Abstract
We investigated the effect of nutritional and environmental factors on Ophiocordyceps longissima mycelial growth. The longest colony diameter was observed on Schizophyllum (mushroom) genetics complete medium plus yeast extract, Schizophyllum (mushroom) genetics minimal medium, and Sabouraud dextrose agar (SDA); however, malt-extract yeast-extract agar, SDA plus yeast extract, yeast-extract malt-extract peptone dextrose agar, SDA, oatmeal agar, and potato dextrose agar showed higher mycelia density. A temperature of 25℃ was optimum and 7.0 was the optimum pH for mycelial growth. Colony diameter was similar under light and dark conditions. Maltose and yeast extract showed the highest mycelial growth among carbon and nitrogen sources respectively. The effect of mineral salts was less obvious; however, K3PO4 showed slightly better growth than that of the other mineral salts tested. Among all nutrition sources tested, complex organic nitrogen sources such as yeast extract, peptone, and tryptone were best for mycelial growth of O. longissima. Ophiocordyceps longissima composite medium, formulated by adding maltose (2% w/v), yeast extract (1% w/v), and K3PO4 (0.05% w/v) resulted in slightly longer colony diameter. In vitro mycelial O. longissima growth was sustainable and the production of fruiting bodies could be used for commercial purposes in the future.
Keywords: Carbon source, Growth characteristics, Mineral salt, Nitrogen source, Ophiocordyceps longissima
Many Cordyceps species grow on nymphs (larvae) of cicada (Cicadidae, Homoptera) [1-3]. One is C. longissima Kobayasi, which was first reported from Japan on Tanna japonensis nymphs [2]. This species was later confirmed in Korea in 1998 [4]. One year later, it was reported in China together with its Hirsutella anamorph [5]. Li et al. confirmed the anamorph as H. longissima Li et al. [6]. Recently, Sung et al. transferred C. longissima to a new genus Ophiocordyceps based on a phylogenetic classification and renamed it O. longissima (Kobayasi) Sung et al. [7].
The stromata of O. longissima are 5~20 cm long, sometimes much longer (Fig. 1). The stroma of O. longissima is characterized by a long stalk with a terminal clavate fertile part without any clear demarcation between the two parts. Perithecia are ovoid to long ovoid, with a short neck, 440~590 × 130~300 µm; asci and secondary spores measure 190~350 × 5~6 µm and 8~11 × 1~1.2 µm respectively (Figs. 2 and 3).
Fig. 1.
Various natural specimens of Ophiocordyceps longissima collected in Korea.
Fig. 2.
Morphological characteristics of Ophiocordyceps longissima. A~C, Apical fertile part of stromata; D~F, Cross-section of stromata showing perithecia; G, H, Ascus heads; I, Threadlike fragmented ascospores.
Fig. 3.
Microscopic structures of Ophiocordyceps longissima. a, fragmented ascospores; b, ascus; c, perithecia (scale bar = 10 mm [a], 25 mm [b], 200 mm [c]).
Cordyceps species, including cicadicolous fungi such as O. sobolifera, are regarded as medicinal mushrooms in oriental society [2, 8-11]. In this context, many researchers have begun to study cultivation characteristics of Cordyceps and allied species [5, 12-23]. Within the past few years, O. longissima specimens have been collected by the Cordyceps Research Institute (CRI), Mushtech, Korea on Mt. Halla at Jeju-do and on Mt. Duryun at Jeollanam-do Korea. In this study, we provide detailed information on mycelial growth characteristics of O. longissima collected in Korea for the first time.
Materials and Methods
Fungal isolates
Multi-ascospore isolates were derived from fresh O. longissima specimens CRI C-6764, CRI C-7080, and CRI C-8587 following the method of Sung et al. [24]. Specimen CRI C-6764 was collected on Mt. Duryun at Jeollanam-do on July 8, 2001. Similarly, two other specimens, CRI C-7080 and CRI C-8587, were collected on Mt. Halla at Jeju-do on July 10 and 12, 2001 respectively. The specimens have been preserved at CRI, Mushtech, Korea. The multi-ascospore isolates, after growing on Sabouraud dextrose agar plus yeast extract (SDAY; dextrose 20 g, yeast extract 5 g, peptone 5 g and agar 15 g per 1,000 mL; pH 5.6) agar plates at 24 ± 1℃ for 30 days, were used in the experiment.
