IDENTIFICATION OF IN SITU 2,4-DICHLOROPHENOXYACETIC ACID-DEGRADING SOIL MICROORGANISMS USING DNA-STABLE ISOTOPE PROBING

Authors: CUPPLES, ALISON; SIMS, GERALD

Submitted to: Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2006
Publication Date: 1/1/2007
Citation: Cupples, A.M., Sims, G.K. 2007. Identification of In Situ 2,4-Dichlorophenoxyacetic Acid-Degrading Soil Microorganisms Using DNA-Stable Isotope Probing. Soil Biology and Biochemistry. 39:232-238.

Interpretive Summary: DNA-based stable isotope probing (SIP) can be used to identify and study the ecology of microorganisms involved in important soil processes without the need to recover the organisms in pure culture. This is an enormous advantage, given that 90-99% of bacteria known to exist in soil have never been isolated in culture. In this study, we used SIP to examine the organisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil. Indigenous soil organisms removed nearly all of the 2,4-D within17 days, whereas little removal occurred in the sterile controls. The use of a technique called terminal restriction fragment length polymorphism (TRFLP) on soil DNA after 17 days indicated a consistent increase in the relative abundance of a bacterium that was later identified (by sequencing its 16S rRNA) as a member of the ' subdivision of Proteobacteria. The SIP technique demonstrated that this organism incorporated a large amount of labeled carbon from the added 2,4-D into its DNA, clearly indicating this was the organism responsible for herbicide degradation. The use of SIP has thus facilitated the identification of unique soil organisms degrading a common herbicide under conditions very similar to those seen in the field. The impact of this work is to allow detailed analysis of the ecology of organisms involved in herbicide fate in the field, which will facilitate development of better strategies to control weeds with less negative effects on the environment.

Technical Abstract: Stable isotope probing (SIP) was used to investigate the microorganisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil samples. Soils were unamended or amended with either unlabelled 2,4-D or UL(ring) 13C-2,4-D. Removal of 2,4-D was complete after 17 days, whereas little removal (89 ± 3 % of applied remained) was observed in the sterile controls. Terminal restriction fragment length polymorphism (TRFLP) on soil DNA after 17 days indicated a consistent increase in the relative abundance of one fragment (217 bp in Hae III digests) in soils spiked with 2,4-D (both unlabeled and labeled samples) compared to the unamended soils. TRFLP profiles from ultracentrifugation fractions of 2,4-D amended samples illustrated the same fragment experienced an increase in buoyant density (BD) in samples spiked with 13C labeled 2,4-D. This increase in DNA BD indicates the organisms represented by this fragment were responsible for degradation and uptake of the herbicide. 16S rRNA sequencing suggests the organisms belong to the ' subdivision of Proteobacteria. Unlike traditional methods of identifying herbicide degrading bacteria typically involving gross manipulation of environmental samples (such as in enrichments), SIP has facilitated the identification of unique soil organisms degrading 2, 4-D under conditions closer to those seen in the field.