Detection of a Methylcarbamate Degradation Gene in Agricultural Soils Using PCR Amplification of Bacterial Community DNA

Abstract

The carbamate insecticide carbofuran (2,3-dihydro-2,2-dimethyl-7-benofurayl-N-methylcarbamate) is biodegraded by a methylcarbamate hydrolase enzyme encoded by a methylcarbamate degradation (mcd) gene cloned from Achromobacter sp. strain WM111. A 0.4-kbp BamHI-KpnI fragment of the mcd gene was used as a DNA probe to monitor soil microbial populations capable of degrading carbofuran in soils from twelve contrasting agricultural sites, representing seven soil series from five U.S. states. Each soil was amended three times with carbofuran (200 μg g⁻¹ dry weight soil) and monitored until 90% of the carbofuran had degraded after each application. Soil bacterial community DNA was extracted and humic acid contaminants removed prior to PCR amplification of mcd. The detection limit for the probe protocol was 10² microorganisms g⁻¹ of soil. Eight soils were mcd positive, and four were negative. Results were independently confirmed using both a Southern blot and slot blot protocol. Of the four negative soils, three exhibited accelerated rates of carbofuran degradation, suggesting that enzymes other than the hydrolase encoded by mcd were active in pesticide removal.

Acknowledgments

The excellent assistance of Dr. Scott Enebak and Mr. Sean Bennette is gratefully acknowledged. We would like to thank Dr. Allan Felsot (IL), Dr. James Kotcon (WV), Dr. Tim Parkin (IA), Dr. Dan Shelton (MD), and Dr. Ron Breakwell (IN) for providing soil samples. This work was supported by USDA/CSRS grant 90-3414-5125 and by a West Virginia University Faculty-Senate grant.