Graduate Thesis Or Dissertation
 

Community profiles of ammonia oxidizers across high-elevation forest-to-meadow transects

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  • 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.
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