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。     

凉山州龙肘山锈红杜鹃与薄叶马银花根部真菌分子检测

【目的】为检验直接分子检测法用于揭示杜鹃花属(Rhododendron)植物根部真菌(Root-associated fungi,RAF)组成的有效性。【方法】采用不依赖于培养的分子检测技术直接从锈红杜鹃(R.bureavii)与薄叶马银花(R.leptothrium)的发根(Hair root)提取DNA,用真菌特异性引物扩增r DNA-ITS区、经克隆后测序,对获得的ITS序列进行分析;通过收集NCBI中与本研究的RAF相似性97%以上的所有序列对应的真菌来源(土壤或根系的身份)数据,分析真菌的生态学特性,并用FUNGuild软件提供的方法划分真菌的生态类型。【结果】从两种杜鹃花根部共检测到15种真菌,其中担子菌门(Basdiomycota)真菌3种,子囊菌门(Ascomycota)真菌12种。柔膜菌目(Helotiales)真菌在两种杜鹃花RAF群落中占据优势,并且在两种杜鹃花根系中均检测到该类真菌。此外,两种杜鹃花根部有多种生态类型的RAF共存,包括曾被频繁报道的杜鹃花类菌根菌Oidiodendron sp.和Rhizoscyphus sp.、内生真菌Phialocephala fortinii、共生一致病过渡型真菌Pezoloma ericae、外生菌根共生菌Meliniomyces sp.,以及腐生型真菌Myceana sp.、Lachnum virgineum、Herpotrichia sp.。【结论】直接分子检测法从两种杜鹃花属植物根部检测到的真菌谱系多样性较高、生态类型复杂,这一方法能较为全面地反映杜鹃花属植物RAF多样性。     

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。     

黔中地区马尾松林下杜鹃根部内生真菌群落组成及其生态功能

探索黔中马尾松(Pinus massoniana)林下杜鹃(Rhododendron simsii)根内生真菌物种多样性和生态功能。采集黔中乌当(WD)、孟关(MG)、龙里(LL) 3个地区马尾松林下杜鹃的发根,提取真菌DNA进行高通量测序。分析真菌Alpha多样性,借助FUNGuild注释平台解析真菌的生态功能类别,探索真菌群落中的核心微生物组,并结合网络图展示菌群之间的关联性。结果,3个地区的杜鹃根部内生真菌多样性非常丰富,WD地区杜鹃内生真菌多样性和丰富度最高。试验共获得有效序列425799条,817个可操作分类单元(OTU),分属于6门19纲52目103科154属,主要隶属于子囊菌门(81.2%)、接合菌门(5.8%)和担子菌门(4.7%);优势纲、目、科分别为:散囊菌纲(43.0%)、散囊菌目(39.2%)、发菌科(39.2%);在属的水平上,青霉属(38.60%)占比最高,其次是木霉属(7.20%)、拟盘多毛孢属(6.10%)。根部的真菌拥有多种生态功能群,如未定义腐生菌(194 OTU)、植物病原菌(20 OTU)、土壤腐生菌(18 OTU)、外生菌(14 OTU)、地衣共生真菌(10 OTU)、杜鹃类菌根真菌(5 OTU)、木腐生菌(5 OTU)、丛枝菌根(4 OTU)、内生菌(2 OTU)、动物病原菌(8 OTU),以及多种混合营养型类群21类,102个Undefined种类在FUNGuild数据库中没有参考信息。根部真菌可以形成生态位共享模式,而且不同功能群之间存在耦合性,核心基因组与关键物种以真菌组形成的生态功能团表现。     

云南杓兰菌根真菌组成及共生关系研究

杓兰属(Cypripedium)植物因具有较高的观赏和药用价值而长期被过度采集,已成为濒危植物。利用菌根技术进行杓兰属植物的保护和人工栽培,需要获得其可培养的菌根真菌。该研究采用分离培养法和共生回接方法,研究了云南杓兰菌根真菌菌群组成及其共生关系。结果表明:(1)从10株云南杓兰300块毛根组织中分离获得126株内生真菌,归属为3个菌属,分别是胶膜菌属(Tulasnella)73株、伏革菌属(Corticium)36株、角担菌属(Ceratobasidium)17株。其中,胶膜菌属为优势菌群,占总菌株数量的57.94%。(2)6株供试菌株中,4株菌株可显著缩短种子的萌发过程,6株菌株对幼苗的生长有显著的促进作用。(3)从中筛选获得一株CY-18高效促生真菌,对云南杓兰种子共生萌发和幼苗共生生长有极显著的促进作用。该研究结果为更好地利用菌根技术进行杓兰属植物资源的保护与可持续利用奠定了基础。     

