Dissertations / Theses on the topic 'ITS (Intergenic Transcribed Spacer)'

Les cyanobactéries sont des organismes procaryotes photosynthétiques capables de produire de nombreux métabolites secondaires. Cette thèse s’intéresse à la caractérisation moléculaire de cyanobactéries productrices de neurotoxines (anatoxine-a et homoanatoxine-a) ainsi qu’à la mise en évidence d’un gène codant pour une activité halogénase dans les clusters de gènes de biosynthèse des métabolites : cyanopeptoline et aéruginosine. De nombreux cas d'intoxications neurotoxiques d'animaux sauvages et domestiques sont régulièrement reportés en France. Nous avons mis en évidence sur deux sites pour lesquels des épisodes mortels ont été rapportés la présence de neurotoxines de cyanobactéries. Les cyanobactéries productrices d’anatoxine-a ont été isolées et déposées dans la collection de cyanobactéries de l’Institut Pasteur PCC. La caractérisation phénotypique et génétique des souches axéniques obtenues montre qu’il s’agit de souches du genre Oscillatoria. Sur la base des séquences codant pour les ARNr16S et ITS (Internal Transcribed Spacer) les relations phylogénétiques des nouvelles souches neurotoxiques au sein du genre Oscillatoria ont été étudiées. Une étude du contenu métabolique en cyanopeptoline et aéruginosine et de la présence des leur clusters de gènes de biosynthèse a été entreprise chez une trentaine de souches de Microcystis. Elle a permis de mettre en évidence la présence d’halométabolites et d’identifier les gènes portant l’activité halogénase au sein des clusters aer et mcn. L’histoire évolutive des clusters aer et mcn au sein du genre Microcystis a été reconstituée par analyse des séquences ITS. Le gène halogénase semble avoir été acquis par la cyanobactérie probablement d'une protéobactérie ancestrale par transfert horinzontal
This work of PhD is cantered around three secondary metabolites produced by cyanobacteria (cyanopeptolin, aeruginosin and anatoxin-a). The first part of this work reports the results about isolating and purifing neurotoxic cyanobacteria from natural samples. Repeated dog deaths occurred in the last years in southern France. Signs of intoxication indicated acute poisoning due to a neurotoxin. Samples were collected at the border of tarn river in summers 2005 and 2006 from six different sites along 30 km. The cyanobacterial neurotoxines anatoxin-a and homoanatoxin-a were detected in extracts of most samples examined by gas chromatography-mass spectrometry. Fifteen filamentous cyanobacteria of the order Oscillatoriales were isolated and displayed four distinct phenotypes. Three of the phenotypes are assignable to the genera Oscillatoria/Phormidium, the fourth to the genus Geitlerinema. The genetic relatedness of the new isolates was evaluated by comparison of the ITS sequences with those of six oscillatorian strains from the PCC collection. Our findings prove that neurotoxic oscillatorian cyanobacteria exist in the Tarn river and thus were most likely implicated in the reported dog poisonings. Furthermore, they re-emphasise the importance of monitoring benthic cyanobacteria in aquatic environments. The second part of this work presents the evolution of the halogenase genes of the strains Microcystis sp. Producing chloreted cyanopeptolin and aeruginosin. Twenty-eight axenic strains of Microcystis aeruginosa from the PCC and NIES culture collections were screened by MALDI-TOF mass spectrometry and their genome analysed by degenerated oligonucleotides. Two putative halogenase genes present in cyanopeptoline and aeruginosin synthetase clusters were identified. Their nucleotide and deduced amino acid sequences were analysed in detail. A phylogenetic study was conducted on thirty halogenase and putative halogenase genes from different microorganisms

The genus Iliamna Greene has a taxonomically complex history. Since its desciption in 1906, the genus was not recognized for some time, several species were initially placed into other genera, and the species status of a few was questioned. Today, eight species of Iliamna are recognized. Six species are located in western North America and two are found isolated to the east. Species in Iliamna are very similar morphologically with only a few characters distinguishing several as separate entities. The need for systematic study became apparent since all but one species are considered rare or endangered. Also, the differentiation between two endangered species, I. corei and I. remota, was unclear in a previous study using random amplified polymorphic DNA fragments. Of the western species, four overlap in distribution (I. crandallii, I grandi ora, I. longisepala, and I. rivularis) and their recognition as separate species has been questioned. The focus of this study was to develop a phylogeny for Iliamna using sequences from the internal transcribed spacer region (ITS) of the nuclear ribosomal RNA genes in order to determine its biogeographical and evolutionary history. Cladistic analysis was performed and the resulting phylogeny is presented. The ITS data provide new insights in the origination of the genus and its distribution. In Iliamna, the ITS region is 677 base pairs long with 120 sites providing information in the formation of phylogenetic trees. Iliamna forms a well-supported clade distinct from related genera and is monophyletic. Three well-supported groups are formed. One contains representatives from the Pacific Northwest. Another contains all of the remaining species with the third clade nested therein. This last clade contains the two eastern species, I. corei and I. remota, but there is not enough variability to support the divergence of these taxa as distinct species. There is also not sufficient variability in the ITS region to identify the western species I. crandallii, I. grandi ora, I. longisepala and I. rivularis as distinct entities.
Master of Science

