FUSARIUM STRAIN IDENTIFICATION IN DIFFERENT WHEAT SAMPLES COLLECTED FROM DIFFERENT

REGIONS FROM THE WEST

PART OF ROMANIA

Fusarium graminearum, Fusarium culmorum

Deoxynivalenol (DON) and Nivalenol (NIV)

Fusarium verticillioides and Fusarium proliferatum

Fumonisin

two chemotaxonomic groups based on production of trichothecenes:

Many of the trichothecene biosynthesis genes are localized in a gene cluster of at least 10 genes.

The genes in this cluster include the genes for:

•trichodiene synthetase (Tri5)•P450 oxygenase (Tri4 and Tri11) •acetyltransferase (Tri3 and Tri7) •transcription factors (Tri6 and Tri10)•a toxin efflux pump (Tri12) •two unidentified hypothetical proteins (Tri8 and Tri9) •another acetyltransferase gene (Tri101) is unlinked to the cluster•the genes Tri13 and Tri14, were found to be under the control of Tri10, but the functions of these genes are not known

TRICHOTHECENES ARE BIOSYNTHESIZED IN A COMPLEX PATHWAY INVOLVING A SERIES OF OXYGENATION, ISOMERIZATION, AND

ESTERIFICATION STEPS

Specie Primers sequence Amplicon size (bp)

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

Tri 7 NIV biosynthetic gene

5’ TGCGTGGCAATATCTTCTTCTA 3’3’ GGTTCAAGTAACGTTCGACAATAG 5’

465

Tri13 NIV biosynthetic gene

5’ CCAAATCCGAAAACCGCAG 3’ 3‘ TTGAAAGCTCCAATGTCGTG 5’

312

FUM 1(PQF5) fumonisin biosynthetic gene

5’ GAGCCGAGTCAGCAAGGATT 3’3’ AGGGTTCGTGAGCCAAGGA 5’

60

Primers for the toxin genes

The DON level in conventional and GMO maize seeds - 2009

THE TOXIN LEVEL IN CONVENTIONAL AND GMO MAIZE –

in previous years

The fumonisin level in conventional and GMO maize seeds - 2009

DON2008 –– low levels for GMO (114,91µg/kg) and conventional (127.09 µg/kg) maize 2009 – the average level for DON was 25 times higher (971,44 µg/kg) compared to GMO (38,59 µg/kg)

FUMONISINThe differences between conventional corn (800 µg/kg) and Bt (512,8 µg/kg) were lower in 2008, compared to 2009 - 1162,01 µg/kg for conventional maize and 462,8 µg/kg for GMO.

Significant variation of the mycotoxins concentration among places, years and genotypes has been reported in other countries, too

1 2 3 4 5 PC

Fusarium graminearum

1 2 3 4 5 PC NC

Fusarium proliferatum

Fusarium verticiloides - negatives

600 bp

MAIZE – conventional and genetically modified - 2010

Fusarium culmorum

1-4 conventional maize

5- Bt (GMO) maize

Fusarium DETECTION

1 2 3 4 5 PC

TRI 7 DON

1 2 3 4 5 PC

TRI 13 DON

TRI 7 NIV - negative

PQF5 – fumonisin - negative

TOXIN GENES DETECTION

WHEAT SAMPLES, collected from last year’s harvest

1 – Fizes Caras Severin county

2 – Sacalaz – Timis county

3 – Beregsau Mare – Timis county

4 – Becicherecu Mic – Timis county

5 – Pecica – Arad county

6 – Ineu – Arad county

7- Barsa – Arad county

Fusarium proliferatum

Fusarium graminearum 1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

Fusarium culmorum

Fusarium verticiloides - negatives

Fusarium DETECTION

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

TRI 7 DON

TRI 13 DON

TRI 7 NIV

PQF5 – fumonisin - negative

TOXIN GENES DETECTION

In general, both DON and NIV chemotypes were reported in Africa, Asia and Europe, while only the DON chemotype was found in the USA

Sample Fg Fc Fp Fv TRI 7 DON

TRI 13DON

TRI 7NIV

PQF5

1 Fizes - Caras Severin county + - - + + - -2 Sacalaz – Timis county + - + - + + - -3 Beregsau Mare – Timis county + - - + + - -4 Becicherecu Mic – Timis county + - - + - + -5 Pecica – Arad county + - - + + + -6 Ineu – Arad county + - - + + + -7 Barsa – Arad county + - - + + - -

The Fusarium presence and toxin producing genes

THE HEAVY METALS INFLUENCE ON TWO ALFALFA

GENOTYPES – A SENSITIVE AND A TOLERANT ONE

Germination on moistened filter paper

The plantlets were transferred in pots (1 week)

