Improvement of Fruiting Body Production in Cordyceps Militaris by Molecular Assessment

Home , Ascospore, Cordyceps militaris, Cordycipitaceae

Arch Microbiol (2013) 195:579–585 DOI 10.1007/s00203-013-0904-8

SHORT COMMUNICATION

Improvement of fruiting body production in Cordyceps militaris by molecular assessment

Guozhen Zhang · Yue Liang

Received: 23 January 2013 / Revised: 16 May 2013 / Accepted: 19 May 2013 / Published online: 12 June 2013 © Springer-Verlag Berlin Heidelberg 2013

Abstract Cordyceps militaris is a heterothallic ascomy- Introduction cetous fungus that has been cultivated as a medicinal mush- room. This study was conducted to improve fruiting body Mushrooms have recently drawn considerable attention as production by PCR assessment. Based on single-ascospore attractive and abundant sources of useful natural products isolates selected from wild and cultivated populations, the (Smith et al. 2002). Among others, Cordyceps species have conserved sequences of α-BOX in MAT1-1 and HMG-BOX traditionally been used not only as a folk tonic food and in MAT1-2 were used as markers for the detection of mat- nutraceutical, but also as a restorative drug for longevity ing types by PCR. PCR results indicated that the ratio of and vitality (Paterson and Russell 2008). Mushrooms of mating types is consistent with a theoretical ratio of 1:1 this genus have also been used for dietary fiber, health sup- (MAT1-1:MAT1-2) in wild (66:70) and cultivated (71:60) plements, and maintenance as well as for prevention and populations. Cross-mating between the opposite mating treatment for human diseases for centuries in East Asian types produced over fivefold more well-developed fruiting countries (Paterson and Russell 2008). bodies than self- or cross-mating between strains within the Cordyceps militaris belongs to Cordycipitaceae, Hypo- same mating type. This study may serve as a valuable refer- creales, Sordariomycetes, and Ascomycota (Sung et al. ence for artificial culturing ofC. militaris and other edible 2007; Zare and Gams 2001). This fungus is entomopatho and medicinal mushrooms and may be useful to develop an genic, which invades and proliferates within larvae and efficient process for the selection, domestication, and man- pupae of various insects species (Clarkson and Charn- agement of strains for industrial-scale production. ley 1996). The medicinal properties and pharmacological effects of C. militaris are similar to other Cordyceps spe- Keywords Cordyceps militaris · Fruiting body · cies. Pharmacological studies of C. militaris began with Heterothallism · Mating type · Stroma the purification of cordycepin (3′-deoxyadenosine), a com- pound with a potential antibacterial, antifungal, antiviral, and antineoplastic activities (Cunningham et al. 1950). Since then, numerous other pharmacological components were isolated from C. militaris, and their constitution and biological activity were studied (Paterson and Russell Communicated by Olaf Kniemeyer. 2008; Zhou et al. 2009). The herbal fruiting bodies of wild C. militaris are not G. Zhang readily and abundantly available in nature because of Department of Plant Pathology, China Agricultural University, Beijing 100193, People’s Republic of China host specificity and the requirement of relatively strict e-mail: [email protected] growth environments (Chen 1986). With dramatically increased consumption of complementary medicine (Pat- * Y. Liang ( ) erson and Russell 2008), harvesting adequate quantities of School of Life Sciences, Chongqing University, Chongqing 400045, People’s Republic of China wild C. militaris fruiting bodies for medicinal purposes is e-mail: [email protected] impractical. In artificial cultivation, extremely optimized

