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The fates of Pseudomonas fluorescens R2fR and its mutant derivative RIWE8, which contains a lacZ reporter gene responsive to wheat root exudate, were compared in a field microplot. Inoculant survival, root colonization, translocation, resistance to stress factors, and reporter gene activity were assessed in bulk and wheat rhizosphere soils. Populations of both strains declined gradually in bulk and wheat rhizosphere soils and on the wheat rhizoplane as determined by specific CFU and immunofluorescence (IF). In samples from both bulk soil and wheat rhizosphere, IF cell counts were up to 3 orders of magnitude greater than the corresponding numbers of CFU after 120 days, indicating the presence of nonculturable inoculant cells. Estimates of RIWE8-specific target DNA molecule numbers in bulk soil samples 3 and 120 days after inoculation by most-probable-number PCR coincided with the corresponding CFU values. Transport of both strains to deeper soil layers was observed by 3 days after introduction into the microplot. Both strains colonized wheat roots similarly, and cells were seen scattered on the surface of 1-month-old wheat seedling roots by immunogold labelling-scanning electron microscopy. On average, reporter gene activity was significantly higher in wheat rhizosphere soil containing RIWE8 cells than in bulk soil or in soils containing R2fR cells. For both strains, resistance to the four stress factors ethanol, high temperature, high osmotic tension, and oxidative stress increased progressively with residence in soil. Cells from the rhizosphere of 11-day-old seedlings showed similar levels of resistance to osmotic and oxidative stresses and enhanced resistance to ethanol and heat as compared to cells from bulk soil. By 37 days, populations of R2fR and RIWE8 in the rhizosphere were significantly more sensitive to osmotic stress than were populations in bulk soil, whereas differences in response to the other stress factors were less evident. Hence, except for the induction of reporter gene expression in strain RIWE8 in the wheat rhizosphere, the data indicated that there were no great differences in the ecological properties in soil between the lacZ-modified and parental strains.

applied and environmental microbiology

Induced Reporter Gene Activity, Enhanced Stress Resistance, and Competitive Ability of a Genetically Modified Pseudomonas fluorescens Strain Released into a Field Plot Planted with Wheat