*2.3. Inoculum Production and Inoculation*

*F. graminearum* and *F. culmorum* are the most important causal agents [72] and two isolates of each species were involved for testing. In the tests, four isolates were used, from *F. culmorum,* the Fc 12375 (1) that were isolated from wheat stalk inside space mycelium from a greenhouse test in the greenhouse of Cereal Research Inst. in 1977. The Fc 52.10 (2) and the two *F. graminearum* isolates, Fg 19.42 (3) and Fg 13.38 (4), originated from naturally contaminated wheat grains (2010). Their monosporic lines were used in the tests. To propagate inoculum the bubble-breeding method was used [1,3,10] on liquid Czapek-Dox medium. As aggressiveness is a variable trait [4,10,69,70], 50% more inocula were produced and the best ones were chosen for use. This way it was possible to put the aggressiveness under control. The aggressiveness of the isolates was done by the Petri dish method [1,3] (Figure 1). The inocula were stored until usage at 4 ◦C. Since the amount of material from the flowering plots was checked on the previous day, only that amount was separated following careful mixing from content of the 10 L balloon, as was necessary for that given day. The rate of the inocula in the mixtures was 50–50% with two components, one-third for three and one fourth with four components. They were made in the afternoon before inoculation. The suspension was fragmented by the Eta Mira household mixer machine (Czech Republic) with a 1 L volume-mixing unit.

**Figure 1.** Aggressiveness test with di fferent isolates of a moderately susceptible genotype No. 1907. Low (B19 **left**), medium (B20 **middle**) and highly aggressive (B22 **right**) isolates of *F. graminearum*. Original inocula, without dilution or mixing (the pictures are illustrations to show the aggressiveness di fferences within *F. graminearum*).

The inoculation was made at full flowering with spray inoculation. First, the control heads were covered at the end of the plot by a polyethylene bag without inoculation, with only sterilized water being sprayed. This was necessary to avoid cross inoculation from the suspension treated groups of heads. Then each plot was inoculated with 15 inocula (isolates 1, 2, 3, 4, 1 + 2, 1 + 3, 1 + 4, 2 + 3, 2 + 4, 3 + 4, 1 + 2+3, 1 + 2+4, 1 + 3+4, 2 + 3+4 and 1 + 2 + 3 + 4) on group of heads within a plot. As the mixtures were mixed *v*/*v* basis (at three components, one third was given from every component [16–18,40,44], in the counting of the e ffect of the mixing, the arithmetical mean was applied. This was applied earlier [2]. This was proportional with the volumes. When no interaction occurs between components, the arithmetical mean functions. If this is not the case, the arithmetical mean serves as control to compare the performance of the mixtures. In this case, as we had aggressiveness data of the participating components, the e ffect of the mixing could be measured. Each group of heads consisted of 15–20 heads and about 50 cm distance between each other to prevent cross inoculation between the isolates. The groups were positioned about 30 cm from the edge in two lines, in each, 8 and 7 groups of heads were spray inoculated (about 15 mL suspension each). Following inoculation, the sprayed heads were covered by polyethylene bags for 48 h [69]. As head size was di fferent, larger heads needed more suspension to have the same coverage as smaller ones. After removing the bags, the groups of heads remained loosely bound at half height of the plants, not to disturb assimilation of the leaf system.

### *2.4. Evaluation of the Disease and Toxin Analysis*

The evaluation of the visual symptoms was done on 10, 14, 18, 22 and 26 days after inoculation. In warmer years, the 22nd day was the last, because of the yellowing, the head symptoms could not be evaluated properly [4,14]. During evaluation, the percentage of the spikelets in the heads of groups were directly estimated as percentage value. Threshing was made carefully to not lose lighter infected grains (Seed Boy, Wintersteiger AG., Ried, Austria); fine cleaning was performed by an Ets Plaut-Aubry air separator (41290 Conan-Oucques, Conan-Oucques, France). In both cases, air speed was regulated so that light Fusarium infected grain remained. Then a visual evaluation of the FDK grains followed, expressed as a percentage value.

For toxin analyses, 6 g of the individual samples was separated for milling by a Perten Laboratory mill (Laboratory Mill 3310, Perten Instruments, 126 53 Hägersten, Sweden). Toxin extraction and DON toxin analysis was done according to Mesterházy et al. [14], where 1 g of fine milled wheat sample of the 6 g milled grain sample was extracted with 4 mL of acetonitrile/water (84/16, *v*/*v*) for 2.5 h in a vertical shaker. All chemicals and toxin standards were purchased from Sigma-Aldrich (1117 Budapest, 23. October Street 6–10). After centrifugation (10,000 rpm, 10 min), 2.5 mL of the extract was passed through an activated charcoal/neutral alumina solid phase extraction (SPE) column (Sigma-Aldrich Supelco, volume 5 mL, filled with 1 g mixture of 20 g Al2O3/Sigma/and 1 g activated carbon coal/Sigma/, prepared in the laboratory) at a flow rate of 1 mL/min. Then, 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*). Liquid chromatographic separation and quantification was made on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a membrane degasser, a binary pump, a standard autosampler, a thermostated column compartment and a diode array detector. DON was separated on a Zorbax SB-Aq (4.6 × 50 × 3.5 μm) column (Agilent) equipped with a Zorbax SB-Aq guard column (Agilent) (4.6 × 12.5 × 5 μm) thermostated at 40 ◦C. The mobile phase A was water, while mobile phase B was acetonitrile. Validation of DON was made by using the DON control toxin from Sigma with a regular dilution grade series. So all concentrations were within the scope of the validation line. 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.
