To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

No description available.

Soybean is an important source of protein in animal feed, and growing demand for meat consumption worldwide has led to increased soybean production. Over 120 million metric tons of soybean were harvested in the United States in 2018, approximately one-third of the world production. In the Mid-Atlantic region, soybean is one of the most valuable field crops. Major foliar diseases that reduce soybean yield in the Mid-Atlantic region are frogeye leaf spot (FLS) and Cercospora leaf blight. In addition to crop rotation and host resistance, foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage these soybean foliar diseases. Yield benefits of foliar fungicides have been inconsistent and this may be the result of low disease pressure, unfavorable environmental conditions for disease development, or the presence of fungal pathogen populations that have developed resistance to fungicides. The objectives of this research were 1) to develop a pyrosequencing-based assay to rapidly quantify QoI resistance frequencies in Cercospora sojina, the causal agent of FLS, 2) to examine the effects of fungicide application timings, disease pressure, and environmental factors on soybean yield, and 3) to develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. using a threshold decision rule. A pyrosequencing assay targeting the G143A mutation was designed, and a Virginia survey of C. sojina populations indicated that the G143A mutation conferring QoI resistance is widespread. In small plot fungicide application timing experiments, five weekly fungicide applications starting at beginning pod (R3) resulted in the greatest yield, but for single fungicide applications, R3 or 1 week after R3 resulted in the greatest yields. There was positive relationship between the cumulative number of disease favorable days (mean daily temperature 20-30°C and ≥ 10 hours of relative humidity >90%) from planting to R3 and disease severity at the full pod stage (r = 0.97, P = <0.01). Higher disease severity was associated with greater yield loss (r2 =0.53, P = 0.10) suggesting foliar fungicide applications are more likely to have yield benefits as the number of disease favorable days prior to R3 increase. A disease favorable-days threshold (FDT) using the environmental parameters indicated above was evaluated in on-farm experiments throughout Virginia, Maryland, and Delaware. Based on decision rules, FDT = 8 three weeks prior to R3 was the best predictor of a yield benefit with an R3 fungicide application. The decision aid was also able to correctly predict when a fungicide application would not be profitable ≥90% of the time. This weather-based decision aid along with monitoring of fungicide resistance development within the region will provide soybean growers in the Mid-Atlantic U.S. with tools to maximize yields and profitability. / Doctor of Philosophy / Soybean is the third most valuable field crop in the world, ranked only behind rice and wheat in value. Over 98% of the soybean crop is used for animal feed due to its high protein content. The United States is the largest soybean producer in the world, responsible for one-third of global production. Soybean is the top cash crop in the Mid-Atlantic region. Foliar fungal diseases can reduce the soybean yield by causing lesions on the leaves that reduce photosynthesis and cause premature defoliation. Frogeye leaf spot (FLS) caused by Cercospora sojina is a major yield reducing soybean foliar diseases in the Mid-Atlantic region. Foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage the disease. However, fungicide efficacy has been inconsistent. Inconsistencies may be due to low disease pressure, improper application timing, or fungicide resistance. The purpose of this research was to investigate the fungicide efficacy inconsistencies and to develop management tools to improve yield and maximize profitability. Our objectives were to 1) develop a molecular assay to quantify frequencies of the mutation conferring fungicide resistance in Virginia populations of C. sojina, 2) examine the effects of fungicide application timings, disease severity, and weather on soybean yield, and 3) develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. The C. sojina fungicide resistance mutation was widespread in Virginia, but overall frequencies were relatively low compared to findings from Midwest and Southern states. In fungicide timing experiments, beginning pod (R3) applications resulted in the most consistent yield benefits, and disease severity and yield loss increased as the number of weather-based disease favorable days prior to R3 increased. We used data from on-farm experiments in Virginia, Maryland, and Delaware to develop a weather-based disease favorable-days threshold that increased the probability that a fungicide application at R3 would have a yield benefit in soybean. The results of our research have led improved fungal disease management recommendations for soybean in the Mid-Atlantic that will maximize yields and profitability.

