The objective of this thesis was to assess the diversity and community structure of fungi in a forest plot in Ontario using a variety of field sampling and analytical methods. Three broad questions were addressed: 1) How do different measures affect the resulting view of fungal diversity? 2) Do fruiting bodies and soil rDNA sampling detect the same phylogenetic and ecological groups of Agaricomycotina? 3) Will additional rDNA sampling resolve the phylogenetic position of unclassified fungal sequences recovered from environmental sampling? Generally, richness, abundance, and phylogenetic diversity (PD) correspond and identify the same dominant fungal groups in the study site, although in different proportions. Clades with longer branch lengths tend to comprise a larger proportion of diversity when assessed using PD. Three phylogenetic-based comparisons were found to be variable in their ability to detect significant differences. Generally, the Unifrac significance measure (Lozupone et al., 2006) is the most conservative, followed by the P-test (Martin, 2002), and Libshuff library comparison (Singleton et al., 2001) with our dataset. Fruiting body collections and rDNA sampling recover largely different assemblages of fungi at the species level; however, both methods identify the same taxonomic groups at the genus-order level as well as ectomycorrhizal fungi as the dominant functional type of Agaricomycotina. This work also shows that the Soil Clone Group I (SCGI) clade is widespread in soils of diverse origins and represents a novel subphylum of Ascomycota.

No description available.

No description available.

abstract: The distinguishing feature of the filamentous fungi is the hyphae - tube-like microscopic cells that exhibit polarized growth via apical extension and allow the fungus to interact with its environment. Fungi elongate at the hyphal apex, through the localized construction of new plasma membrane and cell wall through the exocytosis of secretory vesicles. One population of these vesicles have been identified as chitosomes, containing chitin synthase isoenzymes, which are responsible for the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin, the primary fibrillar component of the fungal cell wall. The chitosomes, in addition to other vesicles, can be observed aggregating in the hyphal tip in most filamentous fungi. In the Ascomycota and Basidiomycota, this collection of vesicles exhibits discrete organization and has been termed a Spitzenkörper. Although accumulations of vesicles can be observed in the hyphal tip of many growing filamentous fungi, some debate continues as to what precisely defines a Spitzenkörper. This study reports the details of three separate projects: first, to document the effects of deleting a single chitin synthase, CHS-1 and CHS-6 in Neurospora crassa with regards to hyphal ultrastructure, cytoplasmic organization, and growth in comparison to the wild-type. Given the importance of chitin synthesis in fungal cell growth, deletion of a critical chitin synthase presumably impacts cell wall structure, fungal growth and cytoplasmic organization. Second, an examination of the ultrastructure of four zygomycetous fungi - Coemansia reversa, Mortierella verticillata, Mucor indicus, and Gilbertella persicaria has been conducted. Utilization of cryofixation and freeze-substitution techniques for electron microscopy has produced improved preservation of cytoplasmic ultrastructure, particularly at the hyphal apex, allowing detailed analysis of vesicle size, contents, and organization. Lastly, hyphal tip organization was reviewed in a broad range of fungi. Previous studies had either focused on a few select fungi or representative groups. Vesicle organization, composition and size do appear to vary among the classes of fungi, but some trends, like the vesicle crescent in the zygomycetous fungi have been documented. / Dissertation/Thesis / Doctoral Dissertation Biology 2015

<p dir="ltr">Spread primarily through sex, members of the microfungal class Laboulbeniomycetes are parasitic on arthropods, where they form tiny, three-dimensional thalli on the host exoskeleton. While the study of Laboulbeniomycetes has had a renaissance in the age of molecular methods, so too has the Red Queen Hypothesis, a coevolutionary theory aimed at characterizing how biotic interactions shape the evolutionary trajectory of “players” over time. While the central dogma of “laboulbeniology” is that labouls do not cause a fitness disadvantage to the host in the wild, here we report the first genome annotation data of a Laboulbeniomycete genome, indicating the possibility of past antagonistic relationships with host species. Some of the genes predicted are homologous with that of highly pathogenic entomopathogenic species, such as those within <i>Cordyceps</i>. To understand why these seemingly benign parasites possess genes associated with pathogenicity, we reviewed recent advances in molecular methods for detecting Red Queen associations, and propose a new model we call “Sleeping Red Queens”. Using Laboulbeniomycete evolutionary history, we predict that antagonists who have adapted to each other over time to a benign state can “re-awaken” or transition to a more antagonistic mode due to environmental changes. The “Sleeping Red Queen” model of Laboulbeniomycetes is supported by research on laboul ecology under artificial conditions. The purpose of this work is to review the history of Laboulbeniomycetes, examine recent advances in molecular methods through the Red Queen lens, report on our new molecular data, and discuss how our new findings can be used to apply Red Queen theory to Laboulbeniomycetes, while also hypothesizing what these future analyses could reveal. With promising advances in next-generation sequencing, we believe our Sleeping Red Queen model will be advantageous for interpretation of future genomic and bioinformatic data of Laboulbeniomycetes.</p>

