Title: Evaluation of PCR-based quantification techniques to estimate the abundance of atrazine chlorohydrolase gene atzA in rhizosphere soils Authors
Submitted to: Journal of Environmental Quality
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 5, 2010
Publication Date: November 1, 2010
Citation: Thompson, B., Lin, C., Hsieh, H., Kremer, R.J., Lerch, R.N., Garrett, H.E. 2010. Evaluation of PCR-based quantification techniques to estimate the abundance of atrazine chlorohydrolase gene atzA in rhizosphere soils. Journal of Environmental Quality. 39(6):1999-2005. Interpretive Summary: The corn herbicide atrazine has been widely used for weed control in U.S. corn production for decades. However, public health and ecological concerns have been raised because of contamination of surface and ground water by atrazine and its breakdown products, which may be toxic to humans and aquatic life. Bioremediation is a strategy for cleaning up contaminated soils using micro-organisms, fungi, green plants or their enzymes. In an effort to achieve complete atrazine degradation in soil, ARS and University of Missouri researchers have begun to investigate the potential of adding a bacterium, Pseudomonas sp. strain ADP, to soils to enhance atrazine degradation. This bacterium contains a series of genes known to be responsible for complete degradation of atrazine. A key challenge when introducing bacteria into contaminated soil is the need to determine if the added bacteria (and their genes) are persisting long enough to degrade the contaminant. In this research, we looked at several methods, from traditional to new DNA-based methods, for quantifying the presence of Pseudomonas sp. strain ADP in soil. The traditional methods require growing the bacteria by culturing soil samples in artificial culture media and counting the viable cells. This approach has the disadvantage of not directly quantifying the genes needed to degrade atrazine, but instead quantifies the viable Pseudomonas sp. strain ADP cells, which may or may not contain the atrazine-degrading genes. The DNA-based methods are more desirable as they can quantify the presence of the gene(s) responsible for degradation and better serve as an indicator of the degradation potential at any given point in time. We investigated two DNA-based methods and show that one of them (the Taqman probe-based real-time PCR assay) possessed the needed sensitivity to quantify an atrazine-degrading gene (atzA) in soils. We then applied the method to soils inoculated with Pseudomonas sp. strain ADP to demonstrate the utility of the method for quantifying the atzA gene in soils over time. This is the first successfully developed method for tracking these atrazine-degrading genes in soils. In addition, this research provides the needed methodology for assessing the effectiveness of Pseudomonas sp. strain ADP when added to soil for the purpose of degrading atrazine in soils. The approach used in this research will benefit environmental scientists in the development of new bioremediation techniques that will result in decreased atrazine contamination of water resources.
Technical Abstract: Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine, (ATR)] is one of the most commonly used herbicides in the Midwestern region of the United States. Many species of bacteria have the ability to degrade soil atrazine into less toxic metabolites, but Pseudomonas sp. strain ADP has the ability to completely degrade atrazine into CO2 and NH3. The addition of atrazine-biodegrading bacteria into contaminated sites to remove entrapped ATR is a promising approach for mitigating the pollution of ATR and its metabolites. There are many challenges in the accurate quantification of catabolic bacterial genes, such as the atrazine-degrading enzyme atzA from Pseudomonas sp. strain ADP, from soil samples. In this study we compared four quantitative methods for determining the optimal means of enumeration of atrazine-degrading bacteria in rhizosphere environments. We compared three qPCR based methods: quantitative competitive PCR and two real-time PCR methods, to traditional dilution-plate counting techniques. We demonstrated the optimal properties of the Taqman probe-based real-time PCR assay for enumeration of atzA. This optimized real-time PCR assay was then utilized to monitor atzA copy number over time to demonstrate the robust atrazine-degrading potential of Pseudomonas sp. strain ADP in a rhizosphere environment.