野生花脸香蘑的分离纯化及ITS序列鉴定

Isolation and identification of wild Lepista sordida by ITS gene sequence

以采自内蒙古根河地区的野生花脸香蘑为材料,采用组织分离法分别对其菌盖处、菌盖与菌柄交界处和菌柄处的组织进行分离纯化;通过测定生长速度和污染率等方法,研究分离纯化的最适培养基和最佳部位;最后将分离物进行ITS序列分析,计算遗传距离,并采用邻接法构建NJ系统发育树。结果表明,分离纯化最适培养基为PDA+子实体煮水培养基;菌盖与菌柄交界处为最佳分离部位,菌丝生长速度快、长势好、污染率低;并且子实体经自然风干2 d后能有效降低污染率;最后分离物经ITS序列测定,系统发育分析证实其为花脸香蘑。

Lepista sordida which was collected from Genhe District in Inner Mongolia was used as materials,tissue isolation was used to isolate the tissue of pileus,stipe,and junction of them. The optimal culture medium and the best tissue for isolation was investigated by measuring growth rate and pollution rate. Then the isolate was identified by internal transcribed spacer（ ITS）,the genetic distance was calculated by Kimura2-parame-ter,and a NJ systematic tree was established by the neighbor-joining method. The results showed that the optimal culture medium was PDA added with sporocarp boiled water,and the junction of pileus and stipe showed fast growth and low pollution,which was the best isolation position. Moreover,the pollution rate could be effectively reduced when the sporocarp had been air-dried for 2 days. At last,the isolate was identified as Lepista sordida by the analysis of phylogenetic relationship of ITS gene sequences.