<p>Forensic science has gained popularity in the last few decades. Many new techniques are being studied and implemented. It includes a wide range of scientific disciplines, such as entomology, physics and biology. An important improvement to forensic science is the development of different DNA techniques which are implemented during an investigation, making DNA a gold standard for forensics. Most of the time DNA is mentioned it is in terms of human DNA, but there are microscopic organisms with useful DNA. In the last decade, with the development of next-generation sequencing (NGS), studies focusing on bacterial communities have been published, but fungal communities have not been extensively studied.</p> For this project, the potential of fungi in forensic science was investigated through three different studies. Human flora was looked at by NGS from thirty-seven human bodies with differing post-mortem intervals (PMIs). The communities were analyzed statistically and quantitatively, resulting in unique operational taxonomic units (OTUs) and genera which were only present in certain PMIs, and in some which were present through the entire PMI time scale. These attributes can help, not only to give a better view on human mycoflora during decomposition, but they can also help in determining fungal signatures during decomposition. These signatures can help in a PMI determination. Moreover, swine carcasses – the model animals for human forensic studies – were investigated as well to create a checklist of fungal flora after five months of winter decomposition in the West Lafayette, Indiana area. Furthermore, due to the increased importance of wildlife forensics, a wildlife study was also conducted using four wildlife species (mute swan, red tailed hawk, river otter, bobcat). The fungal flora from these species were compared within species at the beginning of the study and at skeletonization stage to look at any indicator fungal species and to create a general checklist for wildlife studies in the West Lafayette, Indiana area for future studies. Additionally, the fungal communities were compared across species as well.

