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.     

Molecular identification of mycorrhizal fungi in Neuwiedia veratrifolia (Orchidaceae)

We here apply a previously described method for identification of single peloton orchid mycorrhiza to a key orchid group and extend the usefulness in the heterobasidiomycetes of an existing fungal database for identification of mycorrhizal fungi. We amplified and sequenced mitochondrial ribosomal large subunit DNA from fungi in roots of Neuwiedia veratrifolia (Orchidaceae), a member of the small subfamily Apostasioideae that is sister to the remainder of Orchidaceae, and used the extended database to identify the mycorrhizal fungi. Sequences from fungi cultured from Neuwiedia roots and from direct peloton amplifications were analyzed cladistically with sequences determined from reference fungal collections and published sequences. The fungi from Neuwiedia are referred to the heterobasidiomycetous orders Tulasnellales and Ceratobasidiales, indicating that apostasioids utilize the same fungi as other photosynthetic orchids. The majority of Neuwiedia mycobionts came together in a clade with Tulasnella species, but some were most closely related to Thanatephorus. In some cases members of these two clades were isolated from the same orchid plant, providing another example of multiple mycobionts occurring in a single plant.     

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.     

PCR primers useful for nucleotide sequencing of rDNA of the powdery mildew fungi

Four PCR primers that are useful to determine the nucleotide sequences of the rDNA of the powdery mildew fungi were newly designed. These primers provide both enough stability to work on a wide range of powdery mildews and enough specificity to eliminate contaminating DNA by PCR. DNA sequences of the rDNA ITS region were successfully obtained from specimens that were contaminated by other fungi. In addition, sequence results of the 18S and 28S rDNA were dramatically improved by using these primers in most of the specimens examined.     

Identity and Specificity of Rhizoctonia-Like Fungi from Different Populations of Liparis japonica (Orchidaceae) in Northeast China

Mycorrhizal association is known to be important to orchid species, and a complete understanding of the fungi that form mycorrhizas is required for orchid ecology and conservation. Liparis japonica (Orchidaceae) is a widespread terrestrial photosynthetic orchid in Northeast China. Previously, we found the genetic diversity of this species has been reduced recent years due to habitat destruction and fragmentation, but little was known about the relationship between this orchid species and the mycorrhizal fungi. The Rhizoctonia-like fungi are the commonly accepted mycorrhizal fungi associated with orchids. In this study, the distribution, diversity and specificity of culturable Rhizoctonia-like fungi associated with L. japonica species were investigated from seven populations in Northeast China. Among the 201 endophytic fungal isolates obtained, 86 Rhizoctonia-like fungi were identified based on morphological characters and molecular methods, and the ITS sequences and phylogenetic analysis revealed that all these Rhizoctonia-like fungi fell in the same main clade and were closely related to those of Tulasnella calospora species group. These findings indicated the high mycorrhizal specificity existed in L. japonica species regardless of habitats at least in Northeast China. Our results also supported the wide distribution of this fungal partner, and implied that the decline of L. japonica in Northeast China did not result from high mycorrhizal specificity. Using culture-dependent technology, these mycorrhizal fungal isolates might be important sources for the further utilizing in orchids conservation.     

长茎羊耳蒜菌根真菌类群的研究

研究了广西雅长自然保护区和云南西双版纳自然保护区共3个产地的兰科植物羊耳蒜属长茎羊耳蒜Liparis viridiflora的菌根真菌类群区系组成.根内菌根真菌的核糖体基因内转录间隔区序列(rDNA-ITS)采用PCR技术扩增,克隆,测序并构建系统发育树.结果表明,长茎羊耳蒜根内所检测到的真菌大部分为胶膜菌科Tulasnellaceae真菌;根据序列相似性和系统发育分析,所有真菌可归为12个可操作分类单元(OTU),其中胶膜菌科有7个OTUs,达到总数的90.6%,为优势类群.菌根真菌多样性及区系组成在3个不同产地样本之间存在一定的差异;菌根真菌可能和兰科植物的生境适应性存在一定的相关性.     

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.     

