Mycobiology 38(4) : 282-285 (2010) © The Korean Society of Mycology DOI:10.4489/MYCO.2010.38.4.282 Heterothallic Type of Mating System for Cordyceps cardinalis 1 2 3 4 5 Gi-Ho Sung , Bhushan Shrestha , Sang-Kuk Han , Soo-Young Kim and Jae-Mo Sung * 1 Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Suwon 441707, Korea 2 Green Energy Mission/Nepal, Anam Nagar, Kathmandu P.O. Box 10647, Nepal 3 Division of Forest Biodiversity, Korea National Arboretum, Pocheon 487-820, Korea 4 Donghae Agricultural Technology Center, Donghae 240-030, Korea 5 Cordyceps Institute of Mushtech, Chuncheon 200-936, Korea (Received August 17, 2010. Accepted November 2, 2010) Cordyceps cardinalis successfully produced its fruiting bodies from multi-ascospore isolates. However, subcultures of multiascospore isolates could not produce fruiting bodies after few generations. Fruiting body production also differed from sector to sector of the same isolate. Single ascospore isolates were then co-inoculated in combinations of two to observe the fruiting characteristics. Combinations of certain isolates produced perithecial stromata formation, whereas other combinations did not produce any fruiting bodies. These results show that C. cardinalis is a heterothallic fungus, requiring two isolates of opposite mating types for fruiting body production. It was also shown that single ascospore isolates are hermaphrodites. KEYWORDS : Heterothallism, Homothallism, Mating system, Perithecial stromata, Subculture Cordyceps species are regarded as medicinal mushrooms in East Asian countries. Some of them, for example Ophiocordyceps sinensis (syn. Cordyceps sinensis), have played very important roles in the economic development of local communities [1, 2]. Recently, the mycelium growth and fruiting body formation of Cordyceps species have been studied with great interest with the objective of large-scale cultivation [3-11]. Mating system is an important genetic factor for fruiting body formation in fungi [12-14]. Cordyceps and other clavicipitaceous fungi have been studied in order to determine their mating systems in various environments, such as in nature and culture, or through gene sequencing [15-21]. Most of these Cordyceps species show heterothallism [17-20]. However, homothallic type behavior is also observed in Cordyceps despite apparent heterothallism, e.g., in C. militaris [17, 21]. Besides fruiting body formation, mating type can also help resolve biological species concepts in fungi [22]. In addition to ascospore-derived isolates, conidial isolates of asexual fungi have also led to the development of sexual states of Cordyceps in culture by proper combination of opposite mating types [23]. C. cardinalis is a recently reported fungus [24], and its in vitro fruiting bodies have been successfully produced in Korea [25]. However, the mating system of C. cardinalis is still unknown. In this study, the mating system of C. cardinalis was studied in culture in order for continuous cultivation of its fruiting bodies. Both multi-ascospore and single ascospore isolates were tested for fruiting body for- mation. Different sectors from the same isolate were also employed for fruiting body formation. Materials and Methods Fungal isolates. C. cardinalis specimen CRI C-10735, preserved at the Cordyceps Research Institute (CRI) of Mushtech, Korea was used for multi-ascospore and single ascospore isolations. The specimen was collected from Mt. Dunryu in Jeollanam-do on August 13, 2003. Ascospores were discharged from the fresh specimen on 2% water agar. Multi-ascospore isolates were derived by transferring agar blocks containing numerous ascospores to 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, followed by incubation at 25oC under white fluorescent light for 3 wk. Similarly, 38 single ascospore isolates were also derived, following the method of Shrestha et al. [17], and incubated as above. They were numbered from CRI C-107351 to CRI C-10735-38. Fruiting body formation from multi-ascospore isolates. Liquid inocula of the isolates were prepared by inoculating five mycelial discs (4 mm) in 100 mL of SDAY broth (SDAY without agar). The inoculated SDAY broths were o incubated at 25 ± 1 C for 5 days in a rotary shaker at 