ECOLOGICAL PRE-RELEASE RISK ASSESSMENT OF 2 GENETICALLY-ENGINEERED, BIOLUMINESCENT RHIZOBIUM-MELILOTI STRAINS IN SOIL COLUMN MODEL SYSTEMS

In order to identify potential ecological risks associated with the en vironmental release of two Rhizobium meliloti strains, genetically eng ineered with the firefly-derived luciferase gene (luc), a pre-release greenhouse investigation was conducted. The upper 4 cm of soil columns (30 cm diameter; 65 cm depth), which were filled according to the hor izons of an agricultural field (loamy sand), were inoculated with seed s of Medicago sativa (alfalfa) and R. meliloti cells at approximately 5x10(6) cells.g(-1) soil. Four treatments were tested: inoculation wit h a non-engineered wild type strain (2011), strain L33 (luc(+)), strai n Ll(luc(+), recA(-)) and non-inoculated controls. The fate of the eng ineered strains was followed by two methods: (I) selective cultivation and subsequent detection of bioluminescent colonies and (2) PCR detec tion of the luc gene in DNA, directly extracted from soil. Strain R. m eliloti L33 declined to 9.0x10(4) cfu.g(-1) soil within 24 weeks and t o 2.8x10(3) cfu.g(-1) soil within 85 weeks in the upper 25 cm of the s oil columns. Decline rates for R. meliloti L1 were not significantly d ifferent. Vertical distribution analysis of the recombinant cells afte r 37 weeks revealed that in three of four columns tested, the majority of cells (>98%) remained above 10 cm soil depth and no recombinant ce lls occurred below 20 cm depth. However, in one column all horizons be low 20 cm were colonized with 2.2x10(4) to 6.8x10(4) cfu g(-1) soil. E cological monitoring parameters included organic substance, total nitr ogen, ammonium and nitrate, microbial biomass, culturable bacteria on four different growth media and the immediate utilization of 95 carbon sources (BiologGN) by soil-extracted microbial consortia. None of the parameters was specifically affected by the genetically engineered ce lls.