http://informahealthcare.com/bty Critical Reviews ISSN:0738-8551 (print),1549-7801 (electronic) informa in Biotechnology Crit Rev Biotechnol,Early Online:1-11 2013 Informa Healthcare USA,Inc.DOl:10.3109/07388551.2013.791245 healthcare REVIEW ARTICLE Advances in research of the artificial cultivation of Ophiocordyceps sinensis in China Xuan-Wei Zhou.2,Lin-Jun Li3,and En-Wei Tian4 Plant Biotechnology Research Centre,School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,People's Republic of China,'Tibet Shengniu Biotechnology Co.,Ltd.,Lhasa,Tibet 85000,People's Republic of China,Key Laboratory for Urban Habitat Environment 7000 Science and Technology,School of Environment and Energy,Shenzhen Graduate School of Peking University,Shenzhen 518005,People's Republic of China,and Key Laboratory of Plant Resources Conservation and Sustainable Utilization,South China Botanical Garden,Chinese Academy of Sciences,Guangzhou 510650,People's Republic of China Abstract Keywords Ophiocordyceps sinensis (syn.Cordyceps sinensis),a traditional Chinese medicine called Artificial cultivation,environmental DongChongXiaCao(DCXC)in Chinese,is well known and has been used in Asia countries protection,Hepialus larvae,Hirsutella since the fifteenth century,and it contains some valuable medicinal component defined by sinensis,Ophiocordyceps sinensis modern pharmacological science.DCXC only appears at high altitudes on the Qinghai-Tibetan Plateau.Consequently,it is difficult to find and harvest.Because of its rarity and medicinal History value,DCXC has always been one of the most expensive medicines known.As the price of DCXC has risen in recent years,thousands of migrants have entered into the various grasslands Received 26 April 2012 to search for them in season,which makes ecological environments of the grassland more Revised 21 August 2012 fragile.In order to relieve the environmental pressures and protect this valuable resource,the Accepted 12 February 2013 asn artificial cultivation of DCXC involving two aspects of the genus Hepialus and the fungi of Published online 22 July 2013 the host larvae should be employed and applied at the first available time point.In this article, the reproduction of moth larvae of the genus Hepialus is first described,which includes their ecological characteristics and the methods of artificial feeding.Second,the generation and isolation method of the fungi from DCXC are subsequently summarized,and then the mechanism of fungal spores to attack the moth larvae are restated.Finally,the basic model of artificial cultivation of DCXC is introduced;meanwhile,the potential application of modern 8 biotechnology to the artificial cultivation is analyzed in prospect.This review article will not only expand people's knowledge regarding the artificial cultivation of DCXC,but also hopefully provide an informative reference for the development of this valuable resource and the environmental protection of alpine meadows. Introduction Xin''written by Yiluo Wu in the Qing dynasty (1757 AD)and Ophiocordyceps sinensis (also known as Cordyceps sinensis) "Ben Cao Gang Mu Shi Yi written by Xueming Zhao in is a parasitic complex of a fungus (Hirsutella sinensis)and 1765 AD.In 1843,Berkeley (1843)described the DCXC as caterpillar which belongs to Thitarodes,Hepialidae, the Sphaeria sinensis,and then Saccardo (1878)classified Lepidoptera.The Chinese name,DongChongXiaCao them into the genus of Cordyceps.In western countries,the (DCXC),originates from the growth process of O.sinensis: first person introducing how to use the DCXCwas priest. the larva of some species.Hepialidae is initially infected by doctor Du Halde,who described its Chinese name as Hia fungi in the spring and summer seasons,and turn into"'stiff [sic]Tsao Tong Tchong'(Winkler,2009).The name worm''in winter,so it is called as DongChong (winter Cordyceps sinensis came from the Latin words,i.e.,Cord worm).In the next spring and summer seasons,the stroma denotes"club',ceps is"head'and sinensis means "'made in China''.In 2007.molecular biological methods were germinates and then grows from the head of the larva,which is known as XiaCao (summer grass).This Chinese name is used to modify the classification of Cordycipitaceae and translated from its Tibetan name Yartsa gunbu first recorded Clavicipitaceae,and some Cordyceps spp.were classified into by a Tibetan doctor Zurkhar Namnyi Dorje in the fifteenth a new family,Ophiocordycipitaceae (Sung et al..2007).As century,and then is found in the books of"Bei Cao Cong Ben Cao Cong Xin (New Compilation of Materia Medica) depicted,the DCXC lives as an old silkworm in the winter soil,and its body hair grows out of soil and turns into grass in Address for correspondence:Xuan-Wei Zhou.School of Agriculture and Biology,Shanghai Jiao Tong University.Shanghai 200240.People's the next summer.It tastes "Gan Ping(sweet flat),and its Republic of China.Te:+862134205778.Fax:+862165642425. functions were"beneficial for lung and kidney,activating E-mail:xuanweizhou@sjtu.edu.cn.xuanweizhou@163.com blood circulation to dissipate blood stasis and treating virtual RIGHTS L I N K
2013 http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–11 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2013.791245 REVIEW ARTICLE Advances in research of the artificial cultivation of Ophiocordyceps sinensis in China Xuan-Wei Zhou1,2, Lin-Jun Li3 , and En-Wei Tian4 1 Plant Biotechnology Research Centre, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China, 2 Tibet Shengniu Biotechnology Co., Ltd., Lhasa, Tibet 85000, People’s Republic of China, 3 Key Laboratory for Urban Habitat Environment Science and Technology, School of Environment and Energy, Shenzhen Graduate School of Peking University, Shenzhen 518005, People’s Republic of China, and 4 Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People’s Republic of China Abstract Ophiocordyceps sinensis (syn. Cordyceps sinensis), a traditional Chinese medicine called DongChongXiaCao (DCXC) in Chinese, is well known and has been used in Asia countries since the fifteenth century, and it contains some valuable medicinal component defined by modern pharmacological science. DCXC only appears at high altitudes on the Qinghai-Tibetan Plateau. Consequently, it is difficult to find and harvest. Because of its rarity and medicinal value, DCXC has always been one of the most expensive