1 3 580 Arch Microbiol (2013) 195:579–585 conditions were required (Chen et al. 2002). Moreover, DNA extraction the selection, domestication, and management of C. militaris strains affect industrialized production of this mush- A culture plug bearing actively growing mycelia was trans- room (Lin et al. 2006). Thus, single or a small number of ferred onto freshly-prepared modified PDA. When the strains were generally maintained in a cultivation factory growth of the fungus reached the edge of the plate, myce- or farm. Indeed, C. militaris is a bipolar heterothallic fun- lia were scraped from the surface with a sterilized tooth- gus (Shrestha et al. 2004), i.e., it has two different mating pick and transferred into a 1.5-ml microcentrifuge tube. type (MAT) genes to regulate sexual reproduction (Turgeon Samples were immediately frozen in liquid nitrogen and 1998; Turgeon and Yoder 2000; Yokoyama et al. 2004). In stored at 80 °C until used. For DNA extraction, samples − other fungal species, the alpha box (α-BOX) of MAT1-1 were finely ground in liquid nitrogen and then mixed with and the high mobility group box (HMG-BOX) of MAT1-2 500 μl buffer (50 mM Tris-HCl, 150 mM NaCl, 100 mM are highly conserved (Arie et al. 1997). As a result, the two EDTA pH 7.5, 20 μg/ml RNase A) and 50 μl 20 % SDS. conserved genomic regions have been used to character- The mixture was incubated at 37 °C and inverted every ize MAT genes in numerous heterothallic phytopathogenic 10 min for 1 h. The mixture was mixed with 75 μl 5 M fungi such as Cryphonectria parasitica (McGuire et al. NaCl and 65 μl CTAB/NaCl buffer [10 % CTAB, 0.7 M 2001), Gibberella zeae and G. fujikuroi (Yun et al. 2000), NaCl] and subsequently incubated at 65 °C for 20 min. Mycosphaerella graminicola (Waalwijk et al. 2002), Pyr- After cooling to room temperature, an equal volume of enopeziza brassicae (Foster et al. 1999), and Verticillium phenol/chloroform/isoamyl alcohol (v/v/v; 25:24:1) was dahliae (Usami et al. 2008). In at least one Cordyceps spe- added, and the solution was centrifuged at 12,000g, 4 °C cies, C. takaomontana, the structure of mating type genes for 10 min. The supernatant (~600 μl) was transferred to a was determined (Yokoyama et al. 2003). new sterile 1.5-ml microcentrifuge tube, and an equal vol- The literature describing the production of fruiting bod- ume of chloroform/isoamyl alcohol (v/v; 24:1) was added. ies in C. militaris is limited. To the best of our knowledge, The solution was centrifuged at 12,000g, 4 °C for 10 min. the regulation of the mating system of C. militaris remains The water phase (~400 μl) was collected and mixed with largely unknown. In the present study, we used PCR tech- an equal volume of isopropanol. After precipitation at room nique to assess the mating types of a collection of C. mili- temperature for 30 min, the solution was centrifuged at taris isolates and subsequently demonstrated that mating 12,000g, 4 °C for 15 min. After discarding the supernatant, between isolates with alternative mating types could the resulting pellet was washed twice with 70 % ethanol improve the production of fruiting bodies. and air-dried. The pelleted DNA was dissolved in 40 μl of TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA) and stored at 20 °C. − Materials and methods Polymerase chain reaction (PCR) assay Fungus and cultural conditions The highly conserved sequences of α-BOX in MAT1-1 and Single ascospores were randomly isolated from the dis- HMG-BOX in MAT1-2 have been used as molecular mark- charging fruiting bodies of wild and cultivated C. militaris ers for the detection of mating types in clavicipitaceous and labeled as “cm” followed by an Arabic number. Briefly, fungi (Yokoyama et al. 2004). Therefore, two sets of prim- the freshly collected fruiting body was attached with plastic ers specific for C. militaris were designed and used to deter- tape to the inner side of a sterilized Petri dish (9 cm diam- mine mating types. MAT1-1 (accession no. AB124614) was eter), which was then placed over a 1.6 % (w/v) agar plate, identified using CmM1 (forward, 5′-CCATCTCATCGCG- so that the fruiting body hung above the agar. The moisture GATG-3′; reverse 5′-GGGCAAAACGACCATTG-3′) accumulated in the Petri dish stimulated the discharge of based on the partial α-BOX sequence in the NCBI data- ascospores from the fruiting body. After 2–3 h of incuba- base. PCR was expected to generate a 233-bp product tion at room temperature under continuous light, a tiny agar using the following reaction conditions: 94 °C for 3 min; piece with a single ascospore was excised using a dissect- 35 cycles of 94 °C for 30 s, 53 °C for 30 s, 72 °C for 30 s; ing needle with the aid of a stereomicroscope (60 mag- and a final extension of 72 °C for 10 min. MAT1-2 (acces- × nification) and transferred to a new Petri dish containing sion no. AB124626) was assayed using CmM2 (forward, modified potato dextrose agar [PDA, 20 % (w/v) potato, 5′-ACATACGCTTGTCAAGA-3′; reverse 5′-AGGAGA- 2 % dextrose, 1.6 % agar, 0.3 % peptone, 0.2 % KH2PO4, GCCTTCTTGAT-3′), which was expected to amplify a 0.1 % MgSO 7H O, 0.0025 % vitamin B, pH 6.5–7.0], 191-bp fragment of the HMG-BOX using the following 4· 2 which was incubated at 23 °C under continuous light reaction conditions: 94 °C for 3 min; 35 cycles of 94 °C for (2,000–2,500 lux) for 2 weeks. 30 s, 48 °C for 30 s, 72 °C for 30 s; and a final extension