Effect of medium, temperature, light, and pH on O. longissima mycelial growth
Nine different types of agar media were used to observe the growth characteristics of O. longissima isolates (Table 1). Mycelial discs (5 mm) of all three isolates were inoculated in the center of the agar media and incubated at 25℃ for 30 days. Water agar (2%, WA) was used as the control. Colony diameter (CD) was measured in mm and mycelial density (MD) was qualitatively graded as thin (+), moderate (++), or compact (+++) after the incubation.
Table 1.
Synthetic media composition
WA, water agar; OA, oatmeal agar; MYA, malt-extract yeast-extract agar; MM, Schizophyllum (mushroom) genetics minimal medium; PDA, potato dextrose agar; MCM, Schizophyllum (mushroom) genetics complete medium plus yeast extract; YMA, yeast-extract malt-extract peptone dextrose agar; CDA, Czapek-dox agar; SDAY, Sabouraud dextrose agar plus yeast extract; SDA, Sabouraud dextrose agar.
Schizophyllum (mushroom) genetics minimal medium (MM) and malt-extract yeast-extract agar (MYA) showed better mycelial growth and, hence, were used for selecting the optimum temperature for growth of the O. longissima isolates. Mycelial discs were inoculated on MM and MYA agar plates and incubated at various temperatures ranging from 15~35℃ at regular intervals of 5℃ for 30 days. Similarly, to observe the effect of light on growth, mycelial discs were inoculated on MM agar plates and incubated under continuous light and dark conditions for 30 days at 25℃. CD and MD were recorded after the incubation, as described above.
Liquid MM (100 mL MM without agar) was prepared in 250 mL Erlenmeyer flasks. The pH of the liquid medium was adjusted from 4.0~10.0 at intervals of 1.0 before sterilization. Five mycelial discs were inoculated in the liquid medium with different pH levels and incubated on a rotary shaker at 120 rpm for 30 days at 25℃. The liquid cultures were then filtered through Whatman no. 2 filter paper, the residual mycelia were dried at 60℃ for 24 hr, and the dry weight (DW) of the mycelium was measured in g.
Selection of the optimum carbon source, nitrogen source, mineral salts, and carbon/nitrogen (C/N) ratio
O. longissima isolates were grown on WA supplemented with carbon sources (2% w/v) only. Additionally, 100 mL of MM liquid medium prepared with the carbon sources (2% w/v) in 250 mL Erlenmeyer flasks were inoculated with the isolates. Similarly, isolates were inoculated in WA and MM liquid media supplemented with nitrogen sources (2% w/v) only. Different mineral salts (0.05% w/v) were also tested for their effect on mycelial growth of O. longissima isolates in WA and MM liquid media. Agar cultures and liquid cultures were incubated as described above.
Maltose and yeast extract were the best carbon and nitrogen sources respectively for the mycelial growth of O. longissima isolates. Hence, they were added together in the WA and MM liquid media at different ratios of 20 : 1, 10 : 1, 5 : 1, 2 : 1, 1 : 1, 1 : 2, 1 : 5, and 1 : 10 and inoculated with the isolates. Maltose concentration was fixed at 0.5%, 1.0%, and 2.0% (w/v) for all ratios. Agar and liquid cultures were incubated as described above, and WA medium was used as the control. CD and MD were recorded on agar cultures, and DW was measured in liquid cultures, as described above.
Comparison of O. longissima composite medium (OLCM) with MYA
OLCM was prepared by adding maltose, yeast extract, and K3PO4 at concentrations of 2% (w/v), 1% (w/v), and 0.05% (w/v), respectively, on WA. The O. longissima isolates were inoculated on OLCM and MYA agar plates and observed for CD and MD. WA was used as the control.
Results and Discussion
Selection of optimum medium, temperature, and pH
The CD of O. longissima isolates was relatively longer on Schizophyllum (mushroom) genetics complete medium plus yeast extract, MM, and Sabouraud dextrose agar (SDA); however, compact or moderate density was observed on MYA, SDAY, yeast-extract malt-extract peptone dextrose agar, SDA, potato dextrose agar (PDA), and oatmeal agar (Table 2). CD differed among the isolates; CRI C-7080 and CRI C-8587 grew faster than CRI C-6764. It was clear that media that induced a compact density produced a shorter CD compared to media that produced moderate density (Table 2), as shown in C. militaris isolates [25]. This result indicates that MD and CD of the isolates do not correlate with each other on agar culture. Czapek-dox agar (CDA) produced a thin density and the shortest CD in O. longissima isolates, as reported in other Cordyceps species [26, 27]. CDA does not contain an organic nitrogen source, which may be why it could not support rich growth of the O. longissima isolates. Li et al. [5, 6] showed longer CD of O. longissima isolates on Czapek agar than that on PDA, but did not report the MD. CD of O. longissima isolates in our study was generally longer than that of Li et al. [5, 6].
