Browsing by Author "Badgley, Brian D."

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    Antimicrobial Resistance Mitigation [ARM] Concept Paper
    Vikesland, Peter J.; Alexander, Kathleen A.; Badgley, Brian D.; Krometis, Leigh-Anne H.; Knowlton, Katharine F.; Gohlke, Julia M.; Hall, Ralph P.; Hawley, Dana M.; Heath, Lenwood S.; Hession, W. Cully; Hull, Robert Bruce IV; Moeltner, Klaus; Ponder, Monica A.; Pruden, Amy; Schoenholtz, Stephen H.; Wu, Xiaowei; Xia, Kang; Zhang, Liqing (Virginia Tech, 2017-05-15)
    The development of viable solutions to the global threat of antimicrobial resistance requires a transdisciplinary approach that simultaneously considers the clinical, biological, social, economic, and environmental drivers responsible for this emerging threat. The vision of the Antimicrobial Resistance Mitigation (ARM) group is to build upon and leverage the present strengths of Virginia Tech in ARM research and education using a multifaceted systems approach. Such a framework will empower our group to recognize the interconnectedness and interdependent nature of this threat and enable the delineation, development, and testing of resilient approaches for its mitigation. We seek to develop innovative and sustainable approaches that radically advance detection, characterization, and prevention of antimicrobial resistance emergence and dissemination in human-dominated and natural settings...
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    Assessing Diversity, Culturability and Context-dependent Function of the Amphibian Skin Microbiome
    Medina Lopez, Daniel Christofer (Virginia Tech, 2018-08-17)
    Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation.
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    Balancing the Water Budget: the effect of plant functional type on infiltration to harvest ratios in stormwater bioretention cells
    Krauss, Lauren Marie (Virginia Tech, 2021-01-19)
    Stormwater bioretention cells (BRCs) are a variety of green stormwater infrastructure with the potential to restore pre-urban water balance, provided they can be tailored to infiltrate and evapotranspire (i.e., harvest) urban runoff in proportions consistent with pre-urban hydrologic conditions. This paper evaluates their capacity to do so, focusing on evapotranspirative harvest, which is relatively understudied, and the capacity of CSR (Competitve, Stress-tolerant, and Ruderal) functional type to serve as an overarching framework characterizing the water use strategy of BRC plants. The goal is to determine if harvest (and therefore the ratio of urban runoff infiltrated to harvested; the I:H ratio) might be fine-tuned to meet pre-urban values in BRCs through informed manipulation of plant community composition. This study focuses on 3 critical plant water use traits, the turgor loss point, the point of incipient water stress, and maximum stomatal conductance. A global plant traits meta-analysis identified degree of plant competitiveness and stress tolerance as significant determinants of all three water use traits, with stem type (woody vs herbaceous) also being significant, but only for turgor loss point. Based on these results, six water use scenarios appropriate for plants with different CSR type/stem type combinations were developed. BRC plants spanning the range of CSR types necessary to actionize these scenarios were determined to be available in eight major climate zones of the coterminous US, suggesting that regulating plant water use in BRCs using CSR is likely feasible. Hydraulic simulations (Hydrus 1D) were conducted for each scenario in all eight climate zones and revealed significant differences in evapotranspirative harvest and I:H ratios in simulated BRCs. Competitive woody plants had the highest evapotranspiration and lowest I:H ratios; 1.5-1.8 times more evapotranspiration and a 1.6-2 times lower I:H ratio than stress tolerant herbaceous plants, on average, across climate zones. Despite these significant differences, no simulated BRC in any climate zone was capable of reproducing pre-urban I:H ratios, regardless of plant type. More water was infiltrated than harvested in all scenarios and climates with the inverse being true for all pre-urban conditions. This suggests that absent additional sources of harvest (e.g., use of BRC water for nonpotable purposes such as toilet flushing and outdoor irrigation, or adoption of novel BRC designs that promote lateral exfiltration, stimulating "extra" evapotranspiration from nearby landscapes), BRCs will be unable to restore pre-urban water balance on their own. If true, then using BRCs in combination with other green technologies (particularly those biased towards harvest), may be the best path forward for balancing urban water budgets.
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    Belowground Fungal Community Change Associated with Ecosystem Development
    Pineda Tuiran, Rosana P. (Virginia Tech, 2017)
    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.
