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Molecular and Genomic Methods for Analyzing Hybridization in Aspergillus and other Fungi
Jirkovský, Pavel ; Sklenář, František (advisor) ; Kolařík, Miroslav (referee)
Interpecies hybridization is being studied since the first half of 20. century, when it was described based on phenotypical traits on plant model. Study of this process was historically centred around botanical studies, while its importance in mycology is just being uncovered in recent years. Introgression is a phenomenon that can occur when hybrid backcrosses with one of the parental species. Based on recent studies it is apparent that hybridization played its role in evolution of MAT locus genes of Neurospora, transfer of adaptive traits with Ophiostoma species (invasive pathogen of elm trees). Within Aspergillus family hybridization was described both on intraspecific and interspecific level. More detailed study of this process and improving of used methods could potentially help in clinical application by uncovering adaptive genes responsible for resistance towards antimycotics, higher virulence and other traits.
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Arthroderma in the fur of wild rodents
Moulíková, Štěpánka ; Kolařík, Miroslav (advisor) ; Koukol, Ondřej (referee)
Arthroderma is the most diverse genus of dermatophytes, fungi causing skin infections. However, the genus is most frequently isolated from soil enriched by keratin sources. In my study, I isolated several strains of Arthroderma from the fur of wild rodents from the Czech Republic. Using polyphasic taxonomy, I characterised the 29 strains genetically (ITS rDNA, tubb and tef1α), morphologically, physiologically, and by mating experiments. In the second part of the study, we used data mining of ITS sequence data from GenBank and GlobalFungi Database to further investigate biogeography and ecology of Arthroderma species. In total, eight Arthroderma species were isolated, including four new species. The geographical distribution of the new species was not restricted to the Czech Republic nor rodents, other strains have been isolated from different animal species and soil from Europe, North America and Asia. Data obtained by data mining confirmed that the genus has diverse ecology - some lineages seem to be closely associated with animals (here described A. rodenticum), while others occur typically in soil. However, low numbers of sequence reads of Arthroderma in soil show that the species are not soil generalists but rather dependent on keratin source. This is the first study using data mining to assess...
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Comparative methods for studying adaptive traits of fungal symbionts
Veselská, Tereza ; Kolařík, Miroslav (advisor) ; Voigt, Kerstin (referee) ; Dvořák, Miloň (referee)
The kingdom Fungi encompasses an estimated 2.2 to 6.2 million species that occupy diverse environments, including aquatic, extremely dry, and hot or frosty habitats all over the world. To cope with adverse environmental conditions, fungi have developed numerous adaptations and life strategies, including symbiosis with other organisms, ranging from close, reciprocally beneficial (mutualistic) associations to severe pathogenic infestations. These interactions have an enormous impact on ecosystem functioning, with implications for agriculture and human health. For this reason, understanding the mechanisms enabling the successful development of fungal interactions is necessary for their efficient management. Recent advances in different 'omics' approaches have enabled us to compare species responses to the environment in a more complex way than before and to gain deeper insights into the adaptive mechanisms underlying specific life strategies. My thesis is divided into four main sections. In the first section, I sum up findings about adaptations of fungal symbionts of plants and animals. Then, I introduce two fungal genera, Geosmithia and Pseudogymnoascus, to which I applied comparative methods for tracking adaptive traits. The ecological diversity of the genus Geosmithia allows to trace adaptive...
