Long term evaluation of field-released genetically modified rhizobia

Environ. Biosafety Res. 6 (2007) 167–181 c ISBR, EDP Sciences, 2007  DOI: 10.1051/ebr:2007006 Available online at: www.ebr-journal.org Long term evaluation of field-released genetically modified rhizobia Viviana CORICH1 , Alessio GIACOMINI1 , Elena VENDRAMIN1 , Patrizia VIAN1 , Milena CARLOT1 , Giuseppe CONCHERI1 , Elisa POLONE1 , Sergio CASELLA1 , Marco P. NUTI2 and Andrea SQUARTINI1 * 1 2 Dipartimento di Biotecnologie Agrarie, Università di Padova, Viale dell’Università 16, 35020 Legnaro, Padova, Italy Dipartimento di Biologia delle Piante Agrarie, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy 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. Keywords: rhizobia / GMM / impact / risk assessment / environmental release INTRODUCTION Pseudomonas fluorescens (Basaglia et al., 2003; Corich et al., 1995; Nuti et al., 1997; Resca et al., 2001). This work originated within the framework of the prenormative risk assessment guidelines issued in Europe at the onset of the nineties (Nuti et al., 1994). We have since then devoted our effort to the construction and monitoring of model GMMs whose fate has been investigated in open field trials through a ten-year time span, from the first release to their technical extinction, as defined by the detection limits of available methodologies. We have recently reviewed the collective knowledge gathered after 24 field releases in the Italian territory (Nuti et al., 2003). The present report is the detailed complete outcome of the earliest of these experiments, started in parallel with our previously published joint trials with genetically modified (GM) Azospirillum brasilense and Rhizobium leguminosarum bv. viciae, the nitrogenfixing root nodule microsymbiont of pea and faba bean legume plants, was chosen as host strain for the recombinant genes. It constitutes an example for which a large body of ecological and genetic knowledge is available. In particular, being a widely applied agricultural inoculant, of interest in the areas of biotechnology and marketing, its behavior in soil in relation to massive release, has long been studied. The bacteria were engineered with the same genes to be used as markers for their subsequent tracking. No traits linked to the promotion of crop productivity were at this stage inserted, in order to analyze the sole issue of biosafety. * Corresponding author: The genetic markers were selected to fulfill the requirements of: (1) neutrality with respect to the Article published by EDP Sciences and available at http://www.ebr-journal.org or http://dx.doi.org/10.1051/ebr:2007006 V. Corich et al. environmental selective conditions, in order to not confer advantages or deficiencies to the GMM under normal situations; (2) selectability at the recovery stage, in order to allow a clean and unambiguous monitoring of the released strains when re-isolated from soil; (3) absence of antibiotic resistance genes, to avoid spread of traits prone to jeopardize human clinical therapy. The chosen genes included mercury resistance (the mer operon from Tn1831) and a lacZ gene driven by a synthetic promoter. Such a promoter was designed in order to ensure a high level of lac gene expression. This trait was deemed necessary to differentiate the tagged GMM from background lac+ soil microbiota. It was constructed after aligning sequences of published prokaryotic promoters, and extracting the highest scoring consensus for each base position upstream the transcriptional start site. The resulting construct proved to be very efficient, and its gene expression level was stronger than the lac and tac promoters in Rhizobium and E. coli (Giacomini et al., 1994). The gene cassette including the lac and mer determinants was introduced into Rhizobium leguminosarum either in plasmid-borne form, or integrated into the chromosome. Two alternative regulation modes were devised by either interposing or omitting a lac operator between the synthetic promoter and the reporter gene. Before the field release stage, the resulting GMM strains were tested for over 500 days in microcosms in three different inoculant carrier substrates, assessing their survival, genetic stability and interspecies gene exchange (Corich et al., 1996 and 2000). We found that the stability of these genetic modifications was strongly dependent on the means of insertion into the bacterium genome. Chromosomal integration gave the most stable result, while the absence of operator in a plasmid-borne gene cassette was the one most prone to loss by segregation. The present paper presents a survey carried out over a ten-year period (1994−2004), starting with the open field release of the GMMs in an agricultural setting in the presence of their legume host plants. Their persistence, as well as the impact on resident microbial groups were evaluated. An analysis of the ecological implications was also made by comparing the behavior of a GM Rhizobium isolated in foreign environment (allochthonous) with that of (...truncated)