*2.5. Deoxynivalenol Analysis*

Of the grains in a group of heads, six grams were separated and milled by a Perten Laboratory Mill (Type: 3310, Perten Instruments, 126 53 Hagersten, Sweden). From this, 1 gram was used for toxin extraction for each replicate of each inoculation date in the case of each isolate with 4 mL of acetonitrile/water (84/16, *v*/*v*) for 2.5 h with a vertical shaker. Following centrifugation (10,000 rpm, 10 min), 2.5 mL of the extract was passed through an activated charcoal/neutral alumina SPE column at a flow rate of 1 mL/min. Thereafter, 1.5 mL of the clear extract was transferred to a vial and evaporated to dryness at 40 ◦C under vacuum. The residue was dissolved in 500 μL of acetonitrile/water (20/80, *v*/*v*). HPLC separation and quantification were performed on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, California, USA), which was equipped with a membrane degasser, a binary pump, a standard autosampler, a thermostatted column compartment, and a diode array detector (DAD). DON was separated on a Zorbax SB-Aq (4.6 × 50 × 3.5 μm) column (Agilent) equipped with a Zorbax SB-Aq guard column (4.6 × 12.5 × 5 μm) thermostatted at 40 ◦C. The mobile phase A was water, while mobile phase B was acetonitrile. The gradient elution was performed as follows: 0 min, 5% B; 5 min, 15% B; 8 min, 15% B; 10 min, 5% B; 12 min, 5% B. The flow rate was set to 1 mL/min. The injection volume was 5 μL. DON was monitored at 219 nm. This procedure updated the methodology used by Mesterhazy et al. (1999) [3]. The only di fference was that the measurements were made by a new Agilent Infinity 1260 HPLC system.