The metal treatments were applied after 1 month

Pb and Cd

Cu and Fe

Untreated - control

Pb 50 Cd 1 Cu 100 Fe 100

100 3 250 250

250 10 500 500

500 20

First experimental serie

The Cu and Fe concentration were too high

Pb

Cd

Cu

Fe

Pb

Cd

Cu

Fe

Second experimental serie (ppm concentration)Untreated - control

Pb 50 Cd 1 Cu 5 Fe 5100 3 10 10250 10 50 50

SIGMA

SATELIT

Two month treatment

LSD 5% LSD 1% LSD 0.1 %

25.97 34.27 44.02

The significance of differences between different variants applied to Sigma alfalfa cultivar

No.Variants Growth rate (%)

To experimental mean To control

after 1 week Relative value (%)

Differ./ Signiff.

Relative value (%)

Differ./ Signiff.

1 Control 125.95+7.84 87.04 -18.76 100.00 Control2 Pb 50 131.92+11.16 91.16 -12.79 104.74 5.973 Pb 100 148.09+11.97 102.34 3.38 117.58 22.144 Pb 250 138.49+14.74 95.70 -6.22 109.96 12.545 Cd 1 153.79+11.37 106.28 9.08 122.10 27.84*6 Cd 3 163.95+13.99 113.30 19.24 130.17 38.00**7 Cd 10 150.96+15.67 104.32 6.25 119.86 25.018 Cu 5 152.41+18.04 105.32 7.70 121.01 26.46*9 Cu 10 137.04+14.50 94.70 -7.67 108.81 11.0910 Cu 50 125.12+7.06 86.46 -19.59 99.34 -0.8311 Fe 5 158.4+12.86 109.46 13.69 125.76 32.45*12 Fe 10 150.61+12.17 104.08 5.90 119.58 24.6613 Fe 50 144.48+12.36 99.84 -0.23 114.71 18.53

Exp. mean 144.71+9.12 100.00 Control 114.89 18.76

The highest values of the growth rate with statistic significance were observed after one week for the variants:Cd 3 (38,00**); Fe 5 (32,45*); Cd 1 (27,84*) şi Cu 5 (26,46*).

One week

SIGMA

The significance of differences between different variants applied to Sigma alfalfa cultivar

One month

No.Variants

Growth rate (%)

To experimental mean To control

after 1 month Relative value (%)

Differ./ Signiff.

Relative value (%)

Differ./ Signiff.

1 Control 152.59+11.66 93.69 -10.27 100.00 Control2 Pb 50 151.88+13.56 93.26 -10.98 99.53 -0.713 Pb 100 162.43+13.85 99.74 -0.43 106.45 9.844 Pb 250 150.71+14.99 92.54 -12.15 98.77 -1.885 Cd 1 174.85+13.79 107.36 11.99 114.59 22.266 Cd 3 189.77+20.56 116.52 26.91 124.37 37.187 Cd 10 192.35+22.81 118.11 29.49 126.06 39.768 Cu 5 183.19+20.94 112.48 20.33 120.05 30.609 Cu 10 145.82+16.24 89.54 -17.04 95.56 -6.7710 Cu 50 0.00+0.00 0.00 -162.86000 0.00 -152.59000

11 Fe 5 224.59+28.11 137.90 61.73** 147.19 72.00**12 Fe 10 203.54+21.94 124.98 40.68 133.39 50.95*13 Fe 50 185.52+20.79 113.91 22.66 121.58 32.93

Exp. mean 162.86+15.05 100.00 Control 106.73 10.27

LSD 5% LSD 1% LSD 0.1 %

42.59 56.21 72.21

Compared to the untreated variant, a positive evolution of the growth rate was pointed out for the Fe 5, Cd10 and Cu 5 variants, the increasing being statistically significant only for Fe 5 variant.

Compared to the untreated variant, after one month treatment a superior growth rate was pointed out for all of the variants, except the variants Pb50, Pb250, Cu 10 and Cu 50. The growth was statistical significant only for Fe 5 and Fe 10.

0

145144151158

126 132148

138154

164151 152

137125

163

186204

225

153 152162 151

175190 192

183

146

0

50

100

150

200

250

Contro

l

Pb 50

Pb 10

0

Pb 25

0Cd 1 Cd 3

Cd 10

Cu 5Cu 1

0Cu 5

0Fe 5

Fe 10

Fe 50

Exp. m

ean

Variants

Gro

wth

rate

(%)

1week

1month

Growth rate after 1 week and 1 month after treatment with different metals of Sigma alfalfa cultivar

No.Variants

Growth rate (%) To experimental mean To control

after 1 week Relative value (%)

Differ./ Signiff.

Relative value (%)

Differ./ Signiff.

