Browsing by Subject "Fungi"

This dissertation presents a framework to improve bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs). PAHs are of great concern because they are recalcitrant and toxic. PAHs enter the environment from a variety of sources such as incomplete combustion and coal tar distillation. The PAHs focused on in this dissertation have polluted soils as a result of creosote-based wood treatment operations that took place at Holcomb Creosote and Atlantic Wood Industries, Inc. (AWI) both of which are now classified as Superfund sites. There are numerous sites analogous to these two Superfund sites throughout the world which have been polluted through similar wood-treatment operations, as creosote was once industry’s foremost wood preservative.

There is room for existing PAH treatment options, which are mainly physical and chemical in nature, to be expanded to include more sustainable options. Commonly used technologies include excavation, in situ stabilization, and soil washing. Historically, bioremediation strategies relying on bacteria to transform pollutants have been challenged by the tight sorption of heavy- and middle-weight PAHs to soils, as this restricts aqueous phase transport required for bacterial degradation. Multiple studies have demonstrated fungi to be capable of degrading these inaccessible pollutants and other mixtures of hydrophobic pollutants (mycoremediation). Yet, when fungi have been introduced to polluted soils (mycoaugmentation), they have not been able to outcompete the native microbiota long enough to degrade the contaminants of concern over the long term. It is possible that a thorough characterization of the indigenous fungi at a given site may provide some insights into the development of targeted in situ mycoremediation strategies.

Although incorporating site microbes has been generally acknowledged as important for some time, the techniques enabling thorough assessment of microbial ecosystems are relatively new. Consequently, little is known about PAH-associated microbiomes in general, and even less is known about PAH-associated fungal communities. The work presented in this dissertation aims to address this knowledge gap by leveraging recent advances in high-throughput sequencing technology to design and validate targeted mycoremediation strategies. To this end, the overarching goal of this dissertation was to develop and test a framework for incorporating native fungi into a bioremediation strategy to expand such sustainable remediation options to sites where they have not been relevant in the past.

In the first aim of this dissertation research, advances in high-throughput sequencing were used to identify potential biostimulation targets in soils moderately polluted with PAHs. The next generation sequencing (NGS) platform, Illumina, was utilized to sequence the large sub-unit (LSU) gene commonly used as a marker gene in fungal community studies. Relationships were examined between concentrations of over 31 different polycyclic aromatic hydrocarbons and the pollutant-associated communities to test whether there were any fungi capable of tolerating high levels of these toxic contaminants. In this aim, fungal genera were identified that contained species closely related to known PAHs and petroleum hydrocarbon degraders. In all, this work identified 32 targets for biostimulation, based on Spearman rank correlations between prevalence and mid- and high-molecular weight PAHs. Ascomycetes were found to have higher levels of diversity than any other phylum in this subset of biostimulation targets. These data suggest that ascomycete fungi are more likely to be present in heavily polluted soils than basidiomycete fungi (which had previously been subjects of much interest). Overall, this work illustrates that polluted soils harbor fungal biostimulation targets, specifically within Ascomycota.

The second aim of this thesis research was to use the precision bioremediation assessment in highly polluted soils and then to evaluate a range of amendments with the goal of identifying strategies to stimulate the fungal communities that dominate these PAH-associated fungal communities. Here we applied the approach we fine-tuned in the first aim to the AWI soils, as these soils have some of the highest documented PAH-concentrations. Again, Ascomycota were found to be more prevalent in these soils, so an isolate obtained from AWI was used to compare alternative stimulation techniques between three substrates they are known to grow on: chitin, cellulose, and wood. We used anthracene degradation as a proxy for PAH degradation, which we monitored in sacrificial simplified bioreactors responding to the three amendments. T. harzianum is also known to have enzymes which degrade PAHs, but it is unknown which ecological role uses those enzymes, and thus which ecological role we should promote. T. harzianum was grown in the presence of chitin, cellulose, and wood as substrates in liquid culture with anthracene. Chitin was found to stimulate the highest anthracene removal, with a 0.1% (w/v) amendment resulting in ~93% degradation. While ~13% less than chitin, 1% (w/v) cellulose was also found to stimulate ~46% more anthracene degradation than wood, which had no improvement over the abiotic losses (~33% on average). This is notable because the “go to” method for stimulating fungi in the past has been wood supplements. This work provided insight into alternative stimulation strategies to target specific ecological roles that may better degrade PAHs in situ.

