RESULTS FOR FUSARIUM STRAIN IDENTIFICATION

IN

ARTIFICIAL-INOCULATED WHEAT GENOTYPES

Fusarium graminearum, Fusarium culmorum

Deoxynivalenol (DON) and Nivalenol (NIV)

two chemotaxonomic groups based on production of trichothecenes:

474 control 474 - Fc 12551 474 -Fg 13.05

483 control 483 - Fc 12551 483-Fg 13.05

488 control 488 – Fc 12551 488-Fg 13.05

501 control 501- Fc 12551 501-Fg 13.05

561 control 561- Fc 12551 561-Fg 13.05

BIOLOGICAL MATERIALS

parts

1 grains

2 chaf

3 culm

4 flag leaf

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

Primers for Fusarium identification and toxin genes identification

Specie Primers sequence Amplicon size (bp)

Fusarium graminearum 5’-CTCCGGATATGTTGCGTCAA-3’5’-GGTAGGTATCCGACATGGCAA-3’

450

Fusarium culmorum 5’-ATGGTGAACTCGTCGTGGC-3’

5’-CCCTTCTTACGCCAATCTCG-3’

570

Tri 7 DON biosynthetic gene

5’ TGCGTGGCAATATCTTCTTCCTA 3’

3’ GTGCTAATATTGTGCTAATATTGTGC 5’

381-445

Tri 13 DON biosynthetic gene

5’ CATCATGAGACTTGTGTCAGAGTTTGGG 3’

3’ GCTAGATCGATTGTTGCATTGAG 5’

282

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.

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

1- 5 control 474, 483, 488, 501, 561

6-10 - Fc 12551 474, 483, 488, 501, 561

11-15 - Fg 13.05 474, 483, 488, 501, 561

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

1- 5 control 474, 483, 488, 501, 561

6-10 - Fc 12551 474, 483, 488, 501, 561

11-15 - Fg 13.05 474, 483, 488, 501, 561

16 Fg 13.05

17 Fc 12551

Tri 7 DON biosynthetic gene

Tri 13 DON biosynthetic gene

Fusarium infection

Toxin genes identificationGRAINS

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