Quality Control of Cordyceps sinensis Teleomorph, Anamorph, and Its Products

The fungus is endemic mostly to the alpine shrub-meadow zone of high mountains (3000– 5000 m altitude) or highlands of the south western of China (the Tibet Autonomous Region and the Qinghai, Sichuan, Yunnan and Gansu provinces) and to some countries of the Himalayan south slope (Nepal, Bhutan, and North East India) (Fig. 2). It grows parasitically on Lepidoptera larvae, particularly those belonging to the genus Hepialus (bat moths), which are found in cool weather conditions (Kinjo & Zang, 2001; Pegler et al., 1994). After host larval infection with either meiotic or mitotic spores, the fungus multiplies in the host by yeast-like budding, eventually killing the host. The fungus then grows in the form of stroma

thread-like hyphae.Following overwintering, the fungus ruptures the host body, forming a sexual sporulating structure (a perithecial stroma) that is connected to the dead larva below ground and grows upward to emerge above the soil surface.It is this stroma, either with or without the host larva, that is traditionally used for medicinal purposes (Buenz et al., 2005).Fig. 2. Distribution of Cordyceps sinensis (Buenz et al., 2005)

Medicinal effects of C. sinensis
In Asia, C. sinensis is considered a valuable traditional herb that has several medicinal effects.It has been reported that C. sinensis nourishes the lungs and kidneys (Pegler et al., 1994), strengthens the immune system (Kuo et al., 1996), revitalizes the body after serious illness, acts as an aphrodisiac (Bhattarai, 1993), is a free-radical scavenger (Yamaguchi et al., 2000), exerts an anti-tumour effect (Chen et al., 1997b), and possesses the ability to alter apoptotic homeostasis (Buenz et al., 2005).

Morphology of the C. sinensis teleomorph
C. sinensis has a sexual stage (teleomorph) and an asexual stage (anamorph).It is generally seen in the sexual stage with a stalked fruit body.Recently, an inadequate teleomorph supply has raised its price to around 30,000-50,000 USD per kilogram (depending on size and quality).This high price results in many fake products on the market, e.g.other Cordyceps spp.(e.g. C. cicadicola, C. liangshanensis, C. hawkesii, C. militaris, C. ophioglossoides, C. shanxiensis, C. sobolifera, etc.) or some plant root and stem products; therefore, accurate C. sinensis teleomorph identification is very important (Chen et al., 2009).

C. sinensis anamorph
Due to the serious decline of teleomorph resources, cultured C. sinensis anamorph mycelia have been used as a substitute.However, anamorph culture did not produce a uniform mycelium, and several species have subsequently been reported as possible C. sinensis anamorphs, including Stachybotrys sp.(Kobayasi, 1982), Paecilomyces sinensis (Chen et al., 1984), Scytalidium hepiali (Li & Sun, 1988), Tolypocladium sinensis (Li, 1988), Chrysosporium sinensis (Liang, 1991a), Hirsutella sinensis (Liu et al., 1989), Synnematium sinensis (Yin & Shen, 1990), Cephalosporium sp.(Shen, 1983), Paecilomyces hepiali, Mortierella hepiali, and Scytalidium hepiali (Chen et al., 2009).Many mycelia products made by these species are marketed as 'C.sinensis mycelia products', which confuses consumers.H. sinensis has been confirmed as a C. sinensis anamorph based on microcyclic conidiation (short life cycle) observation (Liang, 1991b;Liu et al., 2001).However, the short life cycle is difficult to observe, and its use is very limited for other species.Similar problems have been encountered with regard to the morphological identification of the desiccated and powdered mycelia products.Thus, development of molecular biological techniques is important in order to extract and analyse fungal DNA even from dead fungi and to infer simple, rapid, and reliable anamorph-teleomorph connections (Egger & Sigler, 1992).

