manner as fresh

PDA preparation. Yeast, malt and RNA

residue were added to PDA at 1.5% (g/100

ml), separately. Each of 50 g of prawnpowder, bovine milk, coconut milk, and

egg were added to distilled water to make

100 ml of individual medium. In every

case, agar concentration was maintained at

1.5 g/100 ml.

Effect of plant hormones. The effect

of indole-3-acetic acid (IAA), indole-3-

butyric acid (IBA), and kinetin (KIN)

was studied in PDA medium for the

production of biomass. PDA was prepared

and autoclaved. Plant growth hormoneswere added to PDA at 2.5 mg/250 ml

concentration separately and poured into

Petri dishes, which were inoculated with

mycelium after the medium had gelled.

Biomass and phenotype of culture was

characterized after 30 days of incubation at

25 C. Fresh PDA (without hormone) was

used as control.

Spore germination. Spores of O.

sobolifera were taken from 20 days old

cultures grown on PDA. Suspensions ofspores were made in PDB (2.4 g/100 ml),

PDB-YM (PDB 2.4 g, yeast 1.5 g/100 ml,

malt 1.5 g/100 ml), 2% glucose solution

(GS), and distilled water (DW) separately.

Each spore suspension was taken (5 ml) in

sterilized watch-glass, and incubated at 25

C for 1 to 5 days. Then, a drop of each spore

suspension was taken on a separate slide

and extra water was soaked with blotting

paper. Thereafter, a drop of lactophenol

cotton blue was added to the sporesuspension on slides. The slides were then

examined under the microscope at 400x to

count spore germination of O. sobolifera.

Another set of spore suspensions was kept

at 4 C for 7 days as a dormancy period.

After incubation at 4 C for 7 days, spore

germination was examined following the

same procedure.

Mycelial growth and branching was

judged visually. After 60 days at 25

C, the growth on each medium wascarefully scrapped off from the surface

of five flasks, each containing 100 ml

of the agar medium. To remove medium

from the mycelium, it was kept in boiling

water for a while. Thereafter, extra water

was soaked with blotting paper and the

biomass was immediately weighed to

determine fresh weight. It was then

placed in an oven for drying at 60 C for

24 h, then weighed and reweighed every

2 h until constant weight, in order todetermine dry weight.

RESULTS AND DISCUSSION

Spore germination. O. sobolifera was


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studied to determine requirements for

spore germination (Table 1, Fig. 1). Four

media (PDB, PDB-YM, GS, DW) were

used and incubation was observed for 1-5

days at 25 C and 4 C. No spore germinationwas observed at 25 C up to 5 days of

incubation in any media. However, when

spore suspensions were kept at 4 C for 7

days to represent a dormancy period, then

incubated at 25 C, less than 50% of spores

were found to be germinated on the rst

day. Spore germination was above 50%,

and 100%, on the second to third days and

fourth to fth days, respectively. PDB-YM

was found to be the best nutrient source for

the spore germination of O. sobolifera. It is well known that nutritional and

environmental factors inuence spore

germination of fungi. Glucose/sucrose

solution and even distilled water are

suitable substrates to germinate fungal

spores. Our study showed no germination

on PDB, PDB-YM, GS and DW of O.

sobolifera spores at 25 C, even after 5

days of incubation. However, when spore

suspensions were kept at a temperature

of 4 C for a week, and then shifted to 25C, spore germination was initiated since

the rst day of incubation. Apparently,

the fungus has a dormancy, which the

low temperature treatment can break. The

natural life cycle of O. sobolifera may

exhibits the same phenomenon because

the fungus grows in mostly snow elds,

where the organism can pass dormancy in

the freezing temperature.

Culture phenotype and ascocarp

formation. O. sobolifera showedexceptional growth and ascocarp

development in the laboratory study. When

PDA and YMA media were inoculated,

callus-like structures (primordia) were

formed and developed a mature ascocarp

once. To conrm its exceptional growth,

Table 1. Factors requirement for spore germination ofOphiocordyceps sobolifera.

Pre-incubation at 4 C Media Germinated spores/day (%)

1 2 3 4 5

Without PDB - - - - -

PDB-YM - - - - -

GS - - - - -

DW - - - - -

With PDB 32 55 96 100 100 PDB-YM 32 61 100 100 100

GS 21 47 88 100 100

DW 14 35 61 89 100

PDB: Potato dextrose broth. PDB-YM: PDB-yeast malt extract (PDB 2.4 g, yeast 1.5 g, malt 1.5 g,

distilled water 100 ml). GS: Glucose solution (2%). DW: Distilled water.


