QUANTITATIVE 16S RDNA-TARGETED POLYMERASE CHAIN-REACTION AND OLIGONUCLEOTIDE HYBRIDIZATION FOR THE DETECTION OF PAENIBACILLUS AZOTOFIXANS IN SOIL AND THE WHEAT RHIZOSPHERE

A molecular method for the detection of Paenibacillus azotofixans in s oil and the wheat rhizosphere was developed. The system consisted of p olymerase chain reaction (PCR) amplification of part of the variable V 1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridizati on with a specific oligonucleotide probe homologous to part of the int ervening region. In vitro specificity tests showed that the detection system worked specifically for P. azotofixans strains, and did not det ect other Paenibacillus species or species of other bacterial genera. Vegetative cells of a rifampicin resistant P. azotofixans derivative w ere trackable in Flevo silt loam (FSL) soil in 24 h experiments using both selective plating and most probable number (MPN)-PCR combined wit h probing, and plate counts parallelled MPN-PCR estimations of numbers of specific targets. MPN-PCR allowed for the detection of down to 10( 2) introduced cells per g of dry soil. Introduced P. azotofixans spore s did not form colonies on selective plates, but were detectable via P CR. The P. azotofixans populations introduced into the silt loam soil suffered a slow decline of the detectable plate count over a period of 14 days. MPN-PCR revealed a similar decline of the number of specific DNA targets. Greater numbers of targets were found in wheat rhizosphe re from Flevo silt loam soil, and these numbers persisted throughout t he experiment. Soil drying resulted in enhanced persistence of the tar get sequences, whereas in a constantly moist soil the numbers of targe t sequences declined. Rewetting of dried soil resulted in declining ta rget sequence numbers. The MPN-PCR detection method is adequate to ass ess the impact of stress conditions affecting P. azotofixans in FSL an d probably other soils, since it abolishes the need for culturing or s pecific markers and is direct and unambiguous due to its high specific ity.