腐生型兰科植物研究进展

腐生型兰科植物又称完全菌根异养型兰(Fully Mycoheterotrophic Orchids),无绿叶,不含叶绿素,不能进行光合作用制造有机物,完全依靠与其共生的真菌提供营养。近年来,腐生兰独特的生活方式引起了生物学家对其生理生态及进化问题的广泛关注,但目前仍缺乏系统深入的研究。为此,作者通过整理《中国植物志》和近年来报道的相关文献,发现分布在中国的腐生型兰科植物约有23属81种,主要集中在天麻属(Gastrodia)(24种)、鸟巢兰属(Neottia)(8种)、无叶兰属(Aphyllorchis)(6种)、山珊瑚属(Galeola)(5种)等。由于腐生兰特殊的营养需求,相比于其他营养类型的兰科植物,其菌根真菌有较大区别。腐生兰的菌根真菌主要有两大类,即外生菌根真菌(Ectomycorrhizal fungi,ECM)和非丝核菌类的腐生菌(Non-rhizoctonia SAP fungi)。该文还综述了腐生兰与其菌根真菌的专一性、营养来源以及系统进化等问题,提出了目前研究存在的问题,并对今后的研究工作进行了展望,以期为腐生兰的资源保护及再生研究提供参考。     

黄精根际及药用部位内生真菌群落组成和生态功能分析

为寻找促进药用植物活性代谢产物合成的微生物,该文以黄精为研究对象,利用高通量测序技术和生态功能预测平台,测定根际土真菌、根茎和根内生真菌的ITS序列,分析其真菌多样性和群落组成,并预测根茎内生真菌的生态功能。结果表明:(1)测序得到1 023个可操作分类单元(OTUs),根际、根茎和根真菌OTU数分别为703、128和141,三种部位真菌群落组成差异显著,根际土存在特有的真菌类群,即壶菌门。(2)根际土、根茎及根共有OTU 41个,子囊菌门占共有真菌的58.15%,丰度最大。(3)根茎内生真菌被划分6个生态功能群,包括未定义腐生菌、菌寄生真菌、动物病原菌、植物病原菌、丛枝菌根真菌和地衣共生真菌,37个undefined种类(34.91%)在FUNGuild数据库中没有参考信息。研究认为根茎中优势菌属Setophoma、新赤壳属等内生真菌可能与活性代谢产物密切相关,可为黄精药用功能菌群的发掘提供数据参考。     

濒危药用植物掌裂兰根部内生真菌和根际土真菌多样性分析

【背景】植物内生真菌对宿主植物促生长、抗旱和增强抗病能力等方面有着重大的研究和利用价值,尤其对兰科植物的生长起到重要的作用。【目的】通过对掌裂兰根部内生真菌和根际土真菌多样性进行系统分析,掌握掌裂兰根部内生真菌与根际土真菌群落结构,为进一步探究掌裂兰植物与真菌共生规律提供参考。【方法】采用Illumina MiSeq高通量测序技术分析掌裂兰根部内生真菌和根际土真菌多样性。【结果】掌裂兰根部内生真菌隶属于7门89属,优势菌属为瘤菌根菌属(Epulorhiza)(16.93%)、头梗霉属(Cephaliophora)(10.41%)、酵母属(Saccharomyces)(5.73%)、角担菌属(Ceratobasidium)(5.32%)和镰刀菌属(Fusarium)(5.12%),其中Epulorhiza和Ceratobasidium为兰科植物菌根真菌;根际土真菌隶属于11门269属,优势菌属为镰刀菌属(Fusarium)(8.09%)、丛赤壳属(Neonectria)(6.79%)、Plectosphaerella (3.39%)和被孢霉属(Mortierella)(3.01%)。通过...     

