The medicinal fungus Cordyceps militaris: Research and development

The medicinal fungus Cordyceps militaris: Research and development

Abstract Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a highly valued medicinal fungus. This entomopath-ogen has a limited distribution, has been overharvested in the wild, and its stromata have not been artificially cultivated. Another entomopathogenic fungus, Cordyceps militaris (commonly known as orange caterpillar fungus), has chemical capacities similar to those of O. sinensis, but unlike O. sinensis, its stromata can be easily cultivated. Consequently, C. militaris is being studied as an alternative to O. sinensis, and the large-scale production of stromata is receiving substantial attention. Significant research has been conducted on the genetic resources, nutritional and environmental.

B. Shrestha
Green Energy Mission/Nepal,
Ghatte Kulo, Anam Nagar,
Kathmandu, P.O. Box 10647, Nepal

W.Zhang
Guangdong Institute of Microbiology,
Guangzhou 510070, China

Y. Zhang
School of Life Sciences, Shanxi University,
Taiyuan 030006, China

X.Liu (*)
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences,
No.3, 1st West Beichen Road, Chaoyang District,
Beijing 100101, China e-mail: liuxz@im.ac.cn
Present Address:

B. Shrestha
Mushroom Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration,
Suwon 441-707, Korea

requirements, mating behavior, and biochemical and pharmacological properties of C. militaris. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biology. The current paper reviews the biological aspects of the fungus including host range, mating system, cytology and genetics, insect- and non-insect nutritional requirements, environmental influence on stroma development, and commercial development.
Keywords Cultivation. Entomopathogenic fungus. Mating system. Pharmaceutical application

Historical review
Medicinal fungi have long been an important part of human culture and civilization, and species in the genus Cordyceps are especially valued (McKenna et al. 2002). Most Cordyceps species s.l. are parasites of insects or other arthropods (Kobayasi 1941, 1982). Ophiocordyceps sinensis (Berk.) G.H. Sung et al. (syn. Cordyceps sinensis (Berk.) Sacc.), for example, is a parasite of caterpillars (Wang and Yao 2011) and is naturally distributed in the Tibetan Plateau of China and surrounding high-altitude grasslands of Nepal, Bhutan, and India (Shrestha et al. 2010; Zhang et al. 2012b). As a highly valued medicinal fungus, O. sinensis has a long history of being collected and traded (Jones 1997; Halpern 1999). Recent studies have shown that the natural populations of O. sinensis are decreasing because of overcollection (Li et al. 2006c; Stone 2008; Zhang et al. 2012b). Because of the scarcity and high price of wild O. sinensis specimens, many counterfeit materials including cultured products are sold in the market, resulting in the

Many Cordyceps species are morphologically or otherwise similar to C. militaris, and these include C. cardinalis G.H. Sung & Spatafora, C. kyusyuensis A. Kawam., C. pseudomilitaris Hywel-Jones & Sivichai, C. rosea Kobayasi & Shimizu, C. roseostromata Kobayasi & Shimizu, C. washingtonensis Mains, and others (Sung and Spatafora 2004; Sung et al. 2007; Wang et al. 2008). Although distinguishing C. kyusyuensis from C. militaris is difficult based on morphology and cultural characteristics (unpublished work), the larvae parasitized by C. kyusyuensis tend to be larger and located deeper in soil than larvae parasitized by C. militaris (Kawamura 1955; Sung 1996; see Table 1). C. kyusyuensis, originally described from Japan on larvae of the lepidopteran Clanis bilineata (family Sphingidae) (Kawamura 1955), has been reported only from China (Kobayasi 1981; Guo and Li 2000) and Korea (Sung 1996), besides Japan. According to Wang et al. (2008), C. kyusyuensis is not a different species, rather a synonym of C. militaris.

Mating system

Only a few cytological studies have been conducted on C. militaris. Earlier studies showed that C. militaris has only two chromosomes (Varitchak 1927; Jenkins 1934), while a recent study reported seven chromosomes (Wang et al. 2010c). Despite the scarcity of cytological studies, the mating system of C. militaris has been studied in many instances at both cultural and molecular levels. Inoculations of two single-ascospore strains together or on opposite sides on a fruiting medium plate are the two major methods for genetic analysis of mating system in ascomycetous fungi, including Cordyceps spp. (Harris 2001). Gao et al. (2000b) reported that single-ascospore strains of C. militaris were unable to produce stromata when growing alone on media. Later, Sung and Shrestha (2002) and Shrestha et al. (2004a) showed that single-ascospore strains of C. militaris produced perithecial stromata when inoculated in combination with a compatible isolate, but produced only deformed stromata without perithecia when inoculated singly. Further, it was shown that primordia developed in the meeting line between two opposite mating-type strains that were inoculated on opposite sides of an agar plate (Shrestha et al. 2004a). These results show that C. militaris is a bipolar heterothallic fungus. The mating type character was found to be stable for as many as 10 generations of subcultures of the original strains (Shrestha et al. 2004a; Sung et al. 2006a). Each single ascospore strain was found to be hermaphroditic but self-incompatible (Shrestha et al. 2004a). In agreement with these studies, Liang et al. (2005) observed that, when growing alone, most of the single-ascospore strains could not produce well-developed stromata or produced only abnormal ones. Gao

and Zheng et al. (2011a) also recently confirmed bipolar heterothallism in C. militaris. A molecular study has demonstrated that C. militaris possesses opposite mating-type idiomorphs, MAT1-1 and MAT1-2 (Yokoyama et al. 2006), or two mating type genes, MAT-a and MAT-HMG (Wang et al. 2010a). Zheng et al. (2011a) recently identified the MAT1-1 mating type locus that included MAT1-1-1 and MAT 1-1-2 genes.