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the callus-like structure was studied under

a microscope and many spores were found

(Fig. 2), showing that it was a sporocarp

of the fungus. Different agar media were

inoculated and formed callus-like structure

and ascocarp.

Twelve media were used to studyascocarp formation and productivity. PDA-

YM, PDAY, and YMA showed greater

growth and branching in the culture.

PDA-YM, PDAY and PDAM showed

the greatest fresh and dry weight. PDA

or YMA alone supported limited growth

of the fungus, while the mixture of yeast,

malt extract and PDA showed exceptional

formation of ascocarp. Addition of malt

extract to PDA was a key factor to accelerate

mycelial growth, whereas addition of yeast

extract was a trigger to develop ascocarps

and branches (Table 2, Fig. 3). Some ofthe media (CDA, CtMA, CMA, PA, EA,

RNAA) were found to be ineffective for

growth and ascocarp development of O.

sobolifera.

To fulll industrial requirements of

biomass and exopolysaccharides, some

Fig. 1. Non-germinated (A), germinated (B), and germination initiation (C) in spores of Ophiocordycepssobolifera.

A

BC


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Fig. 2. Growth and appearance of Ophiocordyceps sobolifera after 30 days incubation (25 C). Callus shaped

sporocarps (A) and mycelia (B) grown on PDA supplemented with 1.5% yeast and malt extracts, respectively.

A B

Table 2. Sporocarp formation and biomass production of Ophiocordyceps sobolifera.

Media Culture Branch/head Fresh weight Dry weight

status (g) (g)

PDA ++ + 5.90 1.27

CDA - - - -

YMA +++ +++ 4.50 1.41

CMA - - - -

CtMA - - - -

PA - - - -

EA + ++ - -

RNAA - - - -

NA + + 2.92 1.05

PDAM ++ - 8.76 1.83

PDAY ++ +++ 9.60 2.20

PDA-YM +++ +++ 11.06 2.87

Data were recorded (n=6) after 60 days of incubation at 25 C. Very strong (+++), strong (++), moderate (+), weak (-).

PDA: Potato dextrose agar. CDA: Carrot dextrose agar. YMA: Yeast-malt agar. CMA: Cow-milk agar. CtMA: Coconut-milk

agar. EA: Egg agar. PA: Prawn agar. RNAA: Residue of ribosomal nucleic acid agar. NA: Nutrient agar. PDAM: PDA-malt

extract. PDAY: PDA-yeast extract. PDA-YM: PDA-yeast malt extract.


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Fig. 4. Effect of plant growth hormones on the growth of Ophiocordyceps sobolifera after 30 days of

incubation at 25 C. Callus-like structure and branched callus-like structure formed on PDA without

hormone (left), and with hormone (right), respectively.

A B

Fig. 3. Growth and appearance of Ophiocordyceps sobolifera after 60 days of incubation at 25 C.

Sporocarps (A) and mycelia (B) grown on PDA supplemented with 1.5% yeast and malt extracts,

respectively.


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attempts have been made by articial

culture. Cell growth and exopolysaccharide

production were found to be enhanced by

submerged and static culture when sodium

dodecyl sulfate (SDS), tween 80, K+

, Ca2+

,Mg2+and Mn2+were added to PDA2.

Effect of plant hormones. The plant

regulator hormones IAA, IBA and KIN

were studied for the production of fungal

biomass. Total production of biomass was

similar with or without hormones in the

PDA medium. However, growth phenotype

of biomass was found to be variable in PDA,

with and without hormones. The cultures

in media with no hormones were found to

form callus-like structure only. Addition ofplant hormones to PDA inuenced branch

formation and proliferation (Fig. 4).

Gryndler et al. revealed a detectable

decrease in the proliferation of hyphae

from the arbuscular mycorrhizal fungus

Glomus stulosum at M of IAA. Plant

hormones can inuence growth and

sporulation of fungi3. Sood demonstrated

that plant growth hormones were adverse

for growth and sporulation of Aspergillus

umbrosus8

. The results from this research showed

conditions for spore germination,

activation of lamentous growth, starting

formation of the sporocarp, and estimation

of biological efcacy on different media,

as well as distinguishable effects of

plant growth hormones on the formation

of ascocarps and branches. All these

data could be helpful for the industrial

manufacture of O. sobolifera.

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2. Cui, J. D. and Y. N. Zhang. 2012. Evaluation of

metal ions and surfactants effect on cell

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Nutritional and environmental requirements for the morphogenesis of Ophiocordyceps sobolifera

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