C. sinensis identification using molecular biology methods
Although C. sinensis has different phenotypes during its life cycle, its genotype during the different stages is unique.Genetic analyses of C. sinensis examined the patterns of genetic variability exhibited by randomly amplified polymorphic DNA (RAPD) markers and nuclear ribosomal DNA (nrDNA) sequence diversity.However, most DNA-based studies have examined genetic differentiation at the population rather than the species level (Buenz et al., 2005).RAPD-polymerase chain reaction (RAPD-PCR) techniques have been used to study the relationship between H. sinensis and C. sinensis (Chen et al., 1999;Li et al., 2000) and those among the geographical populations of C. sinensis (Chen et al., 1999;Chen et al., 1997a).Twenty-nine C. sinensis samples were divided into 3 clusters, i.e. the north population (NP), middle population (MP), and south population (SP) and were considered as different subspecies rather than as different species (Chen et al., 1999;Chen et al., 1997a).The unstable RAPD patterns and large time commitment restricted the application of RAPD-PCR.Using phylogenetic trees and probes based on ITS-region nrDNA sequences (ITS 1, 5.8S, and ITS 2 nrDNA sequences), Chen et al. (2001b) designed 2 C. sinensis-specific probes for species-level identification.A PCR single-stranded conformation polymorphism (PCR-SSCP)-based method was developed in Taiwan to identify C. sinensis and its fermented products (Kuo et al., 2006;Kuo et al., 2005).Kinjo and Zang (2001) suggest that the 17 collections of C. sinensis isolates from 11 southwestern localities in China could be divided into 2 subgroups based on their ITS region sequences.Stensrud et al. (2007) analysed ITS-region nrDNA variations among 71 sequences of C. sinensis made available by the EMBL/GenBank databases.These authors suggested that C. sinensis isolates can be divided into 3 sub-species groups; however, the 2 C. sinensisspecific probes (Chen et al., 2001b), as well as the PCR-SSCP-based method (Kuo et al., 2006;Kuo et al., 2005), could only detect group 2 of Kinjo and Zang (2001) and group A of Stensrud et al. (2007).The objective of this study was to develop an innovative and direct method that can detect all 3 subspecies groups of C. sinensis teleomorph, anamorph, and its derivative products.

Fungal specimens and strains
The specimens and strains used in this study are listed in Table 1.The specimens were washed with sterile water and divided into 3 parts: the stroma (fruiting body), upper part of the sclerotium (head, h), and lower part of the sclerotium (tail, t).Some of the smaller sclerotia (body, b) were used for subsequent experiments.The strains were cultured in 250 mL of potato dextrose broth (PDB; DIFCO, Detroit, MI, USA) in 500-mL flasks and agitated at 100 rpm at 14 °C.The mycelia were harvested after 8 weeks and washed with sterile water.All the specimens and mycelia were then lyophilized and stored at -20°C for subsequent analysis.

DNA preparation
DNA was isolated as described by Moncalvo et al. (1995).In brief, the ground sample (60 mg) was transferred to a 1.5-mL microcentrifuge tube containing 600 μL of lysis buffer (50 mM Tris-HCl, 50 mM EDTA, 3% SDS, and 1% 2-mercaptoethanol; pH 7.2).The tube was incubated in a water bath at 65 °C for 1 h, and the aqueous phase was then extracted twice using 600 μL of PCI (phenol:chloroform:isoamyl alcohol = 25:24:1; Sigma Co., St. Louis, MO, USA).After extraction, the aqueous phase was transferred to a new tube and the precipitated DNA was mixed with 0.1 volumes of 3 M sodium acetate and 0.6 volumes of isopropanol.The DNA was pelleted by centrifugation at 15,000 × g for 5 min, washed twice with cold 70% ethanol, and dried for 30 min in a vacuum oven at 37 °C.The DNA was resuspended in 100 μL of TE buffer (10 mM Tris-HCl and 1 mM EDTA; pH 8.0) containing 2 μL of RNase (500 μg/mL; Roche Applied Science Co., Mannheim, Germany) and incubated in a water bath at 37 °C for 1 h.After addition of 100 μL of chloroform, the aqueous phase was transferred directly into a new tube.DNA was precipitated with 0.1 volumes of 3 M sodium acetate and 0.6 volumes of isopropanol and then pelleted by centrifugation at 15,000 × g for 5 min.The DNA was resuspended in 100 μL of sterile water and stored at -20°C.

