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DON from field to glass

Gary Bergstrom and Paul Schwarz Cornell University and North Dakota State University

Great Lakes Hops and BarleyConference Traverse City, MIMarch 16‐17, 2016

Topics

1. DON – a Fusarium Mycotoxin2. Fusarium Head Blight of Barley and Wheat3. Reducing the DON Risk from Field to

Malthouse4. Fusarium and Mycotoxins in Malting 5. Fusarium and Mycotoxins in Brewing6. Testing of Mycotoxins 7. Conclusions

Mycotoxins are:• Low molecular weight natural products• Produced by filamentous microfungi• Predominantly secondary metabolites• Toxic to vertebrates in low

concentrations

DON is a mycotoxin

Mycotoxins are produced by: Some strains of some species of fungus Under some environmental conditions

Mycotoxins are problematic only when they occur above levels of concern for specific species.

Why do fungi make mycotoxins?

• Increase competitiveness against other microbes• Avoid consumption by insects/or act as insecticides• Enhance survival probability under certain

environmental stresses• Virulence factors in colonizing plants; repress plant

immune responses

• (no one thinks that fungi are making toxins to harm mammals – colateral damage)

G.C. Bergstrom

Gibberella ear rot of cornFusarium graminearum

Mycotoxins produced by Fusarium species

Mycotoxin: Level of concern:

Common effects on animals:

Deoxynivalenol (vomitoxin)

1-3 ppm Feed refusal in monogastric animals; severity increases with level. Swine are the most sensitive species. Adult cattle and poultry tolerate > 10 ppm.

Zearalenone 1-5 ppm Hyperestrogenism and infertility. Swine (gilts) are most sensitive. Adult cattle tolerate 50 ppm.

Fumonisins 5-10 ppm Brain deterioration, death (horses); liver damage (horses, swine, cattle, poultry, others).

Deoxynivalenol• Produced by a number of Fusarium species• Common in grains

• ‘vomitoxin’, feed refusal, diarrhea

R1 R2 R 3 R4 R5

Deoxynivalenol DON OH H OH OH =O

3‐Acetyldeoxynivalenol 3‐ADON OAc H OH OH =O

15‐Acetyldeoxynivalenol 15‐ADON OH H OAc OH =O

Nivalenol NIV OH OH OH OH =O

Fusarenon X FX OH OAc OH OH =O

CH3 O

CH3

R5

R4

CH3

R3

O

H

HR2

R1

CH3 O

CH3

R5

R4

CH3

R3

O

H

HR2

R1

FHB Produces Several Trichothecenes

Proportion of trichothecene genotypes of Fusarium graminearum in 2011 and 2012

n = 101

n = 152

n = 90

n = 95

n = 87

n = 93n = 128

3-ADON15-ADON

Bergstrom et al. unpublishedGary C. Bergstrom, Cornell University

Mycotoxin chemotypes of Fusarium graminearum in wheat in 2007

Schmale, Wood‐Jones, Cowger, Bergstrom, and Arellano. 2011. Plant Pathology 60:909‐917

2011 Survey in New York15% 3-ADON: 85% 15-ADONSame as in 2007

Mycotoxin production on wheat and maize is widely variable among isolates

Wheat kernels

Maize kernelsDON, acetylated derivatives and ZON production in kernels of 16 F. graminearum isolates from wheat spikes and maize ears possessing either a 3-ADON or a 15-ADON trichothecene genotype.

F. graminearum isolates from wheat and maize showed a wide variation in mycotoxin production towards wheat and maize and these isolates could not be structured by their host origin or trichothecene genotype.