Orientador: Ana Maria Goulart de Azevedo Tozzi
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-09T03:16:51Z (GMT). No. of bitstreams: 1 Meireles_JoseEduardodeCarvalho_M.pdf: 5457639 bytes, checksum: 2254e34efd86c3760ca5a245abf9eaf6 (MD5) Previous issue date: 2007
Resumo: Poecilanthe (Leguminosae, Papilionoideae, Brongniartieae) é em gênero sul-americano que inclui atualmente dez espécies. A heterogeneidade morfológica e química encontrada em Poecilanthe dificulta sua circunscrição e coloca em dúvida sua monofilia. Além disso, limites interespecíficos imprecisos e falta de chave de identificação dificultam o reconhecimento das espécies. Este trabalho tem como objetivos testar a monofilia de Poecilanthe e estabelecer as relações entre suas espécies, bem como revisar a taxonomia do gênero. Para tanto, uma análise filogenética de máxima parcimônia baseada em caracteres morfológicos e seqüências de ITS/5.8S (nrDNA) foi realizada. Como subsídio para a análise cladística, foi feito um estudo sobre a morfologia das sementes e embriões de Poecilanthe, que resultou no reconhecimento de quatro padrões distintos de morfologia. Os resultados da filogenia mostram que Poecilanthe não é um gênero monofilético, sendo composto por três clados parafiléticos em relação à tribo. Estes três clados foram caracterizados morfologicamente e considerados como gêneros distintos. Poecilanthe é recircunscrito para incluir apenas as espécies extra-amazônicas (Poecilanthe s.s.), compreendendo então seis espécies. O gênero Amphiodon é restabelecido, e P. ovalifolia combinada neste. Um gênero novo é descrito para incluir P. amazonica e P. hostmannii. Cada um destes gêneros foi tratado taxonomicamente, constando em cada tratamento descrições, ilustrações e chave para a identificação das espécies
Abstract: The genus Poecilanthe (Leguminosae, Papilionoideae, Brongniartieae) currently comprises ten South-American species. The morphological and chemical diversity that is found within this genus renders its circumscription imprecise and brings Poecilanthe¿s monophyly into question. This work aims to test the monophyly of Poecilanthe and to revise the taxonomy of the genus. A parsimony analysis based on both morphological and ITS/5.8S data was carried out. In order to provide characters to the cladistic analysis, the morphology of the seeds and embryos of Poecilanthe was analyzed, and resulted in the identification of four different morphological patterns. The phylogeny does not support Poecilanthe as monophyletic, but resolves three different well-supported lineages that are paraphyletic with respect to the tribe. These clades are morphologically characterized and ranked at the generic level. Poecilanthe is recircumscribed to include the six extra-Amazonian species only. The genus Amphiodon is reinstated and P. ovalifolia is combined. Poecilante amazonica and P. hostmannii are segregated into a new genus. Each genus was revised and descriptions, illustrations and identification key for the species are presented.
Mestrado
Biologia Vegetal
Mestre em Biologia Vegetal

Molecular and morphologic studies were used to determine the evolution, classification and differentiation of Nymphaea odorata. Molecular analyses of the nuclear internal transcribed spacer (ITS) region, the chloroplast trnL-F region, and inter-simple sequence repeat (ISSR) markers determined the variation present between and within two species of Nymphaea. The ITS region resulted in a phylogeny depicting strong separation between species (N. mexicana and N. odorata) and some separation between N. odorataâ s subspecies. The ITS region contained polymorphisms, which upon SAHN clustering and principle coordinate (PCOA) and minimum spanning tree (MST) analyses produced groups similar to the clades in the ITS phylogeny. Sixteen accessions were chosen for trnL-F analysis, where a subspecies-specific molecular marker was found. In most accessions the marker confirmed the original subspecies classification. Molecular analyses using ISSRs characterized among population variation in N. odorata and N. mexicana using five primers. ISSR markers among populations were highly variable within a species and were used in UPGMA, PCOA and MST analysis, which resulted in separation between the subspecies. Both univariate and multivariate analyses were performed on quantitative and qualitative morphological characters. An analysis of variance resulted in six morphological characteristics that were statistically significant (P< 0.05), the majority being leaf blade characteristics. Multivariate statistics of principle component analysis and discriminate analysis resulted in groups for each subspecies, both emphasized the importance of quantitative leaf blade characteristics. Overall, both morphology and molecular characteristics supported the classification of subspecies for ssp. odorata and ssp. tuberosa, due a lack of strong segregation of characteristics.
Master of Science

ABSTRACT COMPARATIVE SEQUENCE ANALYSIS OF THE INTERNAL TRANSCRIBED SPACER 2 REGION OF TURKISH RED PINE (Pinus brutia TEN.) AND NATURAL ALEPPO PINE (Pinus halepensis MILL.) POPULATIONS FROM TURKEY Tozkar, Ö
zge M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya April, 2007, 107 pages Turkish red pine (Pinus brutia) is wide-spread and an important forest tree species in Turkey, occurring mainly in southern, western and north-western Turkey and as small isolated populations in the Black Sea region. Aleppo pine (Pinus halepensis) has naturally found only in Adana and Mugla provinces as small population in mixture with Turkish red pine. Although Turkish red pine and Aleppo pine are morphologically different, Turkish red pine has been regarded as subspecies of Aleppo pine by some taxonomists due to occurrence of natural hybridization between these two species. However, the phylogenic relationship between these species needs to be explored further. In the present study, by sampling overlapped populations of both species from Mugla and Adana provinces (4 populations of Turkish red pine and 3 populations of Aleppo pine), internal transcribed spacer (ITS) region of ribosomal DNA were comparatively studied with sequence analysis. Although ITS1, 5.8s and ITS2 regions of ribosomal DNA were studied with ITS primers, only ITS2 region was successfully amplified with polymerase chain reaction (PCR). The complete data set for this region was analysed using MEGA3.1 and Arlequin softwares. Analysis of molecular variance (AMOVA) demonstrated the highest genetic differentiation between Turkish red pine and Aleppo pine in Mugla with 100 percentage of variation. AMOVA analysis also indicated the possibility of low-level migration of genes between Turkish red pine and Aleppo pine populations in Adana with 50.65 percent of molecular variance. Haplotype comparison revealed that two major haplotypes were represented Based on the results of ITS2 region sequence analysis, Turkish populations of Aleppo pine and Turkish red pine populations could not be fully differentiated. In Mugla province Turkish red pine and Aleppo pine revealed more differentiation due to reproductive isolation. But in Adana province, two species shared more common genetic background due to possible hybridization. Since ITS2 region of nuclear ribosomal DNA revealed a few variable and parsimony informative sites for both species, thus, only ITS2 region of ribosomal DNA does not appear to be sufficient for fully resolving genetic relationships between Turkish red pine and Aleppo pine populations. Further studies including ITS1 and 5.8s regions of ribosomal DNA and populations included from major Aleppo pine distribution areas will be useful to understand the evolutionary relationship between Aleppo pine and Turkish red pine populations in Turkey.