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    Biochar and pH as Drivers of Greenhouse Gas Production in Denitrification Systems
    Davis, James Martin IV (Virginia Tech, 2016-01-05)
    Nitrous oxide (N2O) is a greenhouse gas (GHG) with 300 times the radiative forcing in the atmosphere of carbon dioxide (CO2), and has recently become a subject of great concern because the nitrogen (N) fertilizers which have been necessary to increase agricultural productivity have also dramatically increased N2O emissions from agroecosystems. Many N control practices have been suggested and implemented in agroecosystems, but their ability to simultaneously remove reactive N from the environment and prevent the production of N2O is, at best poorly understood. The goal of this work is to characterize environmental controls on production of N2O in denitrifying bioreactors. The review portion of this work first discusses the geologic history of the N cycle, how its past and present processes differ, and how it is being affected by human activity. It then explores the N cycle's biochemical pathways, reviews the controls for each of its steps, and discusses the environmental drivers of these controls. The review closes with a discussion of environmental N management strategies. The experimental portion of this work further explores these concepts by observing how biochar amendment and the modification of pH affect N2O production in the denitrification pathway in denitrifying bioreactors. Both pH and biochar have previously been shown to affect N2O production and many N management practices utilize biochar or manipulate pH to increase N retention. The objectives of the experiment were to: 1) Examine headspace N2O concentration in sealed, biochar-amended, denitrifying bioreactors; 2) Determine if the effects of pH on N2O production differ in biochar-amended systems versus controls (under acidic, unbuffered, and buffered conditions); 3) Quantify key denitrification genes (nirK, nirS, nosZ) in each treatment combination. Experimental results showed biochar treatment to significantly increase N2O emissions, a result which runs contrary to most, but not all studies regarding its effects on N2O production. Differences between treatments decreased with increasing pH levels. Biochar did not exhibit significant effects on individual denitrification genes, but it did show influence on the ratios of their populations. On the other hand, pH was found to have significant effects on nirS and nosZ populations. Differences in N2O production between biochar and controls were thus explained by biochar's chemical effects, likely its ability to increase denitrification activity. Developing an understanding of the mechanisms behind these differences will require using a combination of isotope tracing, enzyme assays, and mass balance approaches. Future microbial work in biochar-amended systems should attempt to characterize differences in gene expression, overall community structure, and long-term population trends in the genes of interest. The combination of these approaches should allow researchers to better predict where N2O production will occur and develop strategies to mitigate it while simultaneously increasing food production to meet the demands of a growing population.
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    Campylobacter Colonization and Diversity in Young Turkeys in the Context of Gastrointestinal Distress and Antimicrobial Treatment
    Kirchner, Margaret; Miller, William G.; Osborne, Jason A.; Badgley, Brian D.; Neidermeyer, Jeffrey; Kathariou, Sophia (MDPI, 2023-01-19)
    Young turkeys are vulnerable to undifferentiated gastrointestinal distress, including “irritable and crabby syndrome” (ICS), which compromises flock performance and is typically treated with a combination of penicillin and gentamicin (P/G). However, the effects of ICS and P/G treatment on Campylobacter remain poorly understood. We investigated the impact of ICS and P/G treatment on Campylobacter levels and diversity in four flocks from three turkey farms. Cecum and jejunum samples were analyzed weekly from day of hatch to week 4–5. All four flocks became colonized with multidrug resistant (MDR) Campylobacter jejuni and C. coli by week 2–3, and two developed ICS. ICS and P/G treatment did not significantly impact total Campylobacter levels or strain genotypes but impacted species and antimicrobial resistance (AMR) profiles. One flock was raised under antibiotic-free (ABF) conditions while another flock at the same farm was raised conventionally. The ABF flock did not develop ICS while its counterpart did. However, Campylobacter strains, AMR profiles and sequence types were generally shared between these two flocks. Our findings suggest that ICS and P/G treatment impacted Campylobacter population dynamics in commercial young turkey flocks, and that ABF flocks may become readily colonized by MDR strains from non-ABF flocks at the same farm.
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    Characterizing Microbial Community Development in Reclaimed Mine Soils
    Badgley, Brian D.; Sun, Shan (Virginia Tech. Powell River Project, 2014)
    A significant amount of the research to date at the Powell River Project (PRP) has been focused on reforestation, with the assumption that tree growth will ultimately lead to the re-establishment of a fully functioning forest ecosystem. Soil microorganisms are a critical component of this system because they mediate many of the ecosystem services for which forests are valued including carbon sequestration, soil formation, nutrient retention, watershed protection, and groundwater purification. We are characterizing the response of soil microbial communities to land reclamation approaches in the PRP to provide critical information about the restoration of the microbial component of the forest ecosystem. The objectives of this project are threefold: 1) characterize the recovery of soil microorganisms over time; 2) determine if alternate reclamation practices affect microbial diversity and community structure; and 3) compare restored microbial communities to un-mined forest soils to identify potential indicators that ‘healthy’ microbial communities are returning to reclaimed soils. We have identified a variety of reclamation plots within the PRP that represent a range of ages between 5 and 30 years to look at the effects of time. We have also sampled two other sets of to determine effects of reclamation practices: one where soils were amended with biosolids and another that was planted with pines as opposed to the standard hardwood mix. We are using genomic sequencing to fully characterize bacterial and fungal organisms present in soil samples from each plot to determine microbial diversity and community structure. Preliminary results suggest that bacterial communities recover quickly, becoming indistinguishable from communities in undisturbed soils within 10 to 30 years. In addition, certain taxa such as Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes appear to respond to age since reforestation and may contain taxa that can be used to gauge restoration progress.
