Le gène de résistance (Rx) au virus X de la pomme de terre (PVX) code pour une molécule réceptrice qui interagit avec la protéine de capside (CP) du PVX conférant ainsi une résistance à la plante. Les mécanismes de résistance sont encore peu compris. Dans ce projet, nous nous intéressons à la caractérisation de cette interaction. Le premier objectif est de caractériser l’élicitation par la CP. Nous avons d’abord mutagénéisé deux acides aminés clés de la CP du PVX et montré que le niveau de la réponse par Rx est dépendant de l’affinité de l’éliciteur avec le récepteur. Nous montrons également que les CP de virus proches du PVX, présentant des variations naturelles de séquences, sont capables d’induire une résistance par Rx. Le second objectif est d’identifier les partenaires de cette résistance. Une collection de mutants EMS de tomates portant le gène Rx a été générée. Trois plantes présentant un défaut de résistance vis-à-vis du PVX ont été isolées et sont en cours de caractérisation. / In potato, the resistance gene (Rx) encodes a protein that confers resistance against Potato virus X (PVX). The trigger of the resistance is the recognition of PVX coat protein (CP). The mechanisms of this resistance are not well understood. In this project we investigate two different aspects of this interaction. The first goal of this project is to characterize the CP elicitor. In the first approach we mutagenized key amino acids in the PVX CP and showed that the affinity between elicitor and receptor modulates the intensity of the Rx response. In the second approach we showed that other viruses related to PVX with natural sequence variations in the CP are able to induce Rx mediated resistance. The second goal of this project is to identify genes required for Rx mediated resistance a collection of EMS mutants tomato (cv Micro-Tom) carrying the Rx gene has been generated and screened for restored susceptibility to PVX. Three mutants were identified and characterized.

Synthetic fiber reinforced concrete sample sets were exposed to two different environments. One set, of six samples, was exposed to filtered seawater in the lab with wet and dry cycles, while the other set of samples was exposed, on a barge, to the marine environment, in the intracoastal waterways, at SeaTech. The samples were exposed for 8 months, and then removed for experimental and mechanical testing. Upon removal, the barge samples were photographed to observe surface organisms that were attached to each sample. The barge samples, after cleaning, were then exposed to UV light to observe surface bacteria. The barge samples were also taken to Harbor Branch facility for DNA testing, and then sent in for sequencing. This sequencing was used to identify the organisms that were present inside the concrete samples. An Indirect Tensile Strength Test, IDT, was performed on both sets of samples to observe the first crack, max load, and fracture toughness of each sample. The Barge samples had a lower first crack, max load, and fracture toughness, which means that it took less force to break these samples, than the Seawater samples. As the fiber content increased, the Seawater samples grew stronger, while the Barge samples grew weaker. Also, as the fiber content increased, the biodiversity found on the surface of the Barge samples increased as well. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

In order to determine the impact of lesser snow goose (Chen caerulescens caerulescens) herbivory on above and belowground nutrient dynamics, a grazing and nutrient addition experiment was conducted in two habitat types utilized by snow geese near Churchill, Manitoba. Forage plant nutrition, soil microbial biomass, and inorganic and organic soil nutrients were measured in relation to the timing of grazing, over two consecutive summers. Soil was collected from the rhizosphere to determine the influence of foliar herbivory on root-microbe interactions. Primary productivity in both habitats is co-limited by the availability of both nitrogen (N) and phosphorus (P). Aboveground defoliation either caused a reduction or no change in soil microbial biomass nutrients (carbon (C), N, or P). Defoliated shoots had higher N concentrations and did not show compensatory growth within the season. Root biomass was somewhat reduced with grazing, but higher whole plant N content suggests that grazing does not compromise N-uptake.