The present work aims to determine the physiological and sanitary quality of Sweet sorghum (Sorghum bicolor (L.) Moench) seeds, as well as to identify pathogens associated with seed, their transmission to seedlings and the subsequent pathogenicity of isolates obtained, In addition, molecularly identify the fungal species pathogenic to this crop. The experiments were carried out in the Teaching and Seed Research Laboratory (TSRL), of the Plant Engineering Department; In the Elocy Minussi Phytopathology Laboratory, Department of Plant Protection, and at the Biological Institute of São Paulo. Sweet sorghum seeds were used, all without chemical treatment. Sanitary quality was evaluated by sanity test, and physiological characteristics by germination and vigor tests (seedling length, dry mass, emergence, rate of emergence and accelerated aging). It was performed the test of transmission of the pathogens associated to the seeds and the subsequent pathogenicity of the obtained isolates, culminating with the molecular characterization of the identified pathogens, in which were sequenced the Internal Transcribed Spacer (ITS) genomic regions and the Elongation Factor 1 - alpha (TEF1-α) gene. The design used was the completely randomized design, with four cultivars of Sweet sorghum (BRS 506, F19, BRS 511 and BRS 509); For the evaluation of pathogenicity, the factorial scheme is represented by four cultivars and three isolates of Fusarium spp., besides the witness. The seeds of the BRS 509 cultivar were considered to have lower physiological quality than the other cultivars. The DNA sequencing allowed identifying the Fusarium thapsinum species as a pathogenic agent in the sweet sorghum crop, and proven its transmission via seeds.
O presente trabalho teve como objetivo determinar a qualidade fisiológica e sanitária de sementes de cultivares de sorgo sacarino (Sorghum bicolor (L.) Moench), bem como identificar os patógenos associados à semente, sua transmissão às plântulas e a posterior patogenicidade de isolados obtidos, além disso, identificar molecularmente as espécies fúngicas patogênicas a esta cultura. Os experimentos foram realizados no Laboratório Didático e de Pesquisas em Sementes (LDPS), do Departamento de Fitotecnia; no Laboratório de Fitopatologia Elocy Minussi, do Departamento de Defesa Fitossanitária e, no Instituto Biológico de São Paulo. Foram utilizadas sementes de sorgo sacarino, todas sem tratamento químico. A qualidade sanitária foi avaliada pelo teste de sanidade, e as características fisiológicas por meio dos testes de germinação e de vigor (comprimento de plântulas, massa seca, emergência, índice de velocidade de emergência e envelhecimento acelerado). Foi realizado o teste de transmissão dos patógenos associados à semente e a posterior patogenicidade dos isolados fúngicos obtidos, culminando com a caracterização molecular dos patógenos identificados, na qual foram sequenciadas as regiões genômicas Internal Transcribed Spacer (ITS) e o gene do fator de elongação 1-α (TEF1α). O delineamento utilizado foi o inteiramente casualizado com quatro cultivares de sorgo sacarino (BRS 506, Fepagro 19, BRS 511 e BRS 509); já para a avaliação da patogenicidade, o esquema fatorial foi representado pelas quatro cultivares e três isolados de Fusarium sp., além da testemunha. As sementes da cultivar BRS 509 foram consideradas de qualidade fisiológica inferior as demais cultivares. O sequenciamento de DNA permitiu identificar a espécie Fusarium thapsinum como agente patogênico na cultura do sorgo sacarino, sendo comprovada sua transmissão via sementes.

Phacelia purshii Buckley and P. fimbriata Micheaux are two species that are nearly morphologically indistinguishable. Seed germination experiments showed that the high elevation endemic, P. fimbriata requires lower temperatures to trigger germination. Following interspecific crosses, pollen tubes enter ovules and maternal tissue of the gynoecium matures but hybrid diploid and triploid organs fail to develop. DNA sequences from the ribosomal DNA internal transcribed region showed that P. fimbriata and P. purshii comprise a monophyletic clade but that P. fimbriata is more differentiated from related species. In contrast, P. purshii supported significantly higher levels of intraspecific polymorphism. Phacelia fimbriata and P. purshii are sister species with similar morphology but they are unable to hybridize, they are differentiated in physiological characteristics related to environment, and they inhabit different elevations. This pattern of relationship and differentiation suggests P. fimbriata may be the product of ecological speciation.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-21T20:25:51Z No. of bitstreams: 2 Dissertação - Cálita Pollyanna Marques - 2017.pdf: 1468493 bytes, checksum: 90300902d8a48fc5600761dfccf90324 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-22T11:43:21Z (GMT) No. of bitstreams: 2 Dissertação - Cálita Pollyanna Marques - 2017.pdf: 1468493 bytes, checksum: 90300902d8a48fc5600761dfccf90324 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)
Made available in DSpace on 2017-09-22T11:43:21Z (GMT). No. of bitstreams: 2 Dissertação - Cálita Pollyanna Marques - 2017.pdf: 1468493 bytes, checksum: 90300902d8a48fc5600761dfccf90324 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-30
Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
American Cutaneous Leishmaniasis (LTA) is caused by protozoa of the genus Leishmania and it attacks the skin and/or mucous membranes. It is an endemic zoonosis in Brazil being notified in all the Regions. There is a relation between the species of parasite and the clinical manifestations, contributing to the diagnostic complexity, which up to the present, did not exist species-specific diagnosis. In the present work, the proposal was to standardize the analysis of the PCR/RFLP products to the ITS and G6PD genes in polyacrylamide gel. In order to standardize PCR/RFLP analysis on the ITS and G6PD genes, DNA from the reference Leishmania spp. Sample was used and 5%, 6%, 7% 8% and 10% polyacrylamide were tested. The protocol design considered the size of the amplicons generated by the PCR / RFLP, correlating the concentration of the polyacrylamide. To standardize the electrophoresis time the migration of the dyes used in the DNA sample buffer was monitored. To validate the technique, we selected 521 samples from Tocantins patients fixed on slides and 43 samples of parasite isolates stored in Leishbank. The samples from the patients from Tocantins were analyzed by PCR/RFLP to the kDNA and were selected to those positive for the validation. The concentration of the matrix for analysis of the PCR and RFLP products to the ITS gene was standardized in 6% and 8%, respectively, and distinguished the species L. (L.) amazonensis, but the species L. (V.) braziliensis, L. (V.) guyanensis and L. (V.) lainsoni were not distinguished among themselves, occurring the same between L. (V.) naiffi and L. (V.) shawi. The concentration of the matrix for analysis of the PCR products to the G6PD gene was standardized in 6% and distinguished L. (V.) braziliensis from the others. The validation of the polyacrylamide gel analysis of the PCR products to the ITS target from samples of patients fixed in slides was not performed, since of the 256 samples analyzed only 4 amplified. The validation of the analysis of the products of the PCR to the G6PD target in polyacrylamide gel from 48 samples fixed in slides characterized 81% as L. (V.) braziliensis and, characterized 63% of the isolates of parasites as L. (V.) braziliensis. The PCR/RFLP analyzes performed to discriminate Leishmania spp. are complementary and thus contribute to the species-specific diagnosis of LTA.
A Leishmaniose Tegumentar Americana (LTA) é causada por protozoários do gênero Leishmania e acomete pele e/ou mucosas. É uma zoonose endêmica no Brasil sendo notificada em todas as Regiões. Há uma relação entre a espécie de parasito e as manifestações clínicas, contribuindo à complexidade diagnóstica, que até o presente, não existi o diagnóstico espécie-específico. No presente trabalho, a proposta foi padronizar a análise dos produtos da PCR/RFLP aos genes ITS e G6PD em gel de poliacrilamida. Para padronizar as análises dos produtos da PCR/RFLP aos genes ITS e G6PD, foram utilizadas DNA da amostra de Leishmania spp., de referência, sendo testada a poliacrilamida na concentração de 5%, 6%, 7% 8% e 10%. O delineamento do protocolo considerou o tamanho dos amplicons gerados pela PCR/RFLP, correlacionando-o a concentração da poliacrilamida. Para padronizar o tempo da eletroforese foi monitorada a migração dos corantes usados no tampão da amostra de DNA. Para validar a técnica, foram selecionadas 521 amostras de pacientes tocantinenses fixadas em lâminas e 43 amostras de isolados de parasitos armazenados no Leishbank. As amostras dos pacientes tocantinenses foram analisadas pela PCR/RFLP ao kDNA e foram selecionadas àquelas positivas para a validação. A concentração da matriz para analise dos produtos da PCR e RFLP ao gene ITS ficou padronizada respectivamente em 6% e 8% e distinguiu a espécie L.(L.) amazonensis, porém as espécies L. (V.) braziliensis, L. (V.) guyanensis e L. (V.) lainsoni não foram distinguidas entre si, ocorrendo o mesmo entre L. (V.) naiffi e L. (V.) shawi. A concentração da matriz para analise dos produtos da PCR ao gene G6PD ficou padronizada em 6% e distinguiu L. (V.) braziliensis das demais. A validação da análise em gel de poliacrilamida dos produtos da PCR ao alvo ITS a partir de amostras de pacientes fixadas em lâminas não ser realizada, pois das 256 amostras analisadas somente 4 amplificaram. A validação da análise dos produtos da PCR ao alvo G6PD em gel de poliacrilamida a partir de 48 amostras fixadas em lâminas caracterizou 81% como L. (V.) braziliensis e, caracterizou 63% dos isolados de parasitos como L. (V.) braziliensis. As análises pela PCR/RFLP realizadas para discriminar Leishmania spp. se complementam e, assim, contribuem para o diagnóstico espécie-específico da LTA.

