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In JoVE (1)
- Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
Other Publications (38)
- Mycological Research
- Mycological Research
- Mycological Research
- American Journal of Botany
- Methods in Enzymology
- FEMS Microbiology Letters
- Mycological Research
- Mycological Research
- FEMS Microbiology Ecology
- Mycological Research
- FEMS Microbiology Ecology
- Plant Physiology and Biochemistry : PPB / Société Française De Physiologie Végétale
- Mycological Research
- Mycological Research
- Mycological Research
- FEMS Microbiology Ecology
- Mycological Research
- Mycological Research
- Molecular Phylogenetics and Evolution
- Molecular Ecology
- The ISME Journal
- Microbial Ecology
- FEMS Microbiology Ecology
- Fungal Biology
- Lichenologist (London, England)
- International Journal of Systematic and Evolutionary Microbiology
- Fungal Biology
- Fungal Biology
- Fungal Biology
- FEMS Microbiology Ecology
- Microbial Ecology
- FEMS Microbiology Letters
Articles by Martin Grube in JoVE
Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
Barbara Klug1,2, Claudia Rodler1, Martin Koller3, Gernot Wimmer3, Harald H. Kessler2, Martin Grube4, Elisabeth Santigli1
1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
Other articles by Martin Grube on PubMed
Mycological Research. Dec, 2003 | Pubmed ID: 15000242
The ketosynthase domains of putative polyketide synthase (PKS) genes from 15 species in the lichenized genus Lecanora as well as three representatives of other genera were amplified and sequenced using conserved primers. A phylogenetic analysis was carried out with the corresponding amino acid sequences, including those of non-lichenized fungi. The phylogenetic hypothesis places all PKS sequences from Lecanora and the other genera in a clade of PKSs that produce complex aromatic compounds. The PKSs from Lecanora are found in two distinct clades. One of these forms a monophyletic group with PKSs producing precursors of dihydroxy naphthalenes from non-lichenized species. This includes PKSs from the L. rupicola group and several other species which are not closely related. The other clade represents at least one functionally different protein and has no close relationships with known PKSs from other fungi. A comparison between an ITS phylogeny of the species with their DNA sequences of ketosynthase domains reveals similar PKS genes in certain closely related species. Coevolution was unapparent in other clades, suggesting the presence of paralogous genes.
Mycological Research. May, 2004 | Pubmed ID: 15230003
A molecular phylogeny of the Lecanora rupicola group is presented, based on ITS sequence analyses. The study includes saxicolous and corticolous members of the Lecanora rupicola group as well as other Lecanora species with pruinose apothecia. A phylogenetic hypothesis for species in Lecanora s. lat. and various other genera in Lecanoraceae, based on an alignment-free distance estimation technique, shows that the Lecanora rupicola group forms a monophyletic clade within Lecanoraceae. Affinities to the core group of Lecanora are not well supported, likewise the monophyly of Lecanora s. str. with other species groups in Lecanora, such as the lobate taxa (and Rhizoplaca) is not supported. A more detailed analysis involving Lecanora species with pruinose apothecial discs was carried out with model-based Bayesian Markov chain Monte Carlo (B/MCMC) tree sampling. The results suggest the monophyly of the Lecanora species that are characterized by the presence of chromones. Corticolous as well as saxicolous species are included. Lepraria flavescens is closely related to the Lecanora swartzii subgroup, and the new name Lecanora rouxii nom. nov. is introduced for that species. Other Lecanora species with pruinose discs are not closely related to the Lecanora rupicola group.
The Phylogeny of Porinaceae (Ostropomycetidae) Suggests a Neotenic Origin of Perithecia in Lecanoromycetes
Mycological Research. Oct, 2004 | Pubmed ID: 15535063
The family Porinaceae (Trichotheliales) is characterized by perithecial ascomata of ascohymenial origin. The phylogenetic position of this family in the system of ascomycetes has been uncertain and is investigated using mtSSU rDNA sequences. The dataset consists of lichenized representatives of major ascomycete lineages, including those that were previously suspected as relatives of Porinaceae, such as Pyrenulaceae. The dataset was subjected to a Bayesian phylogenetic analysis implementing a Metropolis Coupled Markov Chain Monte Carlo method. The analysis confirms previous classification of the apothecial Gomphillaceae near to Graphidaceae, and suggests that the pyrenocarpous Porinaceae are also close to Graphidaceae, Gyalectaceae, and Stictidaceae. The subclass Ostropomycetidae is here suggested to include the families Gomphillaceae, Gyalectaceae, Graphidaceae (incl. Thelotremataceae), Porinaceae, and Stictidaceae. A special type of hemiangiocarpous ontogeny of the ascomata is shared throughout the Ostropomycetidae, and the closed fruit bodies of Porinaceae are apparently a result of a neotenic ontogeny. This is associated with special hymenial characters: rather thin-walled narrow asci, and a different consistency of the hymenial gels.
