evaluation for genetically modified micro-organisms
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
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
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
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
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
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.
January 1989 December 1990
Università di Pisa (IT)
University College Cork (IE)
University of Bristol (UK)
ICI Seeds SES Italia
Massa Lombarda (IT)