1 3 Arch Microbiol (2013) 195:579–585 581 of 72 °C for 10 min. The PCR products were visualized by Statistical analysis 1.8 % agarose gel electrophoresis and purified with a gel extraction kit (SBS Genetech, Beijing, China). The puri- For each population, the ratio of mating types was tested fied amplicons were ligated into the TA vector pMD18-T with a Chi-square goodness of fit test using SAS software (TaKaRa, Dalian, China) at 16 °C overnight according to (version 9.1). The fresh and dry weights of fruiting bodies the manufacturer’s instructions. Competent DH5α Escheri- were analyzed using the ANOVA (P < 0.05) in SAS. chia coli cells (200 μl) were transformed with 7 μl of the ligation mix and plated on Luria–Bertani agar containing ampicillin (50 μg/ml) for overnight growth at 37 °C. Col- Results ony PCR was performed using randomly selected bacterial colonies, and plasmid DNA from PCR-verified colonies Morphological features of stroma and perithecia was isolated and sequenced (Sangon Biotech, Shanghai, (fruiting bodies) China). The wild C. militaris stromata are orange, stipitate, and Determination and ratio assessment of mating types clavate with a smooth shaft and sparse distribution of semi- embedded perithecia appearing on the hemisphere (Fig. 1a, A binomial test for goodness of fit was conducted to deter- b). While the cultivated C. militaris fruiting body was the mine whether the ratio of mating types (MAT1-1:MAT1-2) same color and shape, the perithecia were more widely within population samples did not deviate from a theoreti- distributed, and most parts of the tapered perithecia were cal ratio of 1:1. Two independent sample populations (a exposed (Fig. 1c, d). The perithecium was flask-shaped and total of over 100 pooled ascospores in each population) thin-walled with a thick ostiole where the thread-like asci for wild and cultivated C. militaris, composed of pooled were arranged parallel to each other (Fig. 1e). Asci were ascospores isolated from three individual fruiting bodies readily released from the perithecium either from the osti- (over 30 ascospores isolated from each fruiting body), were ole by turgor pressure induced with water, as is observed in assessed for ratio of mating types. nature, or from breaks in the wall resulting from excessive pressure (e.g., being crushed) (Fig. 1e). Each ascus con- Fruiting body assessment sisted of eight parallel and multiseptate ascospores (Fig. 1f) with a helmet-like ascal cap observed on top (Fig. 1g). The Two representative single-ascospore strains (cm 1 and cm 7 ascal walls degraded and the thread-like and disarticulat- for MAT1-1; cm 9 and cm 19 for MAT1-2) for each mating ascospores dispersed (Fig. 1f). Stromata and perithecia ing type were selected to examine the fruiting process of were morphologically distinct between wild and cultivated C. militaris. Plugs with actively growing mycelia were C. militaris, especially in the distribution and morphology excised from modified PDA and used to inoculate 30 ml of perithecia. modified potato dextrose broth [PDB, 20 % (w/v) potato,

2 % dextrose, 0.3 % peptone, 0.2 % KH2PO4, 0.1 % PCR assay and ratio of mating types MgSO 7H O, 0.0025 % vitamin B, pH 6.5–7.0] in a 250- 4· 2 ml Erlenmeyer flask. Cultures were agitated on a rotary DNA extracted from different single-ascospore strains shaker (70 rpm) at 23 °C for 3 days. Sterile rice medium was used as templates for PCR amplification, which gen- (10 g rice mixed with 10 ml modified PDB) in a 340-ml erated the expected 233-bp amplicon for MAT1-1 and glass bottle was inoculated with 5 ml liquid culture of each 191-bp amplicon for MAT1-2 (Fig. 2). PCR products strain for self- and/or cross-mating with the same/oppo- were sequenced (data not shown) and determined to site mating type. Cultures were incubated at 23 °C in the be identical to the corresponding MAT1-1 (AB124614) dark until mycelia completely suffused the rice medium or MAT1-2 (AB124626) sequences. The ratio of mat- (2 days); thereafter, cultures were exposed to cycling ing types of wild (136 ascospores in total) and cultivated between light (for 9 h) and dark (for 15 h) until fruiting (131 ascospores in total) population was assessed by PCR. bodies were harvested 45 days later. Three replicates were The null hypothesis (no deviation from a theoretical seg- performed for each treatment (self- and/or cross-mating regation ratio of 1:1, MAT1-1:MAT1-2) in either wild with the same/opposite mating types). The development of (MAT1-1:MAT1-2 66:70, χ2 0.1176, P 0.7316) = = = the fruiting body/stroma was observed (A “ ” indicated or cultivated (MAT1-1:MAT1-2 71:60, χ2 0.9273, + = = well-developed stromata while a “ ” indicated under- P 0.3365) population samples was not rejected using − = developed or undeveloped stromata). The fresh and dry Chi-square analysis, indicating the frequencies of the two (dried at 55 °C overnight) weights of fruiting bodies were mating types occurred equally in wild and cultivated popu- recorded for statistical analysis. lations in our study.