Despite obvious heterothallism, homothallism has been occasionally observed in C. militaris (Sato and Shimazu 2002a; Shrestha et al. 2004a; Liang et al. 2005; Wen et al. 2009; Zheng et al. 2011a). Homothallism in C. militaris could be due to the presence of (1) both genotypes (MAT1-1/2) in the chromosomes, (2) a disomic (diploid) condition with an extra chromosome of the opposite mating type locus, or (3) a heterokaryotic condition with more than one nucleus with opposite mating type loci (Shrestha et al. 2004a; Wen et al. 2009). Intraspecific high genetic variation seems common in C. militaris and may be associated with mating-system instability (Wen et al. 2009). Recently, het-erokaryosis and parasexuality were confirmed in singlespore strains of C. militaris by RAPD analysis (Li et al. 2007a). Mating-type switching can also be another possible reason for homothallism in C. militaris.

In contrast to mating-type loci in homothallism, mating-type loci in heterothallic fungi exist in separate spores. Mating compatibility in heterothallism can be controlled by only one factor (bipolar) or by two factors (tetrapolar). Basidiomycetous fungi are mostly heterothallic, while a majority of ascomycetous fungi studied are homothallic. Heterothallic ascomycetous fungi contain a single mating-type locus with two alternate alleles (bipolar) (Poggeler 2001), while basidiomycetes have evolved multiple mating-type loci (Kothe 2001). To determine mating compatibility in ascomycetes, a researcher must wait until fertile reproductive structures are formed. There is no specific indication in the somatic structure or the asexual reproduction structure in C. militaris strains that they have different abilities of producing fruiting-bodies (Gao et al. 2000b; Wu et al. 2000b). Liang (1990, 2001), however, distinguished fruiting-body formation from ‘Paecilomyces-type’ but not from ‘Acremonium-type’ strains. Li et al. (2006a) also reported that fruiting-bodies were developed from a ‘Lecanicillium-type’ strain but not from an isolate with more densely verticillate phialides. In another study, it was shown that fast-growing isolates produced fewer fruiting-bodies than slow-growing isolates (He et al. 2010).

Although C. militaris lacks distinctive phenotypic characteristics both in the wild and in culture, one obvious phenotypic characteristic in culture is the colony pigmentation that ranges from orange to yellowish-white on nutrient-rich agar media (Shrestha et al. 2006). Either kind of pigmentation was observed in F1 progeny strains when two.

parent strains with contrasting pigmentations (i.e, yellowish-white vs. orange) were crossed (Shrestha et al. 2005a). The F1 progenies either on back-crossing with the parents or sister-crossing among themselves showed that colony pigmentation was independent of or distantly linked with the mating-type locus (Shrestha et al. 2004a, 2005a).

The study of the mating system is more difficult with clavicipitaceous fungi than with many other ascomycetous fungi. Clavicipitaceous fungi are characterized by septate, filamentous ascospores. From a cytological perspective, each filamentous clavicipitaceous ascospore corresponds to a unicellular ascospore of other ascomycetes. Ascospore initials in C. militaris undergo asexual or somatic division seven times and are ultimately filamentous in shape and consist of 128 part-spores (Moore 1964; Ding et al. 1995; Hywel-Jones 2002).

Several characteristics of C. militaris ascospores make single-spore isolation difficult. Firstly, C. militaris asco-spores that consist of as many as 128 part-spores, also known as secondary spores, readily fragment into short filaments upon discharge from mature perithecia, rendering complete unfragmented ascospores difficult to find. Secondly, short filaments of the ascospores tend to overlap or attach end-to-end when released (Shrestha et al. 2005c), which often results in the isolation of multiple ascospores rather than a single one. Thirdly, ascospores tend to germinate quickly in agar media, and germinating hyphae can link the genomes of nearby ascospores (Liang et al. 2005; Shrestha et al. 2005c; Gao 2008). To avoid these problems, researchers should pay attention to ascospore length during isolation and should avoid isolating ascospores that appear abnormally long. Our experience is to leave the ascospores on the isolation medium for a few hours so that they swell but do not germinate. The swelling makes it easier to observe individual ascospore filaments. Although individual ascospore filaments can be isolated after some experience, it is not possible to isolate all the eight ascospores from the same ascus in a Cordyceps (s.l.) species. Isolation of individual part-spores is the final goal that can be achieved to establish a pure culture. Despite difficulty, it is hence urged , in future, to cultures from individual part-spores, probably attainable by the dilution method, in order to overcome the confusion of single-ascospore isolation.