American Journal of Botany. Oct, 2004 | Pubmed ID: 21652303
Based on an overview of progress in molecular systematics of the true fungi (Fungi/Eumycota) since 1990, little overlap was found among single-locus data matrices, which explains why no large-scale multilocus phylogenetic analysis had been undertaken to reveal deep relationships among fungi. As part of the project "Assembling the Fungal Tree of Life" (AFTOL), results of four Bayesian analyses are reported with complementary bootstrap assessment of phylogenetic confidence based on (1) a combined two-locus data set (nucSSU and nucLSU rDNA) with 558 species representing all traditionally recognized fungal phyla (Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota) and the Glomeromycota, (2) a combined three-locus data set (nucSSU, nucLSU, and mitSSU rDNA) with 236 species, (3) a combined three-locus data set (nucSSU, nucLSU rDNA, and RPB2) with 157 species, and (4) a combined four-locus data set (nucSSU, nucLSU, mitSSU rDNA, and RPB2) with 103 species. Because of the lack of complementarity among single-locus data sets, the last three analyses included only members of the Ascomycota and Basidiomycota. The four-locus analysis resolved multiple deep relationships within the Ascomycota and Basidiomycota that were not revealed previously or that received only weak support in previous studies. The impact of this newly discovered phylogenetic structure on supraordinal classifications is discussed. Based on these results and reanalysis of subcellular data, current knowledge of the evolution of septal features of fungal hyphae is synthesized, and a preliminary reassessment of ascomal evolution is presented. Based on previously unpublished data and sequences from GenBank, this study provides a phylogenetic synthesis for the Fungi and a framework for future phylogenetic studies on fungi.
Mycologia. Sep-Oct, 2004 | Pubmed ID: 21148936
The new corticolous species Arthonia isidiata is described from the Pacific lowlands of Costa Rica. A. isidiata is characterized by minute, cylindrical to coralloid isidia produced on the thallus surface. The species currently is known only from the type locality in Corcovado National Park, where it occurs abundantly in the coastal rainforest around Sirena Biological Station.
Methods in Enzymology. 2005 | Pubmed ID: 15865960
Ecological samples of fungal associations pose particular challenges for nucleic acid extraction due to the presence of several genomes. Thorough examination of the samples prior to extraction is important to assess the risks of contamination. If manual separation of symbionts or their axenic cultivation is not feasible, symbiont-specific primers can be applied in PCR experiments. A basic protocol is suggested here which can be optimized for specific applications.
FEMS Microbiology Letters. Jun, 2005 | Pubmed ID: 15927741
Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.
Evolution and Phylogenetic Relationships Within Porinaceae (Ostropomycetidae), Focusing on Foliicolous Species
Mycological Research. Feb, 2006 | Pubmed ID: 16376532
A phylogeny of the lichen family Porinaceae using mitochondrial SSU rDNA sequences is presented, with special focus on foliicolous taxa. Fifty specimens of 28 mostly tropical species, representing eight species groups of Porina as well as the genus Trichothelium, were analysed together with species of other members of Ostropomycetidae, and using Agyriaceae as outgroup. We performed the phylogenetic analyses with a Bayesian approach and under the criterion of maximum parsimony. Four main clades can be distinguished: the P. nitidula-group s. lat. (including Trichothelium, P. papillifera and P. rubescens), the Porina epiphylla-group s. lat. (including the P. radiata-, the P. nucula-, the P. imitatrix- and the P. epiphylla-group s. str.) and two clades of the P. rufula-group. The genus Porina as understood by all recent concepts is paraphyletic, and Trichothelium is nested within the Porina nitidula-group. The non-setose P. repanda forms a monophyletic clade with Trichothelium. The tree does not support a monophyletic origin of substrate preferences or photobiont selection. Species-specific associations with morphologically different trentepohlioid photobionts mapped on the tree suggest that closely related mycobiont species switch between different types of algae.
Mycological Research. Jul, 2006 | Pubmed ID: 16876697
A phylogenetic study of the lichen family Graphidaceae is presented. Most genera of the family, as well as selected representatives of the closely related Thelotremataceae, are included. The results of the Bayesian analysis of combined mt SSU and nuLSU rDNA sequence data were compared with recently introduced concepts of genera. Species of Fissurina and Dyplolabia form a monophyletic group in an unresolved sister-group relationship to other members of Graphidaceae and Thelotremataceae. The family Graphidaceae as currently circumscribed is paraphyletic and we suggest that the name Graphidaceae is used in a broader sense to include members of Thelotremataceae. The concepts of Glyphis, Phaeographis and Platygramme are confirmed by molecular data. Surprisingly, Graphis species are found in two distinct clades, which can only partly be explained by morphology. Hemithecium as recently circumscribed is polyphyletic: H. implicatum, with hyaline spores groups within the Graphis scripta clade, whereas a second Hemithecium species, representing subgenus Leucogramma with brown spores, forms a well-supported clade with other brown-spored species such as 'Sarcographina'lyphiza, Leiorreuma hypomelaenum and Sarcographa ramificans. The evolutionary pattern of morphological characters of the ascomata such as exciple carbonization, paraphyses types, and ascospore characters (colour, septation, and Lugols reaction) are critically discussed.
FEMS Microbiology Ecology. Sep, 2006 | Pubmed ID: 16907761
The bacterial communities associated with 11 different lichen samples (belonging to eight different species) from different habitats were investigated. The culturable aerobic-heterotrophic fraction of the bacterial communities was isolated from nine lichen samples on protein-rich and sugar-rich/N-free media. Thirty-four bacterial isolates were purified and pooled into groups (phylotypes) by analysis of the ribosomal internal transcribed spacer polymorphism. Twenty five phylotypes were identified, each comprising between one and three isolates. One isolate of each phylotype was partially sequenced and the resulting 16S rRNA gene sequences were compared in a phylogenetic analysis. Three genera of Firmicutes, four of Actinobacteria and three of Proteobacteria were identified. Two phylotypes, belonging to the phyla Actinobacteria and Proteobacteria, respectively, were not identified at genus level. Some bacterial taxa were retrieved frequently in different lichen species sampled in the same or different sites. Paenibacillus and Burkholderia phylotypes seem to be common in lichens. Luteibactor rhizovicina was found in three different lichens of two different regions. In a cultivation-independent approach, total DNA was extracted from 11 lichen samples. Molecular fingerprints of the bacterial communities were obtained by PCR-amplification of the internal transcribed spacer region, and sequencing of selected bands indicated the presence of additional bacteria.
