Monday, November 13, 2006 - 3:00 PM

Denitrifying Bacteria from Potato Field Soils: Analysis of Denitrification Genes.

Claudia Goyer1, Catherine E. Dandie1, Sherri Henderson1, David Burton2, Bernie Zebarth1, and Jack T. Trevors3. (1) Agriculture and Agri-Food Canada, 850 Lincoln Road, P.O. Box 20280, Fredericton, NB E3B 4Z7, Canada, (2) Nova Scotia Agric. College, Truro, NS B2N 5E3, Canada, (3) University of Guelph, Edmund C. Bovey Building, Guelph, ON N1G 2W1, Canada

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas.  This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates.  Two different enrichment/isolation procedures were used, yielding different but overlapping denitrifier populations.  Nutrient broth enrichment yielded 31 isolates, mostly Pseudomonas sp. (21), plus Achromobacter (4), Bosea (4), Ensifer (1) and Sinorhizobium (1) sp. isolates.  Direct isolation on modified R2A yielded mostly Bacillus denitrifiers but also similar Pseudomonas sp. as obtained with nutrient broth, and eight other genera, including Chryseobacterium, Rhodanobacter, Staphylococcus and Stenotrophomonas.  Isolates obtained from nutrient broth enrichment were analysed further for denitrification genes by PCR.  The nitrous oxide reductase (nosZ) gene was found in all isolates.  The majority of isolates (~90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene.  Nitrite reductase genes (nirS and nirK) were amplifiable from 25 of the 31 isolates, and were segregated between species similar to previously-isolated denitrifiers.  Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains.  The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes.  Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.  Work is ongoing to characterise the denitrification genes in the novel Bacillus denitrifier isolates obtained in this study, as gene sequences involved in denitrification are poorly represented in current genetic databases.