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。     

Morphological and molecular characterization of Tulasnella spp. fungi isolated from the roots of Epidendrum secundum,a widespread Brazilian orchid

Tulasnella spp. are the main fungal symbionts of Brazilian Epidendrum orchids. The taxonomy of these fungi is largely based on ITS rDNA similarity, but culture dependent techniques are still essential to establish the true biological entity of the mycobiont. The aim of this study was to characterize morphologically and molecularly 16 Tulasnella spp. fungi isolated from three different populations of E. secundum and to test the coincidences between morphological and molecular characterization. Two uninucleate rhizoctonia fungi, obtained from Oncidium barbaceniae, and two phytopathogenic isolates were included as outgroups. Qualitative and quantitative morphological characteristics were analyzed using multivariate statistics and were able to distinguish Ceratobasidium, Tulasnella and Thanatephorus genera and separate the isolates of Tulasnella spp. into two groups. Analysis of RAPD (Random Amplified Polymorphic DNA) and ITS rDNA sequences validated the morphological data. Symbionts of O. barbaceniae presented identity to ITS sequences of Ceratobasidium genus, while E. secundum isolates presented identity to two species of Tulasnella. We observed homogeneity among Tulasnella spp. obtained from a single population and from neighboring populations, but there was higher variability among isolates obtained from populations of regions that were farther apart. Morphological data associated with multivariate statistics proved to be a useful tool in the multi-level taxonomy of these orchid-associated fungi and in estimating the diversity of orchid mycorrhizal fungi.     

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host-switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well-resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event-based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host-switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages.     

Mycorrhizal specificity, preference, and plasticity of six slipper orchids from South Western China

Mycorrhizal fungi of six endangered species, Paphiopedilum micranthum, Paphiopedilum armeniacum, Paphiopedilum dianthum, Cypripedium flavum, Cypripedium guttatum, and Cypripedium tibeticum, from two closely related genera in the Orchidaceae from Southwestern China, were characterized using the nuclear internal transcribed spacer (ITS) and part of the large subunit gene of mitochondrial rDNA (mtLSU) sequences. The most frequently detected fungi belonged to the Tulasnellaceae. These fungi were represented by 25 ITS sequence types and clustered into seven major clades in the phylogenetic analysis of 5.8S sequences. Species of Paphiopedilum and Cypripedium shared no fungal ITS sequence types in common, but their fungal taxa sometimes occurred in the same major clade of the 5.8S phylogenetic tree. Although it had several associated fungal ITS sequence types in a studied plot, each orchid species had in general only a single dominant type. The fungal sequence type spectra of different species of Paphiopedilum from similar habitats sometimes overlapped; however, the dominant sequence types differed among the species and so did the sequence-type spectra within Cypripedium. Orchids of P. micranthum and P. armeniacum transplanted from the field and grown in two greenhouses had a greater number of mycorrhizal associations than those sampled directly from the field. Root specimens from P. micranthum taken from the greenhouses were preferably associated with mycobionts of the Tulasnella calospora complex, while those from the field had mycorrhizal associations of other tulasnelloid taxa. Such plasticity in mycorrhizal associations makes ex situ conservation or even propagation by means of mycorrhization of axenically grown seedlings possible.     

Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand

Three orchid genera, Paphiopedilum, Cymbidium, and Dendrobium, are among the most heavily traded ornamental plants in Thailand. In this study, 27 isolates of Rhizoctonia-like fungi were isolated from root sections of mature orchids in the three orchid genera, collected from diverse horticultural settings in Chiang Mai and Chiang Rai provinces of Thailand. Fungal identification was done by the morphological characterization, the comparison of the internal transcribed spacer and 5.8S ribosomal DNA sequences, and the phylogenetic analysis. Epulorhiza repens was found to be the most common species found in the roots of various species of all three orchid genera, whereas Epulorhiza calendulina-like isolates were strictly found in the roots of Paphiopedilum species. We have also isolated and described an anamorph of Tulasnella irregularis, four new anamorphic species in the genus Tulasnella, and a new anamorphic species in the family Tulasnellaceae. Our study provides information on diversity of root-associated fungi of the orchid genera and at the sampling sites that were rarely addressed in the previous studies.     

Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae)

The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi.     

