辽宁省八种兰科植物根内生真菌多样性

兰科植物的生存强烈依赖菌根真菌,研究兰科菌根真菌对兰科植物的保护有重要作用。本研究以辽宁省的无柱兰(Amitostigma gracile)、二叶舌唇兰(Platanthera chlorantha)、小斑叶兰(Goodyera repens)、蜻蜓兰(Tulotis fuscescens)、山兰(Oreorchis patens)、羊耳蒜(Liparis japonica)、长苞头蕊兰(Cephalanthera longibracteata)和绶草(Spiranthes sinensis) 8种属于极小种群的野生兰科植物为例,利用第二代测序技术对其根内生真菌多样性进行了研究。结果表明:无柱兰、二叶舌唇兰、小斑叶兰和山兰都偏好与角担菌科(Ceratobasidiaceae)真菌共生,羊耳蒜偏好与胶膜菌科(Tulasnellaceae)真菌共生;长苞头蕊兰主要与革菌科(Thelephoraceae)和蜡壳耳科(Sebacinaceae)真菌共生;绶草不仅能与丝核菌属(Rhizoctonia)真菌共生,还能与蜡壳耳科真菌共生;同种兰科植物的不同植株在同一生境下所选择的菌根真菌有差异,而同一地点的不同兰科植物的菌根真菌群落各不相同。由此可见,兰科植物根中菌根真菌的组成并非完全受植物自身控制,但主要菌根真菌仍取决于兰科植物的选择;这8种兰科植物根中的真菌绝大多数为非菌根真菌,而菌根真菌的丰度通常很低,这可能是辽宁地区兰科植物稀少的原因之一。     

兰科植物共生胶膜菌:分类学、多样性、专一性和适应性

兰科植物与丝核菌类真菌,包括胶膜菌科、角担菌科和蜡壳菌科等形成菌根共生体。胶膜菌科真菌作为最广泛分布的共生菌根真菌,表现出与兰科植物的协同进化与密切关系。除了形态学特征分析和比较外,分子技术促进了兰科植物胶膜菌的分类学和多样性研究。兰科植物与胶膜菌的特异性可能限制兰科植物的分布和移栽后的生存能力,但有些兰科植物与胶膜菌的共生关系会因为地理分布或环境变化进行调整,使植物更好地生存,这种适应性为实现无菌苗菌根化来促进兰科植物的迁地保护或繁殖提供可能。本文综述了兰科植物共生菌根真菌胶膜菌在分类学、多样性、特异性和适应性等方面的研究。     

3种杓兰属植物菌根真菌系统发育和多样性分析

兰科植物菌根真菌(Orchid mycorrhizal fungi, OrMF)在兰科植物种子萌发和后续生长发育过程中具有重要作用。该研究采用培养(菌丝团分离)和非培养(克隆文库)2种方法获得同一栖息地3种不同杓兰属植物根中菌根真菌ITS序列并划分可操作分类单元(Operational taxonomic units, OTUs),分析其系统发育关系和多样性。结果表明:(1)所有根段中都有菌丝团定植,共分离出菌根真菌64株,其中63株为胶膜菌科(Tulasnellaceae)真菌,1株为角担菌科(Ceratobasidiaceae)真菌;可划分为7个OUT,每个OTU代表菌株的菌丝都能形成OrMF典型的近球形或椭球形链状排列的念珠状细胞;分离出来的菌根真菌均为无性型菌丝且不产生无性孢子。(2) 非培养法得到的3种杓兰属植物的根中OrMF分别隶属于胶膜菌科(Tulasnellaceae),腊壳菌科(Sebacinaceae)、角担菌科(Ceratobasidiaceae)和革菌科(Thelephoraceae),其中胶膜菌科OTU在种类和数量上占有绝对优势,培养和非培养2种方法得到的OrMF OTU类型和数量均为西藏杓兰(Cypripedium tibeticum)>无苞杓兰(C. flavum)>黄花杓兰(C. bardolphianum),但培养法少于非培养法。(3)对胶膜菌进行系统发育分析显示,优势和非优势OTU均分布在系统发育树的3个不同分支上,这种与多种亲缘关系较远的OrMF共生的现象可能与杓兰属植物对环境的适应性有关,且不同杓兰的OrMF物种丰富度没有显著差异,但群落结构存在差异。     

