3rdMycoKey TechnologicalWorkshop held at Agroscope in Zurich Reckenholz

1. Mycotoxin contamination of
Cereals and “Good agricultural
practice” to reduce fusarium
mycotoxins in cereals
Simon Edwards1 and Ingerd Hofgaard2
1Harper Adams University, Newport, Shropshire, UK
2Norwegian Institute of Bioeconomy Research, Ås, Norway

2. • Mycotoxigenic pathogens and mycotoxins
produced on cereals
• Fusarium pathogens and mycotoxins produced on
cereals
• GAP to reduce Fusarium mycotoxins on small-
grain cereals

3. Aflatoxins
Ochratoxin A
Ergot
Alternaria toxins
Fumonisins
Deoxynivalenol
Zearalenone
HT2 and T2
Which mycotoxins are important in cereals?
Fusarium
mycotoxins
3

4. Mycotoxigenic pathogens (Non-fusarium) and
mycotoxins produced on cereals
Pathogen Primary host Secondary hosts Mycotoxin Region
Aspergillus flavus
(Ear rot)
Maize Aflatoxins Narrow range
during drought
conditions
Penicillium
verrucosum
(Saprophyte)
Small grain cereals Ochratoxin A Northern Europe
Claviceps purpurea
(Ergot)
Rye Other small grain
cereals
Ergot alkaloids
(Ergot sclerotia)
Central/Northern
Europe
Alternaria spp.
(Black point)
Small grain cereals Numerous Tenuazonic acid
Alternariols
Pan Europe

5. Aflatoxins
Difuranocoumarins
Twenty types but only four routinely found in cereals (B1, B2, G1 and
G2).
Cows which consume feed contaminated with Aflatoxin B1
metabolise this to Aflatoxin M1 which is expressed in milk (only
example where biotransfer is significant). Also expressed in
human milk.
Relatively stable molecule that will survive food processing
Aflatoxin B1

6. Aflatoxin – Occurrence
Location: Tropical and sub-tropical climates
Hot continental climates
Food affected: Nuts, dried fruit, cereals, spices
Europe largely limited to maize
Factors impacting on concentration:
Weather (eg drought-stress of maize),
Host plant damage (eg insect damage of maize)
Storage conditions (high temperature/high humidity)
Battilani et al. (2016) Scientific Reports,
6, 24328.

7. 7
Aflatoxin – Occurrence
Three Aspergillus species responsible (A. flavus, A.
parasiticus and A. nominus).
Commonly detected in soil and on decaying plant
material.
Taxonomy not well characterised.
A. flavus
Aspergillus infected maize
(olive brown powdery appearance)

8. Aflatoxin – Legislation
Range of limits depending on the product and if any further physical processing to
occur.
Range of:
0.1-12 μg/kg for B1
4-15 μg/kg for B1+B2+G1+G2
0.025 and 0.05 μg/kg for M1 in infant milk and milk
Complete and complimentary feedingstuffs for livestock – maximum limit ranges
from 5 μg/kg B1 for dairy cows for consumer safety rather than the animals.
COMMISSION REGULATION (EC) No 1881/2006 setting maximum levels for certain
contaminants in foodstuffs
DIRECTIVE 2002/32/EC on undesirable substances in animal feed

9. Aflatoxin – Control - GAP
Maize -
• resistant varieties - fast maturity
• planting dates
• irrigation
• biocontrol – atoxigenic strains
• insect control

10. Ochratoxin A
Several ochratoxins but ochratoxin A (OTA) far more
common than others.
Produced by different species on different crops under
different environmental conditions
Relatively stable molecule with limited reduction during
food processing
Ochratoxin A

11. Different species in different climates/crops.
Both common moulds associated with decaying organic
matter in soils.
Penicillium verrucosum infests cereals in storage in
temperate climates
Ochratoxin A - Occurrence

12. Range of maximum limits set (eg.s only).
Infant food 0.5 μg/kg
Cereal products 3
Unprocessed cereals 5
COMMISSION REGULATION (EU) No 105/2010 amending
Regulation (EC) No 1881/2006 setting maximum levels for certain
contaminants in foodstuffs as regards ochratoxin A
Ochratoxin A - Legislation