Nas últimas décadas, houve aumento progressivo das infecções hospitalares por leveduras principalmente, do gênero Candida. A fonte de infecção pode ser endógena ou exógena, desde que esporos unicelulares de leveduras, permanecem viáveis por meses sobre superfícies bióticas ou abióticas. Diversas espécies de leveduras são encontradas em pele e mucosas de indivíduos sadios. Em estado saprofítico ainda, as leveduras encontram-se no trato gastrintestinal humano mas, a relação entre a presença desses microorganismos e sua patogenicidade está associada a diversos fatores predisponentes, tais como: número e variedade de sítios topográficos colonizados, uso prolongado de antibióticos, infecções associadas causadas por outros microorganismos e particularmente, distúrbios imunológicos ou metabólicos. Leveduras do trato gastrintestinal podem ser transmitidas, via fecal-oral diretamente ou de modo indireto, de indivíduo para indivíduo. A transmissão de uma cepa em estado saprofítico para um hospedeiro suscetível, pode resultar em colonização seguida de infecção. A gravidade do quadro clínico depende de condições do hospedeiro e características do agente etiológico que engloba fatores de virulência e resistência a antifúngicos. Esses atributos são importantes em Candida albicans na qual, enzimas com atividade de fosfolipase e proteinase são marcadores de virulência. De outro modo, fenótipos de resistência, ocorrem, com maior freqüência, em espécies não-Candida albicans. Dada a possibilidade de instalação de doença endógena e a dispersão de cepas virulentas e resistentes, a partir de colonização gastrintestinal, estudos que contribuam para a determinação desses agentes constituintes da microbiota de pacientes internados, são importantes para o conhecimento da história natural das infecções nosocomiais por leveduras. Os objetivos deste trabalho foram avaliar o trato intestinal como fonte potencial de infecção hospitalar por leveduras, descrevendo as espécies prevalentes nas primeiras horas de internação e possíveis alterações temporais, quanto a fenótipo de virulência e resistência a antifúngicos. Foram analisadas 281 amostras de leveduras isoladas de 66 crianças internadas em unidades de pediatria e semi-intensiva de hospitais públicos das cidades de São Paulo e Guarulhos, Brasil. As amostras foram isoladas de fezes coletadas nas primeiras horas de internação e durante o período de internação. A identificação das leveduras quanto a gênero e espécie foram realizadas por métodos tradicionais, analisando aspectos morfológicos e fisiológicos. A capacidade de produção de enzimas, fosfolipase e proteinase, foram verificadas conforme proposto por Price et al. 1982 e Ruchël et al., 1982. A sensibilidade aos antifúngicos: anfotericina B (AMB), fluconazol (FZ), itraconazol (IZ), cetoconazol (CZ) e nistatina (NIS) foram analisada pela técnica de difusão por discos (CECON São Paulo, Brasil). Amostras resistentes ou com sensibilidade intermediária, foram re-avaliadas pelo método de microdiluição segundo NCCL (1997) modificado por EUCAST (2002). As espécies isoladas foram: Candida tropicalis (32,7%), C.albicans (29,9%), C.parapsilosis (27, 1%), Trichosporon cutaneum e T.inkin (3,2%), Rhodotorula mucilaginosa e R.glutinis (0,7%), C.krusei (3,6%), C.guilliermondii (2,1%), C.glabrata (0,4%) e C.kefyr (0,4%). A atividade enzimática foi observada na maioria das 84 amostras de C. albicans, sendo 96% de fosfolipase e 95% de proteinase. Entre as espécies não-albicans do gênero Candida foi verificada atividade em 97% de fosfolipase e 67% de proteinase. Amostras menos sensíveis às drogas azólicas, ou seja, amostras resistentes ou com sensibilidade dependente da dose, foram encontradas em 4,3% das 281 amostras de leveduras, sendo maior porcentagem observada em C.krusei (90%). Conclui-se que existem leveduras de diversas espécies em fezes de população pediátrica hospitalizada, com fenótipos de virulência e resistência a antifúngicos. A manutenção desses fenótipos durante o período de internação pode representar fator de risco para infecção hospitalar endógena, ou ainda, fonte de dispersão de patógenos em potencial, no meio ambiente hospitalar. / At the last decades the nosocomial infections caused by yeasts raised significantly especially by Candida yeasts. The infections source can be endogen or exogenous, since spores of unicellular and multicellular are kept viable for months and several yeasts species are found in skin and mucosa of healthy people. In a saprophytic state yeasts are found in the human gastrointestinal tract but the relationship between the presence of these microorganisms and their pathology is associated with several facts such as: number, variety of sites colonized, effective use of antibiotics, associated infections caused by another microorganisms and mainly disturbance in due to lack of immunity and metabolic. Yeasts in the gastrointestinal tract can be transmitted fecal-oral direct or indirectly from an individual to another. The transmission of a strain in a saprophytic state to a host can result in colony followed by infection. The infection can be serious depending on the host conditions and the etiologic agent that includes virulent factor and resistance to antifungal drugs. These attributes are important to Candida albicans in which enzymes with phospholipase activity are responsible for virulent factors. Resistance phenotypes, otherwise it should occur more frequently in non-albicans species. Concerning the possibility of an endogen disease and the spread of virulent and resistant strains, from the gastrointestinal colony, studies that contribute to determine these agents that constitute the microbiota of patients, are important to know the natural story of nosocomial infections caused by yeasts. This work aims at evaluating the intestinal tract as a source of hospital infections by yeasts describing the remaining species in the first hours and a possible change depending on the time that may happen to virulent phenotypic and resistance to ant fungi. Two hundred eighty one yeast samples from sixty-six children attended in pediatric and semi-intensive units in 2 public hospitals located in São Paulo and Guarulhos cities in Brazil were analyzed. The fecal samples were collected at the first hours after and during their arrival at the hospital. To identify the yeasts according to their gender and species traditional methods were used, analyzing morphological and physiological aspects. The ability to produce enzymes phospholipase and proteinase was verified the same way it was proposed by Price et al. 1982 and Ruchel et al. 1982. The sensibility to antifungals: amphotericin B (AMB), f1uconazole (FZ), ketoconazole (CZ) e nistatin (NIS), was analyzed by the diffusion technical by disks (CECON São Paulo, Brazil). Resistant samples or with intermediate sensibility were confirmed by micro-dilution method according to NCCLS (1997) modified by EUCAST (2002). The isolated species were: Candida tropicalis (30%), C.parapsilosis (27%), C.krusei (4%), Trichosporon cutaneum e T.inkin (3%), Rhodotorula mucilaginosa e R.glutinis (2%), C.guilliermondii (2%), C.glabrata (1%) and C.kefyr (1%). Enzymatic activity was verified in most of the 84 C.albicans samples being 96% of phosfolipase and 95% of proteinase production. Among the non-albicans species of Candida it was observed 97% of phospholipase and 67% of proteinase activity. Less sensitive samples to azoic drugs including resistant or SDD sensibility, which depends on the achieved dose, were found in 4.3% of the 281 samples of yeast. The hugest percentage was observed in C.krusei (90%). We can conclude that different yeast species occur in stools of pediatric population hospitalized, including virulent strains and antifungal resistant phenotypes. The persistent of these phenotypes in the intestinal tract during hospitalization period may represents a risk facto r contributing to endogen infection, or play a role in dissemination of potential pathogens inside a nosocomial environment.

