Ectomycorrhiza synthesis and basidiome formation of an orchid symbiont of the genus Thelephora on Quercus serrata

We induced basidioma formation of a fungus belonging to the Thelephoraceae that was isolated from the orchid Cephalanthera falcata. The mycobiont was isolated from root, cultured on modified Melin-Norkrans medium, and then inoculated onto fine roots of a Quercus serrata (Fagaceae) seedling. After observation of ectomycorrhiza formation, the Q. serrata seedling was grown in a pot. Thirty-six mo after ectomycorrhiza formation, basidioma formation was confirmed at the bottom of the pot. From comparisons in morphological characteristics between the mycobiont and known related Thelephoraceae species, and sequence similarities of internal transcribed spacer region in ribosomal DNA, we identified the mycobiont as Thelephora ellisii sensu lato in Thelephoraceae (Basidiomycota). Phylogenetic analysis indicated that the related sequences came from ectomycorrhizae of trees of the Salicaceae, Pinaceae, and Fagaceae distributed in East Asia, the USA, central Africa, and Europe.     

New species and distribution records for Clavulina (Cantharellales,Basidiomycota) from the Guiana Shield,with a key to the lowland neotropical taxa

Three new and one previously described species of Clavulina (Clavulinaceae, Cantharellales, Basidiomycota) are reported from the central Guiana Shield region from tropical rainforests dominated by ectomycorrhizal trees of the leguminous genus Dicymbe (Fabaceae subfam. Caesalpinioideae). We provide morphological, DNA sequence, habitat, and fruiting occurrence data for each species. The new species conform to a generic concept of Clavulina that includes coralloid, branched basidiomata with amphigenous hymenia, basidia with two or 2−4 incurved sterigmata and postpartal septa present or absent, and smooth, hyaline, guttulate basidiospores. Placements of the new species in Clavulina were corroborated with DNA sequence data from the internal transcribed spacer and large subunit of the nuclear ribosomal repeat, and their infrageneric relationships were examined with phylogenetic analyses based on DNA from the region coding for the second largest subunit of DNA-dependent RNA polymerase II (rpb2). To facilitate future studies of the genus in the neotropics, a key is provided for all Clavulina species described from the lowland neotropics.     

Molecular phylogeny of Rigidoporus microporus isolates associated with white rot disease of rubber trees (Hevea brasiliensis)

Rigidoporus microporus (Polyporales, Basidiomycota) syn. Rigidoporus lignosus is the most destructive root pathogen of rubber plantations distributed in tropical and sub-tropical regions. Our primary objective was to characterize Nigerian isolates from rubber tree and compare them with other West African, Southeast Asian and American isolates. To characterize the 20 isolates from Nigeria, we used sequence data of the nuclear ribosomal DNA ITS and LSU, β-tubulin and translation elongation factor 1-α (tef1) gene sequences. Altogether, 40 isolates of R. microporus were included in the analyses. Isolates from Africa, Asia and South/Central America formed three distinctive clades corresponding to at least three species. No phylogeographic pattern was detected among R. microporus collected from West and Central African rubber plantations suggesting continuous gene flow among these populations. Our molecular phylogenetic analysis suggests the presence of two distinctive species associated with the white rot disease. Phylogenetic analyses placed R. microporus in the Hymenochaetales in the vicinity of Oxyporus. This is the first study to characterize R. microporus isolates from Nigeria through molecular phylogenetic techniques, and also the first to compare isolates from rubber plantations in Africa and Asia.     

Diversity of the rDNA ITS haplotypes of the nematodes Haemonchus contortus (Trichostrongyloidea, Rhabditida) of the same host

Specimens sampled in Central Mongolia have been examined for the intraspecific polymorphism of the nucleotide sequences of ribosomal DNA internal transcribed spacers (ITS1 + 5.8S + ITS2) of the parasitic nematode Haemonchus contortus. A considerable diversity of haplotypes differing in the nucleotide composition of this DNA region has been observed. The phylogenetic relationships between the haplotypes detected in Central Mongolia and the corresponding sequences from other parts of the nematode distribution area (deposited with the NCBI GenBank) have been analyzed. Significantly different sequences have been found along with the haplotypes already observed in H. contortus or differing from them by one-two nucleotides.     

