Pollution induced community tolerance (PICT) and analysis of 16S rRNA genes to evaluate the long‐term effects of herbicides on methanotrophic communities in soil

Abstract

SummaryThere is an increasing interest in agricultural systems in which the use of herbicides is forbidden. Therefore, soils treated with herbicides atrazine and metolachlor for the last 20 years were compared with soil samples from the same field that had never been treated (control soil). We determined the pollution induced community tolerance (PICT) by evaluating the methane oxidation capacity of soil samples after adding increasing amounts of a methane oxidation inhibitor, 2,4‐dichlorophenoxyacetic acid (2,4‐D). Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes assessed whether the soil methanotrophic community differed between the two treatments. Addition of 60 µg 2,4‐D per g soil clearly inhibited methane oxidation in both soils but increased the time needed to oxidize 5% methane in the headspace by 250% for the control soil compared with 175% for the herbicide‐treated soil. This indicates that the soil with a long‐term herbicide history had a greater tolerance to the methane oxidation inhibitor than did the control soil. The DGGE of 16S rRNA genes amplified directly from soil community DNA could also distinguish the two treatments. The banding patterns of the Type I methanotrophs contained fewer bands in the herbicide‐treated soil. It seems that both the PICT approach and DGGE analysis are effective assays to distinguish a long‐term herbicide‐treated soil from an untreated soil.