Fusarium and mycotoxin analysis
AVEQ - WP 5
WP 5: Partners
• P4 Eurofins WEJ Contaminants GmbH (Germany) Scarlett Biselli, Ole Winkelmann, Simone Staiger
• P6 (CRA – MAC) Bergamo (Italy) Rita Redaelli
• P8 (CRA-GPG) Fiorenzuola (Italy) Valeria Terzi, Caterina Morcia
• P15 Agrotest Fyto, Ltd., Kroměříž (Czech Republic) Ivana Polisenska
Methods of analysis?
WP 5: Task and questions
Which Fusarium species?
Which mycotoxins?
Analyse mycotoxins and identify Fusarium species
To dehulle or not to dehulle?
WP 5: Methods I
DON and T-2 (Commission regulation (EC) No. 1881/2006)
ELISA (LOD for DON < 20 ppb, T-2 < 5 ppb)
LC-MS/MS
DON, T-2, HT-2, ZEA, NIV, DAS, 3-ADON, 15-ADON
harmonisation of ELISA laboratories
determination of Fusarium species by PCR (P8)
WP 5: Methods II
samples are not dehulled before analysis
COMMISSION REGULATION (EC) No 1881/2006
Methods III - samples
DON and T-2 by ELISA 100 inoculated accessions from three locations
DON and T-2 by ELISA 100 non inoculated accessions from one location
11 standards in two replications from all locations, both inoculated and non inoculated
DON, T-2, HT-2, ZEA, NIV, DAS, 3-ADON, 15-ADON by LC-MS/MS for standards from all locations both I and NI)
In total: 2200 ELISA, 200 LC-MS/MS, 200 PCR analysis
Harvest years 2008, 2009; each year:
- accessions
(105)
- 2008
- DON
- inoculation
- CZE, GER,ROM
0
500
1000
1500
2000
2500
3000
3500
4000
DON-CZE
0
500
1000
1500
2000
2500
3000
3500
4000
DON-GER
0
500
1000
1500
2000
2500
3000
3500
4000
DON-ROM
Resultsμg/kg
0
50
100
150
200
250
300
350
400
T2-CZE
0
50
100
150
200
250
300
350
400
T2-GER
0
50
100
150
200
250
300
350
400
T2-ROM
μg/kg
Results
- accessions
(105)
- 2008
- T-2
- inoculation
- CZE, GER,ROM
The least contaminated genotypes, 2008
28,529,5GartonSupreme
31,324,7Typhon
36,723,0Joanette
22,320,0Miku
15,315,0Atego
36,710,3Samuel
15,0 6,7Rauhhafer aus Neustadt
Mean rank T-2
Mean rank DON
Ranking at three locations, 103 genotypes
Standards - inoculation, 2008, DON
0
1000
2000
3000
4000
µg/kg
CZE
GER
ROM
Standards - CZE
- 2008, 2009
- DON, T-2
- inoculation
0
50
100
150
200
250
T-2 2008
T-2 2009
0
500
1000
1500
2000
2500
3000
3500
4000
Jaak
Argen
tina
Genzia
na
Auteu
ilM
ina
Belind
a
Evora
Ivory
Krezu
s
Mur
esSau
l
DON 2008
DON 2009
- the lowest Mina, Saul
- the highest Genziana,
Krezus, Evora
μg/kg
-2008, 2009
- DON, T-2
- inoculation0
50100150200250300350400450500
Jaa
k
Arg
en
tina
Ge
nzi
an
a
Au
teu
il
Min
a
Be
lind
a
Evo
ra
Ivo
ry
Kre
zus
Mu
res
Sa
ul
DON 2008
DON 2009
μg/kgStandards - GER
- Mina and Saul in
the middle
- the highest
Genziana and Evora
Standards - natural infection, 2008, DON
0
1750
µg/kg CZE
GER
ROM
ITA
- 2008, 2009
- DON, T-2
- natural infection
Standards - CZE
- low level in 2009
- the lowest Mina and
Saul
0
500
1000
1500
Jaak
Argent
ina
Gen
ziana
Auteu
ilM
ina
Belind
a
Evora
Ivor
y
Krezu
s
Mur
esSau
l
DON 2008
DON 2009
0
20
40
60
80
100
120
140
Jaak
Argent
ina
Gen
ziana
Auteu
ilM
ina
Belind
a
Evora
Ivor
y
Krezu
s
Mur
esSau
l
T-2 2008
T-2 2009
- 2008, 2009
- DON, T-2
- natural infection
Standards - IT
0
100
200
300
400
Jaak
Auteu
il
Genzia
na
Argenti
naM
ina
Belind
aEvo
raIvo
ry
Krezu
s
Mur
esSau
l
DON 2008
DON 2009
0
50
100
150
Jaak
Auteu
il
Gen
ziana
Argent
ina
Mina
Belind
a
Evora
Ivor
y
Krezu
s
Mur
esSau
l
T-2 2008
T-2 2009
- very low in 2009
- Mina and Saul low
Microscopic fungi
inoculum
Different fungi differ in their requirements on environment conditions
substrate
environment conditions
Perithecium Giberella zeae, 14.5.2009
Mycotoxin concentration is dependant on:
Region - Mainly impact of weather- (cropping practices)
Cereal crop - Impact of host resistance to different fusarium pathogens
Year - Impact of weather
Influence is different for different mycotoxins
Field variability, DON
Standards, CZE, inoculation, 2008, 5 replications
0
1000
2000
3000
4000
5000
µg/kg
Field variability, T-2
Standards, CZE, inoculation, 2008, 5 replications
0
100
200
300
Jaak
Argen
tina
Genzia
na
Auteu
ilM
ina
Belind
a
Evora
Ivory
Krezu
s
Mur
esSau
lµg/kg
ELISA vs.
LC-MS/MSy = 0,9558x - 67,715
0
500
1000
1500
2000
2500
3000
3500
4000
0 500 1000 1500 2000 2500 3000 3500 4000
ELISA
LC
-MS
/MS
DON (I, NI) standards
y = 0,8363x - 11,009
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140
ELISA
LC
-MS
/MS
T-2 (I, NI) standards
verification of ELISA results
analysis of other mycotoxins
LC-MS/MS analysis, natural infection, CZE
cultivar DON T2 HT2 ZEA NIV3-
ADON15-
ADON DAS name µg/kg µg/kg µg/kg µg/kg µg/kg µg/kg µg/kg µg/kgJaak 45 21 70 <10 220 <20 <20 10Argentina 1390 59 281 <10 610 70 <20 <10Genziana 610 30 65 <10 97 40 <20 <10Auteuil 300 6 115 <10 268 <20 <20 20Mina <20 24 49 <10 34 <20 <20 25Belinda 820 24 122 19 390 30 <20 20Evora 1100 30 125 22 2760 40 <20 <10Ivory 180 18 52 <10 200 <20 <20 <10Krezus 500 21 69 <10 460 <20 <20 <10Mures 380 23 74 <10 520 <20 <20 <10Saul 25 6 42 <10 91 <20 <20 30mean 535 24 97 514
LC-MS/MS (P4)
Bologna, 2010
Dissemination of the results
SIGA meeting 2010
(Italian Society of
Agricultural Genetics)
Tulln (Austria), Worldwide Mycotoxin Reductionin Food and Feed Chains - ISM Conference 2009
Krakow (Poland), 2009
Brno (CZE), Czech and Slovak
conference about Plant Protection
2009
EUCARPIA meeting,
Cambridge, April 2010
EOC Ystad, 2010