The killing effectiveness of electron beam irradiation has not been completely characterized. The type of microorganisms and the composition of food have a direct effect on the efficiency of this technology. The objectives of this study were to select a surrogate suitable for use in electron beam irradiation studies of fruits and to evaluate the effect of sugar content on the kinetics of microbial damage and recovery. A 2.0 MeV Van de Graaff linear accelerator was used to apply irradiation (up to 5.0 kGy), using different configurations, on gelatin-based systems with the addition of sugars. The systems were inoculated with pathogenic and non-pathogenic bacteria strains (surrogates). Initial studies showed that Escherichia coli K-12 MG1655 is a suitable surrogate that represents the damage induced to common fruit pathogens by irradiation. The reduction in bacteria population can be maintained by storing samples at 4°C. An increase in temperature up to 20°C was enough for the damaged population to recover in 48 hours. Gelatin-based systems proved to be a simple and inexpensive medium to evaluate the effects of irradiation (up to 5.0 kGy) on selected bacteria. Reduction of the system dimensions and their positioning related to the beam source were key factors in increasing the killing effectiveness of irradiation. The sugar levels (up to 8 %) used to mimic the maturity of cantaloupes had no effect on the radiation D10 values and the recovery of the surrogate population quantified as Generation Times. The resistance of the surrogate to irradiation was validated in an optimum configuration and in cantaloupes. Temperature and sugar content caused significantly higher changes to the physical structure of the gel-based systems than irradiation (1.0 kGy). Plate counts and light microscopy techniques demonstrated that the structure of the gelatin-based systems allow for motility of the bacteria in a 3-D array (length, width and depth). When little information was available about the effectiveness of using a low energy linear accelerator, the inoculation of gelatin-based systems proved to be a reliable method to select a suitable surrogate and to predict the effects of irradiation on bacteria as a function of sugar content.

Research on the interactions between microbes and polymeric materials constitutes an important part in antimicrobial identification and provides an insight into microbial response on the polymer surfaces. Herein, a high-content screening method with polymer microarray technology was developed to investigate microbe-polymer interactions, especially in studying adhesion/repellence of microbes (bacteria and parasites). Firstly, the polymer microarray approach was used to successfully identify polymers which either selectively captured or prevented the binding of major food-borne pathogen, Salmonella Typhimurium. A parallel study with a lab strain of Escherichia coli was also carried out, revealing polymers which either displayed a common binding activity or which exhibited species discrimination. Likewise, this polymer microarray technology was applied to more bacterial strains, such as Campylobacter, Clostridium, Streptococcus, Klebsiella and their cocktails to discover families of substrates that displayed strong broad-spectrum bacterial non-binding activity. These synthetic polymers represented a novel class of coating materials which can be used to prevent surface colonisation and subsequent formation of bacterial biofilms. The study of protozoan-polymer interactions was also explored in this thesis. Polymers were identified which either bound or prevented parasites (Crysporidium parvum and Giardia lamblia) binding. Material properties, including wettability, surface roughness and polymer composition were analysed to study correlation of parasite binding and the generation of polymer structure function relationships.

Arsenic is the most pervasive environmental toxic substance. As a consequence of its ubiquity, nearly every organism has genes for resistance to inorganic arsenic. In one project I examined the role of glutaredoxin 2 (Grx2) in reduction of arsenate to arsenite. I demonstrated that Grx2 has both glutaredoxin thiol transfer activity and glutathione S-transferase (GST) activity. In a second project investigated arsenic resistance in a microbiome organism. I discovered that the human gut microflora B. vulgatus has eight continuous genes in its genome and these genes form an arsenical-inducible transcriptional unit. In two other projects I investigated the properties of two As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes and AS3MT in animals). In this project we demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues, and AfArsM from Aspergillus fumigatus methylates only MAs(III) and not As(III). For human, arsenic methylation process is thought to be protective from acute high-level arsenic exposure. However, with long term low-level exposure, hAS3MT is thought to produce intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T and T306I) may be deleterious. I identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C and R251H). I purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis and/or lower stability. I propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases.