New Insights into Classification and Evolution of the Lecanoromycetes (Pezizomycotina, Ascomycota) from Phylogenetic Analyses of Three Ribosomal RNA- and Two Protein-coding Genes
Mycologia. Nov-Dec, 2006 | Pubmed ID: 17486983
The Lecanoromycetes includes most of the lichen-forming fungal species (> 13500) and is therefore one of the most diverse class of all Fungi in terms of phenotypic complexity. We report phylogenetic relationships within the Lecanoromycetes resulting from Bayesian and maximum likelihood analyses with complementary posterior probabilities and bootstrap support values based on three combined multilocus datasets using a supermatrix approach. Nine of 10 orders and 43 of 64 families currently recognized in Eriksson's classification of the Lecanoromycetes (Outline of Ascomycota--2006 Myconet 12:1-82) were represented in this sampling. Our analyses strongly support the Acarosporomycetidae and Ostropomycetidae as monophyletic, whereas the delimitation of the largest subclass, the Lecanoromycetidae, remains uncertain. Independent of future delimitation of the Lecanoromycetidae, the Rhizocarpaceae and Umbilicariaceae should be elevated to the ordinal level. This study shows that recent classifications include several nonmonophyletic taxa at different ranks that need to be recircumscribed. Our phylogenies confirm that ascus morphology cannot be applied consistently to shape the classification of lichen-forming fungi. The increasing amount of missing data associated with the progressive addition of taxa resulted in some cases in the expected loss of support, but we also observed an improvement in statistical support for many internodes. We conclude that a phylogenetic synthesis for a chosen taxonomic group should include a comprehensive assessment of phylogenetic confidence based on multiple estimates using different methods and on a progressive taxon sampling with an increasing number of taxa, even if it involves an increasing amount of missing data.
Mycologia. Jan-Feb, 2007 | Pubmed ID: 17663122
The family Parmeliaceae (Lecanorales, Ascomycota) is possibly the largest, best known and most thoroughly studied lichen family within its order. Despite this fact the relationship between Parmeliaceae and other groups in Lecanorales is still poorly known. The aim of the present study is to contribute to finding the sister group of Parmeliaceae as an aid in future studies on the phylogeny and character evolution of the group. We do this by sampling all potential relatives to the Parmeliaceae that we have identified, i.e. Gypsoplaca, Japewia, Mycoblastus, Protoparmelia, and Tephromela, a good representation of the major groups within the Parmeliaceae s. lat. and a good representation of other taxa in the core Lecanorales. We use molecular data from two genes, the large subunit of the nuclear ribosomal RNA gene (nrLSU) and the small subunit of the mitochondrial ribosomal RNA gene (mrSSU), and a Bayesian analysis of the combined data. The results show that the closest relatives to Parmeliaceae are the two genera Protoparmelia and Gypsoplaca, which are crustose lichens. Parmeliaceae in our sense is a well supported group, including also the family segregates Alectoriaceae, Hypogymniaceae, Usneaceae and Anziaceae.
The New Species Lecanora Bicinctoidea, Its Position and Considerations About Phenotypic Evolution in the Lecanora Rupicola Group
Mycologia. Jan-Feb, 2007 | Pubmed ID: 17663123
A phylogenetic analysis of the Lecanora rupicola group based on combined nITS rDNA and beta-tubulin sequences and a combined dataset of ITS, beta-tubulin and partial sequences of polyketide synthase genes reveals a previously unrecognized species, which here is introduced under the name Lecanora bicinctoidea. The new species is a sister group of the L. swartzii complex (including L. swartzii and L. lojkaeana), which is characterized by eucorticate ascomata, and a morphological diversity that includes also a dwarf-fruticose lineage. The preferential occurrence on vertical to overhanging siliceous rocks corresponds more closely to L. swartzii. A detailed investigation of phenotypic characters reveals that the new species differs from the superficially similar morphospecies L. bicincta in several ways, such as a thallus of comparatively small areoles and broadly sessile ascomata and the development of an amphithecial cortex devoid of algal remnants (i.e. an eucortex). L. bicinctoidea contains methyl 3alpha-hydroxy-4-O-demethylbarbatate, a chemical compound not known from other members of the L. rupicola group. We also discuss the importance of eucortex formation as one of several factors that are involved in the evolution of substrate-detached thallus structures.
Trouble with Lichen: the Re-evaluation and Re-interpretation of Thallus Form and Fruit Body Types in the Molecular Era
Mycological Research. Sep, 2007 | Pubmed ID: 17698333
Following discussions of the definition of the terms 'lichen' and 'thallus', the role of lichenization in the evolution of asco- and basidiomycetes, and divergence and convergence in fruit body types, the morphogenetic interpretation of types of thallus form in lichens is reviewed. Attention is drawn to the various morphogenetic hypotheses proposed to explain the lichen thallus, but it is concluded that it is best interpreted as a novel phenotype with no exact homologue. Similar ascomatal and thallus types are found in lichen-forming fungi of different orders and families, as now revealed by molecular phylogenetic studies. These are interpreted as examples of convergent evolution, strategies by which unrelated fungi either display captured algae to maximize photosynthetic opportunities, or to attach themselves to a substratum. Phenotypic evolution of fruit body and thallus types in the major orders and clades is summarized, and the thallus types known in each order are tabulated. An hypothesis relating the evolution of these structures to hygroscopic movements is proposed, and the critical position of lichens in developing an integrated approach to ascomycete evolution is emphasized.
