Studies On Biology Of Ophiocordyceps Lanpingensis H.Yu&Z.H. Chen

Ophiocordyceps lanpingensis H. Yu&Z. H. Chen was mainly distributed in northwest Yunnan and was a close phylogenetic affinity species of Ophiocordyceps sinensis, a famous and precious Chinese traditional medicine. Biological studies of O. lanpingensis were performed from several aspects, including taxonomic status, ecological investigation, functional component, the separation and extraction of metabolite, and medicinal value. The attempt was to lay the foundation for the development and utilization of O. lanpingensis and it had an important significance for remitting the resources lack of O. sinensis.1The classification of O. lanpingensis was identified based on morphology characters and the molecular data analyses. This species was belonged to Ophiocordyceps genus and characterized by thinner stroma, smaller perithecium, thinner ascospore (multiseptate with short septation). The phylogenetic analyses based on the ITS sequences of O. lanpingensis and its related species in Ophiocordyceps showed that O. lanpingensis had closer evolution relationship with Ophiocordyceps robertsii, Ophiocordyceps rubiginosoperitheciata, Ophiocordyceps stylophora and O. sinensis. The phylogenetic analyses based on5-gene dataset and the mating type gene showed that O. lanpingensis had the closest evolution relationship with O. robertsii and O. sinensis, but still had obvious distances to them. The amino acids sequences of the genes tef-la, rpb1, rpb2and MAT1-2-1were respectively aligned among the3related species. Their sequences of tef-la and rpbl exhibited high identities (98%-99%). However, obvious variations occurred at the rpb2and MAT1-2-1sequences and the heterogeneities were above5%. Both morphological characteristics and systematic analysis supported that O. lanpingensis was a new species of Ophiocordyceps and it was a sister species of O. sinensis.According to Koch’s postulate, the anamorph of O. lanpingensis was Hirsutella lanpingensis H. Yu&Z. H. Chen. Thitarodes yunnanensis larva was inoculated by H. lanpingensis. After the artificial culturation for more than50d, the fruitbody of O. lanpingensis was successfully cultured and the morphology of its stroma was similar to natureal O. lanpingensis.2The ecological environment of O. lanpingensis was investigated. Such as the elevation, vegetation, insect host, entomogenous fungi, soil, air temperature, air humidity, exposure and so on, were surveyed in the present study.O. lanpingensis was mainly spreaded over Hengduan Mountains Region and was a new species generated during the process of orogenic movements. It was distributed in mountain grass of2,600-3,400m elevation. The dominant species of vegetation were herbages such as Galinsoga parviflora, Artemisia argyi, Rumex dentatus and Iris bulleyana. The host of O. lanpingensis was T. yunnanensis and Thitarodes jianchuanensis, whose food was various, mainly the young roots of herbaceous plant, such as Artemisia argyi, Iris bulleyana, Sambucus adnata, Fragaria nilgerrensis, Cirsim chlorolepis, occasionally the humus of the litter layer. Several entomogenous fungi existed in the habitat of O. lanpingensis, such as Isaria tenuipes, Isaria hepiali, Beauveria bassiana and Isaria farinosa. The soil types of its habitat were brown or black, being subacidity, and the moisture content was within34%-42%. The air humidity was from53%to67%. The annual average temperature was10.7-11.7℃, while the temperature in the growth season of O. lanpingensis was10-21℃. O. lanpingensis existed exposure selection of shady slopes and sunny slope with the difference of temperature, air humidity and elevation.3The culture process of H. lanpingensis was optimized. The strains of H. lanpingensis from4sample plots had been conducted active component analysis and the quality isolate OLLJ14was screened to have the optimum functional component. The culture process of the isolate was optimized, and the best culture condition for producing adenosine was selected as the OLM-3medium, culturing at14-16℃constantly for30days. Natural samples of O. lanpingensis should be harvested in mid to late June, when their functional component accumulated to the peak and their quality was the best. The active ingredients of O. lanpingensis and H. lanpingensis were similar to O. sinensis of Tibet.4The metabolic products of H. lanpingensis were separated and extracted with traditional solvent extraction method. The extracts were divided into different parts according to the solvents: petroleum ether, ethyl acetate, absolute ethyl alcohol and water. Seven monomeric compounds were separated and purified. All the seven compounds were known composition, such as β-Sitosterol, ursolic acid and D-mannitol and uridine had important biological function. Ursolic acid was separated from Cordyceps s.1. for the first time.5The results of pharmacology experiment suggested that the crude polysaccharide of H. lanpingensis culture had the effect of delaying senescence. The0.3%crude polysaccharide of H. lanpingensis culture could pull through the decrepit Drosophila melanogaster larva caused by propionic acid, completing the larva their whole life history, no statistical differences comparing with the control. The0.2%crude polysaccharide could delay the average lifetime and the time of half lethal, improve the maximum life-span of male and female D. melanogaster, reaching statistically significant level. It was showed that the sex differences was obvious, and the delay rate of lifetime was much higher in female insect than male insect.The crude extract of H. lanpingensis culture, including petroleum ether part, ethyl acetate part, absolute ethyl alcohol part and water part, all had very significant difference comparing with normal saline on reducing the writhing times in mice induced by HAc. It was suggested that all the polar parts of H, lanpingensis culture had prominent analgesic effects to the peripheral neurogenic pains. The combination of all the polar parts of H. lanpingensis culture was better to restrain the peripheral neurogenic pains.In conclusion, the systematic position of O. lanpingensis was closer to O. sinensis, being as the sibling species of O. sinensis. The active ingredients of O. lanpingensis teleomorph and anamorph culture were both similar to O. sinensis. H. lanpingensis culture had the effect of delaying senescence and analgesia. Therefore, O. lanpingensis was well potential to substitution of O. sinensis.