Evaluation of Different Phyllosphere Sample Types for Parallel Metabarcoding of Fungi and Oomycetes in Vitis vinifera

Metabarcoding is an effective and cost-efficient approach to study environmental microbiomes and has become a standard method in studying microbial community structures and relative species abundance. In grapevine research on leaf microbial communities, two kinds of sample types, either leaf wash sediments representing the phyllosphere microbiome from leaf surfaces or leaf tissue samples, e.g., leaf disks, including phyllosphere and endosphere microorganisms, are used to characterize leaf microbiomes. The goal of this study was to analyze the advantages and disadvantages of these sample preparation methods for the characterization of the phyllosphere microbiome by fungal metabarcoding with both sample types being processed from the exact same set of leaves. We used a metabarcoding strategy, which can detect Fungi and Oomycetes, facilitating the parallel analysis of these communities. At each sampling time point, species richness was shown to be higher in leaf wash samples, and differences in the community structure between samples was smaller for this sample type as well. Furthermore, by comparing read count abundance to qPCR measured relative proportions of selected amplicon sequence variants, a higher congruence was observed for leaf wash samples. Therefore, metabarcoding analyses of leaf samples using leaf wash sediments outperform analyses using leaf disks and should be applied to characterize phyllosphere fungal communities. As a second goal, we show that the direct comparison of metabarcoding libraries of both sample types prepared from the exact same set of leaves also provides a new strategy to identify endophytes that may not be culturable.