Copper and zinc from slurry applications shape grassland soil microbial community structure in a long-term experiment

Soil microbes are crucial in maintaining soil and plant health, underpinning agronomic productivity. This study investigates antimicrobial copper- and zinc-laden cow and pig slurry applications on soil microbial community structures in an approximately 50-year long-term slurry experiment on a permanent grassland located in County Down, Northern Ireland. Illumina and PacBio rRNA sequencing (analysing 16S and ITS genera) were used to investigate soil samples (to 15 cm depth) from the experiment's treatments. Slurry type and application rate were found to alter the soil microbial community structure. The highest slurry application rate resulted in the greatest log-2-fold changes in most relative abundances. Soil pH (ranging from ~6.5 to ~5.8) and carbon content (ranging from ~7% to ~5%) were also significantly elevated in the highest slurry treatments relative to the non-amended control. Soil pH, copper, and zinc concentrations were found to be significant key drivers of soil microbial community shifts (permutation test, p < 0.001). More specifically, copper primarily influenced bacterial (16S; F ≈ 25) and zinc has the greatest influence on fungal (ITS; F ≈ 13) genera. A key finding was that these two community types responded differently to slurry management: Canonical analysis of principal components showed 16S community structure diverged by both slurry type and application rate, whereas ITS community structure was distinguished by application rate alone. Despite this, there is no sign that it has detrimental impacts on grass yield, and indeed, some promoted microbes (Nitrospira spp.) were associated with increased productivity. However, the potentially pathogenic organism (i.e., Fusarium spp.) observed in all treatments is of concern, given the contaminating mycotoxins they produce. The findings illustrate soil microbial responses to sustained and settled long-term organic-amended soils that could not be observed without the long-term nature of this experiment. These findings indicate that long-term Cu and Zn-loaded slurry application restructures the soil microbial community in significant ways with implications for antimicrobial resistance selection and a hypothesised link to mycotoxin-related food safety for further investigation, within a One Health framework linking soil, plant, animal, and human health.