Quantification of Specific Denitrifier Populations in Soil Using Real-Time PCR.
Catherine E. Dandie1, Claudia Goyer1, 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
The role of the size and activity of bacterial populations in controlling denitrification and nitrous oxide emissions is poorly understood. The objective of this study is to develop primer sets for quantification of denitrifier populations in field soils and to use these primers to relate denitrifier population size to soil properties, climatic conditions and management practices as well as to denitrification and nitrous oxide emissions. Culturable denitrifier bacteria isolated from potato field soils were used. Denitrifier gene sequences were obtained for cnorB (nitric oxide reductase gene), aligned and used to design Real-Time PCR primers for quantification of specific denitrifier populations. Two denitrifier populations were targeted: 1) Pseudomonas sp. closely-related to P. mandelii and 2) Bosea sp., which also amplifies target sequence from closely-related strains such as Bradyrhizobium japonicum and Sinorhizobium meliloti. 16S rDNA was also quantified using Real-Time PCR primers and probe. Cloned cnorB gene fragments were used as an external standard for quantification of target copies of denitrifiers. Standard curves were sensitive down to 10-100 copies per reaction and were linear over 4-6 orders of magnitude. Using pure cultures, inoculants could be detected in sterile and non-sterile soil. In non-sterile soil, background populations of Pseudomonas sp. were ~5 x 105 copies per g of dry soil and Bosea sp. ~1 x 105 copies per g of dry soil. Total population counts based on 16S rDNA quantification in this soil were between 108-109 copies detected per g of dry soil. Further studies are underway to determine the effect of denitrification conditions (anaerobiosis, nitrate and glucose additions) on specific denitrifier populations in soil.