120 rpm. Fruiting medium was prepared by mixing 50 g of brown rice, 10 g of silkworm pupa, and 60 mL of distilled water in 1,000 mL of Polypropylene (PP) bottle, folo lowed by sterilization at 121 C for 15 min. Between 15~ *Corresponding author <E-mail : cordyceps@hanmail.net> 282 Mating System in Cordyceps cardinalis 283 20 mL of the liquid inocula was then inoculated into each o PP bottle, followed by incubation at 25 ± 1 C for 60 days under white fluorescent light and humidity of 60~70%. After 60 days of incubation, the fruiting bodies were distinguished by observing perithecia on stromata, following the method of Shrestha et al. [17]. Fruiting bodies that developed perithecia were marked as (+) and those without perithecia as (−). tion, different sectors of the same agar culture produce various types of fruiting bodies [27]. Single ascospore isolates have been shown to possess distinct mating types and cultural characteristics [17, 28]. Usually, original isolates show better fruiting bodies, but the subcultures produce similar or inferior fruiting or no fruiting at all [26]. In this study as well, subcultures demonstrated reduced fruiting body production. Fruiting body formation from subcultures and different sectors. Multi-ascospore isolates were subcultured every 3 wk on SDAY agar plates up to the sixth generation. Subcultures were also inoculated in fruiting medium for fruiting body formation. However, no fruiting bodies could be produced from subcultures after the fourth generation. To observe the effect of sectors on fruiting body formation, 21 different sectors from the same subculture of the fourth generation were inoculated into fruiting medium and observed for fruiting body formation. The sectors were numbered from CRI C-10735-1 to CRI C10735-21. Six sectors, CRI C-10735-2, CRI C-10735-6, CRI C-10735-9, CRI C-10735-13, CRI C-10735-16, and CRI C-10735-20, which could not produce any fruiting bodies, were co-inoculated among themselves into fruiting medium in order to observe the effect of crossing on fruiting body formation. Fruiting body formation from different sectors of subcultures. Subcultures abruptly ceased producing fruiting bodies after the fourth generation. This could have been due to reduced vitality of the subcultures, or the fruiting ability could have differed from sector to sector of the same subculture. A second possibility was demonstrated when six out of 21 sectors from the same subculture of the fourth generation produced fruiting bodies. This phenomenon was again observed when non-fruiting sectors produced fruiting bodies after co-inoculation (Table 1). The combinations of sectors CRI C-10735-2 × CRI C-10735-6, CRI C-10735-2 × CRI C-10735-16, and CRI C-10735-9 × CRI C-10735-20 produced fruiting bodies (Table 1). This shows that the subcultures led to increased homogeneity in the genotypes, depending upon the sectors of the mother isolates that were used for subculturing. It was shown in this study that multi-ascospore isolates of C. cardinalis could not be used to understand mating behavior. Fruiting body formation from single ascospore isolates. All single ascospore isolates were separately inoculated into fruiting media, as described above, and observed for fruiting body formation. Four single ascospore isolates, CRI C-10735-1, CRI C-10735-2, CRI C-10735-3, and CRI C-10735-38, which could not produce perithecial fruiting bodies, were co-inoculated among themselves in order to observe the effect of crossing on fruiting body formation. A combination of isolates CRI C-10735-1 and CRI C-10735-38 produced the best perithecial fruiting bodies; hence, they were selected as tester isolates. These tester isolates were then co-inoculated with each of the remaining single ascospore isolates, and mating behavior was observed. The hermaphroditic nature of single ascospore isolates was also tested using isolates CRI C-10735-34 and CRI C-10735-38 by reciprocal inoculation, following the method of Shrestha et al. [17]. Fruiting body formation from single ascospore isolates. Out of 38 single ascospore isolates, 16 produced fruiting bodies while the rest did not produce any. The fruiting bodies produced from single ascospore isolates were different from those