Methods and detection limits in tracking a genetically modified Pseudomonas sp released in the pine phyllosphere

A method suitable to detect the presence and follow the fate of specific bacteria released on the phyllosphere of conifer trees was devised, tested, and optimised. The procedure was set up using a biocontrol strain that had shown effectiveness and persistence in greenhouse trials against insect pests. The microorganism used is based on a Pseudomonas sp., originally isolated from Pinus nigra and carries the cry9a toxin gene from Bacillus thuringiensis. In order to assess its detectability, specific primers were designed, and the most suitable protocol for DNA isolation from bacteria colonising pine needles was defined upon an experimental comparison of various methods. Different conditions of physical pre-treatments and their combinations with commercially available kits protocols were tested. The most sensitive monitoring (about 102 released cells) was achieved by a procedure based on vortexing in a suspension of glass beads followed by the use of microcolumns designed for a soil DNA extraction kit. The application can be recommended in biosafety studies of released GMMs as well as in ecological surveys of phyllosphere microbiota.