The impact of manure application on the microbiome of grassland

Manure spreading onto land is an important agricultural process. Manure is recycled as organic fertiliser; however it can introduce manure-derived antibiotic resistant bacteria into the environment. Grassland consists of approximately 70 % of global agricultural land and is a vital source of food for livestock. Despite the important role grassland plays in food security, the impact of manure application on its resistome and microbiome is relatively unknown. Antibiotic resistance is a multifactorial issue, involving an intertwining relationship between animals, humans and the environment. Therefore, it is critical to fully understand all potential routes of antimicrobial resistance (AMR) transmission. As the microbiome of grassland is an under-researched area, it is a possible source of AMR transmission to animals which may enter the food chain. A pot trial mesocosm experiment was carried out to investigate the impact of manure application on the microbiome of the phyllosphere of perennial ryegrass (Lolium perenne). Pig slurry was applied to six pots of L. perenne and grass and soil samples were taken two weeks following manure application. Following sonication, viable bacteria were isolated from the soil, manure and grass by plating on selective agars supplemented with antibiotics. Isolates were screened for antibiotic resistant bacteria by antibiotic susceptibility testing. DNA was extracted from the soil, grass and manure and underwent microbial community compositional analysis by 16S rRNA sequencing on the Illumina Miseq platform. The results from this mesocosm experiment will contribute to a further field trial to investigate the impact various manure types have on the microbiome and resistome of grassland.