produced from multi-ascospore isolates in that the former did not produce any perithecia on stroTable 1. Fruiting body formation by different sectors of subculture of Cordyceps cardinalis isolate CRI C-10735 Sector No. 2 6 9 13 16 20 02 06 09 13 16 20 − + − − − − − − − − + − − − − − − + − − − Results and Discussion Fruiting body formation from multi-ascospore isolates and subcultures. The original isolates and subcultures up to the third generation produced perithecial fruiting bodies. Multi-ascospore isolates were, in fact, a mixture of genetically different ascospores. Moreover, they have been shown to produce unstable fruiting bodies as well as degenerating fruiting bodies in C. militaris [26]. In addi- Table 2. Fruiting body formation by single ascospore isolates of Cordyceps cardinalis CRI C-10735 Isolate No. 1 2 3 38 01 02 03 38 − − − − − − + + + − 284 Sung et al. Fig. 1. Fruiting body formation by combinations of single ascospore isolates of Cordyceps cardinalis CRI C-10735. Fig. 2. Fruiting body formation by combinations of single ascospore isolates and tester isolates of Cordyceps cardinalis CRI C10735. Fig. 3. Fruiting body formation by reciprocal inoculations and co-inoculations of single ascospore isolates of Cordyceps cardinalis CRI C-10735-34 and CRI C-10735-38. A, E, Single inoculations; B, D, Reciprocal inoculations between two isolates; C, Co-inoculation of two isolates. mata. Combinations of single ascospore isolates, however, did produce perithecial stromata. Combinations of CRI C-10735-38 × CRI C-10735-1, CRI C-10735-38 × CRI C-10735-2, and CRI C-10735-38 × CRI C-10735-3 produced perithecial fruiting bodies, whereas combinations of CRI C-10735-2 × CRI C-10735-1, CRI C-107353 × CRI C-10735-1, and CRIC-10735-3 × CRI C-10735-2 produced no perithecial fruiting bodies (Table 2, Fig. 1). Thus, isolates CRI C-10735-1, CRI C-10735-2, and CRI C-10735-3 were designated as type A and the other isolate CRI C-10735-38 as type B to indicate that they were of opposite mating type. Most of the remaining single ascospore isolates produced perithecial fruiting bodies when co-inoculated with either CRI C-10735-1 or CRI C-10735-38 (Fig. 2). How- ever, one of them produced fruiting bodies with both tester isolates, whereas the other two did not produce any fruiting bodies with either of the tester isolates. In summary, 23 isolates showed mating type A and 12 isolates showed mating type B. Hermaphroditic nature was shown by single ascospore isolates of opposite mating types CRI C-10735-34 and CRI C-10735-38 when inoculated reciprocally (Fig. 3). Many Cordyceps species have been shown to be heterothallic by analysis of mating type genes [20]. Cultural studies should be conducted more critically to identify the mating system for Cordyceps. C. bassiana shows a very unstable mating system in culture [10, 11]. In nature, Cordyceps species generally show variation in their morphological characteristics. These variations could be due Mating System in Cordyceps cardinalis to heterothallism in their mating systems. Since C. cardinalis has a wider distribution from North America to East Asia [24], heterothallism might be the reason for its adaptation to a wider ecological range. This study showed C. cardinalis as a heterothallic fungus, similar to C. militaris, although the mating type of a few single ascospore isolates could not be determined. We also explained why fruiting body production varies from isolate to isolate of multi-ascospore origin. It might be possible that multi-ascospore isolates occasionally consist of ascospores of the same mating type, and hence do not produce any fruiting bodies. Further, continuous subcultures proliferate only a particular sector of the mother culture, leading to homogeneity in mating type after several subcultures. Such studies can help accumulate more information about the mating systems of Cordyceps in general. Sung et al. [29] has shown that single ascospore isolates of C. militaris degenerate after several subcultures, but not as early as multi-ascospore isolates. Hence, it is necessary to select superior isolates for enhanced fruiting body production, as suggested by Sung et al. [30]. Acknowledgements The authors wish to acknowledge the Cordyceps Research Institute for providing facilities to carry out this study. References 1. Devkota S. Yarsagumba [Cordyceps sinensis (Berk.) Sacc.]; traditional utilization in Dolpa district, Western Nepal. Our Nature 2006;4:48-52. 