RESULTS FOR FUSARIUM STRAIN IDENTIFICATION IN ARTIFICIAL-INOCULATED WHEAT GENOTYPES

1. RESULTS FOR FUSARIUM STRAIN IDENTIFICATION IN ARTIFICIAL-INOCULATED WHEAT GENOTYPES

2. Fusarium graminearum, Fusarium culmorum two chemotaxonomic groups based on production of trichothecenes: Deoxynivalenol (DON) and Nivalenol (NIV)

3. BIOLOGICAL MATERIALS

4. DNA extraction and purification method - CTAB method • 100 mg ground material was mixed with 300 μl water and then 700 μl CTAB buffer was added together with 20 μl RNase solution (10 mg/ml) before incubation at 65 °C for 30 min. And then 10 μl proteinase K solution (20 mg/ml) was added before an incubation at 65 °C for 30 min • samples were centrifuged at 12,000×g for 10 min and the supernatant was transferred to a tube with 500 μl chloroform, vortexed and centrifuged at 12,000 ×g for 15 min. • the upper layer was transferred to a new tube and 2 volumes of CTAB precipitation solution were added and the samples were incubated at RT for 60 min before centrifugation at 12,000 ×g for 5 min. • the pellet was dissolved in 350 μl 1.2 M NaCl and 350 μl chloroform was added before vortexing and centrifugation at 12,000 ×g for 10 min. • the upper layer was precipitated with 0.6 volumes of isopropanol and incubated at RT for 20 min and centrifuged at 12,000 ×g for 10 min. • the pellet was washed in 70% ethanol, vacuum dried and resuspended in 100 μl MQ water. The DNA was quantified by spectophotometric method using NanoDrop 8000 All the DNA samples were diluted to 70 ng/ul

5. Primers for Fusarium identification and toxin genes identification

6. PCR analyses Amplification was performed in a Corbett RESEARCH Thermal Cycler, folowing the indications from literature. • PCR reagents: • Go Taq Green Master Mix PCR kit from Promega 2X. • 20 pmol of each primer. • different concentrations of DNA template. • in a final volume – 25 µl. Amplicons were analyzed by electrophoresis on 2% agarose gel (Promega, USA) and visualized in Ethidium Bromide (0.4 ng/ml) presence.

7. Fusarium graminearum Fusarium culmorum 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Positive control Fg 13.05 Positive control Fc 12551

8. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Tri 7 DON biosynthetic gene Tri 13 DON biosynthetic gene

9. Fusarium infection Toxin genes identification GRAINS

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