Studies of biodegradation in lignins by basidiomycetes yeasts show the conversion of lignin in various degradation products among which vanillin, a valuable substance, suggested to be a strong inhibitor of both fermentation and growth of yeasts, stands. Sporidiobolales yeasts used in these experiments were aimed to be identified by their highly conserved ITS region as well as studied in vanillinsupplemented medium through, vanillin-supplemented plates, TLC and Neubauer’s chamber to find out which, among the several isolates tested, were the most resistant ones, understand how they take up vanillin and how their growth is affected by the presence of the phenolic compound. Two strains were identified as Rhodotorula babjevae. One of them, L4, together with LS22, Rhodosporidium kratochvilovae, could withstand and biodegrade high concentrations of vanillin, producing biodegradation products with Rf values similar to the ones know for vanillic acid and vanillyl alcohol. Better growth in medium supplemented with small doses of vanillin was found, as well as disparity among the same species and their metabolic features, therefore, herbicides resistance was suggested as a reason for strains divergence. Further morphological-species comparison could also describe if there exist a relation between them.
Estudios sobre la biodegradación de ligninas por levaduras basidiomicetes muestran la conversión de lignina en distintos productos de degradación, entre los cuales se encuentra la vainillina, un fuerte inhibidor de la fermentación y el crecimiento de levaduras. Las levaduras Sporidiobolales utilizadas en estos experimentos han intentado ser identificadas a través de la región ETI, muy conservada, además de estudiadas en medios suplementados con vainillina mediante placas suplementadas con vainillina, CCF y cámara de Neubauer para averiguar cuáles son las cepas más resistentes, entender cómo metabolizan la vainillina y cómo su crecimiento se ve afectado por la presencia de dicho compuesto. Dos cepas fueron identificadas como Rhodotorula babjevae. Una de ellas, L4, junto con con la cepa LS22, Rhodosporidium kratochvilovae, pudieron soportar y biodegradar elevadas concentraciones de vainillina, originando productos de biodegradación con valores de Rf similares a los del ácido vanílico y alcohol vanílico previamente conocidos. Se encontró un crecimiento mejor en medios suplementados con pequeñas dosis de vainillina además de una disparidad entre mismas especies y sus características metabólicas, así, herbicidas han sido sugeridos como una posible causa en dicha divergencia. Una futura comparación morfología-especie podrá describir si existe relación entre ambos.

The effect of a free-living stage in host-parasite coevolution: a skink mite phylogenetic study in New Zealand. During the last decade, phylogenetic trees have even been used to compare ecologically related taxa such as parasites and their hosts, and are used to determine their level of coevolution or reciprocal adaptation in time. Diverse coevolutionary events have been detected for this ecological association, where generally the parasite has been regarded as one that feeds exclusively on the host and is likely to cospeciate with it. A different coevolutionary pattern might occur when the parasite has a free-living stage in its life cycle, in which the parasite may have the opportunity to abandon its host and successfully colonise a new species (host-switching) making cospeciation less likely. Many New Zealand skinks are infested with a parasitic mite, Odontacarus sp. (Prostigmata: Leeuwenhoekiidae), which becomes free-living as an adult. The genetic variation of these mites found on four hosts was analyzed for host- parasite coevolutionary events. The hosts were the McCann’s skink and the common skink in coastal Birdling Flat, Canterbury, plus these species and the Grand and Otago skinks in Macraes Flat, Central Otago, South Island, New Zealand. The genetic variation of fast evolving nuclear Internal Transcribed Spacers 2 and mitochondrial Cytochrome c Oxidase I in Odontacarus mites found on these hosts was determined by PCR and DNA sequencing and phylogenetic trees were built using the computer programs PAUP*4 and MrBayes 3. The results show that mite haplotypes only had a significant geographical division and no host-related differences. In Birdling Flat, the COI haplotypes were represented in two groups that infested both regional hosts and had 5.7 % divergence. The same individual mites belonged to a single ITS 2 haplotype, thus indicating a historical geographical division between two populations that now interbreed successfully. The Macraes Flat mites were divided into two COI haplotypes with 2.4% divergence and internal nodes, which showed greater genetic variability than the Birdling Flat populations. The Macraes Flat mites formed two ITS 2 haplotypes with 6% divergence. This greater geographical structure of the Otago mites is probably due to the older age of the mainland area compared to the recently exposed coastal locality of Birdling Flat. The COI haplotypes from the two different regions had a mean distance of 15.5%, with an earlier divergence time than that known for the hosts. For both genes, the haplotypes from different regions had 100% bootstrap support and the parasite showed no host specificity. Mites of the different COI and ITS haplotypes were found on most of the host species that were sampled in Canterbury and Otago. The results of this study suggest that a free-living stage in a parasite’s life cycle can favour coevolutionary events such as inertia (failure to speciate) and host-switching, probably as a result of resource-tracking of the parasite. NB: Electronic files contained on CD to accompany print copy are not included with this version of the thesis.

