Identification of the main Fusarium species contaminating cereals Analysis of fungal genetic diversity and toxinogenic profile, pathogenicity against plant Evaluation of main mycotoxins associated with different species (Aprile 2015)

1. Master Degree in
Industrial and Environmental Biotechnology (2013)
Variabilità e identificazione morfo-molecolare di funghi tossigeni,
isolati da cereali contaminati da micotossine, di diversa provenienza
geografica
PhD in Innovation and management of high- value food
healthy- XXIX Cycle (2014-today)
Genetics, mycotosins and diagnostics of Fusarium species involved in
fusarium head blight of cereals, with special attention to Fusarium
graminearum, for a higher quality and safer cereal production
Veronica Ghionna

2.  Identification of the main Fusarium species
contaminating cereals
 Analysis of fungal genetic diversity and toxinogenic
profile, pathogenicity against plant
 Evaluation of main mycotoxins associated with different
species
Aims of the PhD work

3. Identification and characterization of
Fusarium species isolated from cereals
About 25% of food and feed
products worldwide is
contaminated with mycotoxins
(FAO data)
FUSARIUM HEAD BLIGHT (FHB),
caused by a complex of Fusarium
species,
is one of the most important fungal
diseases on cereals

4.  Austria 2011
150 samples wheat,
barley and other cereals
(Rye, Oat, Sorghum,
Triticale, Corn);
 Austria 2012
64 samples wheat, barley
and other cereals
(Rye, Oat, Corn)
 Germany 2012
47 samples wheat, barley
and other cereals
(Rye, Oat, Corn)
Sample collection
 China 2013
100 samples wheat
465 Cereals samples
BIOMIN
cooperation
MAIN MYCOTOXINS
AUSTRIA- GERMANY
•Beauvericin (BEA)
•Enniatins (ENNs)
•Deoxynivalenol (DON)
•DON-3-glucoside (D3G)
•Zearalenon (ZEN)
MAIN MYCOTOXINS
CHINA
•Enniatins (ENNs)
•Deoxynivalenol (DON)
•DON-3-glucoside (D3G)
•3-acetyl-deoxynivalenol (3A-DON)
•Zearalenon (ZEN)

5. Sample collection
 Italy 2013
40 samples wheat
(Puglia, Campania, Basilicata, Sicilia, Marche)
 Italy 2014
64 samples wheat
(Puglia, Campania, Basilicata, Sicilia, Molise,
Marche, Emilia Romagna, Veneto)
465 Cereals samples
ISCOCEM
project
MAIN MYCOTOXINS
ITALY
•T-2 and HT-2
•Deoxynivalenol (DON)
•Zearalenon (ZEN)

6. Fusarium graminearum Fusarium cerealisFusarium culmorum
Main Fusarium species associated with FUSARIUM HEAD BLIGHT
MAIN MINOR
Fusarium poae Fusarium langsethiae Fusarium sporotrichioides
Fusarium equiseti

7. Main Fusarium species associated with FUSARIUM HEAD BLIGHT
Fusarium avenaceum Fusarium acuminatum
Fusarium tricinctum
MAIN MINOR

8. Description of the activity
 Testing of 100 seeds for each crop sample on selective growth
substrate;
 Morphological characters observation of fungal colonies (for Fusarium
/Alternaria selection);
 Single spore isolation of fungal colonies of interest;
 Morphological and molecular identification at species level for selected
strains;
 Collection of data contamination.
Identification of the main Fusarium and Alternaria spp

9. Morphological
Molecular
Fungal isolates Identification
Morpho-phenotypical
characters
 Housekeeping genes (b-tubulin,
calmodulin, elongation factors...)
Fusarium spp., and belonging to
F. tricinctum/F. acumintum,
F. graminearum clade
gene-sequencing
517 isolates
126/517 isolates

10. Origin Host
Sample
(Nr)
Fusarium Alternaria
Contaminated
samples
Range of
contamination(%)
Contaminated
samples
Range of
contamination(%)
Austria
Wheat 76 72 1 – 68 14 1-80
Barley 91 69 1 – 44 63 1 – 98
Others 47 39 1 – 100 24 1 – 61
TOT 214 180 1 – 100 101 1-98
Germany
Wheat 17 16 3 – 94 17
2-57
Barley 21 18 1 – 54 20
4-68
Others 9 8 1 – 29 8
10-51
TOT 47 42 1-94 45 2-68
China Wheat 100 43 1-68 87 1-100
Analysis of the results

11. Wheat and
barley
• F. poae
• F. graminearum
Maize
• Species belonging to the
F. fujikuroi complex
Fusarium spp mainly occuring on tested crop
samples
Data from morphological identification of the main Fusarium and Alternaria spp
Austria, Germany and China

12. Analysis of the results
Italy
Fusarium contamination (%)
Contaminated samples Range
Sicilia 2/11 1
Campania 10/11 1-8
Puglia 9/11 1-4
Basilicata 6/9 2-8
Molise 2/2 2-3
Emilia Romagna 5/6 3-10
Marche 39/48 1-38
Veneto 5/6 1-12
Fusarium spp mainly occuring on tested crop samples
Durum wheat and
triticum aestivum
• F. poae
• F. graminearum

13. 100
98
96
94
92
90
88
86
84
82
80
78
17125
17126
17106, …., …..
7641
16579
16589
16622
16697
17080, …., …
15480
16506
17104
17105
17107
17108
17109, …, ….
7630
3202
16196
16655
7582
15555
17084
17096
955
15572
15523
16624
17081
17082
17086
17097
16527
17099
17083
7626
7632
17119
17129
5356
16536
16547
16552
16555
17054, …., …
17130
7547
6748
17092
17095
15511
7633
tricinctum
acuminatum
avenaceum
longipes
temperatum
subglutinans
sambucinum
sambucinum
venenatum
langsethiae
culmorum
crookwellense
graminearum
incarnatum
semitectum
equiseti
equiseti
wheat
wheat
wheat
15571
barley
oat
barley
barley
wheat
barley
wheat
wheat
wheat
wheat
wheat
15486
Stringbean
maize
maize
15563
wheat
wheat
Solanum tub.
barley
wheat
wheat
wheat
wheat
wheat
wheat
wheat
15501
15499
wheat
wheat
triticale
wheat
wheat
barley
wheat
wheat
wheat
wheat
Italy
Italy
Italy
Germany
Germany
Germany
Austria
Italy
Austria
Italy
Italy
Italy
Italy
Italy
Philippines
Belgio
Austria
Italy
Italy
Germany
Germany
Italy
Italy
Italy
Italy
Austria
Italy
Italy
Italy
Italy
USA
Austria
Germany
Germany
Germany
China
Italy
Australia
Italy
Italy
12 isolates
Fusarium sp.
23 isolates
8 isolates
68 isolates
Molecular identification
Calmodulin gene (126 isolates)

14. Future activities planned
Verify the identity of species by molecular techniques
(gene sequencing) and multi-locus phylogenetic analysis
Evaluation of the chemotype by using specific primers of
the TRI genes involved in the biosynthesis of
trichothecenes
Detection of the presence of toxins by chemical analysis
with HPLC
Pathogenicity test on plant

15. Conclusions
This wide monitoring of toxigenic fungi in cereals has confirmed that:
 The cereals can be a source of mycotoxins globally very risky for human health;
 The Fusarium head blight of wheat is determined by a number of species of
Fusarium very high, some not defined in terms of taxonomic and mycotoxicological.
 The contamination is often caused by a variety of genera and species, each with a
specific toxinogenic profile which requires a correct identification for a correct
evaluation and management of the risk;