Bacterial communities in the plant phyllosphere harbour distinct responders to a broad-spectrum pesticide

Pesticide application is often accompanied by harmful off-target effects that affect humans, animals, as well as whole ecosystems. However, such effects remain mainly unaddressed in connection with microorganisms, yet they collectively account for a substantial proportion of the total biomass in various ecological niches. We have employed a mechanistic approach where we addressed the effects of a concentration gradient of a broad-spectrum fungicide on non-target bacterial communities in the plant phyllosphere. Following spray application of the chemical pesticide on Nicotiana tabacum L. leaves, we analysed the microbiome by sequencing 16S rRNA gene fragment amplicons. The N. tabacum phyllosphere was characterized by a complex bacterial community with more than 25,000 amplicon sequencing variants (ASVs). It was predominantly colonized by Proteobacteria, with Alphaproteobacteria accounting for up to 33.8% of the indigenous bacterial community. Pesticide application affected the relative ratio between Alphaproteobacteria and Gammaproteobacteria, which represented the second most abundant bacterial class. Detailed statistical analyses indicated that pesticide applications had an effect on core microbiome members and that they selectively affected distinct phyllosphere bacteria. Here, we have identified four positive responders including an ASV assigned to the genus Acinetobacter and 12 negative responders mainly assigned to members of the prevalent Proteobacteria. The overall findings indicate that pesticide use affects the native plant microbiome and that various non-target microorganisms are among the responders. They also indicate that distinct plant-associated bacteria are among the most sensitive responders to the employed fungicide and thus highlight the importance of off-target studies in the context of the plant microbiome.