For the third and final aim of this dissertation research, the two most promising amendments were added with and without Trichoderma harzianum spores to test several mycoremediation treatment strategies in soil bioreactors and compare them with a (no carbon added) nutrient stimulation treatment. Pollutants were added as aged Atlantic Wood Industries soil delivering aged pollutants. Triplicate reactors from each treatment were sequenced at time zero, after two weeks, and after one month. At each sampling time, RNA was extracted, converted to cDNA, and submitted to Illumina MiSeq library preparation targeting the LSU region for fungal community analysis in addition to the V4 region of the 16S rDNA for bacterial community analysis. Statistical analyses using DESeq2 identified responders among the groups of reactors subjected to the different biostimulation treatments. Taxa from both the fungal and the bacterial communities responded differentially to the amendments. Fungi were found to comprise the majority of the significant responders. This work also found that mycoaugmented strains were not successful in establishing themselves as prominent members of the active community. This represents one of the earliest studies to directly measure mycoaugmentation failure. These data propose a hypothesis about functional redundancy inhibiting establishment of augmented fungi as already established fungi outcompete them for freshly added nutrients. Over 90% degradation was observed over the course of one month regardless of treatment-interestingly, the highest degradation was found in the nutrient amendment (no carbon added) treatment. These results show similar degradation across the soil bioreactors, yet different microbial growth, which supports the hypothesis that there is community-level functional redundancy and multiple metabolic food webs that result in the observed pollutant degradation.

Overall, this dissertation work demonstrates how significant advances in sequencing technology can be implemented in design and monitoring stages of bioremediation. This work also suggests that significant advances could be possible through the application of targeted metatranscriptomic analysis. Through incorporating such insights as described in this dissertation, this research brings the field of bioremediation one step closer to successfully engineering microbiomes to degrade contaminants of concern.

Symbioses have shaped our modern world, providing for the air we breathe; for the plant and animal diversity we celebrate; and for the functioning of ecosystems from the tops of mountains to the ocean floor. Here I study symbiosis using fungal bacterial interactions as a model for understanding symbiotic dynamics. In this dissertation I present interpretations of experimental data about fungal bacterial interactions that lend insight into dynamics of symbiotic establishment and consequences of long-term endosymbiosis. More specifically, I examine the interactions of a plant-associated zygomycete, Mortierella elongata, and its interactions with several Betaproteobacteria in the Burkholderiales. I used genome sequencing, comparative genomics, physiological assays, and time-lapse microfluidic videography to ask the following questions; How are bacterial fungal symbioses initiated? How do bacteria and fungi communicate? What resources do these microbes share? Are long-term symbioses essential for one or both partners? What are the impacts of removing long-term endosymbionts for fungal host physiology? What are the effects long-term fungal endosymbiosis on bacterial genome content?

In chapter 1 I present lessons learned from genome sequencing of fungus Mortierella elongata and its primary resident endosymbiont, Mycoavidus cysteinexigens. I tested the hypothesis that genome reduction is a commonality of eukaryotic endosymbionts, and that characteristic genes and pathways are impacted by gene loss and inactivation in endosymbionts. I found that compared to its free-living relatives, M. cysteinexigens has a highly reduced genome and has lost genes coding for the biosynthesis of amino acids and intermediates of glycolysis, among other metabolic pathways. I describe a method for clearing fungi of endosymbionts using antibiotics. I report comparative physiological data for the cleared and uncleared strains and draw conclusions about the nature of their interactions based on the behavior of the fungal host lacking the endosymbiont. I tested the hypothesis that sharing of fungal fatty acids underpins this symbiosis, as suggested by the genome sequences of both microbes. I found that when cleared of endosymbionts, M. elongata grows more rapidly and accumulates fatty acids that are likely used by M. cysteinexigens when present.

In chapter 2 I investigate the transcriptional control of fungal-endosymbiont phenotypes. I continued working with the cleared and uncleared strains developed in chapter 1 and quantified transcript abundance in each isolate. I assigned functions to differentially expressed genes by identifying homologues in the fungal genetic model organism Saccharomyces cerevisiae. I layered on transcriptional data to the patterns that emerged from comparative analyses in chapter 1to better understand fungal response to endosymbiosis. I showed that differential expression of conserved genes underpin the increases in growth and altered metabolism in M. elongata when cleared of M. cysteinexigens. I found that endosymbiont presence is associated with toggling of metabolic programs that result in resources more or less bioavailable to M. cysteinexigens based on metabolic capability predicted by genome annotation. I found that genes with homologues in mating pheromone perception pathways are differentially regulated in cleared isolates of M. elongata, and that this aspect of clearing is shared by other isolates of M. elongata when cleared of their bacterial endosymbionts.

In chapter 3 I examine dynamics of pre-symbiotic signaling events between fungi and bacteria using Mortierella elongata and a free-living bacterium, Burkholderia BT03. Using microbial growth assays and a suite of conditioned medias I showed that growth stimulation is mutual for fungi and bacteria, and that signaling leading up to symbiotic phenotypes involves multiple bi-directional signal exchanges. I designed and used a microfluidic platform along with plate based and liquid culture systems to compare fungal growth rates in response to conditioned medias. By extrapolating rates from microbial growth assays including M. elongata, Burkholderia BT03 and related microbes, I inferred directionality, order, conditionality, specificity, and nature of signal exchange leading to microbial growth stimulation in this system.

