供给天麻种子萌发营养的真菌——紫萁小菇

从天麻种子发芽的原球茎中,共分离到12种可供给天麻种子萌发营养的真菌,其中最优良的01号菌株,诱导培养出子实体,经鉴定为紫萁小菇(Mycena osmundicola Lange),属国内新纪录。用01号菌株及紫萁小菇培养的染菌树叶,伴播天麻种子都可发芽,经酯酶同工酶凝胶电泳分析,二者酶带条数、迁移率(RF值)基本相同,证明鉴定结果可靠(标本保存在医科院药植所真菌研究室)。     

A Cytochenfical Study of Acid Phosphatas in the Mycorrhizal Cells of Gastrodia elata Seedling

A cytochemical study has been made to examine the activity of acid β-glycerophosphatase in the mycorrhizal cells of the seedling of Gastrodia elata BI. using thin sectioning technique in which sections were embedded in glycol mathacrylate (GMA). After the seedling was invaded by the hyphae of Mycena osmundicola Lange, two different kinds of infected cells were formed in its root cortex.the outer 1–2 cell layers namely the hyphae-containing cells (or host cells) contained many coiled hyphae pelotons; the inner comparativly large cell layer or fungus-digesting cells contained a few straight hyphae. Localization of acid phosphatase in hyphae-containing cells showed that only a few senescent hyphae retained the enzyme activity and the plant cells did not release hydrolytic enzyme. So it is considered that the hyphal lysis in hyphae-containing cell may be due to autolysis. In contrast, higher acid phosphatase activity was visualized in many vesicles and small vacuoles of the fungus-digesting cells. When a hypha entered a fungus-digesting cell through a hyphae-containing cell, a number of enzyme granules (i. e, enzymecontaining vesicles) gathered around it. Later on the enzyme granules expanded gradually and became small enzyme vacuoles of 1.6–2.0 μm in diameter. Still later the small enzyme vacuoles fused with each other to form a large vacuole in which a part of an invading hypha was enclosed and gradually digested by hydrolytic enzymes. Finally,the digesting vacuole changed into a residual body containing some metabolic waste. The above results suggest that fungus-digesting cells can actively release hydrolytic enzymes by lysosomal vesicles to digest the invading hyphae, but such function is not present in the hyphae-containing cells,the role of which may be attributed to attracting and controling the invading hyphae.     

天麻球茎不同部位的细胞对蜜环菌侵染的反应

用电镜技术比较研究了天麻球茎皮层细胞和中柱细胞对蜜环菌侵染的反应,皮层细胞在蜜环菌菌丝的刺渺下能产生囊状结构,然后这些小囊将入侵的菌丝包围和消化,而中柱细胞则不能,上述两种细胞在功能上的主要差别就在于此。     

Role of Lysosomal Vesicles in the Digestive Process of Armillaria mellea by the Large Cells of Gastrodia elata

The hyphae of Armillaria mellea Fr. invade the large ceils of Gastrodia elata BI. Through the wall pits of cortical cells. During early stage the plasmalemma of large cell invaginates and the cell wall forms papillary thickenings to restrain the hyphae from invading. When a hypha enters a large cell, it is encircled tightly by the invaginated plasmalemma which is surrounded by a large number of vesicles coated by a unit membrane. As these vesicles fusing with their membranes to the plasmalemma and discharging their contents into the space around the hypha, the space lined by the invaginated plasmalemma enlarges gradually and becomes a digestive vacuole in which a hypha is completely digested. Reaction product form acid phosphatase activities in the vesicles and digestive vacuoles testifies that the vesicles and digestive vacuoles are identical with primary and secondary lysosomes of plant lysosomal system respectively.     

