Tarzan Caterpillar Fungus Resources Multi-gene Molecular Studies

Cordyceps,which belonged to Clavicipitaceae,Hypocreales,Pyrenomucetes,Ascomycota, is a large group of fungi with more than 400 species.Its distribution is global,and there are at less 143 species in china.Three species were found in Shandong province,Cordyceps taishanensis,Cordyceps militaris,Cordyceps ophioglossoides Link forma alba.Three strains were isolated from C.taishanensis and four representative species of Cordyceps collected from Taishan Moutain.The nucleotide sequences of rDNA ITS,18S and 28S rRNA gene from several species of Cordyceps were determined to provide molecular evidences for the asexual satage of three isolates,to examine the phylogenetic relationship among Cordyceps and its probable anamorph and to provide a theoretical basis for the development and utilization of Cordyceps resources.The main results were as follows:1.Based on the Cordyceps fungus universal primers designed by White,the nucleotide sequences of rDNA ITS regions(including ITS1 and ITS2),partial 18S and 28S rRNA genes were amplified by PCR assay in order to clone and sequence.The sequences were analyzed by the application of software such as ClustalX,MEGA4,DNASP4,PAUP and so on.The ITS region,18S and 28S rDNA of seven species of Cordyceps are respectively 567～587 bp,372 bp, 925～928 bp in length.2.Three strains of COA1,COA2,COA3 were isolated from ascocarps of Cordyceps Taishanensis.The entire ITS of COA1 is 528 bp in length,530 bp for COA2,and 548 bp for COA3.According to comparing with database of GenBank,there was only one base pair difference between COA1 and Paecilomyces militaris,COA2 and Beauveria bassiana,COA3 and Paecilomyces tenuipes,and COA1 has a similarity of 99%with Paecilomyces militaris, COA2 has a similarity of 99%with Beauveria bassiana;and COA3 has a similarity of 99%with Paecilomyces tenuipes,which suggested that the asexual stage of COA1 could be Paecilomyces militaris,the anamorph of COA2 could be Beauveria bassiana,and the anamorph of COA3 could be Paecilomyces tenuipes.3.Single-gene analyses were done on ITS sequences and the 18S and 28S rDNA sequences respectively.The maximum parsimony analysis produced three dendrograms derived from the alignment of the ITS sequences.The topology of the dendrogram shows that the KY,COA1, COA4,COA5 and COA6 form a clade,COA2 forms a clade,and COA3 forms a clade.The maximum parsimony phylogenetic tree constructed on 18S rDNA was similar to the MP tree from the ITS sequences at the first clade,but the COA1 was separated from this clade.In the second clade,COA2,COA3 and C.takaomontana together,C.brongniartii,C.bassiana were another clade.The results of the maximum parsimony based on 28S rDNA sequences supported that KY with COA4,COA5,COA6,C.militaris constitute a clade.The relationship between COA3 and C.takaomontan had been fully reflected in the three MP trees.Combined with the ITS,18S,28S rDNA genes,the data of the maximum parsimony analysis supported that COA4,COA5,COA6,C militaris,C.roseostromata,KY constitute a clade,and mergered with COA1 as a sister group.And the COA3 and C takaomontana formed a clade as a sister group,and mergered together with COA2,C.brongniartii,C.bassiana.This is consistent with the results based on ITS sequences.Therefore,Cordyceps distributed in the Taishan Mountain can be roughly divided into 3 categories.4.Fruiting body of C.kyushuensis was studied by light microscopy and scanning electron microscopy techniques.The results indicated that C.kyushuensis and C.taishanensis are extremely similar in the shape of fruit body,the array of asci,the embedding pattem of asci and the shape of perithecium.The ITS region,nrSSU and nrLSU sequences of C.kyushuensis and C. taishanensis were cloned and sequenced.By making a comparison between them,the result showed that the base diversity were less,and the homology up to 99%.Combined morphological and molecular biology characteristics,it deduced that they are synonyms.