medicines known. As the price of DCXC has risen in recent years, thousands of migrants have entered into the various grasslands to search for them in season, which makes ecological environments of the grassland more fragile. In order to relieve the environmental pressures and protect this valuable resource, the artificial cultivation of DCXC involving two aspects of the genus Hepialus and the fungi of the host larvae should be employed and applied at the first available time point. In this article, the reproduction of moth larvae of the genus Hepialus is first described, which includes their ecological characteristics and the methods of artificial feeding. Second, the generation and isolation method of the fungi from DCXC are subsequently summarized, and then the mechanism of fungal spores to attack the moth larvae are restated. Finally, the basic model of artificial cultivation of DCXC is introduced; meanwhile, the potential application of modern biotechnology to the artificial cultivation is analyzed in prospect. This review article will not only expand people’s knowledge regarding the artificial cultivation of DCXC, but also hopefully provide an informative reference for the development of this valuable resource and the environmental protection of alpine meadows. Keywords Artificial cultivation, environmental protection, Hepialus larvae, Hirsutella sinensis, Ophiocordyceps sinensis History Received 26 April 2012 Revised 21 August 2012 Accepted 12 February 2013 Published online 22 July 2013 Introduction Ophiocordyceps sinensis (also known as Cordyceps sinensis) is a parasitic complex of a fungus (Hirsutella sinensis) and caterpillar which belongs to Thitarodes, Hepialidae, Lepidoptera. The Chinese name, DongChongXiaCao (DCXC), originates from the growth process of O. sinensis: the larva of some species. Hepialidae is initially infected by fungi in the spring and summer seasons, and turn into ‘‘stiff worm’’ in winter, so it is called as DongChong (winter worm). In the next spring and summer seasons, the stroma germinates and then grows from the head of the larva, which is known as XiaCao (summer grass). This Chinese name is translated from its Tibetan name Yartsa gunbu first recorded by a Tibetan doctor Zurkhar Namnyi Dorje in the fifteenth century, and then is found in the books of ‘‘Bei Cao Cong Xin’’ written by Yiluo Wu in the Qing dynasty (1757 AD) and ‘‘Ben Cao Gang Mu Shi Yi’’ written by Xueming Zhao in 1765 AD. In 1843, Berkeley (1843) described the DCXC as the Sphaeria sinensis, and then Saccardo (1878) classified them into the genus of Cordyceps. In western countries, the first person introducing how to use the DCXC was a priest, doctor Du Halde, who described its Chinese name as ‘‘Hia [sic] Tsao Tong Tchong’’ (Winkler, 2009). The name Cordyceps sinensis came from the Latin words, i.e., Cord denotes ‘‘club’’, ceps is ‘‘head’’ and sinensis means ‘‘made in China’’. In 2007, molecular biological methods were used to modify the classification of Cordycipitaceae and Clavicipitaceae, and some Cordyceps spp. were classified into a new family, Ophiocordycipitaceae (Sung et al., 2007). As Ben Cao Cong Xin (New Compilation of Materia Medica) depicted, the DCXC lives as an old silkworm in the winter soil, and its body hair grows out of soil and turns into grass in the next summer. It tastes ‘‘Gan Ping’’ (sweet flat), and its functions were ‘‘beneficial for lung and kidney, activating blood circulation to dissipate blood stasis and treating virtual Address for correspondence: Xuan-Wei Zhou, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China. Tel: +86 21 34205778. Fax: +86 21 65642425. E-mail: xuanweizhou@sjtu.edu.cn, xuanweizhou@163.com Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only
2 X.-W.Zhou et al Crit Rev Biotechnol,Early Online:1-11 cough and haemoptysis''.Based on the description of (Liu et al.,1995;Yan,2000).Nowadays,the biological Pharmacopoeia of the People's Republic of China (2005 characteristics of more than 10 species of genus insects have ed.),the DCXC is also functional on curing impotence, been well studied in China,which include H.armodcamu spermatorrhea and the resolving pain in the knee and waist. Oberthar,H.minyuancus (Wang et al.,2001a),H.yushusis Besides,modern medicines have proved that it possesses Chuet Wang,H.lagii Yan (Xu,2004),H.gonggaensis (Yin some functions,such as anti-tumor,anti-hyperglycemia et al.,1995),H.guidera Yan,H.oblifurcus Chuet Wang, (Lo et al.,2006)and significant improvement of the body's H.renzhiensis,H.deginensis,H.alttdcoh W,H.biru immune function (Paterson,2008;Zhou et al.,2009: (Yin et al.,2004),H.pui sp.nov.and Thitarodes pui Zhu et al.,1998).Up to now,the medicinal and economic (Chen et al.,2004;Li et al.,2011a;Sasaki et al.,2008;Wang values of DCXC have been wildly realized and studied in Yao,2011;Yang et al.,2009;Zhang et al.,1989,2007). the world (Weckerle et al.,2010:Winkler.2008.2010: The host insects of the DCXC are mainly located in Zhang et al.,2009b). the zones of the Qinghai-Tibetan Plateau,which have the In recent years,the price of the DCXC has continuously characteristics of regional and vertical distribution. increased and has gradually become the main income source Each insect population has specific geographical location of local farmers and herdsmen.During the harvesting and distribution pattern.The vertical distribution of the genus season of every year,thousands of locals and migrants Hepialus ranges from 2200 to 5000 m above sea level.Most travel to the Sichuan-Tibet and Qinghai-Tibet Plateau of them are located around the altitude of 4000m.Among and search inch-by-inch for the DCXC.This has caused different zones,the range of vertical distribution is the widest serious eco-environmental damage to the local grassland. in Tibet province,followed by Sichuan and Yunnan provinces, Furthermore,the increase of population and human activities, and the narrowest in Qinghai and Gansu provinces.Except for accompanied by some natural factors like drought and water the adult period,the host insects live in the soil,whose eco- uepn d resources shortage,has made the degradation and desertifi- distribution is in respect of many natural factors,such as cation amount of grassland of this area up to 40%of the total terrain,landform,altitude,climate,vegetation and agrotype. grassland area,and meanwhile it is increasing