Sociality and the Rate of rDNA Sequence Evolution in Wasps (Vespidae) and Honeybees (Apis)

Sequence data of mitochondrial 16S ribosomal DNA (mt-rDNA) and nuclear 28S ribosomal DNA (nuc-rDNA) were compared in two honeybee species (Apis mellifera and Apis dorsata) and a selection of 22 wasp species (Vespidae) with different levels of sociality. The averge substitution rates in mt-rDNA and nuc-rDNA were almost-equal in solitary species. In species with larger nests, however, the difference between the nuclear and the mitochondrial substitution rate significantly increased. The average substitution ratio, ψ (nucleotide substitutions in mt-rDNA/nucleotide substitutions in nuc-rDNA) was 1.48 ± 0.12 (SE) among the solitary Eumeninae, 3.70 ± 0.15 among five primitive social Stenogastrinae species, 3.24 ± 0.20 among five Polistinae species, 5.76 ± 0.33 among nine highly eusocial Vespinae, and 12.7 in the two Apis species. The high egg-laying rate and the effective population size skew between the sexes may contribute to the rise of the substitution ratio in the highly eusocial species. Drift and bottleneck effects in the mitochondrial DNA pool during speciation events as well as polyandry may further enhance this phenomenon. Received: 12 January 1998 / Accepted: 28 April 1998     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

Do Sebacinales commonly associate with plant roots as endophytes?

Sebacinales are basal Hymenomycetes with diverse mycorrhizal abilities, ranging from ectomycorrhizae to ericoid and orchid mycorrhizae. Several previous PCR or isolation works raised the possibility that Sebacinales are endophytes in plant roots. We tested this hypothesis in an isolation-independent approach by using specific PCR primers for ribosomal DNA of Sebacinales on AM mycorrhizal or non-mycorrhizal roots. Thirty-nine plant species were sampled on a Caribbean and two European sites (3 repetition per species and site), covering 25 families in monocots and eudicots. PCR signals were obtained from 40 samples (28.9 %) from 27 species (69.2 %) and all sites. Whenever sequencing was successful, a sequence belonging to Sebacinales was recovered. A phylogenetic approach revealed that 13 of them belonged to clade B (encompassing ericoid and orchid mycorrhizal species) and 4 to clade A (usually encompassing only ectomycorrhizal species). These data suggest that Sebacinales may be endophytic in many angiosperm roots, and that this condition is plesiomorphic in Sebacinales. They bridge the gap between physiological studies, inoculating Sebacinales (Piriformospora indica or Sebacina vermifera) on diverse plants and molecular ecology, hitherto restricting Sebacinales to mycorrhizal interactions. Structural and functional aspects of the interaction deserve further studies.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

A new species of Lenzitopsis (Thelephorales,Basidiomycota) and its phylogenetic placement

Lenzitopsis (Thelephorales, Basidiomycota), typified by Lenzitopsis oxycedri, was monotypic before we described the second species of this genus, Lenzitopsis daii in this study. L. daii resembles L. oxycedri in producing lenzitoid hymenophore as well as brown hyphae and echinulate spores, but it differs from the type species by its annual basidiocarps, amyloid spores and growth exclusively on Juniperus chinensis (Cupressaceae). In the phylogenetic perspective inferred with nuclear large subunit ribosomal DNA sequences, the two species were separated from each other and formed a strongly supported clade in the Thelephorales. The two Lenzitopsis species showed a more than 5% difference in internal transcribed spacer sequences. Lenzitopsis species are wood-decaying fungi, and this is the second genus of the order where mycorrhizal life style is unknown, besides of Amaurodon.     

Unique arrangement of coding sequences for 5 S, 5.8 S, 18 S and 25 S ribosomal RNA in Saccharomyces cerevisiae as determined by R-loop and hybridization analysis.

