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WP 5 – Fusarium and mycotoxin analysis
P4 Eurofins Analytik GmbH (Germany) - Scarlett Biselli
P6 Institute for Cereal Research Bergamo (Italy)- Rita Redaelli
P8 Experimental Institute for Cereal Research of Fiorenzuola d´Arda (Italy) - Valeria Terzi
P15 Agrotest Fyto, Ltd. (CR) - Ivana Polisenska
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WP 5 – Fusarium and mycotoxin analysis
- what can we offer?
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P4 (Scarlett) - mycotoxin analyses LC-MS/MS (multitoxin method, DON, T-2, HT-2, zearalenone, diacetoxyscirpenol, 3- and 15- acetyldeoxynivalenol) - 200 samples
P6, P8, P15 (Rita, Valeria, Ivana) - ELISA mycotoxin analyses, DON, T-2, ZEA - 2200 analyses
P8 (Valeria) - Identification of Fusarium species - 200
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WP 5 – Fusarium and mycotoxin analysis
- tasks and questions
1) Which toxins are we to analyse?
2) Which Fusarium species are we to look for?
3) How to manage huge amount of samples?
4) Analyses; Methods
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1) Which toxins are we to analyse?
AVEQ = Avena genetic resources for quality in human consumption
EU legislation setting maximum levels for certain contaminants in foodstuffs: Commission Regulation (EC) 1881/2006
DON (deoxynivalenol), ZEA, T-2 + HT-2
New discussed EU limits for T-2 and HT-2(to be fixed in 200?)
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1) Which toxins are we to analyse?
DON; ZEA; T-2 + HT-2
cereals unprocessed 1250; 100; 100 ppb
oats unprocessed 1750; 100; 500 ppb
cereals intended for 750; 75; 200* ppbdirect human consumption, cereal flour…*oat products
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The maximum level applies to unprocessed cereals placed on the market for first-stage processing. “First-stage processing” shall mean any physical or thermal treatment other than drying.
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2) Which Fusarium species?A
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What should be the purpose ...
A) to analyse naturally infected oats to determine Fusarium species and their mutual proportions (variability in years, locations)
B) to analyse inoculated oats- to confirm that (only) the Fusarium species which was used for inoculation is present ?- quantification of the amount of Fusarium mycelium for studying relationships between mycotoxins content ?
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2) Which Fusarium species?A
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A) to analyse naturally infected oats- which species?
Tekauz and Mueller (2006) - in Canada F. graminearum, F. poae, F. sporotrichoides and F. avenaceum
Parrika et al. (2006) - in Finland F. langsethiae, F. poae, F. culmorum, F. graminearum, F. avenaceum
Imathiu et al. (2006) - in GB F. langsethiae
CR - 2006, 9 oat samples, 16 Fusarium isolates
7x F. sporotrichoides
6x F. poae
2x F. culmorum
?F. langsethiae?
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2) Which Fusarium species?A
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F. langsethiae
- newly described Fusarium species- similar to F. poae and F. sporotrichoides- produces T-2 and HT-2 toxins- isolated from all types of cereals, more common in oats- found in Sweden, Norway, Russia, Austria, Finland- hard artificial infection at flowering
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3) Sample selection for analyses
Inoculated plots:300 accessions + 10 controls each year x 4 locations (Germany, Italy, Romania, CR)______________1240 samples
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In case of 2 (DON, T-2):
550 samples ELISA + 100 HPLC/per year
ELISA potential: 1100 analyses/per year (?number of mycotoxins analysed?)
LC-MS/MS potential: 100 analyses/each year
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3) Sample selection for analyses
LC-MS/MS :- detection limit of 10 ppb- HT-2 toxin- more toxins in one analysis - expensive
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Discussion about LC-MS/MS role
- verification of ELISA results, interesting samples (low visible infection, low ELISA levels…)
- analyses of other mycotoxins
- comparative study - relationship between T-2 and HT-2 (naturally infected, inoculated)
ELISA :- detection limit of 5-50 ppb- HT-2 toxin- one toxin in one analysis- reasonable costs
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4) Analyses, methods A
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- harmonisation of the labs (1-2 reference samples, interlaboratory comparison)
- the same kits for analyses ?
- sample amount per analysis ?
- logistic of sample distribution ?
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Year 2007
CalendarMonth
03 04 05 06 07 08 09 10 11 12
Monthof theproject
1 2 3 4 5 6 7 8 9 10
Year 2008
CalendarMonth
01 02 03 04 05 06 07 08 09 10 11 12
Monthof theproject
11 12 13 14 15 16 17 18 19 20 21 22
Year 2009
CalendarMonth
01 02 03 04 05 06 07 08 09 10 11 12
Monthof theproject
23 24 25 26 27 28 29 30 31 32 33 34
Year 2010
CalendarMonth
01 02 03 04 05 06 07 08 09 10 11 12
Monthof theproject
35 36 37 38 39 40 41 42 43 44 45 46
WP 5 - Time table