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