Evaluation of different phyllosphere sample types for parallel metabarcoding of fungi and oomycetes in grapevine

Leaf wash sediments (W) and leaf disks (L) are used to characterize the mycobiome of the Vitis vinifera phyllosphere by metabarcoding. Both sample types were processed from each leaf pool. Eighteen fully expanded Vitis vinifera cv. Müller-Thurgau leaves per leaf pool were collected at two sampling time points (02.06.2020 (N1) and 30.06.2020(N2)) at an experimental vineyard located near Neustadt an der Weinstraße, Germany. At each time point leaves of untreated (K1-3), Sulphur (S1-3) treated, or Sulphur and Copper (Cu1-3) treated vines were sampled in three repetitions from randomized Blocks resulting in 18x2 amplicon libraries. Leaf disks (one per leaf; 18 per leaf pool) were cut before washing. Negative controls for DNA extraction (K) and template free library preparation (BPCR) as well as an artificial mock community containing equal amounts of DNA from twelve different in vitro cultivated fungal species (Mock) were included. A metabarcoding strategy capable of detecting Fungi and Oomycetes by using the ITS1catta/ITS2ngs (Tedersoo et al. 2017; Tedersoo and Anslan, 2019) primer combination for ITS1 library preparation was applied. Library preparation and sequencing were carried out by AllGenetics & Biology SL (www.allgenetics.eu).