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

Background and Aims The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant.Methods The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field.Key Results Leaf δ¹³C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot.Conclusions The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a ¹³C- and ¹⁵N-enriched fungal source.     

Continent-wide distribution in mycorrhizal fungi: implications for the biogeography of specialized orchids

Background and Aims Although mycorrhizal associations are predominantly generalist, specialized mycorrhizal interactions have repeatedly evolved in Orchidaceae, suggesting a potential role in limiting the geographical range of orchid species. In particular, the Australian orchid flora is characterized by high mycorrhizal specialization and short-range endemism. This study investigates the mycorrhizae used by Pheladenia deformis, one of the few orchid species to occur across the Australian continent. Specifically, it examines whether P. deformis is widely distributed through using multiple fungi or a single widespread fungus, and if the fungi used by Australian orchids are widespread at the continental scale.Methods Mycorrhizal fungi were isolated from P. deformis populations in eastern and western Australia. Germination trials using seed from western Australian populations were conducted to test if these fungi supported germination, regardless of the region in which they occurred. A phylogenetic analysis was undertaken using isolates from P. deformis and other Australian orchids that use the genus Sebacina to test for the occurrence of operational taxonomic units (OTUs) in eastern and western Australia.Key Results With the exception of one isolate, all fungi used by P. deformis belonged to a single fungal OTU of Sebacina. Fungal isolates from eastern and western Australia supported germination of P. deformis. A phylogenetic analysis of Australian Sebacina revealed that all of the OTUs that had been well sampled occurred on both sides of the continent.Conclusions The use of a widespread fungal OTU in P. deformis enables a broad distribution despite high mycorrhizal specificity. The Sebacina OTUs that are used by a range of Australian orchids occur on both sides of the continent, demonstrating that the short-range endemism prevalent in the orchids is not driven by fungal species with narrow distributions. Alternatively, a combination of specific edaphic requirements and a high incidence of pollination by sexual deception may explain biogeographic patterns in southern Australian orchids.     

Four Tulasnella taxa associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata

Of the more than 400 indigenous orchid species in Western Australia, Cryptostylis ovata is the only species that retains its leaves all year round. It exists as a terrestrial herb and occasionally as an epiphyte in forested areas. Like all terrestrial orchids, C. ovata plants associate with mycorrhizal fungi, but their identities have not previously been investigated. Fungi were isolated from pelotons in rhizomes collected from three southern and two northern populations of C. ovata on six occasions over two years. Phylogenetic analysis of ITS sequences temporally and spatially revealed that all the fungal isolates were of Tulasnella species of four distinct groups. One Tulasnella group was present only in the three southern orchid populations, and it closely resembled T. prima isolates previously described from Chiloglottis sp. orchids from eastern Australia. Isolates collected from plants in the two northern populations were of three undescribed Tulasnella groups. Analysis of intra-group diversity using inter-simple sequence repeat markers revealed that plants were usually colonised by a single genotype of Tulasnella at each sampling period, and this genotype usually, but not always, persisted with the host plant over both years tested.     

Diversity and host specificity of endophytic Rhizoctonia-like fungi from tropical orchids

All orchids have an obligate relationship with mycorrhizal symbionts. Most orchid mycorrhizal fungi are classified in the form-genus Rhizoctonia. This group includes anamorphs of Tulasnella, Ceratobasidium, and Thanatephorus. Rhizoctonia can be classified according to the number of nuclei in young cells (multi-, bi-, and uninucleate). From nine Puerto Rican orchids we isolated 108 Rhizoctonia-like fungi. Our isolates were either bi- or uninucleate, the first report of uninucleate Rhizoctonia-like fungi as orchid endophytes. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 26 isolates and identified four fungal lineages, all related to Ceratobasidium spp. from temperate regions. Most orchid species hosted more than one lineage, demonstrating considerable variation in mycorrhizal associations even among related orchid species. The uninucleate condition was not a good phylogenetic character in mycorrhizal fungi from Puerto Rico. All four lineages were represented by fungi from Tolumnia variegata, but only one lineage included fungi from Ionopsis utricularioides. Tropical epiphytic orchids appear to vary in degree of specificity in their mycorrhizal interactions more than previously thought.     

