High-throughput metabarcoding characterises fungal endophyte diversity in the phyllosphere of a barley crop

Over the last decade, the microbiome has received increasing attention as a key factor in macroorganism fitness. Therefore, sustainable pest management requires an understanding of the complex microbial communities existing symbiotically within plants and the way synthetic pesticides interact with them. Fungal endophytes are known to benefit plant growth and fitness and deter pests. Recent advances in next generation sequencing (NGS) technologies have enabled holistic microbiome studies especially in agricultural contexts. Here we profile the fungal endophyte community in the phyllosphere of two barley (Hordeum vulgare) varieties exposed to two systemic foliar fungicides using metabarcoding, an NGS tool that creates community profiles from environmental DNA (eDNA). We used the fungal D2 and ITS2 ribosomal DNA markers and analyzed the data using a novel bioinformatics pipeline introduced here.We found 88 and 128 unique amplicon sequence variants (ASVs) using the D2 and ITS2 metabarcoding assays, respectively. ASV diversity did not change in response to barley cultivar or fungicide treatment, however the structure of the community did change between collection times. To our knowledge, the pipeline we describe for metabarcoding analysis of crop endophytes from surface sterilised phyllosphere material has not been published elsewhere. This workflow can be applied to other microbiome studies in agriculture and we hope it encourages further research into crop microbiomes to improve agroecosystem management.