13. For European cereals: Not present in field.
Only produced on stored crops.
Reduce inoculum Hygiene – efficient cleaning of harvest
equipment and stores when emptied and
again before harvest
Reduce growth Harvest grain at low moisture content
- cool grain
- dry grain
Maintain cool and dry (insect control, store
design, ventilation, monitoring)
Ochratoxin A - Control

14. Ergot
Present as a fungal sclerotia – long-term survival structure
Pathogenic Claviceps spp.
Claviceps purpurea in European cereals.
Main host is rye but also infects other small grain cereals and
grass weeds
Twelve ergot alkaloids and their respective amines
APSnet.org
Ergotamine

15. Ergot – Occurrence
Central and North Europe
Primarily issue on rye and grass weeds
Requires wet weather during flowering so high variation across
regions and seasons

16. Ergot – Legislation
• Unprocessed cereals limit of 0.05 % sclerotia (w/w) (EU, 2015)
• Current discussion limits for sclerotia in unprocessed cereals
and alkaloids in finished cereal products (2019)
• Unprocessed cereals limit of 0.02 % sclerotia (w/w)
Ergot alkaloids µg/kg
Cereal mill products (other than rye) 75
Rye mill products 250
Infant food 20
Recent UK study (AHDB [2019] Project Report 603) has identified ergot alkaloids can
migrate from sclerotia to neighbouring cereals grains in the ear and alkaloids can
transfer to grains post-harvest so even in absence of sclerotia (due to cleaning) mill
products may still exceed proposed alkaloid limits.

17. Ergot – GAP
• Manage grass weeds, especially black-grass
• Harvest field headlands separately from the bulk of the crop
• Plant a non-cereal crop or plough to ensure ergots are
buried to at least 5cm depth
• Avoid varieties with a long flowering period
• Avoid sowing contaminated seed – clean farm-saved seed
thoroughly to remove ergot
• GMP - Post-harvest grain can be cleaned by optical sorters to
remove sclerotia but alkaloids may already be present on
grains

19. Fusarium mycotoxins
100s of different mycotoxins produced by members of the Fusarium
genus – plant pathogens
Many are structurally related and produced within the same
pathway – Trichothecenes
Trichothecenes divided into
Type A (eg HT2 and T2)
Type B (eg deoxynivalenol and nivalenol)
Other important ones include Fumonisins and Zearalenone

20. Mycotoxigenic fusarium pathogens and mycotoxins
produced on cereals in Europe
Pathogen Primary host Secondary hosts Mycotoxin Region
F. graminearum
F. culmorum
Maize (Red ear
rot)
Small grain
cereals (FHB)
DON, ZEA Pan Europe
North Europe
F. langsethiae Oats Small grain
cereals (FHB)
HT2 and T2 toxin North Europe
F. poae Small grain
cereals (FHB)
NIV
DAS
North Europe
F. avenaceum Small grain
cereals (FHB)
ENN
MON
BEA
North Europe
F. verticillioides
F. proliferatum
F. subglutinans
Maize (Pink ear
rot)
FUM
FUM, BEA, MON
BEA, MON
Central and
South Europe
North and
Central Europe

21. Fumonisins
Fumonisins made up of at least 15 closely related chemicals.
Most common is fumonisin B1 (followed by B2 and B3)
Polar metabolites based on a long hydroxylated hydrocarbon chain
Associated with equine leucoencephalomalacia in USA long before isolated in
1980s
Fumonisin B1

22. Fumonisin – Occurrence
Location: Hot continental climates
Food affected: Primarily maize, also rice and sorghum
Factors impacting on concentration:
Host tissue damage (eg insect damage)

23. 23
Fumonisin – Occurrence
Fusarium species, predominantly Fusarium verticilliodes and
Fusarium proliferatum
Commonly detected on maize from hot continental climates
Pink ear rot F. verticilliodes

24. Fumonisin – Legislation
Range of limits depending on the product
4000 μg/kg for unprocessed maize (except for wet milling)
1000 μg/kg for maize intended for direct consumption
800 μg/kg for maize based snack and breakfast cereals
200 μg/kg for infant food
Different level set for maize flour as intermediate depending on
particle size
COMMISSION REGULATION (EC) No 1126/2007 amending Regulation (EC) No
1881/2006 setting maximum levels for certain contaminants in foodstuffs as
regards Fusarium toxins in maize and maize products

