93 × 105 and 3.90× 106 cells g−1 of soil. In addition, qPCR results showed that the lowest number of Pseudomonas was in the soil treated see more with sludge (Table 2). The total number of bacteria in the two soils was estimated to be in the range of 3.43 × 108 and 4.24× 108 cells g−1 of soil using a general qPCR assay targeting the eubacterial
16S rRNA gene (Fierer et al., 2005). Similar to the Pseudomonas data, the total number of bacteria was lowest in the sludge-treated soil. The quantification of Pseudomonas cells in the soils with qPCR (Table 2) showed a significantly higher number of bacteria in the compost-treated soil (P < 0.0001). Detecting 106 Pseudomonas cells g−1 soil is in accordance with previously published data on Pseudomonas in soil (Pallud et al., 2001; Lloyd-Jones et al., 2005). Results from the eubacterial qPCR assay showed the same differences between the soil types as with the genus-specific protocols, highest bacterial counts in the compost-treated soil and a lower in the sludge-treated soil. The sequencing data showed a high diversity of Pseudomonas, identifying c. 200 different OTUs and more than 20 different species at a 3% maximum cluster distance.
If the length of the PCR fragments is taken into consideration, the observed diversity in the Pseudomonas genus is rather high, especially because it is well-documented that the 16S rRNA gene does not LBH589 concentration contain enough genetic variation to identify all Pseudomonas species to species level (Peix et al., 2009). However, in this study, c. 200 different Pseudomonas OTUs, many to species level, were detected by pyrosequencing. Analysis of the Pseudomonas primers using pyrosequencing showed that 99% of the sequences belonged to the genus Pseudomonas. However,
only 8% of the PCR products amplified with Burkholderia primers belonged to the genus Burkholderia and 36% of the sequences were defined as unclassified betaproteobacteria and the remaining divided primarily between Methylotenera, Methylovorus and Thiobacillus. In the Burkholderia sequencing data, several nontarget bacteria were detected. Bacteria like Pseudomonas, Sinobacteraceae, Legionella, Bay 11-7085 Alcaligenaceae, Methylophilaceae and Rhodocyclaceac should not be present. The primer target sequences in all bacteria in NCBI from these groups have a 1–2 bp mismatch to our Burkholderia primers. The most likely explanation is that we used a too low Tm value. The Tm for the Burkholderia primers was set to 60 °C based on a temperature gradient PCR, above 60 °C the bands began to fade. Another explanation could be presence in the soil of bacteria other than Burkholderia with exact match to the primer sequence and that these bacteria are absent from current sequence databases.