The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S₁ nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.     

Developmental stages and secondary compound biosynthesis of mycobiont and whole thallus cultures of <Emphasis Type="Italic">Buellia subsororioides</Emphasis>

Lichen species have unique culture media preferences, and established cultures are known for the synthesis of secondary metabolites. This paper reports observations on the developmental stages and secondary compound biosynthesis by the mycobiont and whole thallus cultures of Buellia subsororioides. It also investigates the suitable media compositions for the culture growth, the role (nutrient or stressor) of sucrose concentrations on the growth stages, biomass, secondary compound profiles, and the quantity of biosynthesized known compounds/g of culture biomass for each treatment using mycobiont cultures. The ascospore-derived mycobiont cultures and thallus macerate-derived whole thallus cultures of B. subsororioides were established and grown using malt yeast extract (MY) medium. Mycobiont cultures were subcultured in MY medium supplemented with sucrose and its concentrations ranging from 0 to 30 % (with 2 % increment between treatments) for 120 days. The molecular identity of cultures was confirmed using nuclear ribosomal Internal transcribed spacer (ITS) DNA sequences obtained from the cultured mycobiont and from the natural thallus. The ITS DNA sequences of the mycobiont showed 99 % similarity with the sequences of the natural thallus. The mycobiont cultures under varying sucrose concentrations initiated as white cottony stages and transformed to brown compact mycelia, with optimum biomass and biosynthesis of nine secondary compounds in MY 10 %. The number of compounds (1–9) varies according to treatments. The whole thallus cultures (MY 0 %) showed a profile of secondary compounds similar to that of the natural thalli along with a trace of one unknown compound. The obtained results are encouraging for the synthesis of the desired quantities of lichen secondary compounds through cultures for relevant applications.     

Divergence Times and Phylogenetic Patterns of Sebacinales,a Highly Diverse and Widespread Fungal Lineage

Patterns of geographic distribution and composition of fungal communities are still poorly understood. Widespread occurrence in terrestrial ecosystems and the unique richness of interactions of Sebacinales with plants make them a target group to study evolutionary events in the light of nutritional lifestyle. We inferred diversity patterns, phylogenetic structures and divergence times of Sebacinales with respect to their nutritional lifestyles by integrating data from fossil-calibrated phylogenetic analyses. Relaxed molecular clock analyses indicated that Sebacinales originated late Permian within Basidiomycota, and their split into Sebacinaceae and Serendipitaceae nom. prov. likely occurred during the late Jurassic and the early Cretaceous, coinciding with major diversifications of land plants. In Sebacinaceae, diversification of species with ectomycorrhizal lifestyle presumably started during the Paleocene. Lineage radiations of the core group of ericoid and cavendishioid mycorrhizal Sebacinales started probably in the Eocene, coinciding with diversification events of their hosts. The diversification of Sebacinales with jungermannioid interactions started during the Oligocene, and occurred much later than the diversification of their hosts. Sebacinales communities associated either with ectomycorrhizal plants, achlorophyllous orchids, ericoid and cavendishioid Ericaceae or liverworts were phylogenetically clustered and globally distributed. Major Sebacinales lineage diversifications started after the continents had drifted apart. We also briefly discuss dispersal patterns of extant Sebacinales.     

New <Emphasis Type="Italic">Clavulina</Emphasis> species from the Pakaraima Mountains of Guyana

Five new species of Clavulina (Clavulinaceae, Cantharellales, Basidiomycota) are described from the Pakaraima Mountains of Guyana, occurring in rain forests dominated by the ectomycorrhizal tree Dicymbe corymbosa (Caesalpiniaceae). These clavarioid fungi have simple (i.e., unbranching) basidiomata, which is a relatively uncommon phenotypic feature for the genus Clavulina. Macromorphological, micromorphological, and habitat data are provided for each taxon, and nuclear ribosomal DNA sequences of the 28S subunit and internal transcribed spacer region were obtained for each holotype collection.     

