Molecular Co‐Existence of Paecilomyces hepiali and Hirsutella sinensis in Caterpillar and Fruiting Bodies of Cordyceps sinensis

Cordyceps sinensis (Cs) is a precious tonic herb used in China for centuries. We reported simultaneous isolation of 2 fungi, Hirsutella sinensis (Hs) and Paecilomyces hepiali (Ph), from fresh Cs and optimized a molecular technique for confirming their co‐existence (FASEB J 19: 2005; 20:A431, 2006). Using a Dual‐PCR technique, we examined fungus DNA in caterpillar and fruiting bodies of 100 pieces of Cs freshly collected in early May on Qinghai‐Tibetan Plateau. ITS4/5 primers were used in the 1st PCR. The amplified DNA segments, a pool of ITS4/5 DNA of fungi, were used as the templates for further identification in the 2nd PCR, where pairs of Ph specific primers and a touch‐down PCR protocol were designed (an annealing temperature initially at 70 degree C with 0.3 degree C decrement for each cycle, for 35 cycles). The Dual‐PCR products were examined by DNA sequencing and endonuclease digestions with AvaI and AluI for Hs, or BamHI and AccI for Ph. As the result, Ph DNA segments were amplified from genomic DNA isolated from 98 pieces of caterpillar bodies and 98 pieces of fruiting bodies of freshly collected Cs. Our results further evidenced molecular existence of Ph DNA in both caterpillar and fruiting bodies of natural Cs, in addition to Hs, strongly supporting the multi‐fungi hypothesis for natural Cordyceps sinensis.