at an annual Among them,the soil microclimate and foodstuffs are the rate of 3-5%.The enormous environmental pressure and most important factors closely related to the distribution of ecological problems have attracted the attention of many host insects.The host insects of Hepialus distribute every- scientists and governments (Bian et al.,2008;Li et al., where in the alpine,sub-alpine meadow and alpine shrubs,the 2011b).How to implement sustainable utilization of the soil types of which are the alpine meadow soil and sub-alpine DCXC resource on the Qinghai-Tibet Plateau has become an meadow soil (Boesi Cardi,2009;Li et al.,2011c:Liu et al.. urgent issue to be solved.In the recent 20 years,some 2007;Yanm,2001;Yang et al,1992,1996). research institutes and universities from China have paid a lot of efforts and made some progress under the support of the Biological characteristics Chinese government,such as the Major State Basic Research Development Program of China(973 Program),the National The host insects Hepialus of the DCXC are of complete High-tech R&D Program of China (863 Program)and the metamorphosis.There are four stages in its life cycle,i.e.egg, National Natural Science Foundation of China.Beside some larva,pupa and adult Chen et al.(1973).Although there are rules and regulations on the measures in policy and law,all some differences on the biological and ecological characters attentions have been focused on the following aspects: among the different Hepialus,they all have a long larva stage artificial generation of the moth larvae of Hepialus,artificial and an obvious alternate of generation as shown in Figure 日 generation of O.sinensis fungal strains,how the fungal spores 1(a).The insects of Hepialus need to take 3-4 years or even attack the moth larvae and artificial cultivation of DCXC >5 years in general to complete a life cycle: Meanwhile,a conclusion has been made that the eco- (1)About 500 eggs were laid by each female from June to environmental pressure of the grasslands can be relieved by August.Under the moisturizing and incubating condi- means of artificial intervention in the sustainable use of the tions,the developmental duration of eggs takes about 30- DCXC resource (Hu et al.,2005;Liu et al.,2010a,b; 40 days in Sichuan,Yunnan,Qinghai,Gansu and 70 days Zhu et al..2009) in Naqu of Tibet region.Under natural conditions,the hatchability of the larvae is about 80%in Naqu of Tibetan Breeding of the moth larvae of Hepialidae region,and the chorion from egg appearing to incubation goes through a change from white to black. Ecological characteristics of the family Hepialidae 2) The larval stage has six to eight instars,and different larval instars may bring about some differences on the Species and distribution duration of larval development.Since the larvae suffer Up to now,more than 50 species of the family Hepialidae various hazards from natural enemies during the entire have been found and identified in the main producing areas of developmental duration,then the survival rate of the the DCXC,among which,12 species appear in Sichuan,20 larvae in natural conditions are usually less than 10%. species in Yunnan,9 species in Qinghai,14 species in Tibet With the growth of the larvae,the head capsule will and 3 species in Gansu.In addition,some kinds of other change from milky white to light red or deep yellow family insects have also been discovered in these areas, during the adult (or mature)larvae stage.The larvae are including Hepialiscus,Forkalus,Bipectius and Magnificus. generally distributed in aggregation at a depth of 5-25 cm They may be related to the host insect of the DCXC in the soil.The change from larvae to pupa takes about RIGHTS L I N K
cough and haemoptysis’’. Based on the description of Pharmacopoeia of the People’s Republic of China (2005 ed.), the DCXC is also functional on curing impotence, spermatorrhea and the resolving pain in the knee and waist. Besides, modern medicines have proved that it possesses some functions, such as anti-tumor, anti-hyperglycemia (Lo et al., 2006) and significant improvement of the body’s immune function (Paterson, 2008; Zhou et al., 2009; Zhu et al., 1998). Up to now, the medicinal and economic values of DCXC have been wildly realized and studied in the world (Weckerle et al., 2010; Winkler, 2008, 2010; Zhang et al., 2009b). In recent years, the price of the DCXC has continuously increased and has gradually become the main income source of local farmers and herdsmen. During the harvesting season of every year, thousands of locals and migrants travel to the Sichuan-Tibet and Qinghai-Tibet Plateau and search inch-by-inch for the DCXC. This has caused serious eco-environmental damage to the local grassland. Furthermore, the increase of population and human activities, accompanied by some natural factors like drought and water resources shortage, has made the degradation and desertification amount of grassland of this area up to 40% of the total grassland area, and meanwhile it is increasing at an annual rate of 3–5%. The enormous environmental pressure and ecological problems have attracted the attention of many scientists and governments (Bian et al., 2008; Li et al., 2011b). How to implement sustainable utilization of the DCXC resource on the Qinghai-Tibet Plateau has become an urgent issue to be solved. In the recent 20 years, some research institutes and universities from China have paid a lot of efforts and made some progress under the support of the Chinese government, such as the Major State Basic Research Development Program of China (973 Program), the National High-tech R&D Program of China (863 Program) and the National Natural Science Foundation of China. Beside some rules and regulations on the measures in policy and law, all attentions have been focused on the following aspects: artificial generation of the moth larvae of Hepialus, artificial generation of O. sinensis fungal strains, how the fungal spores attack the moth larvae and artificial cultivation of DCXC. Meanwhile, a conclusion has been made that the ecoenvironmental pressure of the grasslands can be relieved by means of artificial intervention in the sustainable use of the DCXC resource (Hu et al., 2005; Liu et al., 2010a,b; Zhu et al., 2009). Breeding of the moth larvae of Hepialidae Ecological characteristics of the family Hepialidae Species and distribution