evaluation for genetically modified microbial inoculants
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.
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.
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.
October 1991 September 1993
Università di Pisa (IT)
University College Cork (IE)
University of Bristol (UK)