Visualization and direct counting of individual denitrifying bacterial cells in soil by nirK-targeted direct in situ PCR

Abstract

The abundance of denitrifying bacteria in soil has been determined primarily by the conventional most probable number (MPN) method. We have developed a single-cell identification technique that is culture-independent, direct in situ PCR, to enumerate denitrifying bacteria in soils. The specificity of this method was evaluated with six species of denitrifying bacteria using nirK as the target gene; Escherichia coli was used as a negative control. Almost all (97.3%-100%) of the nirK-type denitrifying bacteria (Agromonas oligotrophica, Alcaligenes faecalis, Achromobacter denitrificans, Bradyrhizobium japonicum, and Pseudomonas chlororaphis) were detected by direct in situ PCR, whereas no E. coli cells and only a few cells (2.4%) of nirS-type denitrifying bacteria (Pseudomonas aeruginosa) were detected. Numbers of denitrifying bacteria in upland and paddy soil samples quantified by this method were 3.3 × 108 to 2.6 × 109 cells g-1 dry soil. These values are approximately 1,000 to 300,000 times higher than those estimated by the MPN method. These results suggest that direct in situ PCR is a better tool for quantifying denitrifying bacteria in soil than the conventional MPN method.