DNA sequencing and analyses
The PCR-amplified products were sequenced by the Mission Biotech Company (Taipei, Taiwan) (Chen & Hseu, 2002).The sequences were analyzed by an autosequencer (Applied Biosystems) using a Terminator Cycler Sequencing Ready Reaction Kit (Applied Biosystems).The sequences were imported into the BioEdit Sequence Alignment Editor version 7.0.9.0 (Hall, 1999) and aligned using the CLUSTAL W (Thompson et al., 1994) option.

Restriction fragment length polymorphisms (RFLPs)
For RFLP analysis, 20 μL of the PCR products was digested with 1 μL each of the restriction enzyme CfoI (GCG↓C; 10 U/μl; Roche Applied Science Co.) and RsaI (GT↓AC; 10 U/μL; Roche Applied Science Co.), 5 μL of 10× SuRE/Cut buffer L (Roche Applied Science Co.), and distilled water to a final volume of 50 μL.Tubes were incubated at 37 °C for at least 4 h before separation on a 2.0% agarose gel and visualization by staining in ethidium bromide and UV transillumination (Chen & Hseu, 1999) 1).This result suggested that the stroma and sclerotium from each isolate originated from the same species.

Phylogenetic analysis
The phylogenetic tree based on the ITS region sequences is illustrated in Fig. 4. It was constructed using the sequencing data collected in this study as well as from GenBank.The C. memorabilis strain ATCC 36743 was set as the out-group.Kinjo and Zang (2001), and the bootstrap level was 1000.
Group C, which we discovered earlier (Chen et al., 2004), included the following: (1) Tibet isolate Cs1014C(b) and ( 2) Sichuan isolate W1023(f).The bootstrap level of Group C was 1000.Although this group was remote from C. sinensis Groups A and B, the 2 isolates were identified to be C. sinensis based on morphological observations.Group D included the following isolates/strains: GenBank Accession Nos.AF122030 (strain BCRC 36421), AF291749 (strain MPNU 8002), and AB067720 (isolate SHANGHAI).The bootstrap level of Group D was 1000.Although these isolates were scientifically named C.  (Hodge et al., 1996).Another possibility was that the 3 strains might be contaminants or associated fungi of C. sinensis because many contaminant anamorphic fungi were associated with Cordyceps species (Kinjo & Zang, 2001).Group E included only 1 isolate that had the GenBank Accession No. AB067719 (isolate SANMEI).It was not accurately represented in the GenBank format by Kinjo.The isolate was not considered a C. sinensis anamorph strain because of the large identity difference in its 18S nrDNA sequences and those of C. sinensis specimens (discussed below).According to the above-mentioned findings, Groups A, B, and C were considered the real C. sinensis.Group A included C. sinensis isolates from 5 sources (Tibet, Sichuan, Qinghai, Yunnan, and Gansu); Group B, 3 sources (Tibet, Sichuan, and Qinghai); and Group C, 2 sources (Tibet and Sichuan).This indicated that the intra-group isolates from different geographic regions were identical subspecies.However, the inter-group isolates were different subspecies.The identity between Groups A and B was 86.0%-89.4% as compared to 63.2%-66.0%between Groups A and C and 56.6%-57.8% between Groups B and C.Only Group A members could be detected using the 2 probes developed by Chen et al. (2001a).
The ITS region and NS5/NS6 region sequences of C. sinensis in the present study and GenBank (Table 1) were aligned and analysed.The identity between each ITS region sequence was 56.6%-100%; however, the NS5/NS6 region sequences of the isolates were almost completely identical.Therefore, the NS5/NS6 region sequence of C. sinensis was compared to those of other Cordyceps spp. to determine the diversity therein.Obviously, C. sinensis had 2 restriction sites-CfoI and RsaI (the signature sequence)-that could be used to differentiate C. sinensis from other Cordyceps spp.(Fig. 5).To ensure specificity of the signature sequence, the GenBank database was searched using the search phrase 'Cordyceps 18S rRNA gene'.Simultaneously, the NS5/NS6 region sequence was uploaded to GenBank and the database was searched for the sequences that were mostly closely related using the Basic Local Alignment Search Tool (BLAST) program (Altschul et al., 1990).None of the sequences contained both restriction sites (data not shown); thus, C. sinensis could be definitively identified based on the 2 restriction sites.
The PCR-RFLP method based on the signature sequence was developed and used to characterize C. sinensis fermented products in Taiwan (Table 3).a The marketing sample content descriptions were provided by the respective suppliers.
b Full company names related to the marketing samples are not shown here.
Table 3. Cordyceps sinensis mycelium fermented products collected in the present study Among the 12 fermented products, only AV and L matched the signature sequence and were, therefore, considered genuine C. sinensis mycelium products (Fig. 6).Another 8 products-A, B, C, G1, Gen, NT, PH, and 4B, none of which were digested by CfoI and RsaI-were considered fake products.The product DP, whose content was labeled C. sinensis and Ganoderma lucidum mycelia, had 2 corresponding polymorphism patterns; however, the product P, whose content was labeled only as C. sinensis mycelium, must not exclusively comprise pure C. sinensis mycelium.