Gary C. Bergstrom, Cornell University

Kuhnem, P.R., Del Ponte,E.M., Dong, Y,. and Bergstrom, G.C. Phytopathology 22 Oct 2014 First Look

Mycotoxin genotype prevalence in three NYS locations

© G.C. Bergstrom

Aurora 2012 Aurora 2013

Belmont 2013

Willsboro 2013

LAND USE GENOTYPE

N=281 N=428 N=319

N=379

15‐ADON

3‐ADON

Trichothecene genotype frequency by niche in New York in 2013

Kuhnem, Del Ponte, and Bergstrom See Poster No. 29

• Several Billion $$ direct and indirect loss in North America• Contamination of grain with deoxynivalenol (DON) produced by F. graminearum• Livestock feed refusal, weight loss resulting from vomiting• DON reduction is the current main focus of management efforts

Unmilled grainFood and beverage products

Baby food products

Current US FDAGuidelines

2.0 1.0 -

Current ECGuidelines

- 0.5 0.1

Proposed FAO-Codex-

Alimentarius- 0.5 0.1

Fusarium head blight - the problem

Fusarium graminearum sensu stricto (lineage 7)

Predominates on wheat, barley, and corn in North America

A United Effort to Fight an Enemy of Wheat and Barley: Fusarium Head Blight

Marcia McMullen, Gary Bergstrom, Erick De Wolf, Ruth Dill-Macky, Don Hershman, Greg Shaner, and Dave Van Sanford

December 2012 Plant Disease Feature Article

Review of epidemics and progress in US since 1997

www.scabusa

Since 1999:

•$61.9 Million invested in research and outreach

•1,628 research projects

•Involvement of 174 scientists

2015 Spring Malting Barley Integrated Fusarium Management Trial

variety

PowderyMildew

% Leaf Blotch

%

FHB Incidence

% FHB Severity

% FHB Index

%DONppm

Yield bu/A

@14.5%

AAC Synergy 1.0 C 1.8 B 3.3 A B 1.0 B 0 B 0.9 B 43.4 A

Lacey 31.9 A 5.0 A 4.7 A 3.0 A 0 A 2.4 A 44.6 A

Newdale 0.9 C 0.7 B 1.7 B 0.5 B 0 B 0.7 B 33.8 A

Quest 18.7 B 6.6 A 2.7 B 2.8 A 0 B 0.7 B 37.8 A

LSD 7.23 2.09 1.76 0.50 0.04 0.60 NS

CV 120.1 101.4 75.6 68.9 92.8 87.7 40.0

Jaime Cummings, R.J. Richtmyer, Gary Bergstrom, and Russ Hahn

Foliar fungicides for management of FHB and DON

April

Early May

Late May

Early June

Spray with an effective triazole based on risk of

FHB. Effective spray window from full head

emergence through the next 5-7 days

Michael Wunsch

Fungicidal suppression of FHB & DON – meta-analysis of 100 U.S. test environments*

% Suppression compared to non‐treated

Triazole fungicide: Fusarium head blight disease

DON toxin

metconazole 86% 50 45

prothioconazole 41%

48 43

prothioconazole 19% & tebuconazole 19%

52 42

tebuconazole 38.7% 40 23

propiconazole 41.8% 32 12

*Paul et al. 2008. Phytopathology 98:999-1011

The overall mean percent control

K. T. Willyerd et al. Plant Disease. 2012. 96:957-967.

2015 Spring Malting Barley Integrated Fusarium Management Trial

Treatment

PowderyMildew

%

Leaf Blotch%

FHB Incidence%

FHB Severity

% FHB index

% DONppm

Yield bu/A @ 14.5%

Nontreated 18.6 A 3 A 2.5 A 2 A 0.1 A 1.6 A 35.6 A

Prosaro 10.6 A 3 A 2.8 A 2 A 0.1 A 0.9 A 44.6 A

Caramba 10.1 A 4 A 4 A 2 A 0.1 A 1.0 A 39.5 A

LSD NS NS NS NS NS NS NS

CV 120.1 101.4 75.6 68.9 92.8 87.7 40.0

Jaime Cummings, R.J. Richtmyer, Gary Bergstrom, and Russ Hahn

Risk prediction systems for management of FHB and DON

http://www.wheatscab.psu.edu/riskTool.html

Fungicide sensitivity: Effective concentration of tebuconazole and metconazole that reduces mycelial growth by 50% (EC50)

EC50 = 8.09 mg/l


What should occur as a consequence of these findings?


Use proactive management strategies that reduce risk of selection for resistance in pathogen populations.

•Integrated disease management (cultural, varietal, fungicidal methods).

•Alternate or combine triazole active ingredients at flowering; use other fungicide (mode of action) at earlier growth stages.

