<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.

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Research regarding the distribution and structure of fungal communities in caves is lacking. The current study examines fungal communities in Kartchner Caverns, a mineralogically diverse cave located in the Whetstone Mountains, Arizona, USA. The first study examines culturable fungal diversity from speleothem surfaces. Twenty-one fungal genera represented by 43 genotypes and 53 distinct morphological taxonomic units (MTU) were recovered from 15 speleothems. Analysis of DGGE profiles indicated a significant effect of sampling site on community structure. The second study examined fungal diversity from speleothem and rock wall surfaces using the 454 FLX Titanium sequencing platform using the rDNA internal transcribed spacer 1 (ITS1) as a genetic marker. Fungal diversity was estimated and compared between speleothem and rock wall surfaces and its variation with distance from the natural entrance of the cave was quantified. Effects of environmental factors and nutrient concentrations in speleothem drip water at different sample sites on fungal diversity were also examined. Sequencing revealed 2219 fungal operational taxonomic units (OTUs) at 95% similarity. Speleothems supported a higher fungal richness and diversity than rock walls, but community membership and the taxonomic distribution of fungal OTUs did not differ significantly. OTU richness and diversity were negatively correlated with distance from the natural cave entrance. Community membership and taxonomic distribution of fungal OTUs differed significantly between the front and back of the cave. There was no observed effect of drip water nutrient concentration on fungal community structure. The third study examined fungal community structure from bat guano over the course of a year. There was no significant difference in fungal OTU richness, diversity, or community membership and taxonomic affiliations among sampling times. There were no significant differences in nutrient concentrations of guano samples among sampling times. Nutrient concentration did have a significant effect on community structure, especially the level of nitrogen and calcium.

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Numerous studies have looked at biotic succession at the aboveground level; however, there are no studies describing fungal community change associated with long-term ecosystem development. To understand ecosystem development, the organisms responsible for shaping and driving these systems and their relationships with the vegetation and soil factors, it is critical to provide insight into aboveground and belowground linkages to ultimately include this new information into ecosystem theory. I hypothesized that fungal communities would change with pedogenesis, that these changes would correlate with vegetation community change, and that they should show change of composition and diversity as the seasons change. Chapter 1 discusses the main topics related to this dissertation. Chapter 2 includes a publication draft that describes a study of sand-dune soil samples from northern Michigan that were analyzed to pinpoint the structural change in the fungal community during the development of the ecosystem. The samples were analyzed by pyrosequencing the soil DNA, targeting the internal transcribed spacer region. Chapter 3 contains a coauthored published paper that describes plant invasion of fields in Virginia to determine how they impact soil bacterial and fungal communities. The bacterial and fungal communities that were invaded by 3 different plant species exhibited similar changes, regardless of plant species, suggesting that some functional traits of invasives may have similar impacts on belowground communities. Chapter 4 remarks the conclusions of this research. / Master of Science / Ecosystems, including the soils underneath, are the environments that surround us perform a large number of critical human-relevant functions (playing roles in production of food, filtration of water for drinking, sequestration of carbon and nitrogen to build soil organic matter, and buffer against flooding). Yet, how these systems naturally develop over time are still in need of detailed study. One particular area of interest and need is the study of belowground fungal communities. It is not commonly known, but plants and ecosystems are highly dependent on the underground web of fungal hyphae that transform nutrients and provide water to plants. A first step in gaining this understanding utilized a natural ecosystem development gradient known as a chronosequence. It was expected that fungal communities would change as soil and ecosystem development progressed and that they would mimic changes in soil and vegetative properties. Discerning if these linkages occur is the first step to assessing how they work together to create ecosystems and their valuable environmental services. Chapter 1 provides a discussion of the main topics in this dissertation. Chapter 2 is at the heart of the dissertation via a study of fungal communities in a developmental soil ecosystem in northern Michigan in addition, in Chapter 3, I include a coauthored published paper that describes plant invasion of fields in Virginia. Chapter 4 remarks on the major conclusions of this Master thesis, supporting the role that vegetation and fungal community change in soil are associated with one another.