Up to now, more than 50 species of the family Hepialidae have been found and identified in the main producing areas of the DCXC, among which, 12 species appear in Sichuan, 20 species in Yunnan, 9 species in Qinghai, 14 species in Tibet and 3 species in Gansu. In addition, some kinds of other family insects have also been discovered in these areas, including Hepialiscus, Forkalus, Bipectius and Magnificus. They may be related to the host insect of the DCXC (Liu et al., 1995; Yan, 2000). Nowadays, the biological characteristics of more than 10 species of genus insects have been well studied in China, which include H. armodcamu Oberthar, H. minyuancus (Wang et al., 2001a), H. yushusis Chuet Wang, H. lagii Yan (Xu, 2004), H. gonggaensis (Yin et al., 1995), H. guidera Yan, H. oblifurcus Chuet Wang, H. renzhiensis, H. deginensis, H. alttdcoh W, H. biru (Yin et al., 2004), H. pui sp. nov. and Thitarodes pui (Chen et al., 2004; Li et al., 2011a; Sasaki et al., 2008; Wang & Yao, 2011; Yang et al., 2009; Zhang et al., 1989, 2007). The host insects of the DCXC are mainly located in the zones of the Qinghai-Tibetan Plateau, which have the characteristics of regional and vertical distribution. Each insect population has specific geographical location and distribution pattern. The vertical distribution of the genus Hepialus ranges from 2200 to 5000 m above sea level. Most of them are located around the altitude of 4000 m. Among different zones, the range of vertical distribution is the widest in Tibet province, followed by Sichuan and Yunnan provinces, and the narrowest in Qinghai and Gansu provinces. Except for the adult period, the host insects live in the soil, whose ecodistribution is in respect of many natural factors, such as terrain, landform, altitude, climate, vegetation and agrotype. Among them, the soil microclimate and foodstuffs are the most important factors closely related to the distribution of host insects. The host insects of Hepialus distribute everywhere in the alpine, sub-alpine meadow and alpine shrubs, the soil types of which are the alpine meadow soil and sub-alpine meadow soil (Boesi & Cardi, 2009; Li et al., 2011c; Liu et al., 2007; Yan, 2001; Yang et al., 1992, 1996). Biological characteristics The host insects Hepialus of the DCXC are of complete metamorphosis. There are four stages in its life cycle, i.e. egg, larva, pupa and adult Chen et al. (1973). Although there are some differences on the biological and ecological characters among the different Hepialus, they all have a long larva stage and an obvious alternate of generation as shown in Figure 1(a). The insects of Hepialus need to take 3–4 years or even 45 years in general to complete a life cycle: (1) About 500 eggs were laid by each female from June to August. Under the moisturizing and incubating conditions, the developmental duration of eggs takes about 30– 40 days in Sichuan, Yunnan, Qinghai, Gansu and 70 days in Naqu of Tibet region. Under natural conditions, the hatchability of the larvae is about 80% in Naqu of Tibetan region, and the chorion from egg appearing to incubation goes through a change from white to black. (2) The larval stage has six to eight instars, and different larval instars may bring about some differences on the duration of larval development. Since the larvae suffer various hazards from natural enemies during the entire developmental duration, then the survival rate of the larvae in natural conditions are usually less than 10%. With the growth of the larvae, the head capsule will change from milky white to light red or deep yellow during the adult (or mature) larvae stage. The larvae are generally distributed in aggregation at a depth of 5–25 cm in the soil. The change from larvae to pupa takes about 2 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
D0L:10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis (a) (b) Adult insect Development of DCXC Pupa Egg-masses Infective larvae (3rd-5 th instar） Adult DCXC 5th instar larvae Young larvae Spread of spores Forming spore 3rd instar larvae Figure 1.The life cycle of the insect of the genus Hepialus(a)and the development process of the DCXC(b). mH有032 harc.con 2-4 years in light of the different species involved.For Dasiphora fruticosa (L.)Rydb.and Astragalus membranaceus example,H.yushusis larvae need to take about 996 days (Fisch.Bge.In addition,dozens of the tender roots from to become the pupa,H.kangdingensis 1000 days and H. different plant families can also be chewed,including gonggaensis 875-1040 days.The larvae with different Cyperaceae,Poaceae,Gentianaceae,Primulaceae and instars can be found in the soil all the year around. Juncaceae (Gu et al.,2006;Wu et al.,2007;Zhuo et al., (3)A part of larvae turn into the pupa at the end of every 2008) May,and the pupa stage for most of the larvae is from June to July.During this period,the developmental Artificial culture of the host larvae of Hepialus duration of the pupa needs to take 40 days,when the temperature is 10-15C and the soil relative humidity is The insects Hepialus of the DCXC need take 3-4 years to 40-45%.The peak period of the adult emergence differs finish an alternation of generations.The larval stage generally has six instars,or even seven to eight instars under special from the altitudes.The color variation of Hepialus moth environmental conditions.But,under the conditions of from pre-pupa to emergence is from light to dark. complete artificial rearing,the larvae only take 1-2.2 years (4)The emergence of pupa often happens at 17:00-20:00 to complete an alternation of generations (Yang et al.,2009). every day during June to August.In most producing If we want to implement the artificial cultivation of the areas,the amount of the female adult insects is larger than that of the males.In general,the peak of mating DCXC,the key techniques will be the artificial culture of the host larvae of Hepialus under the condition of soilless,the differs from the altitudes,and meanwhile the number of growth nutrition required and the prevention of diseases. mating is one for females and two to three for males.The g Based on the previous literature,the developmental rate of the duration from opulating to ovipositing is 5-40 min,and eggs may come to 86.75%under the conditions of relative the copulation is beneficial to the ovipositing of the humidity 50%and temperature 15C (Liu et