Table 2.
Effect of medium on Ophiocordyceps longissima mycelial growth
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density; MCM, Schizophyllum (mushroom) genetics complete medium plus yeast extract; MM, Schizophyllum (mushroom) genetics minimal medium; SDA, Sabouraud dextrose agar; MYA, malt-extract yeast-extract agar; OA, oatmeal agar; PDA, potato dextrose agar; SDAY, Sabouraud dextrose agar plus yeast extract; YMA, yeastextract malt-extract peptone dextrose agar; CDA, Czapek-dox agar; WA, water agar.
The longest CD was observed at 25℃ (Fig. 4), agreeing with previous studies [5, 14, 24, 26, 28, 29]. O. longissima isolates had a similar CD at 20℃ and 30℃, which was similar to Metacordyceps yongmunensis and O. heteropoda [27, 29]. However, this was in contrast to C. cardinalis that showed no growth at 30℃ and above [26]. Almost no mycelial growth of O. longissima isolates occurred at 15℃ and 35℃ (Fig. 4).
Fig. 4.
Effect of temperature on mycelial growth of Ophiocordyceps longissima isolates cultured on malt-extract yeast-extract agar (MYA) and Schizophyllum (mushroom) genetics minimal medium (MM). CRI, Cordyceps Research Institute.
No obvious difference was observed in the CD of O. longissima between light and dark conditions (Fig. 5). The prime effect of light is the induction of pigmentation [25]. In our study, the O. longissima isolates produced reddish white pigmentation under light, similar to that observed by Li et al. [5, 6]. In addition to pigmentation, light also controls fruiting morphology, such as elongation and branching in culture [14]. Cycles of dark/light periods may be critical in some species to induce fruiting bodies [16]. A pH of 7.0 produced the highest DW, followed by pH 8.0, which was similar to previous studies (Fig. 6) [14, 26, 28].
Fig. 5.
Effect of light on colony diameter of Ophiocordyceps longissima isolates cultured on Schizophyllum (mushroom) genetics minimal medium. CRI, Cordyceps Research Institute.
Fig. 6.
Effect of pH on mycelial dry wt. of Ophiocordyceps longissima isolates cultured in liquid Schizophyllum (mushroom) genetics minimal medium. CRI, Cordyceps Research Institute.
Selection of optimum carbon source, nitrogen source, mineral salts, and C/N ratio
It was very difficult to observe the effect of carbon source on mycelial growth of O. longissima in agar culture because the CD was too short in all carbon sources tested, particularly for the CRI C-6764 isolate (Table 3). Furthermore, all carbon sources produced only a thin density of mycelia, showing that carbon sources do not sustain rich mycelial growth (Table 3). Among the carbon sources tested, maltose produced the highest DW in liquid culture, whereas the remaining carbon sources produced less than half of that of maltose. Based on DW, maltose was the best carbon source among those tested.
Table 3.
Effect of carbon source on Ophiocordyceps longissima mycelial growth
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density; DW, dry wt. of mycelium; WA, water agar.
Among the nitrogen sources tested, yeast extract produced the highest mycelial growth, followed by peptone and tryptone in both agar and liquid cultures (Table 4). Yeast extract, peptone, and tryptone also produced much better mycelial growth than the carbon sources. NaNO3, KNO3, glycine, and ammonium tartrate all produced short, thin densities, as with WA (Table 4). However, L-asparagine produced moderate density and a similar CD as NaNO3, KNO3, glycine, and ammonium tartrate. These results showed that complex organic nitrogen sources such as yeast extract, peptone, and tryptone sustain favorable growth of O. longissima, as in other Cordyceps and allied species [25-27, 29]. Yeast extract was selected as the best nitrogen source for mycelial growth of O. longissima isolates and was used for further tests. Mineral salts showed the poorest mycelial growth among all nutritional sources tested (Table 5). All mineral salts tested produced thin MD and CD, similar to WA. K3PO4 showed slightly better mycelial growth than that of other mineral salts. Notably, CuSO4·5H2O showed a rather shorter CD than that of WA in all isolates (Table 5).
Table 4.