四种杓兰的菌根结构及其周年动态

对黄花杓兰、云南杓兰、西藏杓兰和紫点杓兰四种高山杓兰成年植株的根进行了一个生长周期的切片观察。发现四种杓兰的的菌根结构及其在一个生长周期中的动态变化有以下共同特征:在其成年生活期的各个时期,都有真菌入侵形成内生菌根结构,菌丝在其根部皮层细胞内有四种存在状态:(a)咖啡色或黄色、菌丝扭结缠绕而成的大菌丝结;(b)灰褐色或黄色、由片断菌丝组成的大菌丝结;(c)无色、零散的菌丝;(d)橘红色和黄色、菌丝形态模糊的小菌丝结。真菌的入侵-消解在其成年生活期中周而复始地进行,四种状态的菌丝是处于入侵-消解循环不同阶段的菌丝。并由此对高海拔环境下二者的共生关系进行了初步的探讨:真菌侵入杓兰根部以获得自身生存和繁衍所需的营养物质,杓兰利用真菌菌丝消解后的营养物质,进行混合营养。     

青藏高原冰川棘豆(Oxytropis glacialis)内生菌核心微生物组的界定及其互作网络分析

【背景】植物内生菌(endophyte)对寄主植物的益生作用有利于植物的生存与扩散,而菌群互作网络为内生菌生态功能的实现提供了基础保障。【目的】了解影响西藏畜牧业发展的主要疯草冰川棘豆(Oxytropis glacialis)内生菌核心微生物组及其菌群互作网络,为青藏高原疯草类有毒植物的科学治理和利用提供基础参考依据。【方法】利用高通量测序技术分析冰川棘豆中内生菌核心微生物组,构建内生菌相关性网络,并以与苦马豆素代谢相关内生菌为例分析冰川棘豆内生菌的互作模式。【结果】内生细菌测序共得到175791条有效序列,注释到428个细菌OTU,分属于19个门和267个属;内生真菌测序共得到757 113条有效序列,注释到391个真菌OTU,分属于7个门和149个属。Venn图分析表明,根、叶组织的核心内生细菌菌属数目(54、62)大于核心内生真菌(22、13),根组织中的核心内生菌种类与叶组织相当(76、75)。系统发育树分析表明,冰川棘豆中存在产生苦马豆素内生真菌链格孢属(Alternaria)和降解苦马豆素的内生细菌短波单胞杆菌属(Brevundimonas)。相关性网络分析表明,内生菌菌群间以正向反馈的互作网络关系为主,核心菌群可能主要通过间接性的互作方式将影响传递到微生物群落,其中链格孢属与短波单胞杆菌属作为核心菌属通过间接性的显著相关关系(|ρ|0.6,P0.01)参与菌群间互作网络。【结论】冰川棘豆核心菌群以间接的方式参与内生菌菌群间的互作网络,高度的菌属连接性为内生菌生态功能的实现提供可能。     

A diverse fungal community associated with Pseudorchis albida (Orchidaceae) roots

In addition to orchid mycorrhizal fungi (OrMF), the roots of orchids harbour plant fungal endophytes termed root-associated fungi (RAF). In the present study, the endangered photosynthetic orchid Pseudorchis albida was screened for OrMF and RAF using culture-dependent (isolations from root sections and pelotons) and culture-independent (cloning from root sections) techniques. The efficiency of the different approaches for detecting the fungi and the effect of the sampling season (summer or autumn) were evaluated. In total, 66 distinct OTUs of mycorrhizal and non-mycorrhizal fungi were found, which, to our knowledge, is the highest diversity of RAF that has yet been detected in a single orchid species. The OrMF community was dominated by Tulasnella species, which were mainly detected by isolation from pelotons or cloning from root sections. The roots and tubers showed higher mycorrhizal colonization in summer, corroborating the frequent reports of Tulasnella from pelotons in this season. In contrast, two helotialean fungi, Varicosporium elodeae and Leohumicola sp., the latter of which was repeatedly isolated from pelotons, were significantly more abundant in the autumn.     

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.     

