Human gastrointestinal (GI) tract inhabits huge amount of microorganisms which contribute to the immune response in the gut. An immune system disorder disease inflammatory bowel disease (IBD) was believed to be associated with several factors including gut bacteria, immunological responses, and genetic characteristic. Yet, the etiology of the IBD is not clear. Experiments on animal models and human samples had supported that commensal bacteria play an important role in the IBD pathogenesis. To better understand the pathogenesis of IBD, in this dissertation, bacteria rRNA gene populations were examined and associations related to the disease were analyzed in mouse model and human samples.

Evidences showed that several bacteria inculding Lachnospiraceae and Clostridium Ramosum were significant related to the colitis. It was not sure that those bacteria were the causal agents of the disorder in the immune system or their populations changed were the result of being targeted by the immune response disorder. Predominant bacteria in human mucosal tissue were examined in the disease sample comparing the healthy controls to address that the bacteria shift in the mucosal layer related to the IBD. Ruminococcus, also a member of Lachnospiraceae, was significant in the mucosal sample reduce in the disease subjects. A mucin-degrading bacteria Akkermansia was first found in this study related to the IBD. Faecalibacterium was only reduced specifically in Crohn's disease while keep similar lever in healthy control and Ulcerative colitis. Bacteria community two mouse model with different genetic background were investigate and different bacteria were presented in fecal and tissue samples.

Overall, consistent evidence in this study indicated bacteria levels of several species were reduced in the disease because of an immune response toward bacteria in this niche. This study provided a better clue to lead a greater understanding of the etiology of the IBD.

The research described in this dissertation examined peach replant disease and two nematode biological control systems using traditional plant pathology methods and molecular microbial ecology methods.

In Chapter 1, the experiments identified microbes associated with peach replant disease in soils with various levels of disease symptoms. To identify bacteria, fungi and oomycetes associated with the replant disease, culture and culture-independent analyses were performed on DNA extracted from plant roots. Among the most abundant bacterial operational taxonomic units, 27 were negatively correlated with peach top weights while 10 were positively correlated. Among the most abundant fungi and oomycetes, negative and positive associations were identified between P. vexans and Trichoderma spp. and peach top weights, respectively, and verified with sequence-selective quantitative PCR analyses.

In Chapter 2, the population dynamics between Dactylella oviparasitica and Heterodera schachtii were investigated. Higher initial D. oviparasitica populations were associated with lower final H. schachtii populations. Regression models showed that the initial densities of D. oviparasitica were only significant when predicting the final densities of H. schachtii J2 and eggs as well as fungal egg parasitism, while the initial densities of J2 were significant for all final H. schachtii measurements. H. schachtii-associated D. oviparasitica populations were greatly reduced in nematodes collected from soil compared to nematodes collected from roots. Finally, phylogenetic analysis of rRNA genes suggested that D. oviparasitica belongs to a clade of nematophagous fungi with a large geographical distribution.

In Chapter 3, three strains of Pochonia chlamydosporia var. chlamydosporia were genetically characterized and examined for their biocontrol efficacies against Meloidogyne incognita. All strains exhibited different patterns with the enterobacterial repetitive intergenic consensus (ERIC) PCR analysis. Strains 1 and 4 were similar in the PCR analyses of ß-tubulin and the rRNA internal transcribed spacer. In greenhouse trials, all strains reduced the numbers of nematode egg masses. Strain 4 reduced almost 50% of the eggs, and reduced the numbers of J2 and root-galling. A newly developed small subunit rRNA-based PCR analysis differentiated strain 4 from the others, and could potentially be used as a screening tool for identifying other effective biocontrol strains of P. chlamydosporia var. chlamydosporia.

Bacteria are present in nearly all terrestrial environments and play varied and important roles. Understanding their impacts on the environments and hosts where they reside is greatly aided by an accurate estimation of the number and types present. We have adapted polony technology to Oligonucleotide Fingerprinting of Ribosomal rRNA Genes (OFRG), a hybridization-based method for clustering similar 16S rDNA sequences. We present a new OFRG probe set design method that utilizes the available taxonomic information of training sequences to improve the clustering of fingerprints into biologically meaningful groups. A software tool is presented that quickly and accurately identifies randomly placed polonies in microarray images. The polony OFRG method is applied to DNA from a mock bacterial community created from a clone library, as well as to PCR amplicons made from the same mock community to examine PCR bias. We also examine several natural bacterial communities, making polonies starting directly from genomic DNA templates. The method successfully clusters the known bacterial community and reveals the presence of artifacts in template from the mock community PCR. Natural bacterial communities are differentiated using a weighted UniFrac analysis. Due to the initial spatial separation of sample DNA strands, polonies are essentially free of the PCR bias and chimeric sequence formation that occurs in mixed-template PCR reactions. An additional benefit of the polony format is that sequences of near full-length rDNA can be obtained when desired - a feature not possible with current high-throughput sequencing methods. We anticipate polony OFRG may be an invaluable tool for microbial population studies where these two characteristics are required.