al.,2009).The females.Similarly,to the mating,the peak of ovipositing nutritional components required by the larvae with five instars also varies with the altitude.After copulation,the females can be met by the artificial feeding formula,consisting of can lay about 5-45 eggs once on a nearby meadow.The Rheum pumilum 5.0,sorbic acids 0.5,soybean 15,corn flour longevity of the females is longer than that of the males, 10,wheat bran 8,yeast powder 5.0,agar 10 and water 100 but the females die soon after laying eggs (Liu et al., (g/L).The weight of larvae can be put on by adding the 2008:Wang et al.,2001a;Wang Yao,2011;Yang natural plants of Rheum pumilum,while sorbic acid and Jiang,1995:Yang et al.,1991;Zhu et al.,2007). nipagin esters,which will not affect the growth of larvae, Plant groups of the food of the host larvae can be added as antiseptic agents in the artificial feeds (Wang,2002). The host larvae of Hepialus are the omnivorous insects,which Since the 1970s,there are more than 10 departments lead a troglodytic life in the soil,and prefer to feed on the studying the artificial cultivation of DCXC,but only a few tender roots of plants.There are many plant species fed on by species of host insects were successfully raised in laboratory the host insects and the well-known underground stems of 15 conditions in China.For example,the Hepialus oblifurcus species plants of 10 families can be served as the food of the larvae and Hepialus gonggaensis larvae were artificially larvae.Among them,the larvae prefer to the tender root of raised at low-altitude area in the DCXC research base in Polygonaceae plants,such as Polygonum sphaerostachyum Kangding County by Chongqing Academy of Chinese Meisn,P.viviparum L.and Rheum pumilum Maxim.They can Materia Media.Meanwhile,Hepialus gonggaensis larvae also graze the tender root of Rosaceae plants,such as were also artificially raised at Zhejiang Agriculture RIGH T S L I N K
2–4 years in light of the different species involved. For example, H. yushusis larvae need to take about 996 days to become the pupa, H. kangdingensis 1000 days and H. gonggaensis 875–1040 days. The larvae with different instars can be found in the soil all the year around. (3) A part of larvae turn into the pupa at the end of every May, and the pupa stage for most of the larvae is from June to July. During this period, the developmental duration of the pupa needs to take 40 days, when the temperature is 10–15 C and the soil relative humidity is 40–45%. The peak period of the adult emergence differs from the altitudes. The color variation of Hepialus moth from pre-pupa to emergence is from light to dark. (4) The emergence of pupa often happens at 17:00–20:00 every day during June to August. In most producing areas, the amount of the female adult insects is larger than that of the males. In general, the peak of mating differs from the altitudes, and meanwhile the number of mating is one for females and two to three for males. The duration from opulating to ovipositing is 5–40 min, and the copulation is beneficial to the ovipositing of the females. Similarly, to the mating, the peak of ovipositing also varies with the altitude. After copulation, the females can lay about 5–45 eggs once on a nearby meadow. The longevity of the females is longer than that of the males, but the females die soon after laying eggs (Liu et al., 2008; Wang et al., 2001a; Wang & Yao, 2011; Yang & Jiang, 1995; Yang et al., 1991; Zhu et al., 2007). Plant groups of the food of the host larvae The host larvae of Hepialus are the omnivorous insects, which lead a troglodytic life in the soil, and prefer to feed on the tender roots of plants. There are many plant species fed on by the host insects and the well-known underground stems of 15 species plants of 10 families can be served as the food of the larvae. Among them, the larvae prefer to the tender root of Polygonaceae plants, such as Polygonum sphaerostachyum Meisn, P. viviparum L. and Rheum pumilum Maxim. They can also graze the tender root of Rosaceae plants, such as Dasiphora fruticosa (L.) Rydb. and Astragalus membranaceus (Fisch.) Bge. In addition, dozens of the tender roots from different plant families can also be chewed, including Cyperaceae, Poaceae, Gentianaceae, Primulaceae and Juncaceae (Gu et al., 2006; Wu et al., 2007; Zhuo et al., 2008). Artificial culture of the host larvae of Hepialus The insects Hepialus of the DCXC need take 3–4 years to finish an alternation of generations. The larval stage generally has six instars, or even seven to eight instars under special environmental conditions. But, under the conditions of complete artificial rearing, the larvae only take 1–2.2 years to complete an alternation of generations (Yang et al., 2009). If we want to implement the artificial cultivation of the DCXC, the key techniques will be the artificial culture of the host larvae of Hepialus under the condition of soilless, the growth nutrition required and the prevention of diseases. Based on the previous literature, the developmental rate of the eggs may come to 86.75% under the conditions of relative humidity 50% and temperature 15 C (Liu et al., 2009). The nutritional components required by the larvae with five instars can be met by the artificial feeding formula, consisting of Rheum pumilum 5.0, sorbic acids 0.5, soybean 15, corn flour 10, wheat bran 8, yeast powder 5.0, agar 10 and water 100 (g/L). The weight of larvae can be put on by adding the natural plants of Rheum pumilum, while sorbic acid and nipagin esters, which will not affect the growth of larvae, can be added as antiseptic agents in the artificial feeds (Wang, 2002). Since the 1970s, there are more than 10 departments studying the artificial cultivation of DCXC, but only a few species of host insects were successfully raised in laboratory conditions in China. For example, the Hepialus oblifurcus larvae and Hepialus gonggaensis larvae were artificially raised at low-altitude area in the DCXC research base in Kangding County by Chongqing Academy of Chinese Materia Media. Meanwhile, Hepialus gonggaensis larvae were also artificially raised at Zhejiang Agriculture Figure 1. The life cycle of the insect of the genus Hepialus (a) and the development process of the DCXC (b). DOI: 10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 3 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only.
