Rhizosphere effects on biodegradation of 2,5-dichlorobenzoate by a bioluminescent strain of root-colonizing Pseudomonas fluorescens

A recombinant strain of bioluminescent Pseudomonas fluorescens 2–79 RLD containing a catabolic pathway for degradation of 2,5-dichlorobenzoate (2,5-DCB) was monitored in soil microcosms to examine the influence of plants on its growth and activity in a contaminated soil. Recombinant P. fluorescens 2–79 RLD was generated by mating a versatile chlorobenzoate utilizer, P. putida P 111, containing plasmid pPB111, with a bioluminescent strain of P. fluorescens that had been transformed previously with a Tn7-luxCDABE marker. Plasmid pPB111 contains genes encoding for a chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to their corresponding catechols. DNA hybridization experiments and cell-free extract assays with parental and recombinant P. fluorescens 2–79 RLD suggested that the reaction product of the plasmid pPB111 encoded chlorobenzoate dioxygenase was degraded by an endogenous catechol dioxygenase in P. fluorescens 2–79. After introduction of P. fluorescens 2–79 RLD into soil containing 10 mg kg−1 2,5-DCB, normally recalcitrant 2,5-DCB was degraded rapidly over a period of 2 to 4 days in soil with plants. In contrast, 2,5-DCB disappearance in nonplanted soil was significantly slower, requiring 7 days in one experiment, and more than 2 weeks in a second experiment. Population numbers of the degrader were similar in planted and nonplanted soil for the first 7 days, but declined in nonplanted soils by day 14. Physiological status, measured using an assay based on lag-phase duration, was similar in planted and nonplanted soils at day 2, but rapidly declined in nonplanted soil by day 7. At day 14, plasmid stability in P. fluorescens 2–79 RLD was significantly greater in rhizosphere soil; only 10% of P. fluorescens 2–79 RLD cells in rhizosphere soil had lost the ability to degrade 2,5-DCB, versus 94% of the cells in nonplanted soil. The plasmid also was transferred to indigenous bacteria in both planted and nonplanted soils, as determined by the appearance of novel degraders. The results demonstrate that the presence of plants promoted rapid degradation of DCB and provided a niche that enhanced maintenance of plasmid pPB111 in the degrader bacterium.