Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors

The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one riparian zone soil. The samples were amended with atrazine, and mineralization was measured in biometers as 14CO2 evolution in samples spiked with [ring-U-14C]-labeled atrazine. Atrazine-degrading microorganisms were enumerated by a most-probable-number (MPN) method, which was based on loss of atrazine (HPLC assay) in acetate media. Dot-blot hybridization assays were performed on total DNA extracted from the MPN samples. The DNA probes used in dot-blot assays were the genes atzA (atrazine chlorohydrolase from Pseudomonas ADP), atrA (cytochrome P-450 from Rhodococcus TE1), and trzD (cyanuric acid amidohydrolase from Pseudomonas NRRLB-12228). The herbicide amendment enhanced the subsequent rate of mineralization of atrazine in all three soil samples. The MPN numbers were in the range of 100–102 cells g−1 dry wt. soil, indicating that atrazine-degrading microorganisms could not be quantitatively enumerated by this technique. Positive hybridization signals of DNA extracted from MPN samples were frequent with the atzA probe; the atrA dot blots had fewer positive signals. The trzD signals were negligible or undetectable, although the parallel mineralization studies showed fast and extensive 14CO2 evolution from [ring-U-14C]-atrazine. The results suggested that trzD, the only gene known to encode s-triazine ring-cleavage, is not dominant among the atrazine-degrading populations of these soils. Streptomycin and cycloheximide were added to soil samples in biometers to determine the relative contributions of bacteria and fungi in the soils to the mineralization of atrazine. The relative suppression of mineralization in the presence of the bacterial or fungal specific inhibitor was approximately the same, indicating that both groups contributed to the mineralization of atrazine.