碩士
中華醫事學院
生物科技研究所
93
There are three species of bacterial can infect the local Phalaenopsis, three of them are Pectobacterium chrysanthemi, Acidovorax avenae subsp. cattleyae and Burkholderia gladioli. In this study, we use the in-house bacterial internal transcribed spacer database to analysis the sequence between the bacterial that can infect the Phalaenopsi. We design primers AAC-ITS and BG-ITS based on the results of the analysis of Acidovorax avenae subsp. cattleyae and Burkholderia gladioli ITS sequence, and the primers pecS、pecM and idgA designed by indigoidine related gene, to detect the Pectobacterium chrysanthemi. The primers we use in this study reveals perfect results to their on response bacterial. In the sensitivity test, primer idgA can detect the Pectobacterium chrysanthemi with 1.2x104 cells, primer AAC-ITS can detect Acidovorax avenae subsp. cattleyae with1x102 cells and primer BG-ITS can detect Burkholderia gladioli with 1.2x104 cells. The primers designed in this study can obviously and accurately detect the bacterial that can infect the local Phalaenopsis.

Vischeria and Eustigmatos are closely related genera occurring in terrestrial habitats. These genera were distinguished by the differences in the features of the cell wall (projections and ridges of different form, smooth surface respectively). Up to date three species of the genus Eustigmatos and twelve species of the genus Vischeria have been described, but nine of the Vischeria species have been rarely, if ever, observed since the original description. This work is focused on evaluating molecular variability, diversity, and taxonomy of the Vischeria/Eustigmatos complex. Ninety seven strains, obtained from public algal collections or newly isolated from localities from all over the world, were studied, including the type strains of two Eustigmatos species and three Vischeria species. Phylogenetic analyses of the ITS2 rDNA and rbcL sequences showed that these genera are not genetically separated. The five types strains each represented a separate evolutionary lineage. Some of the additional lineages included strains morphologically corresponding to the species Eustigmatos magnus. Some of the newly isolated strains are according to the markers examined genetically indistinguishable from known strains from public algal collections. However, some of them are new lineages. Only one of the phylogenetic...

碩士
國立中興大學
生命科學院碩士在職專班
99
With the rapid development of molecular biology techniques, the taxonomy of fungi has been transformed from by morphological phenotype into a new era of molecular classification. In this study, we use of polymerase chain reaction (PCR) technology to amplify both the consensus sequence of the laccase gene and the ribosomal DNA internal transcribed spacer sequences (ITS) of 15 Ganoderma strains (including four BCRC standard strains and 11 of wild type isolates) and three exceptionally genus strains. The specific 595~915 bp of PCR product of TS1-ITS2 fragement and 1401~1651 bp of laccase gene fragments have been amplified, then cloning by TA- strategy for DNA sequencing. Each DNA sequence has been applied into NCBI database for sequence alignment by similarity analysis to compare the naming of molecular identification and traditional morphological classification. And, respectively, alignment by CLUSTAL program to establish the phylogenetic tree.

碩士
國立中興大學
植物病理學系
87
Colletotrichum gloeosporioides (Cg), the causal agent of anthracnose on various important crops, is the most notorious post-harvest pathogens greatly deteriorating the quality and shelf-life of agricultural products. The traditional way for identification of Colletotrichum spp. (C-spp) is quite often frustrating and time consuming mainly due to the wide host range of the pathogen, the great morphological variation; and what even worse was that cross infection might occur among different Colletotrichum spp. To cope with the increasing need of rapid detection and identification of this fungal pathogen from various agricultural products after the country become a member of the World Trade Organization (WTO), the potential application of rDNA ITS sequence characteristics as a biochemical tool was explored. The full length ITS rDNAs of a total of 29 C-spp isolates, and 7 non-C-sp isloates, were produced by polymerase chain reaction (PCR) amplification using the universal primer pair ITS1/ITS4 (White et al.1990). The Colletotrichum species tested included C. gloeosporioides (Cg), C. acutatum (Ca), C. musae (Cm), C. graminicola (Cgr), C. coccodes (Cco), C. capsici (Cc), C. dematium (Cd), C. lindemuthianum (Cl), and Colletotrichum sp. (C-sp) that species status awaited to be identified. The non- Colletotrichum species tested included Rhizopus oryzae, Penicillium digitatum, Pestalotia sp., Fusarium solani, Alternaria brassica, Ustilago esculenta, and Trichoderma reesici. The 600 bps amplicons were generated from all C-spp isolates tested as was expected; the amplicons from 7 non-C-spp isolates ranged from 500-800bps. Fifteen of the C-spp amplicons which include 7 from Cg; 2 each from Cc, Cgr; and one each from Cm, Cd, Cl and C-sp, were cloned and sequenced. The full length of these amplicons ranged from 535 to 555 bps; in which ITS1 region was from 160 to 181bps, ITS2 region was from 152 to 153bps; whereas the 5.8S rDNAs all appeared to be 160bps. Comparison of the inter-specific sequence identity indicated the existence of much greater divergence among ITS1 (identity reached only 53.9%) as compared to that among ITS2 region (identity was approximately 81.8%). Comparative analysis of the ITS full length and the ITS1 sequence data by the distance matrix method and the parsimony method both concluded these 15 tested isolates into 5 distinctive species groups namely, CG (C. gloeosporioides), CA (C. acutatum), CM (C. musae), CL (C. lindemuthianum), and CC (C. capsici). The sequence data also revealed that the ITS1 sequence of tested CG, CA and CC isolates were 99-100% identical to each compared isolate available in the GenBank/EMBL data bank; whereas for ITS2 region, only those from CA and CG isolates were greater than 98% in identity. For the rapid detection and identification of Colletotrichum spp. by PCR, the efficacy of the species specific primer pairs developed by Mills et al. (1992) were examined; the results appeared to be non-satisfactory. To resolve the problem, the heteroduplex mobility assay (HMA) was attempted using the ITS-rDNA amplicons of all 29 tested C-spp isolates as the targets. The analysis by the above described distance matrix method using the distance value estimated from the mobility changes concluded the 29 isolates into 5 distinctive species groups same as that by sequence data analysis. Moreover, from the repeatable band distribution characteristics, a total of 6 heteroduplex patterns (HP) were established. By the established HP fingerprint, the species group CG can be further divided into CG1 and CG2; while the other 4 species groups remain unchanged. In the established species group system, Cgr1, Cgr2 and Cd1 isolates, originally identified as C. graminicola and C. dematium respectively, were reclassified as member of CC since their ITS sequences were 98-100% identical to that of C. capsici. Likewise, the species status of Cg8 isolate was changed from C. musae to a member of CG; Cg2, Ca1, Cg9 and C-sp1 isolates were changed from C. gloeosporioides to be members of CA; and Cc1 isolate was changed from C. capsici to a member of CG2. Among these isolates that original identification were disputable, the species status of Cc1 was reexamined by the original author lately and proved the previous assignment as C. capsici might be a misinterpretation. The results discussed in this investigation, although only limited numbers of fungal isolates had been explored, appeared to provide evidence that sequence analysis of ITS rDNAs a dependable tool in the species level identification of Colletotrichum spp. Also the combination of the genus- or species-specific PCR together with HMA and HP analysis is easy to process, not time consuming, and provides repeatable and distinct data bases useful for species identification. The use of the established techniques for practical application in rapid detection and pathogen identification of anthracnose, and even other fungal diseases, are thus recommended.