Effect of nitrogen source on Ophiocordyceps longissima mycelial growth
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density; DW, dry wt. of mycelium; WA, water agar.
Table 5.
Effect of mineral salt on Ophiocordyceps longissima mycelial growth
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density; WA, water agar.
O. longissima isolates produced compact MD at all C/N ratios (Table 6). However, C/N ratios of 2 : 1 and 1 : 1 showed the longest CD (Table 6). Nitrogen favors mycelial growth but higher nitrogen ratios slowed CD, probably due to higher nutrient concentrations; thus, increasing the water potential of the medium and consequently decreasing the amount of water available to the isolates. A 2 : 1 C/N ratio was selected with maltose and yeast extract at concentrations of 2% and 1%, respectively, to formulate the OLCM.
Table 6.
Effect of carbon/nitrogen (C/N) ratio on Ophiocordyceps longissima isolate CRI C-7080 mycelial growth
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density.
Comparison of OLCM with MYA
Both OLCM and MYA produced compact MD (Table 7). However, OLCM produced slightly longer CD than that of MYA. A higher concentration of yeast extract is likely favorable for O. longissima mycelial growth, as in C. militaris [25]. The medium is the most important factor for mycelial growth and fruiting body formation in Cordyceps species. Commonly used mycological media can support profuse mycelial growth in Cordyceps species. In contrast, cereals such as rice, supplemented with pupal power, sawdust, peptone, and yeast extract are used for fruiting body production [5, 14]. Fruiting medium is much more complex than mycelial growth medium. Obviously, fruiting body formation is a much more complex physio- and morpho-genetic process. As with mycelial growth, nitrogen sources are likely the most important nutritional factor for fruiting body formation. Fruiting body formation of O. longissima has been reported by Li et al. [5, 6] in rice medium. It is concluded that various nutritional and environmental factors should be tested to induce profuse fruiting bodies in O. longissima, so that it can be commercially utilized as a health and medicinal food.
Table 7.
Comparison between Ophiocordyceps longissima composite medium (OLCM) and malt-extract yeast-extract agar (MYA)
CRI, Cordyceps Research Institute; CD, colony diameter; MD, mycelial density; WA, water agar.
Acknowledgements
We acknowledge the Cordyceps Research Institute (CRI), Mushtech, Korea for providing facilities to conduct this study.
References
- 1.Kobayasi Y. On the genus Cordyceps and its allies on Cicadidae from Japan. Bull Biogeogr Soc Jpn. 1939;9:145–176. [Google Scholar]
- 2.Kobayasi Y, Shimizu D. Monographic studies of Cordyceps 2: Group parasitic on Cicadidae. Bull Natl Sci Mus Tokyo. 1963;6:286–314. [Google Scholar]
- 3.Sung JM. The insects-born fungus of Korea in color. Seoul: Kyohak Publishing Co., Ltd.; 1996. [Google Scholar]
- 4.Lee JB, Oh DC. Higher fungi of Cheju-do (1): unrecorded mushrooms. Korean J Mycol. 1998;26:538–550. [Google Scholar]
- 5.Li CR, Huang B, Fan MZ, Li ZZ. Cordyceps longissima and its Hirsutella anamorph. J Anhui Agric Univ. 1999;26:374–377. [Google Scholar]
- 6.Li CR, Fan MZ, Huang B, Li ZZ. Hirsutella longissima sp nov, the anamorph of Cordyceps longissima. Mycosystema. 2001;20:29–34. [Google Scholar]
- 7.Sung GH, Hywel-Jones NL, Sung JM, Luangsa-ard JJ, Shrestha B, Spatafora JW. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Stud Mycol. 2007;57:5–59. doi: 10.3114/sim.2007.57.01. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Kiho T, Ukai S. Tochukaso (Semitake and others), Cordyceps species. Food Rev Int. 1995;11:231–234. [Google Scholar]
- 9.Mizuno T. Medicinal effects and utilization of Cordyceps (Fr.) Link (Ascomycetes) and Isaria Fr. (Mitosporic fungi) Chinese caterpillar fungi, "Tochukaso" (Review) Int J Med Mushrooms. 1999;1:251–261. [Google Scholar]