As a whole this thesis explores how comparative microbial genomics and phenotypic assays can provide mechanistic insight into symbiotic establishment and the effects of long-term symbioses. The results presented here provide novel insights into biotic and abiotic factors dictating symbiotic establishment. Second, they suggest long-term endosymbionts of eukaryotic cells experience convergent gene loss. Lastly they emphasize that long-term endosymbionts strongly impact host metabolism, and that host-microbe metabolic intertwining is a commonality of many symbioses. The use of a systems biology approach to generate comparative genomics data on multiple levels that enable insight into the consequences of fungal bacterial symbioses is a novel contribution for the field.

Fungal endophytes are ubiquitous inhabitants of plants and can have a wide range of effects on their hosts, from pathogenic to mutualistic. These fungal associates are important drivers of plant success and therefore contribute to plant community structure. The majority of endophyte studies have focused on seed plants, but in order to understand the dynamics of endophytes at the ecosystem scale, as well as the evolution of these fungal associations, investigations are also necessary in earlier-diverging clades of plants, such as the non-vascular bryophytes (mosses, liverworts, and hornworts). This dissertation presents a survey of the diversity of fungal endophytes found in the liverwort Marchantia polymorpha L. and develops a gnotobiotic experimental system for testing the effects of these fungi on their liverwort host. The survey reveals a diverse community of fungi in M. polymorpha, with some fungi that are associated with this host across geographically distant sites. The laboratory experiments demonstrate that culturable endophytes of M. polymorpha can, in isolation, cause positive, negative, or neutral effects on host success and that these effects change in response to nutrient levels and the presence of multiple endophytes. The experimental system developed in this dissertation has great potential in the growing field of plant microbiota research to answer questions that range in scale from molecular mechanisms to ecosystem function.

Rising atmospheric CO2 concentration, currently about 390 ppm, causes climate change and is expected to reach 500 ppm or higher this century due to human activities. Soils are the largest terrestrial pool of carbon, and changes in soil carbon storage due to plant and microbial activities could affect atmospheric CO2 levels. This dissertation studies soil carbon and microbial responses to an experimental preindustrial-to-future CO2 gradient (250-515 ppm) in a grassland ecosystem. Two contrasting soil types are studied in the gradient, providing insight on how natural ecosystem variation modifies CO2 effects.

Although total soil organic carbon (SOC) did not change with CO2 treatment after four growing seasons, fast-cycling SOC pools did respond to CO2, particularly in the black clay soil. Microbial biomass increased 18% and microbial activity increased 30% across the CO2 gradient in the black clay, but neither factor changed with CO2 in the sandy loam. Similarly a one-year laboratory soil incubation showed that a fast-cycling SOC pool increased 75% across the CO2 gradient in the black clay. Size fractionation of SOC showed that coarse POM-C, the youngest and most labile fraction, increased four-fold across the CO2 gradient in the black clay, while it increased 50% across the gradient in the sandy loam. CO2 enrichment in this grassland increased the fast-cycling soil organic carbon pool as in other elevated CO2 studies, but only in the black clay soil.

CO2 also induced changes in microbial community composition, and we explored the functional consequences in a microcosm experiment. Soil collected in the third growing season of CO2 treatment was used to inoculate Indiangrass seedlings grown in the lab. The elevated CO2 soil inoculum had higher microbial biomass C/N (C/N = 21) than the subambient CO2 soil inoculum (C/N = 16), suggesting a difference in community composition. Mean plant height in elevated CO2 soil inoculum (475 ppm) was 57% greater than in subambient CO2 soil inoculum (300 ppm), but the difference was not statistically significant. Similarly, total leaf N from plants in elevated CO2 soil was 28% greater on average than in subambient CO2 soil, but not significantly different. CO2-induced microbial effects on plant growth were either negligible or occurred at finer microbial taxonomic levels, making them difficult to resolve at the whole-community level.

Soil fungi decompose soil organic matter, and studying fungal community responses to CO2 could improve our understanding of soil carbon responses. We studied fungal communities in the CO2 gradient using Sanger sequencing and pyrosequencing of rDNA. As in our soil C study, fungal community responses to CO2 were mostly linear, and occurred mostly in the black clay soil. Fungal species richness increased linearly with CO2 treatment in the black clay. The relative abundance of Chytridiomycota (chytrids) increased linearly with CO2 in the black clay, while the relative abundance of Glomeromycota (arbuscular mycorrhizal fungi) increased linearly with CO2 in the sandy loam. Increased labile C availability at elevated CO2 and/or decreased inorganic N may explain the increase in fungal species richness and Chytridiomycota abundance in the black clay, while increased P limitation may explain the stimulation of Glomeromycota at elevated CO2 in the sandy loam. Across both soils, fungal species richness increased linearly with soil respiration, an index of decomposition rate (p = 0.01, R2 = 0.46). Adding fungal species may have improved decomposition efficiency, but it is also possible that species richness and decomposition increased due to another factor such as C quantity. Soil type strongly structured both fungal community and arbuscular mycorrhizal fungal community composition.

Together, these studies suggest that soil C and fungal community responses to CO2 were mostly linear, and were most apparent in the black clay soil. Soil type strongly influenced fungal community composition as well as which phyla responded to CO2. Therefore, soil type could be a useful addition to predictions of soil carbon and microbial responses to future CO2 levels.

