*2.3. Seed Quality*

For the evaluation of germination ability, 3 × 50 seeds from each experimental plot (180 samples) were sown in plastic boxes between two layers of moistened (to 60% WR) filter paper. After sowing, the samples were prechilled at 7 ◦C for 3 days and placed in Sanyo growth chamber (Sanyo Electric Co., Ltd., Osaka, Japan) at constant temperature 20 ◦C. After four days, first count (germination energy) was made. The normal seedlings were counted and share in percent was evaluated. According to present International Seed Testing Association Rules [22] after eight days, the final count (germination capacity) and evaluation of normal seedlings, abnormal seedlings (AS), dead seeds (DS), and fresh ungerminated (FUS) seeds were made.

### *2.4. Fusarium DNA Quantification with Real-Time PCR*

### 2.4.1. Isolation of Total DNA from Grain

DNA was extracted according to Doyle and Doyle [23] protocol with modifications of Department of Phytopathology and Molecular Mycology UTP.

Ten grams of grain was homogenized to fine powder and 100 mg of such prepared sample was taken for DNA isolation. Samples were transferred into 2.0 mL tubes and poured with 600 μL of the extraction bu ffer containing CTAB 5.0%, EDTA 0.5 M, NaCl 5.0 M, Tris-HCl (pH 8.0) 1.0 M, β-mercaptoethanol, PVP (2.0%), and water. DNA was purified by addition of phenol:chloroform: isoamyl alcohol mixture (25:24:1) followed by centrifugation and taking the upper phase (supernatant) to the new tube, where equal volume of chloroform:isoamyl alcohol mixture (24:1) was added, mixed by inverting and centrifuged. Supernatant was taken and DNA was precipitated with cold ethanol. DNA pellet was washed with 70% cold ethanol, left to dry for 25–30 min and poured with TE bu ffer or sterile water to dissolve. Samples were stored at −20 ◦C for further analyses.