Out-crossing has been successfully achieved for C. mil-itaris in culture. For example, Sung et al. (2006b) reported the formation of perithecial stromata from crossing between different specimens of C. militaris. Although it has been frequently studied, the mating system of C. militaris has been described as unknown in fungal textbooks (e.g., Webster and Weber 2007).

It is clear that mating compatibility not only produces fertile perithecia but also affects developmental and morphological patterns of stromata. Stromata produced by

successful mating are regular, club-shaped, or cylindrical, whereas those produced without mating are deformed and abnormal in size (Shrestha et al. 2004a; Liang et al. 2005). Usually, the cortex in the fertile clava of stromata becomes loose and spongy at maturity whereas, in those produced without mating, it remains hard and compact (unpublished data). The reason might be that substantial materials and energy are used for the development of perithecia in the case of mating, leaving the interstitial cortex tissue weak. Although the differences of biochemical composition between perithecial and non-perithecial stromata remain to be determined, Wen et al. (2005) showed that the amounts of medicinal components such as polysaccharide, adenosine, cordycepin, and cordycepic acid differ between sclerotium and stroma of C. militaris.

In addition to clarifying the life cycles of fungi, mating studies are also important for strain improvement (Kothe 2001; Poggeler 2001). In addition, the mating system also has evolutionary and taxonomic significance in Cordyceps. Although researchers continue to debate whether homothal-lism preceded heterothallism or vice versa (Geiser et al. 1998; Yun et al. 1999), it is generally assumed that the mating systems might have switched from one to the other type more than once in the course of evolution. Whether most Cordyceps species are homothallic or heterothallic remains unknown. A molecular study has shown that several Cordyceps species studied contain opposite mating-type idiomorphs, MAT1-1 and MAT1-2 (Yokoyama et al. 2006). A recent study of the mating system in O. sinensis has detected only the mating-type gene MAT1-2-1 of the idio-morph MAT1-2 (Zhang et al. 2011), suggesting that O. sinensis could be homothallic. The study of the mating system in O. sinensis is hindered by inadequate understanding of its biology, slow hyphal growth, difficulty in inducing stromata in culture, and the scarcity of genetic and genomic resources (Zhang et al. 2011).

In addition to increasing our understanding of fruiting-body formation, information about mating compatibility can also help with the identification of similar species, such as C. militaris and C. kyusyuensis, according to a “biological species concept”. It is important to understand the biological species concept of C. militaris in the context of its worldwide distribution and wide host range.

Nomenclature and status of the anamorph

Cordyceps militaris produces asexual spores (conidia) on the tips of little-differentiated phialides and eight filamentous sexual spores (ascospores) in an ascus (within a peri-thecium) during its sexual phase (Kobayasi 1941; Feng et al. 1990; Liang 1990; Ding et al. 1995). Naming of the conidial structure or anamorph began from the early phase of research on C. militaris, but the name has frequently changed.

Gams (1971) removed C. militaris anamorph from Paecilo-myces (now mainly Isaria) (Gams et al. 2005; Hodge et al. 2005) or Cephalosporium/Acremonium and transferred it to Verticillium sect. Prostrata. Zare and Gams (2001) later erected a new genus Lecanicillium to substitute for Verticil-Hum sect. Prostrata including the anamorph of C. militaris. It is a characteristic of this fungus that conidia can arise in the same strain either in chains or in heads which may be partially modified by different media. The phialides can either arise singly or in whorls again possibly dependent on the medium, but no mutation is required for such a change.

Finally, starting on 1 January 2013, the confusion over multiple naming of holomorphic fungi will be resolved through the recent revision of Article 59 of the International Code of Botanical Nomenclature (ICBN) [recently changed to the International Code of Nomenclature for algae, fungi, and plants (ICN)] that will validate only one name in a clade based on priority (Hawksworth 2011; Miller et al. 2011; Norvell 2011). The revision will help establish the concept of one fungus = one name (Hawksworth 2011). Different anamorph names associated with C. militaris can be stated as morphological stages, e.g., acremonium, lecanicillium, paecilomyces, verticillium, etc., with a lower case initial letter and normal non-italic type, following Cannon and Kirk (2000) and Hawksworth (2011).

