【作者】 周国英；

【导师】 谢碧霞；

【作者基本信息】 中南林学院 ， 森林培育学， 2002， 博士

【摘要】 松乳菇(Laclarius deliciosus)，又名美味松乳菇，系红乳菇科、乳菇属真菌。其肉质细嫩，味道鲜美独特，营养丰富，可食用也可药用，被视为山中珍品，市场上十分走俏。它与马尾松共生，是一种菌根食用菌，因有特殊的生态条件、营养方式和子实体分化条件，目前尚不能人工栽培，市场供应全依赖于野生采集，由于市场紧缺，过度采伐，导致产地自然资源破坏严重，产量逐渐减少。为保护食用菌资源的多样性，菌根食用菌的半人工栽培是目前国内外研究的热点。半人工栽培的关键问题是生产出菌根化苗木，而生产菌根化苗木需要大量菌根真菌，故培养菌根食用菌菌丝体是开发利用菌根真菌的先决条件。本论文对松乳菇表型(phenotype)，组织分离及菌丝固体纯培养，菌丝液体纯培养，分离物的DNA指纹鉴别以及纯培养物菌丝接种马尾松苗木合成菌根化苗情况进行了系统研究。主要研究结果如下： 1、松乳菇菌丝有锁状联合，有隔，分枝少，粗为3.0～3.3μm；担子和担孢子发育过程大致可分为担子膨大期、担孢子形成期和担子及担孢子成熟期三个阶段；成熟担子表面有较明显的条状突起，成熟担孢子椭球形，表面有较明显的不完整的网纹，大小为6～7×5～6μm，内部有一个大油滴。 2、松乳菇子实体和菌丝体含有多种功能成分。有多种人体必需的矿质元素，尤其是人体容易缺乏的微量元素铁、锌含量极高，分别为：灰绿色菌盖679.4mg／kg、382.87mg／kg，橘红色菌盖398.02mg／kg、275.40mg／kg，菌丝体893.60 mg／kg、418.11mg／kg，菌丝体的铁、锌含量比子实体要高。含有6种脂肪酸，子实体中硬脂酸在总脂肪酸中的含量最高，为56.10％；而菌丝中很低仅为4.59％，但菌丝中亚油酸含量最高，达到53.72％，亚麻酸含量也差不多是子实体的4倍。菌丝体中的多糖提取率远大于子实体，是子实体的4倍，达到9.36％。 松乳菇子实体营养丰富，含有17种氨基酸，人体必需的8种氨基酸齐全，其中含量最高的是丙氨酸，占氨基酸总量的11.14％，其次是亮氨酸，占氨基酸总量的9.96％。菌盖和菌柄中所含氨基酸种类相同，组成相近，但氨基酸总量不同，分别占子实体干重的12.75％和8.98％。子实体中必需氨基酸含量相当高，明显高于FAO／WHO模式的标准蛋白，第一限制氨基酸是蛋氨酸。采用了多种蛋白质评价方法，松乳菇的化学评分和氨基酸比值系数分，两项指标均居第一位；必需氨基酸指数、生物价和营养指数三项指标均居第二位；只有氨基酸评分居第三位，可见松乳菇的营养价值是相当高的。 3、采用12种培养基配方，对两种不同颜色、6个不同来源的松乳菇子实体的不同部位进行组织分离，结果从菌盖与菌柄交叉部位获得了慢生型的菌丝体分离菌株(来源于灰绿色子实体)和快生型的菌丝体分离菌株(来源于橘红色于实体)。子实体不同的组织部位对分离效果影响不大，在不同部位取得的组织均能萌发出菌丝；而不同培养基对分离效果影响较大，PDA是分离松乳菇较好的培养基。培养基中加松针煮出汁能促进松乳菇菌丝的生长。 松乳菇菌丝固体纯培养特性：菌丝生长温度范围7～37℃，适宜的生长温度是15～30℃，最佳生长温度是27℃；喜中性偏酸性环境，适宜生长的pH值为5～7，最适pH值为6；对碳源利用范围广，蔗糖最好，葡萄糖次之，果糖最差；对氮源的利用，有机氮中，单一氮源优于复合氮源，其中尿素不能利用； 学 位 论 文无机氮源中，铰态氮优于硝态氮，以硫酸铰效果最好，亚硝酸盐不能利用；松乳菇对盐浓度适应能力很强，在盐浓度为14g／L条件下也具有生长能力，适宜生长的盐浓度范围为0－6g／L。 4、松乳菇菌丝液体纯培养的优化工艺：正交筛选的培养基配方为糊精2．5％，牛肉膏2％，蔗糖＋甘露醇3％，，MgSO。0．01％，KTI％，烟酸＋核黄素（0．5％＋0．l％）；培养条件为温度26．5℃，转速130r／min。进行液体纯培养25d，得到菌丝产量和多糖产量分别为0．44979／100ml和0．6759／100ml。 温度和摇床每大振荡的时间对松乳菇菌丝生长和多糖产量的影响起诀定作用，PH对松乳菇多糖得率的影响不大，但PH对松乳菇菌丝生长起决定作用。选择松乳菇产多糖的最佳培养温度为27”C，最佳振荡时间为Sh／d，最适pH为7，经过15d便可使菌丝产量达到324mg／100ml，多糖产量达到695。g／100ml。 5、针对松乳菇分离菌丝体目前难出菇现实，运用RAPD技术，对从供试松乳菇子实体进行组织分离的纯培养物进行DNA指纹鉴别。采用筛选到的25个扩增效果好、重复性稳定的引物介导子实体与5个松乳菇分离纯培养物菌株进行扩增，均得到清晰可辨的mA指纹图谱：分子数据分析表明：供试菌株2、3、4号与松乳菇于实体1号之间的DNA相似性极高，供试菌株5、6号与子实体1号之间的DNA相似性也高，5株供试菌株在 80 ％以上相似水平上聚成一类，属于松乳菇菌种（Laclarlus delicfosus人但分离物的遗传稳定性有待进一步研究。 6、松乳菇纯培养物接种马尾松苗木，能与马尾松苗形成菌根，在马尾松容器育苗实验中菌根形成率为73．3％；与马尾松形成菌根后促进了马尾松苗木的生长更多还原

