Fitness of genetically modified Pseudomonas fluorescens in competition for soil and root colonization

Abstract

The cryIVB gene of Bacillus thuringiensis var morrisoni and the Tn5-derived nptII marker gene were inserted into the chromosome of a rifampicin-resistant derivative of the grass root isolate Pseudomonas fluorescens R2f, via homologous recombination. In addition, nptII alone was inserted. Southern hybridization analysis revealed that single copies of nptII or nptII-cryIVB had been inserted into the genomes of clones Ar-1 and Art-3, respectively. The growth rates of both modified derivatives in different liquid media were similar to that of the parent strain. Further, neither of the two modified strains showed altered fitness during intermittent growth in different liquid media in competition with the parent strain. Separately introduced parent and modified strains showed a similar rate of decline in loamy sand soil microcosms. In soil inoculated with parent/modified strain mixtures, both modified strains showed impaired survival compared to that of the parent. Strain Art-3 carrying nptII-cryIVB showed reduced fitness similar to that of strain Ar-1, which carried nptII. Starvation of modified strain and parent prior to mixed introduction into soil resulted in enhanced survival of the total added poppulation as compared to that of a population of vegetative cells. However, starvation did not affect the competitive disadvantage of the modified strain. Studies on the colonization of the rhizosphere and rhizoplane of young wheat plants by parent/modified strain mixtures similarly showed a disadvantage for both modified strains, since these progressively made up smaller portions of the introduced P. fluorescens populations. Kanamycin added to soil containing the modified/parent strain mixes did not affect the proportion of the modified strain in the introduced P. fluorescens populations in soil. There was, however, an effect on rhizosphere and rhizoplane colonization. A drying/rewetting cycle applied to the soil inoculated with the modified/parent strain mixes revealed an ability to withstand moisture stress of the modified strain similar to that of the parent.