Ultrastructural and Genetic Characteristics of Endolithic Cyanobacterial Biofilms Colonizing Antarctic Granite Rocks
FEMS Microbiology Ecology. Feb, 2007 | Pubmed ID: 17328119
The precise identification of the cyanobacteria that comprise an endolithic biofilm is hindered by difficulties in culturing the organisms found in these biofilms and a lack of previous molecular and ultrastructural data. This study characterizes, both at the ultrastructural and molecular level, two different cyanobacterial biofilms found in fissures of granite from continental Antarctica. Electron microscopy revealed structural differences between the two biofilms. One was only loosely adhered to the substrate, while the other biofilm showed a closer association between cells and rock minerals and was tightly attached to the substrate. Cells from both biofilms where ultrastructurally distinct, displaying, for instance, clear differences in their sheaths. The amounts of EPS and their organization associated with the cyanobacteria may determine the differences in adhesion and effects on the lithic substrate observed in the biofilms. By sequencing part of the 16S rRNA gene, the two cyanobacteria were also genetically characterized. The gene sequence of the cells comprising the biofilm that was tightly attached to the lithic substrate showed most homology with that of an endolithic cyanobacterium from Switzerland (AY153458), and the cyanobacterial type loosely adhered to the rock, clustered with Acaryochloris marina, the only organism unequivocally known to contain chlorophyll d. This study reveals the presence of at least two different types of endolithic biofilm, dominated each by a single type of cyanobacterium, able to withstand the harsh conditions of the Antarctic climate.
Alterations in Secondary Metabolism of Aposymbiotically Grown Mycobionts of Xanthoria Elegans and Cultured Resynthesis Stages
Plant Physiology and Biochemistry : PPB / Société Française De Physiologie Végétale. Feb, 2007 | Pubmed ID: 17344057
HPLC analyses of Xanthoria elegans cultivated on different media and either aposymbiontically or with its photobiont revealed that the carbon source and the presence of the algal partner have an impact on the secondary metabolism of the mycobiont. The aposymbiotically (without photobiont) grown mycobiont contained up to 70% more of the main compounds in its thallus than in resynthesis stage. Although this is speculative, the induction of the polyketide pathway may be a feedback mechanism to the absence of the photobiont. All cultures produce a variety of substances which were not detectable in the voucher specimen. Besides physcion (the major substance), we were able to identify emodin as well as physcion-bisanthrone, teloschistin monoacetate and derivatives. A strong inducible effect on the production of physcion, physcion-bisanthrone and on their precursors and derivatives was found for mannitol. By contrast, supplementation of ribitol had negligible effects, if any, on polyketide quantities although it is the main carbon source for the mycobiont in free-living lichens with Trebouxia photobiont.
The Sterile Microfilamentous Lichenized Fungi Cystocoleus Ebeneus and Racodium Rupestre Are Relatives of Plant Pathogens and Clinically Important Dothidealean Fungi
Mycological Research. Jan, 2008 | Pubmed ID: 18207379
The phylogenetic positions of the always-sterile microfilamentous lichens Cystocoleus ebeneus and Racodium rupestre were studied in a phylogenetic framework using sequence data of 5' nuSSU, nuLSU, and mtSSU rDNA. The analysis reveals that both genera are ascomycetes and belong to Dothideomycetidae: they are not close to lichenized members within the subclass, but rather belong to Capnodiales. The macroscopically scarcely distinguishable C. ebeneus and R. rupestre do not form a monophyletic group. The well-supported clade of R. rupestre is basal to the one in which C. ebeneus is close to Mycosphaerellaceae. This study provides another example of ascomycetes with very different life-styles and ecologies being closely related.
A Combined Molecular and Morphological Approach to Species Delimitation in Black-fruited, Endolithic Caloplaca: High Genetic and Low Morphological Diversity
Mycological Research. Jan, 2008 | Pubmed ID: 18222679
A revision based on the morphological and genetic analyses of 133 specimens of black-fruited, endolithic Caloplaca belonging to subgenus Pyrenodesmia is presented. The material was collected in 16 sites distributed along a transept from Gargano (Central Italy) to the southeastern Alps, from sea level to ca 1500m. The nuclear ITS was sequenced for all the mycobionts and selected representatives of photobionts. Except for the sorediate C. erodens, all species share the same algal lineage of Trebouxia as photobiont. The haplotype analysis of the mycobionts revealed an unexpected, high genetic heterogeneity. Three main morphotypic clusters were recognized among five species: C. albopruinosa (syn. C. agardhiana auct.), C. alociza, C. badioreagens, C. erodens, and C. variabilis. A phylogenetic analysis, including already available Caloplaca sequence data, revealed that these lichens form a monophyletic group within the genus. For each species, notes on ecology, distribution in Italy, and nomenclature are given.
