猪苓菌丝形成菌核伴生菌的发现及应用

在野生猪苓 (Grifolaumbellata (Pers .)Pil偄ｔ)菌核生长穴中首次分离到猪苓菌丝形成菌核所必需的伴生菌(Grifolasp .)。实验证明 :纯培养的猪苓菌丝不能形成菌核 ,但其与伴生菌共培养 ,无论在实验室培养基上或用树棒栽培 ,猪苓菌核形成很快且发育正常 ;伴生菌是猪苓菌丝形成菌核的关键生物因子。另外 ,伴生菌菌丝和猪苓菌菌丝二者形态差别较大 ,前者多为细长的薄壁菌丝 ,后者多为细胞直径较大的薄壁和厚壁菌丝。     

猪苓菌丝形成菌核结构的特点

对猪苓(Grifola umbellata(Pers.)Pilat)菌丝在人工条件下形成菌核及繁殖过程、人工菌核与野生菌核及培养基上未形成菌核的猪苓菌丝的显微结构进行了系统研究.研究证明:人工菌核的结构与野生菌核的结构相似,均具有菌髓和皮层结构.人工菌核中的菌丝与培养基表面未形成菌核的猪苓菌丝存在着显著的差异,人工菌核是由培养基上纯培养的菌丝分化为膨大菌丝再由此形成有高度组织分化的猪苓菌核.     

猪苓菌丝形成菌核结构的特点(英文)

对猪苓(Grifolaumbellata(Pers.)Pilat)菌丝在人工条件下形成菌核及繁殖过程、人工菌核与野生菌核及培养基上未形成菌核的猪苓菌丝的显微结构进行了系统研究。研究证明人工菌核的结构与野生菌核的结构相似,均具有菌髓和皮层结构。人工菌核中的菌丝与培养基表面未形成菌核的猪苓菌丝存在着显著的差异,人工菌核是由培养基上纯培养的菌丝分化为膨大菌丝再由此形成有高度组织分化的猪苓菌核。     

从ITS序列探讨猪苓与其伴生菌的亲缘关系

对猪苓菌丝、野生猪苓子实体、野生猪苓菌核和其伴生菌的 5.8SrDNA及其两侧的ITS 1区和ITS2区进行了序列分析。发现猪苓与其伴生菌的ITS序列同源性达 99.36% ,说明猪苓与其伴生菌有着极高的分子亲缘关系。     

猪苓菌核结构性质的研究

利用光学显微镜和电子显微镜对猪苓菌核的结构及其发育进行了研究。组成猪苓菌核的菌丝与平板培养或发酵培养的猪苓菌丝比较,具有多分枝、融合频率高、菌丝形态不规则等特点。猪苓菌核表现了高度的结构分化,有表皮、表皮下层、疏松菌丝层和结构菌丝之分,结构菌丝是组成菌核的主要成分,疏松菌丝层类似一般菌核的髓,但大小、位置在菌核中变化较大。菌核的个体繁殖是由母体菌核的一束或几柬菌丝突破表皮而萌发产生新的菌核;较系统的观察了猪苓菌核细胞分裂及其菌丝内部结构的变化。     

蜜环菌侵染后猪苓菌核防御结构的发生及功能

蜜环菌通过猪苓菌核表皮侵染菌核时,菌核表皮下层的菌丝具特异的拮抗反应,如细胞中有结晶颗粒出现,厚壁菌丝形成,部分薄壁菌丝有质壁分离现象。蜜环菌的侵染诱导了菌核防御结构的发生：离侵染点一定距离的部位出现由少量木质化菌丝和厚壁菌丝形成的疏松带状;蜜环菌侵入后,上述菌丝增多并紧密排列形成菌核的初级隔离腔,入侵的蜜环菌和部分菌核被隔离在腔中;在初级隔离腔形成的同时,外围的次级隔离腔开始发育。蜜环菌环菌和部     

蜜环菌侵染后猪苓菌核防御结构的发生及功能

蜜环菌通过猪苓菌核表皮侵染菌核时,菌核表皮下层的菌丝具特异的拮抗反应,如细胞中有结晶颗粒出现,厚壁菌丝形成,部分薄壁菌丝有质壁分离现象。蜜环菌的侵染诱导了菌核防御结构的发生:离侵染点一定距离的部位出现由少量木质化菌丝和厚壁菌丝形成的疏松带状;蜜环菌侵入后,上述菌丝增多并紧密排列形成菌核的初级隔离腔,入侵的蜜环菌和部分菌核被隔离在腔中;在初级隔离腔形成的同时,外围的次级隔离腔开始发育。蜜环菌菌索和皮层菌丝分枝可突破初、次级隔离腔的壁,再以菌索产生的侵染带侵染菌核较外部的最后防线即三级隔离腔。本文较系统的阐述了蜜环菌侵染后菌核各防御结构的发生特点及功能。     

猪苓与蜜环菌的关系

蜜环菌Armilla riella mcllea(Vahl：Ff．)Kgtst·侵入猪苓Gri[ola umbe』zd‘4(PetsFr．)PilOt)菌核,激活了猪苓菌抵御异体侵染免疫反应的本能,猪苓菌丝细胞木质化,形成与菌核表皮结构相似的隔离腔,将蜜环菌素和部分猪苓菌丝包围。在隔离腔中蜜环菌消化分隔在腔中的猪苓菌丝,另外猪苓菌丝也可侵入或附着在蜜环菌索及侵染带细胞间隙吸收其代谢产物,猪苓菌核即可萌发出新苓正常生长。当隔离腔中的猪苓菌丝被消化后,蛮环菌生活力也减弱,解体后被猪苓菌吸收利用,隔离腔变成空腔。从广义角度看,仍可把蜜环菌与猪苓菌寄生与反寄生的营养关系概括为共生关系。     