INTRODUÇÃO: Onicomicose é a infecção fúngica das unhas e é considerada a onicopatia mais frequente em adultos. Representa até 50% das lesões ungueais, sendo necessária confirmação diagnóstica com exame complementar que demostre a presença do fungo na unha, sendo o exame micológico direto e a cultura para fungos os mais utilizados. A dermatoscopia é um exame não invasivo, rápido e de baixo custo cujos padrões para onicomicose relatados na literatura em estudos retrospectivos chegam a 100% de sensibilidade e especificidade. Deste modo realizamos um estudo prospectivo para comparar a dermatoscopia frente ao exame micológico. MÉTODOS: Estudo prospectivo, transversal avaliando 109 pacientes e 202 unhas com suspeita diagnóstica de onicomicose. Os padrões dermatoscópicos encontrados foram descritos através de fotografias digitais. A sensibilidade e especificidade dos padrões de onicomicose distal-lateral foram determinados de acordo com o resultado do exame micológico. RESULTADOS: Foram significativos (p < 0,05) para o diagnóstico de onicomicose distal lateral e tiveram sensibilidade/especificidade calculadas os padrões: borda recortada (80,2% / 65,3%), borda linear (12,6% / 42,9%), estrias irregulares (81,1% / 65,3%), estrias finas/regulares (9,9% / 59,2%); e as cores branco (93,7% / 18,4%), amarelo (63,1% / 71,4%) e laranja (10,8% / 100%). Após análise multivariada stepwise forward os padrões de estrias irregulares, borda linear e cor amarela mantiveram significância estatística (p < 0,05). DISCUSSÃO: Os achados deste trabalho prospectivo estão de acordo com a literatura mostrando que há correlação entre o exame micológico e a dermatoscopia. Todavia, não ratifica a sensibilidade e especificidade de 100% encontrada em estudo retrospectivo para os padrões de borda recortada e borda linear. Também não demonstrado na literatura, as cores amarela, branca e laranja foram também estatisticamente significativas para o diagnóstico. CONCLUSÃO: A dermatoscopia correlaciona bem com a história natural da infecção fúngica e com o exame micológico, sendo um exame promissor para o diagnóstico de onicomicose. Sugerimos que futuros estudos comparem a dermatoscopia com exames considerados padrão-ouro (ex: microscopia de fluorescência e PCR) para detectar exames falso negativos / BACKGROUND: Onychomycosis is defined as a fungal infection of the nail and is considered the most common onychopathy in adults. It represents up to 50% of nail diseases and demonstration of the fungal pathogen is necessary for diagnostic confirmation. Direct mycological examination and fungal culture are commonly used for this purpose. Dermoscopy is a noninvasive, fast and inexpensive exam, reaching 100% diagnostic sensitivity and specificity for onychomycosis in retrospective studies. Thus, we conducted a prospective study to compare dermoscopy with mycological examination. METHODS Prospective, cross-sectional study with 109 patients and 202 nails evaluated. Dermoscopic patterns were described using digital photography and their sensitivity and specificity for distal-lateral onychomycosis were determined. RESULTS: Statistically significant (p < 0.05) patterns and colors for the diagnosis of distal-lateral onychomycosis and respective sensitivity / specificity: jagged edge (80.2% / 65.3%), linear edge (12.6% / 42 , 9%), longitudinal irregular streaks (81.1% / 65.3%), longitudinal fine / regular streaks (9.9% / 59.2%); white color (93.7% / 18.4%), yellow color (63.1% / 71.4%) and orange color (10.8% / 100%). After a stepwise forward multivariate analysis irregular streaks, linear edge and yellow color remained statistically significant (p < 0.05). DISCUSSION: Findings of this prospective study are in agreement with the literature showing that there is correlation between mycological examination and dermoscopy. However, this study does not agree with 100% sensitivity and specificity found in retrospective studies for jagged edge and linear edge patterns. In addition, white, yellow and orange colors were also statistically significant for the diagnosis of onychomycosis. CONCLUSION: Dermoscopy correlates well with the natural history of fungal nail infection and mycological examination, and we consider it a promising method for the diagnosis of onychomycosis. We suggest that future studies compare dermoscopy with a gold standard exam (ex: fluorescence microscopy, PCR) to detect false negative cases

This work describes progress toward developing a model system to investigate <i>in plantae</i> suppression of human pathogens by <i>Stemphylium</i>-like fungal endophyte strains.

