Molecular Identification For Connection Of Cordyceps And ITS Allies

The host ranges of Cordyceps genus fungi are narrow.However,there are two different stages,i.e.teleomorph and anamorph,on its life history.The Koch's postulates are the most reliable methods to confirm the relationship between teleomorph and anamorph.It is hard to obtain the same fruiting bodies,even inoculating the anamorphic isolate to the same host,because it is difficulty to simulate the natural growth conditions of many Cordyceps spp.Several other biological methods have different limitations as well.However,in recent years,molecular biology tools show good prospects for determination of the teleomorph-anamorph connections.ITS area is a part of transcription unit,including two non-coding ITS1 and ITS2 spacers.The ITS region is short and is easy relatively for PCR amplification. The region has the conservative length and high variability of nucleotide sequence. Therefore,the teleomorph-anamorph connections can be conformed by analysing and comparing the ITS sequences of the Cordyceps specimens and the assumptive anamorphic isolates and make up for some shortcomings of biological methods.In this paper,the relationships between teleomorph and anamorph of Cordyceps were studied by ribosomal ITS sequence(rDNA ITS) analysis on the basis of biological and morphological investigations and morphological features of several anamorphs of Cordyceps were observed as well.The main results are reported as follows:Cordyceps formosana and its anamorph Hirsutella huangshanensis have the same ITS sequences and the molecular biological evidence confirms the morphological results.In the phylogenetic tree including Hirsutella huangshanensis and some other Hirsutella spp,C.formosana and H.huangshanensis are in a same clade with 100% bootstrap support.Phylogenetic tree can be divided into two main clades,one including C.sinensis,H.sinensis,C.coehlidiicola,C.heteropoda and H.rhossiliensis, the other including of C.formosana,H.huangshanensis and C.heteropoda.The results showed that C.formosana and H.huangshanensis are the same species at different stages of growth,i.e.H,huangshanensis is the anamorph of C.formosana, and the conidial state of C.formosana belonged to the genus Hirsutella is reasonable.C.heteropoda var.langyashanensis and its anamorph H.heteropoda are the same ITS sequences too.In the phylogenetic tree including H.heteropoda and some other Hirsutella spp,C.heteropoda var.langyashanensis and H.heteropoda are in a same clade with 100%bootstrap support.The relationship between C.heteropoda var. langyashanensis and C.heteropoda are the closest of all,and the differences are obvious with other Hirsutella spp,Beauveria and Metarhizium.The results indicated that H.heteropoda is the anamorph of C.heteropoda var.langyashanensis and it is reasonable to be belonged to the genus Hirsutella.The molecular biological evidence confirms the morphological results.Meanwhile,seven species of Cordyceps and its allies,i.e.Mariannaea pruinosa, Hirsutella formicarum,Nomuraea atypicola,Hirsutella longissima,Paecilomyces tenuipes and the two assumptive anamorphs of C.kusanagiensis and C.martialis were also sequenced and compared.The results showed that the former 5 species were the anamorphs of C.pruinosa,C.formicarum,C.cylindrica,C.longissima and C. takaomontana.The isolation of assumptive anamorphs of C.kusanagiensis and C. martialis are correct.C.bifusispora identified by in our laboratory several years ago might probably be C.pruinosa.The sequences between Paecilomyces farinosus and P. hepialid have high similarity and might probably be the synonym.We also found that the sequence of C.martialis is similar to the genus Pochonia by comparison to GenBank.The findings supported the morphological results too.The sequence of C. kusanagiensis is similar to Verticillium Nees and Metarrhizium by comparison to GenBank and it is real taxonomic status need more further study.