The Insect Pathogenicity of Cordyceps militaris - a molecular, metabolic, ecological, and phylogenetic study

虫草素（Cordycepin），或3 '-脱氧腺苷（3'-deoxyadenosine），是由世界性昆虫病原“冬虫夏草”（Cordyceps millitaris）及其近缘物种分泌的次级代谢产物。该代谢物以其抗炎作用而闻名，因此几个世纪以来一直被用作传统中药，近年来也是药理学研究的焦点。然而，虫草素和其他次级代谢产物在昆虫幼虫宿主感染中的生态学作用以及这些物质对这一过程后发生的性发育的参与的研究尚处于起步阶段。培养退化是在广泛的子囊菌中观察到的一种现象，是在随后的营养状态下继代培养后丧失有性发育和进行部分次级代谢的能力。本文以蛹虫草（Cordyceps militaris，CM 2）为材料，在马铃薯葡萄糖培养基上进行了虫草素高产菌株的继代培养。通过液相色谱（LC）-偶联质谱（MS）对虫草素和相关代谢物喷司他丁进行靶向分析，结合使用RT-qPCR的基因表达分析，提供了性发育和次级代谢物生物合成的表型和基因表达之间存在联系的证据。虫草素生产大大减少退化，多次传代培养的样品，虽然生产部分恢复后，这些长期转移到一个新的媒体类型的基础上的主机基板（丝蛹粉琼脂）。性发育基因的表达显着下降，以及虫草素和相关的生物合成基因也被检测到。进行非靶向LC-MS分析以比较退化和非退化菌株。

Cordycepin, or 3'-deoxyadenosine, is a secondary metabolite secreted by the cosmopolitan entomopathogenic "caterpillar fungus" Cordyceps millitaris and closely related species. The metabolite is well known for its anti-inflammatory effects and thus has been used as both a traditional Chinese medicine for centuries and a focus of pharmacological research in recent years. However, studies on the ecological roles of cordycepin and other secondary metabolites in the infection of the insect larval host and the involvement of these on the sexual development which occurs following this process are in their infancy. Culture degeneration, a phenomenon observed in a broad range of ascomycete fungi, is the loss of ability to develop sexually and perform parts of secondary metabolism following subsequent subculturing in the vegetative state. This is a significant problematic issue in cordycepin production from laboratory and industrial-based cultures.Here, a Chinese strain of Cordyceps militaris, CM2 (MycoMedica) with high cordycepin production was successively sub-cultured on potato dextrose agar. Targeted analysis of cordycepin and associated metabolite pentostatin by liquid chromatography (LC)-coupled mass spectrometry (MS), combined with a gene expression analysis using RT-qPCR provided evidence of a link between phenotypes and gene expression of both sexual development and secondary metabolite biosynthesis. Cordycepin production was greatly reduced in degenerated, multiply-subcultured samples; although production was partially restored following long-term transfer of these to a new media type based on the host substrate (silk pupa powder agar). Significant decreases in the expression of a sexual development gene, as well as cordycepin and related biosynthesis genes were also detected. Untargeted LC-MS analysis was performed to compare degenerated and non-degenerated strains.