Purifying Selection is a Prevailing Motif in the Evolution of Ketoacyl Synthase Domains of Polyketide Synthases from Lichenized Fungi
Mycological Research. Feb, 2008 | Pubmed ID: 18280127
We analysed ketoacyl synthase domains of type I polyketide synthase (PKS) gene fragments of 163 lichenized and 51 non-lichenized fungi in a Bayesian phylogenetic framework. Lichenized taxa from several unrelated taxonomic groups, some of which produce identical secondary metabolites, were included. We found 12 clades of non-reducing PKS genes, which represent monophyletic PKS paralogues. PAML and SELECTON analyses indicated that purifying selection is the prevailing selective force in the evolution of the keto synthase domain of these paralogues. We detected no unambiguous correlation between PKS clades and the distribution of lichen substances. Together with the strong evidence for purifying selection, the wide distribution of certain paralogues in ascomycetes suggested early gene duplication events in the evolutionary history of this gene family in the Ascomycota.
In Situ Analysis of the Bacterial Community Associated with the Reindeer Lichen Cladonia Arbuscula Reveals Predominance of Alphaproteobacteria
FEMS Microbiology Ecology. Oct, 2008 | Pubmed ID: 18631179
The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.
Bartheletia Paradoxa is a Living Fossil on Ginkgo Leaf Litter with a Unique Septal Structure in the Basidiomycota
Mycological Research. Nov, 2008 | Pubmed ID: 18657613
Bartheletia paradoxa, a basidiomycete growing on fallen leaves of Ginkgo biloba, is redescribed. In autumn a rapidly developing anamorph is formed on freshly fallen leaves and subsequently a teleomorph with hemispherical pustules of thick-walled resting spores (teliospores) that germinate after a resting period of one year with stipitate, longitudinally septate, statismosporic phragmobasidia. The basidia produce several basidiospores on each sporogenous locus. Inoculation experiments and observations in the field suggest that the basidiospores infect the freshly fallen leaves of G. biloba so that the life cycle is completed. The extraordinarily rapid development has also been confirmed in cultures on agar media and in inoculation experiments. Inoculation experiments also indicate that the fungus is specific to G. biloba. The septa of the hyphae have no central pores, but multiple plasmodesma-like perforations. The basidiospores and conidia are uninucleate, but an assessment of the karyology is still pending. A molecular phylogenetic hypothesis based on nuSSU rDNA sequences suggests that the fungus belongs to the Agaricomycotina, clustering in an unresolved position at the basal branching of the group. The family Bartheletiaceae fam. nov. is proposed to accommodate Bartheletia paradoxa in the Agaricomycotina. The name B. paradoxa is validated by a Latin diagnosis and by the designation of types.
Mycological Research. Jan, 2009 | Pubmed ID: 18822374
We characterize the transcript of a polyketide synthase gene (PKS) from the cultured mycobiont of Xanthoria elegans (XePKS1) using SMART-rapid amplification of cDNA ends (RACE) cDNA synthesis. Sequence analysis of the cloned cDNA reveals an open reading frame of 2144 amino acid residues. It contains features of a non-reducing fungal type I PKS with an N-terminal starter unit: acyl carrier protein (ACP) transacetylase domain, ketosynthase, acyltransferase, two acyl carrier protein domains, and a thioesterase domain. XePKS1 was the only paralogue detected in the cDNA and the genomic DNA of the cultured X. elegans mycobiont by using a degenerate PCR approach targeted at the conserved regions of non-reducing type I PKS genes. The hypothetical protein is phylogenetically related to genes that are basal to a clade of dihydroxynaphthalene synthases (non-reducing clade II) and anthraquinone type synthases of non-lichenized fungi (non-reducing clade I). According to hplc and tlc analyses, the cultured mycobiont exclusively produced anthraquinones and its precursors. Therefore, we discuss whether the characterized paralogue is involved in anthraquinone production, which raises the possibility of a paraphyletic origin of lichen anthraquinone biosynthesis. The cDNA of XePKS1 was the first full-length coding sequence of a lichen PKS to be published. This proves SMART RACE to be a suitable tool for obtaining full-length coding sequences of genes from environmental samples and organisms, which are hardly amenable to standard molecular approaches or genomic sequencing.
Repeated Evolution of Closed Fruiting Bodies is Linked to Ascoma Development in the Largest Group of Lichenized Fungi (Lecanoromycetes, Ascomycota)
Molecular Phylogenetics and Evolution. Jul, 2009 | Pubmed ID: 19328858
Fruiting bodies are responsible for the effective dispersal of meiospores in ascomycetes. Different fruiting body types include open (apothecia) or closed (perithecia, cleistothecia) forms, which have traditionally been used as key paradigms for ascomycete classification. Molecular phylogenies show that most fruiting body types have multiple phylogenetic origins within the phylum, and are not suitable for the circumscription of classes. One exception are perithecia that are restricted in non-lichenized fungi to the monophyletic class Sordariomycetes. However, lichenized fungi with perithecioid fruiting bodies were found to belong to three other classes unrelated to Sordariomycetes. One of these is Lecanoromycetes, which includes the bulk of lichenized fungi. To understand the evolution of perithecioid fruiting bodies in the mostly apotheciate Lecanoromycetes, we assembled a combined data set of nuclear and mitochondrial ribosomal, and RPB1 DNA sequences, and traced the evolution of two morphological characters (fruiting body type and fruiting body development). We reconstructed ancestral character states using maximum likelihood and Bayesian methods. Additionally, we tested for correlation of character changes in a combined Bayesian/maximum likelihood framework. The results suggest that perithecia have evolved in unrelated groups of lichen-forming fungi. Within Lecanoromycetes they have evolved independently several times from apotheciate ancestors. Further, our analyses support a correlation between the type of fruiting body and the type of ascoma ontogeny. The evolution of angiocarpous ascoma development in Lecanoromycetes is a pre-adaptation for the repeated gain of perithecia. This finding is consistent with the hypothesis of a neotenic origin of perithecioid fruiting bodies in Lecanoromycetes.