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Fig. 1 Fruiting bodies and perithecia of Cordyceps militaris. a wild fruiting body; b perithecia on wild fruiting body under a stereomicroscope ( 12 magnification);c cultivated× fruiting body; d perithecia on cultivated fruiting body under a stereomicroscope ( 12 magnification); e perithecia× and crushed perithecia with asci; f single ascus indicated by arrow; g helmet-like ascal cap indicated by arrow

stromata. Moreover, some strains (e.g., strain cm1) formed stromata but not well developed (defined as stromata without ascospores) or did not produce stromata during self- mating (Fig. 3; Table 1).

Discussion

There are very few studies focused on the correlation Fig. 2 PCR assay for the determination of C. militaris mating types. between mating types and the improvement of fruiting body Representative strains (cm1, cm7, cm9, and cm19) were assayed production. Mating type genes have been reviewed for vari- using specific primer sets for each mating type, and each representative strain only can amplify either 233 bp for MAT1-1 or 191 bp for ous homothallic and heterothallic species of filamentous MAT1-2; (M) molecular ladder (100–600 bp) ascomycetes (Turgeon and Yoder 2000). The molecular characterization of the mating type genes from C. takaomontana provides the first description of structures of the Assessment of stroma production mating type loci from a heterothallic Cordyceps species (Yokoyama et al. 2003). Similar to the MAT idiomorphs Representative strains of MAT1-1 (strain no. cm1 and cm7) of other members of Clavicipitaceae, each MAT locus of and MAT1-2 (strain no. cm9 and cm19) mating types deter- C. militaris harbors a single mating type gene, encoding an mined by PCR were selected for fruiting body assessment α-box or HMG-domain protein (Yokoyama et al. 2004). (Fig. 2). The morphological characteristics of fruiting bod- The differences in morphological features of a fruiting ies/stromata were observed and recorded (Fig. 3). Fresh body/stroma under natural and artificial conditions were and dry weights of fruiting bodies/stromata from each treat- presented in this study and complement the results provided ment were measured and statistically analyzed (Table 1). in a previous study in which only C. militaris grown on arti- Results indicated that cross-mating with the opposite mat- ficial medium was studied (Yahagi et al. 2004). The charac- ing types produced more well-developed (characterized terization of perithecia observed from the present study was as fully developed with straight, clavate stromata) fruiting consistent with the phylogenetic classification of Cordyceps bodies; however, cross-mating with the same mating type (Sung et al. 2007). Additionally, the observed ascospore produced fewer stromata, which were undeveloped (i.e., arrangement, size, and behavior at maturity were similar to the medium was completely covered by mycelia, and fewer those previously described (Sung et al. 2007). Furthermore, stromatal initials were formed without the production of the distribution and morphological differences (e.g., perithe- perithecia and ascospores), while self-mating produced cia) between wild and cultivated C. militaris may have been under-developed (i.e., anamorphotic stromata without the caused by differences in environmental and nutritional con- formation of peritehica and ascospores) and immature ditions during growth (Kues and Liu 2000).

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Fig. 3 Assessment and development of C. militaris fruiting bodies. Representative strains (cm1 and cm7 for MAT1-1 and cm9 and cm19 for MAT1-2) underwent self- and/or cross- mating with the same and opposite mating types. The resulting data are shown in Table 1