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。     

Production of indole-3-acetic acid by Thai native orchid-associated fungi

Thirteen endophytic fungi were isolated from roots of three orchid species, Spathoglottis affinis, Paphiopedelum bellatulum and Phaius tankervilleae. Of these, three fungal isolates produced high levels of indole-3-acetic acid (IAA) in culture medium supplemented with 2 mg/ml of L-tryptophan, and were selected for further analysis. Morphological characteristics and a phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that the fungal isolates CMU-SLP 007 and CMU-NUT 013 belonged to family Tulasnellaceae, genus Tulasnella (the anamorphic genus Epulorhiza) and the fungal isolate CMU-AU 006 belonged to Colletotrichum gloeosporioides. These three fungal isolates produced maximum levels of IAA when grown in a culture medium supplemented with 4 mg/ml of L-tryptophan (C. gloeosporioides CMU-AU 006, 243.56 μg/ml and Tulasnella sp. CMU-SLP 007, 155.63 μg/ml) and 6 mg/ml of L-tryptophan (Tulasnella sp. CMU-NUT 013, 104.03 μg/ml). Thin layer chromatography revealed that all fungal IAA presented the same R_f value as the standard IAA. The biological activity of fungal IAA showed that it increased the length of stem forming roots and the number of roots of kidney bean (Phaseolus vulgaris), promoted seed germination, the length of roots and root to shoot ratio of corn (Zea mays) and increased the elongation of rice (Oryza sativa) coleoptiles when compared with all controls (water and culture medium treatments). In addition, the results of all biological activities using fungal IAA indicated that the quality of fungal IAA were similar to standard IAA.     

Cryptostylis species (Orchidaceae) from a broad geographic and habitat range associate with a phylogenetically narrow lineage of Tulasnellaceae fungi

While many Australian terrestrial orchids have highly specialized mycorrhizal associations, we tested the hypothesis that the geographically widespread orchid genus Cryptostylis associates with a diversity of fungal species. Using fungal isolation and molecular approaches, we investigated the mycorrhizal associations of five Australian Cryptostylis species (27 sites sampled) and included limited sampling from three Asiatic Cryptostylis species (two sites). Like related orchid genera, Tulasnellaceae formed the main fungal associations of the Cryptostylis species we sampled, although some ectomycorrhizal, ericoid and saprotrophic fungi were detected infrequently. Each species of Australian Cryptostylis associated with three to seven Tulasnella Operational Taxonomic Units (OTUs), except for C. hunteriana where only one Tulasnella OTU was detected. In total, eleven Tulasnella OTUs associated with Australian Cryptostylis. The Asiatic Cryptostylis associated with four different Tulasnella OTUs belonging to the same lineage as the Australian species. While five Tulasnella OTUs (T. australiensis, T. prima, T. warcupii, T. densa, and T. punctata) were used by multiple species of Australian Cryptostylis, the most commonly used OTU differed between orchid species. The association with different Tulasnella fungi by Cryptostylis species co-occurring at the same site suggests that in any given environmental condition, Cryptostylis species may intrinsically favour different fungal OTUs.     

Defining species in Tulasnella by correlating morphology and nrDNA ITS-5.8S sequence data of basidiomata from a tropical Andean forest

The genus Tulasnella comprises important orchid mycobionts. Molecular phylogenetic studies on nrITS-5.8S sequences of Tulasnella species previously isolated from mycorrhizas of epiphytic orchids from a tropical Andean forest showed genomic variability among clones which was difficult to interpret as intra- or interspecific variations or to correlate with described Tulasnella species. To improve this situation, we collected basidiomata of Tulasnella in an Andean forest, studied part of the sequences of fungal ribosomal genes and correlated molecular data with the morphology of the specimens. Within five basidiomata displaying slight morphological variability, we found inter-specimen nrITS1-5.8S-ITS2 variability corresponding to proportional differences of less than 1% except for one clone with 5.1% divergence. Results indicate that the slightly variable basidiomata should be considered as one species, which is morphologically tentatively assigned to the Tulasnella pruinosa complex. However, comparison of nrITS1-5.8S-ITS2 sequences, including sequences of T. pruinosa from other origins, indicate that Tulasnella sp. is only distantly related to the T. pruinosa specimens included in the analyses. Sequences of all morphologically similar and taxonomically well-identified species are required to decide whether the basidiomata analyzed in the present study represent a new species. The new sequences are rather similar to sequences obtained previously from mycorrhizae of epiphytic orchids of the same area indicating mycorrhizal potential of this fungus.     

