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
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/28834 |
| Date | 02 July 2002 |
| Creators | Mintie, Ann |
| Contributors | Bottomley, Peter J. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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