1. 1 WP 5 – Fusarium and mycotoxin analysis P4 Eurofins Analytik GmbH (Germany) - Scarlett Biselli P6Institute for Cereal Research Bergamo (Italy) - Rita Redaelli P8Experimental Institute for Cereal Research of Fiorenzuola d´Arda (Italy) - Valeria Terzi P15Agrotest Fyto, Ltd. (CR) - Ivana Polisenska AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007

2. 2 WP 5 – Fusarium and mycotoxin analysis - what can we offer? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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

3. 3 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 AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007

4. 4 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?) AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007

5. 5 1)Which toxins are we to analyse? DON; ZEA; T-2 + HT-2 cereals unprocessed1250; 100; 100 ppb oats unprocessed1750; 100; 500 ppb cereals intended for 750; 75; 200* ppb direct human consumption, cereal flour…*oat products AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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.

6. 6 2)Which Fusarium species? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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 ?

7. 7 2)Which Fusarium species? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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?

8. 8 2)Which Fusarium species? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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

9. 9 3)Sample selection for analyses Inoculated plots: 300 accessions + 10 controls each year x 4 locations(Germany, Italy, Romania, CR) ______________ 1240 samples AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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

10. 10 3)Sample selection for analyses LC-MS/MS : - detection limit of 10 ppb - HT-2 toxin - more toxins in one analysis - expensive AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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

11. 11 4)Analyses, methods AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 - harmonisation of the labs (1-2 reference samples, interlaboratory comparison) - the same kits for analyses ? - sample amount per analysis ? - logistic of sample distribution ?

12. 12 WP 5 - Time table