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Invasion of Phytophthora alni in Europe
Pecka, Štěpán ; Černý, Karel (advisor) ; Kolařík, Miroslav (referee)
Species complex Phytophthora alni s. l. (Peronosporomycetes: Stramenopila) is a group of invasive pathogens of alder trees. It spreads mainly via water and causes root and collar rot often resulting in the death of attacked trees. Especially in riparian stands it often causes significant losses of alder and therefore remarcable changes of ecosystems and watercourses. The species complex is very polymorphic and divided into three species - P. ×alni, P. uniformis and P. ×multiformis. The most widespread in Europe and in the Czech Republic is the P. ×alni species. The aim of this thesis is to summarize information about P. alni complex (and especially of the most important taxon P. ×alni), to describe history of its spread and development of its area in Europe, genetic variability and inner structure of its populations, ecology and pathways of spread and, based of the known results, decribe the current situation of the invasion. Key words: Phytophthora alni, Peronosporomycetes, Alnus, pathogen, hybridization, area, riparian stands
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Ecology of yeasts in forest soils
Mašínová, Tereza ; Baldrian, Petr (advisor) ; Marečková, Markéta (referee) ; Kolařík, Miroslav (referee)
Microbial communities inhabiting upper soil horizons represent an important component of forest ecosystems. However, despite the evidence that yeasts represent an integral part of topsoil fungal communities, their role in forest ecosystems received so far little attention. The aims of my PhD thesis were to describe yeast communities in soil and litter of a temperate forest using high- throughput sequencing of environmental DNA, identify dominant yeast species and to explore how the composition of yeast communities reflects the biotic and abiotic factors of the environment. I also aimed to isolate yeasts from forest topsoil, describe novel yeast taxa abundant according to the environmental DNA survey and screen representative isolates for the traits relevant to their involvement in organic matter transformation. I have demonstrated that in forest topsoil, yeasts represent a substantial proportion of fungal communities with higher relative abundance in soil than in litter. In litter, yeast communities differ significantly among beech, oak and spruce-dominated stands. Drivers of community assembly are probably more complex in soils and comprise the effects of soil chemistry and vegetation. Even though there are similarities in the response of the communities of yeasts and filamentous fungi to...
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Pyrosequencing analysis of fungal assemblages and the effect of ITS rDNA intragenomic variation on diversity estimates
Zelenka, Tomáš ; Kolařík, Miroslav (advisor) ; Větrovský, Tomáš (referee)
The ITS region of nuclear ribosomal DNA has recently become a frequently studied region for its use as a barcode marker. It is employed in environmental metagenomic analyses, the determination of fungi and in studies of fungal phylogenetics. In genomes, rDNA occurs in the form of large multicopy tandem arrays. Thus common Sanger sequencing leads to a consensus of many copies which in fact conceals most of the potential intragenomic variation. However 454 pyrosequencing reveals the sequence of single copies. Although it is believed that most of the variation among copies is reduced during a process called concerted evolution, some variation might still be conserved, including variation of non-functional pseudogenes. As a consequence, when using 454 pyrosequencing, we are not able to discriminate between this variation and real diversity. This could have a huge impact on the estimation of real diversity as well as on the correct assessment of pyrosequence studies. This thesis reviews current knowledge of intragenomic variation among fungi and summarizes some papers applying pyrosequencing in the research of fungal diversity. At the same time it indicates intragenomic variation as a potential cause of untrue diversity in diversity studies. Up until now there hasn't existed any experimental survey covering...
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Cellulolytic fungi and their diversity on plant litter
Gálová, Diana ; Baldrian, Petr (advisor) ; Kolařík, Miroslav (referee)
Litter decomposition requires the presence of corresponding degradative enzymes, produced mainly by fungi. Forest soils show considerable spatial heterogeneity of distribution of these enzymes at diferent scales. Moreover, enzyme pruduction varies during the year, usually accompanied by the change in fungal community composition. In this work I examined if this spatial heterogeneity can be seen even at a scale of an individual leaf and whether the fungal community differs among enzyme activity hotspots and inactive parts of the leaves. Another goal was isolation of celulytic fungi from cellulose litterbags incubated on forest floor using particle filtration and dilution-to-extinction method. In a broadleaved forest dominated by oak leaves at different stages of decay were collected: senescent leaves on twigs, and leaves after 2, 10 and 22 months of decomposition. Ten leaves per season were taken for analysis of cellobiohydrolase activity over the leaf surface. Leaves were attachmed onto melted agarose plate and leaf surface was covered with low melting point agarose containing fluorescently labelled substrate. For each leaf a map of enzyme activity was created and area with the high and low enzyme activity was identified. From both sites a square of approx. 1 cm2 was cut out, DNA was extracted and fungal...