四川黄龙沟优势兰科植物菌根真菌多样性及其季节变化

在自然条件下,兰科菌根真菌对兰花的种子萌发和植株生长都是必不可少的。为了解高原兰科植物菌根真菌的多样性状况及其季节性变化规律,选取了四川黄龙沟的两种生境中生长的8种优势兰科植物,分别于植株的萌芽期(4月份)、生长期(7月份)和果期(9月份)采集营养根进行菌根真菌的多样性研究。其中,黄花杓兰(Cypripedium flavum)、少花鹤顶兰(Phaiusdelavayi)、二叶匍茎兰(Galearis diantha)和广布小蝶兰(Ponerorchis chusua)分布在开阔生境;筒距兰(Tipularia szechuanica)、小花舌唇兰(Platanthera minutiflora)、珊瑚兰(Corallorhiza trifida)和尖唇鸟巢兰(Neottia acuminate)则分布在密林生境。通过对分离所得的50个菌株进行形态观察和ITS序列测定相结合的鉴定,共获得菌根真菌41种。对担子菌和子囊菌分别进行的系统发育树构建结果显示,子囊菌为优势种类(35种),以柔膜菌目(Helotiales)、炭角菌目(Xylariales)和肉座菌目(Hypocreales)内的种类为主,担子菌则以胶膜菌(Tulasnellaceaesp.)为主。在8种兰科植物中,二叶匐茎兰表现出极高的专一性,其菌根真菌均属于Hypocrea。其余兰科植物的菌根真菌分别属于不同的科,专一性相对较低。物种丰富度和Simpson多样性指数分析结果表明,密林生境的兰科植物的菌根真菌多样性在各生长季节基本高于开阔生境。此外,两种生境的优势兰科植物的菌根真菌物种多样性随生长季节转变所呈现的变化规律是相似的:萌发期和生长期的多样性均较高,峰值出现在生长期,到果期时则大幅下降。这与高原兰科植物的生长特性及营养供求规律基本相符。     

广西喀斯特地区毛唇芋兰根内与根际土壤真菌群落组成分析

为探索兰科(Orchidaceae)植物毛唇芋兰(Nervilia fordii)根内和根际土壤真菌群落多样性，该研究采用Illumina MiSeq高通量测序技术，分析了大新(DX)和龙州(LZ)两个样地(简称两地)毛唇芋兰根内和根际土壤的真菌组成。结果表明：(1)两地的毛唇芋兰根内和根际土壤真菌多样性很丰富，根际土壤真菌多样性均高于根内，主根的真菌多样性高于走茎。(2)通过测序共获得有效序列118 040条，207个可操作分类单元(OTUs)涉及8门19纲42目86科123属。(3)担子菌门(Basidiomycota)真菌是两地毛唇芋兰根内真菌的共同优势菌群，涉及胶膜菌科(Tulasnellaceae)、Trimorphomycetaceae、角担菌科(Ceratobasidiaceae)、马拉色菌科(Malasseziaceae)和小皮伞科(Marasmiaceae)等，其中优势科和优势属分别是胶膜菌科(75%)和瘤菌根菌属(Epulorhiza)(56%),而土壤中的优势菌属则是镰刀菌属(Fusarium)。综上认为，毛唇芋兰根内与根际土壤中的优势菌群既差异显著也存在一些共同...     

