*2.1. Plant Material*

Seven genotypes were selected with di ffering resistance to FHB, including four winter wheat cultivars from the variety breeding program at Szeged without strong selection for FHB, Hungary, and three lines chosen from our FHB resistance program (Table 1). They had similar flowering times, but di ffered in FHB resistance level. According to the previous tests, two genotypes proved to be susceptible, one was rated as moderately susceptible, two were rated as moderately resistant, and two were rated as resistant.


**Table 1.** Genotypes, their abbreviations and resistance classification Szeged, 2013–2014.

### *2.2. Field Experiments for Disease Evaluation*

The plant material was evaluated in the nursery of the Cereal Research Nonprofit Ltd. in Szeged, Hungary, (46◦1424" N, 20◦539" E) over two seasons (2012/2013 and 2013/2014). The field experiments were conducted in three replications in a randomized complete block design. A plot (genotype, replicate) consisted of 12 rows, 1.5 m long with a 20 cm row spacing. For the six inoculation times, two row subplots were used. For control, the first inoculation served as the mid-anthesis inoculation (Figure 1). The seven cultivars and three replicates produced 21 plots for an experiment. Each genotype per replications was sown in six (one plot per inoculation date) two-row plots of 1.5 m length in mid-October, using a Wintersteiger Plot Spider planter (Wintersteiger GmbH, Ried, Austria). The width of the plots was approximately 40 cm.

**Figure 1.** Map of a plot in the experiment. Isolates: 1, 2, 3, 4, inoculated bunches about 50 cm from each other.

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

Fungal suspensions were made in 10 L heat-stable glass flasks filled with 9.4 L liquid Czapek–Dox medium [26]. They were aerated at room temperature for a week. Suspensions were then stored at 4 ◦C until they were used for inoculation. Inocula contained mycelium and conidia as well. Each genotype was inoculated individually with two *F. culmorum* (F.c. 12375 /1/, F.c. 52.10 /2/) and two *F. graminearum* (F.g. 19.42 /3/, Fg. 13.38 /4/) isolates. The same isolates were used for studies in 2013 and 2014. The cited authors used a different conidium concentration/mL. Schroeder and Christensen [8] applied an inoculum with 5–8 × 10<sup>6</sup> conidia/mL, Hart et al. [9] used 6.6 × 10<sup>4</sup> conidia /mL, Cowger and Arrellano [12] used 1 × 10<sup>5</sup> conidia/mL, and Siou et al. used 2 × 10<sup>4</sup> conidia/mL [13]. This suggests that there is no agreemen<sup>t</sup> for which concentration is optimal. The authors did not report the reason for using the given concentration. Theoretically, it should have been some relation with aggressiveness, but none intended to give an explanation. This means that chances were low to find a good solution. However, an aggressiveness test [26] was developed to measure directly the aggressiveness of the given inoculum. This solved the problem and we could test this very important feature. A test needed 10 Petri dishes, 5–5 for two genotypes in seedling stage. Sterile double layer filter paper was placed in 12 cm diameter dishes, 9 mL suspension was uniformly spread on the surface and 25 seeds were placed into the dish in a 5 × 5 binding. Besides the original concentration, 1:1, 1:2, and 1:4 dilutions were also applied, along with a control with sterile distilled water. The rating was made daily from the second to sixth day. The number of healthy germs was evaluated. From the test, the aggressiveness could be directly seen. Therefore, the selection of the inocula was made in this system. As we did not want to relate the aggressiveness of the isolates used to each other, the standardized conidium concentration was not necessary. Even so, the four isolates differed significantly from each other.

Six inoculation dates were tested, the first at anthesis at Feekes growth stage 10.5.1 [27], second at four days, third at eight days, fourth at 11 days, fifth at 13 days, and the sixth 16 days later. Inoculation started on about 10 May. The inoculation was performed with the spray inoculation method [23]. Bunches of 15–25 spikes were sprayed from all sides with a handheld sprayer with the use of 15–20 mL fungal suspension for each sample, as described by Mesterhazy [23,28].

After inoculation, bunches were covered for 48 h in a transparent polyethylene bag, sealed to maintain 100% relative humidity and promote infection. After removing the bags, the plants were loosely bound with a label for identification at half-plant height to allow the leaves to photosynthesize freely.