4 X.-W.Zhou et al. Crit Rev Biotechnol,Early Online:1-11 University in Hangzhou by Gao Zuxun and his colleagues Determination of anamorph Subsequently,many problems related to the alternation of generations of H.armodcamu which include the pupa Anamorphic types emergence,adult mating,eggs hatching and breeding larvae In recent years,the artificial cultivation of the fungal strains were explored by the Grassland Research Institute of isolated from DCXC through the fermenting method has Academy of Animal and Veterinary Sciences of Qinghai become an intensive research topic.More than 30 species of Province.For over 40 years,a significant amount of research fungal strains belonging to 13 genera,which are involved in was focused on raising host larvae,but the average survival the anamorphic types of the DCXC,have been reported for raising host larvae in the experimental conditions was only in China.According to general international naming rules 0.1~7.7%.The survival rate of the host especially in large- of the botany,such varieties have been reported as Hirsutella scale raising conditions is too low.Therefore,the DCXC hepiali,Hirsutella sinensis,Paecilomyces hepiali, cannot be cultured in large-scale culture under artificial Paecilomyces sinensis,Mortierella hepiali,Cephdosporium conditions (Zhang et al.,2009a). sinensis, Scytalidium hepiali,Tolypocladium sinensis, Chrysosporium sinensis and Stachybotry.Among these Generation of the fungi isolated from the DCXC strains,Paecilomyces sinensis and Mortierella hepiali have X77000 been found not to be of the anamorphic types.Besides,five Isolation of fungal strains strains are not identified at the level of species and only have The primary separation of fungi from the DCXC materials their genus names (Jiang Yao,2003). is a relatively simple operational process.The fungi So many strains and all kinds of names have brought can be isolated from various tissues and organs in about a dispute regarding the classification standardization different development durations of the DCXC,such as of anamorphic types of the DCXC.At present,there has fruiting body,mummified caterpillar,living larvae and been some evidence implying that Hirsutella sinensis ascospore. X.J.Liu,Y.L.Guo,Y.X.Yu and W.Zeng belongs to the anamorph of the DCXC (Li Zeng,1998:Li et al., 2000;Liang,2001;Liu et al.,2001,2003a;Mo Zhang, Isolated from fruiting body,mummified caterpillar and 2001:Zhao Li.1999).However.the relationship ascospores between the anamorph and the DCXC needs to be further The fresh and complete DCXC,or the mummified cater- studied asn pillar dug in the soil,are generally selected as materials. The isolation procedure of both materials is the same with Identification of anamorphic types each other.First,the surface of the materials is sterilized with alcohol.Then the materials are carefully placed in In natural conditions,a number of filamentous fungi can water agar media and cultured at 20C.When the natural combine with the fruiting body of the DCXC.Among fruiting body with ascospore is selected as the material,the these fungi,some hetero-fungi are similar to the DCXC 8 common isolation method is to sterilize the fruiting body strain on ecological characteristics.Besides,it is possible with ascospore and then put the two extremes of stroma that two or more than two fungal strains grow in the same hang over the medium or the inclined plane of the potato host insect.Most of these fungi are usually difficult to dextrose agar (PDA)in cuvette.When the spores drop artificially culture.Therefore,many identification methods on the medium and begin to germinate,they are cultivated from nature are developed to determine the anamorph of g at20°C. the DCXC. (1)Identification of the anamorph based on the correlation to Isolated from the living larvae infected with fungi the teleomorphic of the DCXC.Koboyasi suggested five criteria for identification of the anamorphic:D having Conduct a microscopic examination to the body fluids of the some portion of stroma of the DCXC or asexual conidial bat moth larva which is collected in the field.After sterilizing fructification of branch;forming the asexual conidial the body surface of the larva,cut off the pleopod and squeeze fructification on the mycelium of the fruiting body in out the body fluids into the PDA medium.The body fluids are DCXC:3 forming the asexual conidial fructification on cultivated at 20C.After the primary clone emerging, the same DCXC host growing the fruiting body: individual hyphal tips are cut and placed on PDA medium, @forming the asexual conidial fructification on the and the morphology characteristics of mycelium are observed same insects but different DCXC fruiting bodies in the and identified (Zhou et al.,2010a). same region;forming the asexual conidial fructifica- Both the position of the strains separated and the isolation tion when the ascospore of the DCXC is inoculated on the method employed play an important role in the formation of media (Koboyasi,1949). the fruiting body.Compared with some other positions like (2)Identification of the anamorph based on the microcycle the sclerotium and stroma petiole,the strains separated from conidiation.Microcycle conidiation is a repeated con- the fruiting body are much easier to form the fruiting body. idiation phenomenon,in which no hypha or merely Besides,the strains derived from the ascospore are much scrannel grows after the conidiospore germinating of more advantageous in forming the fruiting body than those filamentous fungus.It is relatively simple,reliable and derived from other tissues (Li et al.,2006). practical to identify the anamorph of the DCXC based RIGHTS L I N K
University in Hangzhou by Gao Zuxun and his colleagues. Subsequently, many problems related to the alternation of generations of H. armodcamu which include the pupa emergence, adult mating, eggs hatching and breeding larvae were explored by the Grassland Research Institute of Academy of Animal and Veterinary Sciences of Qinghai Province. For over 40 years, a significant amount of research was focused on raising host larvae, but the average survival for raising host larvae in the experimental conditions was only 0.1 7.7%. The survival rate of the host especially in largescale raising conditions is too low. Therefore, the DCXC cannot be cultured in large-scale culture under artificial conditions (Zhang et al., 2009a). Generation of the fungi isolated from the DCXC Isolation of fungal strains The primary separation of fungi from the DCXC materials is a relatively simple operational process. The fungi can be isolated from various tissues and organs in different development durations of the DCXC, such as fruiting body, mummified caterpillar, living larvae and ascospore. Isolated from fruiting body, mummified caterpillar and ascospores The fresh and complete DCXC, or the mummified caterpillar dug in the soil, are generally selected as materials. The isolation procedure of both materials is the same with each other. First, the surface of the materials is sterilized with alcohol. Then the materials are carefully placed in water agar media and cultured at 20 C. When the natural fruiting body with ascospore is selected as the material, the common isolation method is to sterilize the fruiting body with ascospore and then put the two extremes of stroma hang over the medium or the inclined plane of the potato dextrose agar (PDA) in cuvette. When the spores drop on the medium and begin to germinate, they are cultivated at 20 C. Isolated from the living larvae infected with fungi Conduct a microscopic examination to the body fluids of the bat moth larva which is collected in the field. After sterilizing the body surface of the larva, cut off the pleopod and squeeze out the body fluids into the PDA medium. The body fluids are cultivated at 20 C. After the primary clone emerging, individual hyphal tips are cut and placed on PDA medium, and the morphology characteristics of mycelium are observed and identified (Zhou et al., 2010a). Both the position of the strains separated and the isolation method employed play an important role in the formation of the fruiting body. Compared with some other positions like the sclerotium and stroma petiole, the strains separated from the fruiting body are much easier to form the fruiting body. Besides, the strains derived from the ascospore are much more advantageous in forming the fruiting body than those derived from other tissues (Li et al., 2006). Determination of anamorph Anamorphic types In recent years, the artificial cultivation of the fungal strains isolated from DCXC through the fermenting method has become an intensive research topic. More than 30 species of fungal strains belonging to 13 genera, which are involved in the anamorphic types of the DCXC, have been reported in China. According to general international naming rules of the botany, such varieties have been reported as Hirsutella hepiali, Hirsutella sinensis, Paecilomyces hepiali, Paecilomyces sinensis, Mortierella hepiali, Cephdosporium sinensis, Scytalidium hepiali, Tolypocladium sinensis, Chrysosporium sinensis and Stachybotry. Among these strains, Paecilomyces sinensis and Mortierella hepiali have been found not to be of the anamorphic types. Besides, five strains are not identified at the level of species and only have their genus names (Jiang & Yao, 2003). So many strains and all kinds of names have brought about a dispute regarding the classification standardization of anamorphic types of the DCXC. At present, there has been some evidence implying that Hirsutella sinensis X. J. Liu, Y. L. Guo, Y. X. Yu and W. Zeng belongs to the anamorph of the DCXC (Li & Zeng, 1998; Li et al., 2000; Liang, 2001; Liu et al., 2001, 2003a; Mo & Zhang, 2001; Zhao & Li, 1999). However, the relationship between the anamorph and the DCXC needs to be further studied. Identification of anamorphic types In natural conditions, a number of filamentous fungi can combine with the fruiting body of the DCXC. Among these fungi, some hetero-fungi are similar to the DCXC strain on ecological characteristics. Besides, it is possible that two or more than two fungal strains grow in the same host insect. Most of these fungi are usually difficult to artificially culture. Therefore, many identification methods from nature are developed to determine the anamorph of the DCXC. (1) Identification of the anamorph based on the correlation to the teleomorphic of the DCXC. Koboyasi suggested five criteria for identification of the anamorphic: A having some portion of stroma of the DCXC or asexual conidial fructification of branch; B forming the asexual conidial fructification on the mycelium of the fruiting body in DCXC; C forming the asexual conidial fructification on the same DCXC host growing the fruiting body; D forming the asexual conidial fructification on the same insects but different DCXC fruiting bodies in the same region; E forming the asexual conidial fructification when the ascospore of the DCXC is inoculated on the media (Koboyasi, 1949). (2) Identification of the anamorph based on the microcycle conidiation. Microcycle conidiation is a repeated conidiation phenomenon, in which no hypha or merely scrannel grows after the conidiospore germinating of filamentous fungus. It is relatively simple, reliable and practical to identify the anamorph of the DCXC based 4 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.
