High-Throughput Metabarcoding Characterizes 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. Sustainable pest management requires an understanding of the complex microbial endophyte communities existing symbiotically within plants and the way synthetic pesticides interact with them. Fungal endophytes are known to benefit plant growth and fitness and may deter pests and diseases. Recent advances in high-throughput sequencing (HTS) have enabled integrative microbiome studies, especially in agricultural contexts. Here, we profile the fungal endophyte community in the phyllosphere of two barley (Hordeum vulgare) cultivars exposed to two systemic foliar fungicides using metabarcoding, an HTS tool that constructs community profiles from environmental DNA. We studied the fungal nuclear ribosomal large subunit D2 and internal transcribed spacer 2 (ITS2) DNA markers through a bioinformatics pipeline introduced here. We found 88 and 128 unique amplicon sequence variants (ASVs) using the D2 and ITS2 metabarcoding assays, respectively. With principal coordinate analysis and permutational analysis of variance, ASV diversity did not change in response to barley cultivar or fungicide treatment; however, the community structure of unsprayed plants did change between two collection times 8 days apart. The workflow described here can be applied to other microbiome studies in agriculture and we hope it encourages further research into crop microbiomes to improve agroecosystem management.