Transcript Slide 1

FUSARIUM STRAIN IDENTIFICATION IN
DIFFERENT WHEAT SAMPLES
COLLECTED FROM DIFFERENT
REGIONS FROM THE WEST
PART OF ROMANIA
Fusarium graminearum, Fusarium culmorum
two chemotaxonomic groups based on production of trichothecenes:
Deoxynivalenol (DON) and
Nivalenol (NIV)
Fusarium verticillioides and Fusarium proliferatum
Fumonisin
TRICHOTHECENES ARE BIOSYNTHESIZED IN A COMPLEX PATHWAY
INVOLVING A SERIES OF OXYGENATION, ISOMERIZATION, AND
ESTERIFICATION STEPS
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
Primers for the toxin genes
Specie
Primers sequence
Amplicon size
(bp)
Tri 7 DON biosynthetic
gene
5’ TGCGTGGCAATATCTTCTTCCTA 3’
3’ GTGCTAATATTGTGCTAATATTGTGC 5’
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
381-445
THE TOXIN LEVEL IN CONVENTIONAL AND GMO MAIZE –
in previous years
The DON level in conventional and GMO maize seeds - 2009
The fumonisin level in conventional and GMO maize seeds - 2009
DON
2008 –– 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)
FUMONISIN
The 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
MAIZE – conventional and genetically modified - 2010
Fusarium DETECTION
1-4 conventional maize
1
2
3
4
5
PC
5- Bt (GMO) maize
Fusarium culmorum
Fusarium graminearum
1
2 3
4
600 bp
5 PC NC
Fusarium verticiloides - negatives
Fusarium proliferatum
TOXIN GENES DETECTION
TRI 13 DON
1
TRI 7 DON
1
2
3 4 5 PC
TRI 7 NIV - negative
PQF5 – fumonisin - negative
2
3
4
5
PC
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 DETECTION
1
2
3
4
4
5
5
6
Fusarium graminearum
1
2
3
4
5
6
7
1
2
3
Fusarium verticiloides - negatives
Fusarium culmorum
6
7
7
TRI 7 DON
TOXIN GENES DETECTION
1
2
3
4
5
6
7
TRI 13 DON
1
2
3
4
5
6
TRI 7 NIV
1
2
3
4
5
6
7
PQF5 – fumonisin - negative
7
The Fusarium presence and toxin producing genes
1
2
3
4
5
6
7
Sample
Fg
Fc
Fizes - Caras Severin county
Sacalaz – Timis county
Beregsau Mare – Timis county
Becicherecu Mic – Timis county
Pecica – Arad county
Ineu – Arad county
Barsa – Arad county
+
+
+
+
+
+
+
-
Fp
+
Fv
-
TRI 7
DON
+
+
+
+
+
+
+
TRI 13
DON
+
+
+
+
+
+
TRI 7
NIV
+
+
+
-
PQF5
-
In general, both DON and NIV chemotypes were reported in Africa, Asia
and Europe, while only the DON chemotype was found in the USA
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
First experimental serie
Untreated - control
Pb
50
Cd
1
Cu
100
Fe
100
100
3
250
250
250
10
500
500
500
20
The Cu and Fe concentration
were too high
Pb
Pb
Cd
Cd
Cu
Fe
Cu
Fe
Second experimental serie (ppm concentration)
Untreated - control
Pb
50
Cd
1
Cu
5
Fe
5
100
3
10
10
250
10
50
50
Two month treatment
SIGMA
SATELIT
The significance of differences between different
variants applied to Sigma alfalfa cultivar
No.
Variants Growth rate (%)
after 1 week
1 Control
2
Pb 50
3
Pb 100
4
Pb 250
5
Cd 1
6
Cd 3
7
Cd 10
8
Cu 5
9
Cu 10
10 Cu 50
11
Fe 5
12 Fe 10
13 Fe 50
Exp. mean
LSD 5%
25.97
125.95+7.84
131.92+11.16
148.09+11.97
138.49+14.74
153.79+11.37
163.95+13.99
150.96+15.67
152.41+18.04
137.04+14.50
125.12+7.06
158.4+12.86
150.61+12.17
144.48+12.36
144.71+9.12
LSD 1%
34.27
To experimental
mean
Relative Differ./
value (%) Signiff.
87.04
-18.76
91.16
-12.79
102.34
3.38
95.70
-6.22
106.28
9.08
113.30
19.24
104.32
6.25
105.32
7.70
94.70
-7.67
86.46
-19.59
109.46
13.69
104.08
5.90
99.84
-0.23
100.00
Control
SIGMA
To control
Relative
value (%)
100.00
104.74
117.58
109.96
122.10
130.17
119.86
121.01
108.81
99.34
125.76
119.58
114.71
114.89
Differ./
Signiff.
Control
5.97
22.14
12.54
27.84*
38.00**
25.01
26.46*
11.09
-0.83
32.45*
24.66
18.53
18.76
One week
LSD 0.1 %
44.02
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*).
The significance of differences between different
variants applied to Sigma alfalfa cultivar
No.
