bars with different colors are different at the tukey’s hsd test (p=0.05) avena genetic resources...

Avena genetic resources for quality

in human consumption (AVEQ)

WP5,Fusarium and Mycotoxins analysis.

1. Samples from artificial inoculation of Fusarium spp. in field tests of Germany,

Czech Republic and Romania.

2. Natural infection in Northern Italy field test.

3. ELISA test for DON and T-2 detection

4. qPCR analysis on a subset of samples to quantify Fusarium

CRA-GPG (ex Experimental Institute for Cereal Research), Fiorenzuola d’Arda,

Italy

Valeria Terzi, Caterina Morcia

ELISA test for mycotoxin detection

127 samples from Fusarium Screening 2008 in Gross Lusewitz

ELISA tests for DON on these 127 samples are ongoing

ELISA tests for T2 on these 127 samples?

Starting from the results obtained we can select a set of genotypes grown in Northern Italy,

under natural infection, and evaluate them for DON

TOX>255

20<TOX254

15<TOX203

10<TOX152

TOX101

AnnoClasse

TOX>255

20<TOX254

15<TOX203

10<TOX152

TOX101

AnnoClasse

SpecieClasse

Grano duro3

Grano tenero2

Orzo1

SpecieClasse

Grano duro3

Grano tenero2

Orzo1

Varietà

San Carlo Blasco - SagittarioExpress4

Creso - Duilio - Simeto

Amarok - Centauro - Eureka -Guadalupe - Serio - Soissons

Federal3

OrobelBilancia - Isengrain - Mieti - Mol

- VaioletAmillis -Mattina

2

Colosseo -Neodur

Bolero - Bologna - Enesco -Nobel - Paderno - Pandas

Baraka1

Grano duroGrano teneroOrzoClasse

Varietà




Federal3



2

Colosseo -Neodur


Baraka1


Cereali3

Rinnovo – Prato - Altro2

Ortaggi1

PrecessioneClasse

Cereali3


Ortaggi1

PrecessioneClasse

Semina su sodo –minimum tillage

3

Lavorazione senza rivoltamento della zolla

2

Aratura - Scasso1

Tipo di lavorazione del suoloClasse


3


2

Aratura - Scasso1

Tipo di lavorazione del suoloClasse6Lavorazione

5Precessione

4Varietà

3Specie

2Zona

1Andamento stagionale

54321

Classi (colonne)Variabile (righe)

6Lavorazione

5Precessione

4Varietà

3Specie

2Zona


54321


Rete agrometeo

Modello FHB-risk

Indice TOX

TOX>255

20<TOX254

15<TOX203

10<TOX152

TOX101

AnnoClasse

TOX>255

20<TOX254

15<TOX203

10<TOX152

TOX101

AnnoClasse

SpecieClasse

Grano duro3

Grano tenero2

Orzo1

SpecieClasse

Grano duro3

Grano tenero2

Orzo1

Varietà




Federal3



2

Colosseo -Neodur


Baraka1


Varietà




Federal3



2

Colosseo -Neodur


Baraka1


Cereali3


Ortaggi1

PrecessioneClasse

Cereali3


Ortaggi1

PrecessioneClasse


3


2

Aratura - Scasso1

Tipo di lavorazione del suoloClasse


3


2

Aratura - Scasso1

Tipo di lavorazione del suoloClasse6Lavorazione

5Precessione

4Varietà

3Specie

2Zona


54321


6Lavorazione

5Precessione

4Varietà

3Specie

2Zona


54321


Rete agrometeo

Modello FHB-risk

Indice TOX

Rete agrometeo

Modello FHB-riskModello FHB-risk

Indice TOX

Barley

Fiorenzuola Tolentino

Triticale

DON survey in other cereals, 2008

Specie fungina

Sequenza 5’-3’ Riferimento bibliografico

F.culmorum Primer Forward TCACCCAAGACGGGAATGAPrimer Reverse GAACGCTGCCCTCAAGCTTprobe CACTTGGATATATTTCC

Waalwijk C, van der Heide R, de Vries I, van der Lee T, Schoen C, Costel-de Corainville G, Hausen-Hahn I, Kastelein P, Kohl J, Lonnet P, Demarquet T, Kema GHJ (2004) Eur J Plant Pathol 110: 481-494

F.graminearum Primer Forward GGCGCTTCTCGTGAACACAPrimer ReverseGGCTAAACAGCACGAATGCprobe AGATATGTCTCTTCAAGTCT

Waalwijk C, van der Heide R, de Vries I, van der Lee T, Schoen C, Costel-de Corainville G, Hausen-Hahn I, Kastelein P, Kohl J, Lonnet P, Demarquet T, Kema GHJ (2004) Eur J Plant Pathol 110: 481-494