En Europe de l’Ouest, S est devenu un élément limitant pour la croissance des plantes. Ainsi, des carences en S apparaissent de plus en plus fréquemment sur des cultures tel que le colza. Dans le sol, 95% de S est sous formes organiques, non disponibles pour les plantes. L’intervention de la microflore est indispensable pour assurer la minéralisation du S organique en sulfates, assimilables par la plante. Nos objectifs ont été de caractériser la communauté fongique impliquée dans la minéralisation des esters de S, forme majoritaire de S organique, via une activité arylsulfatase (ARS), dans les rhizosphères de colza et d’orge. La communauté fongique est composée de plusieurs genres affiliés principalement aux Ascomycètes. Chez les souches fongiques isolées de la rhizosphère de colza et d’orge, une activité ARS a été détectée dans différents compartiments cellulaires. La régulation de ces activités ARS semble dépendante du taxon considéré. Nous avons par ailleurs montré que l’environnement rhizosphérique n’influence pas toujours la taille de la communauté fongique ARS. Dans les sols, si les activités ARS totale et intracellulaire semblent négativement corrélées avec les quantités de sulfates, l’activité ARS extracellulaire semble indépendante de la disponibilité en sulfates. En conclusion, l’ensemble des expérimentations suggère que la communauté fongique fonctionnelle joue un rôle dans la dynamique du S dans les sols agricoles. Des approches d’écologie fonctionnelle permettraient de mieux cerner leur implication dans la disponibilité en S minéral pour la plante / In Western Europe, sulfur (S) deficiency occurs in certain crops, including crucifers and cereals. Therefore, S becomes limiting for crop production and plants exhaust S mainly from soil organic S. In soil, 95% of S is in organic form that is not readily available for plants. This organic form containing principally ester S requires microbial mineralization to sulfate by arylsulfatase (ARS) enzyme. Our objectives were to characterize the fungal community having the ARS activity in the rhizosphere of rape versus that of barley. Functional fungal community comprised several genera principally belonging to Ascomycota. In different fungal strains, ARS activity was detected in different cellular compartments. The regulation of ARS was mostly dependent on microbial taxa. The density of the functional fungal community was not influenced by rhizospheric compartment.In soils, total and intracellular ARS activities were negatively correlated with soil sulfates whereas soil extracellular ARS activity was independent of sulfates. The overall results suggest that the functional fungal community could play a role in the dynamics of S in agricultural soils. Further approaches should be developed to allow a better understanding of their potential involvement in S nutrition of crops

The incomplete degradation of bio-based and biodegradable plastics (BBPs) in soils causes multiple threats to soil quality, human health, and food security. Plastic residuals can interact with soil microbial communities. We aimed to link the structure and enzyme-mediated functional traits of a microbial community composition that were present during poly (butylene succinate-co-butylene adipate (PBSA) decomposition in soil with (PSN) and without (PS) the addition of nitrogen fertilizer ((NH4 )2SO4 ). We identified bacterial (Achromobacter, Luteimonas, Rhodanobacter, and Lysobacter) and fungal (Fusarium, Chaetomium, Clonostachys, Fusicolla, and Acremonium) taxa that were linked to the activities of ß-glucosidase, chitinase, phosphatase, and lipase in plastic-amended soils. Fungal biomass increased by 1.7 and 4 times in PS and PSN treatment, respectively, as compared to nonplastic amended soil. PBSA significantly changed the relationships between soil properties (C: N ratio, TN, and pH) and microbial community structure; however, the relationships between fungal biomass and soil enzyme activities remained constant. PBSA significantly altered the relationship between fungal biomass and acid phosphatase. We demonstrated that although the soil functions related to nutrient cycling were not negatively affected in PSN treatment, potential negative effects are reasoned by the enrichment of plant pathogens. We concluded that in comparison to fungi, the bacteria demonstrated a broader functional spectrum in the BBP degradation process