The kingdom Fungi is one of the major groups of the plant microbiome(Hardoim et al., 2015; Vandenkoornhuyse et al., 2015; Peay et al., 2016). Of the various plant-fungus interactions, mycorrhizal fungi that form mutualistic associations with host plants are the best studied symbiotic system(Bonfante & Genre, 2010; van der Heijden et al., 2015). Fungal endophytes represent another major type of plant-fungus symbioses(Rodriguez et al., 2009; Porras-Alfaro & Bayman, 2011). Defined as endosymbionts inhabiting a wide range of plant and lichen hosts without causing obvious symptoms, endophytes are now considered both ubiquitous and hyperdiverse (Stone, 2004; Rodriguez et al., 2009; U'Ren et al., 2012). Yet most of these fungi have to be identified using a phylogenetic approach (Arnold et al., 2009; Gazis et al., 2012; Chen et al., 2015) and remain unknown at lower taxonomic ranks (e.g., genus and species) and undefined in terms of their function in their symptomless hosts(Arnold et al., 2003; Busby et al., 2016). It is now understood that some endophytes are capable of switching to pathogenic(Wipornpan Photita et al.; Ávarez-Loayza et al., 2011) or saprotrophic(U'Ren et al., 2010; Zuccaro et al., 2011; Kuo et al., 2014) modes, but the genetic mechanisms of these switches remain unexplored. Bryophytes are a major component of the vegetation in boreal and arctic regions, where ecosystems are most vulnerable to global climate change(Turetsky et al., 2012; Jassey et al., 2013). It has been proposed that early land plants adopted a terrestrial lifestyle with the help of fungi(Heckman et al., 2001; Field et al., 2015). Mosses do not have mutualistic fungal symbionts such as mycorrhizal fungi(Davey & Currah, 2006; Field et al., 2015), but they are known to harbor diverse fungal endophytes of uncertain functions(U'Ren et al., 2010; Davey et al., 2012; Davey et al., 2013). The growth form of the moss Dicranum scoparium provided an ideal system for studying functional transitions between endophytism and saprotrophism across a senescent gradient. My PhD thesis focuses on the evolutionary history (Chapter 1) and functionality (Chapter 2, 3) of endophytic fungi.

In Chapter 1, I investigated the phylogenetic placements of fungal endophytes within the pharmaceutically and agriculturally important class Eurotiomycetes. The class Eurotiomycetes (Pezizomycotina, Ascomycota) includes various fungi with different ecological traits, including animal pathogens, saprotrophs, ectomycorrhizae, plant pathogens, rock-inhabiting fungi, lichens and endophytes(Geiser et al., 2006; Schoch et al., 2009; Gueidan et al., 2015). Phylogenetic affiliations of eurotiomycetous fungal endophytes with their ecologically diverse relatives had not been evaluated, leaving a gap in our understanding of the major evolutionary trends and ecological breadth of Eurotiomycetes as a whole. To fill this gap, we recently inferred the phylogenetic and taxonomic affinities of representatives of class 3 endophytes within Eurotiomycetes (Chen et al., 2015). Our results based on seven loci and 157 taxa revealed an undescribed new order (Phaeomoniellales) composed mainly of fungal endophytes and plant pathogens, and to a lesser extent, endolichenic and lichen-forming fungi. However, most of the deep nodes within this order were poorly supported. Interestingly, while described species of the order Phaeomoniellales are mostly plant pathogens on angiosperms (e.g., Genera Vitis, Nephelium and Prunus(Groenewald et al., 2001; Damm U. et al., 2010; Rossman et al., 2010; Thambugala et al., 2014)), endophytes within this order were mostly isolated from leaves of gymnosperms (Fig.1). These results, first-authored by the Co-PI, have been published in the journal Molecular Phylogenetic and Evolution(Chen et al., 2015).

In Chapter 2, I used metatranscriptomes of fungal ribosomal RNA to detect active fungal communities across a gradual gradient of senescence in wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution and the active component of fungal communities at a given time in adjacent living, senescing, and dead tissues. My results suggested that Ascomycota generally were more prevalent and active in living tissues, whereas Basidiomycota were more prevalent and active in senescing and dead tissues. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of metatranscriptomics and amplicon sequencing of cDNA is promising for studying symbiotic systems with complex microbial diversity, allowing simultaneous detection of microbial presence, abundance and metabolic activity in symbiotic systems.

In Chpater3, I investigated the functions of D. scoparium across its naturally occurring senescence gradient and the associated fungal nutrient transporter (carbon, amino acid, phosphorus and nitrogen) activities. Higher fungal nutrient-related transporter activities were detected toward the bottom layer of the moss gametophytes. Among the four fungal nutrient types (Amino acid, carbon, nitrogen, phosphorus), the activities of nitrogen-related transporters had a drastic increase proportionally toward the bottom layer. In parallel, nitrogen breakdown was detected as the most enriched Gene Ontology term of D. scoparium for those transcripts having higher expression in the bottom layers. I analyzed the most abundant fungal nitrogen-related transporters in my dataset, the ammonium transporters, using a phylogenetic approach. I revealed that all ammonium transporters actively expressed in association with D. scoparium belong to the MEPg clade. Different sets of potential plant-microbe communication/defense/symbiosis-related genes are highly expressed in top vs. bottom layers, which suggest different mechanisms are involved in plant-fungus associations in photosynthetic vs. decomposing tissues.