This dissertation documents efforts to evaluate metabolic variables and the fecal microbiome in adult horses fed grass hay. In the first study, eight Arabian geldings limit-fed an 18% vs. 12% non-structural carbohydrate (NSC) hays in a cross-over design during two 28-day periods were included to evaluate the influence of grass hay NSC on serum insulin and plasma glucose concentrations. Serum insulin concentrations was higher in geldings fed the 18% NSC hay; however, this difference was only detected on day 7 and none of the geldings developed hyperinsulinemia. Blood glucose concentrations did not differ between hay groups. The second and third studies were extensions of the first and were conducted to use denaturing gradient gel electrophoresis (DGGE) and real-time PCR in evaluating the effect of forage carbohydrates on equine fecal bacteria diversity and abundance, respectively. Fecal microbiomes were similar (80.5-87.9%) between geldings. The abundance of bacteria belonging to the Firmicutes phylum increased (p = 0.02) in the feces of geldings fed 12% NSC hay (mean 8.06 range [8.03-8.11] log10 copies/g feces) compared to the feces of the same geldings when fed the 18% NSC hay (7.97 [7.97-7.98] log₁₀ copies/g feces). The Firmicutes (43.7%), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%) phyla dominated the fecal microbiomes. This work was the first to report the presence of the Actinobacteria, Cyanobacteria, and TM7 phyla in the equine fecal or gut microbiome. There was a high abundance (38%) of unclassified bacterial sequences in the gelding fecal microbiome. In the fourth study, 5 overweight adult mixed-breed mares and 5 adult mixed-breed mares in moderate condition, limit-fed a grass hay, were used to evaluate the effect of body condition on diet digestibility, plasma and fecal volatile fatty acid (VFA) concentrations, and fecal bacterial abundance. Hay, fecal, and blood samples were taken daily for 4 days after a 10 day adaptation period. A difference in hay digestibility, fecal VFA concentration, or bacterial abundance was not detected between overweight mares and mares in moderate condition. Plasma acetate, a product of microbial fermentation of fiber, was higher in the overweight mare group. / Ph. D.

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.

Yes / Type 2 diabetes mellitus prevalence is growing globally, and the leading cause of mortality in these patients is cardiovascular disease. Epigenetic mechanisms such as microRNAs (miRs) and DNA methylation may contribute to complications of type 2 diabetes mellitus. We discovered an aberrant type 2 diabetes mellitus–smooth muscle cell phenotype driven by persistent up-regulation of miR-145. This study aimed to determine whether elevated expression was due to changes in methylation at the miR-145 promoter. Smooth muscle cells were cultured from saphenous veins of 22 non-diabetic and 22 type 2 diabetes mellitus donors. DNA was extracted, bisulphite treated and pyrosequencing used to interrogate methylation at 11 CpG sites within the miR-145 promoter. Inter-patient variation was high irrespective of type 2 diabetes mellitus. Differential methylation trends were apparent between non-diabetic and type 2 diabetes mellitus–smooth muscle cells at most sites but were not statistically significant. Methylation at CpGs −112 and −106 was consistently lower than all other sites explored in non-diabetic and type 2 diabetes mellitus–smooth muscle cells. Finally, miR-145 expression per se was not correlated with methylation levels observed at any site. The persistent up-regulation of miR- 145 observed in type 2 diabetes mellitus–smooth muscle cells is not related to methylation at the miR-145 promoter. Crucially, miR-145 methylation is highly variable between patients, serving as a cautionary note for future studies of this region in primary human cell types.

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

L'ancienne mine de plomb et de zinc de Carnoulès (Gard, France) a généré 1.5 Mt de déchets d'où émerge un drainage de mine aux eaux acides alimentant un ruisseau, le Reigous. Ce site fournit un exceptionnel exemple d'adaptation à un environnement extrême en raison des eaux acides (pH~3) et de très fortes concentrations en métaux et métalloïdes, particulièrement en As. Dans les 30 premiers mètres du ruisseau, l'activité bactérienne conduit à un phénomène de remédiation naturelle avec la co-précipitation de 20 à 60% de l'As dissous avec du fer. Les bactéries présentes dans les sédiments du ruisseau ont dans un premier temps été décrites par clonage/séquençage du gène de l'ARNr 16S, puis les membres actifs des communautés bactériennes ont été révélés par une approche de métaprotéomique. L'étude des Archaea au sein des sédiments a révélé la présence de groupes impliqués dans la méthanogénèse ou dans l'oxydation de l'ammoniac qui pourraient participer au cycle du carbone ou de l'azote. Les eucaryotes ont été caractérisés pour la première fois sur ce site par pyroséquençage, mettant en évidence une forte proportion de champignons (60%). Enfin, l'étude des variations spatiotemporelles des populations bactériennes dans les eaux a conduit à l'identification de 6801 OTUs dont des phyla encore jamais identifiés sur ce site. La concentration en arsenic, la température et le potentiel redox semblent jouer un rôle dans la structuration de ces communautés. Ce travail de thèse a ainsi contribué à une meilleure connaissance des microorganismes présents (Bactéries, Archaea, Eucaryotes) et de leurs dynamiques spatiotemporelles en relation avec les paramètres physicochimiques du milieu. / Acidic mining drainage generated at Carnoulès, a former Pb-Zn mine (Gard, France) coincides with the spring of the Reigous Creek. This site provides an exceptional example of adaptation to extreme environments due to its acidic water (pH~3) and very high concentrations of metals and metalloids, particularly arsenic. During the first 30 m of downflow in Reigous Creek, natural remediation occurred, with co-precipitation of 20 to 60% of the dissolved arsenic with iron, mediated by bacteria. Bacterial communities inhabiting the creek sediments were first described by cloning/sequencing of the 16S rRNA genes and the active members were identified by a metaproteomic approach. A survey of the archaeal community in the sediment highlighted the presence of sequences phylogenetically related to methanogenic Archaea and to ammonia oxidizers, which could be involved in carbon and nitrogen biochemical cycling. The Eukaryotic communities were studied for the first time at this site by pyrosequencing, revealing that around 60% of the sequences belonged to Fungi. Finally, the study of the spatiotemporal variations of the water bacterial communities allowed the identification of 6801 OTUs including sequences of taxa never detected before. The environmental variables significantly correlated with bacterial community dynamics appear to be arsenic concentration, temperature and Eh. This PhD work has contributed to a better understanding of the spatiotemporal dynamics of microorganisms (Bacteria, Archaea, Eukaryotes) in relation with the physicochemical parameters of their environment.