Cavity-excavating birds, such as woodpeckers, are ecosystem engineers and are often assumed to rely upon wood decay fungi to assist in softening the wood of potential excavation sites. Endangered red-cockaded woodpeckers (Picoides borealis; RCWs) are the only birds known to solely excavate through the sapwood and into the heartwood of living pine trees and generally take many years to complete their excavations. These birds may have developed a partnership with wood-inhabiting fungi to facilitate the excavation process. Past attempts to understand the complex relationships between cavity excavators and fungi relied on visual surveys of fruiting bodies, or evidence of decay, resulting in a one bird, one fungus paradigm. Using molecular methods, I investigated the relationships between RCWs and fungi, and found that the relationships between cavity-excavators and fungi involve multiple fungal species and are far more complex than previously imagined. Through a field survey, I showed that RCW excavations contain distinct communities of fungi, and propose two hypotheses to explain this result, (1) RCWs select trees with distinct fungal communities (tree selection hypothesis), or (2) RCWs promote distinct fungal communities via their excavations (bird facilitation hypothesis). By swabbing the birds, I found that RCWs carry fungal communities similar to those found in their completed excavations, demonstrating that RCWs may directly facilitate fungal dispersal during the excavation process. Through a test of the bird introduction hypothesis which implemented human-made experimental drilled cavity starts (incomplete excavations), half of which were inaccessible to the birds, I showed that RCW accessibility influences fungal community development in excavations. This experimental evidence demonstrates that the relationship between RCWs and fungal communities is a multipartite symbiosis may be mutualistic. Finally, by tracking fungal community development in experimental cavity starts through time, I also demonstrated that the fungal communities found in RCW excavations undergo succession, and that this process is influenced by the birds. The relationships described in this body of work provide the basis for future studies on cavity excavators and fungi, and also have implications for a diverse community of secondary cavity nesters, wood-inhabiting fungi, forest ecology, and the conservation of biodiversity. / Ph. D.

The growing demand for strawberries has imposed challenges, especially regarding the control of pests. Many farmers report problems with reduced chemical control efficiency, probably due to selection of resistant populations of insects and mites. An alternative is the use of biological control using pathogenic fungi as a tool in integrated pest management. Metarhizium spp. (Hypocreales: Clavicipitaceae) are generalist entomopathogenic fungi with worldwide distribution and can cause diseases in a large number of hosts. Many studies on the development of Metarhizium as a biological control agent were performed, but this bulk of knowledge is in remarkable contrast to the lack of research on the fundamental ecology of Metarhizium in agroecosystems. This thesis aimed to evaluate the establishment, persistence and dispersal of these entomopathogenic fungi in strawberry crop soil in Inconfidentes, Minas Gerais, Brazil; and to study the diversity and abundance of species of Metarhizium isolated from organic and conventional strawberry crop soils, and the field margins in Brazil and Denmark. The effectiveness of new species of Metarhizium recently found in Brazil, was evaluated against two spotted spider mite, Tetranychus urticae. Applied isolates of M. anisopliae (ESALQ1037) and M. robertsii (ESALQ1426) were able to persist for up to 12 months after the application within the soil, and disperse to other plots and colonize the rhizosphere of strawberry plants. In the plots where ESALQ1037 and ESALQ1426 were applied, 25% and 87.5% of the isolates recovered after 12 months consisted of the same isolates inoculated. A new taxonomically unassigned lineage, referred to as Metarhizium sp. Indet. 