**2.1. Field experiments**

Two winter common wheat cultivars (Bohemia and Darwin, both quality group A) and two winter spelt wheat cultivars (Ceralio and Rubiota) from the exact field plot trials, conducted in the years 2010/2011 and 2011–2012 at the experimental station of the Czech University of Life Sciences in Prague (295 m above sea level, average annual temperature 8.4°C, average sum of precipitation 575 mm), were used for the evaluation of the effect of FHB infestation on the *Fusarium* mycotoxins [DON, deoxynivalenol-3-β-d-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON) and ZON] content in grain, standard technological quality parameters of grain, and rheological parameters of dough. Field plot trials were carried out by the method of randomized blocks in three replications; an average size of experimental plot was 12 m<sup>2</sup> . Exact field experiments were conducted in an organic farming system on experimental area, certified for organic farming and in comparison also in usual conventional system, using herbicide treatment and nitrogen fertilization −120 kg N ha−1, applied in two doses −60 kg N ha−1 after wheat overwintering, 60 kg N ha−1 at the beginning of shooting. Treatments with natural *Fusarium* spp. contamination were evaluated in both growing systems; to ensure a higher level of *Fusarium* infestation, artificial inoculation of flowering ears was used too.

### **2.2. Artificial inoculation**

The isolates of *Fusarium culmorum* and *F. graminearum* used for the artificial inoculation were obtained from the mycological collection of the Crop Research Institute in Prague and cultivated on sterile wheat grains. Wheat grains with the cultures of *F. culmorum* and *F. graminearum* were put into a vessel with water and shaken for 15 min in a laboratory shaker to release the spores into water. The obtained suspension was filtered through the gauze. Then, artificial inoculation was made using the suspension of *F. culmorum* and *F. graminearum* spores in the ratio of 1:1, 10<sup>7</sup> of spores ml−1 (Bürker chamber was used for the verification of inoculums density), 2 l of suspension per experimental plot (12 m<sup>2</sup> ). The suspension was dosed with a hand sprayer at the beginning of the wheat flowering [14]. Harvested grain samples were used for *Fusarium* mycotoxins determination.

#### **2.3.** *Fusarium* **mycotoxins determination**

A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was used for the isolation of analytes from the wheat grain. It is the multiresidue determination of wide range of mycotoxins applicable within different matrices (cereals, feed, and others), based on the extraction of analytes with acetonitrile; water and further purification of the extracts consists of the division between the two phases by means of inorganic salts (NaCl, MgSO<sup>4</sup> ). Analytes were transported into the upper acetonitrile layer, while the polar co-extract matrix (e.g. sugars or amino acids) remained in the aqueous phase [15]. The ultra-high performance liquid chromatograph Acquity UPLC System (Waters, USA), coupled with the tandem mass spectrometer LCT Premier XE (Waters, USA) with analyzer time-of-flight MS (TOFMS) was used for the identification and detection of analytes. Wider scale of *Fusarium* mycotoxins was determined deoxynivalenol (DON) and its conjugated forms, zearalenone (ZON) and its metabolites, enniatins, and so forth, but some of them only in trace amount. Only the mycotoxins, which were detected most frequently in our wheat grain samples—DON, deoxynivalenol-3-β-d-glucoside (D3G), 3-acetyldeoxynivalenol (3-ADON), and ZON—are presented in this study.

### **2.4. Standard technological quality parameters**

Crude protein content (CP) in grain dry matter according to the Kjeldahl method (EN ISO 20483; ICC-Standard No. 105/2), wet gluten content (WG) in grain dry matter and gluten index (GI) using the apparatus Glutomatic Perten (ISO 5531), falling number (FN)—ISO 3093, sedimentation index—Zeleny test (ZS)—ISO 5529, volume weigh (VW)—ISO7971-2, and TKW (thousand kernels weight) were determined with the frame of the baking quality.

#### **2.5. Rheological characteristics**

Protein and starch characteristics of the wheat flour (dough development, protein weakening, starch gelatinization, diastatics activity, and anti-stalling effect) were determined by the apparatus Mixolab (Chopin, Tripette et Renaud, Paris, France) according to the Mixolab protocol Chopin+ [16]. Evaluated flour was obtained by milling the cereal grain samples on a Bühler mill automat MLU 202.

A typical Mixolab curve, which is shown in **Figure 1**, is separated to the five stages represented by five (C1–C5) points [16].

**Figure 1.** Standard Mixolab curve.

The two first stages of the Mixolab curve correspond to the rheological characteristics of proteins—stability, elasticity, and water absorption, whereas the other stages relate mainly to the starch and amylolytic activity. Evaluated characteristics from measured Mixolab curve are C1 (Nm) marks maximum torque during mixing, used to determine water absorption; C1 (min) time required to achieve the maximum torque; C2 (Nm) measures the weakening of the protein based on the mechanical work and the increasing temperature; C3 (Nm) indicates the rate of starch gelatinization; C4 (Nm) represents the stability of the hot-formed gel; C5 (Nm) expresses starch retrogradation during the cooling period; difference C1–C2 represents the protein network strength under the increasing heating; difference C3–C4 shows diastatic activity and relates with falling number; difference C5–C4 correlates with the anti-stalling effects, represents the shelf life of end products; and DS indicates the stability of the dough before weakening [16].

Results were statistically evaluated by the analysis of variance (ANOVA) and correlation analysis with the statistical significance expression on the level α = 0.05 and α = 0.01 in the "Statistica 9.0 CZ".
