fusarium strain identification in different wheat samples collected from different regions from the...
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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.
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