•Avoid unnecessary sprays – especially at early growth stages or those that target cereal debris

Scab Smart web site on management of FHB and DON

www.scabsmart.org

Harvest and post‐harvest management of FHB and DON

•Timely harvest at acceptable moisture level

•Combine adjustment (high fan)

•Grain drying and custom cleaning

What is / can be the contribution of cultural control to integrated management of FHB/DON?

ResistantCultivars

Fungicides /PredictionTools

CulturalPractices

???

?

How big is the ‘cultural’ slice of the management pie?

What gap is left in the absence of cultural control?

Cultural control of Fusarium head blight is based on …

avoiding or limiting exposure of cereal spikes to spores.

Macroconidia

Ascospores

Management of overwintered cereal residues: Regional impact and benefits in individual wheat fields

•Less debris decomposition and higher inoculum pressure in cold winter regions

•FHB severity declined during era of the moldboard plow, 1940s through 1970s

•Regional increases in FHB, predominance of Fusarium graminearum as causal fungus associated with increased acreage of corn

Horizontal transport of spores over km distancesin the planetary boundary layer (60 m)

Maldonado-Ramirez, Schmale, Shields, and Bergstrom. 2005. Agric. Forest Meteorol. 132:20-27

Deposition of spores:Landing of viable spores of G. zeae by gravitational settling or by rainfall

Similar magnitude over variable landscapes

Day

Night

Schmale, Shields, and Bergstrom. 2006. Can. J. Plant Pathol. 28:100-108.

Local distance infection by released clones of G. zeae

3% Clonal source

Control

% Clones

Keller, Waxman, Bergstrom, and Schmale. 2010. Plant Disease 94: 1151‐1155.

100 ft

Suggested a 3 to 6 meter (10 to 20 feet) interplot to avoid significant interplot interference from inoculum-bearing cereal residue

Collaborators: Carl Bradley, Ann Hazelrigg, Don Hershman, Martin Nagelkirk, Laura Sweets, Stephen Wegulo

Commercial-scale wheat after corn strip trials (no-till vs moldboard-plowed) in seven states (2011 - 2013)


Commercial-scale wheat after corn strip trials (no-till vs moldboard-plowed) experimental design

Mead, Nebraska

Aurora, New York

Average increase in DON of 22% (0.24 ppm) associated with no-till corn residue in wheat strips

00.51

1.52

2.53

3.54

4.55

IL KY MO NE NY VT MI IL KY MO NE NY VT MI IL NY NE

DON (p

pm)

Moldboard Plow No‐till Chisel Plow

Average 17% (0.38 ppm) increase when background level > 0.50 ppm

**

*

* *

2013

2011 2012

Conclusions about management of inoculum sources for FHB

•Spores liberated from within-field debris may provide a significant fraction of inoculum for a given field, though often less than 30% (most important in FHB-limiting environments)

•Regional, atmospheric spore populations generally provide more inoculum than within-field sources (especially under FHB-conducive environments)

•Inoculum (debris) management strategies in individual fields may result in incremental reductions of FHB/DON, and thus contribute to integrated management

What is the contribution of cultural control to integrated management of FHB/DON?

ResistantCultivars

Fungicides /PredictionTools

CulturalPractices

?

For wheat/barley within corn-growing regions in the north central and northeastern U.S., generally less than 30% contribution to DON reduction.

No single answer for all environments and cropping systems.

Reducing the Fusarium / DON risk for malting barley in the Northeast:

Steuben Co. NY 7/3/14

New York Barley Grower Survey question: Rank your biggest grain quality issue, with 1 being the highest.

#1 Ranking contribution #2 Ranking contribution #3 Ranking contribution #4 Ranking contribution

Ranking of Grain Quality Issues by Growers

Protein out of optimal range (optimal range being 9‐12%)

Low germination and pre‐harvest sprouting

Plumpness and uniformity of grain

Mold and mycotoxin (DON)

6‐Row malting barley FHB symptoms

Surface sterilized Incubated 5 days

Darkened florets indicative of FHB Pink Fusarium colonies grow from symptomatic florets

Dark molds are saprophytes not associated with symptoms

• Soil Tests• Malt Quality• DON & Fusarium Content• Foliar Disease

Survey of 40 commercial barley fields in 2015

Acknowledgements: Kevin Ganoe, Mike Stanyard, Justin O’Dea, Aaron Gabriel, Jaime Cummings