猪苓菌丝形成菌核差异蛋白质组学研究

药用真菌猪苓菌丝形成菌核分子机制一直是人们关注的热点问题。本研究首次对猪苓进行了系统的蛋白质组学研究。基于液质联用技术并在数据依赖性采集模式下,成功鉴定了猪苓菌丝形成菌核的初始期、菌核生长期和成熟期的蛋白质组,共鉴定蛋白1 391个(global FDR 1%)。采用先进的SWATH MSALL的非标记定量方式对初始期菌丝和菌核进行了差异蛋白质组学研究,从中定量蛋白1 234个,占鉴定蛋白数量的88.7%。猪苓菌丝形成菌核过程中有378个蛋白差异表达。差异蛋白GO注释结果表明,在猪苓菌丝形成菌核过程中,差异蛋白参与的蛋白功能和生物学过程多样,以催化和结合为主,还参与感知环境刺激、信号转导、电子转移、过氧化活性、菌丝生长及胞壁合成等功能。进一步推测分析表明,猪苓菌丝形成菌核可能是由于缺氧或温差等多种环境胁迫诱导所致,生成活性氧并形成氧化应激状态。信号通过丝裂原活化蛋白激酶通路(MAPK通路)途径向下游传递,可能调控了胞壁蛋白糖基化修饰,进而促进猪苓菌丝极性生长和菌丝形态学变化而形成菌核。KEGG代谢通路分析表明,差异蛋白还主要参与了糖、脂质和氨基酸等初级产物以及甾醇、多聚乙酰等次级代谢物的合成与代谢。因此,对差异蛋白质组的深入解析和研究有利于揭示猪苓菌丝形成菌核的蛋白分子机制,具有较强的理论和现实意义。     

猪苓、伴生菌及蜜环菌共培养的形态学研究*

本文对猪苓、伴生菌及蜜环菌两两共培养及三者共培养进行了宏观形态观察及细胞学水平上的研究。结果表明,猪苓与伴生菌共培养时,在二者之间形成一致密拮抗线,猪苓菌落表面菌丝分化产生大量菌丝束;猪苓与蜜环菌共培养时,猪苓能阻止蜜环菌菌索对其自身的进一步侵袭,互作区中的双方菌丝及菌索均停止生长;蜜环菌与伴生菌共培养时,蜜环菌能穿透整个伴生菌菌落,在伴生菌菌落下方产生大量分枝;三者共培养后,猪苓对蜜环菌的防御能力有所下降,伴生菌对蜜环菌的耐受力有所提高,蜜环菌产生的新分枝均向伴生菌一侧生长,猪苓与伴生菌之间并不形成致密拮抗线,只可见双方菌丝的白色交融区。 猪苓与伴生菌均能在蜜环菌菌索皮层上形成侵入位点。     

猪苓菌核结晶及厚壁细胞的起源与发育

猪苓菌核的含晶细胞发生于菌丝中间或顶端,该细胞具有体积大、细胞质丰富等特点;结晶是由细胞质中的微小颗粒沉积于液泡中逐渐发育而成,液泡周围常有数量较多的线粒体分布,结晶发育至一定大小时细胞壁破裂释放出结晶,单个结晶在菌核中可聚集成大的棱状晶体。厚壁细胞产生于菌丝中间,与两端细胞以横隔膜相隔,细胞质收缩的同时胞壁加厚,厚壁细胞发育至仅留很小胞腔或完全被加厚物质充满时,可与相邻菌丝细胞分离;猪苓菌核厚壁细胞与有些真菌无性厚壁孢子的形成类同,但其大小不等在5～30μm之间。     

Morphological characteristics of sclerotia formed from hyphae of <Emphasis Type="Italic">Grifola umbellata</Emphasis> under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.     

猪苓、伴生菌及蜜环菌共培养的形态学研究

本文对猪苓、伴生菌及蜜环菌两两共培养及三者共培养进行了宏观形态观察及细胞学水平上的研究。结果表明,猪苓与伴生菌共培养时,在二者之间形成一致密拮抗线,猪苓菌落表面菌丝分化产生大量菌丝束;猪苓与蜜环菌共培养时,猪苓能阻止蜜环菌菌索对其自身的进一步侵袭,互作区中的双方菌丝及菌索均停止生长;蜜环菌与伴生菌共培养时,蜜环菌能穿透整个伴生菌菌落,在伴生菌菌落下方产生大量分枝;三者共培养后,猪苓对蜜环菌的防御能力有所下降,伴生菌对蜜环菌的耐受力有所提高,蜜环菌产生的新分枝均向伴生菌一侧生长,猪苓与伴生菌之间并不形成致密拮抗线,只可见双方菌丝的白色交融区。 猪苓与伴生菌均能在蜜环菌菌索皮层上形成侵入位点。     

Nutrient Source of Sclerotia of Grifola umbellata and Its Relationship to Armillaria mellea

Ways nutrient-uptake of sclerotia of Grifola umbellata and the relationship between G. umbellata and Armillaria mellea were studied. At the primary stage of sclerotia of G. umbellate infected by A. mellea, the hyphae of G. umbellata could obtain nutrients by invading the one- to three-layer-cells of A. mellea cortex which existed in the sclerotia of G. umbellata, ar late stage of A. mellea infection, the nutrient source of sclerotia of G. umbellata mainly depended on its hyphae, adhering on the intercellular space of A. mellea, to suck the metabolic products of A. mellea. After being nourished the hyphae of sclerotia of G. umbellata outside of the rhizomorph of A. mellea began to reproduce, as their nuclear divisions were well observed. The results suggested that the mutual assimilation between G. umbellata and A. meIlea could be defined as a form of special Symbiotic relation.     

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.