1. Craigiebuckler, Aberdeen, AB15 8QH, UK “ENDEGRADE” Endophytic bacteria for improving phytoremediation Fiona Porteous Moore, Colin Campbell, Edward Moore & E.U. partners

2. ENDEGRADE 7 project partners SCOTLAND BELGIUM IRELAND DENMARK Macaulay IT Carlow Institute LUC VITO DEC-NV DTU NERI (INDUSTRIAL PARTNER)

3. PHYTOREMEDIATION Ideally plants must have  deep roots  perennial  large water use Plants  breakdown/volatilise compounds  store them in leaves/shoots 4 methods  phytovolatilisation  phytoextraction  rhizofiltration  phytostabilisation

4. Problems with some current remediation strategies Phytoremediation >pollutants kill the plants >volatilisation through leaves >partial degradation leading to toxic products Soil bioremediation >indigenous population overcomes introduced degraders >nutrient applications often needed >pollutants can be taken up by plants faster than soil microbes can degrade them

5. Endegrade - the concept enhance plant survival enhance degradation reduce volatilisation

6. Project aims Isolate & identify bacteria from Willow and Poplar Screen for natural degradation potential for target compounds Equip endophytic bacteria with degradation plasmids Assess re-colonisation and phytoremediation efficiency Risk assessment for field use

7. What is known about endophytic bacteria? neutral or beneficial effects on the plant - direct plant growth-promoting activity - N 2 -fixation - disease suppression - enhanced pest control exist in all plant species examined 10 3 - 10 5 cfu/gm plant tissue highest numbers observed in the roots bacteria remain localised in specific tissues

8. We do not know… community dynamics & diversity colonisation potential plant specific relationships?

9. Pollutants problematic for phytoremediation...

10. Bacterial endophyte isolates analysed to date

11. Relative frequencies of bacterial isolates, classified to the genus level, on the basis of 16S rDNA sequence analysis

12. Relative frequencies of isolates classified as Pseudomonas species, based upon 16S rDNA sequence analysis

14. Distribution of endophytic bacterial isolates in Poplar

15. Multivariate analysis - presence / absence of isolates in different species of trees. Effect of tree species

16. Napthalene degradation in Pea (proof of concept) Work by: Germaine et al, IT Carlow

17. Plant protection Iris & VM1450 - 2,4-D protection (Germaine et al, IT Carlow) Pea & PCB degraders - 4-chlorobiphenyl protection (Germaine et al, IT Carlow) Lupine & VM1330- Toluene protection, reduced volatilisation, enhanced growth (Barac et al, LUC) Toluene & TCE degrader constructs from Brassica napus - to test in plantae degradation (Borremans et al, VITO)

18. Re-inoculation K.Germain et al, FEMS Microbiol.Ecol, in press. 3 endophytic isolates gfp/Kan marked and re-inoculated into Poplar (original host) All strains colonised roots, and 2 strains colonised stems and leaves after 10 weeks

19. VM1449 (Ps. veronii) colony on root xylem of poplar tree 10 weeks after inoculation x1000

20. Poplar used in phytoremediation field trial in Belgium

21. Current work Endophytic construct in Poplar exposed to 2,4-D - does in degrade/protect? >2,4-D degradation genes & colonisation Seasonal community dynamics of endophytes - affects inoculation time/method >intra/inter species variation, seasonal dynamics, compartmentalisation

22. Acknowledgements Macaulay Institute - Renate Wendler, Duncan White NERI - Denmark - Uli Karlson DTU - Denmark - Stefan Trapp VITO - Belgium - Daniel van der Lelie, Brigitte Borremans LUC- Belgium - Jaco Vangronsveld, Licy Oeyen, Tanja Barac IT Carlow- Ireland - David Dowling, David Ryan, Keiran Germaine, Elaine Keogh DEC NV- Belgium - Siegried D’Haene, Gunther de Becker