药用植物绶草（兰科）的菌根真菌群落组成分析

兰科菌根真菌（OMF）被认为是影响兰科植物物种丰度和分布的一个重要因素。对广域分布兰科植物的菌根区系进行研究有助于人们更深入地了解兰科植物分布格局的形成机制。本研究以我国广域分布的兰科药用植物绶草Spiranthes sinensis为材料,采用Illumina Miseq高通量测序技术对北京、上海、江西、广西、云南、甘肃6个样地的绶草菌根区系进行了研究。一共检测到51个OMF分类单元,其中角担菌科Ceratobasidiaceae真菌是绶草菌根的主要类群,约有1/3的角担菌科种类存在于所有的样本中,说明该类真菌亦广域分布;胶膜菌科Tulasnellaceae、肉丝耳科Serendipitaceae、红菇科Russulaceae、革菌科Thelephoraceae、口蘑科Tricholomataceae等真菌亦有发现,只是其相对多度较低,且较多种类表现出明显的地域特异性;6个样地的绶草菌根区系组成存在显著性差异,且这种差异与地理距离之间并未表现出明显的相关性,暗示菌根区系组成更多受生境因素的影响。本研究结果可为进一步采用菌根技术实现该类兰科药用植物的种质保育及栽培生产提供理论参考。     

独花兰菌根真菌的分离及rDNA ITS序列在其分类鉴定中的应用

独花兰(Changnienia amoena Chien)为我国特有的独属独种兰科植物,仅生长于长江中下游及陕西南部的山地林下及沟谷中,是中国特有兰科物种之一,被列为国家二级保护植物.以浙江天目山自然保护区的野生独花兰中分离获得的菌根真菌为对象,应用传统的形态结构鉴别与rDNA ITS分子生物学手段相结合的研究方法,进行了菌株的分类鉴定研究,确定该菌株为兰科菌根真菌之一的胶膜菌属(Tulasnella)真菌.研究结果对于全面了解兰科植物菌根真菌的特征和有效保护独花兰植物资源均具较大意义和参考价值.     

辽宁省9种兰科植物根内与根际土壤中真菌群落结构的差异

兰科植物的生存及生长高度依赖其根中的共生真菌, 其中的菌根真菌更是对兰科植物的种子萌发与后续生长有着非常重要的作用, 研究兰科植物根中的真菌, 尤其是菌根真菌, 对兰科植物的保护有重要作用。该研究利用第二代测序技术, 对中国辽宁省境内的9种属于极小种群的兰科植物的根、根际土和根围土中的真菌群落和菌根真菌组成进行了研究。结果显示, 兰科植物根中的真菌群落和根际土、根围土中的真菌群落具有显著差异。兰科植物根中的总操作分类单元(OTU)数目远小于根际土和根围土中的总OTU数目。同时, 兰科植物根中菌根真菌的种类和丰度与根际土、根围土中菌根真菌的种类与丰度没有明显联系。FunGuild分析结果显示, 丛枝菌根真菌在根际土与根围土中的丰度非常高, 但在兰科植物的根中却数量极少。这些结果表明, 兰科植物根中的真菌群落与土壤中的真菌群落在一定程度上是相互独立的。     

辽宁省9种兰科植物根内与根际土壤中真菌群落结构的差异

兰科植物的生存及生长高度依赖其根中的共生真菌,其中的菌根真菌更是对兰科植物的种子萌发与后续生长有着非常重要的作用,研究兰科植物根中的真菌,尤其是菌根真菌,对兰科植物的保护有重要作用。该研究利用第二代测序技术,对中国辽宁省境内的9种属于极小种群的兰科植物的根、根际土和根围土中的真菌群落和菌根真菌组成进行了研究。结果显示,兰科植物根中的真菌群落和根际土、根围土中的真菌群落具有显著差异。兰科植物根中的总操作分类单元(OTU)数目远小于根际土和根围土中的总OTU数目。同时,兰科植物根中菌根真菌的种类和丰度与根际土、根围土中菌根真菌的种类与丰度没有明显联系。FunGuild分析结果显示,丛枝菌根真菌在根际土与根围土中的丰度非常高,但在兰科植物的根中却数量极少。这些结果表明,兰科植物根中的真菌群落与土壤中的真菌群落在一定程度上是相互独立的。     

