蛹虫草菌株退化特征及鉴别方法

蛹虫草菌株在继代培养和低温长期保藏过程中极易退化导致子实体产量下降，对产业造成重大影响。本研究以正常菌株、PDA斜面长期4 ℃保藏导致退化的菌株和连续继代培养的菌株为材料，观察其子实体、菌落、菌丝形态和分生孢子数量，对菌丝细胞核、线粒体、活性氧积累和芽生孢子内脂滴进行染色观察；并对菌丝中虫草素、腺苷和麦角甾醇含量进行比较分析。结果表明PDA斜面长期4 ℃保藏和连续继代培养导致的蛹虫草菌株退化表型大多数一致，即退化菌株较正常菌株子实体产量降低、菌丝粘连打结、分生孢子数量显著降低、菌丝活性氧含量升高、细胞内线粒体数量减少、芽生孢子脂滴由弥散的小脂滴融合为大脂滴。然而，在菌落见光转色方面，长期低温保藏退化菌株基本不转色，而继代培养退化菌株转色不稳定；长期保藏退化菌株菌丝细胞核数目无明显变化，继代培养退化菌株细胞核数量明显减少；长期保藏退化菌株菌丝中虫草素、腺苷和麦角甾醇含量较正常菌株降低58%、41%和70%，继代培养退化菌株麦角甾醇含量无明显变化。因此，显微观察菌丝是否出现粘连打结及氮蓝四唑NBT检测菌丝活性氧含量，操作简单、用时较短，可用于大规模生产中蛹虫草菌株退化的检测手段；线粒体和脂滴染色也可作为退化菌株的鉴别方法；而生产中常用的通过菌落见光转色判断菌株优劣的方法需要慎重。继代培养菌株第4代开始出现明显的退化特征，因此在生产中使用的菌株最好控制在继代培养3代以内。

Characteristics and identification of Cordyceps militaris strain degeneration

Strain degeneration occurs often in Cordyceps militaris during subculture and long-term storage at low temperature, resulting in a decrease in yield and a great loss in income. In this study, normal strains and degenerated strains due to long-term storage at 4 °C PDA slant and continuous subculture of C. militaris were observed to survey the morphological variation of fruiting bodies, colonies and mycelia and sporulation capacity of conidia. The nuclei, mitochondria, reactive oxygen species accumulation in cell, and lipid droplets in blastospore were observed by staining method, and the content of cordycepin, adenosine and ergosterol in mycelia was analyzed. The results showed that most of the phenotypical variation of degenerated strains caused by long-term storage at 4 °C PDA slant and continuous subculture were consistently displayed as: the decrease of fruiting body yield, the presence of adhesive and knotted hyphae, significant decrease of conidia, increased level of reactive oxygen species, reduction of mitochondria in the mycelia, mergence of dispersed and small lipid droplets in the blastospores into large droplets. There is no color change after being exposed to light for the colony of degenerated strains caused by long-term storage at 4 °C, whereas color change is unstable during the continuous subculture. The number of nuclei in the mycelia of degenerate strains undergoing continuous subculture is reduced whereas no change in that undergoing long-term storage at 4 °C. The content of cordycepin, adenosine and ergosterol in the mycelia of degenerated strains of long-term preservation was reduced by 58%, 41% and 70% as compared with that of the normal strains, but the ergosterol content of the degenerated strains of continuous subculture was unchanging. Microscopic observation of adhesive and knotted mycelia, and staining the mycelia by nitrogen blue tetrazolium (NBT) to detect the reactive oxygen species are easy to operate and not time-consuming. This method can be used to detect the degeneration of C. militaris strains on a large scale. Mitochondrial and lipid droplet staining can also be used to identify the degenerated strains. The commonly used method of judging the quality of spawn by color conversion after light irradiation needs to be cautious. Obvious degeneration of the strains usually appears in fourth generation during subculture, so the strains used for cultivation should be selected within three generations.