【Abstract】 Laclarius deliciosus is a edible mycorrhizal fungus. It was chosen to be studied on morphology, nutrition analyse and assessment, tissue isolation and Solid pure culture, liquid fermentation, RAPD molecule identify and the effect of L deliciosus on the growth of Pinus massoniana seedlings. The main results of the research paper are as follows:l.The phenotype analysis of L deliciosus was made, which is different from two colour. Mycelium, basidium and basidiospore were observed with scanning electron microscope and transmission electron microscope. The results showed that four basidiospores on basidium evenly, the shapes were elliptical, 6-7X5-6 pm in size, with clamp connection in growing mycelium of 3. 0-3. 3 urn in width.2. L deliciosus is a high protein and low fat health food. It was concluded that contents of mineral elements , fatty acids and polysaccharides of two different colour fruitbody are not the same. After the compoment analysis and compared with other kinds of edible fungi, the nutrition position of L. deliciosus was defined . The international adopted nutrient value assessment method was applied to the overall assessment of the protein nutrient value of L. deliciosus. The conclusion was drawn that the protein content of L. Deliciosus is comparatively high, the trace elements humanbody needed are rich in fruitbody ang mycelia of L. deliciosus, and it can be regarded as good source of useful elements, such as P, Ca> Zn, Ni. There are six kinds of fatty acid in the grey pileus and mycelia. The content of setearic acid in fuitbody is high, which is 56. 10% of the total fatty acid. The poly-unsaturated fatty acid is 56. 07% of the total fatty acid in the mycelia, higher than Rivinosa and peanut. The fruitbody of L. deliciosus 17 kinds of amino acids, the total content of which in pileus and stipe is respectively 12. 75% and 8. 98%. The essential amino acid makes up 48.39% of the total amino acids. The first amino acid is met. CS, MS, EAAI, BV, NIand SRCAA of which is respectively 77. 6, 64.9, 98.6, 95.8, 23.2 and 74.19. It was proved that CS and SRCAA possess the first place, EAAI> BY and NI the second place,only AAS the third place .By comparision,the nutrient value in pileus of L. deliciosus is a little higher than that of L. edodes. Polysaccharide production rate in mycelia is 9. 36%, much higher than fruitbody too.3. The tissue isolation for L. deliciosus were made with 12 media from different positions of 6 basidiocarps of different source in the studies. The slow-growing mycelia and fast-growing mycelia were got .The results showed that different place of taking tissue had little effects on the result of tissue isolation from L. deliciosus. Among media were taken in experiment of tissue isolation, PDA was the best medium. Mycelia grew fastest on PDA and Yeast-glucose -gallium. Pine leaives extract was put into media tested, the growth of mycelia increased atdifferent levels.Mycelia had wide temperature range for growth, from 7 to 37"C , the suitable temperature range was 15 to 30 癈 , and the optimun was 27 癈, stopped growing at 5 癈 and 40 癈 . Mycelia had wide pH range for growth, 4 to 9 , the suitable pH range were 5 to 7, the optimum was 6. Mycelia could absorb all the carbon sucroses tested (fructose, glucose, sucrose, maltose, lactose, starch, dextrin), the sucrose was the best utilized carbon nutrition, secondly was glucose and the last was fructose. Mycelia could not utilize NaNOj and Urea, the others could be utilized at different levels. Among organic nitrogens, simple nitrogen, such as glycine were better utilized than the complex nitrogens, such as peptone and yeast extract. Among inorganic nitrogens, ammonium nitrogen was better utilized than nitrate nitrogen. Mycelia could endure salt concentration powerfully, mycelia had wide salt concentration range for growth, from 0 to 14g/L, suitable salt concentration range for growth were 0 to 6 g/L, mycelia was growing slow from 8 to 14 g/L .4. Liquid fermenttation of mycelia was investigated on the effect of carbon s更多还原