Environmental risk assessment at the microbial level: utilizing total RNA sequencing to evaluate the non-target impact of biochemicals in agriculture

The use of biochemicals in agriculture has become crucial to meeting the global demand for food production. Agrochemicals, such as fertilizers and pesticides, are widely applied to enhance crop efficiency. Additionally, other chemicals like nitrification inhibitors offer the potential to mitigate the environmental impact caused by the excessive use of fertilizers. While their effects on targeted microbial taxa are known, their broader environmental risk to the entire microbial community remains poorly understood.

Considering the urgency of assessing these risks, extraction and deep sequencing of total RNA offers a powerful approach to unveil non-target effects without favoring specific taxa or introducing bias from dead or dormant biomass. By simultanously analysing rRNA and mRNA, it is possible to investigate negative effects not only on the activity of specific taxa but also on critical ecosystem functions providing the potential to discover previously unknown effects.

Here, we demonstrate how total RNA analysis can enhance our understanding of non-target effects of agrochemicals on microbial soil communities. In the GENEPEASE II project, the impact of the commercial fungicide Prosaro was examined using soil microcosms over a 6 months period. Besides the expected decline in fungal taxa, significant effects on the overall microbial community were observed, as indicated by shifts in the rRNA and the gene expression profiles (all ANOVAs p < 0.05). Ongoing in-depth analyses will identify individual taxa affected by the fungicide. By linking these taxa to their ecological roles in natural settings and identifying up- and downregulated genes, we aim to pinpoint ecosystem functions that are potentially affected by the use of the fungicide.

Moving forward, this method will be applied to a field trial investigating the non-target effects of nitrification inhibitors nitrapyrin and DMPP. Both compounds have the potential to reduce emissions of the greenhouse gas N₂O. In this context, the analysis of mRNA provides an opportunity to investigate potential impacts on enzymes closely related to ammonia monooxygenase, the primary target of nitrification inhibition. Such effects could potentially impact greenhouse gas-regulating processes, such as methane oxidation, and therefore counteract the positive environmental benefits of nitrification inhibition.

Ultimately, refining this method for the presented experiments will help to develop a robust approach for utilizing total RNA in various agrochemical applications. Thus, total RNA analysis can serve as a crucial tool for incorporating microbial community data into environmental risk assessments, therefore contributing to a more sustainable future in agriculture.