Long term evaluation of field-released genetically modified rhizobia

Abstract

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.

Preparing synthetic biology for the world

“…Currently, we have methods to predict and test the environmental impact of an engineered microbe on indigenous microbial populations in soil (Corich et al, 2007; reviewed in Urgun-Demirtas et al, 2006). However, this work is limited to a few representative cases.…”

Beneficial microorganisms for soybean (Glycine max (L.) Merr), with a focus on low root-zone temperatures

“…Introduced strains are often poor competitors and populations never recover from the initial drop (Corich et al 2007;Hirsch and Spokes 1994). However, even small populations continue to affect the rhizosphere.…”

“…Corich et al (2007) found that stability of a genetic marker ranged from 85 to 100 % over the course of a growing season in a field study of R. leguminosarum and pea. Transfer of genetically modified traits to autochthonous microorganisms would be predicted to occur, but has rarely been observed in practice .…”

Methods and detection limits in tracking a genetically modifiedPseudomonas sp. released in the pine phyllosphere

“…The importance of microbe-tracking and rescuing protocols has also been stressed at political level in Europe, through the guidelines issued at both EU and national levels . Long-term approaches on risk assessment have been undertaken through joint programs, which have yielded comprehensive data covering several fields (Jansson et al, 2000;Nuti et al, 2003;Corich et al,. 2007).…”