- 10.Kinjo N, Zang M. Morphological and phylogenetic studies on Cordyceps sinensis distributed in southwestern China. Mycoscience. 2001;42:567–574. [Google Scholar]
- 11.Shrestha B, Zhang W, Zhang Y, Liu X. What is the Chinese caterpillar fungus Ophiocordyceps sinensis (Ophiocordycipitaceae)? Mycology. 2010;1:228–236. [Google Scholar]
- 12.Yahagi N. Cultivation of Isaria japonica and inoculation to silkworm. Touchu-Kasou. 1985;5:27–30. [Google Scholar]
- 13.Harada Y, Akiyama N, Yamamoto K, Shirota Y. Production of Cordyceps militaris fruit body on artificially inoculated pupae of Mamestra brassicae in the laboratory. Trans Mycol Soc Jpn. 1995;36:67–72. [Google Scholar]
- 14.Yamanaka K, Inatomi S. Cultivation of Isaria japonica fruit bodies on mixed plant/insect media. Food Rev Int. 1997;13:455–460. [Google Scholar]
- 15.Yamanaka K, Inatomi S, Hanaoka M. Cultivation characteristics of Isaria japonica. Mycoscience. 1998;39:43–48. [Google Scholar]
- 16.Kana-uchi A, Fukatsu T. Light-induced fruit body formation of an entomogenous fungus Paecilomyces tenuipes. Mycoscience. 1999;40:349–351. [Google Scholar]
- 17.Chen R, Ichida M. Infection of the silkworm, Bombyx mori, with Cordyceps militaris. J Insect Biotechnol Sericology. 2002;71:61–63. [Google Scholar]
- 18.Sato H, Shimazu M. Stromata production for Cordyceps militaris (Clavicipitales: Clavicipitaceae) by injection of hyphal bodies to alternative host insects. Appl Entomol Zool. 2002;37:85–92. [Google Scholar]
- 19.Yahagi N, Yahagi R, Takano F, Fushiya S, Tanaka T, Murakami K, Ohta T. Growth of ascocarps from cultured Cordyceps militaris (L.:Fr.) Fr. and Cordyceps formicarum Kobayasi in an agar medium. Nippon Kingakkai Kaiho. 2004;45:15–19. [Google Scholar]
- 20.Li CR, Nam SH, Geng DG, Fan MZ, Li ZZ. Artificial culture of seventeen Cordyceps spp. Mycosystema. 2006;25:639–645. [Google Scholar]
- 21.Lee JO, Shrestha B, Kim TW, Sung GH, Sung JM. Stable formation of fruiting body in Cordyceps bassiana. Mycobiology. 2007;35:230–234. doi: 10.4489/MYCO.2007.35.4.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Lee JO, Shrestha B, Sung GH, Han SK, Kim TW, Sung JM. Cultural characteristics and fruiting body production in Cordyceps bassiana. Mycobiology. 2010;38:118–121. doi: 10.4489/MYCO.2010.38.2.118. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Kim SY, Shrestha B, Sung GH, Han SK, Sung JM. Optimum conditions for artificial fruiting body formation of Cordyceps cardinalis. Mycobiology. 2010;38:133–136. doi: 10.4489/MYCO.2010.38.2.133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Sung GH, Shrestha B, Park KB, Sung JM. Cultural characteristics of Shimizuomyces paradoxus collected from Korea. Mycobiology. 2010;38:189–194. doi: 10.4489/MYCO.2010.38.3.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Shrestha B, Lee WH, Han SK, Sung JM. Observations on some of the mycelial growth and pigmentation characteristics of Cordyceps militaris isolates. Mycobiology. 2006;34:83–91. doi: 10.4489/MYCO.2006.34.2.083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Sung GH, Shrestha B, Han SK, Kim SY, Sung JM. Growth and cultural characteristics of Cordyceps cardinalis collected from Korea. Mycobiology. 2010;38:274–281. doi: 10.4489/MYCO.2010.38.4.274. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Sung GH, Shrestha B, Han SK, Sung JM. Cultural characteristics of Ophiocordyceps heteropoda collected from Korea. Mycobiology. 2011;39:1–6. doi: 10.4489/MYCO.2011.39.1.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Sasaki F, Miyamoto T, Tamai Y, Yajima T. Optimum temperature and pH for mycelial growth of Cordyceps nutans Pat. (Ascomycetes) Int J Med Mushrooms. 2005;7:301–304. [Google Scholar]
- 29.Sung GH, Shrestha B, Sung JM. Characteristics of Metacordyceps yongmunensis, a new species from Korea. Mycobiology. 2010;38:171–175. doi: 10.4489/MYCO.2010.38.3.171. [DOI] [PMC free article] [PubMed] [Google Scholar]