The pathogen strain identification The pathogen strain identification based on toxin biosynthetic genes based on toxin biosynthetic genes in the Romanian wheat genotypesin the Romanian wheat genotypes

Fungal control DNA

DNA extraction: FastDNA SPIN Kit for Soil Quiagen

Permits a rapid isolation of PCR-ready genomic DNA from fungal samples.

Culture media:

Potato Extract Glucose Agar

Cereal samples preparation

DNA extraction and purification method - CTAB method

Quantification of the extracted DNA by spectrophotometric method

Thermo Scientific NanoDrop 8000

100 mg ground material was pre-treated with Celulace (Promega) for 2 hours at 65°C then 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.

Amplification was performed in a Corbett RESEARCH Thermal Cycler, folowing the indications from literature.

Amplicons were analyzed by electrophoresis on 2% agarose gel (Promega, USA) and visualized in Ethidium Bromide (0.4 ng/ml) presence.

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.

PCR analyses

Romanian wheat samples

DON 7 biosynthetic gene

DON 13 biosynthetic gene

Romanian wheat samples

NIV 13 biosynthetic gene

Romanian wheat samples

NIV 13 biosynthetic gene

Romanian wheat samples

Fusarium graminearum

Fusarium culmorum

Corn samples

15 artificially infected samples

14 GM corn samples

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