Tidying up international nucleotide sequence databases: ecological, geographical and sequence quality annotation of its sequences of mycorrhizal fungi

Sequence analysis of the ribosomal RNA operon, particularly the internal transcribed spacer (ITS) region, provides a powerful tool for identification of mycorrhizal fungi. The sequence data deposited in the International Nucleotide Sequence Databases (INSD) are, however, unfiltered for quality and are often poorly annotated with metadata. To detect chimeric and low-quality sequences and assign the ectomycorrhizal fungi to phylogenetic lineages, fungal ITS sequences were downloaded from INSD, aligned within family-level groups, and examined through phylogenetic analyses and BLAST searches. By combining the fungal sequence database UNITE and the annotation and search tool PlutoF, we also added metadata from the literature to these accessions. Altogether 35,632 sequences belonged to mycorrhizal fungi or originated from ericoid and orchid mycorrhizal roots. Of these sequences, 677 were considered chimeric and 2,174 of low read quality. Information detailing country of collection, geographical coordinates, interacting taxon and isolation source were supplemented to cover 78.0%, 33.0%, 41.7% and 96.4% of the sequences, respectively. These annotated sequences are publicly available via UNITE (http://unite.ut.ee/) for downstream biogeographic, ecological and taxonomic analyses. In European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/), the annotated sequences have a special link-out to UNITE. We intend to expand the data annotation to additional genes and all taxonomic groups and functional guilds of fungi.     

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。     

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.     

Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.     

A Conserved Motif in the 5.8S Ribosomal RNA (rRNA) Gene is a Useful Diagnostic Marker for Plant Internal Transcribed Spacer (ITS) Sequences

The nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has become an important nuclear locus for molecular systematic investigations of angiosperms at the intergenic and interspecific levels. Universal PCR primers are positioned on the conserved rRNA genes (18S, 5.8S, 26S) to amplify the entire ITS spacer region. Recent reports of fungal and algal contaminants, first described as plant ITS sequences, stress the need for diagnostic markers specific for the angiosperm ITS region. This report describes a conserved 14 base pair (bp) motif in the 5.8S rRNA gene that can be used to differentiate between flowering plants, bryophytes, and several orders of algae and fungi, including common plant pathogenic and non-pathogenic fungi. A variant of the motif (found in fungi and algae) contains a convenient EcoRI restriction site that has several applications for eliminating problematic contaminants from plant ITS preparations.     

Isolation of TC/AG Repeat Microsatellite Sequences for Fingerprinting Rice Blast Fungus and Their Possible Horizontal Transfer to Plant Species

Genome fingerprinting has been a major role in characterization of population structure and analysis of the variability in phytopathogenic fungi. In order to characterize Korean rice blast fungal isolates, the genomic DNAs were digested with Alu I endonuclease and subsequent PCR amplifications using random decamer primers with combinations of microsatellite primers had been carried out. This Alu-Inter SSR technique revealed high polymorphism among the Korean blast fungal isolates. Then, fragments from the Alu-Inter SSR analysis were isolated to be used as probes in Southern hybridization, which also revealed high polymorphism between isolates to distinguish individuals. The sequences of the isolated fragments contained TC/AG tandem repeats interspersed with a 30 bp direct repeat. In gel blot analysis, the isolated TC/AG repeat microsatellite sequences were proved to be useful for characterizing the isolates in blast fungi in addition to the conventional MGR (Magnaporthe grisea repeat) probes. One interesting point was that the rice blast fungus derived TC/AG repeat microsatellite sequences were abundant in non-rice blast fungi and plant species, but not in other fungi and yeasts. A discussion on the possible horizontal gene transfer between phytopathogenic fungi and host plants is presented.     

Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases.     

A PCR-based method for detecting the mycelia of stipitate hydnoid fungi in soil

To reduce the reliance on sporocarp records for conservation efforts, information on the below-ground distribution of specific fungal species, such as stipitate hydnoid fungi, is required. Species-specific primers were developed within the internal transcribed spacer (ITS1 and ITS2) regions for 12 hydnoid fungal species including Bankera fuligineoalba, Hydnellum aurantiacum, H. caeruleum, H. concrescens, H. ferrugineum, H. peckii, Phellodon confluens, P. melaleucus, P. niger, P. tomentosus, Sarcodon glaucopus and S. squamosus. The specificity of the primer pairs was tested using BLAST searches and PCR amplifications. All primers amplified DNA only of the target species with the exception of those designed for P. melaleucus. In order to assess the ability of the primers to detect DNA from mycelium in soil, DNA extracted from soil samples taken from around solitary H. peckii sporocarps was amplified with the H. peckii primer 1peck and ITS2. H. peckii DNA was detected in 70% of all soil samples and up to 40 cm away from the base of individual sporocarps. The development of these species-specific primers provides a below-ground alternative for monitoring the distribution of these rare fungi.     

