乌蒙山区小菇属萌发菌的分离鉴定及其菌种生产优化  

Isolation and Identification of Mycena of Pleurotus in Wumeng Mountain Area and Optimization of Its Production Conditions

作　　者：王绘 袁青松 周涛 袁媛[2] 徐娇 江维克 张进强 王晓[3] 刘大会[4] 张光文 潘成 WANG Hui;YUAN Qing-song;ZHOU Tao;YUAN Yuan;XU Jiao;JIANG Wei-ke;ZHANG Jin-qiang;WANG Xiao;LIU Da-hui;ZHANG Guang-wen;PAN Cheng(Guizhou University of Traditional Chinese Medicine(TCM),Guiyang 550025,China;National Resource Center for Chinese Meteria Medica,Chinese Academy of Chinese Medical Sciences,Beijing 100700,China;Shandong Analysis and Test Center,Ji'nan 250014,China;Hubei University of Chinese Medicine,Wuhan 430065,China;Guizhou Wumengteng Fungus Industry Limited Company,Dafang 551600,China)

机构地区：[1]贵州中医药大学,贵阳550025 [2]中国中医科学院中药资源中心,北京100700 [3]山东省分析测试中心,济南250014 [4]湖北中医药大学,武汉430065 [5]贵州乌蒙腾菌业有限公司,贵州大方551600

出　　处：《中国实验方剂学杂志》2020年第19期43-52,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81960694);中央本级重大增减支项目(2060302);贵州省科技计划项目(黔科合平台人才[2019]5611号);贵州省百层次创新型人才项目(黔科合平台人才[2018]5638);贵州省教育厅创新群体重大研究项目(黔教合KY字[2018]022);现代农业产业技术体系建设项目(CARS-21)。

摘　　要：目的:分离鉴定小菇属菌,扩展萌发菌种质资源和优化萌发菌培养条件,为天麻萌发菌菌种的生产提供资料和指导。方法:采用米麻组织块分离法和转接纯化培养技术进行菌株的分离纯化,运用传统形态学、显微技术对分离菌株的菌落、菌丝、孢子等形态特征进行鉴定,通过聚合酶链式反应(PCR)扩增rDNA(Ribosomal DNA)内转录间隔区(ITS)进行测序分析,进一步与美国国家生物信息中心(NCBI)数据库进行同源性检索比对,利用MEGA6软件采用最大似然法(Maximum Likelihood,M-L)构建系统进化树,对分离菌株进行分类鉴定;同时,采用正交试验优化萌发菌生长条件。结果:分离获得86株菌株,分别属于12个属的21个物种,其中WMMFJ,SHXG,WMM-21和MFJ8103菌株鉴定为Mycena purpureofusca,ZT01-6和ZT01-8菌株鉴定为M.cf.purpureofusca。萌发菌在麦麸培养基上的生长速率显著快于PDA培养基,且萌发菌生长的最佳培养基组成为土豆100 g,麦麸150 g,玉米粉100 g,葡萄糖20 g;间苯三酚的2种浓度对WMMFJ菌株有显著促进生长作用,对WMM-21和ZT01-6菌株有促进作用;2-甲氧基酚的2种浓度对WMMFJ菌株也具有促进作用。结论:新分离鉴定6株萌发菌菌种,其中4株为M.purpureofusca,2株为M.cf.Purpureofusca,采用的分离方法提高了萌发菌菌株的分离效果。Objective:Isolate and identify Mycena,expand the resources of geminating fungus of Gastrodia elata and optimize the culture conditions of Mycena,in order to provide information and guidance for the production of geminating fungus of G.elata.Method:Juvenile tuber tissue mass transfer separation and purification technology was used for the separation and purification of strains,traditional morphology microscopy was used to isolate the colony mycelia spores morphological characteristics,such as identification,polymerase chain reaction(PCR)amplification rDNA(Ribosomal DNA)internal transcribed spacer(ITS)was used for sequencing analysis and further homology with NCBI database retrieval,MEGA6 software was used to establish Phylogenetic tree by the Maximum likelihood method(MaximumLikelihood,M-L),so as to classify and identify isolated strains.At the same time,orthogonal test was used to optimize the optimal growth conditions of Mycena.Result:A total of 86 strains were isolated,which belong to 21 species in 12 genera.WMMFJ,SHXG,WMM-21 and MFJ8103 were identified as M.purpureofusca,and ZT01-6 and ZT01-8 were identified as M.cf.purpureofusca.The growth rate of Mycena in wheat bran medium was significantly higher than in PDA medium.The optimal medium composition for the growth of germinating bacteria was 100 g potato,150 g wheat bran and 20 g corn flour,100 g glucose.And 1,3,5-Trihydroxybenzene significantly promoted the growth of WMMFJ,and played a role in promoting the growth of WMM-21 and ZT01-6,and 2-Methoxyphenol promoted the growth of WMMFJ.Conclusion:Six strains of Mycena were isolated and identified,four of them are M.purpureofusca,and two of them are M.cf.Purpureofusca.The separation method improved the separation effect of germinating bacteria.

关 键 词：天麻 萌发 小菇属菌 分离 鉴定 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学]