Polycyclic aromatic hydrocarbons (PAHs) are a class of over 100 chemicals found at various EPA Superfund sites across the United States formed through the incomplete combustion of organic compounds. These environmental contaminants are of concern due to their carcinogenicity, mutagenicity, and teratogenicity. Bioremediation using microorganisms is an economically efficient and environmentally sustainable process for PAH transformation in the environment. However, bacterial bioremediation schemes are limited in their ability to transform high molecular weight (HMW) PAHs. Through exploiting non-specific extracellular fungal enzymes in a mixed fungal-bacterial consortia, HMW PAHs have the ability to become bioavailable to bacteria, and ultimately be transformed. This approach is not widely used in the field of bioremediation, as there is limited understanding around interkingdom relationships between bacteria and fungi. There is limited knowledge of the array of extracellular enzymes that may assist with the transformation of PAHs, and whether these enzymes can be biostimulated or antagonized to increase the removal of PAHs in the environment. Additionally, a knowledge gap exists with regards to the survivability of introduced fungal and bacterial isolates, and whether a mixed fungal-bacteria consortia will provide an advantage in soil communities. This dissertation work will focus on developing a joint fungal-bacterial consortia that will exploit the cross-kingdom interactions of fungi and bacteria to assist in the removal and transformation of PAHs. This will be examined through isolating and identifying fungal isolates from creosote contaminated soil and assessing them for their ability to be biostimulated to produce extracellular enzymes that can be analogized for PAH transformation. Once determined, fungal and bacterial isolates will be assessed together for their ability to produce a joint biofilm. Lastly, fungal, and bacterial isolates will be assessed for their transformation capability and survivability amongst the microflora community in PAH soils.

This dissertation's first research objective was to identify and characterize indigenous fungi for a biostimulation/bioaugmentation scheme targeting PAHs. The goal of this objective was to find promising indigenous fungal isolates for further investigation in the transformation of PAHs. First, creosote contaminated soil was diluted and plated onto an array of growth agars to provide a representative overview of the fungal soil community. Fungi were isolated, cultured, and screened for the enzymatic production of laccase (Lac), manganese peroxidase (MnP), and tannase, along with the ability to degrade cellulose and starch. Fungal isolates were then incubated alongside complex amendments and Lac production will be measured through absorbance. Fungal isolates and complex amendments were lastly incubated alongside PAHs to measure transformation. From this objective, it was found that wide variety of fungal isolates native to creosote contaminated sediment were found to be able to produce extracellular enzymes that may assist in the biotransformation of PAHs. It was also found that complex amendments may also be used to increase extracellular enzymatic production to assist with PAH transformation. The complex grasshopper amendment is shown to be the most promising complex amendment to use in a future scheme.

This dissertation's second objective was to observe and characterize intra- and interkingdom behaviors in a mixed fungal-bacterial consortium for the improved transformation of PAHs. This research goal aimed to investigate the interactions between fungi and bacteria that could aid in the transformation and degradation of PAHs, as well as understand the potential for antagonistic and mutualistic interactions within soil communities. In this objective, a series of fungal permutations were performed from a select group of previously isolated indigenous fungi. The fungal permutations were visually assessed for unique growth patterns and assessed for changes in enzymatic functions. Permutations were then examined in the presence of a complex amendment to compare enzymatic production of fungal consortia to monoculture isolates. A select group of fungal isolates were then incubated alongside PAH degrading bacteria and examined for biofilm formation using optical density. Finally, fungal-bacterial mixed consortia and select model PAHs were incubated with the model complex amendment and measured for transformation capability. It was found that fungi will develop different morphological responses in the presence of other isolates. From this objective it was found that a mixed consortia between Novosphingobium pentaromativorans and filamentous fungi such as Penicilium.513 and Trichoderma.508 were deemed to be more advantageous for bacterial cell adhesion than yeast and yeast-like fungi such as Scheffersomyces.502 and Aureobasidium.509. When placed against model PAHs: FLA, PHE, PYR, and BaP, it was found that nearly all the fungal isolates saw improvements in their ability to degrade when placed alongside the bacteria. Additionally, when co-cultures were provided with a grasshopper amendment, increases in PAH degradation were observed.