5 in this study, was found in strawberry crop margins. The dominant species of Metarhizium in Brazil and Denmark was Metarhizium robertsii and M. brunneum respectively. Further, Metarhizium pemphigi was first detected in Denmark in this study. Soil in organically grown strawberries harbored a more diverse population of Metarhizium spp. compared with conventionally grown strawberries. These studies showed for the first time the potential of new species of Metarhizium as spider mite biological control agents, the lowest median lethal time (LT50 = 4 ± 0.17 days) was observed in mites treated with the isolate ESALQ1638 of Metarhizium sp. indet. 1. The best isolates were ESALQPL63 of B. bassiana, ESALQ1608 and ESALQ1638 of Metarhizium sp. indet. 1 and ESALQ3069 and ESALQ3222 of M. pingshaense based on the survival curve, total mortality, percentage of sporulated cadavers and LT50. Knowledge of the diversity of Metarhizium spp. and persistence in strawberry soil generated in this study may be useful in developing conservation strategies and maximize the natural biological pest control. / A crescente demanda por morangos vem impondo desafios, especialmente quanto ao controle das pragas. Muitos agricultores relatam problemas com a redução da eficiência do controle químico, provavelmente devido à seleção de populações resistentes de insetos e ácaros. Uma alternativa é o uso de controle biológico com fungos entomopatogênicos como ferramenta dentro do manejo integrado de pragas. Metarhizium spp. (Hypocreales: Clavicipitaceae), são fungos entomopatogênicos generalistas com distribuição cosmopolita e que podem causar doenças em um grande número de hospedeiros. Muitos estudos sobre o desenvolvimento de Metarhizium como agente de controle biológico foram realizados, mas este leque de conhecimento está em contraste com a notável falta de investigação sobre a ecologia de Metarhizium nos agroecossistemas. Esta tese teve como objetivo avaliar o estabelecimento, persistência e dispersão destes fungos entomopatogênicos em solo de morangueiro em Inconfidentes, Minas Gerais, Brasil; bem como estudar a diversidade e abundância de espécies de Metarhizium isolados do solo de cultivos orgânico e convencional de morangueiro, e das margens das plantações no Brasil e Dinamarca. A eficácia de novas espécies de Metarhizium, encontradas recentemente no Brasil, foi avaliada contra o ácaro rajado, Tetranychus urticae. Os isolados inoculados de M. anisopliae (ESALQ1037) e M. robertsii (ESALQ1426) foram capazes de persistir por até 12 meses após a aplicação no solo, além de dispersar para outras parcelas e colonizar a rizosfera dos morangueiros. Nas parcelas onde ESALQ1037 e ESALQ1426 foram aplicados, 25% e 87,5% dos isolados recuperados após 12 meses consistiam dos mesmos isolados inoculados. Uma nova linhagem não taxonomicamente identificada, referida neste trabalho como Metarhizium sp. Indet. 5, foi encontrada nas margens de morangueiros cultivados. A espécie dominante de Metarhizium no Brasil e Dinamarca foi Metarhizium robertsii e M. brunneum, respectivamente. Além disso, Metarhizium pemphigi foi detectado pela primeira vez na Dinamarca neste estudo. Solos de cultivo orgânico de morangueiro em geral apresentaram uma diversidade maior de Metarhizium do que solos de cultivos convencionais. Estes estudos revelaram pela primeira vez o potencial de novas espécies de Metarhizium como agentes de controle biológico do ácaro rajado, sendo o menor tempo letal mediano (TL50= 4 ± 0.17 dias) observado em ácaros tratados com o isolado ESALQ1638 de Metarhizium sp. indet. 1. Os melhores isolados foram ESALQPL63 de B. bassiana, ESALQ1608 e ESALQ1638 de Metarhizium sp. indet. 1 e ESALQ3069 e ESALQ3222 de M. pingshaense baseado na curva de sobrevivência, mortalidade total, porcentagem de cadáveres esporulados e TL50. O conhecimento da diversidade de Metarhizium spp. e persistência em solos de morango, gerados neste estudo, poderão ser úteis no desenvolvimento de estratégias de conservação e maximizar o controle biológico natural de pragas.