This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ß-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement (“self-impact”), and how much the two rhizobia would additively contribute to nodulation.

Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

Concerns have been raised in Europe about the efficiency, sustainability, and environmental impact of the first genetically modified crops. The committees and regulators in charge of approving procedures have encouraged a field trial approach for safety assessment studies under current agronomic conditions. We describe the gene flow from sugar beet (Beta vulgaris L.) in a multi-year and multi-crop monitoring study on farmers' fields at two locations that has been carried out since 1995. We analyzed two sugar beet lines that have been genetically transformed for herbicide resistance. One sugar beet has resistance to glufosinate and the other to glyphosate. Large differences among lines, years and locations were observed. These differences provided a broad range of situations to estimate the risks. Sugar beet bolters produced the majority (86%) of the herbicide-resistant seeds harvested in the field. Direct pollen flow from sugar beet bolters to weed beets that were growing within the same field as well as in a neighboring field that was left fallow accounted for only 0.4% of the resistant seeds released over the years and locations. Descendants of the hybrids between the sugar beet and the weed beet produced the remaining 13.6% of resistant seeds. Herbicide-resistant seeds from the progeny of the weed beet were recorded up to 112 m away from the closest transgenic pollen donor. Indications were observed of non-randomness of the weed beet producing resistant progeny. We also analyzed pollen flow to male-sterile bait plants located within and outside of the sugar beet field. Herbicide-resistant pollen flow was recorded up to 277 m, and fitted with an inverse power regression. Using sugar beet varieties with no, or very low, sensitivity to bolting and destroying bolters are two necessary measures that could delay gene flow.

Risk-assessment studies of virus-resistant transgenic plants (VRTPs) focussing on recombination of a plant virus with a transgenic sequence of a different virus should include a comparison of recombination frequencies between viruses in double-infected non-transgenic plants with those observed in singly infected transgenic plants to estimate recombination incidence in VRTPs. In this study, the occurrence of recombination events was investigated in non-transgenic plants double-infected with two different potyviruses, as well as in potyviral genomes in singly infected transgenic plants expressing potyvirus sequences. Different potyviruses, namely Potato virus A (PVA), Tobacco vein mottling virus (TVMV), two strains of Potato virus Y (PVY-O, PVY-H) and two strains of Plum pox virus (PPV-NAT, PPV-SK68), were used in three combinations for double infection of a common host. Furthermore, transgenic plants expressing either potyviral coat protein (CP), helicase (CI) or polymerase (NIb) coding sequences (PPV-NAT-CP, PVY-CI, PVY-NIb) were singly-infected with a heterologous potyvirus, which was not targeted by the respective transgenic resistance. To identify recombinant potyviral sequences, a sensitive RT-PCR was developed to detect up to one recombinant molecule out of 106 parental molecules. In 304 mixed infected non-transgenic plants, 92 mixed and 164 single infected transgenic plants screened for recombinant sequences no recombinant potyviral sequence was found. These results indicate that recombination events between different potyviruses in mixed infections and between a potyvirus infecting a potyvirus-resistant transgenic plant are likely to be very infrequent.