title: Fungal endophytes in Dactylis glomerata sample D61.S2011 taken in 2011
design: The fungal communities in soil and endosphere were assessed by a nested PCR approach targeting the ITS region as described in Wemheuer and Wemheuer (Wemheuer and Wemheuer, 2017). In the first PCR, the primers ITS1-F_KYO2 (5-TAGAGGAAGTAAAAGTCGTAA-3) (Toju et al., 2012) and ITS4 (5- TCCTCCGCTTATTGATATGC-3) (White et al., 1990) were used to suppress co-amplification of plant-derived ITS regions. The PCR mixture (25 l) contained: 5 l of 5-fold Phusion GC buffer, 200 M of each of the four deoxynucleoside triphosphates, 4 M of each primer, 5% DMSO, 25 mM MgCl2, 0.5 U of Phusion High Fidelity DNA polymerase (Thermo Scientific) and approximately 10 ng DNA sample as template. Negative controls were performed using the reaction mixture without template. Genomic DNA of Aspergillus nidulans was used as template in the positive control for the fungal product. The following thermal cycle scheme was utilized: initial denaturation at 98C for 30 s followed by 6 cycles of denaturation at 98C for 15 s, annealing at 53C for 30 s decreasing 0.5C in each cycle, followed by extension at 72C for 30 s and 29 cycles of denaturation at 98C for 15 s, annealing at 50C for 30 s, followed by extension at 72C for 30 s. The final extension was carried out at 72C for 2 min. Obtained PCR products were subjected to nested PCR. The ITS2 region was subsequently amplified as described for the first PCR using approximately 50 ng product of the first PCR and the primers ITS3_KYO2 (Toju et al., 2012) and ITS4 (White et al., 1990) containing the MiSeq adaptors (underlined): MiSeq-ITS3_KYO2 (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATGAAGAACGYAGYRAA-3) and MiSeq-ITS4 (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC-3).