How Fungicide Alters the Hidden Mycobiome of a Restored Prairie System

Fungal Endophytes are microscopic fungi that live inside plant tissues and form a symbiotic relationship that influences the fitness of both parties. Fungicides are a widely used method of crop disease control in agriculture, but fungicides can be carried into other environments by water and wind. This experiment looks at how long-term fungicide exposure affects diversity of fungal endophytes that are grown in vitro as well as screens them for phosphate solubilization ability. Phosphate is a vital macronutrient that is essential for making nucleic acids (DNA, RNA) as well as playing a vital role in energy transfer throughout the plant\u27s cells. Phosphate solubility allows the plants to develop higher efficiency for water and nutrients use. Microbes that can solubilize phosphate help plants receive readily available phosphate

GEMS Student: Biofilm Production in Rhizobia Influences Clover Drought Response

Rhizobia serve as model system for examining how phenotypic changes in rhizobia influence the plant. Rhizobium-legume symbioses result in the formation of nodules on the root systems of host plants. Compositional and functional changes in microbial communities facilitate the host plants’ response to environmental stressors (i.e.; drought stress). Physiological effects of soil moisture on microbial communities result in specialized communities that can tolerate much low soil-moisture habitats while others are limited to high soil-moisture environments. This suggests microbial communities can assist in maintaining plant fitness when exposed to nonideal environmental conditions

Investigating the spatiotemporal dynamics of Bacillus subtilis community biofilms

In nature, microbial communities are composed of diverse, coexisting bacteria. The soil-dwelling bacterium Bacillus subtilis forms structured communities of cells encased in an extracellular matrix, known as biofilms. Within a biofilm, genetically identical B. subtilis cells can differentiate into phenotypically distinct subpopulations, which may serve as a survival mechanism during times of stress. B. subtilis also produces numerous specialized metabolites, some of which can induce B. subtilis cellular differentiation. However, the spatial arrangement and relationships of cell subpopulations and specialized metabolites remains generally unknown. Furthermore, how B. subtilis differentiation is altered in more complex, environmentally-relevant microbial communities is unclear. I hypothesize B. subtilis gene expression and spatial organization of individual phenotypes within biofilm communities is impacted by specialized metabolites and coculture with a second bacterium. The overall goals of my thesis project are to determine the spatial organization of B. subtilis phenotypes and establish the molecular determinants of B. subtilis community biofilms. Finally, my dissertation will lay the groundwork for manipulating biofilm structure and cellular differentiation in B. subtilis.Doctor of Philosoph

Post-termination Effects of Cover Crop Monocultures and Mixtures on Soil Inorganic Nitrogen and Microbial Communities on Two Organic Farms in Illinois

Cover crops can continue to affect agricultural systems even after they have been terminated by influencing nitrogen dynamics and by altering soil microbial communities. These post-termination effects can influence soil fertility, weed pressure, and the dynamics of potential plant pathogens in the narrow window of time between cover crop termination and cash crop emergence. We evaluated the post-termination effects of 12 different spring-sown cover crop mixtures and monocultures on soil nitrogen and microbial communities on two different organic farms in Central Illinois (on Lawson silt loam soil) and Northern Illinois (on Virgil silt loam soil). In comparison to control plots with no cover crops, all cover crop treatments significantly reduced soil nitrate levels but increased the potentially mineralizable nitrogen pool following termination. Nitrate levels of cover crop plots approached those of controls after 2 and 4 weeks, respectively, but potentially mineralizable nitrogen levels in cover plots remained elevated for at least 4 weeks following termination. Monocultures of Brassica cover crops showed the greatest decrease in soil nitrate, while Brassicas and unplanted control plots containing high biomass of weeds showed the greatest increase in potentially mineralizable nitrogen in comparison to plant-free control plots. In contrast to their effect on soil nitrogen, cover crops had very limited impact on the composition of soil microbial communities. Overall microbial community composition varied across sites and years, and only soil fungi significantly responded to cover cropping treatments. Nevertheless, we found that some highly correlated groups of soil microbes showed significant responses to soil nitrate and to high plant biomass. Key members of these correlated groups included ammonia-oxidizing organisms and saprotrophic fungi. Our results suggest that cover crops may reduce the potential for springtime nitrogen leaching losses by retaining nitrogen in the soil organic pool, and they may also have impacts on the soil microbial community that are particularly relevant for nitrogen cycling and decomposition of plant residues

Soil Bacteria and Fungi Respond on Different Spatial Scales to Invasion by the Legume Lespedeza cuneata

The spatial scale on which microbial communities respond to plant invasions may provide important clues as to the nature of potential invader–microbe interactions. Lespedeza cuneata (Dum. Cours.) G. Don is an invasive legume that may benefit from associations with mycorrhizal fungi; however, it has also been suggested that the plant is allelopathic and may alter the soil chemistry of invaded sites through secondary metabolites in its root exudates or litter. Thus, L. cuneata invasion may interact with soil microorganisms on a variety of scales. We investigated L. cuneata-related changes to soil bacterial and fungal communities at two spatial scales using multiple sites from across its invaded N. American range. Using whole-community DNA fingerprinting, we characterized microbial community variation at the scale of entire invaded sites and at the scale of individual plants. Based on permutational multivariate analysis of variance, soil bacterial communities in heavily invaded sites were significantly different from those of uninvaded sites, but bacteria did not show any evidence of responding at very local scales around individual plants. In contrast, soil fungi did not change significantly at the scale of entire sites, but there were significant differences between fungal communities of native versus exotic plants within particular sites. The differential scaling of bacterial and fungal responses indicates that L. cuneata interacts differently with soil bacteria and soil fungi, and these microorganisms may play very different roles in the invasion process of this plant

Expanding Molecular Coverage in Mass Spectrometry Imaging of Microbial Systems Using Metal-Assisted Laser Desorption/Ionization

Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial ( approximately 67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific bacteroidales Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 Bacteroidales 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific “Bacteroidales” Populations

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 “Bacteroidales” 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution

An Affinity–Effect Relationship for Microbial Communities in Plant–Soil Feedback Loops

Feedback loops involving soil microorganisms can regulate plant populations. Here, we hypothesize that microorganisms are most likely to play a role in plant–soil feedback loops when they possess an affinity for a particular plant and the capacity to consistently affect the growth of that plant for good or ill. We characterized microbial communities using whole-community DNA fingerprinting from multiple "home-and-away" experiments involving giant ragweed (Ambrosia trifida L.) and common sunflower (Helianthus annuus L.), and we looked for affinity–effect relationships in these microbial communities. Using canonical ordination and partial least squares regression, we developed indices expressing each microorganism's affinity for ragweed or sunflower and its putative effect on plant biomass, and we used linear regression to analyze the relationship between microbial affinity and effect. Significant linear affinity–effect relationships were found in 75% of cases. Affinity–effect relationships were stronger for ragweed than for sunflower, and ragweed affinity–effect relationships showed consistent potential for negative feedback loops. The ragweed feedback relationships indicated the potential involvement of multiple microbial taxa, resulting in strong, consistent affinity–effect relationships in spite of large-scale microbial variability between trials. In contrast, sunflower plant–soil feedback may involve just a few key players, making it more sensitive to underlying microbial variation. We propose that affinity–effect relationship can be used to determine key microbial players in plant–soil feedback against a low "signal-to-noise" background of complex microbial datasets