Phylogeny and taxonomy of the genus Phylloporia (Hymenochaetales)

Based on 28 taxa, including six species of Phylloporia, and respectively one representative of 17 genera of the Hymenochaetales, a phylogenetic analysis of a region of the large subunit of the nuclear encoded ribosomal DNA was performed. Molecular sequence data as well as morphological and anatomical features show the genus to be monophyletic. Next related to Phylloporia is Fulvifomes. The phylogenetic relationships of Phylloporia are discussed. In addition the genus Phylloporia is monographed; 12 species are accepted and described with a key.     

Assessment of soil fungal communities using pyrosequencing

Pyrosequencing, a non-electrophoretic method of DNA sequencing, was used to investigate the extensive fungal community in soils of three islands in the Yellow Sea of Korea, between Korea and China. Pyrosequencing was carried out on amplicons derived from the 5′ region of 18S rDNA. A total of 10,166 reads were obtained, with an average length of 103 bp. The maximum number of fungal phylotypes in soil predicted at 99% similarity was 3,334. The maximum numbers of phylotypes predicted at 97% and 95% similarities were 736 and 286, respectively. Through phylogenetic assignment using BLASTN, a total of 372 tentative taxa were identified. The majority of true fungal sequences recovered in this study belonged to the Ascomycota (182 tentative taxa in 2,708 reads) and Basidiomycota (172 tentative taxa in 6,837 reads). The predominant species of Ascomycota detected have been described as lichen-forming fungi, litter/wood decomposers, plant parasites, endophytes, and saprotrophs: Peltigera neopolydactyla (Lecanoromycetes), Paecilomyces sp. (Sordariomycetes), Phacopsis huuskonenii (Lecanoromycetes), and Raffaelea hennebertii (mitosporicAscomycota). The majority of sequences in the Basidiomycota matched ectomycorrhizal and wood rotting fungi, including species of the Agaricales and Aphyllophorales, respectively. A high number of sequences in the Thelephorales, Boletales, Stereales, Hymenochaetales, and Ceratobasidiomycetes were also detected. By applying high-throughput pyrosequencing, we observed a high diversity of soil fungi and found evidence that pyrosequencing is a reliable technique for investigating fungal communities in soils.     

Taxonomic and phylogenetic analysis of the Oomycota mosquito larvae pathogen Crypticola clavulifera

Beside the taxonomic features displayed by Crypticola clavulifera in culture and unpublished data on its phylogeny, little is known about the main phylogenetic features of this unusual mosquito pathogen. We have PCR amplified the 18S SSU rDNA, ITS, and the partial coding regions of COXII and COXI to study the phylogenetic features of this pathogen within the tree of life. Our phylogenetic data showed C. clavulifera clustered among homologous DNA sequences of the Oomycota class Saprolegniomycetes. Our study support previous taxonomic and unpublished molecular analysis, placing this unusual mosquito larvae pathogen as part of the earliest diverging cluster within the Saprolegniomycetes.     

Phylogenetic Analysis of Ruminant Theileria spp. from China Based on 28S Ribosomal RNA Gene

Species identification using DNA sequences is the basis for DNA taxonomy. In this study, we sequenced the ribosomal large-subunit RNA gene sequences (3,037-3,061 bp) in length of 13 Chinese Theileria stocks that were infective to cattle and sheep. The complete 28S rRNA gene is relatively difficult to amplify and its conserved region is not important for phylogenetic study. Therefore, we selected the D2-D3 region from the complete 28S rRNA sequences for phylogenetic analysis. Our analyses of 28S rRNA gene sequences showed that the 28S rRNA was useful as a phylogenetic marker for analyzing the relationships among Theileria spp. in ruminants. In addition, the D2-D3 region was a short segment that could be used instead of the whole 28S rRNA sequence during the phylogenetic analysis of Theileria, and it may be an ideal DNA barcode.     