INTRODUÇÃO: Onicomicose é a infecção fúngica das unhas e é considerada a onicopatia mais frequente em adultos. Representa até 50% das lesões ungueais, sendo necessária confirmação diagnóstica com exame complementar que demostre a presença do fungo na unha, sendo o exame micológico direto e a cultura para fungos os mais utilizados. A dermatoscopia é um exame não invasivo, rápido e de baixo custo cujos padrões para onicomicose relatados na literatura em estudos retrospectivos chegam a 100% de sensibilidade e especificidade. Deste modo realizamos um estudo prospectivo para comparar a dermatoscopia frente ao exame micológico. MÉTODOS: Estudo prospectivo, transversal avaliando 109 pacientes e 202 unhas com suspeita diagnóstica de onicomicose. Os padrões dermatoscópicos encontrados foram descritos através de fotografias digitais. A sensibilidade e especificidade dos padrões de onicomicose distal-lateral foram determinados de acordo com o resultado do exame micológico. RESULTADOS: Foram significativos (p < 0,05) para o diagnóstico de onicomicose distal lateral e tiveram sensibilidade/especificidade calculadas os padrões: borda recortada (80,2% / 65,3%), borda linear (12,6% / 42,9%), estrias irregulares (81,1% / 65,3%), estrias finas/regulares (9,9% / 59,2%); e as cores branco (93,7% / 18,4%), amarelo (63,1% / 71,4%) e laranja (10,8% / 100%). Após análise multivariada stepwise forward os padrões de estrias irregulares, borda linear e cor amarela mantiveram significância estatística (p < 0,05). DISCUSSÃO: Os achados deste trabalho prospectivo estão de acordo com a literatura mostrando que há correlação entre o exame micológico e a dermatoscopia. Todavia, não ratifica a sensibilidade e especificidade de 100% encontrada em estudo retrospectivo para os padrões de borda recortada e borda linear. Também não demonstrado na literatura, as cores amarela, branca e laranja foram também estatisticamente significativas para o diagnóstico. CONCLUSÃO: A dermatoscopia correlaciona bem com a história natural da infecção fúngica e com o exame micológico, sendo um exame promissor para o diagnóstico de onicomicose. Sugerimos que futuros estudos comparem a dermatoscopia com exames considerados padrão-ouro (ex: microscopia de fluorescência e PCR) para detectar exames falso negativos / BACKGROUND: Onychomycosis is defined as a fungal infection of the nail and is considered the most common onychopathy in adults. It represents up to 50% of nail diseases and demonstration of the fungal pathogen is necessary for diagnostic confirmation. Direct mycological examination and fungal culture are commonly used for this purpose. Dermoscopy is a noninvasive, fast and inexpensive exam, reaching 100% diagnostic sensitivity and specificity for onychomycosis in retrospective studies. Thus, we conducted a prospective study to compare dermoscopy with mycological examination. METHODS Prospective, cross-sectional study with 109 patients and 202 nails evaluated. Dermoscopic patterns were described using digital photography and their sensitivity and specificity for distal-lateral onychomycosis were determined. RESULTS: Statistically significant (p < 0.05) patterns and colors for the diagnosis of distal-lateral onychomycosis and respective sensitivity / specificity: jagged edge (80.2% / 65.3%), linear edge (12.6% / 42 , 9%), longitudinal irregular streaks (81.1% / 65.3%), longitudinal fine / regular streaks (9.9% / 59.2%); white color (93.7% / 18.4%), yellow color (63.1% / 71.4%) and orange color (10.8% / 100%). After a stepwise forward multivariate analysis irregular streaks, linear edge and yellow color remained statistically significant (p < 0.05). DISCUSSION: Findings of this prospective study are in agreement with the literature showing that there is correlation between mycological examination and dermoscopy. However, this study does not agree with 100% sensitivity and specificity found in retrospective studies for jagged edge and linear edge patterns. In addition, white, yellow and orange colors were also statistically significant for the diagnosis of onychomycosis. CONCLUSION: Dermoscopy correlates well with the natural history of fungal nail infection and mycological examination, and we consider it a promising method for the diagnosis of onychomycosis. We suggest that future studies compare dermoscopy with a gold standard exam (ex: fluorescence microscopy, PCR) to detect false negative cases