L’esca est une maladie de dépérissement du bois de la vigne conduisant à la mort des ceps. Actuellement le vignoble mondial est atteint, et au niveau français, cette maladie ne cesse de progresser. Ainsi, 8% des ceps dans le Jura et 4,5% dans la région de Bordeaux manifestent des symptômes d’esca, selon les parcelles des chiffres beaucoup plus élevés sont obtenus, certains cépages sont aussi beaucoup plus sensibles que d’autres. Plusieurs champignons seraient impliqués dans l’esca mais leur rôle ainsi que la détermination de la microflore responsable de cette maladie est encore sujette à interrogation. Dans ce contexte, l’objectif de cette thèse a été de caractériser et de comparer les microflores fongiques et bactériennes colonisant le bois de ceps de vigne ayant exprimé ou non des symptômes foliaires d’esca. Dans un premier temps, nous avons prélevé des ceps (cultivar Cabernet Sauvignon) relativement jeunes (10 ans d’âge) car ils présentaient l’intérêt d’être peu dégradés au niveau du bois du tronc, les symptômes foliaires étant associés à la présence d’amadou (une nécrose typique de l’esca) uniquement dans les bras. Une grande diversité dans les communautés fongiques (674 OTUs) et bactériennes (222 OTUs) colonisant le bois a été observée. Cette diversité est plus importante dans le bois sain de la vigne que dans celui partiellement ou totalement nécrosé. Les techniques utilisées, i.e. isolement/séquençage de souches, empreinte moléculaire (Single Strand Conformation Polymorphism, SSCP) et pyroséquençage 454, ont montré que les communautés bactériennes ou fongiques étaient différentes dans les tissus dégradés comparés à ceux qui ne l’étaient pas. Des changements de microflores en fonction du temps (expérimentation durant 1 année) ont aussi été observés. D’une façon générale, les espèces de champignons impliquées dans l’esca sont déjà présentes dans le bois apparemment sain de ceps esca-foliaires symptomatiques mais aussi des asymptomatiques. Il n’a pas été possible de différencier ces 2 types de microflores au niveau du bois sain des plants, cette différentiation se faisant au niveau des nécroses, qui sont plus abondantes dans les ceps esca-symptomatiques. Pour la première fois nous avons montré que des communautés bactériennes spécifiques étaient associées à l’esca, leurs aptitudes trophiques étant différentes selon les tissus où elles étaient prélevées. Les espèces isolées suggèrent que certaines pourraient avoir un rôle dans la protection du végétal, d’autres dans la dégradation des structures du bois, e.g. de la lignine, préparant ainsi le terrain aux champignons dégradateurs des tissus ligneux, déjà présents à l’intérieur des ceps. Nous avons aussi étudiés des ceps plus âgés (cultivar Baco blanc), de 42 et 58 ans, qui avaient un rendement acceptable et n’avaient pas manifesté de symptômes d’esca ou eutypiose (une autre maladie du bois) l’année du prélèvement. Au niveau des tissus fonctionnels du bois, les communautés fongiques étaient caractéristiques de plants atteints par l’eutypiose (ceps de 42 ans) ou de l’esca (ceux de 58 ans). La non expression par les ceps de ces 2 maladies pourrait cependant être associée à la forte présence de champignons mycoparasites et protecteur du végétal, comme Trichoderma spp., dans ces tissus fonctionnels. Les interactions au sein des communautés fongiques créant un équilibre où le pathogène ne se développerait pas de façon extensive. Les caractéristiques du Baco blanc, un hybride, moins sensible à certaines maladies de la vigne, pourrait aussi expliquer ce résultat. Ainsi la présence d’une microflore bénéfique naturellement présente dans le bois des ceps associée à des plants ayant une tolérance à ces maladies pourrait ouvrir de nouvelles perspectives pour lutter l’esca, voire l’eutypiose, pour lesquelles aucun moyen de protection n’existe aujourd’hui. / Esca is a Grapevine Trunk Disease (GTD) that induces a decline in grapevine vigour that generally leads up with the death of the plants. Nowadays, vineyards worldwide are attacked by esca and, in France this disease increases steadily. In the Jura, 8% of the grapevines are esca-foliar symptomatic and approximately 4.5% in the Bordeaux region. However, some vineyards are more severely attacked by esca, and certain cultivars are more susceptible than others. Although several pathogenic fungi are associated with esca, their individual roles and their interaction with other microorganisms for