海南濒危树种保育圃白木香与降香黄檀幼苗根部真菌谱系与生态型多样性比较

【背景】根部真菌是影响植物幼苗存活、定植和生长的重要因子之一,但是苗圃培育的幼苗根部真菌物种组成与生态学特性尚不清楚。【目的】研究苗圃培育的白木香(Aquilaria sinensis)与降香黄檀(Dalbergia odorifera)幼苗根部真菌群落谱系与生态型多样性,以及宿主植物对根部真菌群落结构的影响。【方法】采集幼苗根尖样品提取基因组DNA,用真菌通用引物与丛枝菌根真菌(AMF)特异性引物扩增真菌r DNA-ITS区,经克隆、测序、序列分析鉴定真菌。通过基于核酸与Metadata数据关联分析的FUNGuild软件,划分根部真菌的营养型和共位群。采用非公制多维尺度分析法(NMDS)研究幼苗根部真菌群落物种组成差异与宿主植物物种及形态指标的关系。【结果】白木香与降香黄檀幼苗根部真菌物种丰富,达51个OTU;谱系多样性较高,涉及毛霉菌门(Mucoromycota,51%)、子囊菌门(Ascomycota,43%)以及担子菌门(Basidiomycota,6%)。这些根部真菌涉及不同的营养型与共位群,包括共生型真菌29种,频度较高的如Glomeromycetes sp.2、Rhizophagus irregularis等,二者均属于AMF共位群;腐生营养型真菌5种,如Talaromyces pinophilus、Rhizopycnis vagum等;病原型真菌2种,是Mycoleptodiscus sp.和Fusarium phaseoli;还有15种其生态类型不确定。NMDS分析结果表明,宿主植物物种、株高、地径、叶面积对根部真菌群落物种组成的影响不显著。然而,株高对AMF群落的物种组成有较弱的影响。【结论】本苗圃条件下,土壤中本土性根部真菌繁殖体较为充足,白木香与降香黄檀幼苗根部真菌群落谱系多样性较高,多种营养型与共位群的根部真菌共存;此外,采用真菌通用引物对ITS1F/ITS4研究根部真菌群落物种多样性时,AMF多样性可能会被极度低估。     

Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities

Fungal communities play a key role in ecosystem functioning. However, only little is known about their composition in plant roots and the soil of biomass plantations. The goal of this study was to analyze fungal biodiversity in their belowground habitats and to gain information on the strategies by which ectomycorrhizal (ECM) fungi form colonies. In a 2-year-old plantation, fungal communities in the soil and roots of three different poplar genotypes (Populus × canescens, wildtype and two transgenic lines with suppressed cinnamyl alcohol dehydrogenase activity) were analyzed by 454 pyrosequencing targeting the rDNA internal transcribed spacer 1 (ITS) region. The results were compared with the dynamics of the root-associated ECM community studied by morphotyping/Sanger sequencing in two subsequent years. Fungal species and family richness in the soil were surprisingly high in this simple plantation ecosystem, with 5944 operational taxonomic units (OTUs) and 186 described fungal families. These findings indicate the importance that fungal species are already available for colonization of plant roots (2399 OTUs and 115 families). The transgenic modification of poplar plants had no influence on fungal root or soil communities. Fungal families and OTUs were more evenly distributed in the soil than in roots, probably as a result of soil plowing before the establishment of the plantation. Saprophytic, pathogenic, and endophytic fungi were the dominating groups in soil, whereas ECMs were dominant in roots (87%). Arbuscular mycorrhizal diversity was higher in soil than in roots. Species richness of the root-associated ECM community, which was low compared with ECM fungi detected by 454 analyses, increased after 1 year. This increase was mainly caused by ECM fungal species already traced in the preceding year in roots. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localized ECM fungi.     

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.     

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。     

High specificity generally characterizes mycorrhizal association in rare lady's slipper orchids, genus Cypripedium

Lady's slipper orchids (Cypripedium spp.) are rare terrestrial plants that grow throughout the temperate Northern Hemisphere. Like all orchids, they require mycorrhizal fungi for germination and seedling nutrition. The nutritional relationships of adult Cypripedium mycorrhizae are unclear; however, Cypripedium distribution may be limited by mycorrhizal specificity, whether this specificity occurs only during the seedling stage or carries on into adulthood. We attempted to identify the primary mycorrhizal symbionts for 100 Cypripedium plants, and successfully did so with two Cypripedium calceolus, 10 Cypripedium californicum, six Cypripedium candidum, 16 Cypripedium fasciculatum, two Cypripedium guttatum, 12 Cypripedium montanum, and 11 Cypripedium parviflorum plants from a total of 44 populations in Europe and North America, yielding fungal nuclear large subunit and mitochondrial large subunit sequence and RFLP (restriction fragment length polymorphism) data for 59 plants. Because orchid mycorrhizal fungi are typically observed without fruiting structures, we assessed fungal identity through direct PCR (polymerase chain reaction) amplification of fungal genes from mycorrhizally colonized root tissue. Phylogenetic analysis revealed that the great majority of Cypripedium mycorrhizal fungi are members of narrow clades within the fungal family Tulasnellaceae. Rarely occurring root endophytes include members of the Sebacinaceae, Ceratobasidiaceae, and the ascomycetous genus, Phialophora. C. californicum was the only orchid species with apparently low specificity, as it associated with tulasnelloid, ceratobasidioid, and sebacinoid fungi in roughly equal proportion. Our results add support to the growing literature showing that high specificity is not limited to nonphotosynthetic plants, but also occurs in photosynthetic ones.