6 X.-W.Zhou et al Crit Rev Biotechnol,Early Online:1-11 13 DCXC strains from three distribution regions on the carbon source is glucose,the nitrogen source is peptone and Qinghai-Tibetan Plateau and found that among them the correct amount of yeast extract is also required.In there exists a large genetic differentiation.This phenom- addition,if a little magnesium sulfate and potassium enon may result from the large geographic distance dihydrogen phosphate are added,the mycelia will grow Based on this,the taxonomists are suggested to conduct faster and better (Wang et al.,2001b;Wang et al.,2004). further study on whether they belong to different Up to now,there are many patented technologies for subgenera and even species (Chen et al.,1997). isolation of fungal strain from DCXC.The various preserva- With the rapid development of science and technology,a tion methods have been also developed,and the ideal one is variety of advanced technologies have applied in the study of to use the vacuum cryoapplication (Wang et al.,1991),or to the DCXC.Especially,molecular biology technology has simulate natural conditions for preserving strain.Fang and increasingly connected with other disciplines,such as plant, colleagues conducted a test for preserving the DCXC strains, animal and microbiology.It can reveal the internal identity respectively,under natural conditions and in a freezer. between anamorph and teleomorph of fungi,which is used to After preserving the strains in simulated natural conditions identify and select the anamorphic strains that have the largest for 30 days,at 10-15C,in light at daytime and darkness at genetic similarity in sexual phase.It has become the most night,the quality of mycelial was much better than in a 7000 advanced method at present (Jiang Yao,2004;Zhou et al., traditional freezer (Fang et al.,2011). 2005).This undoubtedly provides much more objective It is worth noting that the number of ascospores in each scientific evidence for identifying the anamorph of the ascus of the DCXC varies from different regions (Liu et al., DCXC.However,further research should be conducted on 2003b),and so does the size of ascospore and the number whether the H.sinensis is the real and unique anamorph of of septa (Shrestha et al.,2010).The characteristics and the DCXC. morphology of cultured conidiogenous cells of the isolated H.sinensis derived from DCXC in different habitats have Cultivation of the O.sinensis fungi been reported in several papers (He et al.,2011;Liang et al., uepn Kq wo'aoyeuoju woy Ophiocordyceps sinensis is an ascomycete whose life cycle 2010:Liu et al,.1989). includes the conidium stage and ascospore stage,also called as the anamorph and teleomorph stages.The culturing process The mechanism of fungal spore attacking the moth of O.sinensis is not different from other filamentous fungi, larvae which include such these steps as strains screening,strains activation,intermediate culture and batched culture(shown in The moth larva is infected to death by fungal spores in the Figure 2).Based on the physical and chemical property of soil,and in the next year it will turn into a fruiting body,i.e. media,two kinds of culturing methods can be used,i.e.one is DCXC.Some studies inferred that early in every August,the the liquid-state culture (LSC)(Liu et al.,2009)and the other infection rate of the moth larvae with four to five instars is the is the solid-state culture (SSC)(Ge et al.,2009).In the culture largest,ie.approximately 90%,while few of the moth larvae process,the key factors to successfully culturing and gaining with six instars are infected and the moth larvae under three the effective products are the temperature,relative humidity, instars are hardly infected.The O.sinensis fungi,which can oxygen and nutritional components (Chen Feng,1992; infect the host insects,should be mature sexual reproduction Shang Wang,1996;Wang et al.,2009).In general,the spores and the mycelium cultured in later stage.During the optimum growth temperature of H.sinensis is at 18-20C, infection stage,soil structure,temperature and humidity,and and the mycelia can grow rapidly at >20C,but is restrained atmosphere temperature may affect the infection of moth to grow at >25C.Its optimum pH value is 5-6,the best larvae (Yang et al.,1989).Meanwhile,the internal amino acids and microelements of the larvae as well as the DCXC fungi correlate closely to the infection rate of the larvae Selecting Screening (Yang et al..1990). media strains After the O.sinensis attacking the hemocoelom of the host larvae,they will break into long fusiform hyphal fragments. The thallus propagates by means of apiculus proliferation,and Sterilization Activating the gemma is morphologically similar to the precursor.When and cooling strain the gemma grows up and its length and width are similar to those of the precursor,it breaks off and forms an independent individual.In the specific external conditions,the hyphal Aspesis inoculation fragment propagates inside the host larva to a certain density and then enters into the differentiation phase.Meanwhile,the thallus starts to break one by one:first.a tabula comes out of SSC LSC the central thallus and part it into two segments;then,two segments start to break from their centre and part the thallus into four segments;finally,similarly,the thallus is parted into Mass of eight segments.After that,the differentiation will go to the mycelia end.Some parts of the thallus will be out of shape or broken, while the other parts of the thallus will link to each other and Figure 2.Fundamental procedure for breeding of Hirsutella sinensis. the cell wall will be assimilated,which makes the protoplasts RIGH T S L I N K13 DCXC strains from three distribution regions on the Qinghai-Tibetan