Molecular Ecology. May, 2009 | Pubmed ID: 19389164
Lowland tropical habitats harbour an unexplored genetic diversity of epiphyllous fungi. In the shade of rainforest understoreys, lichenized fungi are specialized to an ephemeral habitat where they produce little vegetative biomass and develop reproductive structures early. In a first population genetic study of epiphyllous lichen fungi, we analysed the intraspecific genetic diversity of five leaf-colonizing lichen mycobiont species. Sampling focused on a lowland perhumid forest plot in Costa Rica, with additional collections from other localities throughout the country. In all species we detected sympatric occurrence of highly diverged haplotypes. Haplotypes belonging to distinct clades in networks were also found on the same leaf, clearly indicating multiple independent colonization events on single leaves. Despite the unusually high genetic diversity of these leaf-colonizing tropical fungi, we did not detect pronounced spatial structure of the haplotype distribution between geographical regions. The observed patterns suggest that the diversity of foliicolous lichens could be much higher than expected, with several cryptic genetic lineages within each morphologically characterized species.
The ISME Journal. Sep, 2009 | Pubmed ID: 19554038
Lichens are generally considered as mutualisms between fungi and green algae or cyanobacteria. These partnerships allow light-exposed and long-living joint structures. The unique organization of lichens provides still unexplored environments for microbial communities. To study lichen-associated bacterial communities, we analyze samples, by a polyphasic approach, from three lichen species (Cladonia arbuscula, Lecanora polytropa and Umbilicaria cylindrica) from alpine environments. Our results indicate that bacteria can form highly structured, biofilm-like assemblages on fungal surfaces and reach considerable abundances of up to 10(8) cells per gram fresh weight. Fluorescence in situ hybridization reveals the predominance of Alphaproteobacteria. Microbial fingerprints performed by PCR-single-strand conformation polymorphism analysis using universal and group-specific primers show distinct patterns for each lichen species. Characterization of cultivable strains and presence of functional genes in the total fraction suggest the involvement of associated bacteria in nutrient cycling. Ubiquitous nifH genes, which encode the nitrogenase reductase, show a high diversity and are assigned to Alphaproteobacteria and Firmicutes, for example, Paenibacillus. Cultivable strains mainly belonging to the genera Acinetobacter, Bacillus, Burkholderia, Methylobacterium and Paenibacillus show lytic (chitinolytic, glucanolytic, and proteolytic) activities, hormone production (indole-3-acetic acid) as well as phosphate mobilization and antagonistic activity toward other microorganisms. The traditional concept of lichens has to be expanded to consider multiple bacterial partners.
Microbial Ecology. Jan, 2010 | Pubmed ID: 19834639
Dispersal of symbiotic partners by joint propagules is considered as an efficient strategy to maintain successful associations and to circumvent low symbiont availability. Joint dispersal is widespread in diverse symbioses and a particularly common reproductive mode in lichens. We were interested in the implications of joint symbiont dispersal on population genetic structure and investigated patterns of symbiont association in populations of two closely related lichen species in the genus Physconia, with similar range of compatible algal partners. One of the lichen species is characterized by joint dispersal of both symbionts, whereas the other species propagates by meiotic fungal spores alone. The latter species must reestablish the symbiotic stage with appropriate algae sampled from the environment. Both fungal species have specialized on photobionts representing a monophyletic lineage of the algal genus Trebouxia. The results indicate no correlated association of symbiont genotypes in the species with joint symbiont dispersal. We rather show that algal gene diversity in populations of lichenized fungi with different propagation strategies is not necessarily different. The association with algae that differ from the co-dispersed genotypes during the vegetative development of the thalli is the most likely explanation for the observed pattern. Maintenance of symbiotic associations is an option but not a strict consequence of joint symbiont dispersal in lichens.
FEMS Microbiology Ecology. Jan, 2010 | Pubmed ID: 19878320
Our planet offers many opportunities for life on the edge: high and low temperatures, high salt concentrations, acidic and basic conditions and toxic environments, to name but a few extremes. Recent studies have revealed the diversity of fungi that can occur in stressful environments that are hostile to most eukaryotes. We review these studies here, with the additional purpose of proposing some mechanisms that would allow for the evolutionary adaptation of eukaryotic microbial life under extreme conditions. We focus, in particular, on life in ice and life at high salt concentrations, as there is a surprising similarity between the fungal populations in these two kinds of environments, both of which are characterized by low water activity. We propose steps of evolution of generalist species towards the development of specialists in extreme habitats. We argue that traits present in some fungal groups, such as asexuality, synthesis of melanin-like pigments and a flexible morphology, are preadaptations that facilitate persistence and eventual adaptation to conditions on the ecological edge, as well as biotope switches. These processes are important for understanding the evolution of extremophiles; moreover, they have implications for the emergence of novel fungal pathogens.
