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 KUniversity 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 large￾scale 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 cater￾pillar 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 fructifica￾tion 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 con￾idiation 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.