兰科菌根研究综述

兰科菌根是一种内生菌根,主要寄生于兰科（Orchidaceae）植物的种子及根系上。对兰科菌根真菌的分类及真菌资源多样性、兰科菌根的形态和菌根对兰科植物的效应等最新研究进展进行了综述。目前研究已知,感染兰科植物根部并能与之共生的真菌绝大多数属于担子菌门（Basidiomycota）和半知菌门（Deuteromycotha）,也有部分属于子囊菌门（Ascomycota）;兰科菌根的形成可分为两种情况：一是对兰科植物种子的侵染;二是对成长新根的侵染。菌根真菌对兰科植物的种子萌发及植株生长发育均有一定影响。     

Molecular identifications uncover diverse fungal symbionts of Pleione (Orchidaceae)

Fungal mutualisms are essential for the evolution and diversification of Orchidaceae, yet the fungal symbionts of Pleione orchids are poorly understood because molecular data are unavailable for this genus. Based on ITS-rDNA sequencing for mycobionts of 15 Pleione species (both wild and cultivated plants were included), we conducted phylogenetic analyses for the most dominant mycobionts, and compared the operational taxonomic units (OTUs) of mycorrhizal fungi among species within Pleione. Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (Sebacinales), Atractiellales, and Auriculariales were reported as putative mycobionts of Pleione. In particular, the mycorrhizal associations between subtropical orchids and Atractiellales have not been observed before. For the dominant mycobionts in the roots of Pleione and its related genera, Bletilla and Coelogyne, we detected no fungal OTU that was shared. Within Pleione, species with a sympatric distribution showed preferences for different fungi. Epiphytic and lithophytic individuals of Pleione albiflora shared OTUs of Tulasnellaceae but harbored different OTUs of Sebacinales, indicating some degree of fungal specificity toward certain habitats. These findings provide new insights into the ecological adaptation and evolution of orchids, and will contribute to the conservation and utilization of species resources.     

Variation in Mycorrhizal Associations with Tulasnelloid Fungi among Populations of Five Dactylorhiza Species

Background

Orchid species rely on mycorrhizal symbioses with fungi to complete their life cycle. Although there is mounting evidence that orchids can associate with several fungi from different clades or families, less is known about the actual geographic distribution of these fungi and how they are distributed across different orchid species within a genus.

Methodology/Principal Findings

We investigated among-population variation in mycorrhizal associations in five species of the genus Dactylorhiza (D. fuchsii, D. incarnata, D. maculata, D. majalis and D. praetermissa) using culture-independent detection and identification techniques enabling simultaneous detection of multiple fungi in a single individual. Mycorrhizal specificity, determined as the number of fungal operational taxonomic units (OTUs), and phylogenetic diversity of fungi were compared between species, whereas discriminant analysis was used to compare mycorrhizal spectra across populations and species. Based on a 95% cut-off value in internal transcribed spacer (ITS) sequence similarity, a total of ten OTUs was identified belonging to three different clades within the Tulasnellaceae. Most OTUs were found in two or more Dactylorhiza species, and some of them were common and widespread, occurring in more than 50% of all sampled populations. Each orchid species associated with at least five different OTUs, whereas most individuals also associated with two or more fungal OTUs at the same time. Phylogenetic diversity, corrected for species richness, was not significantly different between species, confirming the generality of the observed orchid mycorrhizal associations.

Conclusions/Significance

We found that the investigated species of the genus Dactylorhiza associated with a wide range of fungal OTUs from the Tulasnellaceae, some of which were widespread and common. These findings challenge the idea that orchid rarity is related to mycorrhizal specificity and fungal distribution.     