碩士
國立屏東科技大學
獸醫學系
91
The final goal of this study is for the fast diagnosis of different types of pathogenic Escherichria coli including EHEC ( Enterohemorragic E.coli )、ETEC ( Enterotoxigenic E.coli )、EIEC ( Enteroinvasive E.coli )、 EPEC ( Enteropathogenic E.coli ) and EAEC ( Enteroaggregative E.coli ). Moreover , the target genes employed for the differentiation of these kind of pathogenic E. coli are VTⅠ, VTⅡ, ST, LT, ial , pCVD432, eaeAgen , H7 and uidA. However, the resulting data could provide with that the pathogenic E. coli can be diagnosed by using three tubes-one step multiplex PCR ( polymerase chain reaction ). The diagnostic process just took as short as two and half hours in terms of fast- diagnosis. The ITS ( Internal transcribed spacer ) region of DNA fragment was cloned and sequenced from EHEC, ETEC, EPEC, EIEC and EAEC respectively. After a phylogenic analysis by using DNA star softwire, three groups of distinct characteristics of E. coli were demonstrated as groupⅠ: ETEC and EHEC were characterized by secreting their specific enterotoxins or cytotoxins in clinics; group Ⅱ: EAEC alone was not found in humans other than animals; group Ⅲ: EIEC and EPEC: the pathogenesis of this group of E. coli was not proposed by the mechanism of toxin-producing. The resulting data showed that 10% divergence in DNA sequences between groupⅠand groupⅡ, 41.5% divergence between groupⅠand groupⅢand the same result as shown between groupⅡand groupⅢ. However, The sequences of ITS could be used for differentiation of different types of pathogenic E. coli based on phylogenic analysis and also coincided with the characteristics of the different pathogenic E. coli which are grouped described above.

碩士
國立臺灣大學
獸醫學研究所
90
Chicken coccidia is a protozoan parasite of the genus Eimeria. It is economically important that causes loses in the poultry industry globally. The various species can be distinguished on the basis of morphology of the oocyst and the location of parasite within intestinal cells, but these criteria could be unreliable. Arbitrarily-primed PCR has been utilized to fingerprint avian Eimeria species, but this method does not always give reproducible results due to low specificity. For this reason, PCR assays have been employed for the detection of Eimeria species in this study. Five sets of primer derived from the internal transcribed spacer 1 (ITS-1) regions were used to distinguish the five Eimeria species including Eimeria tenella, E. necatrix, E. brunetti, E. acervulina and E. maxima. We use vaccine as template for the five species. The similarities of five species sequences between the vaccine and Genbank are 94%~100%. Analysis for E. tenella ITS-1 partial sequence of Taiwan and Genbank, the similarity is 99.6%. PCR sensitivity test of E. tenella in Taiwan is 50 oocysts. Five sets of primer won''''t amplify any non-specific band of chicken genome or intestinal contents.

Garry oak (Quercus garryana Douglas ex. Hook) is a white oak (Quercus sect. Quercus) with a geographic range extending from southwestern BC to south-central California. It is the only native white oak in BC and Washington, and is the northernmost species of the California Floristic Province-Pacific Northwest white oak clade. I used molecular methods to address the following questions: 1) What are the patterns of genetic variation within Garry oak? 2) How do these patterns vary geographically, and how did the spatial distribution of the gene lineages come to occupy its current geographical range? 3) Does Garry oak show evidence of genetic interaction with other white oak species in western North America? 4) Is there morphological or genetic evidence to support the three described varieties of Garry oak? I obtained samples of Garry oak from 117 localities over its geographic range, as well as samples of two other California white oaks (Q. lobata and Q. douglasii) and a Rocky Mountain species (Q. gambelii). Analyses of DNA sequence data from four plastid DNA regions revealed 24 distinct molecular variants (haplotypes) in Garry oak. These show a strong south-to-north decrease in genetic diversity, consistent with post-glacial northward expansion. Haplotypes present in the northern part of the range provide evidence of two separate northward migrations, only one of which reached the northern range limit of Garry oak in BC. I found that Garry oak shared plastid DNA haplotypes with two other white oak species, indicating that it hybridizes with other oaks in the southern part of its range. The nuclear ribosomal ITS phylogeny showed poor resolution, but both cpDNA and nrDNA may indicate that Q. garryana is more closely related to the white oaks of central North America than was previously thought. My findings also suggest that the three currently recognized varieties of Garry oak (var. garryana, breweri and semota) are not well differentiated genetically, but show morphological variation at the regional level. This study shows the phylogeographic patterns within Q. garryana. In addition, it contributes to conservation efforts in Garry oak ecosystems by indicating regions of high genetic diversity in Garry oak, including genetically unique populations that may be especially worthy of preservation.
Graduate

