Birch (Betula) and hybrid aspen (Populus) for rhizoremediation: depicting the plant associated microbiome and the catabolic capacity

The adequacy of birch and hybrid aspen for phytotechnology was studied in green house experiments using either polyaromatic hydrocarbons (PAHs) or petroleum hydrocarbons as pollutant. The tree-associated microbes from the different plant compartments were studied in order to take advantage of ecosystem services to benefit bioremediation. Below ground microbial communities were depicted by analysis of functional extradiol dioxygenase genes and structural 16S rRNA genes. The ectomycorrhizal (ECM) fungal diversity was analysed by PCR of the ITS region from the root tips. The PCR products were fingerprinted with PCR-RFLP and T-RFLP. Endophytic and epiphytic bacteria were isolated from all compartments of hybrid aspen and grouped in haplotypes by 16S rRNA analysis. PAH degradation with fifty endophytic bacterial strains was studied for evaluation of their biodegradative potential. In our rhizoremediation project the molecular tool box was successfully used together with bacterial isolation to describe the aspen microbiome. Woody plant rhizoremediation was carried out and the degradation of PAHs was analysed (gas chromatography/masspectrometry). Biodegradation of PAHs was observed in planted as well as unplanted microcosms, showing differential degradation of individual PAHs. Genetic aromatic ring-cleavage potential was detected in rhizosphere soil demonstrated by higher diversity of extradiol dioxygenase genes than in bulk soil with no plant and Burkholderia were dominant in PAH-polluted aspen rhizosphere. The Wales birch clone tolerated high concentrations of PAHs (1200 ppm), whereas aspen was more sensitive to the pollutant, but still tolerated 900 ppm of PAHs. Tolerance tests with petroleum hydrocarbons showed that 10% oil was lethal, whereas 1% was tolerated by the aspen seedlings. ECM mycorrhizal diversity was moderate with 5-7 morphotypes per aspen family. They included Thelephora, Cenococcum geophilum and Cortinarius. Thirteen aspen associated bacterial strains were able to grow on PAHs as sole source of carbon and energy. All of them were isolated from roots and belonged to the Burkholderia genera and same types were part of uncultured bacterial community in PAH-amended soil. Ten strains grouped with B. fungorum species, one with B. sordidicola and one with Burkholderia sp R-701. The behavior of these Burkholderia strains toward dibenzothiophene is similar to another and to a very closely related strain: Burkholderia sp DBT1. Unexpectedly an amplicon of correct size using two pairs of primers specific for DBT1 p51 and pH1A operons, was obtained for all ten B. fungorum and R-701 strains isolated from aspen plants growing on PAHs. The subsequent RFLP analysis of the amplicons generated by PCR from pH1A operon showed the same pattern in all the stains. The findings improve our understanding of rhizoremediation associated microbes and may benefit bioremediation research by facilitating the development of molecular tools to detect and monitor populations involved in plant maintenance and degradative processes.