Bruns, M. 1996. Nucleic acid probe analysis of autotrophic ammonia-oxidizer populations in soils. Ph.D. Dissertation, Michigan State University, East Lansing, Michigan, USA.
Nitrification, the microbial oxidation of ammonium to nitrate, leads to significant nitrogen (N) losses from soils. Nitrification rates in soils have been related to population sizes of ammonia-oxidizing bacteria, but population structure effects are poorly understood. This study employed nucleic acid-based methods to determine how ammonia oxidizer populations differed in cultivated, never-tilled, and successional soils. Hybridizations were conducted between genomic DNA from pure cultures and probes from Nitrosomonas europaea genes for ammonia monooxygenase and hydroxylamine oxidireductase. These probes produced 24 to 80% less hybridization signal in genomic DNA from four other ammonia oxidizers under low-stringency conditions. This indicated that N. europaea functional probes would not provide quantitative size estimates for heterogeneous populations. Sizes of ammonia oxidizer populations in soils were determined by Most Probable Number (MPN) enumeration in media containing 10, 100, or 2000 ppm NH4-N. MPN counts were consistently one-tenth lower in never-tilled soils than in cultivated soils. Population structures were compared by using PCR to amplify 16S rRNA genes of ammonia oxidizers from bacterial community DNA, cloning the PCR products, and comparing cloned sequences for percent similarity over a 318-base region. The 16S rRNA gene sequences obtained from never-tilled soils were four times more diverse than sequences obtained from cultivated soils. A phylogenetic tree showed that sequences from cultivated soils fell within one distinct cluster of Nitrosospira; sequences from never-tilled soils fell into two clusters of Nitrosospira and one of Nitrosomonas. Denaturing gradient gel electrophoresis (DGGE) patterns differed between samples from never-tilled and cultivated soils; patterns from samples from replicate plots of each treatment were very similar in each of two successive years. Probe hybridizations of DGGE blots showed that sequences from the single subgroup of Nitrosospira spp. predominated in samples from cultivated or fertilized successional soils; these were not detected in samples from never-tilled or unfertilized successional soils. Differences in populations were reflected in mean NH4-N concentrations that were tenfold higher in never-tilled soils that in cultivated soils. Different influential factors in these soils include pH, allelopathy, microsite distribution, or direct effects of cultivation and fertilization.
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