鼓槌石斛的地理分布与菌根真菌区系组成的相关性

本文研究了兰科石斛属鼓槌石斛的地理分布与菌根真菌区系组成的相关性。采用克隆文库技术对云南省西双版纳和临沧市两个地区的8份野生鼓槌石斛根部样品进行了分析。结果表明,共从两个地区的鼓槌石斛根部获得了14个真菌的可操作分类单元（OTU）,主要隶属于胶膜菌科Tulasnellaceae和小纺锤菌目Atractiellales。分析鼓槌石斛对菌根真菌的专一性和偏好性发现,鼓槌石斛对菌根真菌专一性较低,每个植株都能同时与多种菌根真菌共生;不同地理分布的鼓槌石斛菌根真菌类群存在明显差异,两个地区的鼓槌石斛的菌根真菌中只有1个OTU是相同的,其余均不同。说明菌根真菌可能和鼓槌石斛的生态适应存在一定的相关性。     

三种杓兰根相关真菌多样性和生态功能

【背景】除了菌根真菌(Orchid mycorrhizal fungi,OrMF)外,兰科植物根中还有其它内生真菌,称为根相关真菌(Root-associated fungi,RAF)。【目的】采用分离培养的方法获得同一栖息地针叶林和灌木林两种不同生境西藏杓兰、黄花杓兰和无苞杓兰的RAF菌株,研究其真菌谱系、多样性和生态功能结构。【方法】从杓兰根碎屑中分离RAF,通过总DNA提取、PCR扩增及测序得到ITS(Internaltranscribedspacer)序列;进行系统发育和多样性分析,并通过NCBI数据库比对得到相似性最高序列的注释信息来分析RAF生态学特性。【结果】共分离得到278株RAF,25种OTU类型,包括23个子囊菌门OTU,2个毛霉菌门OTU。RAF物种丰富度分析发现西藏杓兰的较黄花杓兰高,不同生境没有显著差异;不同杓兰物种较不同生境的RAF群落分化程度高。生态功能分析显示25个OTU包括共生型、腐生型和致病型3种营养型,以及外生菌根菌群、植物病原菌群、内生真菌群、动物病原菌群、真菌寄生菌群、杜鹃花类菌根群、未定义的腐生菌群和不确定型8种共位群。【结论】阐明不同生境采集的不同杓兰中RAF的分布特点和生态功能,为未来研究RAF与杓兰属植物的共生关系奠定基础。     

High diversity of root-associated fungi isolated from three epiphytic orchids in southern Ecuador

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum, Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens. Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens. No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi.     

不同海拔的三种杓兰属植物与菌根真菌群落组成相关性

本研究采集了四川黄龙沟沿海拔梯度3 170-3 400m上4个不同杓兰居群中3种杓兰植物根,利用克隆文库方法获得菌根真菌ITS序列,研究同一栖息地（黄龙沟）不同海拔梯度和不同杓兰对菌根真菌多样性和群落结构的影响。共得到18个可操作分类单元（OTU）,其中14个OTU隶属胶膜菌科Tulasnellaceae,为优势类群（99.6%）;2个OTU隶属腊壳菌科Sebacinaceae,2个OTU隶属亡革菌科Thelephoraceae。随着海拔升高,西藏杓兰菌根真菌多样性减少,而黄花杓兰和无苞杓兰没有明显变化;海拔对3种杓兰菌根真菌群落结构均无显著影响。3种杓兰之间菌根真菌群落结构差异显著且指示物种互不相同,说明在同一栖息地,杓兰对菌根真菌的偏好性显著影响其菌根真菌群落结构。这些研究结果利于了解环境变化对杓兰属植物菌根真菌区系组成的影响,为进一步探索菌根真菌与杓兰属植物的互作机制奠定基础。     

A rare temperate terrestrial orchid selects similar Tulasnella taxa in ex situ and in situ environments