青藏高原食草动物粪栖真菌的多样性

分别利用真菌通用引物和厌氧真菌特异引物构建了西藏地区3种反刍动物和1种单胃动物共8份新鲜粪样的ITS克隆文库,以通过系统发育分析解析其中好氧真菌与厌氧真菌的多样性。通用引物ITS文库测得324条真菌序列,分别属于子囊菌门Ascomycota 3个目、担子菌门Basidiomycota 2个目、接合菌门Zygomycota的1个目和严格厌氧的新丽鞭毛菌门Neocallimastigomycota,共24个OTUs。其中,子囊菌门相对丰度最高,占80.6%;新丽鞭毛菌门相对丰度最低,仅占0.6%。大部分OTUs与已知真菌属、种关系较远。厌氧真菌特异引物文库测得661条序列,全部属于新丽鞭毛菌门,包括所有已知的厌氧霉属Anaeromyces、盲肠菌属Caecomyces、肠霉属Cyllamyces、新丽鞭毛属Neocallimastix、奥式霉属Orpinomyces、胃梨囊霉属Piromyces 6个属和3个未培养的属级类群(NG9、NG10、NG11),共29个OTUs。其中3个已知的单中心属存在于所有反刍动物样品中,并以Piromyces相对丰度最高(37.4%)。单胃动物马粪样中全部为NG9类群。NG9是本研究新发现的属级类群,研究中同时揭示有多个未培养种和潜在的新种。研究结果证明青藏高原反刍动物粪栖真菌多样性较高,并存在丰富的未培养种和潜在的新属及新种。     

Identification of root rot fungi in nursery seedlings by nested multiplex PCR.

The internal transcribed spacer (ITS) of the ribosomal DNA (rDNA) subunit repeat was sequenced in 12 isolates of Cylindrocladium floridanum and 11 isolates of Cylindrocarpon destructans. Sequences were aligned and compared with ITS sequences of other fungi in GenBank. Some intraspecific variability was present within our collections of C. destructans but not in C. floridanum. Three ITS variants were identified within C. destructans, but there was no apparent association between ITS variants and host or geographic origin. Two internal primers were synthesized for the specific amplification of portions of the ITS for C. floridanum, and two primers were designed to amplify all three variants of C. destructans. The species-specific primers amplified PCR products of the expected length when tested with cultures of C, destructans and C. floridanum from white spruce, black spruce, Norway spruce, red spruce, jack pine, red pine, and black walnut from eight nurseries and three plantations in Quebec. No amplification resulted from PCR reactions on fungal DNA from 26 common contaminants of conifer roots. For amplifications directly from infected tissues, a nested primer PCR using two rounds of amplification was combined with multiplex PCR approach resulting in the amplification of two different species-specific PCR fragments in the same reaction. First, the entire ITS was amplified with one universal primer and a second primer specific to fungi; a second round of amplification was carried out with species-specific primers that amplified a 400-bp PCR product from C. destructans and a 328-bp product from C. floridanum. The species-specific fragments were amplified directly from infected roots from which one or the two fungi had been isolated.     

Analysis of fungal diversity in <Emphasis Type="Italic">Orchis tridentata</Emphasis> Scopoli

We have assessed the identities of fungi associated with Orchis tridentata, an endangered orchid species growing in open woodland and poor grassland of Central and Southern Europe. Fungal diversity in ten O. tridentata adult individuals collected in two protected areas of Central Italy was analysed by means of morphological and molecular methods. Sequencing of the cloned ITS fungal inserts corresponding to the dominant PCR products obtained from amplification of total root DNA with ITS1F and ITS4 primers revealed a variety of fungal species occurring in O. tridentata roots. Among them, members of the basidiomycete families Ceratobasidiaceae, Tulasnellaceae and Hymenogastraceae were recovered, together with ascomycetes belonging to Leptodontidium and Terfezia. The implications of these results in the understanding of O. tridentata biology and for the conservation of this threatened orchid species are discussed.