Synonymous terms for stromata

A stroma is a compact, somatic structure or a cushion-like matrix of mainly ascomycetous fungi on which or in which spores or fruiting bodies are usually formed (Alexopoulos et al. 1996; Kirk et al. 2001). The stroma is generally distinguished from the synnema, which carries conidia or asexual spores at the tips of conidiogenous cells (phialides) or on indistinct hyphae. Stromata of C. militaris are clavate, clubshaped, or cylindrical with a lower sterile stipe and upper fertile clava, also known as the apical part or head, and are orange or yellow in color. In the wild, stromata are solitary to few or in some cases gregarious. The literature on the cultivation of Cordyceps spp., however, uses a variety of terms interchangeably with stroma, and these include fruiting-body (or fruitbody, fruit body, fruit-body), artificial stroma, perithecial stroma, sporophore, sexual sporophyte, sporocarp, ascostroma, and others. Among them, ‘fruit-body’, ‘fruit body’, and ‘fruiting-body’ are most commonly used and may be preferred to stroma for communication with farmers, businessmen, and consumers. Stroma and stromata, however, are the preferred terms for scientific reports. Different words have also been used to describe stromata produced in culture, and these include normal versus abnormal, complete versus incomplete, regular versus irregular, stable versus unstable, mature versus immature, perfect versus imperfect, and uniform versus deformed.

Cytology, genetics, and genomics

Only a few studies have been conducted on the cytology and genetics of C. militaris. Moore (1964) showed that the manner of somatic nuclear division is similar to that of cell division. Electrophoresis karyotype analysis showed that its chromosomal number is seven, and that chromosome size ranges between 2.0 and 5.7 Mb (Wang et al. 2010c). However, recent whole genome sequencing of C. militaris has shown that its total genome is exactly 32.2 Mb (Zheng et al. 2011a), which is smaller than that of two other entomopa-thogenic fungi, Metarhizium anisopliae (39.04 Mb) and M. acridum (38.05 Mb) (Gao et al. 2011). Conditions affecting the formation and regeneration of protoplasts from C. mili-taris have been extensively studied (Ma et al. 2008; Liu et al. 2009; Zhou and Luo 2009; Li et al. 2011), and mutants with superior traits, e.g., high production of cordycepin, polysaccharide, and fruiting-bodies, have been obtained by irradiation induction (Che et al. 2004; Zhou and Bian 2007; Zhou et al. 2009a; Li et al. 2011).

The study of fungal genetics requires an efficient transformation system. Zheng et al. (2011b) developed and optimized Agrobacterium tumefaciens-mediated transformation for C. militaris, which can facilitate the identification of functional genes. Expressed sequence tag (EST) analysis revealed different transcriptional patterns for C. militaris in mycelia growing in liquid culture or on solid rice medium, and in fruiting-bodies produced on rice medium or on silkworm pupae (Xiong et al. 2010). Further analysis showed that genes involved in cell metabolism, energy metabolism, and stress responses were upregulated during asexual development, and that genes associated with cell wall structures were upregulated during sexual development (Xiong et al. 2010). Many studies have described how conditions affect fruiting-body formation and metabolite production (e.g., cordycepin, polysaccharide) (Cui and Zhang 2011; He et al. 2011b), but the genetic basis for these processes is still unclear.

Fortunately, the genome of C. militaris has recently been sequenced (Zheng et al. 2011a). A total of 9,684 proteincoding genes have been predicted, and 13.7 % of these genes are species-specific, which is significantly higher than the percentage for M. anisopliae (4.8 %) or M. acridum (3.5 %). About 16 % of the C. militaris genes (1,547) are related to pathogen-host interactions. No orthologs of known human mycotoxins have been detected (Zheng et al. 2011a), which is consistent with its safe usage as a medicine. More than 63 % of the total 9,684 genes were expressed during both mycelial growth and fruiting-body

formation. The Zn2Cys6-type transcription factors and MAPK pathway were induced during fruiting, but not the PKA pathway, which differs from the induction of these pathways in other fungi. The complete sequencing of the C. militaris genome will facilitate functional studies of interesting genes and elucidate the genetic background for biosynthesis of bioactive components, insect pathogenicity, and isolate degeneration (Zheng et al. 2011a).

Significant genetic differences between wild-type strains and degenerate strains have been detected (Li et al. 2003, 2007a). However, unlike the significant intraspecific genetic diversity of O. sinensis (Zhang et al. 2009), the genetic distance among C. militaris isolates from different localities is extremely low based on nrDNA ITS sequences (K2P distance value <0.01) (Wang et al. 2008).

Some C. militaris proteins have been purified, and some C. militaris genes have been cloned. The purified proteins include an extracellular trypsin-type serine protease P-1-1 (Hattori et al. 2005), a cytotoxic and antifungal protease CMP (Park et al. 2009), an antifungal peptide cordymin (Wong et al. 2011), a haemagglutinin with anti-proliferative activity towards hepatoma cells (Wong et al. 2009), a lectin (CML) that exhibits haemagglutination activity in mouse and rat erythrocytes (Jung et al. 2007), fibrinolytic enzymes (Kim et al. 2006; Cui et al. 2008; Choi et al. 2011), and superoxide dismutase (Wang et al. 2005a). The cloned genes include a glyceraldehyde-3-phosphate dehydrogenase (GPD) gene (Gong et al. 2009), superoxide dismutase genes (Park et al. 2005; Wang et al. 2005b), and the |3-1,3-glucan synthase catalytic subunit gene (Ujita et al. 2006).