Ultrastructural studies and molecular characterization of root-associated fungi of $$\textit{}$$ (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.     

Ceratobasidiaceae mycorrhizal fungi isolated from nonphotosynthetic orchid <Emphasis Type="Italic">Chamaegastrodia sikokiana</Emphasis>

Mycorrhizal fungi were isolated from the nonphotosynthetic orchid Chamaegastrodia sikokiana and identified as members of Ceratobasidiaceae by phylogenetic analysis of the internal transcribed spacer (ITS) region of ribosomal deoxyribonucleic acid. The ITS sequences were similar among geographically separated samples obtained from Mt. Kiyosumi in Chiba Prefecture and Mt. Yokokura in Kochi Prefecture. One of the isolated fungi, KI1-2, formed ectomycorrhiza on seedlings of Abies firma in pot culture, suggesting that tripartite symbiosis exists among C. sikokiana, mycorrhizal fungi, and A. firma in nature, and carbon compounds are supplied from A. firma to C. sikokiana through the hyphae of the mycorrhizal fungi. To our knowledge, this is the second study to suggest the involvement of Ceratobasidiaceae fungi in tripartite symbiosis with achlorophyllous orchids and photosynthetic host plants.     

Variation in mycorrhizal performance in the epiphytic orchid <Emphasis Type="Italic">Tolumnia variegata in vitro</Emphasis>: the potential for natural selection

Symbiotic seed germination is a critical stage in orchid life histories. Natural selection may act to favor plants that efficiently use mycorrhizal fungi. However, the necessary conditions for natural selection – variation, heritability, and differences in fitness – have not been demonstrated for either orchid or fungus. With the epiphytic orchid Tolumnia variegata as a model system, we ask the following questions: (1) Do seeds from different individuals in a population differ in germination and seedling development in the presence of the same fungi? (2) Do different mycorrhizal fungi (Ceratobasidium spp.) differ in ability to stimulate seed germination and growth in T. variegata? And (3) are the Ceratobasidium isolates that best induce seed germination and seedling development more closely related to each other than to isolates that are less effective? We performed symbiotic seed germination experiments in vitro. The experiments were done using mycorrhizal fungi isolated from T. variegata; relationships among the fungi were inferred from nuclear ribosomal ITS sequences. We found significant variation for both symbiotic germination and seedling growth among biparental seed crops obtained from a population of T. variegata plants. Differences among Ceratobasidium fungi in seed germination were significant. The fungi that induced highest seed germination and seedling development belonged to two of four clades of Ceratobasidium. The two experiments show that there is potential for natural selection to act on orchid–fungus relationships. Given that orchids vary in performance, and that mycorrhizal fungi are not geographically distributed homogeneously, mycorrhizae may affect population size, distribution and evolution of orchids.     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

Low specificity and nested subset structure characterize mycorrhizal associations in five closely related species of the genus Orchis

Most orchid species rely on mycorrhizae to complete their life cycle. Despite a growing body of literature identifying orchid mycorrhizal associations, the nature and specificity of the association between orchid species and mycorrhizal fungi remains largely an open question. Nonetheless, better insights into these obligate plant–fungus associations are indispensable for understanding the biology and conservation of orchid populations. To investigate orchid mycorrhizal associations in five species of the genus Orchis (O. anthropophora, O. mascula, O. militaris, O. purpurea, and O. simia), we developed internal transcribed spacer‐based DNA arrays from extensive clone library sequence data sets, enabling rapid and simultaneous detection of a wide range of basidiomycetous mycorrhizal fungi. A low degree of specificity was observed, with two orchid species associating with nine different fungal partners. Phylogenetic analysis revealed that the majority of Orchis mycorrhizal fungi are members of the Tulasnellaceae, but in some plants, members of the Thelephoraceae, Cortinariaceae and Ceratobasidiaceae were also found. In all species except one (O. mascula), individual plants associated with more than one fungus simultaneously, and in some cases, associations with ≥3 mycorrhizal fungi at the same time were identified. Nestedness analysis showed that orchid mycorrhizal associations were significantly nested, suggesting asymmetric specialization and a dense core of interactions created by symmetric interactions between generalist species. Our results add support to the growing literature that multiple associations may be common among orchids. Low specificity or preference for a widespread fungal symbiont may partly explain the wide distribution of the investigated species.