碩士
國立中興大學
食品科學系
91
ABSTRACT Staphylococcal enterotoxins (SEs), first identified in 1959, are a group of extracellular proteins produced by some strains of Staphylococcus aureus. While Vibrio parahaemoliticus (860 cases) has been the leading cause of foodborne diseases, Staphylococcus aureus and related enterotoxins are the second most frequently encountered cause of foodborne diseases (247 cases) in Taiwan. However, the aetiology of a high percentage of the of staphylococcal foodborne poisoning (SFP) cases remains unclear. SEs are classified as members of the pyrogenic toxin (PT) superantigen family because of their biological activities and structural relatedness. They are unlike the other members of the PT family, the SEs have the unique ability to induce staphylococcal food poisoning, a common form of gastroenteritis. Twelve serologically distinct enterotoxins, i.e. enterotoxin A (SEA), SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL and SEM, have been identified. The SEC serotype is heterogenous and contains several antigenic and sequence molecular variants, designated SEC1, SEC2, SEC3, SECbovine, and SECovine. The classification is based on the minor antigenic differences and the animal host with which they are infected. Amino acid sequences of the SEC1, SEC2 and SEC3 and the nucleotide sequence of these SEC genes have been identified. However, additional SEC variants, other than SEC1, SEC2 and SEC3, do exist. Commercially available immunoassay kits, such as the staphylococcal enterotoxins reverse phase latex agglutination (SET-RPLA) kit, and the staphylococcal enterotoxin ELISA (SET-EIA) kit, only allow the detection of classical SEs (i.e. SEA, SEB, SEC, SED and SEE) but not the specific individual subtypes of SEC. DNA probes and the polymerase chain reaction (PCR) primers are the two methods available for DNA detection. These probes and primers are specific for the detection of various staphylococcal enterotoxin genes. For SEC subtypes, there is only one report regarding the method for detection and differentiation of SEC subtypes. Due to the high homology of the amino acid sequence in SEC1, SEC2 and SEC3, the method for differentiation of SEC subtypes is not yet commercially available. Based on the nucleotide sequences for these enterotoxins, we have designed SEC subtype specific primers for the detection of these SEC genes individually. We have used those SEC subtype specific primers to investigate the distribution of SEC1, SEC2 and SEC3 S. aureus strains in staphylococcal food poisoning outbreaks occurred in the central Taiwan in 1995-2000. Despite the high homology (97%) in their gene sequences, we designed a new set of PCR primers with which we were able to differentiate each specific SEC subtypes. We have used these primers for the differentiation of 39 strains of SEC S. aureus isolated from patients suffered with food poisoning between 1995-2000 in the central Taiwan. In the recent years, the existence of new types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, and SEM) has also been reported. However, the relationship between these new SEs and food poisoning is not fully understood at the present time. It is estimated that about 95% of staphylococcal food poisoning outbreaks are caused by SEA to SEE. The remaining 5% of outbreaks may therefore be associated with the other newly identified SEs. To clarify the role played by these newly identified SEs in food poisoning, the development of reliable methods of detection of SE proteins is essential. However, characterization of these SEs has been hindered because of the low levels of production, difficulties associated with purification, the lack of simple and practical methods for their detection. The only reliable assay for the unidentified SEs is the monkey feeding test. However, this test cannot differentiate serologically different SEs. Among the techniques used to identify toxin genotypes, PCR have been reported to be very successful and reliable. Our laboratory previously designed specific primers for the successful and reliable detection of SEs, ETA, ETB, and TSST-1. Purification of an unidentified SE will allow the development of a specific and sensitive PCR assay for rapid and reliable detection. Therefore, we developed a PCR procedure that will rapidly assess whether staphylococcal isolates harbor seg, seh, and sei, encoding the SEs. A total of 116 clinical isolates of S. aureus obtained from patients suffered with food poisoning and 55 clinical isolates other of S. aureus negative to the classical enterotoxins (SEA® SEE) detection were selected for testing. All isolates were identified with the BAM method and SET-RPLA system. PCR assays were performed for the classical enterotoxins (SEA® SEE) gene sequences. In this report, novel PCR primers specific for the detection of SEG, SEH and SEI genes, ie, seg, seh and sei, were designed and used for the assay of 55 human isolates of S. aureus negative to the classical enterotoxins (SEA® SEE) detection. Human salmonellosis occurs in a variety of forms including gastroenteritis, organ focal infection, or systemic febrile infection. Salmonellae are widely distributed in the environment and are harboring in both wild and domestic animals. A number of salmonella serotypes may be transferred to poultry from various sources, such as feedstuffs, breeding flocks, rodents, and wild birds. The conventional methods for detection and identification of Salmonella spp. include the preculture process, selective enrichment and biotypes identification and the serotyping process for O, Vi and H antigens. According to the O, Vi and H sero grouping, strains of Salmonella spp. can then be identified. Since these procedures are laborious, the development of rapid identification methods is important. The purpose of this study is to develop a PCR system that can be used for the detection of Salmonella spp. and S. Enteritidis. Detection sensitivity and reliability for these systems and the conditions for application of such systems on food and clinical samples will also be investigated. S. Enteritidis is one of the most common Salmonella species that may cause food-borne disease and salmonellosis. The Centers for Disease Control and Prevention (USA) reported a fivefold increase in S. Enteritidis isolation rates in the northeastern and central Atlantic states between 1976 and 1985. In this study, we would design the PCR system specific for S. Enteritidis detection. From the insertion sequence (Accession no. Z83734), PCR primers specific for S. Enteritidis detection is designed IS1/IS2. Only S. Berta and S. Gallinarum may generate false results. The results indicated that the primers could be used as a molecular tool for future field survey of S. Enteritidis both in foods and in clinical samples. The feasibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was also investigated by direct sequencing of polymerase chain reaction-amplified DNA from all ITS regions in a collection of 40 strains of 25 different serotypes of S. enterica, and by sequencing individual ITS region from a single strain. The sequence contained internal transcribed spacer fragments of 485—490 bases in length. Based on the sequences in the amplified ITS regions, we designed a pair of PCR primers (ITSF and ITSR) which could be used for the specific detection of Salmonella spp. with all serovars. The uniqueness of these primer sequences was confirmed. Molecular size of the PCR product is 312 bp and all the non-Salmonella strains tested generated negative PCR results. The sensitivity for boiled (97℃, 30 min) S. Typhimurium whole bacteria cell was N ×100 (N = 1~9) cells per assay. When this PCR primer pair was used for the detection of Salmonella cells contaminated in cow milk and raw chicken meat, the detection limit was 103 cfu per g or ml of the food samples. For food samples spiked with N ×100 (N = 1~9) CFU per g or ml S. Enteritidis (ATCC 13076) cells, homogenised, inoculated into 1﹪buffered peptone water and grown at 37℃ for 8 hours, the detection of S. Enteritidis using the PCR was successful.

