Graduation date: 1997

Graduation date: 1992

In recent years considerable interest has been shown in the diversity of ammonia-oxidizing bacteria in soil communities. The majority of the research has been carried out in Northern Europe where soils have received high atmospheric inputs of nitrogen over the past two centuries. In contrast, although much work has been conducted on nitrogen cycling processes in nitrogen limited forest ecosystems in western North America, no studies have examined the characteristics of ammonia-oxidizing communities in those environments. I was interested in measuring nitrification potential along a high-elevation temperate meadow-to-forest gradient, and characterizing the ammonia-oxidizing communities along that gradient using both molecular and culturing methods. Two experimental sites (Lookout and Carpenter) were chosen in the H.J. Andrews Experimental Forest, located in the western Cascade Range of Oregon, at elevations of approximately 1500 meters. Although nitrification potential rates (NPRs) between sites were not significantly different (P=0.544), variation was observed both within and between sites for specific vegetation types. NPRs were significantly lower in forest (F) soil samples than in meadow (M) soil samples, averaging 5 and 2% of meadow NPRs at Lookout and Carpenter, respectively. In meadow soil samples, most probable number (MPN) population densities of ammonia-oxidizers ranged from 0.6 to 2.6 x 10⁴ cells gram⁻¹ of oven dry soil and 0.9 x 10³ to 1.1 x 10⁵ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. In forest soil samples, population densities ranged from undetectable to 1.1 x 10⁴ cells g⁻¹ OD soil, and 0.9 x 10² to 2.3 x 10³ cells g⁻¹ OD soil at Lookout and Carpenter, respectively. Microbial community DNA was amplified using primers to the ammonia monooxygenase subunit A. Terminal restriction fragments polymorphism analysis with three different restriction enzymes (CfoI, TaqI, and AluI) revealed community profiles dominated by Nitrosospira species. One fragment from CfoI (66 bp) and one fragment from AluI (392-bp) were prominent in 47 soil samples from both sites, and represented between 32 to 100% of the Genescan fragment analyses of PCR products. A full length fragment from AluI digests (491-bp), and three fragments from CfoI (68, 100, and 135- bp) were found sporadically in fewer soil sample T-RFLPs, and within those samples represented smaller percentages of total peak areas. The CfoI 135-bp fragment length was associated primarily with M and meadow/forest (M/F) soils where it was observed in approximately 58 and 100% of the respective transect locations. Eight isolates recovered from soil samples were analyzed using the same molecular methods as the field samples. The T-RFLP patterns of the isolates corresponded with many of those found in the community fingerprints. Four unique amoA sequences were identified among these isolates, including one that possessed the dominant T-RFLP amoA fingerprint in soil samples. This sequence shared 99.8% similarity with Nitrosospira sp. Ka4, a cluster 4 ammonia oxidizer isolated in Norway. Sequence analysis phylogenetically associated the other three isolates (with unique amoA sequences) near Nitrosospira sp. Nsp 1 and Nitrosospira briensis, both cluster 3 ammonia oxidizers. Cloning and sequencing of soil DNA confirmed that ammonia oxidizers with these amoA sequences were present in the soil samples. Two additional amoA sequences were identified in clones that were 95% similar and paraphylogenetically positioned between representatives of clusters 3 and 4. So far, these sequences have not been found in any of the isolates analyzed. / Graduation date: 2003

Mat-forming ectomycorrhizal (EcM) fungi represent a prevalent constituent of many temperate forest ecosystems and create dramatic changes in soil structure and chemistry. EcM mat soil have been shown to have increased microbial respiration rates and have been hypothesized to harbor unique assemblages of fungi and bacteria. The objectives of this dissertation were to characterize and examine the fungal and bacterial communities associated with EcM mats in old-growth forests of the H.J. Andrews Experimental Forest located in the Oregon Cascades. Additionally, this work assessed the application of traditional, emerging, and novel molecular sampling techniques for determining microbial communities of environmental samples. This research investigated the microbial communities associated with two common EcM mat genera found in old-growth Douglas fir stands in the Pacific Northwest; Piloderma (Atheliales, Basidiomycota) and Ramaria (Gomphales, Basidiomycota). Soil samples were collected from Piloderma and Ramaria mats and surrounding non- mat soil for molecular analysis of nucleic acids. First, a comparative study was conducted to determine the most appropriate rDNA molecular sampling technique for microbial community characterization. Two next-generation sequencing methods, Roche 454 pyrosequencing and Illumina-based environmental sequencing, the latter developed by the author, were compared to a more traditional sequencing approach, i.e., Sanger sequencing of clone libraries. These findings informed the subsequent sampling of the fungal ITS and bacterial 16S rDNA fragment with 454 pyrosequencing to determine the microbial communities within mat and non-mat soils. Second, this work utilized a pyrosequencing approach to explore fungal community structure in EcM mat and non-mat soils. This work concluded that differences in microbial communities do exist between Piloderma mat, Ramaria mat, and non-mat soils, but the differences are largely quantitative with relatively few distinct taxonomic shifts in microbial constituents. Piloderma, Ramaria and Russula, in addition to being the dominant taxa found on mycorrhizal root tips, were found to be the most abundant taxa in bulk soils within their respective mat types or non-mat sample. The background fungal communities within the EcM mats in this study exhibited considerable taxonomic overlap with the exception of Piloderma vs. non-mat comparisons; Russula species dominated nonmat soils but tended to be excluded or significantly underrepresented in Piloderma mats. Lastly, this study explored the bacterial communities associated with Piloderma and Ramaria mats using lower- coverage 454-Jr pyrosequencing. Bacterial communities exhibited significant structure as a function of mat-type, soil horizon and pH, but this finding should be interpreted with respect to the nonrandom distribution of Piloderma-mats in the O- horizon and the Ramaria-mats in the A-horizon, and the tendancy for EcM mats to be more acidic than surrounding soils. Nonetheless, the total microbial (bacterial and fungal) community was typically dominated by the mat-forming taxa, or Russula, in the case of non-mat soils. While the presence of Piloderma mats did enrich or restrict some bacterial groups, soil pH was also found to be a significant driver of bacterial richness and taxonomic diversity. Fungal and bacterial richness were also found to be positively related to one another, regardless of soil horizon or EcM mat type. This work, taken together, contributes to the understanding of hyperdiversity and heterogeneity of microbial communities of temperate forest soils and highlights the potential for fungal and bacterial communities to be influenced by the presence of EcM mats. / Graduation date: 2012