25. Fumonisin – Control - GAP
FUM producing Fusarium species generally infect through
wounds.
Most wounds on maize caused by insect damage (eg European
corn borer).
Control FUM indirectly by controlling insects.
1. Efficient insecticide program
2. Insect resistant GM maize (BT-maize)

26. Fusarium head blight
Fungal disease complex of small grain cereals
Important disease on wheat, barley and oats world-wide
Barley and oats are less susceptible in most growing regions
Disease dependent on weather at specific crop growth stages
Range of Fusarium species able to infect cereals
Different species produce different mycotoxins
Different species occur on different cereals and in different climates
Mycotoxin profile varies by cereal, region and season
Mycotoxin profile changes over decades and continents

27. Fusarium disease cycle
HGCA Fusarium Guide,
Summer 2007

28. Fusarium Disease Conditions
Warm dry spring induces spore production on crop
debris
Heavy rainfall in June splashes spores onto ears
Infection occurs mainly at flowering under warm
humid conditions
High rainfall/humidity through summer allows
infection to spread, particularly once the crop ripens

29. Fusarium epidemiology
DON and ZON are produced by F. culmorum and F.
graminearum on cereal crops pre-harvest during head
blight infections.
DON is produced primarily during infection (ie
anthesis/milky ripe)
ZON is produced pre-harvest (dough/ripe)

30. Mycotoxin production during FHB infection
0
20
40
60
80
100
120
Mycotoxinconcentration
(%harvestvalue)
Wheat growth stage
DON
ZON
Flowering Milky Ripe Dough Ripe Harvest
Based on data from Matthaus et al. (2004) Progression of mycotoxin and nutrient concentrations in wheat after
inoculation with Fusarium culmorum. Archives of Animal Nutrition 58: 19-35.
Ripening
phase

31. Deoxynivalenol (DON)
DON is the most common trichothecene
Type B trichothecene
Close relative is nivalenol – less common but more toxic
Acetylated versions occur at low frequency 15- and 3-acetylDON
Known in US as vomitoxin as induces vomiting in pigs
DON is a virulence factor
DON

32. DON – Occurrence
Location: Temperate and continental climates
Food affected: Cereal (Maize, Wheat, Oats and Barley)
Factors impacting on concentration:
Weather
Fusarium inoculum (previous crop and cultivation)
Host resistance
Fungicide
Lodging
Harvest delays

33. 33
DON – Occurrence
Fusarium species, predominantly Fusarium
graminearum and F. culmorum
Commonly detected on maize and wheat across
all growing regions
Red ear rot Fusarium head blight
F. graminearum
F. culmorum

34. DON – Legislation
1250 μg/kg for unprocessed cereals except
1750 μg/kg for unprocessed durum wheat, oats and maize (except for dry milling)
750 µg/kg for cereals for direct consumption, mill fractions and pasta (Different
level set for maize flour as intermediate depending on particle size)
500 μg/kg for cereal products
200 μg/kg for infant food
COMMISSION REGULATION (EC) No 1126/2007 amending Regulation (EC) No
1881/2006 setting maximum levels for certain contaminants in foodstuffs as
regards Fusarium toxins in maize and maize products

35. DON – Control - GAP
For Maize:
• Previous crop – avoid maize or wheat
• Cultivation - ploughing
• Early harvesting
• Resistant varieties – early maturing
- silk resistance
- kernel resistance
• Balanced nutrition
• Canopy density – avoid thick crops

36. DON – Control - GAP
For Wheat:
• Previous crop – avoid maize
• Cultivation - ploughing
• Early harvesting
• Resistant varieties
• Balanced nutrition
• Avoiding lodging – use of PGR
- optimum N inputs

37. Zearalenone (ZON)
ZON is an oestrogenic mycotoxin
ZON has an unknown function in the fungus.
Production is linked to transition from pathogen to
saprophytic lifestyle
Very low water solubility
ZON

38. ZON – Occurrence
Location: Temperate and continental climates
Food affected: Cereal (Maize, Wheat, Oats and Barley)
Factors impacting on concentration:
As for DON BUT …
Harvest delays are critical – Infected crops routinely have high
DON but if ripening and harvest conditions are hot and dry then
ZON remains very low