Table 1 Yield of fruiting bodies/stromata by self- and/or cross-mating with the same and opposite mating types Treatment Mating type Fresh weight (g)a Dry weight (g)b Developmentc cm1 cm9 1 2 12.86 0.77 A 2.58 0.21 A × × ± ± + cm1 cm19 1 2 12.62 1.50 A 2.56 0.25 A × × ± ± + cm7 cm9 1 2 13.74 1.43 A 2.56 0.23 A × × ± ± + cm7 cm19 1 2 15.49 0.43 A 2.68 0.04 A × × ± ± + cm1 cm7 1 1 0.00 0.00 C 0.00 0.00 C × × ± ± − cm9 cm19 2 2 0.32 0.32 C 0.05 0.05 C × × ± ± − cm1 1 1.18 1.18 C 0.25 0.25 C ± ± − cm7 1 0.80 0.42 C 0.19 0.11 C ± ± − cm9 2 6.64 1.23 B 1.30 0.19 B ± ± − cm19 2 2.35 0.13 C 0.45 0.01 C ± ± − a Data are expressed as the mean standard error of triplicate measurements on a fresh weight basis. Values marked by the same letters within each treatment in each column are ±not significantly different P( < 0.05) b Data are expressed as the mean standard error of triplicate measurements on a dry weight basis. Values marked by the same letters within each treatment in each column are not± significantly different P( < 0.05) c The development of fruiting bodies/stromata. A “ ” indicates well-developed fruiting bodies/stromata while A “ ” indicates under-developed or undeveloped stromata + −

Cordyceps militaris is a bipolar heterothallic ascomy- fungal stocks for industrial-scale production, which may cetous fungus, leading to the theoretical equal frequency lead to less chance for mating through strains with differ- of both mating types in a population (Ji et al. 2004; Arie ent mating types. Therefore, heterothallism in this fungus et al. 1997; Shrestha et al. 2004). In this study, the ratio of is artificially interfered with during industrial production. mating types in wild and cultivated populations was con- Moreover, the ratio of mating types from wild and culti- sistent with the theoretical ratio (MAT1-1:MAT1-2 1:1). vated populations in this study indicated that a large-size = However, the strains or colonies with unknown and/or sin- population of strains is necessary, resulting in more possi- gle genetic background are often isolated and supplied as bility to obtain different mating types (Shrestha et al. 2004;

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Lin et al. 2006). However, some studies proposed that the results presented in this study could effectively facilitate predominance of one mating type may be important as a the screening of strains. Additionally, mating type deter- reason for degeneration of strains due to strains with a par- mination could enhance fruiting body production by cross- ticular mating type competing to survive. Additionally, an mating with strains of the opposite mating type. Finally, unequal prevalence of mating types may have some ecolog- this practical procedure may lead to efficient selection, ical or functional importance (Lee et al. 2010). For exam- domestication, and management of strains to decrease the ple, one mating type (MATα) of Cryptococcus neoformans possibility of degeneration and also provide a theoretical was predominant in nature and in clinical strains because reference for the industrial-scale production of C. militaris of the inherent genetic differences between the two mating and even other Cordyceps fungi. types, which suggested the strains containing MATα were suitable for survival (McClelland et al. 2004). Moreover, Acknowledgments This study was supported by research Grants the prevalent MAT1-1 mating type was recently described from the National Natural Science Foundation of China (No. 30770011), the Fundamental Research Funds for the Central Uni- in an individual progeny population isolated from a single versities (No. CDJRC10290005). We are also grateful to Dr. William strain, which may draw attention to the skewed ratio of Yajima for critically reading this manuscript, Mr. Haiwang Zhang and mating types in a single genetic background (Zheng et al. Xi Wang for technical assistance. 2011). Therefore, the occurrence of a skewed ratio of mating types in wild and cultivated C. militaris for industrial- References scale production should be monitored further. This could help to avoid degeneration of strains and provide homog- Arie T, Christiansen SK, Yoder OC, Turgeon BG (1997) Efficient enous genetic populations for strain screening because dis- cloning of ascomycete mating type genes by PCR amplifica- tortion is a common phenomenon of genetic elements in tion of the conserved MAT HMG Box. Fungal Genet Biol nature and may be affected by several factors (Lyttle 1991). 21(1):118–130 Obviously, the fruiting process of C. militaris is not only Chen S (1986) Knowledge of Cordyceps. Entomol Knowl 3:133–135 Chen Y, Piao R, Jin Y, Yan H (2002) Study on the artificial good qual- associated with the mating type system, but also with fun- ity and high output cultivation technique of Cordyceps militaris. gal characteristics and environmental conditions, similar Edible Fungi China 21(5):20–22 to other fungi (Pöggeler et al. 2006). A previous investiga- Clarkson JM, Charnley AK (1996) New insights into the mechanisms tion also indicated that strains produce under-developed or of fungal pathogenesis in insects. Trends Microbiol 4(5):197–203 Cunningham KG, Manson W, Spring FS, Hutchinson SA (1950) undeveloped stromata by self-mating (Liang et al. 2005). Cordycepin, a metabolic product isolated from cultures of Therefore, we suggest the use of cross-mating with differ- Cordyceps militaris (Linn.) Link. 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