CHARACTERIZATION OF RHIZOSPHERE COLONIZATION BY LUMINESCENT ENTEROBACTER-CLOACAE AT THE POPULATION AND SINGLE-CELL LEVELS

A bioluminescence marker system was used to characterize colonization of the rhizosphere by a bacterial inoculum, both in terms of populatio n activity and at the single-cell level. Plasmid pQF70/44, which conta ins luxAB genes under the control of a strong constitutive phage promo ter, was introduced into the rhizobacterium and model biocontrol agent Enterobacter cloacae. Light output from the lux-modified strain was d etected by luminometry of samples from growing cultures of E. cloacae and from inoculated soil and wheat root samples. The minimum detection limits for fully active cells under optimum conditions were 90 and 43 5 cells g(-1) for liquid culture and soil, respectively. The metabolic activities of the lux-marked population of E. cloacae, characterized by luminometry, contrasted in rhizosphere and nonrhizosphere soil. Cel ls in the rhizosphere were active, and there was a linear relationship between light output and cell concentration. The activity of cells in nonrhizosphere soil could not be detected unless the soil was supplie d with substrate, Novel use of a charge-coupled device is reported for the spatial characterization of rhizosphere colonization by E. cloaca e (pQF70/44) at the single-cell and population levels. Used macroscopi cally, the charge-coupled device identified differences in colonizatio n due to competition from indigenous soil organisms. The lux-marked ba cterium was able to colonize all depths of roots in the absence of com petition but was restricted to the spermosphere in the presence of com petition (nonsterile soil). The combined use of luminometry, dilution plate counts, and charge-coupled device imaging (macroscopic and micro scopic) demonstrates the potential of luminescence-based detection as a powerful system for assessment of rhizosphere colonization by lux-ma rked microbial inocula.