Master of Science / Department of Biology / Lydia H. Zeglin / Anthropogenic actions have significantly increased biological nitrogen (N) availability on a global scale. In tallgrass prairies, this phenomenon is exacerbated by land management changes, such as fire suppression. Historically, tallgrass prairie fire removed N through volatilization, but fire suppression has contributed to increased soil N availability as well as woody encroachment. Because soil microbes respond to N availability and plant growth, these changes may alter microbial composition and important microbially-mediated functions. Grassland management affects the soil environment on multiple time scales including short (fertilization or fire event), seasonal (growing vs. non-growing season), and long-term (decadal plant turnover and nutrient accumulation), therefore my goal was to understand community variability at different time scales affecting the population and community dynamics of soil microbes. I predicted soil microbes would be sensitive to environmental changes at all time scales, seasonal variation would reflect increased plant rhizodeposit-supported populations during summer and decomposers during winter, and long-term fire suppression and chronic fertilization would drive soil microbial community turnover associated with accumulation of plant litter and N. To address these predictions, soils were collected from the Belowground Plot Experiment (BGPE) at Konza Prairie Biological Station: a 30-y factorial field manipulation of N fertilization and burning. Surface soils (0-15 cm) were sampled monthly between Nov 2014 – Dec 2015, including one week post-fire (April) and post-fertilization (June). Genomic DNA was extracted from each sample for qPCR and PCR for Illumina MiSeq library sequencing of the prokaryotic 16S rRNA gene and fungal ITS, to estimate population and community dynamics of soil microbes. Soil environmental characteristics and plant communities were measured in July 2015 to evaluate correlations between plant and microbial communities, and environmental variability. Soil microbial responses to short-term fire/fertilization events were minimal, while microbial population sizes fluctuate seasonally and synchronously, and microbial community composition varied more with management history than at shorter time scales. Bacterial populations increased 10x during growing-season plant rhizodeposition, while fungal populations were less dynamic, but decreased in fall, possibly reflecting a shift to subsistence on soil organic matter. In contrast, microbial community composition was seasonally stable, but distinct between long-term management treatments, which may indicate accumulation of niche-defining plant or soil properties over decades. Prokaryotic communities responded to altered N availability via both fertilization and loss due to fire, with the highest abundance of "copiotrophic" (r-selected) taxa in unburned, fertilized soils. Fungal communities responded to N fertilization with higher abundance of arbuscular mycorrhizal fungi, pathogens, and saprotrophs, possibly due to changes in nutrient stoichiometry and litter availability in fertilized plots. However, fungal response to fire was largely independent of N availability, and plant community differences were correlated with fungal, but not bacterial, community composition, highlighting the likely nutritional codependence of fungi and plants, and fungal competitive advantages for plant litter substrates. The timing of changes in soil microbial communities is critical for plant nutrition and nutrient cycling in prairies, and this novel dataset on the temporal resolution of microbial responses to environmental variability contributes to the broader understanding of ecosystem responses to global change.

The rapid expansion of medical discoveries has been met with growing number of deaths from nosocomial multidrug-resistant bacteria. The dramatic rise of these antibiotic-resistant microorganisms has been placed on the World Health Organization’s watchlist as one of the biggest threats to the future of healthcare. There continues to be a shortage of effective antibiotics with the rise of these “superbugs”. With the growing number of deadly pathogens, the future of medicine relies on scientific findings of novel compounds to combat multidrug-resistant bacteria. The Appalachian Highlands Region holds the potential for discovering these new compounds. As the most biodiverse temperate forest region in North America, the Smoky Mountains contains a plethora of microorganisms that have become genetically diversified over millions of years. In order to compete with one another, many of these soil bacteria naturally produce their own antibiotics. With the wide variation of natural bacteria, Appalachia serves as a testing ground to harness the power of natural antibiotics and understand how these compounds can aide in clinic use. A gram of soil contains more than 10,000 different species of bacteria. The biodiversity of these microbes is still largely unknown, as almost 99% of these species cannot be cultured in a normal lab setting. Utilizing the 16S genomic region of microbes, this pilot project will lay the foundations of discovering Appalachia’s microbiome, which has, thus far, never been cataloged.

Indiana University-Purdue University Indianapolis (IUPUI) / Nitrous oxide (N2O) is an atmospheric constituent that contributes to climate warming and stratospheric ozone depletion. A large fraction of the anthropogenic N2O emission originates from agricultural soils suggesting therefore a strong connection between N2O accumulation in the atmosphere and agricultural land management. During the last 2-3 decades, no-till (NT) farming and integration of cover crops into crop rotation represent two major developments in agriculture, but much remains to be learned about the impact of these management approaches on N2O emission and underlying biological soil factors. This dissertation focuses on the contribution of different components of the soil microflora to N2O production, and how different types of cover crops (legume vs grass) affect the soil microbial community composition, mineral N availability, and N2O emission in plowed (PT) and NT soils. To address these questions, several laboratory and greenhouse experiments were conducted. Results of these experiments documented soil microbial community responses to cover crop addition and could inform the selection of cover crops most suitable to soils under different tillage practices.