The final objective of this dissertation work was to assess the performance of a mixed fungal bacterial consortia in a PAH-spiked soil microflora community. The purpose of this research objective is to assess the viability of a mixed fungal bacterial consortia in a soil reactor that mimics field conditions. Reactors were developed to assess community changes in PAH-spiked soil and non-spiked soil. Reactors were also inoculated with either monocultures or cocultures to explore how each variation affects the microbial community over time. Reactors were also biostimulated with a complex grasshopper amendment. Over a 60-day period, reactors were sacrificed at various points. RNA was extracted out of the soil at each time point and used to analyze the bacterial and fungal communities. Furthermore, soil samples were evaluated for PAH degradation at every time interval. No correlation between mixed fungal-bacterial consortia and PAH degradation was observed based on this objective. In comparison to their naturally attenuated counterparts, soil reactors that were subjected to biostimulation, bioaugmentation, and biostimulation-bioaugmentation analyses did not demonstrate appreciable variations in community structure.

This dissertation work will help engineers and researchers create efficient and sustainable PAH remediation strategies. This dissertation addresses long-standing knowledge gaps in mixed consortia fungal behavior and fungal-bacterial interactions. This work provides a framework for future investigations into cross-kingdom interaction, improved bioaugmentation methods, and optimized biostimulation for target organisms.

The Piedmont region of the southeastern United States has undergone considerable land-use change since settlement by Europeans and Africans. Forests were cleared for agriculture, followed centuries later by land abandonment. Following abandonment, natural recruitment, plantings for erosion control, and plantation forestry have resulted in a large area of the region covered by loblolly pine, Pinus taeda. Today, the Piedmont is a mosaic of farm fields, pastures, pine forests, and relic woodlots. The Calhoun Experimental Forest, located in Union County, SC, has provided a unique history of land use change's alteration of soil properties and processes, the ability of reforestation to restore or deplete soil fertility, and provided insights into the effects this change has on biological diversity.

In this work, the diversity of fungi living in soil is examined in the context of land-use change and soil biogeochemical change in and around the Calhoun Forest. This study uses molecular tools to identify fungal species from soil and to identify mycorrhizal associates of loblolly pine in a bioassay of propagule diversity, and proposes a novel use of quantitative PCR to quantify the relative abundance of major fungal families affected by land-use change.

Fungal diversity in soils is high in all land uses, but fungal communities shift from agricultural field communities largely comprised of unicellular ascomycetes and basal lineages to forest communities dominated by saprophytic and symbiotic basidiomycetes. In addition to this shift across a land use gradient, fungal communities are also responding to changes in carbon quantity and quality, biologically available nitrogen and phosphorus, pH, acidity and texture.

ECM propagule communities also differ across a land use gradient of cultivated fields, grasslands, pine forests, and mixed hardwood stands. There are few ECM propagules able to associate with loblolly pine in cultivated and grassland soils. There is a trend towards higher ECM diversity in the hardwood and pine soils, and both of those soil communities are distinct from each other as well as from soils from field treatments.

Quantitative PCR, coupled with a nested set of taxon-specific, fungal primers, is a potential way to estimate the abundance of the given taxon relative to all fungi in an environmental DNA. Primers specific to several taxonomic level of fungi were tested to confirm amplification in PCR, then were tested for taxonomic specificity by generating clone libraries with environmental DNA. Several of the successful primers were tested with soil DNA extracts in QPCR and the calculated ratios of fungal abundance varied widely by method of analysis. The results suggest that many repeated measurements and many replicates are required for a robust estimate of the relative abundance of a specific taxon.

Although the majority of the population carries Candida spp as normal components of their microflora, these species are important human pathogens that have the ability to cause disease under conditions of immunosuppression or altered host defenses. The spectrum of disease caused by these species ranges from cutaneous infections of the skin, mouth, esophagus and vagina, to life-threatening systemic disease. Despite increases in drug resistance, the antifungal armamentarium has changed little over the past decade. Thus increasing our understanding of the life cycles of these organisms, not only how they propagate themselves, but also how genetic diversity is created within the population is of considerable import. Additionally expanding our knowledge of key signal transduction cascades that are important for cell survival and response to stress will add in developing new antifungal therapies and strategies.

This thesis addresses both of these key areas of fungal pathogenesis. In the first chapter, we use genome comparisons between parasexual, asexual, and sexual species of pathogenic Candida as a first approximation to answer the question of whether examining genome content alone can allow us to understand why species have a particular life cycle. We start by examining the structure of the mating type locus (MAT) of two sexual species C. lusitaniae and C. guilliermondii. Interestingly, both species are missing either one or two (respectively) canonical transcription factors suggesting that the control of sexual identity and meiosis in these organisms has been significantly rewired. Mutant analysis of the retained transcription factors is used to understand how sexual identity and sporulation are controlled in these strains. Secondly, based on the observation that these species are missing many key genes involved in mating and meiosis, we use meiotic mapping, SPO11 mutant analysis, and comparative genome hybridization to demonstrate that these species are indeed meiotic, but that the meiosis that occurs is occasional unfaithful generating aneuploid and diploid progeny.

