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 Kon 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 compos￾ition and pharmacology, the preliminary results sug￾gested 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 identifica￾tion, 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 con￾idiation, 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 identify￾ing 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 fermenta￾tion products of isolation fungi with the native DCXC, confirming whether they have similar chemical compos￾ition and pharmacological function, or the same protein band after running the sodium dodecyl sulphate–poly￾acrylamide 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 consist￾ent 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. takaomon￾tana, 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 polymer￾ase 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), sequence￾related amplified polymorphism, single nucleotide poly￾morphism 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 finger￾printing 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