Cultivation of C. militaris

Nutritional requirements for stroma growth and production

Insects

The artificial growth and stroma production of C. militaris has been studied in the laboratory on various insect pupae and larvae, most often on the silkworm Bombyx mori, (Gu and Liang 1987; Liang and Gu 1987; Gu et al. 1988; Gong et al. 1993; Zhou et al. 2000; Chen and Ichida 2002; Li 2002; Pan et al. 2002; Sato and Shimazu 2002b; Zhang et al. 2003; Liu 2004; Wen et al. 2004; Li et al. 2006a; Zheng et al. 2008a, b; Chai et al. 2010; Hong et al. 2010; Mu et al. 2010). Other insects used for artificial stroma production are Antherea pernyi (Gu and Liang 1987; Liang and Gu 1987; Gu et al. 1988; Yuan 1988, 1989; Feng et al. 1990; Wang et al. 2002), Mamestra brassicae (Harada et al. 1995; Sato and Shimazu 2002b), Tenebrio molitor (Sato and Shimazu 2002b; Lin et al. 2005), Ostrinia nubilalis (Liang and Gu 1987), Heliothis virescens, H. zea and Spodoptera.

frugiperda (Sanchez-Pena 1990), Andraca bipunctata (Panigrahi 1995), Philosamia cynthia (Jiang and Xun 1996), Spodoptera litura (Sato and Shimazu 2002b), and Clanis bilineata (Song 2009). Chen and Ichida (2002) documented a higher rate of infection and stroma formation in silkworm pupae than in silkworm larvae. Among three varieties of silkworm (Daeseungjam, Bae-gokjam, and Keumokjam), the Daeseungjam variety was found to be the most suitable for stroma formation of C. militaris (Hong et al. 2010).

Natural organic substrates

Because insects are expensive and not always available (Lin et al. 2006c), and because insects can be difficult to handle and thus prone to microbial contamination, alternative organic substrates have been tested for commercial production of C. militaris stromata. Fortunately, cereals with the addition of some organic substances have proven to be good substitutes for insects. Kobayasi (1941) documented stroma production of C. militaris on rice substrate. Since then, rice has been used as the principal ingredient for growing stromata of C. militaris (Basith and Madelin 1968; Chen and Wu 1990; Liang 1990; Ma and Chen 1991; Sung etal. 1993, 1999; Pen 1995; Sung 1996; Wu et al. 1996; Zhang and Liu 1997; Choi et al. 1999; Li 2002; Zhang 2003; Li et al. 2006d; Lin et al. 2006b; Wen et al. 2008b; Chen et al. 2011b).

The porosity of the fruiting medium affects mycelial growth and fruiting-body yield. Porosity increases with grain size and declines with a higher ratio of water to grain during rice medium preparation. In the absence of interstices, mycelia mostly grow only on the surface of the medium and thereby cannot absorb enough nutrition from the substrate. Interstices permit hyphae to grow inside the medium and to obtain sufficient nutrition from the substrate (Kobayasi 1941). A ratio of rice to water from 1:1 to 1:1.35 or slightly higher has been reported to be optimal for stroma production (Sung et al. 1999, 2002; Lin et al. 2006b; Zheng et al. 2008c; Yue 2010), but the optimal ratio may depend upon the rice cultivar and its glutinous quality. Husked rice (popularly known as brown or unpolished rice) is usually used for cultivation of C. militaris. Maximum fruiting-body yield has been obtained with whole rice grain (Wen et al. 2008b).

Other organic materials used for the production of C. militaris stromata include bean powder, corn grain, corn cobs, cotton seed coats, jowar, millet, sorghum, fragments of sunflower floral disks, and wheat grain (Chen and Wu 1990; Zhang and Liu 1997; Li 2002; Li et al. 2004a; Zhao et al. 2006a; Gao and Wang 2008; Wei and Huang 2009). Rice mixed with silkworm pupae has proven to be superior to other substrates and is now routinely used (Ren 1998; Chen.

et al. 2002; Shrestha et al. 2004a, b, 2005a, b; Sung et al. 2002, 2006a, b; Zhao et al. 2006a; Jin et al. 2009). C. militaris strains require a relatively low level of nitrogen, and excessive nitrogen might suppress differentiation of the fruiting-body (Gao et al. 2000a). This probably explains why yields have been observed to be less on insects than on cereals in the culture. Xie et al. (2009a, b) have also shown that brown rice, malt, and soybean are better sources of nutrition for C. militaris than chemically defined media. Agar media are usually not suitable for stroma production (Basith and Madelin 1968; Yahagi et al. 2004).

Hormones and mineral components of the media

Plant hormones such as 2, 4-D, citric acid triamine, colchicines, and others may enhance stroma production by C. militaris (Li et al. 2004a; Wang et al. 2010b; Xiao et al. 2010). Similarly, mineral salts such as K+, Mg +, and Ca2+ at a concentration of 0.1 g/l may increase fruiting yield (Li et al. 2004a). Some elements may enhance the production of bioactive compounds of C. militaris in culture (Dong et al. 2012).