In the second and third chapters we examine the calcineurin signaling pathway, which is crucial for mediated tolerance to cellular stresses including cations, azole antifungals, and passage through the host bloodstream. First, we show that clinical use of calcineurin inhibitors in combination with azole antifungals does not result in resistance to the combination, suggesting that if non-immunosuppressive analogs could be further developed this combinatorial strategy may have great clinical efficacy. Second, we use previous studies of the calcineurin signaling pathway in S. cerevisiae to direct a candidate gene approach for elucidating other components of this pathway in C. albicans. Specifically, we identify homologs of the RCN1, MID1, and CCH1 genes, and use a combination of phenotypic assays and heterologous expression studies to understand the roles of these proteins in C. albicans. Although the mutant strains share some phenotypic properties with calcineurin deletion strains, none completely recapitulate a calcineurin mutant.

In the last chapter, we examine the plausibility of targeting the homoserine dehyrogenase (Hom6) protein in C. albicans and C. glabrata as a novel antifungal strategy. Studies in S. cerevisiae had demonstrated a synthetic lethality between hom6 and fpr1, the gene encoding FKBP12 a prolyl-isomerase that is the binding target of the immunosuppressant FK506. Thiss synthetic lethality was due to the buildup of a toxic intermediate in the methionine and threonine biosynthetic pathway as a result of deletion of hom6 and inhibition of FKBP12. We deleted HOM6 from both C. albicans and the more highly drug-resistant species C. glabrata. Studies suggest that regulation of the threonine and methionine biosynthetic pathway in C. albicans has been rewired such that the synthetic lethality between hom6 and FKBP12 inhibition no longer exists. However, in C. glabrata preliminary analysis suggest that similarly to S. cerevisiae hom6 and inhibition of FKBP12 can result in cell death.

Eukaryotic human pathogens present a serious threat to global health, causing hundreds of millions of infections with high death rate each year. Fungi and protozoa are two major classes of eukaryotic pathogens. Fungi Cryptococcus neoformans, Candida albicans, and protozoa Plasmodium falciparum are important pathogens from these classes. Although the therapeutics treating infections caused by these species are available, the options of front-line drugs are limited and the drug resistance is emerging and spreading. Therefore, there is a need for new therapeutics. Protein prenylation catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase) is essential to the survival of Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum. The previous biophysical and biochemical studies of FTase and GGTase from these species illustrate their divergence from the human enzymes, providing opportunities to develop species specific FTase or GGTase inhibitors for treating infectious diseases.In this dissertation, we choose to target FTases from Cryptococcus neoformans, Candida albicans, and Plasmodium falciparum by repurposing and derivatizing the well-studied human FTase inhibitors. We first derivatized human FTase inhibitor L-778,123, leading to a novel compound that shows potent inhibition of Cryptococcus neoformans growth with MIC value of 3 µM. The IC50 of the compound is 130 nM in the presence of physiological concentration of phosphate. Crystal structures of the compound bound to Cryptococcus neoformans FTase (CnFTase) shows a distinct binding mode from the starting compound, explaining the inhibition mechanism. Additionally, the compound does not exhibit significant mammalian cell toxicity up to 200 µM in cell based assays. We also derivatized and evaluated another human FTase inhibitor Tipifarnib. The derivatives showed the improved antifungal activity against Cryptococcus neoformans and Candida albicans. Finally, we have developed a new system to produce Plasmodium falciparum FTase for future inhibitor development. The data present in this dissertation could advance the future development of novel treatment for infections caused by eukaryotic human pathogens. Additionally, we report two protein engineering studies. The first addresses stability and overexpression of the telomerase riboprotein complex. Here we engineered the catalytic core complex and the RNA binding domain, and evaluated the capability of using these materials for inhibitor development. In the second study, an intein was inserted into DNA polymerases to produce temperature controlled enzymes. The intein controlled DNA polymerases only showed activities after intein splicing triggered by high temperature (>60oC), enabling the capability of conducting “hot-start” reactions by themselves. We demonstrated that using intein controlled DNA polymerases could reduce the nonspecific amplifications in PCR reactions.

