【作者】 何苏琴；

【导师】 王生荣；

【作者基本信息】 甘肃农业大学 ， 畜牧学， 2011， 博士

【摘要】 冬虫夏草Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora=Cordyceps sinensis Berk.是我国特有名贵的滋补强壮中药之一,主要分布于我国的青海、西藏、甘肃、四川、云南等省区,北起祁连山一带,南至滇西北高山,东到川西高原山地,西达喜马拉雅山的大部分地区。青藏高原北缘及祁连山东端是甘肃冬虫夏草的主要分布区。年贮藏量8.1吨。甘南藏族自治州是甘肃冬虫夏草的主产区,产量占甘肃省冬虫夏草的产量的90%以上。本研究通过对甘肃省冬虫夏草主产区虫草种类、冬虫夏草子座形态及其无性型中国被毛孢形态的研究,对甘肃省3种虫草上的主要真菌进行了分离鉴定,取得了如下成果:1、对采自甘肃玛曲、碌曲、夏河、天祝及青海贵南的多年、多批次的百余枚冬虫夏草标样进行了分离,统计结果显示,中国被毛孢Hirsutella sinensis总的分出率在80%以上。除中国被毛孢外,分出率较高的尚有被孢霉Mortierella spp.、茎点霉Phoma sp.、玫红裸囊菌Pseudogymnoascus roses、拟青霉Paecilomyces spp.等14个类群的真菌,但在不同批次的冬虫夏草样本分离中,分离频次不定。2、采自甘肃的冬虫夏草,少数病虫生2-4个子座,或子座叉状分枝。中国被毛孢Hirsutella sinensis在培养条件下产生无色和近黑色两种颜色的分生孢子,近黑色孢子大小的平均值比无色孢子稍大。这在前人对中国被毛孢的原始描述及以往的文献中是没有见到的。显示出丰富的形态学多样性。3、除冬虫夏草Ophiocordyceps sinensis外,从甘肃甘南藏族自治州的碌曲县和夏河县采集到的另外2种寄生于蝠蛾幼虫的虫草,依据形态学和分子生物学鉴定结果,2种虫草分别被鉴定为细虫草Ophiocordyceps gracilis和峨眉虫草Ophiocordyceps emeiensis。两种虫草均为甘肃省新纪录虫草。采自甘肃夏河的细虫草Ophiocordyceps gracilis的内菌核上,羽束梗孢Paraisaria dubia的分出率达100%;采自甘肃碌曲的峨眉虫草Ophiocordyceps emeiensis的子座和内菌核上,被毛孢Hirsutella sp.的分出率分别达100%和60%,轮枝状真菌Verticillium spp.在细虫草上分出率达50%。拟青霉Paecilomyces spp.在两种虫草上均有较高的分出率。4、培养性状及生物学研究表明,Hirsutella sinensis、Geomyces pannorum和Pseudogymnoascus roseus 3种菌均为为嗜低温菌。Hirsutella sinensis菌株Cordy-007菌丝适宜生长温度为15-20℃,25℃菌丝稍有萌动菌落不扩展,30℃2周即致死;Geomyces pannorum菌株Geo-6的适宜生长温度为15-20℃,30℃和35℃下菌丝不生长。35℃1周即致死;Pseudogymnoascus roseus菌株Pseu-F的适宜生长温度为17.5-20℃,30℃和35℃下菌丝不生长。35℃1周即致死。5、对2株与冬虫夏草无性型相关真菌的代表性菌株进行了形态学和分子生物学比对鉴定,表明:中国被毛孢是冬虫夏草O.sinensis的无性型,峨眉虫草Ophiocordyceps emeiensis的无性型是被毛孢Hirsutella sp.。6、用正交试验法对2个分离自冬虫夏草子座的具有潜在利用价值的Geomyces pannorum菌株Geo-6和Pseudogymnoascus roseus菌株Pseu-F进行了液体发酵培养基筛选试验,试验因素包括马铃薯、黄豆、蔗糖+葡萄糖、蛋白胨、酵母膏、矿物盐、维生素等,筛选出最佳液体发酵培养基分别为:蔗糖20g+葡萄糖10g+酵母浸出物5g+大豆25g+马铃薯100g+Co.B 0.2g(1000ml培养基);和蔗糖20g+葡萄糖10g,蛋白胨10g,酵母浸出物5g,黄豆50g,马铃薯100g。在优化配方的液体培养基中培养,2菌株的菌丝体干重收率分别达17.36g/L(培养8天)和36.32~36.86g/L(培养4~5天)。更多还原

