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。     

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。     

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。     

Cytological Studies on the Process of Armillaria Mellea Infection Through the Sclerotia of Grifola Umbellata

Adhering to the sclerotium of Grifola umbellata, Armillaria mellea could invade the sclerotium in a manner of rhizomorph without capsule, after which the sclerotium formed a deep coloured stereoscopic septate cavity outside of the rhizomorph. At the early stage of infection, segmentation was visualized either in the cortex or the apex of A. mellea rhizomorph to form a new rhizomorph which penetrated another parts of G. umbellata sclerotium. At the late stage of infection, the cortical hyphae of A. mellea rhizomorph produced a branch to invade the wall of the septate cavity of G. umbellata sclerotium and, in a manner of hyphae, it could further form new rhizomorph after its penetration through that wall. An alternate way of expanding A. mellea infection in sclerotium was to form a invading band which was composed of a few rolls of round ceils derived from cortical hyphae of A. mellea rhizomorph. The band could invade sclerotia to a farther distance and then could connect with each other.     

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。     

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.     

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.     

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。     

蜜环菌菌索多糖的免疫增强作用研究

研究了野生蜜环菌菌索多糖(polysaccharide from the rhizomorph of Armillaria mellea,AMP)对小鼠免疫功能的影响.结果表明对小鼠分别灌胃(ig)剂量为100、200、300mg/kg·d的AMP均能增加小鼠体重、改变免疫器官重量、抵抗环磷酰胺(CY)对小鼠外周血白细胞数量的影响.实验显示灌胃AMP能提高小鼠单核巨噬细胞系统的吞噬功能,增强小鼠迟发型变态反应,促进溶血素的生成.上述结果表明AMP能增强机体免疫功能.     

蜜环菌提取物对佐剂性关节炎大鼠的治疗作用

本文通过对佐剂性关节炎大鼠原发性和继发性足肿胀度的测量、全身关节炎指数评分、体重及体态变化、免疫脏器指数、炎性踝关节切片的形态以及血清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6、前列腺素E2、一氧化氮和一氧化氮合成酶等指标来考察蜜环菌Armillaria mellea的5个不同提取物对佐剂性关节炎大鼠的影响及作用机理。结果表明,蜜环菌甲醇提取物对原发性和继发性足肿胀有明显的抑制;甲醇组能明显抑制佐剂性关节炎大鼠脾脏和胸腺萎缩,并能抑制血清中肿瘤坏死因子α、白细胞介素1β、白细胞介素6和前列腺素E2的分泌,抑制大鼠踝关节炎细胞的形成。因此蜜环菌提取物中甲醇提取物对佐剂性关节炎大鼠治疗效果最显著。     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

野生和人工培养的蜜环菌菌索营养成分比较

对野生和人工培养的蜜环菌菌索主要营养成分及氨基酸含量进行了测定和比较。结果表明：人工培养的蜜环菌菌索蛋白质、脂肪、多糖含量均高于野生菌索,二者的氨基酸组成相似,但人工培养的菌索氨基酸总量高于野生菌索。     

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。     

蜜环菌糖苷水解酶家族基因在菌索与菌丝间的差异表达分析

蜜环菌是一种兼性腐生和寄生的真菌,通过降解伴栽基质并为药用植物(天麻)或菌物(猪苓)提供营养物质,而糖苷水解酶是这一过程的主要酶类.本研究从蜜环菌Armillaria mellea 541菌株转录数据库中共获得糖苷水解酶家族基因170个,分布于39个亚家族.进一步分析发现,这些家族基因编码的糖苷水解酶家族蛋白(glyc...     

蜜环菌菌索分化的差异蛋白质组学研究

菌索是蜜环菌与宿主互作的组织结构,蜜环菌菌丝形成菌索的分子机制尚不清楚.本研究采用SWATH-MSALL非标记定量蛋白质组学技术,首次对Armillaria mellea菌丝形成菌素过程的蛋白质组学进行了系统研究.在蜜环菌菌丝和菌索中共鉴定蛋白1724个(global FDR 1％),定量蛋白1179个.与菌丝相比,蜜...     

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.