Fencing off critical source areas reduces faecal contamination and reshapes microbial communities in agricultural headwater catchments.

Agricultural headwater catchments are critical sources of microbial contamination to surface waters, yet the effectiveness of mitigation practices in these small, dynamic systems is not well defined. A paired-catchment experimental design was used to assess the effects of permanent fencing of critical source areas (CSAs) in ephemeral streams within deer-grazed pastures. Faecal, soil, and freshwater samples were collected over four years, comprising two years of baseline monitoring and two years following mitigation. Viable Escherichia coli concentrations were measured in faeces, soil and water alongside high-resolution microbiome profiling and microbial source tracking. Microbiomes of freshly voided faeces were dominated by anaerobic taxa (Bacteroidia, Clostridia and Spirochaetia) which were significantly reduced (p < 0.001) in aged faeces and replaced by aerotolerant Actinobacteria, Alphaproteobacteria and Gammaproteobacteria (p < 0.001). Fencing reduced E. coli concentrations in CSA soils by 87.4% (p < 0.0001) and by 47.0% (p = 0.041) in storm event flows, with the strongest effects in water during baseflow conditions with a 60.6% (p < 0.0004) reduction in E. coli. Freshwater microbial alpha diversity increased in the treated catchment, while soil microbiomes remained stable. Beta diversity analyses indicated distinct shifts in freshwater communities after the permanent fenced treatment, but shifts in the CSA soil microbiomes after fencing were not observed. Source tracking indicated reduced CSA soil contributions (p < 0.001), with a greater relative influence of aged faeces during storm events (p < 0.0001). These results demonstrate that fencing off CSAs can lower faecal inputs and alter aquatic microbial communities. Culture-based and sequencing approaches provided complementary insights into contamination patterns, linking observed reductions in E. coli concentrations to changes in microbial diversity and community structure. This work highlights how mitigation targeting CSAs can influence both water quality and freshwater microbiomes, and underscores the value of integrating traditional monitoring with molecular methods in evaluating agricultural management practices.