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 of C. 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. mili- taris 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.