天麻生长过程中酸性磷酸酶的研究

以蜜环菌(Armillaria mellea)伴生的天麻(Gastrodia elata)块茎为材料,研究了天麻生长发育过程中酸性磷酸酶(ACP)活性及其同工酶的变化。结果表明,在天麻生长90d时,其酶谱中有3条谱带,且均显色较弱;生长至120d时,其酶谱与生长90d的酶谱相同,但谱带显色加深;而在生长150d时,酶谱中缺少了一条Rf 0.852的谱带。在90~150d的天麻生长发育过程中,酸性磷酸酶(ACP)活性呈现先升高后下降的趋势。讨论了酸性磷酸酶(ACP)同工酶变化的生理学意义。     

天麻消化紫萁小菇及蜜环菌过程中细胞超微结构变化的研究

本文对天麻种子消化入侵的紫萁小菇菌丝及营养繁殖茎消化蜜环菌过程中,细胞超微结构的变化进行了研究。观察结果表明:紫萁小菇侵入种胚后,染菌胚细胞的细胞器逐渐消失,其细胞质产生囊状体起消化菌丝的作用,存在于胚细胞中的紫萁小菇菌丝有脱壁或失去细胞质成为空腔等变化;种子萌发形成的原球茎消化紫萁小菇的方式同胚萌发阶段类同。蜜环菌侵入原球茎分化的营养繁殖茎后,皮层细胞产生消化酶类颗粒或囊状体包围并分解蜜环菌菌丝;被皮层细胞消化的菌丝残物或部分菌丝进入皮层内面的大型细胞,此时大型细胞的代谢功能显著增强,该细胞中的各种水解酶颗粒及液泡等完成对菌体物质的最终同化。紫萁小菇及蜜环菌先后在天麻有性繁殖和无性繁殖阶段侵染供给其营养,但菌丝被消化过程中的细胞形态变化、被消化方式不完全一致。     

天麻种子／原球茎和营养繁殖茎被菌根真菌定殖后的细胞分化

天麻（Gastrodia elata Bl.)种子可与紫萁小菇（Mycena osmundicola Lange),兰小菇（M.orchidicola Fan et Guo)等一类小菇属真菌共生萌发形成原球茎。侵入种皮的菌丝集结在柄状细胞外周的胚柄残迹中,首先侵入胚的柄状细胞,然后自柄状细胞侵入其他原胚细胞。原胚细胞发生功能分化,形成菌丝结细胞和消化细胞,初被菌丝定殖的原胚细胞具有消化菌丝的功能,随后,部分原胚细胞逐渐被菌丝充满,充育成菌丝结细胞。菌丝由菌丝结细胞进一步侵入消化细胞后最终被消化。由原球茎分化形成的营养繁殖茎（以下简称营繁茎）进一步被蜜环菌（Armilariella mellea(Vahl.Fr.)Karst.)定植,蜜环菌与紫箕小菇的菌丝同时存在于营繁茎中,但两菌相遇时都停止蔓延,互不交错侵染。     

Cytodifferentiation of the Seeds (Protocorms) and Vegetative Propagation Corms Colonized by Mycorrhizal Fungi

The stipecell, subepidermal parenchyma cells and inner cortical parenchyma cells were differentiated from Gastrodia elata Bl. seed and protocorm cells when they were colonized by the fungal hyphae of Mycena osmundicola Lange and M. orchidicola Fan et Guo. The hyphae aggregated in the suspensor remnant surrounding stipecell, primarily penetrated the stipecell, and then colonized the embryo of seed. Stipecell is the unique invading site of the hyphae. Subepidermal parenchyma cells containing pelotons of hyphae is also a kind of passage cells of hyphae, but, when primarily colonized by hyphae, they can degenerate a little of hyphae. The hyphae colonizing inner cortical parenchyma cells were totally degenerated, and the function of inner ocrtical parenchyma cells is digestive. The vegetative propagation corms, which differentiated from protocorms, were recolonized by Armilariella mellea (Vahl:Fr.) Karst., and the hyphae of A. mellea and M. osmundicola were found in the same cell, but there is a layer of cells uncolonized by mycorrhizal fungal hyphae. This means the two fungal species can not crisscross colonize the cell of G. elata.     