F.culmorum Primer Forward ATGGTGAACTCGTCGTGGCPrimer Reverse CCCTTCTTACGCCAATCTTCG

Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Physiol Mol Plant Pathol 53: 17-37

Demeke T, Clear RM, Patrick SK, Gaba D (2005) Int J Food Microbiol 103: 271-284

F.graminearum Primer Forward CTCCGGATATGTTGCGTCAAPrimer Reverse GGTAGGTATCCGACATGGCAA

Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Physiol Mol Plant Pathol 53: 17-37

Brandffass C, Karlovsky (2006) BMC Microbiology 6:4 doi:10.1186/1471-2180/6/4Demeke T, Clear RM, Patrick SK, Gaba D (2005) Int J Food Microbiol 103: 271-284

F.graminearum Primer Forward GGTCTCGACAGCAATGGTGTTPrimer Reverse GCTTGTGTTTTTCGTGGCAGTprobe ACAACGGCACCTCTGAGCTCCAGC

Reischer GH, Lemmens M, Farnleitner A, Adler A, Mach RL (2004), 59(1): 141-146

F.culmorum Primer Forward GATGCCAGACCAAGACGAAGPrimer Reverse GATGCCAGACGCACTAAGAT

Schilling AG, Moller EM, Geiger HH (1996) Phytopathol 86:515-522Brandffass C, Karlovsky (2006) BMC Microbiology 6:4 doi:10.1186/1471-2180/6/4

F.culmorumF.graminearum

Primer Forward CAAAAGCTTCCCGAGTGTGTCPrimer Reverse GGCGAAGGTTCAAGGATGAC

Leisova L, Kucera L, Chrpova J, Sykorova S, Sıp V, Ovesna J (2006) J Phytopathol 154: 603–611

F.culmorum Primer Forward TTCACTAGATCGTCCGGCAGPrimer Reverse GAGCCCTCCAAGCGAGAAGprobe AAAGAAGTTGCAATGTTAGTG


F.culmorum Primer Forward GTACCAGAGCAC GGC GTTGPrimer Reverse TCGCTCTCGATCAAAAGAAGGprobe ACAAGTCCCCTCGACGTG


F.graminearum Primer Forward AGGGACAATAAGTGCAGAPrimer Reverse ACTGTGCACTGTCGCAAGTG

Niessen L, Vogel RF (1997) Syst Appl Microbiol 20: 111-123

Fusarium spp. Primer Forward GCTGCTCATCACTTTGCTCAGPrimer Reverse CTGATCTGGTCACGCTCATC

Niessen L, Vogel RF (1997) Syst Appl Microbiol 21: 618-631 Schnerr H, Niessen L, Vogel R F (2001) Int Jf Food Microbiol 71:53–61

Fusarium spp. Primer Forward TGATTTACATGGAGGCCGAATCTCAPrimer Reverse TTCGAATGTTGGTGATTCATAGTCGTTprobe TTGCCCCTCTTTGATCGAGTTGCGTCTCCC

Bluhm BH, Cousin MA, Woloshuk (2004) J Food Protect 67(3): 536-543

Kimura et al, 2007

tacttcgtcg acatggggga gtaaccgccg tttcccgccc cttgttaagc taagcgtttt 2581 taatatggaa aacggagttc atctacagta gagtcgacaa gatctgcaat ctatcagtgc 2641 ttaaatgcag ttccagctac gcgactttca ggcaccggca atggagatct tggctcagat

2701 tctagtctaa ctaacacata ggaaaggggt tgtagatcgt acagctaaat tcagagcctc 2761 ctgctaaacc taaaagggct aagttgccca aactttattc aacgcagttg cagtgcattc

2821 gggagccaac tccgcgggat atatgtgatg gccggcagtt tcgtagtgct gatcataaaa 2881 gtggtcatat ttaaggcctg tcgcccaggt agacttttgc cagggcacaa tataacaccg 2941 cggctaccta ggtaagtgag gctttcttct gtgttgatag acggtcgttt cactagtcca

3001 aacacagacc acggacaatg accgaactca atatcccgat ccaaggattg atccctagat 3061 ttaggcctac

F. graminearum: MPVP74, ITEM2, ITEM124 , ITEM126,

ITEM646, ITEM2724, ITEM4576, ITEM6350, ITEM6403,ITEM6477 ,

ITEM6484, ITEM6613

F. culmorum: MPVP69, MPVP70, MPVP71, ITEM 2, ITEM 3,

ITEM 5, ITEM 6, ITEM7, ITEM8, ITEM 9, ITEM 11, ITEM12, ITEM 13, ITEM 15, ITEM 16, ITEM 17, ITEM 18, ITEM 19,ITEM 21, ITEM 22,

