This project aims to study the role of poplar associated bacteria in phytoremediation of soils contaminated with organic pollutants and heavy metals using poplar. TCE and BTEX will be used as model components for the organic pollutants and Ni for the heavy metals. In a first step the bacterial population of Populus trichocarpa x deltoids (Hoogvorst), planted on a site with a Ni, TCE and BTEX contaminated groundwater plume, will be isolated from the rhizosphere soil, root, leaf and shoot samples. This bacterial population will be identified and characterized. The bacterial strains will be identified using BOX-PCR genomic DNA fingerprinting followed by 16S-rDNA sequence analysis. For the characterization, the Ni resistance including the Ni minimum inhibiting concentration (MIC) and the potential precipitation on the celwall (SEM-EDX and TEM-EDX), the growth on BTEX with the potential cometabolization of TCE (PCR with specific primers), and the siderophore production will be analyzed. In a second part the dominant endophytes will be modified, using natural gene transfer, to endophytes capable to degrade TCE and BTEX and/or to precipitate Ni on their cell wall. This part of the project will take place in the Biology department of Brookhaven National Laboratories, USA in cooperation with Dr. Safiyh Taghavi and Dr. Daniel van der Lelie. The degradation-pathways for TCE and BTEX will be induced by the pTOM plasmide. MiniTn5 transposons with the ncc-nre gene and the pMOL222 plasmide will be used to obtain Ni-resistant bacteria. In a next step cuttings of Populus trichocarpa x deltoides (Hoogvorst) will be inoculated with a selection of endophytes out of the collection of natural endophytes and the modified strains. The colonization efficiency, the phytoremediation efficiency and the phytotoxicity will be analysed by a greenhouse experiment (hydroculture and non sterile sandy soil). In order to be able to analyse the colonisation efficiency, the endophytes will be genetically labelled via the introduction in their chromosome of a canamycin-gfp cassette. Epifluorescence microscopy will be used for the in planta vizualisation of the gfp labelled endophytes. The phytoremediation efficiency will be analysed by following of the degradation and the evapotranspiration of toluene and TCE, and the Ni-uptake and precipitation on the celwall (SEM-EDX and TEM-EDX). To follow TCE, toluene and Ni phytotoxicity plants growth and the photosynthesis activity will be studied. Following, the potential horizontal gene transfer that can occur between inoculated and endogenous endophytes will be controlled in this greenhouse study. For this purpose the toluene- and TCE degradation and/or Ni resistance/sequestration will be tested for the non-labelled endophytes. The endophytes with a positive test will be genomic fingerprinted using BOX-PCR, PCR with specific primers and 16S-rDNA sequence analysis. In the last part some non-GMO endophytes will be inoculated in situ in the Populus trichocarpa x deltoids (Hoogvorst) growing on a site with a Ni, TCE and toluene contaminated groundwater plume and this after consultation and permission of the authorities responsible for the use of GMO. The Ni-, TCE- and BTEX concentrations in the groundwater will be measured regular. The Ni-uptake will be evaluated by measuring the Ni-concentration in leaf, root and shoot and the BTEX and TCE evapotranspiration will be measured. Additionally, in situ horizontal gene transfer will be checked.