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    Comparative genomics of the core and accessory genomes of 48 Sinorhizobium strains comprising five genospecies
    Sugawara, Masayuki; Epstein, Brendan; Badgley, Brian D.; Unno, Tatsuya; Xu, Lei; Reese, Jennifer; Gyaneshwar, Prasad; Denny, Roxanne; Mudge, Joann; Bharti, Arvind K.; Farmer, Andrew D.; May, Gregory D.; Woodward, Jimmy E.; Médigue, Claudine; Vallenet, David; Lajus, Aurélie; Rouy, Zoé; Martinez-Vaz, Betsy; Tiffin, Peter; Young, Nevin D.; Sadowsky, Michael J. (2013-02-20)
    Background The sinorhizobia are amongst the most well studied members of nitrogen-fixing root nodule bacteria and contribute substantial amounts of fixed nitrogen to the biosphere. While the alfalfa symbiont Sinorhizobium meliloti RM 1021 was one of the first rhizobial strains to be completely sequenced, little information is available about the genomes of this large and diverse species group. Results Here we report the draft assembly and annotation of 48 strains of Sinorhizobium comprising five genospecies. While S. meliloti and S. medicae are taxonomically related, they displayed different nodulation patterns on diverse Medicago host plants, and have differences in gene content, including those involved in conjugation and organic sulfur utilization. Genes involved in Nod factor and polysaccharide biosynthesis, denitrification and type III, IV, and VI secretion systems also vary within and between species. Symbiotic phenotyping and mutational analyses indicated that some type IV secretion genes are symbiosis-related and involved in nitrogen fixation efficiency. Moreover, there is a correlation between the presence of type IV secretion systems, heme biosynthesis and microaerobic denitrification genes, and symbiotic efficiency. Conclusions Our results suggest that each Sinorhizobium strain uses a slightly different strategy to obtain maximum compatibility with a host plant. This large genome data set provides useful information to better understand the functional features of five Sinorhizobium species, especially compatibility in legume-Sinorhizobium interactions. The diversity of genes present in the accessory genomes of members of this genus indicates that each bacterium has adopted slightly different strategies to interact with diverse plant genera and soil environments.
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    Development of Fungal Bioreactors for Water Related Treatment and Disinfection Applications
    Umstead, Russell Blake (Virginia Tech, 2016-08-23)
    Wastewater, recycled irrigation water, and agricultural runoff can contain high levels of pathogenic bacteria, which pose a threat to human and ecosystem health. The use of a bioreactor containing mycelial mats of filamentous fungi is a novel treatment technology that incorporates physical, biological, and biochemical processes to remove bacterial pathogens from influent water. Although a relatively new concept, fungal bioreactors have demonstrated the ability to dramatically reduce fecal coliform bacteria in water, but no studies have attempted to explicitly identify the bacterial pathogen removal mechanisms exhibited by the fungi. This study evaluated several different species of fungi for use in fungal bioreactor systems and aimed to identify the modes of action responsible for the removal of bacterial pathogens. The species evaluated were Daedaleopsis confragosa, Pleurotus eryngii, and Piptoporus betulinus. Experimental results showed that all species of fungi assessed were capable of removing E. coli in a synthetic water solution. Significant concentrations of hydrogen peroxide, an antiseptic, were produced by all species of fungi evaluated. The fungal bioreactors containing P. eryngii produced the highest concentrations of hydrogen peroxide, generating a maximum concentration of 30.5 mg/l or 0.896 mM. This maximum value exceeds reported minimum concentrations required to demonstrate bacteriostatic and bactericidal effects when continually applied, providing evidence that a major bacterial removal mode of action is the production of antimicrobial compounds. In addition to its promising application to improve water quality, fungal bioreactors are a low cost and passive treatment technology. The development a hyper-functional system could be a have a substantial impact on the use of recycled irrigation water and on the water/wastewater treatment industry, for both municipal and agricultural wastewater.