【Abstract】 Ophiocordyceps sinensis is a specific traditinal chinese medicine.It is mainly distributed in Qinghai-Tibetan Plateau, include Tibet, Qinghai, Gansu, Sichuan, Yunnan Province, and marginal region of Himalaya, such as Nepal, the Kingdom Bhutan and India.The marginal region of northern of Qinghai-Tibetan Plateau and the end of Qilian Mountain is major producing region of Gansu Province, hold in store 8100 Kg per year. Gannan Tibetan Autonomous Prefecture is major producing region of Gansu Province, the yield of Ophiocordyceps sinensis are more than 90% of the total yield of Gansu Province.In this paper, by dentification of major fungi isolated from Ophiocordyceps spp., in Gansu province, obtained the following results:1. Over one hundred samples of Ophiocordyceps sinensis, from Maqu, Luqu, Xiahe, Tianzhu of Gansu Province and Guinan of Qinghai Province, were isolated for many years and batches, the general isolated ratio of Hirsutella sinensis reached more than 80%. Mortierella spp., Phoma sp. , Pseudogymnoascus roses and Paecilomyces spp. etc. 14 fungi populations were also frequently isolated, but the ratios were unsteady in different batches.2. The morphological diversity of Ophiocordyceps sinensis stroma and condia of Hirsutella sinensis was researched. A few samples of Ophiocordyceps sinensis born 2-4 stromata per endosclerotium or formed dichotomous stroma, in major producing areas of Ophiocordyceps sinensis of Gansu Province. Hirsutella sinensis can produced colourless and near black condia in culture, different from original description of the species and prior report.3. Ophiocordyceps gracilis and Ophiocordyceps emeiensis were new records in Gansu Province. They were collected in Xiahe and Luqu of Gannan Tibetan Autonomous Prefecture. The ratio of Paraisaria dubia isolated from endosclerotium of Ophiocordyceps gracilis reached 100%, the ratios of Hirsutella sp. isolated from endosclerotium and stroma of Ophiocordyceps emeiensis reached 100% and 60% respectively. The ratios of Verticillium spp. from Ophiocordyceps gracilis reached 50%, the ratios of Paecilomyces spp. were high for two species of Ophiocordyceps.4. Cultural and biological characters shows, Hirsutella sinensis strain Cordy-007, Geomyces pannorum strain Geo-6 and Pseudogymnoascus roseus strain Pseu-F was psychrophilic fungi, optimum growth temperature≤20℃, the lethal temperatures of mycelium of three strains were 30℃two weeks, 35℃one week, 35℃one week , respectively.5. The results of comparative research of morphology and molecular biology, reconfirmed that Hirsutella sinensis is anamorph of Ophiocordyceps sinensis, and the anamorph of Ophiocordyceps emeiensis is Hirsutella sp..6. Submerged culture media were screened by means of orthogonal design experiments to Geomyces pannorum strain Geo-6 and Pseudogymnoascus roseus strain Pseu-F, the potential resources fungi.Test factors included sucrose, glucose, peptone, yeast extract, soybean, potato, mineral salt and Co.VB. Based on the results of tests, for strain Geo-6, the best combinations of culture medium are sucrose 20g, glucose 10g, yeast extract 5g, soybean 25g, potato 100g , Co.VB 0.2g, in 1000ml liquid medium; for strain Pseu-F, the best combinations of culture medium are sucrose 20g, glucose 10g, peptone 10g, yeast extract 5g, soybean 50g, potato 100g, in 1000ml liquid medium. Mycelia yield (dry weight) of strain Geo-6 and strain Pseu-F came up to 17.36g/L(cultured 8d) and 36.32~36.86g/L(cultured 4-5d) respectively, in optimal formula liquid medium.更多还原