Mycologia. Jul-Aug, 2010 | Pubmed ID: 20648751
Most members of Verrucariales, as in other lichenized lineages, form typical thallus morphologies, including crustose, squamulose, foliose and rarely subfruticose thalli. Some members occur in humid habitats; however some evolved unusually delicate thallus morphologies, such as minute shell-like disks, microfilamentous cushions or brittle flakes. In this study we aimed to elucidate the phylogenetic placement of such morphologically outstanding taxa. We included in our analyses Agonimia spp., Flakea papillata, Normandina spp. and Psoroglaena spp. and used a multilocus sequence dataset of the Verrucariales (SSU rDNA, LSU rDNA, mtSSU, RPB1). The resulting hypothesis revealed genus Normandina as a monophyletic group. Lauderlindsaya was confirmed as a synonym of Normandina. Normandina acroglypta is nested in N. pulchella. Psoroglaena abscondita does not cluster with other species of this genus (P. biatorella and P. stigonemoides) and is related to Verrucula and Placocarpus. Four species of Agonimia form a monophyletic group together with Norrlinia, whereas A. repleta represents its own clade. Apart from this latter relationship the phylogenetic relationship of Flakea and Normandina with other lineages in Verrucariaceae remains elusive with our multilocus dataset. More sequence data of protein-coding loci are required to increase phylogenetic resolution because morphological evolution seems to be dynamic in Verrucariales.
Fungal Biology. Apr, 2010 | Pubmed ID: 20943148
Lichenized and non-lichenized fungi produce a wide range of secondary metabolites. So far, type I polyketide synthases (PKSs) are the suggested catalysts for the biosynthesis of lichen compounds. We were interested whether lichen mycobionts also contain type III PKSs, representing a class that was only recently discovered in fungi. With an alignment of known type III CHS-like genes we applied the CODEHOP strategy to design degenerate PCR primers. We further screened available fungal genomes for type III PKS genes and aligned these sequences for a phylogenetic analysis. Type III-like genes from lichen mycobionts are closely related to those known from non-lichenized fungi, but not to those of bacteria and/or plants. We conclude that type III PKS genes are ubiquitous in fungi. They are present in diverse unrelated lichen mycobionts, but their function in lichens is so far unclear.
A Contribution to the Taxonomy of the Genus Rinodina (Physciaceae, Lichenized Ascomycotina) Using Combined ITS and MtSSU RDNA Data
Lichenologist (London, England). Sep, 2010 | Pubmed ID: 22121298
To test the phylogenetic position of phenotypically peculiar species in the Physciaceae we generated 47 new sequences (26 of nrITS region and 21 of mtSSU rDNA) from 19 crustose taxa of Physciaceae mainly from the genus Rinodina. Phylogenetic analysis confirmed the Buellia and Physcia groups. The analysis revealed a considerable variability of characters traditionally used for classification, especially in the delimitation of the genera Buellia and Rinodina. While ascus types agree well with the distinction of the Buellia and Physcia groups, none of the other traditional characters, including excipulum type and ascospore thickening, were consistent within subclades of the Physcia group. We suggest that both excipulum type and ascospore characters are rather dynamic in the evolution of Rinodina species and only appear consistent in morphologically more complex foliose and fruticose groups, which are characterized by thallus characters not present in the crustose groups. Two recent taxonomic changes are supported by molecular characters: Endohyalina insularis (syn. 'Rinodina' insularis) and Rinodina lindingeri (syn. 'Buellia' lindingeri). In addition Rinodina parvula (syn. 'Buellia' parvula) is reinstated. New records for Endohyalina brandii, E. diederichii, E. insularis and Rinodina albana are presented.
Frondihabitans Cladoniiphilus Sp. Nov., an Actinobacterium of the Family Microbacteriaceae Isolated from Lichen, and Emended Description of the Genus Frondihabitans
International Journal of Systematic and Evolutionary Microbiology. Dec, 2011 | Pubmed ID: 21296927
A novel actinobacterium, designated strain CafT13(T), was isolated from the thallus of the reindeer lichen Cladonia arbuscula sampled in the Austrian Alps (Koralpe). The organism was aerobic, with rod- to irregular-shaped cells (often forming dense clusters of cells when grown in liquid medium), Gram-stain-positive, oxidase-negative, catalase-positive and non-motile. It was able to grow at 1 °C and at low to neutral pH, but not above 30 °C or at high pH. The peptidoglycan type was B2β with ornithine as the diagnostic diamino acid. The menaquinones were MK-7 and MK-8. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids, three unidentified glycolipids and one unidentified aminolipid. The predominant fatty acids were C(18:1), C(14:0) 2-OH, C(17:1)ω9c, C(16:0) and anteiso-C(15:0). The mean DNA G+C content of strain CafT13(T) was 69.0±0.17 mol%. 16S rRNA gene sequence analysis showed that strain CafT13(T) belongs to the family Microbacteriaceae, within the genus Frondihabitans. The mean level of DNA-DNA relatedness between strain CafT13(T) and the type strain of Frondihabitans australicus was 35.2±5.23%. The enzyme spectrum of strain CafT13(T) differentiated it from recognized species of the genus Frondihabitans. Based on molecular, chemotaxonomic and physiological data, strain CafT13(T) is considered to represent a novel species of the genus Frondihabitans, for which the name Frondihabitans cladoniiphilus sp. nov. is proposed; the type strain is CafT13(T) (=DSM 23273(T)=LMG 25550(T)).