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    Does it pay to be mature? Assessing the performance of a mature bioretention cell seven years post-construction
    Willard, Lory Lee (Virginia Tech, 2014-10-29)
    Bioretention cells (BRCs) are low-impact development stormwater management structures that integrate water quantity and quality management. Although BRCs have a predicted design life of about 25 years, most current research focuses on performance of cells less than two years old. This project evaluated the effectiveness of a BRC installed in 2007 to treat a 0.16-ha parking lot in Blacksburg, VA. After installation, this BRC was monitored for five months to determine initial flow reduction and total suspended solids, and nutrient removal. By monitoring for the same parameters, changes in cell performance since installation were quantified. ISCO automated stormwater samplers collected inflow and outflow composite samples from the cell, which were then analyzed for fecal indicator bacteria (total coliforms, E. coli, and enterococci), total suspended solids (TSS), total nitrogen (TN), and total phosphorus (TP). To determine if denitrification is occurring within the BRC, media samples taken throughout the cell were analyzed using qPCR. The bioretention media was also sampled to quantify changes in media nutrient content and particle size over the past seven years. Results indicate the bioretention media has not accumulated nitrogen and phosphorus since installation, and that the BRC remains effective at reducing flow volume and peak flow rates, as well as TSS, TN, TP, total coliforms, E. coli, and enterococci loads. Bacterial analysis of the media show most of the denitrifiers are present in the top layers of the bioretention media, despite an internal water storage layer and the bottom of the cell designed specifically for denitrification.
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    Effect of Water Chemistry, Pipe Material, Temperature and Flow on the Building Plumbing Microbiome and Opportunistic Pathogen Occurrence
    Ji, Pan (Virginia Tech, 2017-10-12)
    The building plumbing microbiome has important implications, especially in terms of its role as a reservoir and conduit for the spread of opportunistic pathogens (OPs), such as Legionella pneumophila. This dissertation applied next-generation DNA sequencing tools to survey the composition of building plumbing microbiomes and assessed hypothetical factors shaping them. A challenge to identifying key factors shaping building plumbing microbiomes is untangling the relative contributions of influent water quality, provided by drinking water utilities, and those of building-level features, such as pipe materials. To this end, standardized pipe rigs were deployed at the treatment plants and in distal portions of the water distribution system at five water utilities across the eastern U.S. Source water and treatment practices appeared to be the overarching factors shaping the microbial taxonomic composition at the tap, with five key water chemistry parameters identified (total chlorine, pH, P, SO42- and Mg2+). Hot water plumbing is of particular interest because OPs tend to proliferate in warm water environments and can be inhaled in aerosols when showering. Two identical lab-scale recirculating hot water rigs were operated in parallel to examine the combined effects of water heater temperature set point, pipe orientation, and water use frequency on the hot water plumbing microbiome. Our results revealed distinct microbial taxonomic compositions between the biofilm and water phases. Importantly, above a threshold of 51 °C, water heater temperature, pipe orientation, and water use frequency together incurred a prominent shift in microbiome composition and L. pneumophila occurrence. While heat shock is a popular means of remediating L. pneumophila contamination in plumbing, its broader effects on the microbiome are unknown. Here, heat shock was applied to acclimated lab-scale hot water rigs. Comparison of pre- versus post- heat shock samples indicated little to no change in either the microbial composition or L. pneumophila levels at the tap, where both water heater temperature and water use frequency had the most dominant effect. Overall, this dissertation contributes to advancing guidance regarding where to most effectively target controls for OPs and also advances research towards identifying the features of a 'healthy' built environment microbiome.
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    The Effects of dairy cattle antibiotics on soil microbial community cycling and antibiotic resistance
    Hedin, Matthew Lowell (Virginia Tech, 2018-05-11)
    Antibiotic use in agricultural ecosystems has the potential to increase resistance to antibiotics in soil microbial communities since 40-95% of an antibiotic dose administered to livestock is excreted intact or as metabolites. Exposure to antibiotics is also known to alter microbial community composition, biomass, and physiology, but the potential influences of antibiotic residues on the essential ecosystem processes that microbes regulate, e.g., carbon and nitrogen cycling are not well understood. I investigated the effects of antibiotic residues associated with dairy cattle operations on soil microbial communities and the ecosystem processes they regulate. I examined the effects of antibiotic exposure on the biogeochemical functioning of soil microbial communities by measuring the activity of extracellular enzymes associated with organic matter processing and nutrient mineralization in soils collected from dairy cattle operations across the United States. At each experimental station paired sites were identified by local managers that represented sites with high and low stocking rates of dairy cows who had been treated prophylactically with antibiotics to prevent mastitis. Responses varied among individual enzymes, but I found an overall significant decrease in total hydrolytic enzyme activity under high cattle stocking rates indicating a change in the functioning of the microbial community in soils exposed to antibiotic laden manure. Principle components analysis suggest that while some of the variation in enzyme activities are associated with the abundance of antibiotic resistance genes, soil organic matter (total organic, mineralizable, and particulate organic carbon) was the most significant variable accounting for differences in enzyme activities. This reflects an inherent challenge in studies of antibiotic exposure in agricultural landscapes: the difficulty of distinguishing direct effects of antibiotic residues from the organic matter and nutrient subsidy associated with manure applications. To address this concern I conducted a series of incubation experiments manipulating soils to isolate the influences of antibiotics, manure resource subsidies, and bovine microbiome inoculants into soils. Specifically, I examined soil respiration and antibiotic resistance gene counts using qPCR following treatment with cephapirin, pirilimycin and a positive and negative control. I found that pre-exposure to antibiotics and manure is important in modulating the response of microbial communities (soil respiration, and gene copy numbers of AmpC and TetO) to further antibiotic exposure. I conclude that antibiotics themselves have a direct effect on soil communities and their functioning that is additive to the effect of manure (i.e., as a resource subsidy). This effect is mediated by the history of previous exposure to antibiotics, i.e., cattle stocking density. These results suggest that antibiotic residues from dairy cattle operation may have significant effects on microbial communities and the biogeochemical cycling they regulate in agricultural ecosystems.
