Sequences were deposited in the NCBI database under accession numbers: GU139965–GU140004. To design two diagnostic primer/probe sets for esyn1 homologues present in genomes of F. avenaceum/F. tricinctum and F. poae, the sequences were aligned with geneious pro 4.0.0 (Biomatters Ltd, Auckland, New Zealand). Sequences of the esyn1 gene of F. avenaceum/F. tricinctum (EF029060, EF026103, AF351600, AF351597, AF351594, AF351591, AF351588, AF351585, EF040582) and F. scirpi (Z18755) used for alignment were obtained from the NCBI database. Conserved nucleotide sites within esyn1 homologues present PLX4032 cost in genomes of

F. avenaceum/F. tricinctum and F. poae, respectively, were used to design primers and probes using primer express 3.0 (Applied Biosystems) (Table 2). esyn1 probes, conjugated with an MGB group, were labeled at the 5′-end with FAM, while the IPC probe was labeled at the 5′-end with VIC. All primers were synthesized by genomed (Warsaw, Poland), while MGB probes were ordered from ABI PRISM Primers and TaqMan Probe Synthesis Service. TaqMan reaction conditions were used for each esyn1 homologue, including fungal IPC control as recommended by the manufacturer in the fast PCR protocol: 95 °C for 20 s (95 °C for 3 s, 60 °C for 30 s) × 36. TaqMan reagents were optimized

as follows: 2 μL DNA, 10 μL H2O, GSK458 5 μL Real-Time 2 × PCR Master Mix Probe [Taq DNA polymerase 1 U μL−1, reaction buffer (2 ×), MgCl2 (10 mM), dNTP mix (0.5 mM each), stabilizers], 0.2 μL ROX 50 × (A&A Biotechnology, Gdynia, Poland), 1.8 pM of each IPC primer, 0.5 pM of IPC probe, 6 pM of either F. avenaceum/F. tricinctum or F. Fenbendazole poae esyn1-based primers and 1.7 pM of either F. avenaceum/F. tricinctum or F. poae esyn1-based probe. All PCR amplifications were carried out in a 7500 Fast Real-Time PCR System (Applied Biosystems) in a final volume of 17 μL. The threshold value was 0.1 U. To determine the efficiency and sensitivity of the assay, 10.5 ng of genomic DNA of F. avenaceum and 35 ng of F. poae DNA isolates were

serially diluted by a factor of 5 with water and used as a template. In order to eliminate false-positive results, a PCR reaction was considered positive only if the CT value was <35. The relationships between the quantified DNA and enniatins A+B concentrations were determined by Pearson’s correlation analysis using statistica software (Data Analysis Software System, ver. 6.1; StatSoft Inc., 2003, http://www.statsoft.com). Samples with enniatins values below limit of quantifications (LOQs) were considered as LOQs values. Original enniatins values were transformed using Box–Cox transformation, with λ=−0.73 and 0.29 for F. avenaceum/F. tricinctum and F. poae esyn1 amounts, respectively. The coefficients of the Pearson correlation (R) were calculated. Because of the high polymorphism of the esyn1 gene, two pairs of primers/probes potentially specific for F. avenaceum/F. tricinctum and F.