Combined use of a specific probe and PCAT medium to study Burkholderia in soil

Abstract

Due to its pathogenic traits and agricultural benefits, there is some challenge in detecting Burkholderia in the soil environment. In this perspective, an existing semi-selective medium, (PCAT), was combined with a Burkholderia specific molecular probe. Using the complete 16S rRNA sequences of all available Burkholderia species type strains, we selected the following sequence: 5′-ACCCTCTGTTCCGACCATTGTATGA-3′. The probe was validated against GenBank sequences, with dot blots and colony hybridization tests. A diversity study of all strains growing on a PCAT plate after plating a soil dilution (75 strains) was carried out with ARDRA analysis and colony hybridization tests. All the hybridizing strains belonged to genus Burkholderia. The major type of non-hybridizing isolates belonged to Pseudomonas (16S rRNA sequencing). Both tools were combined to compare the Burkholderia populations in a rhizosphere (maize) and a non-rhizosphere soil. Based on hybridizing PCAT isolates, we were able to show an increase in Burkholderia populations in the maize rhizosphere. This genus represented 2% and 16% of the total cultivable microflora in the non-rhizosphere and rhizosphere soils, respectively. Although PCAT was shown not to be appropriate to routinely enumerate Burkholderia populations in soil, it allowed environmental investigations at the genus level, when combined with a molecular specific probe.