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    Effects of Freshwater Salinization and Associated Base Cations on Bacterial Ecology and Water Quality
    DeVilbiss, Stephen Edward (Virginia Tech, 2021-01-05)
    Anthropogenic freshwater salinization, which is caused by numerous human activities including agriculture, urbanization, and deicing, impacts an estimated 37% of the contiguous drainage area in the United States. High salt concentrations in brackish and marine environments (~1,500 – 60,000 µS cm-1) influence aquatic bacteria. Less is known about the effects of freshwater salt concentrations (≤ 1,500 µS cm-1) on bacterial ecology, despite the pervasiveness of freshwater salinization. Bacteria perform many fundamental ecosystem processes (e.g. biogeochemical cycling) and serve as indicators of human health risk from exposure to waterborne pathogens. Thus, to understand how salt pollution affects freshwater ecosystems, there is a critical need to understand how freshwater salinization is impacting bacterial ecology. Using a series of controlled mesocosm experiments, my objectives were to determine how (1) survival of fecal indicator bacteria (FIB), (2) the diversity of native freshwater bacterial communities, and (3) bacterial respiration and nutrient uptake rates responded across a freshwater salinity gradient of different salt profiles. Survival rates (t90) of Escherichia coli, the EPA recommended freshwater FIB, increased by over 200% as salinity increased from 30 to 1,500 µS cm-1. Survival rates were also significantly higher in water with elevated Mg2+ relative to other base cations, suggesting that different salt sources and ion profiles can have varied effects in FIB survival. Thus, freshwater salinization could cause accumulating concentrations of FIB even without increased loading, increasing the risk of bacterial impairment. Diversity of native bacterial communities also varied across a freshwater salinity gradient, with a general increase in species richness as salinity reached 1,500 µS cm-1. Community variability (β-diversity) was greatest at intermediate salinities of 125 – 350 µS cm-1 and decreased towards the upper and lower extremes (30 and 1,500 µS cm-1, respectively). These diversity patterns suggest that osmotic stress is an environmental filter, but filtering strength is lowest at intermediate salinities causing a change from more deterministic to more stochastic assembly mechanisms. Different salt types also produced distinct bacterial community structures. Lastly, bacterial respiration doubled as salinity increased to 350 – 800 µS cm-1, revealing a subsidy-stress response of bacterial respiration across a freshwater salinity gradient. Corresponding changes in nitrogen and phosphorus uptake increased N:P ratios in ambient water, especially in mesocosms with elevated Ca2+, which could affect nutrient limitation in salinized streams enriched with Ca2+. Bacterial community structure based on Bray-Curtis dissimilarity was not correlated to pairwise changes in respiration rates but was linked to net nitrogen and phosphorus uptake after five days. Collectively, these results establish that freshwater salinization alters bacterial ecology at the individual population, whole community, and ecosystem process scales. Further, different salt types (e.g., CaCl2, MgCl2, NaCl, KCl, sea salt) had varying effects on bacteria at all levels and should be considered when predicting the effects of salinization on freshwater ecosystems. Developing more nuanced salt management plans that consider not only amount, but different types, of salts in freshwaters could help improve our ability to predict human health risk from waterborne pathogens and mitigate/ reduce salinity-induced impacts to freshwater ecosystem processes and services.