D0L:10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 5 on microcycle conidiation (Mo Zhang,2001).Chen However,so far,only a few Cordyceps species such as and his colleagues studied the anamorph using the tissue C.brongniartii,C.norvegica,C.militaris,C.takaomon- isolation techniques,and then selected the colony tana,C.pruinosa,C.clavulata,C.sobolifera and producing conidiation coremium to make the purification C.tuberculate have been reported,and they can produce by means of amerosporous isolation.The purified strains a sexual fruiting body (Liu Liang,1996;Mo Zhang, were used to study their pharmacological functions. 2001;Shimazu et al.,1988).Although there are many Compared with the natural DCXC on chemical compos- difficulties in the artificial inducement of the fruit bodies ition and pharmacology.the preliminary results sug- of DCXC,actually,it is not necessary for practical gested that the Paecilomyces sinensis is the anamorph of application to strictly determine the anamorph of the the DCXC(Chen et al.,1984).Using the same methods, DCXC.However,it is necessary for us to determine the Li Sun (1988)reported the other two strains, real relationship between teleomorph and anamorph. Scytalidium hapiali and Tolypocladium sinensis are also (5)Confirmation based on the protein fingerprinting.The the anamorph.Through isolation,culture and identifica- protein fingerprinting used for the determination of tion,H.sinensis was found to be the anamorph of the the DCXC anamorph includes a series of processes as DCXC in Naqu Tibet (Chen Zhou,2001;Zhang et al., follows:isoelectric focusing,SDS-PAGE,non-coherent 2010).As a result of the conditional differences between acetic acid urea-polyacrylamide gel electrophoresis, secondary ascospore germination and microcycle con- matrix-assisted laser desorption ionization-time of flight idiation,it is difficult to operate microcycle conidiation mass spectrometer and peptide mass fingerprinting.Chen on a small amount of ascospores.In addition,before the Dai (1995)compared more than 10 strains isolated complete DCXC are cultured in batches with the sexual from natural DCXC through the isoenzyme and all strains,it is difficult to determine the anamorph of the proteinase fingerprinting techniques,and the results DCXC (Liang,1991). provided the reliable evidence for identifying DCXC (3)Identification based on the isolation and culture of species.Some researchers have also studied the protein isolates.The isolation and culture methods for identify- electrophoresis within the asexual phase of the DCXC, ing anamorph include:validating each other after and found that the isolated products of the strains isolation and cultivation with many batches and various correlated with the natural DCXC in sexual phase.To paths;picking the germinated monospores for the some extent,the protein fingerprinting reflects their purifying culture:validating each other after obtaining intrinsic relationships.Thus,the fungi collected in many the cultural products of mycelia isolating using tissue and batches and paths can be analyzed using SDS-PAGE to spore isolation techniques;comparing the fermenta- assist in identifying the controversial fungi(Yang,2005). tion products of isolation fungi with the native DCXC. (6)Confirmation based on the DNA fingerprinting.DNA confirming whether they have similar chemical compos- fingerprinting mainly depends on the molecular markers ition and pharmacological function,or the same protein of different species,and the DNA fragment from all kinds band after running the sodium dodecyl sulphate-poly- of biological specimens can be synthesized by polymer- acrylamide gel electrophoresis(SDS-PAGE),or the same ase chain reaction techniques,while the fragment size antigenicity and crossing-over serology relationship by and number are different in various organisms.In the past means of rocket line immune-electrophoresis analysis: 10 years,the standard and reliable methods at the validating whether pure culture products are consist- molecular level have been developed for identifying ent with the native DCXC in pharmacological functions traditional Chinese medicine (TCM),mainly including (Wang et al.,1998).Although these indirect methods the random-amplified polymorphic DNA (RAPD), cannot be used to accurately identify the real relationship simple sequence repeat (SSR),restriction fragment between the anamorph and teleomorph of the DCXC, length polymorphism (RFLP),amplified fragment their alternate use and mutual validation are very helpful length polymorphism,sequence characterized amplified to avoid misidentifying of the anamorph. regions,inter-simple sequence repeat (ISSR),sequence- The anamorph separation of ascospore is a traditional related amplified polymorphism,single nucleotide poly- and reliable method,but is not suitable for the fungus hard to morphism and telomeric repeat amplification protocol culture.For some species whose ascospore is hard to (Zhou et al.,2006).Using the RAPD technique,Li et al. germinate,the anamorph of them can be obtained by tissue (2000)isolated the H.sinensis from the DCXC fruiting isolation method.But it needs to be separated in many batches body in Qinghai,and gained the genomic DNA finger- and various paths and it is difficult to avoid the probability of printing of the DCXC and H.sinensis,respectively,the misidentification. similarity rate of which reached up to 96%.This result (4)Confirmation based on artificial induction of fruiting suggested H.sinensis is the anamorph of the DCXC (Li body.According to Koch's postulation,the determination et al.,2000).Using the internal transcribed spacer(ITS) of the DCXC anamorph under the experimental condition sequences of ribosomal DNA (rDNA)as marks,Zhao is to induce postulate anamorph artificially infected Li (1999)also studied the anamorph and teleomorph insect(or medium)to form the DCXC fruiting body with of the DCXC which grow in Tibet.Based on comparison mature perithecium.Based on this thought,people have and analysis of the sequences of rDNA ITS,the made tireless efforts to culture the DCXC fruiting body results also suggested that H.sinensis is the anamorph with mature perithecium to determine the anamorph of DCXC.Using RAPD technique,Chen et al.analyzed RIGH T S L I N K