A Cytological Study on the Nutrient Uptake Mechanism of a Saprophytic Orchid Gastrodia elata

There are three type cells infected by the mycorrhizal fungus, ArmiUaria mellea (Vahl ex Fr. ) Karst in the tube cortex of Gastrodia elata BI., namely the passage cells, host cells and digesting cells. Ultrastructural study demonstrated that the passage cells were distroyed by the hyphae, the host cells kept symbiotic with the hyphae, but the digesting cells could become inversely parasitic on the hyphae from which nutrient were being uptaken. The detail process of the digesting cells obtaining nutrient'from the fungus is described as follows: Firstly the digesting cells began to attack the invading hyphae by releasing numerious electron-transparent vesicles of lysosomal property, secondly they took up the soluble organic material leaked out from the digested hyphae by forming many electron-dense endocytic tubes and vesicles, and finally they endocytosed and hydrolysed the insoluble hyphal walls by forming large digesting vacuole in which a piece of hyphal wall was completely enveloped.     

天麻的营养繁殖茎及其抑菌功能

天麻9Gastrodia elata Bl.）与蜜环菌（Armillaria mellea (Vahl.ex Fr.)Quel.)营共生生活,在正常情况下蜜环菌只侵染种麻及分化生长出的营养繁殖茎的表皮、皮层及大型细胞层。种麻的大型细胞层及营养繁殖茎隔离区的空腔细胞层和木栓细胞层,都是种麻与新生麻的防御结构,它们保护新生麻不遭蜜环菌病理侵染而正常生长。冬季,天麻进入冬期,隔离区的木栓细胞层形成断裂层,此层细胞是新生麻能够安全越冬的最后一道防御结构。     

The Structure and Antifungal Functions of Vegetative Propagation Corm of Gastrodia elata

The growth of Gastrodia elata Bl. and Armillaria mellea (Vahl. ex Fr.) Quel. shares a special symbiotic relation. In general, A. mellea invades the G. elata , the epidermal cells, the cortical cells and the large cells of the growing vegetative propagation corm of G. elata . The empty cavity cells, the cork cells of the isolation in the vegetative propagation corms and the large cells of G. elata were the defensive structure, protecting the new G. elata from pathological invasion by A. mellea . In winter, G. elata enters the stage of hibernation.　The faulting layer derived from the cork cells of the isolation was the last defensive structure by which new G. elata could safely live through the winter.     

乌天麻Gastrodianin基因的鉴定及其在天麻发育进程中的表达

基于相似性克隆策略和RT-PCR,从兰科植物乌天麻(Gastrodia elataf.glauca)球茎克隆得到两个新的天麻抗真菌蛋白基因,命名为gastrodianin-4A(ga4A)和gastrodianin-4B(ga4B),并首次用Northern杂交研究了该基因在植物不同部位的转录表达。结果表明,天麻个体的不同部位只转录表达同一种Gastrodianin基因,纯化自球茎的Gastrodianin蛋白质的肽质量指纹谱与推导的Ga4A和Ga4B成熟蛋白的氨基酸序列相吻合;Northern杂交证明天麻的地上器官Gastrodianin基因转录表达量远远高于地下球茎,而次生球茎皮层组织的表达量比中柱和整个营繁茎的都高一些。天麻球茎Gastrodianin的外周表达模式可能是天麻在地下抵御蜜环菌(Armillaria mellea Karst)入侵球茎皮层内部的防卫机制之一,但该基因在天麻地上部分的高丰度表达暗示Gastrodianin可能蕴藏其它生理功能。     

天麻矿物质吸收及其营养机理探讨

探讨了天麻吸收矿物质过程和天麻的营养机理。研究蜜环菌在天麻的金属元素、非金属元素吸收中起的作用, 阐明天麻的有机营养吸收过程。提出了天麻吸收非金属元素的两个途径,分析了天麻吸收金属元素的“泵”的作用机理。指出了天麻蜜环菌之间的游离氨基酸特别是天冬氨酸和谷氨酸的交流是天麻和蜜环菌生长发育的关键。     

蜜环菌菌种分离新法——天麻组织分离法

报道了一蜜环菌菌种分离方法——天麻组织分离法,并对此法与常用分离法——菌索分离法进行比较试验。结果发现,天麻组织分离法分离成功率高（78％）,远高于常用的菌索分离法（16％）,且前者操作简便、难度低,所得菌种生活活力、生长形态均优于后法。     

Studies on the Character istic of the Structures in the Cortical Cells of Gastrodia clara after Infection of Armillaria mellea

Aided by the techniques of thin and ultrathin sectioning and electron microscopy, the characteristic of structures in the cortical cells of Gastrodia elata was further investigated after infection of Armillaria mellea. It was found that the “hyphal coils”, observed with light microscope, in the cortical cells of G. elata were saccate structures deriv- ed from the cytoplasm of cortical cells and enclosed hyphae. And the cell walls of hyphae were digested in these sacs. Then, these hyphae without cell wall were cut into protoplast fragments in inner-most cortical cells. The results indicated that the cortical cells of G. elata possess digestive function.     