Structure and Function of the Symbiosis Partners of the Lung Lichen (Lobaria Pulmonaria L. Hoffm.) Analyzed by Metaproteomics
Proteomics. Jul, 2011 | Pubmed ID: 21604374
Environmental proteomics, also referred to as metaproteomics, is an emerging technology to study the structure and function of microbial communities. Here, we applied semi-quantitative label-free proteomics using one-dimensional gel electrophoresis combined with LC-MS/MS and normalized spectral counting together with fluorescence in situ hybridization and confocal laser scanning microscopy to characterize the metaproteome of the lung lichen symbiosis Lobaria pulmonaria. In addition to the myco- and photobiont, L. pulmonaria harbors proteins from a highly diverse prokaryotic community, which is dominated by Proteobacteria and including also Archaea. While fungal proteins are most dominant (75.4% of all assigned spectra), about the same amount of spectra were assigned to prokaryotic proteins (10%) and to the green algal photobiont (9%). While the latter proteins were found to be mainly associated with energy and carbohydrate metabolism, a major proportion of fungal and bacterial proteins appeared to be involved in PTMs and protein turnover and other diverse functions.
Fungal Biology. Oct, 2011 | Pubmed ID: 21944204
Fungal Biology. Oct, 2011 | Pubmed ID: 21944210
In this study we investigate bacterial communities in association with an enriched black-fungal community in the plant phyllosphere to test whether these fungi create an environment for specific bacteria. Under organic conditions of agriculture, grapevine plants (Vitis vinifera) display an increased occurrence of the black fungi Aureobasidium pullulans and Epicoccum nigrum. Their enrichment agrees with the tolerance of these fungi to copper and sulphate, both used as main fungicides in organic viticulture. Both fungi also intrude the plant material to grow endophytically. Bacterial communities associated with black fungi of the plant surface and endosphere showed no differences compared to those found in conventionally managed V. vinifera plants. This suggests that despite an increase of these black fungi in organic practice, they do not shape bacterial diversity in grapevine plants. Nevertheless, dual cultures revealed a negative effect of Aureobasidium on the growth of certain bacilli, whereas growth of Aureobasidium was impeded by one Pseudomonas strain. Such singular effects are either not apparent in the natural black-fungal--bacterial community of the grape phyllosphere or are of rather localized effect.
Fungal Biology. Oct, 2011 | Pubmed ID: 21944213
Specific indoor environments select for certain stress-tolerant fungi and can drive their evolution towards acquiring medically important traits. Here we review the current knowledge in this area of research, focussing on the so-called black yeasts. Many of these melanised stress-tolerant organisms originate in unusual ecological niches in nature, and they have a number of preadaptations that make them particularly suited for growth on human-made surfaces and substrates. Several pathogenic species have been isolated recently from various domestic habitats. We argue that in addition to enriching for - potentially - pathogenic species, the selection pressure and stress acting on microorganisms in indoor environments are driving their evolution towards acquiring the missing virulence factors and further enhancing their stress tolerance and pathogenic potential. Some of the polyextremotolerant fungi are particularly problematic: they can grow at elevated temperatures, and so they have a higher potential to colonise warm-blooded organisms. As several species of black fungi are already implicated in health problems of various kinds, their selection and possible evolution in human environments are of concern.
Photobiont Association and Genetic Diversity of the Optionally Lichenized Fungus Schizoxylon Albescens
FEMS Microbiology Ecology. Feb, 2011 | Pubmed ID: 21133956
The fungus Schizoxylon albescens occurs both as lichen and as saprobe. Lichenized colonies grow on the bark of Populus tremula; saprotrophic morphs grow on dead Populus branches. We wanted to (1) test whether lichenized and saprotrophic S. albescens are genetically distinct, (2) investigate photobiont association and diversity, (3) investigate the interactions between fungi and algae that occur during co-cultivation and (4) test whether Schizoxylon shows algal selectivity during lichenization. Fungal and algal genetic diversity were investigated for three markers. Algae from lichenized thalli were isolated in axenic cultures, and isolate sequence diversity was compared with algae amplified directly from thallus fragments. Co-culture experiments of fungi and algae were performed to study the morphological interaction patterns. Two distinct phylogenetic units are revealed in S. albescens, which are interpreted as phenotypically cryptic species. The algae are related to Coccomyxa and Pseudococcomyxa, and form two distinct sister clades separating samples isolated in cultures from those amplified directly from thallus fragments, indicating that more easily cultured strains of algae are not necessarily major components of the lichens. Schizoxylon albescens interacts with isolated algal strains, similar to fungal-Coccomyxa symbioses in nature. As the system is maintained without difficulty in culture, it can potentially be an easily controlled lichen symbiosis study system under laboratory conditions.
Microbial Diversity Inside Pumpkins: Microhabitat-specific Communities Display a High Antagonistic Potential Against Phytopathogens
Microbial Ecology. Feb, 2012 | Pubmed ID: 21947430
Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.
Bacterial Taxa Associated with the Lung Lichen Lobaria Pulmonaria Are Differentially Shaped by Geography and Habitat
FEMS Microbiology Letters. Jan, 2012 | Pubmed ID: 22268428
The correlation between the taxonomic composition of Alphaproteobacteria, Burkholderia and nitrogen fixers associated with the lichen Lobaria pulmonaria and the geographical distribution of the host was studied across four sites in Europe. Results proved that the diversity of Alphaproteobacteria is affected by geography, while those of Burkholderia and nitrogen fixers were mostly driven by local habitat. This difference indicates a higher stability of the association between Alphaproteobacteria and the lichen host.