2. Winkler D. Yartsa Gunbu (Cordyceps sinensis) and the fungal commodification of Tibet’s rural economy. Econ Bot 2008;62:291-305. 3. Liang ZQ. Anamorph of Cordyceps militaris and artificial culture of its fruitbody. Southwest China J Agric Sci 1990;3: 1-6. 4. Liu JL, Liang ZQ, Liu AY. Artificial culture of fruiting bodies of Cordyceps gunnii. Southwest China J Agric Sci 1990; 3:6-10. 5. Liang ZQ. Cordyceps and its artificial culture. J Guizhou Agric Coll 1994;13:1-21. 6. Pen X. The cultivation of Cordyceps militaris fruitbody on artificial media and the determination of SOD activity. Acta Edulis Fungi 1995;2:25-8. 7. Sung JM. The insects-born fungus of Korea in color. Seoul: Kyohak Publishing Co., Ltd.; 1996. 8. Shrestha B, Han SK, Lee WH, Choi SK, Lee JO, Sung JM. Distribution and in vitro fruiting of Cordyceps militaris in Korea. Mycobiology 2005;33:178-81. 9. Li CR, Nam SH, Geng DG, Fan MZ, Li ZZ. Artificial culture of seventeen Cordyceps spp. Mycosystema 2006;25:639-45. 10. Lee JO, Shrestha B, Kim TW, Sung GH, Sung JM. Stable formation of fruiting body in Cordyceps bassiana. Mycobiology 2007;35:230-4. 285 11. Lee JO, Shrestha B, Sung GH, Han SK, Kim TW, Sung JM. Cultural characteristics and fruiting body production in Cordyceps bassiana. Mycobiology 2010;38:118-21. 12. Coppin E, Debuchy R, Arnaise S, Picard M. Mating types and sexual development in filamentous ascomycetes. Microbiol Mol Biol Rev 1997;61:411-28. 13. Kothe E. Mating-type genes for basidiomycete strain improvement in mushroom farming. Appl Microbiol Biotechnol 2001;56:602-12. 14. Pöggeler S. Mating-type genes for classical strain improvements of ascomycetes. Appl Microbiol Biotechnol 2001;56: 589-601. 15. White JF Jr, Owens JR. Stromal development and mating system of Balansia epichloë, a leaf-colonizing endophyte of warmseason grasses. Appl Environ Microbiol 1992;58:513-9. 16. Yokoyama E, Yamagishi K, Hara A. Structures of the matingtype loci of Cordyceps takaomontana. Appl Environ Microbiol 2003;69:5019-22. 17. Shrestha B, Kim HK, Sung GH, Spatafora JW, Sung JM. Bipolar heterothallism, a principal mating system of Cordyceps militaris in vitro. Biotechnol Bioprocess Engin 2004;9:440-6. 18. Yokoyama E, Yamagishi K, Hara A. Development of a PCRbased mating-type assay for Clavicipitaceae. FEMS Microbiol Lett 2004;237:205-12. 19. Yokoyama E, Yamagishi K, Hara A. Heterothallism in Cordyceps takaomontana. FEMS Microbiol Lett 2005;250:145-50. 20. Yokoyama E, Arakawa M, Yamagishi K, Hara A. Phylogenetic and structural analyses of the mating-type loci in Clavicipitaceae. FEMS Microbiol Lett 2006;264:182-91. 21. Wen TC, Li MF, Kang JC, Lei BX. A molecular genetic study on the fruiting-body formation of Cordyceps militaris. KSM Newsl 2009;21(2):76-95. 22. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 2000;31:21-32. 23. Lee JO, Shrestha B, Sung GH, Han SK, Sung JM. Successful development of Cordyceps bassiana stromata from Beauveria bassiana. Mycobiology 2010;38:13-6. 24. Sung GH, Spatafora JW. Cordyceps cardinalis sp. nov., a new species of Cordyceps with an east Asian-eastern North American distribution. Mycologia 2004;96:658-66. 25. Kim SY, Shrestha B, Sung GH, Han SK, Sung JM. Optimum conditions for artificial fruiting body formation of Cordyceps cardinalis. Mycobiology 2010;38:133-6. 26. Shrestha B, Park YJ, Han SK, Choi SK, Sung JM. Instability in in vitro fruiting of Cordyceps militaris. J Mushroom Sci Prod 2004;2:140-4. 27. Shrestha B, Nam IS, Kim HK, Sung JM. Effect of sectors of isolates on fruiting of Cordyceps militaris. KSM Newsl 2002;14:98. 28. 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. 29. Sung JM, Park YJ, Lee JO, Han SK, Lee WH, Choi SK, Shrestha B. Effect of preservation periods and subcultures on fruiting body formation of Cordyceps militaris in vitro. Mycobiology 2006;34:196-9. 30. Sung JM, Park YJ, Lee JO, Han SK, Lee WH, Choi SK, Shrestha B. Selection of superior strains of Cordyceps militaris with enhanced fruiting body productivity. Mycobiology 2006;34:131-7.