Duration of stroma production and stromata yield

Commercial production must take into account the duration of stroma production. Stromata are usually produced over a period of 35-70 days (Zhang and Liu 1997; Sung et al. 1999; Yue 2010; Du et al. 2010). Zhang and Liu (1997) reported the production period of 35-45 days on rice but 40-70 days on other substrates such as maize, millet, and rice-tussah. Culture duration, however, depends upon the shape and volume of the culture container and the amount of medium. Stroma production has been quantified in some studies. Wu et al. (1996) obtained 25 g of fresh fruit bodies of C. militaris from 50 g of rice medium, while Zhang and Liu (1997) reported a biological transformation rate of 61 % on rice, 58-59 % on millet and rice-tussah media, and 42 % on maize. Production of 18.0 g of stromata (fresh wt.) has been recently obtained from 20 g of rice (Lin et al. 2006a). Nearly 9 g of dry stromata (equivalent to about 68 g of fresh wt.) was produced from 60 g of brown rice supplemented with 10 g of silkworm pupae (Sung et al. 2006b).

Effect of environment

Whereas hormones govern morphogenetic and developmental changes in plant tissue culture, it is environmental factors that govern the change from the somatic phase to the reproductive phase in fungi. The optimal environmental factors for growth of C. militaris stromata are briefly discussed below.

Temperature

Temperature greatly affects C. militaris stroma production. High temperatures (about 25 °C) lead to maximal mycelial growth, but lower temperatures induce and sustain stroma production, viz. temperatures within the range of 18-22 °C are reported as optimal (Sung et al. 1999, 2002; Gao et al. 2000a; Zhao et al. 2006a; Du et al. 2010; Sato and Shimazu 2002b), although a lower range of 14-17 °C (Du et al. 2007) or a higher temperature of 25 °C (Li et al. 2004a; Yue 2010) have also been reported to be appropriate for stroma production. The maturation period of stromata is shorter at 25 °C than at 20 °C (Sato and Shimazu 2002b).

Light

Light is the most important environmental factor affecting C. militaris stroma production and no stromata are produced in darkness (Sato and Shimazu 2002b). Gao et al. (2000a) obtained stromata under light intensities as high as 4,500 lx, but Sato and Shimazu (2002b) indicated that the upper limit was 1,400 lx; in general, 500-1,000 lx is considered optimal (Sung et al. 1999; Gao et al. 2000a; Sato and Shimazu 2002b; Li et al. 2004a; Zhao et al. 2006a; Du et al. 2010), with a 12-h light/dark cycle of 500-1,000 lx (Sung et al. 2002; Chen et al. 2011b). It has been found that 18 h of light at 200 lx was optimal for pigmentation and primordium initiation, whereas 10 h was suitable for fruiting-body growth and yield (Mu et al. 2010). Longer and wider fruiting-bodies were produced with light intensities of 500-1,000 lx than with an intensity of 100 lx (Hong et al. 2010).

Air exchange and humidity

High air exchange in the culture container favors mycelial growth, primordium formation, and biomass yield (Zhang et al. 2010a). Among materials tested for covering culture bottles containing C. militaris, a hydrophobic fluoropore membrane was the best (Zhang et al. 2010a). A high humidity of 70-90 %, which probably matches the humidity experienced by the fungus in nature, favors stroma production. Low humidity causes the medium to dry too quickly. Especially in dry, indoor environments, a humidifier will be required to maintain sufficient humidity.

Inoculum preparation

For production of C. militaris stromata, media can be efficiently inoculated with a liquid culture of the fungus (Sung 1996). The inoculum in liquid culture consists of conidia, hyphal fragments, and hyphal pellets affected by nutrients, culture duration, mode of culture, etc. The liquid culture.

medium usually contains simple sources of carbon (usually in the form of carbohydrates) and nitrogen (inorganic as well as organic) along with mineral salts. The optimum temperature and pH for liquid culture of C. militaris are 20-25 °C and 6.0-8.0, respectively. While C. militaris my-celia produce yellowish-white to orange pigments on solid media in light (Shrestha et al. 2006), liquid cultures of the fungus usually remain colorless. Pellets in liquid culture increase in size as the culture ages (Han et al. 2009). Pellets are not a suitable form for inoculation purposes for three reasons. First, and most importantly, pellets do not grow as fast as conidial or hyphal suspensions on the fruiting medium. Second, pellets frequently fail to induce stroma primor-dia. Third, inoculum consisting of pellets is difficult to quantify. For large-scale preparation of liquid inoculum, shaking and aeration of the liquid are required for the homogeneous growth of mycelium. The quantity of inoculum added to the fruiting medium depends on the volumes of the culture container and the fruiting medium (Sung et al. 2002; Lin et al. 2006b).

Industrial and commercial development

Although more than 400 Cordyceps s.l. species have been described, only about 36 species have been artificially cultivated for the production of fruiting-bodies (Wang 1995; Sung 1996; Li et al. 2006b). Of those species that have been artificially cultivated, only C. militaris has been commercially cultivated; commercial development has focused on C. militaris because of its excellent pharmaceutical effect and short production period (Li et al. 2006b).