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    Effects of Glyphosate Herbicide on Phytophthora cinnamomi and Mine Soil Microbial Communities
    Klopf, Sara K.; Holliday, Jason A.; Badgley, Brian D. (Virginia Tech. Powell River Project, 2019)
    The American Chestnut (Castanea denata) was once the dominant hardwood species within the forests of the Appalachians and an important resource for people and wildlife. In the early 1900s, a fungal blight (Cryphonectria parasitica) was introduced from imported ‘Japanese Giant’ nursery trees that caused topkill of American chestnuts (Tallamy 2007). Trees infected with C. parasitica die back, then continually resprout from the roots. Since 1983, the American Chestnut Foundation has been conducting a backcross breeding program to produce hybrid trees resistant to the blight, and more recently, various research institutions have been working to develop transgenic varieties of American chestnut which express genes that may give the trees resistance to the blight. Additional pathogens, such as the fungus-like oomycete which causes root rot (Phytophthora cinnamomi), have furthered threatened C. dentata, and research of transgenic American chestnuts has included the identification of genes that may provide resistance to P. cinnamomi.
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    Effects of land management and climate change on soil microbial communities in Appalachian forest ecosystems
    Osburn, Ernest D. (Virginia Tech, 2021-03-26)
    In terrestrial ecosystems, microorganisms are the dominant drivers of virtually all ecosystem processes, particularly cycling of carbon (C), nitrogen (N), and phosphorus (P). These microbial functions are critical for promoting ecosystem services that support human well-being, such as provisioning of clean drinking water, nitrogen retention, and carbon storage. In forests of the Appalachian region of the eastern US, these ecosystem services are threatened by multiple anthropogenic influences, including present and past land use activities (e.g., logging, conversion to agriculture) and climate change (e.g., intensifying droughts). However, despite the central importance of microbial communities in promoting ecosystem functions, impacts of land management and climate change on soil microorganisms remain poorly understood in the region. This dissertation seeks to address the following questions: 1) How does a new forest management practice, Rhododendron understory removal, influence the ecosystem functions of soil microbial communities? 2) Do historical land management activities have long-term legacy effects on the structure and ecosystem functions of soil microbial communities? And 3) Does historical land use influence responses of soil microbial communities to intensifying drought? In chapter 2, I show that experimental Rhododendron understory removal increased soil C and N availability, thereby promoting increased total microbial biomass. This increased microbial biomass resulted in elevated production of microbial extracellular enzymes, which increased rates of C and N cycling in soils following Rhododendron removal. In chapter 3, I examined soils across several historically disturbed and adjacent undisturbed reference forests and show that historical management activities, e.g., logging, conversion to agriculture, have long-term effects on soil microbial communities 4-8 decades after management activities occurred. These effects included increased bacterial diversity, increased relative abundance of r-selected bacterial taxa, and increased abundance of arbuscular mycorrhizal fungi. In chapter 4, I show that key soil biogeochemical processes, i.e., C mineralization, N mineralization, and nitrification, exhibit generally higher rates in historically disturbed forests relative to adjacent reference forests. Further, I attributed these changes in ecosystem process rates to changes in key aspects of microbial communities, including microbial biomass, extracellular enzyme activities, and bacterial r- vs K-selection. In chapter 5, I conducted a drought-rewetting experiment and show wide-ranging effects of experimental drought on soil microbial communities, including altered diversity, community composition, and shifts in the relative abundances of several specific taxa. Further, drought responses were particularly evident in soils from historically disturbed forests, indicating influences of land management on responses of soil communities to climate change. Finally, in chapter 6, I show that the experimental drought also influenced several ecosystem-scale properties of soils, including increased soil N pools and increased respiratory C loss. Overall, my dissertation reveals wide-ranging effects of anthropogenic activities on soil microorganisms and shows that microbial communities will influence forest responses to global change at the ecosystem scale.
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    Effects of Microbial Community Stress Response and Emerging Contaminants on Wastewater Treatment Plants
    Metch, Jacob W. (Virginia Tech, 2017-04-13)
    As the population in water stressed areas increases, it is critical that wastewater treatment plants (WWTPs) continue to replenish depleted water supplies, and serve as an alternative water source. WWTPs depend on microorganisms in activated sludge to remove pollutants from wastewater and therefore an understanding of how these microorganisms are affected by various conditions and pollutants is needed. Also, as consumer products and industrial processes evolve, so do the pollutants they discharge to wastewater. In order to keep pace with these changes, understanding the effects of emerging contaminants to WWTP processes is essential. The research herein assesses microbial community dynamics of the response of nitrifying microorganisms in activated sludge to variation in ammonia concentration and evaluates the impact of engineered nanoparticles on activated sludge microbial communities and other emerging pollutants, such as antibiotic resistance genes and disinfection by-products. In order to assess microbial community dynamics of the response of nitrifying microorganisms to removal of ammonia in the feed, nitrifying activated sludge reactors were operated at various relevant temperatures and the nitrifying microbial community was characterized using activity assays and bio-molecular techniques. We found that Nitrospira spp. were the dominant nitrifying microorganisms, exhibiting stable relative abundance across multiple trials and over a range of temperatures. These results indicate the possibility of comammox bacteria in the system and highlight the complexity of nitrifying microbial communities in activated sludge relative to past understanding. Both microbial and chemical impacts of engineered nanoparticles on WWTP processes were also investigated. Metagenomic analysis of DNA extracted from activated sludge sequencing batch reactors dosed with gold nanoparticles with varied surface coating and morphology indicated that nanoparticle morphology impacted the microbial community and antibiotic resistance gene content more than surface coating. However, nanoparticle fate was controlled by surface coating more than morphology. Disinfection by-product formation in the presence of nanoparticles during WWTP disinfection was assessed using silver, titanium dioxide, ceria, and zero valent iron nanoparticles. Silver nanoparticles were found to enhance trihalomethane formation, which was attributed to the citrate coating of the nanoparticles. These studies both raise concern over the relationship between engineered nanoparticles and other emerging concerns in WWTPs, and take a step towards informing nanoparticle design in a manner that limits their associated environmental impact.
