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WP 5 – Fusarium and mycotoxin analysis. AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007. P4 Eurofins Analytik GmbH (Germany) - Scarlett Biselli P6 Institute for Cereal Research Bergamo (Italy) - Rita Redaelli
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WP 5 – Fusarium and mycotoxin analysis AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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
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
WP 5 – Fusarium and mycotoxin analysis - tasks and questions AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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
1) Which toxins are we to analyse? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 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?)
1) Which toxins are we to analyse? AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 DON; ZEA; T-2 + HT-2 cereals unprocessed 1250; 100; 100 ppb oats unprocessed 1750; 100; 500 ppb cereals intended for 750; 75; 200* ppb direct human consumption, cereal flour…*oat products 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.
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 ?
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?
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
3) Sample selection for analyses AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 Inoculated plots: 300 accessions + 10 controls each year x 4 locations (Germany, Italy, Romania, CR) ______________ 1240 samples ELISA potential: 1100 analyses/per year (?number of mycotoxins analysed?) LC-MS/MS potential: 100 analyses/each year In case of 2 (DON, T-2): 550 samples ELISA + 100 HPLC/per year
3) Sample selection for analyses AVEQ -1st project meeting Clermont Ferrand, 19.-21.9. 2007 LC-MS/MS : - detection limit of 10 ppb - HT-2 toxin - more toxins in one analysis - expensive ELISA : - detection limit of 5-50 ppb - HT-2 toxin - one toxin in one analysis - reasonable costs 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)
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 ?