Genetically modified Pseudomonas associated with plants: aspects for environmental risk assessment

Front cover: Double staining of P. syringae Cit7sp using FITC and CTC stains. In epi-fluorescence microscopy using 450-480 nm exciter filter and 515 nm barrier filter, cells stained green by FITC and respiring cells contained intracellular yellow CTC-formazan crystals. Original publications This thesis is based on the following publications, which in the text are referred to by their Roman numerals. High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations. Colonization strategies and conjugal gene transfer of inoculated Pseudomonas syringae on the leaf surface. The papers I-III are reprinted by kind permission from the publishers. Abbreviations ANOVA analysis of variance Ap ampicillin CCD coupled charged device CFU colony forming unit CLPP community level physiological profiles CTC 5-cyano-2,3-ditolyl tetrazolium DGGE denaturing gradient gel electrophoresis EU European Union FITC fluorescein isothiocyanate GM genetically modified GMO genetically modified organism Hg mercury IPTG isopropylthiogalactoside kb kilobase pair Km kanamycin Nal nalidixic acid OECD Organisation for economic cooperation and development PCR polymerase chain reaction RFLP restriction fragment length polymorhpism Rif rifampicin Sp spectinomycin Tc tetracycline UPGMA unweighted pair group method with arithmetic means VBNC viable but non-culturable SUMMARY Bacteria belonging to the genus Pseudomonas can be used as biological control agents in association with plants, for protection against plant diseases or frost damage. Biological control is considered to be more environmentally friendly than the use of traditional chemical agents. In planned commercial products, there is however, often a need to improve the used bacterial strains by genetic engineering techniques. Because little is known about the behaviour and impact of introduced genetically modified bacteria in the environment, legislation requires a risk assessment to be performed, proving the strain to be safe to use, before genetically modified bacteria are allowed to be released into the environment. In this work, the survival and impact of introduced Pseudomonas bacteria were studied in two habitats likely to be the target for future biological plant protecting agents; the leaf surface and the roots. The bacteria were genetically modified by tagging with marker genes to facilitate monitoring. Experiments were performed in laboratory setups mimicking environmental conditions. Introduced Pseudomonas bacteria survived on the leaf surface regardless of the prevailing humidity. In soil and in the root system of birch, the introduced bacteria did not survive as large populations. In particular, at high soil temperatures, the bacteria disappeared when measured by plating techniques, but were detected using molecular techniques. This …