**2. Materials and Methods**

### *2.1. Plant Material and Field Trial*

The entire field experiment was conducted from October 2018 to July 2019 at Osijek (45◦32 N, 18◦44 E), Croatia. The soil type is a eutric cambisol. The average annual precipitation in the growing season is 531.3 mm, and the average annual temperature is 10.9 ◦C. Plots consisted of eight row plots with a 2.5 m length and a 1.08 m width at a sowing rate of 330 seeds/m2. The weed control was conducted with the herbicide Sekator (100 g/<sup>L</sup> amidosulfuron and 25 g/<sup>L</sup> iodosulfuronmethyl-sodium) at wheat tillering (GS 31). A total of 170 kg N/ha was applied to the plots as a granular ammonium nitrate fertilizer and was split between GS 23 and 45. The experiment was conducted with two experimental replications in two treatments (trials): naturally-infected and *Fusarium*-inoculated. In each treatment, the same four winter varieties were used (Ficko and Pepeljuga originated from the Agricultural Institute Osijek, Croatia; Mv Karizma and Mv Kolompos originated from the Agricultural Institute in Martonvasar, Hungary). The grains were taken by harvesting the whole plot with a Wintersteiger cereal plot combine-harvester. The harvested grains were mixed thoroughly from two replications in each treatment, and 100 g grain samples were taken from each plot to analyze the mycotoxin content.

### *2.2. Inoculum Production and Inoculation Procedure*

The *Fusarium* species used in this experiment were two the most prevalent casual agents of FHB: *F. graminearum* strain (PIO 31), previously isolated from winter wheat collected in East Croatia and *F. culmorum* strain (IFA 104), obtained from IFA, Tulln. Conidial inoculum of the *F. graminearum* was produced in mung bean medium [34], while *F. culmorum* spores were produced by a mixture of wheat and oat grains [35]. Conidial concentrations of both species were determined using a hemocytometer and were set to 1 × 10<sup>5</sup> mL−1. The spore suspensions were set to a concentration so that a single bottle of one strain contained a su fficient amount of suspension (>900 mL), which could be diluted in 100 L of water immediately before inoculation (100 mL/m2). One treatment was grown according to standard agronomical practice with no usage of fungicide and without misting treatment, while another treatment was subjected to two inoculation events using a tractor-back sprayer with *Fusarium* spp. at the time of flowering (Zadok's scale 65) [36]. Misting was provided by spraying with a tractor back-sprayer on several occasions.