Plateau and found that among them there exists a large genetic differentiation. This phenom￾enon may result from the large geographic distance. Based on this, the taxonomists are suggested to conduct further study on whether they belong to different subgenera and even species (Chen et al., 1997). With the rapid development of science and technology, a variety of advanced technologies have applied in the study of the DCXC. Especially, molecular biology technology has increasingly connected with other disciplines, such as plant, animal and microbiology. It can reveal the internal identity between anamorph and teleomorph of fungi, which is used to identify and select the anamorphic strains that have the largest genetic similarity in sexual phase. It has become the most advanced method at present (Jiang & Yao, 2004; Zhou et al., 2005). This undoubtedly provides much more objective scientific evidence for identifying the anamorph of the DCXC. However, further research should be conducted on whether the H. sinensis is the real and unique anamorph of the DCXC. Cultivation of the O. sinensis fungi Ophiocordyceps sinensis is an ascomycete whose life cycle includes the conidium stage and ascospore stage, also called as the anamorph and teleomorph stages. The culturing process of O. sinensis is not different from other filamentous fungi, which include such these steps as strains screening, strains activation, intermediate culture and batched culture (shown in Figure 2). Based on the physical and chemical property of media, two kinds of culturing methods can be used, i.e. one is the liquid-state culture (LSC) (Liu et al., 2009) and the other is the solid-state culture (SSC) (Ge et al., 2009). In the culture process, the key factors to successfully culturing and gaining the effective products are the temperature, relative humidity, oxygen and nutritional components (Chen & Feng, 1992; Shang & Wang, 1996; Wang et al., 2009). In general, the optimum growth temperature of H. sinensis is at 18–20 C, and the mycelia can grow rapidly at420 C, but is restrained to grow at 425 C. Its optimum pH value is 5–6, the best carbon source is glucose, the nitrogen source is peptone and the correct amount of yeast extract is also required. In addition, if a little magnesium sulfate and potassium dihydrogen phosphate are added, the mycelia will grow faster and better (Wang et al., 2001b; Wang et al., 2004). Up to now, there are many patented technologies for isolation of fungal strain from DCXC. The various preserva￾tion methods have been also developed, and the ideal one is to use the vacuum cryoapplication (Wang et al., 1991), or to simulate natural conditions for preserving strain. Fang and colleagues conducted a test for preserving the DCXC strains, respectively, under natural conditions and in a freezer. After preserving the strains in simulated natural conditions for 30 days, at 10–15 C, in light at daytime and darkness at night, the quality of mycelial was much better than in a traditional freezer (Fang et al., 2011). It is worth noting that the number of ascospores in each ascus of the DCXC varies from different regions (Liu et al., 2003b), and so does the size of ascospore and the number of septa (Shrestha et al., 2010). The characteristics and morphology of cultured conidiogenous cells of the isolated H. sinensis derived from DCXC in different habitats have been reported in several papers (He et al., 2011; Liang et al., 2010; Liu et al., 1989). The mechanism of fungal spore attacking the moth larvae The moth larva is infected to death by fungal spores in the soil, and in the next year it will turn into a fruiting body, i.e. DCXC. Some studies inferred that early in every August, the infection rate of the moth larvae with four to five instars is the largest, i.e. approximately 90%, while few of the moth larvae with six instars are infected and the moth larvae under three instars are hardly infected. The O. sinensis fungi, which can infect the host insects, should be mature sexual reproduction spores and the mycelium cultured in later stage. During the infection stage, soil structure, temperature and humidity, and atmosphere temperature may affect the infection of moth larvae (Yang et al., 1989). Meanwhile, the internal amino acids and microelements of the larvae as well as the DCXC fungi correlate closely to the infection rate of the larvae (Yang et al., 1990). After the O. sinensis attacking the hemocoelom of the host larvae, they will break into long fusiform hyphal fragments. The thallus propagates by means of apiculus proliferation, and the gemma is morphologically similar to the precursor. When the gemma grows up and its length and width are similar to those of the precursor, it breaks off and forms an independent individual. In the specific external conditions, the hyphal fragment propagates inside the host larva to a certain density and then enters into the differentiation phase. Meanwhile, the thallus starts to break one by one: first, a tabula comes out of the central thallus and part it into two segments; then, two segments start to break from their centre and part the thallus into four segments; finally, similarly, the thallus is parted into eight segments. After that, the differentiation will go to the end. Some parts of the thallus will be out of shape or broken, while the other parts of the thallus will link to each other and the cell wall will be assimilated, which makes the protoplasts Selecting media Sterilization and cooling Screening strains Activating strain Aspesis inoculation SSC Mass of mycelia LSC Figure 2. Fundamental procedure for breeding of Hirsutella sinensis. 6 X.-W. Zhou et al. Crit Rev Biotechnol, Early Online: 1–11 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only.