Mycorrhizal symbiosis in orchids is unique in that fungal presence is considered a requirement for germination as well as for further development. Additionally, orchid fungal associations can exhibit high specificity in nature. Yet, an important ecological question remains unanswered: ‘With which orchid mycorrhizal fungi (OMF) do un-inoculated orchid seedlings form symbiosis when cultured ex situ?’ Simultaneously, it is asserted that orchid conservation efforts involving ex situ plant culture should exclusively utilize natural symbionts of the respective orchid taxa. We present a first comparison of OMF communities within the roots of asymbiotically cultured plants of the rare orchid Platanthera chapmanii grown ex situ (ES), and those occurring naturally in situ (IS). Nuclear ribosomal internal transcribed spacer (nrITS) barcoding region was used to identify peloton forming OMF from roots collected between 2012 and 2014 from both growing environments. Our 114 sequences clustered into 11 operational taxonomic units (OTUs) belonging to four closely related clades of the fungal family Tulasnellaceae. Shannon–Wiener (H) and Simpson diversity (D) indices were similar (p = 0.81 for both) for ES and IS OMF communities. Beta diversity comparisons also showed similarity between ES and IS treatments based on weighted (p = 0.10) and unweighted (p = 0.20) Bray–Curtis dissimilarity matrices. Bayesian and Maximum Likelihood (ML) phylograms clustered ES and IS derived fungal OTUs into the same clades. Our data suggest that P. chapmanii: (1) forms symbiosis with taxonomically similar fungi in ex situ culture and in its native soil, and (2) exhibits a narrow phylogenetic breadth of mycorrhizal fungal OTUs within the Tulasnellaceae.     

Phylogenetic constrains on mycorrhizal specificity in eight Dendrobium(Orchidaceae) species

Plant phylogeny constrains orchid mycorrhizal(OrM) fungal community composition in some orchids. Here, we investigated the structures of the OrM fungal communities of eight Dendrobium species in one niche to determine whether similarities in the OrM fungal communities correlated with the phylogeny of the host plants and whether the Dendrobium-OrM fungal interactions are phylogenetically conserved. A phylogeny based on DNA data was constructed for the eight coexisting Dendrobium species,and the OrM fungal communities were characterized by their roots. There were 31 different fungal lineages associated with the eight Dendrobium species. In total, 82.98% of the identified associations belonging to Tulasnellaceae, and a smaller proportion involved members of the unknown Basidiomycota(9.67%). Community analyses revealed that phylogenetically related Dendrobium tended to interact with a similar set of Tulasnellaceae fungi. The interactions between Dendrobium and Tulasnellaceae fungi were significantly influenced by the phylogenetic relationships among the Dendrobium species. Our results provide evidence that the mycorrhizal specificity in the eight coexisting Dendrobium species was phylogenetically conserved.     

Shifts in mycorrhizal fungi during the evolution of autotrophy to mycoheterotrophy in Cymbidium (Orchidaceae)

? Premise of the study: Mycoheterotrophic plants, which completely depend upon mycorrhizal fungi for their nutrient supply, have unusual associations with fungal partners. The processes involved in shifts in fungal associations during cladogenesis of plant partners from autotrophy to mycoheterotrophy have not been demonstrated using a robust phylogenetic framework. ? Methods: Consequences of a mycorrhizal shift were examined in Cymbidium (Orchidaceae) using achlorophyllous and sister chlorophyllous species. Fungal associates of the two achlorophyllous mycoheterotrophs (C. macrorhizon and C. aberrans), their close relatives, the chlorophyllous mixotrophs (C. goeringii and C. lancifolium) and an outgroup, the chlorophyllous autotroph C. dayanum, were identified by internal transcribed spacers of the nuclear ribosomal DNA sequences. ? Key results: Molecular identification of mycorrhizal fungi revealed: (1) the outgroup autotroph is predominantly dependent on saprobic Tulasnellaceae, (2) the mixotrophs associate with the Tulasnellaceae and ectomycorrhizal groups including the Sebacinales, Russulaceae, Thelephoraceae and Clavulinaceae, and (3) the two mycoheterotrophs are mostly specialized with ectomycorrhizal Sebacinales. ? Conclusion: Fungal partners in Cymbidium have shifted from saprobic to ectomycorrhizal fungi via a phase of coexistence of both nutritional types of fungi. These three phases correspond to the evolution from autotrophy to mycoheterotrophy via mixotrophy in Cymbidium. We demonstrate that shifts in mycorrhizal fungi correlate with the evolution of nutritional modes in plants. Furthermore, gradual shifts in fungal partners through a phase of coexistence of different types of mycobionts may play a crucial role in the evolution of mycoheterotrophic plants.