39. ZON – Legislation
100 μg/kg for unprocessed cereals except
350 μg/kg for unprocessed maize (except for dry milling)
75 µg/kg for cereals for direct consumption, mill fractions and pasta (Different level
set for maize for direct consumption, maize oil and maize-based products)
50 μg/kg for cereal products
20 μg/kg for infant food
COMMISSION REGULATION (EC) No 1126/2007 amending Regulation (EC) No
1881/2006 setting maximum levels for certain contaminants in foodstuffs as
regards Fusarium toxins in maize and maize products

40. ZON - GAP
For Maize and Wheat:
As for DON except …..
Early harvesting is critical

41. HT-2 and T-2 toxins (HT2 +T2)
Type A trichothecenes
Close relative is neosolaniol, diacetoxyscirpenol, T2 triol
and T2 tetraol
Co-occur as all part of same pathway
Less common but more toxic than DON
T2

42. HT2+T2 – Occurrence
Location: Temperate climates
Food affected: Cereal (Maize, Wheat, Oats and Barley)
Factors impacting on concentration:
• Weather
• Fusarium inoculum (previous crop and cultivation)
• Host resistance

43. 43
HT2+T2 – Occurrence
Predominantly Fusarium langsethiae
Occasional on maize but more common on Nordic cereals, French
barley and UK Oats
FHB on barley FHB on oats
F. langsethiae

44. Currently no legislative limits within EU.
Indicative limits set for continued monitoring in 2013 including 1000 ug/kg
HT2+T2 for unprocessed oats
Member States with active involvement of food and feed business operators
should:
• Continue monitoring of HT2 and T2.
• Submit results to EFSA
• Investigate as to why exceedance of indicative levels occurred
• Investigate as to how exceedance of indicative levels can be avoided
New discussion limits set include 500 µg/kg for unprocessed oats, 50 for
barley and 20 for wheat (EC Unpub, Feb 2019)
HT2+T2 – Legislation

45. HT2+T2 - GAP
For Oats:
Previous crop – Minimise other cereals in rotation
Resistant varieties
Organic production

46. Identification of GAP using modelling of mycotoxin
concentration in harvested commercial grain against
associated agronomy data
Collect large sample size over multiple years
Model using linear regression
Force year and region to front of model (accounts for
temporal and spatial variation)
Include polynomial sub-model for harvest week (based
on long-term local average, -2 = 2 weeks early, +2 = 2
weeks late)

47. Change d.f. s.s. m.s. v.r. F pr.
+ Year 7 449.5 64.2 349.0 <.001
+ Region 7 32.7 4.7 25.4 <.001
+ Year.Region 49 45.2 0.9 5.0 <.001
+ Precrop 9 8.8 1.0 5.3 <.001
+ Cultivation 1 0.6 0.6 3.3 0.071
+ Precrop.Cult. 9 7.0 0.8 4.2 <.001
+ Variety 26 14.9 0.6 3.1 <.001
+ POL(HarvestWeek) 2 4.5 2.3 12.2 <.001
Residual 1074 197.6 0.2
Total 1184 760.9 0.6
69
2.2
2.0
0.6
Modelling of DON by year, region and agronomy

48. 0
100
200
300
400
500
600
2006 2007 2008 2009 2010 2011 2012 2013
DON(ppb)
Year
South East South West
East East Midlands
West Midlands Yorkshire and Humberside
Temporal and spatial variation

49. 0
500
1000
1500
2000
2500
DON(ppb)
Previous crop
Plough
Min-till
Predicted DON based on previous crop and cultivation

50. Minimum tillage after forage maize

51. Direct drilled after grain maize

52. 0
50
100
150
200
250 Cordiale
Gallant
Gladiator
Grafton
Malacca
Oakley
Battalion
Claire
Consort
Duxford
Einstein
Glasgow
Hereward
Humber
Invicta
JBDiego
KWS-Santiago
Robigus
Scout
Solstice
Viscount
Xi19
Zebedee
Alchemy
Istabraq
Soissons
DON(ppb)
Variety6 75
Predicted DON based on variety

53. Statistical analysis of harvest date
Delayed harvests of more than 2 weeks have a major impact on DON and ZON
concentration in wheat
-4
400
200
-2
250
2
300
350
50
150
4
100
0
0
harvest_week
20
-4
30
0
40
4
50
60
70
0
-2
10
2
ZON_ug_kg
harvest_week
y=-7.5+50(1.52x)
r2=0.89
y=1.96+2.48(2.0x)
r2=0.97

54. Fungicide reduction of DON
Not significant in model
Know highly dependent on accurate timing
Field experiments show effective reduction - but can be misleading effective if Fusarium
inoculum and fungicide applied within a short time frame.
We use inoculated oat grain applied to the ground in Spring to provide a more natural
inoculum with spores released over a long time frame.