on microcycle conidiation (Mo & Zhang, 2001). Chen and his colleagues studied the anamorph using the tissue isolation techniques, and then selected the colony producing conidiation coremium to make the purification by means of amerosporous isolation. The purified strains were used to study their pharmacological functions. Compared with the natural DCXC on chemical composition and pharmacology, the preliminary results suggested that the Paecilomyces sinensis is the anamorph of the DCXC (Chen et al., 1984). Using the same methods, Li & Sun (1988) reported the other two strains, Scytalidium hapiali and Tolypocladium sinensis are also the anamorph. Through isolation, culture and identification, H. sinensis was found to be the anamorph of the DCXC in Naqu Tibet (Chen & Zhou, 2001; Zhang et al., 2010). As a result of the conditional differences between secondary ascospore germination and microcycle conidiation, it is difficult to operate microcycle conidiation on a small amount of ascospores. In addition, before the complete DCXC are cultured in batches with the sexual strains, it is difficult to determine the anamorph of the DCXC (Liang, 1991). (3) Identification based on the isolation and culture of isolates. The isolation and culture methods for identifying anamorph include: A validating each other after isolation and cultivation with many batches and various paths; B picking the germinated monospores for the purifying culture; C validating each other after obtaining the cultural products of mycelia isolating using tissue and spore isolation techniques; D comparing the fermentation products of isolation fungi with the native DCXC, confirming whether they have similar chemical composition and pharmacological function, or the same protein band after running the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), or the same antigenicity and crossing-over serology relationship by means of rocket line immune-electrophoresis analysis; E validating whether pure culture products are consistent with the native DCXC in pharmacological functions (Wang et al., 1998). Although these indirect methods cannot be used to accurately identify the real relationship between the anamorph and teleomorph of the DCXC, their alternate use and mutual validation are very helpful to avoid misidentifying of the anamorph. The anamorph separation of ascospore is a traditional and reliable method, but is not suitable for the fungus hard to culture. For some species whose ascospore is hard to germinate, the anamorph of them can be obtained by tissue isolation method. But it needs to be separated in many batches and various paths and it is difficult to avoid the probability of misidentification. (4) Confirmation based on artificial induction of fruiting body. According to Koch’s postulation, the determination of the DCXC anamorph under the experimental condition is to induce postulate anamorph artificially infected insect (or medium) to form the DCXC fruiting body with mature perithecium. Based on this thought, people have made tireless efforts to culture the DCXC fruiting body with mature perithecium to determine the anamorph. However, so far, only a few Cordyceps species such as C. brongniartii, C. norvegica, C. militaris, C. takaomontana, C. pruinosa, C. clavulata, C. sobolifera and C. tuberculate have been reported, and they can produce a sexual fruiting body (Liu & Liang, 1996; Mo & Zhang, 2001; Shimazu et al., 1988). Although there are many difficulties in the artificial inducement of the fruit bodies of DCXC, actually, it is not necessary for practical application to strictly determine the anamorph of the DCXC. However, it is necessary for us to determine the real relationship between teleomorph and anamorph. (5) Confirmation based on the protein fingerprinting. The protein fingerprinting used for the determination of the DCXC anamorph includes a series of processes as follows: isoelectric focusing, SDS–PAGE, non-coherent acetic acid urea–polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometer and peptide mass fingerprinting. Chen & Dai (1995) compared more than 10 strains isolated from natural DCXC through the isoenzyme and all proteinase fingerprinting techniques, and the results provided the reliable evidence for identifying DCXC species. Some researchers have also studied the protein electrophoresis within the asexual phase of the DCXC, and found that the isolated products of the strains correlated with the natural DCXC in sexual phase. To some extent, the protein fingerprinting reflects their intrinsic relationships. Thus, the fungi collected in many batches and paths can be analyzed using SDS–PAGE to assist in identifying the controversial fungi (Yang, 2005). (6) Confirmation based on the DNA fingerprinting. DNA fingerprinting mainly depends on the molecular markers of different species, and the DNA fragment from all kinds of biological specimens can be synthesized by polymerase chain reaction techniques, while the fragment size and number are different in various organisms. In the past 10 years, the standard and reliable methods at the molecular level have been developed for identifying traditional Chinese medicine (TCM), mainly including the random-amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism, sequence characterized amplified regions, inter-simple sequence repeat (ISSR), sequencerelated amplified polymorphism, single nucleotide polymorphism and telomeric repeat amplification protocol (Zhou et al., 2006). Using the RAPD technique, Li et al. (2000) isolated the H. sinensis from the DCXC fruiting body in Qinghai, and gained the genomic DNA fingerprinting of the DCXC and H. sinensis, respectively, the similarity rate of which reached up to 96%. This result suggested H. sinensis is the anamorph of the DCXC (Li et al., 2000). Using the internal transcribed spacer (ITS) sequences of ribosomal DNA (rDNA) as marks, Zhao & Li (1999) also studied the anamorph and teleomorph of the DCXC which grow in Tibet. Based on comparison and analysis of the sequences of rDNA ITS, the results also suggested that H. sinensis is the anamorph of DCXC. Using RAPD technique, Chen et al. analyzed DOI: 10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 5 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only