Structural dynamics of bacterial ribosomes. III. Quantitative conversion of vacant ribosome couples into an initiation complex with R17 RNA as messenger

The arrangement of the DNA sequences coding for the ribosomal 5.8 S RNA in the genome of Xenopus laevis has been studied. In Xenopus the 5.8 S cistrons, like the ribosomal 28 S and 18 S cistrons, are reiterated some 600-fold (Clarkson et al., 1973a). When banded in caesium chloride, the 5.8 S cistrons separate from somatic DNA of high molecular weight and band as a distinct satellite, indicating a clustered arrangement in the genome. The buoyant density of this satellite (1.723 g cm^?3) corresponds to that of the ribosomal DNA satellite.It has previously been shown that the ribosomal DNA sequences have been deleted from the genome of the anucleotide Xenopus mutant. Our findings, first that the anucleolate mutant does not synthesize 5.8 S RNA and second that somatic DNA from this mutant does not detectably hybridize with 5.8 S RNA, demonstrate that the 5.8 S cistronic complement has been similarly deleted. This finding supports our contention that 5.8 S sequences are clustered on chromosomal DNA and further suggests that they are located close to or within the rDNA complements in the nucleolus organizer region.Pre-hybridization to saturation with unlabelled 5.8 S RNA results in only a slight increase in the buoyant density of denatured 5.8 S coding sequences from low molecular weight DNA. Since a contiguous arrangement of the 5.8 S sequences would give rise to a much larger increase in density, it follows that, although clustered, the sequences must be intercalated within stretches of other DNA. By contrast, pre-hybridization of the somatic DNA with unlabelled 28 S or 18 S ribosomal RNAs results in large shifts in the buoyant density of the 5.8 S sequences. These shifts indicate that the 5.8 S sequences are closely linked to both 28 S and 18 S coding sequences.It is concluded that the 5.8 S cistrons are interspersed along the ribosomal DNA sense strand and that each is located together with a 28 S and an 18 S cistron in a ribosomal repeat unit. Estimates, obtained from the pre-hybridization experiments, of the separations between the 5.8 S and the 28 S and 18 S sequences, are combined in a model of the ribosomal repeat unit. In this model the 5.8 S cistron is located within the transcribed spacer which links the 28 S and 18 S coding sequences.     

Experimental evidence of ericoid mycorrhizal potential within Serendipitaceae (Sebacinales)

The Sebacinales are a monophyletic group of ubiquitous hymenomycetous mycobionts which form ericoid and orchid mycorrhizae, ecto- and ectendomycorrhizae, and nonspecific root endophytic associations with a wide spectrum of plants. However, due to the complete lack of fungal isolates derived from Ericaceae roots, the Sebacinales ericoid mycorrhizal (ErM) potential has not yet been tested experimentally. Here, we report for the first time isolation of a serendipitoid (formerly Sebacinales Group B) mycobiont from Ericaceae which survived in pure culture for several years. This allowed us to test its ability to form ericoid mycorrhizae with an Ericaceae host in vitro, to describe its development and colonization pattern in host roots over time, and to compare its performance with typical ErM fungi and other serendipitoids derived from non-Ericaceae hosts. Out of ten serendipitoid isolates tested, eight intracellularly colonized Vaccinium hair roots, but only the Ericaceae-derived isolate repeatedly formed typical ericoid mycorrhiza morphologically identical to ericoid mycorrhiza commonly found in naturally colonized Ericaceae, but yet different from ericoid mycorrhiza formed in vitro by the prominent ascomycetous ErM fungus Rhizoscyphus ericae. One Orchidaceae-derived isolate repeatedly formed abundant hyaline intracellular microsclerotia morphologically identical to those occasionally found in naturally colonized Ericaceae, and an isolate of Serendipita (= Piriformospora) indica produced abundant intracellular chlamydospores typical of this species. Our results confirm for the first time experimentally that some Sebacinales can form ericoid mycorrhiza, point to their broad endophytic potential in Ericaceae hosts, and suggest possible ericoid mycorrhizal specificity in Serendipitaceae.