55. Fungicide reduction of DON
Metconazole
Prothioconazole
Tebuconazole
Epoxiconazole + dimoxystrobin
Epoxiconazole (weak)
Carbendazim (variable)
Compared by rate, prothioconazole is best.
Some strobilurins can have a negative impact (increase DON)
Control commercially more variable than in field trials as
fungicide, inoculum and moisture timing are all critical

56. 0
2000
4000
6000
8000
10000
12000
DON(ppb)
Untreated at T3
0
2000
4000
6000
8000
10000
12000
DON(ppb)
Untreated at T3
Prothio at T3
Factorial fungicide reduction of DON with
prothioconazole
56

57. No fungicide T3 prothioconazole Full prothioconazole
(No T1 or T2) (T1, T2 and T3)

58. Seed
Treatment
T1
T2
T3
Targeting Fusarium throughout season

59. Statistical analysis of ZON
Modelling ZON concentration against year, region and agronomy
conducted based on over 1000 wheat samples collected at harvest from
2007-2013.
58% of variance accounted for by year and region (mainly weather)
3% of additional variance accounted for by agronomy
39% of variance not accounted for (some of unaccounted variation would
be due to differences in weather within regions)
Weather is predominant factor
Agronomy data very similar to DON except harvest timing
has a greater impact

60. Good Agricultural Practice to minimise
Fusarium mycotoxins in milling wheat
• Fusarium resistant varieties
• Good rotation - avoid maize as previous crop
• Cultivation - plough in crop debris following a cereal
crop (particularly maize)
• Use a high rate of a good FHB fungicide at correct
timing (GS 59).
• Avoid lodging
• Timely harvest

61. Impact of GAP for mycotoxin control
Ferrigo et al (2016) Molecules 21:627VH, very high; H, high; S, significant; L, low

62. • 23 partners
• > 40% industry participation
• 5 end users from the industry
The Consortium

63. WorkPackage1
Pre-harvest objectives
• Alternatives to triazole
fungicides including
biopesticides (suitable for
organic farming) for cereals
• Biofumigation and accelerated
biodegradation combined with
minimum tillage targeting
Fusarium
• Aflatoxin Resistant maize
hybrids
• Atoxigenic Aspergillus strains to
obtain aflatoxin free maize in
the EU

64. Biopesticides
All encompassing definition of all pest control
mechanisms other than conventional pesticides
Includes: Biocontrol agents, botanicals, plant defence
stimulants, simple salts and metals
Increasing interest in these as alternatives to
conventional pesticides due to:
• Suitable for organic farming (not all)
• Increasing cost of registration of pesticides
• Development of resistance to pesticides
• Increasing reliance on fewer pesticides

65. Field experiments under high disease pressure (inoculated
experiments) Oats in Norway Wheat in UK
Selected biopesticides for screening based on:
1) Previous evidence of activity against Fusarium
2) Availability of product in UK and Norway
Slight difference between countries based on authoritive bodies
permissions to use products in field experiments
16-17 biopesticides including:
Biocontrol agents, copper compounds, zinc compounds,
botanicals, plant stimulants, phosphites

66. DON concentration in harvested wheat grain - HAU 2016
a
b b
bc
cd
cd
cd cd
d d d d d d d d
d d d d
0
1000
2000
3000
4000
5000
6000
7000
8000
DON(µg/kg)
Biopesticide treatments

67. 0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
16,000
DON(µg/kg)
DON concentration in harvested oat grain - NIBIO 2016

68. Summary
• No currently available biopesticides within Europe
have good activity against Fusarium head blight.
• Currently reliant on triazole fungicides
• Old fungicide chemistry of limited benefit
• New fungicide chemistry with very good Fusarium
activity in pipeline due 2022
• Alternative control strategies targeting the inoculum
showing promising results but needs to be translated
into field control

69. This project has received funding from the European Union's Horizon 2020 research
and innovation programme under grant agreement No 678012.
Thank you for your attention!