Cordyceps militaris has been cultivated in liquid media for harvesting mycelia and on solid media for induction of fruiting-bodies. While conditions for submerged cultivation of C. militaris inoculum have been optimized (Kim et al. 2003; Liu et al. 2008), large-scale production of C. militaris fruiting-bodies currently uses only solid media consisting of artificial substrates or insects (e.g., the silkworm B. mori). Because cultivation on insects is costly, fruiting-bodies of C. militaris are mainly cultivated on artificial media in which rice is the main component. Substrates used for industrial cultivation of C. militaris in China have recently been reviewed (Wang et al. 2009b). They include media for stock culture, pre-culture spawn, and spawn. For each purpose, the ingredients differ depending on the company (Wang et al. 2009b). Although the cost for cultivation of several insects has recently decreased greatly, fruiting-bodies cultivated on insects (1,000 RMB/kg) are twice as expensive to produce as biomass cultivated in artificial media (500 RMB/kg) (Li et al. 2007b). Compared with the extremely high price of O. sinensis, however, the price of C. militaris is affordable.

In the cultivation of C. militaris fruiting-bodies, four pivotal growth periods are usually identified: mycelial culture, pigment induction, stromata stimulation, and fruiting-body production (Lu et al. 2005). Successful cultivation requires proper control of temperature, humidity, and light (Ren et al. 2009).

Cordyceps militaris cultures have two main uses. First, the fruiting-bodies can be directly consumed as food. C. militaris can be used in stewed chicken, stewed duck, soup, hot pot, tea, and so on. Use of C. militaris in soup is very popular in Southeast Asia, especially in Guangdong, Hong Kong, and Taiwan, China. This use has been shown to be safe if consumption is less than 2.5 g/kg of body weight (Che 2003). Second, C. militaris fruiting-bodies and myce-lia can be used as health products and drugs. In China, many health products contain C. militaris, and these include oral liquids, capsules, wines, vinegars, teas, yogurt, and soy sauce (Wang and Yang 2006). Cultures of C. militaris are also used to produce drugs for maintenance of kidney and lung function, anti-aging, regulation of sleep, and chronic bronchitis (Dai et al. 2007). Currently, more than 30 kinds of C. militaris health products and drugs are available on the market (Huang et al. 2010).

Problems and prospects

Degeneration of isolates

Degeneration of isolates is the main problem in C. militaris cultivation. Degeneration is manifested as reduced growth rate, mycelial density, pigmentation, and fruiting-body yield, and also as changes in fruiting-body shape and size. Degeneration can also be evident in the reduced production of desired compounds from fermented cultures. Degeneration in stroma production is related to the kind of material that was used for isolation. If the isolate was derived from multiple ascospores (multi-spores) or tissue, decline in stroma production occurs soon after one or two subcultures (Shrestha et al. 2004b). Degeneration can be delayed if cultures are obtained from single ascospores (Shrestha et al. 2004a; Sung et al. 2006a).

Compatible pairs of single-ascospore or single-conidium strains should be used to study the effects of biotic and abiotic factors on stroma production. With isolates derived from multiple ascospores, multiple conidia, or tissue, stroma production will vary even when culture conditions are constant (Shrestha et al. 2002, 2004b; Liu et al. 2006). Unfortunately, most publications concerning C. militaris culture do not indicate how the isolate was obtained.

Degeneration in colony pigmentation has been observed in C. militaris strains after several subcultures (Sung et al. 2006a). Lin et al. (2010) observed reduced dehydrogenase

activity and pigment in degenerated strains but no change in mating-type or evidence of dsRNA infection. In addition, the formation of heritable white synnemata has been reported from C. militaris isolates in culture (Sato et al. 1997; Wang et al. 2009a). Formation of white synnemata in C. militaris isolates may provide an opportunity for new strain development as is the case for other cultivated mushrooms such as Flammulina velutipes. A few studies have shown that preservation of C. militaris isolates at 4-10 °C helps to maintain the ability of fruiting-body production for up to 6 months (Sung et al. 2006a; Geng et al. 2009). An increased mutation frequency at the DNA level has been associated with degeneration of C. militaris isolates (Li et al. 2003).

The genes involved in C. militaris degeneration have not been identified. In C. militaris, identification of genes and their function is mostly lacking (Zheng et al. 2011b). So far the glyceraldehyde-3-phosphate dehydrogenase gene, the |3-1,3-glucan synthase catalytic subunit gene, and the superoxide dismutase Cu, Zn-SOD gene are known to be involved in phosphorylation, cell wall constitution, and defense against oxidative damage, respectively (Wang et al. 2005b; Ujita et al. 2006; Gong et al. 2009). Recent research has demonstrated that Agrobacterium tumefa-ciens-mediated transformation is useful to elucidate the function of genes in C. militaris (Zheng et al. 2011b); this method should also help in determining which genes are responsible for degeneration of isolates.

