Measurement of the competitiveness index of Rhizobium tropici strain CIAT899 derivatives marked with the gusA gene

Abstract

The gusA gene encoding β-glucuronidase has been adapted for use as a marker gene for ecological analysis of Gram-negative bacteria. A key requirement for such marker genes is that they should not directly affect the ecological behaviour being studied. In this paper, we examine the effects of introduction of the gusA gene on a representative and easily studied ecological phenotype, rhizobial nodulation competitiveness. Five independent isolates of Rhizobium tropici strain CIAT899 marked with the gusA gene on mini-transposon mTn 5SS gusA10 were characterized for nodulation characteristics and competitive abilities on common bean ( Phaseolus vulgaris). Insertion of mTn 5SS gusA10 did not significantly change the nodulation or nitrogen fixation behaviour of R. tropici CIAT899. However, the competitiveness index of different mTn 5SS gusA10-marked derivatives varied relative to the parental strain, both between three independent experiments and between isolates. One isolate was less competitive than the wild-type in all three experiments. The competitiveness indices of the other four gusA-marked strains varied between experiments, but in each case, they appeared either equally competitive or more competitive than the parental strain. The data obtained enabled highly statistically significant calculations of competitiveness indices, as all the nodules on each plant could be analysed for nodule occupancy. These experiments indicate that, although, primary selection of marked strains is essential, gusA-marked Rhizobium derivatives with competitive abilities indistinguishable from the parental strain, can be readily obtained.