碩士
國立中興大學
植物病理學系
89
As a major fungal pathogen causing vascular wilt disease, Fusarium oxysporum (F. o.) has fairly wide spectrum of host range. The Fusarium wilt of tomato, caused by F. o. f. sp. lycopersici (FoLy) is one of the limiting factors of tomato cultivation in Taiwan. In tradition, the identification of Fusarium spp. is mainly based on the morphological characteristics, the pathogenicity, and the vegetative compatibility. However, as the spore morphology of the fungus varies greatly and the sporulation were greatly affected by various environmental factors, the identification of Fusarium wilt pathogen remains quite often frustrating and discouraging especially for the beginners. In order to develop methodology and protocol practically useful for fast identification of members of F. o., the polymerase chain reaction (PCR) approach with rDNAs as explored target was initiated. A total of 62 Fusarium isolates which including 32 of FoLys, 20 of other formae speciales of F. o., and 10 of other different species of Fusarium, were used for this investigation. By PCR amplification using the universal primer pair ITS1 and ITS4, an amplicon around 550 bps in size was consistently obtained from the genomic DNA preparations of these 62 Fusarium isolates. Among them, 41 which including 13 FoLys of representative collected locations were cloned and sequenced. The 41 cloned fragments have molecular weight ranging from 504 to 530 bps. Analysis of the data obtained indicated the existence of extremely high sequence identity among different formae species of F. o. tested. That of F. o. f. sp. pisi (CCRC35290) was an exception, however. Its sequence data showed only 93.27 to 93.66% identities with other formae speciales of F. o. tested, whereas a 99.8% and 100% identity was found as comparing to that of F. roseum and F. verticillioides respectively. The exceptional low sequence identity comparising to that of other formae speciales of F. o. indicated the need of reexamination on the taxonomical status of their purchase isolate. A followed phylogenetic study by Maximum-likelihood (ML) and Neighbor-joining (NJ) approaches, respectively, indicate that all these 13 FoLy isolates tested may be classified in the same group, although the bootstrap value was only 66%. A further comparison of the full-length sequence data of these 41 isolates with 193 Fusarium spp. ITS sequences available on GenBank/EMBL was proceeded by the ML method and the NJ method respectively. The phylogram constructed by ML method concluded the covered isolates into 7 different groups, whereas that constructed by NJ method classified them into only 4 groups. In the former classification, all the FoLys tested were classified in group-IV, and F. o. f. sp. pisi, F. roseum and F. verticillioides were in group-III. Also noted in the phylogram data was that all F. o. formae speciales isolates tested were concluded in the same group (iii) by the NJ method. For the fast identification of the studied Fusarium spp., both heteroduplex mobility assay (HMA) and random amplified polymorphic DNA analysis were applied. The results of HMA in which ITS rDNA amplicon of FoLy-14 isolate was used as a standard to react with that of all other tested Fusarium isolates concluded 5 characteristic heteroduplex patterns (HP). In followed RAPD analysis using total DNA as a template, the electrophoretogram obtained from an attempt using S111 as random primer demonstrated a nearly identical banding pattern of FoLys, indicating the existence of evolutionary close-relatedness among isolates of different geographical origins. Moreover, a 900 bps fragment specific to FoLy isolates was identified from PCR using S115 as a random primer. The specific fragment was cloned, sequenced; and a FoLy-specific primer pair FoLyF/FoLyR was adapted. The FoLy-specific DNA probe (pFoLy) was prepared by nick-translation using the 150 bps PCR products obtained from FoLyF/FoLyR specific amplification. The specificity of pFoLy was demonstrated by southern blot analysis and as well by PCR, using genomic DNAs obtained from all tested Fusarium isolates as templates. The detection limit by PCR using FoLyF/FoLyR primer pair reached 1 ng (genomic DNA); a follow-up southern blotting by Dig-labelled pFoLy application brought the detection limit further down to 100 pg level. It seems evident that for the fast identification and detection of FoLy, the specific primer pair FoLyF/FoLyR with the aid of specific probe DNA pFoLy would serve as valuable molecular tools. As for HP analysis, the methodology appeared to be useful for species level rather than for the formae speciales level differentiation.

Dead wood plays an important role in forest ecosystems in the context of C dynamics, nutrient cycling, forest regeneration and biodiversity. Decaying wood sustains biodiversity by providing habitats and energy for fungi, bacteria, invertebrates, and many other organisms. Dead wood is resistant to decomposition and its decay is driven mainly by filamentous fungi. Community structure of wood- inhabiting fungi changes during decomposition, but the relationship between substrate quality and decomposer community is still poorly understood. This work studied fungal community composition with respect to tree species, stage of decay, volume and physico-chemical properties (such as pH, carbon and nitrogen content) of dead wood. Fungi were identified using next generation sequencing approaches - 454-pyrosequencing and Illumina MiSeq sequencing. Tree species, volume of dead wood (branches x logs) and stage of decay were the main variables affecting fungal community composition. Higher enzyme activities and content of fungal biomass indicate faster colonization of small branches than tree trunks by fungi. Fungal community composition, wood chemical properties and enzyme activities changed during decomposition. Both content of nitrogen and fungal biomass increased during decomposition. Enzyme activites peaked...

Understanding of carbon cycling in coniferous forests that represent a large carbon sink is crucial for our understanding of natural processes under global climate change. Recognition of fungi as fundamental decomposers can contribute to this understanding. Fungi are able to decompose numbers of substrates and possess a variety of enzymes to do so In this study I present litter decomposing fungi in mountain spruce forest from national park Šumava. The aim of my thesis was to follow succession and community changes of fungi from the early stages of decomposition of Picea abies needles until degradation of organic matter in the organic horizon of the soil. This aim was accomplished partly by recording the extracellular enzyme production of fungi in different stages of decomposition from needles attached to the twigs of a fallen tree to a litter material in later stages of decomposition on the soil surface. In addition to testing of fungi on their natural substrata - needle litter, enzyme activities were also measured in laboratory agar cultures, which allow comparison of diverse fungi with different origins. Enzyme activities were aimed at enzymes decomposing cellulose and compounds found in litter. Although ecology of endophytic and saprothrophic fungi suggest differences in enzyme production, these...