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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 microbial inoculants

Background and objectives

Soil microbes may be modified genetically for agricultural applications by traditional methods or using new recombinant DNA technology. In either case, little is known about the survival, persistence and dispersal in soil of genetically modified microbes. It is also unclear how such microbes are likely to be affected by environmental and agronomic factors. Monitoring the fate of microbes released into an open soil environment may be difficult, particularly if the microbe concerned establishes a low-density population. However, such monitoring is essential for risk assessment and environmental impact analysis, as required by European Directive EC/220/90.


Approach and methodology

Tools were developed for the identification and characterisation of rhizobial inoculants. The fate of genetically modified rhizobia was studied during inoculant manufacture and storage and in the field, to identify factors affecting the survival of the inoculant. Attempts were also made to develop and to evaluate intrinsically biosafe soil microbial inoculants.


Main findings and outcome

Reporter gene systems showed that the genetically modified organisms behaved similarly to the unmodified parental strain in test systems. However, population dynamics depended on the intrinsic stability of the construct used. Field experiments showed no correlation between the physicochemical properties of the soil and survival but did reveal that changes in the water table affected the survival of the introduced microbes during the first twelve months, whether or not the host plant was present. PCR techniques were used to check for changes in the strain after inoculation. In general, it was possible to show that the strains isolated were derived from the strain inoculated. However, in one case, the available information concerning the inoculant was found to be incorrect as the strain isolated was completely different from that expected. At another site, two populations had developed from the original strain. A vector system, the thy vector system, was developed and tested under field conditions. This system decreased nodulation efficiency but not symbiotic efficiency and was found to result in the stable maintenance of the genetic trait in field conditions.
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Conclusions

New systems were developed for the transformation of rhizobial microbes, making it possible to transform microbes without the use of antibiotic resistance cassettes, thereby reducing the risk of transfer of these cassettes to other organisms, and providing opportunities for the containment of genetically modified microbes. Methods were also developed for monitoring genetic changes in the population. The only factor found to affect the survival of genetically modified microbes in the field was the height of the water table.

 

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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imageResearch project
 

Contract number
BIOT-CT91-0283

Period
October 1991 – September 1993

Coordinator
M.P. Nuti
Università di Pisa (IT)

 
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Partners


F. O’Gara
University College Cork (IE)

M. Basaglia
Heligenetics S.P.A.
Gaiba (IT)

J.E. Beringer
University of Bristol (UK)

 
 
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