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    Evaluating Campylobacter spp at the human-wildlife interface
    Medley, Sarah E. (Virginia Tech, 2019-11-05)
    Campylobacter spp. infections are an increasing global concern responsible for a significant burden of disease every year. Wildlife and domestic animals are considered important reservoirs, but little is known about host-factors driving pathogen infection dynamics in wild mammal populations. In countries like Botswana, there is significant spatial overlap between humans and wildlife with a large proportion of the population vulnerable to Campylobacter infection, making Botswana an ideal location to study these interactions. This thesis reviews mammalian wildlife species that have been identified as carriers of Campylobacter spp., identifies life-history traits (urban association, trophic level, and sociality) that may be driving Campylobacter infection, and utilizes banded mongoose (Mungos mungo) (n=201) as a study species to illuminate potential Campylobacter spp. transmission at the human-wildlife interface in northern Botswana. Results of the latter study suggest that human-landscapes are critical to C. jejuni infection in banded mongooses, as mongooses utilizing man-made structures as dens had significantly higher levels of C. jejuni than mongooses using natural dens (p=0.019). A similar association was found across all wild mammals with significantly greater number of urban dwelling species positive for C. jejuni than urban avoiders (p = 0.04). Omnivorous and social mammals were significantly associated with C. coli presence (p=0.04 and p<0.00 respectively), but not with C. jejuni indicating there may be important differences in transmission dynamics between Campylobacter species. These results suggest that landscape features and life-history traits can have important influences on Campylobacter species exposure and transmission dynamics in wildlife.
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    Evaluation of Digital PCR (dPCR) for the Quantification of Soil Nitrogen Turnover Bacteria in Wetland Mesocosms in Response to Season, Fertilization, and Plant Species Richness
    Shah, Parita Raj (Virginia Tech, 2019-02-11)
    Excess nutrients from nonpoint sources are an ongoing problem that is expected to worsen as population and fertilizer usage rise. Conventional centralized treatment systems are not well suited to address nonpoint source pollution. More distributed best management practices (BMPs) like constructed wetlands are a promising alternative and have been widely implemented in the US since the 1970's. Constructed wetlands are multi-functional systems that can effectively store and transform harmful contaminants using primarily natural processes. However, the removal of pollutants like nitrogen by wetlands is highly variable, likely due to a combination of factors such as plant species-specific assimilation behavior, the effects of plant communities on microbial diversity and function, and variable nitrogen inputs. In this study, the effect of plant species richness (i.e., number of plant species in a system) and seasonal nutrient loading (i.e., nitrogen fertilization) on the microbial community responsible for regulating nitrogen turnover in wetland mesocosm soils was investigated. The chip-based QuantStudio 3D digital PCR (QS3D dPCR) system was used to quantify ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), comammox, anammox, and denitrifiers. Principal component analysis (PCA) was used to identify dominant patterns in the microbial community and nitrogen species. Resampling-based analysis of variance (ANOVA) was used to assess statistical significance of any observed differences caused by nitrogen fertilization or plant species richness. Results indicated that fertilization or season, which was convolved with fertilization, was the dominant factor influencing the microbial community in the study environment (27% variance explained), as indicated by the disparate clustering of fall (fertilized) and spring (unfertilized) samples about principal component 1 (fall: negative PC1, spring: positive PC1). Because unplanted unfertilized controls sampled in November clustered within the season in which they were collected rather than with other unfertilized samples collected in May, season may have influenced microbial community shifts more than fertilization for unplanted systems. This finding should be interpreted cautiously, however, given the small number of unplanted unfertilized controls (N = 2) and the absence of similar controls in the planted systems. The most abundant bacterial groups detected in May (November) were AOB, nirK, anammox, and Nitrospira spp. NOB (AOB, anammox, Nitrospira spp. NOB, and nosZ). The effects of plant species richness were more nuanced, with greater richness significantly impacting the abundance of only a subset of bacterial groups (i.e., the nitrifying bacteria AOB, Nitrospira spp. NOB, and comammox, but not the denitrifying bacteria). Different relationships between richness and microbial abundance were observed in different seasonal nutrient loadings (i.e., interaction effects between richness and fertilization were detected for some bacterial groups).