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Fungal genetics, genome evolution and use of flow cytometry in study of DNA
Würtherlová, Tereza ; Kolařík, Miroslav (advisor) ; Kostovčík, Martin (referee)
The fungal genome is a dynamically changing structure. By its remodelling, the organism can respond to the environmental changes and develop itself. The genome expansion is often accompanied by transition to parasitic or mutualistic way of life. The genome expansion can be caused by the multiplication of some chromosomes (aneuploidisation), the whole genome duplication (polyploidisation) or the spreading of repetitive sequences. The impact of genome size to the ecology and life strategy of the organisms is more and more studied in recent years. In mycology, nevertheless, it escapes sufficient attention. The flow cytometry (FCM) is a modern and progressively developing method that enables to determine the genome size and estimate CG/AT base ratio. The combination of FCM with knowledge of the fungal ecology and forces that form the genome enable to discover a general trends of the evolutionary processes. My study summarises knowledge about the processes leading to changes in the size/structure of the fungal genome, the correlations with genome size and presents the principle of flow cytometry and its application in mycology.
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Taxonomy, diversity and clinical relevance of the genus Aspergillus
Hubka, Vít ; Kolařík, Miroslav (advisor) ; Labuda, Roman (referee) ; de Beer, Wilhelm (referee)
Aspergillus is a speciose genus encompassing nearly 400 species that has significant economic impacts on human health, the food industry, biotechnology and pharmacology. The research included in this thesis focuses on current issues related to the generic concept, subgeneric classification and species delimitation in Aspergillus. It addresses the need for revisions of several sections or species complexes. It provides novel information regarding etiology of aspergillosis as well as the antifungal susceptibilities of several less common opportunistic pathogens. The taxonomic section of the thesis contributes to the taxonomic stability and the new concept of the genus Aspergillus, which changed in response to the discontinuation of dual nomenclature in fungi. Sufficient arguments were collected (e.g., verification of monophyly, unifying phenotypic characters) for maintaining a broad concept of the genus and avoiding splitting it into several genera. All genera typified by sexual morphs and having Aspergillus asexual states were synonymized with Aspergillus and the appropriate names adopted; new combinations were made for teleomorphic species that lacked Aspergillus names. This thesis also contributed to infrageneric taxonomy of the genus via the proposal of four new sections in the subg. Circumdati,...
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The effect of microbiota on pathogenesis of gut diseases
Galanová, Natalie ; Kolařík, Miroslav (advisor) ; Hudcovic, Tomáš (referee)
Gut microbiota is considered an important factor in the development of various diseases including inflammatory bowel disease (IBD, n = 127), Ulcerative colitis, Crohn's disease, and colorectal cancer (CRC, n = 64). A part of this thtesis is to prepare clinical material of different sorts (stool, biopsy) for sequencing on Illumina Miseq platform. This is achieved trough DNA isolation, amplification of 16S and internal transcribed spacer (ITS), normalization and ligation of sequencing adaptors. The aim of this project is to describe the differences between microbiota in healthy and diseased subjects in case of IBD or unimpaired and tumorous tissue for CRC patients. This research is also being based on cultivation, where a fresh stool samples (n = 3) are cultivated in a broad range of conditions, which enables us to obtain ecophysiological and species diversity of these samples by traditional and molecular methods. The cultivable fungi are also assigned reliable taxonomy by amplification of relevant genes (ITS1, β tubulin, second largest subunit of RNA polymerase II, RPB2) followed by both-sided Sanger sequencing. Selected species of fungi are processed into lysates, which are used for stimulation of mice macrofage cell line (RAW). Therefore the impact on immunity response is studied in vitro and...
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