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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Risk evaluation for genetically modified micro-organisms

Background and objectives

The recent developments in gene technology have led to a situation where it is possible to produce genetically modified micro-organisms (GMMs). These GMMs can be used for a variety of commercial purposes in agriculture. Some of these GMMs will be used in the open and are therefore not contained and liable to escape. Little is known about the survival, persistence and spread of micro-organisms in the environment, and it is therefore difficult to judge the risks associated with the release of micro-organisms into the environment, whether or not genetically modified. The aim of this project was to increase our knowledge in this area of pre-normative research, with a special focus on agriculturally relevant soil and seed inoculants.

Approach and methodology

The project aimed to develop appropriate marker genes for routine detection and unambiguous identification of Rhizobium and Bradyrhizobium subspecies in soil and the rhizosphere (around plant roots). These markers were then used to assess the probability that released strains, including genetically modified strains, will multiply, survive and persist in soil and rhizosphere environments. Particular emphasis was placed on the development of marker genes other than the genes for resistance to antibiotics commonly used for this purpose. Standard recombinant DNA and gene fusion techniques were exploited to create novel combinations of expression systems responding to unique environmental signals. The project used soybean, recently introduced into cultivation in Italy, to provide a baseline study: this involved analysis of soil properties, of the microbial community profile and cropping history. The release of GMMs was then studied in this system at the microcosm level.

Main findings and outcome

The catabolic ß-galactosidase genes (lacZY) and the genes for mercury resistance were selected as appropriate reporter genes for use in Rhizobium and Bradyrhizobium subspecies. The sensitivity of detection of these genes was substantially enhanced by using strong synthetic promoters or by replacing the lac promoter by inducible promoters responding to an unrelated environmental signal, for example the presence of dicarboxylic acids. An elegant solution to the problems associated with the development of stable vector systems was developed using chromosomally integrated thymidilate synthase genes taken from a food grade micro-organism.

The second part of the project was the assessment of risks associated with the release of these GMMs. Methods were developed to allow accurate monitoring of released Rhizobia, both wild type and genetically modified. One method was pulsed field electrophoresis of DNA digested with rarely-cutting restriction endonucleases. Pyrolysis mass spectrometry was also used. This involves igniting samples of the bacteria and passing the volatile combustion products through a mass spectrometer. This approach is rapid and sensitive and allows bacteria isolated from different soil types to be compared to each other and to the inoculant strains. It can thus be used to study genetic drift. Wild-type Rhizobia were found to survive well in most soils tested, and there were no obvious soil characteristics that affected survival. The genetically modified Rhizobia released in soil at the microcosm level, behaved in the same way as the wild strains.


The vector system developed is a widely applicable and environmentally friendly vector system. It can be used for introducing marker genes into Rhizobia. Furthermore, it can be used to introduce genes of potential benefit to Rhizobia-legume symbiosis. Powerful techniques were developed to monitor the fate of released micro-organisms: the use of high constitutive gene expression allowed unambiguous detection of introduced genes; and pyrolysis mass spectrometry allowed analysis of genetic drift. It was found that genetically modified Rhizobia did not behave any differently to wild-type Rhizobia following release into the soil.


Major publications

Corich V., Bosco F., Giacomini A., Basaglia M., Squartini A., Nuti M.P., “Fate of genetically modified Rhizobium leguminosarum biovar viciae during prolonged storage of commercial inoculants”.
Journal of Applied Bacteriology, 81, 1996, pp. 319-328.

Kay H.E., Coutinho H.L.C., Fattori M., Manfio G.P., Goodacre R., Nuti M.P., Basaglia M., Beringer J.E., “The identification of Bradyrhizobium japonicum strains isolated from Italian soils”.
Microbiology, 140, 1994, pp. 2333-2339.

Giacomini A., Ollero F.J., Squartini A., Nuti M.P., “Construction of multipurpose gene cartridges based on a novel synthetic promoter for high-level gene expression in Gram-negative bacteria”.
Gene, 144, 1994, pp. 17-24.

Nuti M.P., Basaglia M., Bonfante P., Casella S., Corich V., Dal Maistro L., Giacomini A., Martini I., Peruch U., Poggiolini S., Squartini A., Vian P., “Field release of genetically modified biofertilizers and phitostimulators”, in The Biosafety Results of Field Tests of Genetically Modified Plants and Micro-organisms, S. Matsui, S. Miyazaki, K. Kasamo (eds.), Japan Int. Res. Center for Agricultural Sciences JIRCAS Publ., 1997, pp. 101-111.

Struffi P., Corich V., Giacomini A., Benguedouar A., Squartini A., Casella S., Nuti M.P., “Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium”.
J. Applied Microbiology, 84, 1998, pp. 81-89.

Corich V., Giacomini A., Vendramin E., Vian P., Carlot M., Squartini A., Nuti M.P., “The field release and Monitoring of Rhizobial Strains Marked with lacz and Mercury Resistance Genes”, in Tracking Genetically Engineered Micro-organisms, J.K. Jansson, J.D. Van Elsas, M.J. Bailey (eds.), Landes Biosciences Texas Publ., 2000, pp. 139-144.
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Contract number

January 1989 – December 1990

M.P. Nuti
Università di Pisa (IT)


F. O’Gara
University College Cork (IE)

J.E. Beringer
University of Bristol (UK)

M. Fattori
ICI Seeds – SES Italia
Massa Lombarda (IT)

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