Huanglongbing (HLB) is a significant global concern for citrus orchards. However, certain “Survivor Trees” have demonstrated tolerance and recovery from the disease, presenting potential avenues for HLB cures and prevention. This study delves into the mechanisms behind this resilience. My findings indicate that the Survivor Tree Phenotype might be primarily influenced by host-microbe interactions. Additionally, microbial dynamics reveal that certain microbes might exacerbate or mitigate HLB effects. Another key insight is the potential role of callose, a polysaccharide associated with defense mechanisms, which may interact with the putative pathogen 'Candidatus Liberibacter asiaticus' (CLas) and the enzyme β-1,3-glucanase from Streptomyces spp. to influence the Survivor Tree Phenotype. Transcriptomic analyses further highlight unique gene expression patterns related to disease resistance and callose synthesis. In sum, this study offers insights into HLB resilience, suggesting areas for further exploration, particularly around the interactions of callose, CLas, and Streptomyces, to enhance our understanding of HLB tolerance mechanisms.

Chapter 1. Bacteria have been implicated as contributing factors in colorectal cancer (CRC), yet identifying the causal bacteria has remained challenging. To identify such organisms, we examined the host-associated bacteria in an inflammatory bowel disease (IBD) mouse model (IL-10-/-) that exhibits increased levels of CRC and systemic DNA damage in the peripheral blood. High-throughput sequence analysis of 16S rRNA genes identified several intestinal bacteria that were differentially abundant between the IL-10-/- and control mice, including Lactobacillus johnsonii, Lactobacillus animalis, and Bacteroides vulgatus. These bacteria were tested for their ability to affect DNA damage in cell culture and animal experiments. Oral administration of the bacterium that was more abundant in the control mice (L. johnsonii) decreased systemic DNA damage in mice treated with dextran sodium sulfate (DSS) while the bacteria that were more abundant in the IL-10-/- mice increased DNA damage in cell culture (L. animalis, B. vulgatus) and wild type mice (L. animalis). We posit that examining the host-associated intestinal bacteria allowed us to identify these functionally important microorganisms, and we anticipate that this approach may be useful for identifying bacteria involved in other diseases where bacteria play a causal role.

Chapter 2. Obesity is becoming an epidemic that affects one-third of adults in the USA and which has multiple causes including genes, diet, activity level and intestinal bacteria. Using a diet-induced obesity model, we compared the host-associated intestinal bacteria of mice fed four different diets: a standard vivarium chow and three high-fat diets. Mice fed the vivarium chow gained significantly less weight over the course of the experiment compared to mice fed the high fat diets. By using a high-throughput sequence analysis of the rRNA-ITS gene region, we identified several bacteria, including E. faecium, L. animalis, SFB and C. innocuum, whose abundance varied by diet type. We predict that the intestinal bacteria that are higher in abundance in mice fed the high fat diets compared to mice fed the vivarium chow play a causative role in the diet-induced obesity observed in this experiment, and that the bacteria that are higher in abundance in mice fed the vivarium chow compared to mice fed the high fat diets inhibit obesity. Future Koch's postulates like experimentation using germ-free mice will be used to test these predictions.

This dissertation describes two agricultural disease microbiomes and the novel utilization of a Universal Assay primer to sequence all three domains of life. Chapter 1 reviews the current literature on Replant Disease, a multi-kingdom agricultural plant disease with no proven causal agents. In Chapter 2, the first fungal ITS sequencing of Coffee Potato Taste Defect disease highlights the importance of multiple domains of life in identifying causal agents in agricultural microbiome diseases. Coffee beans with varying levels of Potato Taste Defect were sequenced. This project identified four fungi as putative causal agents: Aspergillus versicolor, Penicillium cinnamopurpureum, Talaromyces radicus, and Thermomyces lanuginosus. Two putatively beneficial fungi were also identified: Clavispora lusitaniae and Kazachstania humilis. Chapter 3 introduces a novel Illumina amplicon assay designed to characterize organisms across all three domains of life: Archaea, Bacteria, and Eukarya. Samples from commonly studied microbiomes are used to illustrate the value of a single Illumina assay as a first look at complex microbiome questions. Environmental samples that are rich in some or all microbial kingdoms were chosen, including animal gut, plant roots, soil, and ocean water. Chapter 4 focuses on Walnut Replant Disease. Through the Universal Assay and qPCR, this chapter identifies the nematode Pratylenchus vulnus as a putative causal organism. A single Mucoromycota fungal ASV along with numerous bacteria are also significantly correlated to the disease.

The ability of Dactylella oviparasitica and Fusarium oxysporum to suppress Heterodera schachtii numbers was examined in field microplots. Fungi were individually added to fumigated field soil that was seeded with sugar beet. Four weeks later, soils were infested with H. schachtii second-stage juveniles (J2). At two harvests, 11 weeks and 19 weeks (1,469 and 2,547 degree days (base 8°C), respectively) after nematode infestation, H. schachtii cyst and egg numbers were assessed. At both time points, D. oviparasitica reduced H. schachtii population densities to those in the naturally suppressive soil, even when additional H. schachtii J2 were added to the microplots after the first harvest. Although F. oxysporum did not alter H. schachtii population densities after 11 weeks, significant reductions were detected after 19 weeks. The sustainability of the H. schachtii suppressiveness created by single applications of the fungi at the beginning of the microplot trials was further examined in a greenhouse study. Soil collected at the completion of the microplot trials was potted and seeded with sugar beet. Four weeks later, each pot was infested with H. schachtii J2. Approximately 16 weeks (1,389 degree days) after seeding, the D. oviparasitica-amended soil produced greater fresh root weights and considerably smaller nematode population densities than the nonamended control.