Growth rate
(%)
Variants
after 1 month
1
2
3
4
5
6
7
8
9
10
11
12
13
Control
Pb 50
Pb 100
Pb 250
Cd 1
Cd 3
Cd 10
Cu 5
Cu 10
Cu 50
Fe 5
Fe 10
Fe 50
Exp. mean
LSD 5%
42.59
152.59+11.66
151.88+13.56
162.43+13.85
150.71+14.99
174.85+13.79
189.77+20.56
192.35+22.81
183.19+20.94
145.82+16.24
0.00+0.00
224.59+28.11
203.54+21.94
185.52+20.79
162.86+15.05
To experimental
To control
mean
Relative Differ./
Relative
Differ./
value (%) Signiff. value (%) Signiff.
93.69
-10.27
100.00
Control
93.26
-10.98
99.53
-0.71
99.74
-0.43
106.45
9.84
92.54
-12.15
98.77
-1.88
107.36
11.99
114.59
22.26
116.52
26.91
124.37
37.18
118.11
29.49
126.06
39.76
112.48
20.33
120.05
30.60
89.54
-17.04
95.56
-6.77
0.00
-162.86000
0.00
-152.59000
137.90 61.73**
147.19
72.00**
124.98
40.68
133.39
50.95*
113.91
22.66
121.58
32.93
100.00
Control
106.73
10.27
One month
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.
LSD 1% LSD 0.1 %
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.
250
225
204
200
152
162
163
151
146
150
148
126
132
186
183
175
153
164
154
151
158
152
138
137
151
144
145
125
100
1week
1month
50
Fe
10
Fe
5
Fe
50
C
u
10
C
50
Ex
p.
m
ea
n
Variants
u
5
u
C
10
C
d
3
d
C
C
d
1
0
on
tro
l
Pb
50
Pb
10
0
Pb
25
0
0
C
Growth rate (%)
192
190
Growth rate after 1 week and 1 month after treatment with
different metals of Sigma alfalfa cultivar
The significance of differences between different variants
applied to Satelit alfalfa cultivar
One week
No.
Growth rate
To experimental mean
To control
Variants
(%)
Relative Differ./
Relative
Differ./
after 1 week
value (%) Signiff. value (%) Signiff.
1 Control 120.68+6.58
88.89
-15.09
100.00
Control
2
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
-12.08
4 Pb 250 120.65+9.10
88.86
-15.12
99.98
-0.03
5
Cd 1
141.81+19.05
104.45
6.04
117.51
21.13
6
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
24.62
8
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
-8.79
10 Cu 50 137.87+28.93
101.55
2.10
114.24
17.19
Exp.
135.77+6.03
100.00
Control
112.51
15.09
mean
LSD 5%
29.19
LSD 1% LSD 0.1 %
38.52
49.48
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*).
The significance of differences between different variants
applied to Satelit alfalfa cultivar
One month
No.
Variant
s
1
2
3
4
5
6
7
8
9
10
Control
Pb 50
Pb 100
Pb 250
Cd 1
Cd 3
Cd 10
Cu 5
Cu 10
Cu 50
Exp.
mean
LSD 5%
44.73
Growth rate
(%)
To experimental mean
after 1 month
Relative
value (%)
Differ./
Signiff.
153.54+21.44
171.66+18.71
203.57+32.56
172.30+19.08
177.37+21.03
201.41+26.06
169.32+15.09
211.90+30.14
140.85+12.83
0.00+0.00
95.85
107.16
127.08
107.56
110.72
125.73
105.70
132.28
87.93
0.00
-6.65
11.47
43.38
12.11
17.18
41.22
9.13
51.71*
-19.34
-160.19000
160.19+19.14
100.00
Control
LSD 1% LSD 0.1 %
59.03
75.83
To control
Relative
Differ./
value
Signiff.
(%)
100.00 Control
111.80
18.12
132.58 50.03*
112.22
18.76
115.52
23.83
131.18 47.87*
110.28
15.78
138.01 58.36*
91.74
-12.69
0.00 -153.54000
104.33
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*).
6.65
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.
250
211.9
203.57
200
1week
201.41
1month
172.3 177.37
171.66
169.32
Growth rate (%)
153.54
140.85
150
159.8
152.27
100
120.68
158.87
145.3
141.81
160.19
137.87 135.77
120.65
111.89
108.6
50
0
n
m
ea
50
Ex
p.
C
u
u
10
5
u
C
d
C
Variants
C
10
3
C
d
1
d
C
25
0
Pb
10
0
Pb
50
Pb
C
on
tro
l
0
Growth rate after 1 week and 1 month after treatment with different
metals of Satelit alfalfa cultivar
170
163.95
Sigma
160
159.8
158.87
Satelit
153.79
Growth rate (%)
150
152.27 150.96
152.41
148.09
145.3
141.81
140
137.87
138.49
137.04
131.92
130
125.95
120
110
120.68
125.12
120.65
111.89
108.6
100
Control Pb 50 Pb 100 Pb 250
Cd 1
Cd 3
Cd 10
Cu 5
Cu 10
Cu 50
Variants
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.
220
212
204
200
Sigma
201
192
Satelit
Growth rate (%)
190
183
180
172
172
177
175
169
162
160
154
153
152
151
146
141
140
120
100
Control Pb 50 Pb 100 Pb 250
Cd 1
Cd 3
Variants
Cd 10
Cu 5
Cu 10
Cu 50
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 !