M ORPHOGENESIS OF OPHIoCoRDYCEPS MICOLOGIA A PLICADA INTERNATIONAL , 26(1), 2014, pp.

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2014, BERKELEY, CA, U.S.A. www.micaplint.com

NUTRITIOnAL AnD EnVIROnMEnTAL REQUIREMEnTS FOR THE MORPHOGEnESIS OF OPHIOCORDYCEPS

SOBOLIFERA

A. IMTIAJ1,2 AnD S. OHGA1*

1

Division of Forest Environmental Sciences, Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka 811-2415, Japan. 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 of Ophiocordyceps 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. sobolifera on PDA and yeast-malt agar resulted in mycelial growth and calluslike 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: ohga@forest.kyushu-u.ac.jp

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ural slow growth pattern of wild O. sobolifera can not supply the industrial requirement and purposes. Moreover, its presence in nature is restricted to specific areas and the size of sporocarps is extremely small. It is essential to develop methods for commercial production of biomass. Some attempts have been made to obtain biomass by submerged culture. Submerged culture gives potential advantages of higher mycelial production in a compact space and shorter time with less chance of contamination1. There are differing synonyms for O. sobolifera including different species, morphic status, and growth types leading to confusion about its taxonomic identification. Therefore, DNA sequences were obtained for authentication of the fungal species. Thereafter, a phenotypic study including key requirements for mycelial growth, spore germination and ascocarp formation was carried out, which can be useful for industrial applications of O. sobolifera. MATERIALS AND METHODS Collection and identification 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 at 25 C for further study. Identification was confirmed 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.

INTRODUCTION Ophiocordyceps sobolifera (Hill ex Watson) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora is an insect parasitic fungus belonging to Ophiocordycipitaceae, Ascomycota. This fungus is found in China, Cuba, Japan, Korea, Madagascar, Mexico and Sri Lanka; it is also synonymously known as Cordyceps sobolifera, Clavaria sobolifera, Sphaeria sobolifera and Torrubia sobolifera. Beauveria sobolifera is the anamorph (asexual reproductive stage) of O. sobolifera9. It is assumed that the synanamorphic fungus O. sobolifera has anamorphic, teleomorphic and holomorphic phases. Most of the members of the family are known worldwide as herbal folk medicinal mushroom, due to the presence of important bioactive compounds, such as adenosine, cordycepin and exopolysaccharides7. The chemical constituents for most species include cordycepin (3-de-oxyadenosine), ergosterol, polysaccharides, a glycoprotein and peptides containing -aminoisobutyric acid. Polysaccharides account for anti-inflammatory, antioxidant, anti-tumour, anti-metastatic, immunomodulatory, hypoglycemic, steroidogenic and hypolipidaemic effects, whereas cordycepin contributes to the anti-tumour, insecticidal and antibacterial activities6. Liquid culture filtrate of Cordyceps species can inhibit the growth of bacteria4. It is also reported that this fungal species may boost energy and ameliorate nephrotoxicity-induced renal dysfunction in rats via antioxidant, anti-apoptosis, and anti-autophagy mechanisms10. In general, the morphological biomass of Cordyceps species 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-

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Ltd., Tokyo, Japan (www.genenet.co.jp). DNA extraction, PCR, gel electrophoresis, sequencing and analysis of DNA sequences was carried out following the methods of our previous work 5. The identified mycelial pure 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 prawn powder, 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-3butyric acid (IBA), and kinetin (KIN) was studied in PDA medium for the production of biomass. PDA was prepared and autoclaved. Plant growth hormones were 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 of spores 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 spore suspension 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 was carefully 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 to determine dry weight. RESULTS AND DISCUSSION Spore germination. O. sobolifera was

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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 25 C, spore germination was initiated since the first 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 fields, where the organism can pass dormancy in the freezing temperature. Culture phenotype and ascocarp formation. O. sobolifera showed exceptional 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 confirm its exceptional growth,

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 germination was 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 first day. Spore germination was above 50%, and 100%, on the second to third days and fourth to fifth 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 influence spore germination of fungi. Glucose/sucrose solution and even distilled water are suitable substrates to germinate fungal spores. Our study showed no germination

Table 1. Factors requirement for spore germination of Ophiocordyceps sobolifera. Pre-incubation at 4 C Media Germinated spores/day (%) 1 2 3 4 5 Without With PDB - - - - PDB-YM - - - - GS - - - - DW - - - - 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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Fig. 1. Non-germinated (A), germinated (B), and germination initiation (C) in spores of Ophiocordyceps sobolifera.

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 study ascocarp formation and productivity. PDAYM, 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 of the media (CDA, CtMA, CMA, PA, EA, RNAA) were found to be ineffective for growth and ascocarp development of O. sobolifera. To fulfill industrial requirements of biomass and exopolysaccharides, some

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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.

Table 2. Sporocarp formation and biomass production of Ophiocordyceps sobolifera. Media Culture Branch/head status Fresh weight (g) Dry weight (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. 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.

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.

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by Cordyceps sinensis 16 in submerged culture. Bioresource Technology 98: 165168. Cui, J. D. and Y. N. Zhang. 2012. Evaluation of metal ions and surfactants effect on cell growth and exopolysaccharide production in two-stage submerged culture of Cordyceps militaris. Applied Biochemistry and Biotechnology 168: 1394-1404. Gryndler, M., H. Hrelov, I. Chvtalov and J. Jansa. 1998. The effect of selected plant hormones on in vitro proliferation of hyphae of Glomus fistulosum. Biologia Plantarum 41: 255-263. Imtiaj, A. and T. S. Lee. 2007. Screening of antibacterial and antifungal activities from Korean wild mushrooms. World Journal of Agricultural Sciences 3: 316-321. Imtiaj, A., T. S. Lee and S. Ohga. 2011. Sequence variation of Pleurotus species collected from Eastern Asia. Micologia Aplicada International 23: 1-10. Ng, T. B. and H. X. Wang. 2005. Pharmacological actions of Cordyceps, a prized folk medicine. Journal of Pharmacy and Pharmacology 57: 1509-1519. Song, C. H., B. K. Yang, K. S. Ra, D. H. Shon, E. J. Park, G. I. Go and Y.H. Kim. 1998. Hepatoprotective effect of extracellular polymer produced by submerged culture of Ganoderma lucidum WK-003. Journal of Microbiology and Biotechnology 8: 277 279. Sood, M. 2011. Cultural physiology: effect of plant growth hormones on the growth and sporulation of Aspergillus umbrosus. Journal of Phytology 3: 27-29. Sung, G. H., N. L. H. Jones, J. M. Sung, J. J. Luangsaard, B. Shrestha and J. W. Spatafora. 2007. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Studies in Mycology 57: 5-59. Wu, M. F., P. C. Li, C. C. Chen, S. S. Ye, C. T. Chien and C. C. Yu. 2011. Cordyceps sobolifera extract ameliorates lipopolysaccharideinduced renal dysfunction in the rat. American Journal of Chinese Medicine 39: 523-535.

attempts have been made by artificial 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 of plant hormones to PDA influenced branch formation and proliferation (Fig. 4). Gryndler et al. revealed a detectable decrease in the proliferation of hyphae from the arbuscular mycorrhizal fungus Glomus fistulosum at M of IAA. Plant hormones can influence 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 filamentous growth, starting formation of the sporocarp, and estimation of biological efficacy 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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LITERATURE CITED
1. Cha, S. H., J. S. Lim, C. S. Yoon, J. H. Koh, H. I. Chang and S. W. Kim. 2007. Production of mycelia and exo-biopolymer from molasses

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