Fate and Ecological Interactions of Firefly Luc Gene-Tagged Sinorhizobium Meliloti 2011-Bacteria in Soil Inhabited by High Levels of Indigenous Alfalfa Nodulating Populations

Abstract

A field study was conducted with genetically modified sinorhizobium meliloti strains L1 (RecA-) and L33 (RecA+), both tagged with the firefly luciferase luc gene as an identification marker. The strains' fate was studied over a time period of five years. Both strains were rapidly outcompeted for alfalfa nodulation by an indigenous population. In summary, this study demonstrates the usefulness of tagging bacteria designed for environmental releases by the firefly luciferase gene and the high resilience of soil bacteria to allow the establishment of foreign bacterial populations.Key words: alfalfa, Sinorhizobium meliloti, luc gene-tagging, nodulation competitiveness, indigenous populations, field releases of genetically modified bacteria. FATE AND ECOLOGICAL INTERACTIONS OF FIREFLY LUC GENE-TAGGED sINORhIzObIUm melIlOTI 2011-BACTERIA IN SOIL INHABITED BY HIGH LEVELS OF INDIGENOUS ALFALFA NODULATING POPULATIONSA field study was conducted with genetically modified Sinorhizobium meliloti strains L1 (RecA -) and L33 (RecA + ), both tagged with the firefly luciferase luc gene as an identification marker. The strains were released as peat-based inocula at densities of approx. 10 6 cfu g -1 soil in replicate and randomised field plots at a field site in Bavaria (Germany). The strains' fate was studied over a time period of five years. During the first six weeks after inoculation, the viable count of both luc tagged strains sharply dropped for more than two orders of magnitude. Thereafter, the titer of both strains more or less steadily declined within the next five years to approx. 10 2 cfu g -1. No significant differences in the survival of strains L1 (RecA -) and L33 (RecA + ) were noted at most sampling dates demonstrating that the recA gene function was dispensable under the prevalent environmental conditions. Both strains were rapidly outcompeted for alfalfa nodulation by an indigenous population which was well-established in the field site soil. RecA -strain L1 was slightly faster outcompeted than its isogenic RecA + counterpart L33. In order to mimic normal agricultural practice, one year after the field release the non-host plant rye was grown in a total of 21 of the 49 field plots. Cultivation of rye had neither a negative nor a positive effect on the survival of strains L1 and L33 in these plots, respectively. Both strains showed the same survival capabilities, comparable to that of the field plots were the host plant alfalfa was grown. The diversity of the indigenous population was characterized by employing the Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR fingerprint method. Typing of more than 340 indigenous isolates recovered from alfalfa root nodules throughout the monitoring period of five years revealed the presence of five dominant strain types accounting for approximately 67 % of the isolates of the indigenous alfalfa nodulating population. Nearly one third of the dominant isolates displayed the Rhizobium sp. Or191 fingerprint pattern. IS fingerprinting of representatives of the various s...