天麻内生真菌菌群组成的初步研究

从采自贵州、陕西、四川和云南的天麻组织中共分离获得62株内生真菌。经形态学与分子生物学相结合鉴定,62株内生真菌分属于3个亚门、5纲、7目、9科、13属。研究结果还表明采自不同地点的天麻以及天麻不同部位的内生真菌的类群与分布存在明显差异。     

四株昭通乌天麻共生蜜环菌的生理生化特征及其分子鉴定

目的 对4株昭通乌天麻（Gastrodia elata）共生蜜环菌（Armillaria mella）的生理生化活性进行研究,并鉴定其分类地位。方法 通过测定不同碳源和氮源对蜜环菌生长的影响以及胞外酶活性,掌握该蜜环菌生理生化特性,再结合rDNA-IGS序列分析方法进行鉴定。结果 不同蜜环菌需要不同的碳源、氮源;3种胞外酶活性中木聚糖酶活性最大,其次是羧甲基纤维素酶,漆酶活性最小;供试蜜环菌与云南、贵州地区天麻共生蜜环菌以及猪苓共生蜜环菌的亲缘关系比较远。结论 蜜环菌能否作为天麻共生菌与其分类地位无关。     

不同蜜环菌菌株对乌天麻产量的影响

用不同的蜜环菌(Amillaria mellea)菌株与长白山乌天麻(Gastrodia elata)进行伴栽试验,以探索不同蜜环菌菌株对乌天麻产量的影响。结果表明:不同蜜环菌菌株对乌天麻产量的影响不同,其中菌株Ar-4能显著提高天麻产量,仿野生栽培和室外箱栽试验均表现出良好的伴栽效果。     

人工种植天麻的影响因子优化研究

天麻是一种名贵的中草药,可用于治疗多种疾病。自然条件下,天麻通过与真菌蜜环菌的共生从腐木获取营养进行生长。为了对人工大规模种植天麻的影响因子进行优化研究,利用正交设计方法,在昭通小草坝地区开展了一个样本量较大的种植实验。在测试的4种因子中,接种蜜环菌对种植天麻影响最大：其次,在露天灌木丛地种植的天麻产量明显高于在林地下的种植。此外,开展了12个树种对蜜环菌生长以及天麻种植的影响研究。结果发现十齿花（Dipentodonsinicus）对蜜环菌的生长最好,同时,也在天麻种植实验中有最高的产量;然而,添加磷、钾元素并不提高天麻的产量。     

Study of Factors for Cultivating the Orchid Species Gastrodia elata,a Traditional Chinese Medicine

The orchid speices Gastrodia elata is a valuable traditional Chinese medicine and has been widely applied for treating a variety of diseases. The yield of wild Gelata is very limited since its vegetative growth is exclusively dependent on its symbiosis with the fungus Armillaria mellea, from which Gelata is able to obtain nutrients from rotten wood in the forest. To develop a standard for cultivating Gelata in large quantities, four factors that may influence the yield of Gelata need to be further investigated, including cultivation environment, inoculation volume of Armillaria, wood from different tree species, and the effect of fertilizer on the maturation of Gelata. To optimize these factors, a large scale orthogonal experiment was performed in the farmland of Xiaocaoba, Zhaotong, Yunnan Province. Among the four factors tested, inoculation of Armillaria played the most important role in the maturation of Gelata. The yield of mature tubers, in terms of both tuber weight and quantity, on open ground is greater than that in forested areas. Of the 12 tree species tested, Dipentodon sinicus stimulated the largest amount of growth of Armillaria and produced the greatest yield of Gelata in the farmland. In comparison to the other factors tested, fertilizer showed no effect on the yield of G-elata.