GENE PROBE ANALYSIS OF SOIL MICROBIAL-POPULATIONS SELECTED BY AMENDMENT WITH 2,4-DICHLOROPHENOXYACETIC ACID

Soils with a history of 2,4-dichlorophenoxyacetic acid (2,4-D) treatment at field application rates and control soils with no prior exposure to 2,4-D were amended with 2,4-D in the laboratory. Before and during these treatments, the populations of 2,4-D-degrading bacteria were monitored by most-probable-number (MPN) enumeration and hybridization analyses, using probes for the tfd genes of plasmid pJP4, which encode enzymes for 2,4-D degradation. Data obtained by these alternate methods were compared. Several months after the most recent field application of 2,4-D (approximately 1 ppm), soils with a 42-year history of 2,4-D treatment did not have significantly higher numbers of 2,4-D-degrading organisms than did control soils with no prior history of treatment. In response to laboratory amendments with 2,4-D, both the previously treated soils and those with no prior history of exposure exhibited a dramatic increase in the number of 2,4-D-metabolizing organisms. The MPN data indicate a 4- to 5-log population increase after one amendment with 250 ppm of 2,4-D and ultimately a 6- to 7-log increase after four additional amendments, each with 400 ppm of 2,4-D. Similarly, when total bacterial DNA from the soil microbial community of these samples was analyzed by using a probe for the tfdA gene (2,4-D monooxygenase) or the tfdB gene (2,4-dichlorophenol hydroxylase) a dramatic increase in the level of hybridization was observed in both soils. Probes to the tfdC, -D, -E, and -F genes did not hybridize to the bacterial community DNA to any significant extent before or after 2,4-D treatment, indicating that pathways different from the canonical pJP4-encoded pathway at the DNA sequence level, and possibly at the functional level, account for the degradative activity in these soils. Quantitative hybridization data and MPN values were in agreement, indicating that most of the 2,4-D-degrading populations were detected by the tfdA and tfdB gene probes. The hybridization patterns detected in Southern analyses of bacterial community DNA indicated that a dominant 2,4-D-degrading population was selected and maintained in these soils.