Concurrent stimulation of diflufenican biodegradation and changes in the active microbiome in gravel revealed by Total RNA

The use of slowly degrading pesticides poses a particular problem when these are applied to urban areas such as gravel paths. The urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. We, therefore, endeavoured to stimulate the degradation of the pesticide diflufenican added to an urban gravel microcosm amended with dry alfalfa to increase microbial activity. In the present study, the formation of the primary diflufenican metabolite 2-[3-(Trifluoromethyl)phenoxy]nicotinic acid (commonly abbreviated as AE-B) was stimulated by the alfalfa amendment. The concurrent changes of the active microbial communities within the gravel were explored using shotgun metatranscriptomic sequencing of ribosomal RNA and messenger RNA. Our results showed, that while the active microbial communities in the gravel were dominated by bacteria with a relative abundance of 87.0 - 98.5 %, the eukaryotic groups, fungi and micro-eukaryotes, both had a 4-5 fold increase in relative abundance over time in the alfalfa amended treatment. Specifically, the relative abundance of microorganisms involved in degradation of complex carbon sources, Bacteroidetes, Verrucomicrobia, Sordariomycetes, Mortierellales, and Tremellales, were shown to increase in the alfalfa amended treatment. Further, the functional gene profile showed an increase in genes involved in increased activity and production of new biomass in the alfalfa treatment compared to the control, as well as pointing to genes potentially involved in biodegradation of complex carbon sources and the biotransformation of diflufenican.