Discussion
ITS-region nrDNA, which consists of the 2 variable non-coding regions ITS 1 and ITS 2, was more broadly used in phylogenetic analysis than 18S nrDNA.However, the 2 C. sinensis- specific probes (Chen et al., 2001b) and the PCR-SSCP method (Kuo et al., 2006;Kuo et al., 2005) based on the ITS region nrDNA sequence could detect only group A in Fig. 4. Thus, the 18S nrDNA was used in the present study instead of the ITS region to develop a method by which to differentiate C. sinensis.
The signature sequence based on the CfoI and RsaI restriction sites of the NS5/NS6 region was an innovative and species-level genetic marker of C. sinensis.It could be broadly used to determine C. sinensis teleomorph, anamorph, and the identification and differentiation of derivative products.In addition, the C. sinensis was further divided into 3 intra-species groups, based on the ITS-region sequences.These molecular systematic indicators could serve as the foundation for further research and applications.Many teleomorph and anamorph fermented products in the market do not comprise C. sinensis (i.e.fake products), and some of them are not made exclusively of pure C. sinensis (Fig. 6).They might be made of or mixed with other Cordyceps spp.-C.sinensis-related anamorph strains, plant powders, or other materials.Although many of these ingredients were reported as having various biological functions, the fake or impure products, labelled as 'C.sinensis mycelium products', might cause serious problems with regards to food safety.Thus, the signature sequence will be a powerful tool in assaying C. sinensis fermented products and performing quality control measures.
On the basis of morphological observation and signature sequence confirmation, strain RS3 was identified as C. sinensis anamorph, i.e. H. sinensis.According to the ITS-region nrDNA sequence, it belongs to group A of Fig. 4.However, the genomic statuses of the anamorph strains of groups B and C remain unclear.Further work should be undertaken to collect live C. sinensis specimens for group B and group C anamorph strain isolation and for research into the morphological characteristics and medicinal effects of each group isolate.

Conclusion
In the present study, we successfully developed a molecular method that can detect all 3 sub-species groups of C. sinensis.This innovative method can be applied to C. sinensis teleomorph and anamorph identification and can be used to improve quality control.

Acknowledgment
This study was supported by a grant (Grant No. DOH-91-TD-1163) from the Department of Health, Executive Yuan, Taiwan, Republic of China.

Fig. 4 .
Fig. 4. Phylogenetic tree resulting from the Neighbour-Joining Method (NJ) of the nrDNA ITS1, 5.8S, and ITS2 region sequences of Cordyceps sinensis and other related fungi.NJ bootstrap percentage values are shown at each branch