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    Factors Affecting Denitrification Potential and the Microbial Ecology of Established Bioretention Cells Across the Eastern Mid-Atlantic Region
    Waller, Lucas John (Virginia Tech, 2016-06-30)
    Increases in impervious surfaces caused by urbanization has led to higher volumes and rates of stormwater runoff that transports urban pollutants directly into natural waterways. Bioretention cells (BRCs) are vegetated soil systems designed to intercept stormwater runoff and reduce loads of water and contaminants discharged to surface waters. Nitrogen removal efficiency is highly variable and improvements are constrained by a poor understanding of the physical, biological, and chemical processes that occur within a BRC. The objectives of this study are to characterize and quantify the microbial communities in a range of existing BRCs, and determine which design factors have the greatest impact on denitrification, a microbial process responsible for removing nitrogen from stormwater. We sampled 23 BRCs throughout MD, VA, and NC, and quantified patterns in populations of denitrifying bacteria, denitrification potential, and microbial community structure within the soil medium. We found the greatest denitrifier populations and denitrification potential in the upper layer of the soil medium, which does not coincide with the internal water storage zone that is engineered to harbor anaerobic conditions favorable to denitrifying bacteria at the bottom of recent BRC designs. Results indicate that BRC vegetative cover, soil media nitrogen, and organic carbon concentrations are among the variables that facilitate nitrifying and denitrifying bacteria populations in BRCs. Bacterial community composition was most different between the top and bottom samples of the BRCs while fungal community composition differed most by BRC vegetative cover. Both fungal and bacterial community compositions were influenced by nitrogen and carbon concentrations.
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    Genomic, transcriptomic, and metagenomic approaches for detecting fungal plant pathogens and investigating the molecular basis of fungal ice nucleation activity
    Yang, Shu (Virginia Tech, 2022-02-02)
    Fungi play important roles in various environments. Some of them infect plants and cause economically important diseases. However, many fungal pathogens cause similar symptoms or are even spread asymptomatically, making it difficult to identify them morphologically. Therefore, culture-independent, sequence-based diagnostic methods that can detect and identify fungi independently of the symptoms that they cause are desirable. Whole genome metagenomic sequencing has the potential to enable rapid diagnosis of plant diseases without culturing pathogens and designing pathogen-specific probes. In my study, the MinION nanopore sequencer, a portable single‐molecule sequencing platform developed by Oxford Nanopore Technologies, was employed to detect the fungus Calonectria pseudonaviculata (Cps), the causal agent of the devastating boxwood blight disease of the popular ornamental boxwood (Buxus spp.). Various DNA extraction methods and computational tools were compared. Detection was sensitive with an extremely low false positive rate for most methods. Therefore, metagenomic sequencing is a promising technology that could be implemented in routine diagnostics of fungal diseases. Other fungi may play important roles in the atmosphere because of their ice nucleation activity (INA). INA is the capacity of some particles to induce ice formation above the temperature that pure water freezes (-38°C). Importantly, INPs affect the ratio of ice crystals to liquid droplets in clouds, which in turn affects Earth's radiation balance and the intensity and frequency of precipitation. A few fungal species can produce ice nucleating particles (INPs) that cause ice formation at temperatures ≥ –10°C and they may be present in clouds. Two such fungal genera are Fusarium and Mortierella but little is known about their INPs and the genetic basis of their INA. In my study, F. avenaceum and M. alpina were examined in detail. INPs of both species were characterized and it was found that strains within both species varied in regards to the strength of INA. Whole genome sequencing and comparative genomic studies were then performed to identify putative INA genes. Differential expression analyses at different growth temperatures were also performed. INP properties of the two species shared similarities, both appearing to consist of secreted aggregates larger than 30 kDa. Low temperatures induced INA in both species. Lists of candidate INA genes were identified based on their presence in the strains with the strongest INA and/or induction of their expression at low temperatures and because they either encode secreted proteins or enzymes that produce other molecules known to have INA in other organisms. These genes can now be characterized further to help identify the fungal INA genes in both species. This can be expected to help increase our understanding of the role of fungal INA in the atmosphere.