This dissertation is an investigation of the systematics, phylogeography, and ecology of a globally distributed fungal family, the Elaphomycetaceae. In Chapter 1, we assess the literature on fungal phylogeography, reviewing large-scale phylogenetics studies and performing a meta-data analysis of fungal population genetics. In particular, we examined the possible effects of asexuality, trophic niche, dispersal method, and ocean barriers on population structure. In Chapter 2, we examine the systematics and phylogeography of the Elaphomycetaceae, a family consisting of the truffle genus Elaphomyces and the stalked genus Pseudotulostoma, hypothesizing that the mammal-dispersed truffle would show evidence of dispersal limitation. Using DNA sequence data, we determined that Pseudotulostoma is derived from a lineage of Elaphomyces, indicating that Elaphomyces as currently defined is paraphyletic. The distribution of each subgenus of Elaphomyces is nearly global; representative species have been found on every continent save Africa and Antarctica. This biogeographic pattern does not follow the pattern expected by a scenario of continental vicariance. Dating analysis in BEAST confirmed that broadly distributed clades are, in most cases, too young for this pattern to be explained by continental vicariance, indicating that occasional long-distance dispersal has been a significant component in the biogeographic history of the Elaphomycetaceae. This finding contradicts our initial hypothesis that the mammal-dispersed truffles would be dispersal- limited. In Chapter 3, we investigate the role of Elaphomyces as a host for the fungal parasite Elaphocordyceps, a parasite derived from insect pathogens that attacks both insect larvae and Elaphomyces, its only fungal host. We examined the biogeography of Elaphocordyceps isolated from Elaphomyces specimens in order to test whether it, like its host, showed recent connections between the Southern and Northern Hemispheres. We also evaluated the pathogenicity of infection as determined by a visual rubric for the truffle gleba, the phylogenetic distribution of Elaphocordyceps species on its host, testing for seasonal, climate, and host-parasite effects. In Chapter 4, based on the phylogeographic pattern seen in Elaphomyces that resembles that of some air-dispersed fungi, we used theoretical and experimental methods to test whether Elaphomyces could be dispersed by air. We tested the capacity for air dispersal with an experimental test of passive air dispersal on the powdery spores of Elaphomyces morettii and found that these large spores could disperse over a short distance (10 m) in comparable numbers with the spores of the giant puffball Calvatia cyathiformis, which is known to be air-dispersed. The major findings of this thesis are that 1) fungi in general show high dispersal ability, but that trophic niche and dispersal mode may affect population structure, 2) that Pseudotulostoma, a stalked genus, is derived from the truffle Elaphomyces, 3) that the Elaphomycetaceae have experienced frequent long-distance dispersal despite 4) that the fitness of Elaphomyces as indicated by glebal development varies with host-parasite interactions based on species identity, but not with climate or season, and that 5) Elaphomyces spores, should they be released into the air, can remain in the air long enough to be dispersed long distances by the wind. The overall conclusion of this thesis is that, while Elaphomyces is clearly reliant on animal vectors for excavation and dispersal, its past history of long-distance dispersal and current spore trajectories indicate it can be passively air-dispersed as well.

Fungal pathogens likely play an important role in regulating populations of tree seedlings and preserving forest diversity, due to their ubiquitous presence and differential effects on survival. Host-specific mortality from natural enemies is one of the most widely tested hypotheses in community ecology to explain the high biodiversity of forests. The effects of fungal pathogens on seedling survival are usually discussed under the framework of the Janzen-Connell (JC) hypothesis, which posits that seedlings are more likely to survive when dispersed far from the parent tree or at low densities due to pressure from host-specific pathogens (Janzen 1970, Connell 1971). One of the key challenges to assessing the importance of JC effects has been to identify and quantify the effects of the large numbers of potential pathogens required to maintain host diversity. The primary objectives of this research were to (1) characterize the fungi associated with seedling disease and mortality for a number of important southeastern US forest tree species; and (2) determine if these associations are consistent with the Janzen-Connell hypothesis in terms of differential effects on seedling survival.

Culture-based methods and ribosomal DNA (rDNA) sequencing were used to characterize the fungal community in recently dead and live seedlings of thirteen common tree species in a temperate mixed hardwood forest (North Carolina, USA), with the goal of identifying putative seedling pathogens. Cultures were initially classified and grouped into 130 operational taxonomic units (OTUs) using 96% internal transcribed spacer (ITS) sequence similarity; 46% of all OTUs were found only once. Using rarefaction, it was concluded that the richness of the system was not fully sampled and likely included over 200 taxa (based on non-parametric richness estimators). Species richness did not differ between sampling sites or among the five most common hosts sampled. The large ribosomal subunit (LSU) region of rDNA was then sequenced for representative samples of common OTUs and refined identifications using a constrained maximum likelihood phylogenetic analysis. Phylogenetic placement verified strong BLAST classifications, and allowed for placement of unknown taxa to the order level, with many of these unknowns placed in the Leotiomycetes and Xylariales (Sordariomycetes).

Next, a hierarchical Bayesian model was developed to predict the effects of multiple putative fungal pathogens on individual seedling survival, without forcing the effects of multiple fungi to be additive. The process of disease was partitioned into a chain of events including incidence, infection, detection, and survival, and conditional probabilities were used to quantify each component individually, but in the context of one another. The use of this modeling approach was illustrated by examining the effects of two putative fungal pathogens, Colletotrichum acutatum and Cylindrocarpon sp. A, an undescribed species of Cylindrocarpon, on the survival of five seedling hosts in both a maximum likelihood and Bayesian framework.

Finally, the model was used to assess the impacts of these fungi on seedling survival, alone and in combination, using data on five potential fungal pathogens and five hosts. Multi-host fungi had differential effects on seedling survival depending on host identity, and multiple infections may impact survival even when single infections do not. Evaluating these interactions among multiple plant and fungal species generates a set of targeted hypotheses of specific plant-fungal combinations that could help us better understand pathogen-driven diversity maintenance at larger scales than previously possible. Building on these results, some recommendations are provided as to how the Janzen-Connell hypothesis can be re-evaluated with respect to host specificity, pathogen distribution, and environmental context.