1 Control 120.68+6.58 88.89 -15.09 100.00 Control2 Pb 50 159.80+25.99 117.70 24.03 132.42 39.12**3 Pb 100 108.60+8.44 79.99 -27.17 89.99 -12.084 Pb 250 120.65+9.10 88.86 -15.12 99.98 -0.035 Cd 1 141.81+19.05 104.45 6.04 117.51 21.136 Cd 3 152.27+16.58 112.15 16.50 126.18 31.59*7 Cd 10 145.30+14.39 107.02 9.53 120.40 24.628 Cu 5 158.87+22.57 117.01 23.10 131.65 38.19*9 Cu 10 111.89+6.73 82.41 -23.88 92.72 -8.7910 Cu 50 137.87+28.93 101.55 2.10 114.24 17.19

Exp. mean 135.77+6.03 100.00 Control 112.51 15.09

LSD 5% LSD 1% LSD 0.1 %

29.19 38.52 49.48

The significance of differences between different variants applied to Satelit alfalfa cultivar

Compared to the untreated variant, most of the variants, except Pb 100, Pb 250 şi Cu 10, had a positive influence on the growth rate. The highest values, statistically significant, were pointed out for the variants Pb50 (39,12**), Cu 5 (38,19*) şi Cd 3 (31,59*).

One week

No.

Variants

Growth rate (%)

To experimental mean To control

after 1 month Relative value (%)

Differ./ Signiff.

Relative value (%)

Differ./ Signiff.

1 Control 153.54+21.44 95.85 -6.65 100.00 Control2 Pb 50 171.66+18.71 107.16 11.47 111.80 18.123 Pb 100 203.57+32.56 127.08 43.38 132.58 50.03*4 Pb 250 172.30+19.08 107.56 12.11 112.22 18.765 Cd 1 177.37+21.03 110.72 17.18 115.52 23.836 Cd 3 201.41+26.06 125.73 41.22 131.18 47.87*7 Cd 10 169.32+15.09 105.70 9.13 110.28 15.788 Cu 5 211.90+30.14 132.28 51.71* 138.01 58.36*9 Cu 10 140.85+12.83 87.93 -19.34 91.74 -12.69

10 Cu 50 0.00+0.00 0.00 -160.19000 0.00 -153.54000

Exp. mean 160.19+19.14 100.00 Control 104.33 6.65

LSD 5% LSD 1% LSD 0.1 %

44.73 59.03 75.83

The significance of differences between different variants applied to Satelit alfalfa cultivar

One month

Compared to the untreated variant, a superior growth rate was pointed out for all of the variants, except Cu 10. The highest values, statistically significant were register for the variants: Cu 5 (58,36*); Pb 100 (50,03*) şi Cd 3 (47,87*).

Compared to the experience mean, all the treatments, except Pb 50 and the control, had a positive and progressive effect in time.The same trend was observed compared to the untreated variant, too.

0

160.19

120.68

159.8

108.6120.65

141.81152.27

145.3158.87

111.89

137.87 135.77

153.54

171.66

203.57

172.3 177.37

201.41

169.32

211.9

140.85

0

50

100

150

200

250

Contro

l

Pb 50

Pb 100

Pb 250

Cd 1 Cd 3

Cd 10

Cu 5Cu 1

0Cu 5

0

Exp. m

ean

Variants

Gro

wth

rate

(%)

1week

1month

Growth rate after 1 week and 1 month after treatment with different metals of Satelit alfalfa cultivar

125.95

131.92

148.09

138.49

153.79

163.95

152.41

137.04

120.68

159.8

108.6

120.65

141.81

152.27

145.3

158.87

111.89

150.96

125.12

137.87

100

110

120

130

140

150

160

170

Control Pb 50 Pb 100 Pb 250 Cd 1 Cd 3 Cd 10 Cu 5 Cu 10 Cu 50

Variants

Gro

wth

rate

(%)

Sigma

Satelit

Growth rate for studied alfalfa cultivar 1 week after treatment with different metals

Considering the interaction genotype x treatment, after one week treatment we observed significant differences between the growth rate of the two alfalfa genotypes for the following treatments: Pb 50, Pb 100, Pb 250 şi Cu 10.

153 152

162

151

175

190183

146

154

172

204

172177

201

169

212

141

192

100

120

140

160

180

200

220

Control Pb 50 Pb 100 Pb 250 Cd 1 Cd 3 Cd 10 Cu 5 Cu 10 Cu 50Variants

Gro

wth

rate

(%)

Sigma

Satelit

Growth rate for studied alfalfa cultivar 1 month after treatment with different metals

After one month treatment the effect of the interaction genotype x treatment on the growth rate was reduced, statistically significant differences being observed only for Pb 100 and Cu 5 variants.

THANK YOU FOR YOUR

ATTENTION !