ITEM 23, ITEM 24, ITEM 25, ITEM 32

F. poae: MPVP90F. avenaceum: MPVP66

F. verticilloides: MPVP228Microchodium nivale: MPVP77

0

1

2

3

4

5

6

59 61 65 71 75 85

Growth stages (BBCH)

DN

A (

ln p

g)

F 2F 1

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 6 8 10

DNA (ln pg)

DO

N (

ln p

pm

)

F. culmorum

F. graminearum

3 bread wheat cv3 durum wheat cv2 yearsInoculation with F. graminearum (2 strains)

Inoculation with F. culmorum (3 strains) 6 developmental stages

anno di coltivazione (21%)

specie di Fusarium (6%)

stadio di crescita del frumento (32%)

varietà di frumento (4%)

anno x stadio di crescita (5%)

anno x varietà (8%)

specie di Fusarium x stadio di crescita

(4%)

varianza residua (20%)

Parametri di regressione 1 Campioni

N di casi

Coefficiente di

correlazione a b P2 R2

adj3

Totale dei campioni

44 0.93 458.3 10.11 < 0.001 0.86

con DON 500 μg/kg

28 0.91 997.7 9.77 < 0.001 0.83

Granella 11 0.97 -120.0 11.11 < 0.001 0.92

Farina integrale

11 0.89 -47.9 7.99 < 0.001 0.77

Farina 11 0.99 -49.6 12.60 < 0.001 0.98

Pane 11 0.87 -266.1 91.27 0.001 0.72

Cultivar Località Contenuto in DON (μg/kg)

A 30.217 B 29.630 C 15.685 D 2.288 E 247

Guadalupe

F 11 G 3.559 H 14.495 I 2.173 L 285

Serio

M 140

0 20 40 60 80 100

bread

flour

wholemeal

grainsqPCR based Fusarium

traceability in raw materials

and food.

Fusarium traceability for early diagnosis

Fun

gus

Hou

rs

Bila

ncia

Sag

ittar

io

Dui

lio

S.C

arlo

06

121824487214421628806

1218244872144216288

F.

culm

orum

F.

gram

inea

rum

2 bread wheat cv2 durum wheat cvIn potsInoculation with F. graminearum Inoculation with F. culmorumFlowering stage (65 BBCH)Samples from 6hours from inoculation to 12 days

Avena genetic resources for quality in human consumption (AVEQ)

qPCR based Fusarium traceability

1.We have checked our qPCR assay on strains fromCzech Republic and Germany. It works for F.culmorum and graminearum.

2.Preliminary qPCR analysis was done on a set of oat samples from Czech Republic.

Avena genetic resources for quality in human consumption (AVEQ)

qPCR based Fusarium traceability

1.2. We’ll apply the assay to 11 standard cv, inoculated and non

inoculated

3. PCR results will be gathered with ELISA results

4. Starting from the results, it will be evaluated to expand the PCR assays.

Standards, PCR identification, Kromeriz (Czech Rep.), harvest 2008

InoculatedSaul 419 Saul 09-1082Saul 446 Saul 09-1083CPVO19960125 380 Belinda 09-1087CPVO19960125 424 Belinda 09-10888 376 Argentina 09-10928 382 Argentina 09-1093CPVO19981528 366 Evora 09-1097CPVO19981528 408 Evora 09-1098ROM007-16701 199 Mures 09-1102ROM007-16701 392 Mures 09-110300004 378 Jaak 09-110700004 384 Jaak 09-110830040 187 Auteuil 09-111230040 303 Auteuil 09-1113700 349 Genziana 09-1117700 354 Genziana 09-1118Krezus 174 Krezus 09-1122Krezus 356 Krezus 09-1123A7BM0001 351 Mina 09-1127A7BM0001 454 Mina 09-1128CPVO20040091 391 Ivory 09-1132CPVO20040091 444 Ivory 09-1133spring wheat 379 Aranka 09-1137spring wheat 407 Aranka 09-1138Non-inoculated00004 950 Jaak 09-114030040 946 Auteuil 09-1142700 952 Genziana 09-11448 955 Argentina 09-1146A7BM0001 954 Mina 09-1148CPVO19960125 949 Belinda 09-1150CPVO19981528 951 Evora 09-1152CPVO20040091 947 Ivory 09-1154Krezus 945 Krezus 09-1156ROM007-16701 953 Mures 09-1158Saul 948 Saul 09-1160spring wheat 927 Aranka 09-1162

bars with different colors are different at the tukey’s hsd test (p=0.05) avena genetic resources...

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