the esca have still to be determined. In this context, the objective of the present PhD study is to characterize and compare the bacterial and fungal microflora that colonize the wood tissues of esca-foliar symptomatic and asymptomatic grapevines. First, we sampled young (10 year-old) grapevines (Cabernet Sauvignon cultivar) because they had only few necroses in the trunk and white-rot (also called amadou) was only present in the cordons of symptomatic plants. Great diversity in the fungal (674 OTUs) and bacterial (222 OTUs) communities was observed. This diversity was higher in the apparently healthy wood than in the partially or totally necrotic wood tissues. The methods used isolation/sequencing of microbial strains, a molecular fingerprinting method (Single Strand Conformation Polymorphism, SSCP) and 454 pyrosequencing showed that the fungal and bacterial communities of the necrotic and healthy wood tissues were different. Changes in the microflora over time (over a one-year period) have been observed. Fungal species involved in esca are already present in the apparently healthy wood of esca-foliar symptomatic plants but also in the asymptomatic ones. It was not possible to differentiate these 2 microflora. Only microflora from the necroses differed from those of the healthy wood with these necroses being more developed in the esca-foliar symptomatic grapevines. For the first time, we were able to determine that specific bacterial communities are associated with esca. Depending on the wood tissues, different types of bacteria were isolated, with different trophic behaviour. Two roles could be assigned to the species isolated from the various wood tissues: (i) a positive role, due to the biocontrol potential that many species have; (ii) a negative one, by predisposing the wood of grapevines to fungal attacks. We also studied, old (42 and 58 year-old) grapevines of the cultivar, Baco blanc, that produced regular harvests. The plants had no expressed foliar symptoms of esca or eutypa dieback during the sampling year. Many plant pathogens colonized the functional wood tissues, but in 58 year-old plants they were associated with esca, and in 42 year-old plants, with eutypa dieback. The absence of GTDs expression could be linked to the numerous plant protectant mycoparasites, such as Trichoderma spp., that colonized the functional wood tissues. Interactions between species within the fungal communities may create a balance that is unfavourable to the development of the pathogens. The use of Baco blanc, a hybrid less susceptible to certain grapevine diseases could also explain this result. So, because no means of protection are currently available, the combination of beneficial microflora within the garpevine wood tissues with plants that are tolerant to esca, or even eutypa dieback, could be helpful to control those diseases.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ari Jumpponen / Traditional fallow periods in the Bolivian highlands are being shortened in an effort to increase short-term crop yields, with potential long-term impacts on soil communities. Using 454-pyrosequencing, we characterized fungal and bacterial community responses to (1) the length of fallow period and (2) the presence of the plants Parasthrephia sp. or Baccharis sp. (both locally known as ‘thola’). Thola is widely considered by farmers as beneficial to soil health, although it is also frequently harvested as a source of fuel by farmers. Soils in one study area, Ancoraimes, had higher levels of organic matter, nitrogen and other macronutrients compared to the other study area, Umala. In our analyses, Ancoraimes soils supported more diverse fungal communities, whereas Umala had more diverse bacterial communities. Unexpectedly, the longer fallow periods were associated with lower fungal diversity in Umala and lower bacterial diversity in Ancoraimes. Fungi assigned to genera Verticillium, Didymella, and Alternaria, and bacteria assigned to genera Paenibacillus, Segetibacter, and Bacillariophyta decreased in abundance with longer fallow period. The presence of thola did not significantly affect overall soil fungal or bacterial diversity, but did increase the frequency of some genera such as Fusarium and Bradyrhizobium. Our results suggest that fallow period has a range of effects on microbial communities, and that the removal of thola from the fields impacts the dynamics of the soil microbial communities.