Strain development and large-scale cultivation of C.

militaris

Cordyceps militaris cultivars with desirable properties such as high production of stromata and high cordycepin content have recently been developed (Sun et al. 2009; Du et al. 2010). Du et al. (2010) reported a new cultivar of C. mili-taris with high cordycepin yield (24.98 mg/g of fruiting-body dry wt.). Che et al. (2004) obtained a higher yielding and more stable strain of C. militaris by UV mutagenesis. Recently, regeneration of C. militaris from its protoplasts has been studied (Zhou and Bian 2007; Liu et al. 2009; Zhou and Luo 2009). C. militaris isolates from different regions contain different concentrations of active compounds (Wen et al. 2008a). Thus, superior isolates can be selected for propagation.

Cordyceps militaris is usually cultured in small containers (0.5-1.0 l) because the fungus requires high humidity and moisture, and because small containers such as bottles and trays seem to provide an excellent environment for the growth of primordia and stromata. Reusable light plastic containers can reduce the cost of culture. Substantially increasing production and reducing costs will require the development of bed cultivation or other cultivation methods.

Xiong et al. (2010) considered insect-grown C. militaris to be far superior to cereal-grown C. militaris. Huang et al. (2009), however, reported that fruiting-bodies had higher contents of cordycepin and adenosine when cultivated on rice medium than on silkworm chrysalid or wheat medium. The latter authors also observed that cordycepin and adenosine contents were higher in cultivated fruiting-bodies of C. militaris than in natural O. sinensis fruiting-bodies, and that the contents in cultured mycelium of C. militaris were similar to those in O. sinensis fruiting-bodies. Thus, they proposed that rice-grown C. militaris was the best substitute for O. sinensis (Huang et al. 2009). Brown rice along with malt and soybean has been shown to provide sufficient nutrition for C. militaris (Xie et al. 2009a, b). Wu et al. (2012) compared polysaccharides of C. militaris fruiting-bodies cultivated on rice medium and silkworm pupae, and found that the components and structures of polysaccharides differ in fruiting-bodies cultivated in those two types of media. Similarly, metabolomic studies at regular durations is also useful in order to determine the optimum cultivation period of C. militaris (Choi et al. 2010). There are diverse views, however, regarding the medicinal properties of wild and cultivated C. militaris as well as those grown on various organic substances such as cereals and insects (Li et al. 2004b; Huang et al. 2009; Xiong et al. 2010). Future studies on C. militaris will likely identify new organic substrates that are economical and that support high stroma production and a high concentration of bioactive compounds. The sequencing of its complete genome (Zheng et al. 2011a) will facilitate research on the molecular basis of the biology and medicinal qualities of C. militaris.

The phylogenetic classification has divided the genus Cordyceps into five genera in three families that fit the present concept of the fungal tree of life (Sung et al. 2007; Kepler et al. 2012b). Phylogenetic classification is, however, in a state of flux and has resulted in the frequent transfer of Cordyceps species from one genus to another in different families (Sung et al. 2007; Kepler et al. 2012a). Furthermore, Cordyceps spp. growing on cicadas have been phylo-genetically placed under three different genera (Sung et al. 2007; Sato et al. 2012). Outside the mycological world, the transfer of C. sinensis from Cordyceps to the newly established genus Ophiocordyceps has caused some confusion among researchers in the biochemical and pharmacological communities, because of the belief that O. sinensis (syn. C. sinensis) would be different from Cordyceps spp. at the generic level, though they have adapted to the same level of insect-dependent nutrition. All Cordyceps spp. have similar life cycles and have developed mechanisms to invade insects and grow on them; the major difference being the locality where they grow and the host insect they infect.

Given the diversity of host insects and geographical regions, Cordyceps spp. differ widely in shape, size, color, texture, position of fertile parts, and other micromorphological characters. Among all Cordyceps spp. s.l., O. sinensis has a distinct ecological niche in that it grows at high altitudes in the alpine region. Cordyceps s.l. is a broad genus that encompasses clavicipitaceous fungi that grow on insects as well as on fungi. Cordyceps species that grow on fungi have been phylogenetically placed in the new genera Elaphocor-dyceps and Tyrannocordyceps (Sung et al. 2007; Kepler et al. 2012b). The rest of the Cordyceps species grow on insects and spiders. Cordyceps species remain dormant in soil until they get in contact with a host. They all grow inside the host body and form endosclerotium before emerging from the host body in the summer. Besides their morphological diversity, they also differ widely in cultivability. C. militaris is the most successfully cultivated species (Kobayasi 1941; Sung 1996). As stated above, we believe that C. militaris is worth promoting as a medicinal fungus, being the best described and cultivable Cordyceps species.

Acknowledgments The first author acknowledges the support of the Green Energy Mission/Nepal, Kathmandu, Nepal and a grant from the Next-Generation BioGreen 21 Program (PJ008154 and PJ008321), Rural Development Administration, Republic of Korea, during the preparation of the manuscript. The second author acknowledges the support of the National Natural Science Foundation of China (No. 30870450). The authors also sincerely thank the unknown reviewer for the helpful comments and suggestions.

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