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

16 19 12 36 14 27 22 40 35 33 39 1 30 13 3 18 31 37 38 29 17 25 7 15 21 6 24 5 8 20 34 10 2 32 26 28 4 9 23 11

ppm

Sample Number

DON

16

65

20

9

11

0

5

10

15

20

25

Less than 1 1‐2 More than 2

Samples

ppm

DON

2014

2015

Different DON results by ELISA vs chromatography

© G.C. Bergstrom

0

1

2

3

4

5

6

7

33 35 22 16 27 40 36 12 29 39 18 14 1 13 19 38 31 30 25 37 21 6 8 3 17 15 5 7 24 32 2 10 20 11 26 28 34 4 9 23

DON (p

pm)

Sample #

DON Comparison on 40 field samples measured by ELISA and GC‐MS

GC-MS

ELISA

How good a predictor is grain DON of viable Fusarium content?

© G.C. Bergstrom

0

10

20

30

40

50

60

70

0

1

2

3

4

5

6

7

33 35 22 16 27 40 36 12 29 39 18 14 1 13 19 38 31 30 25 37 21 6 8 3 17 15 5 7 24 32 2 10 20 11 26 28 34 4 9 23

% Fusarium kerne

ls

DON (p

pm)

Sample #

DON vs Fusarium kernels

UMN DON % Fusarium kernels

Viable Fusarium content before, during, and after craft malting?

MaltGrain

© G.C. Bergstrom

Farmhouse Malt Subversive Malting

East Coast Malts

Queen City Malting

Niagara Malt

NY Craft Malt

Pioneer Malting Inc.

Empire Malt

Empire Malt

On to the malt house …

© G.C. BergstromMadison Co. NY 6/24/14

Fusarium and Mycotoxins in Malting

COFCO Malt (Jiangyin) Co., Ltd


Key Concerns:• DON is transferred from malt to beer

– Worldwide surveys show DON is commonly present in beer, albeit at low (ppb) levels

– Safety/public health concerns– Public perception concerns

• Fusarium can grow during malting, although exact behavior is difficult to predict.– Production of additional DON– DON on malt


Key Concerns:• Fusarium can damage grain components and impact malt quality.– Increased FAN and soluble protein are common with FHB

• Fusarium produces metabolites that can cause gushing in bottled beer.

• DON may be present on by‐products (rootlets, dust and clean‐out).


Fusarium on barley: symptoms of FHB are not always visible


Fusarium on germinating barley


• The normal pattern is to see DON decrease in the steep, and remain low on the finished malt.

• DON is solubilized or DON/Fusarium are rinsed off with dust, etc.


• However, this only works to a point.

• DON levels in more heavily infected samples generally will not be reduced to satisfactory levels by steeping.


• In some cases, the Fusarium will grow during malting.– Additional DON is

produced• The finished malt can

contain significant levels of DON and DON3G.

• Often a problem when grain is malted too soon after harvest – Fusarium is still viable


• Barley DON levels are not good predictors of the level on malt– Barley DON x Malt DON (0‐60 ppm) r=0.84(0‐10 ppm) r=0.80(0‐5 ppm) r=0.68 (Schwarz et al 1995).

• Should test malt as well as barley

DON‐3‐Glucoside: A Masked Mycotoxin

Dexynivalenol‐3‐Glucoside

• D3G is a masked (or bound) mycotoxin:– Meaning that it is not detected by normal analytical procedures.

• The plant uses an enzyme to attach glucose to DON.– A detoxification or defense mechanism of the plant.

• D3G can increase during malting– Germinating seed converts some DON to D3G

Dexynivalenol‐3‐Glucoside

• DON3G has lower toxicity and is not currently measured or regulated.– Measurement requires LC‐MS– May interfere with some ELISA methods

• Leads to over‐estimation of DON in malt?• ASBC has found several test kits not to be suitable for measurement of DON in malt

Fusarium and Mycotoxins in Wheat

• FHB infected wheat and rye seem very prone to develop DON during malting (especially right after harvest).

• Empirical evidence, however, suggests that “pink grains” do not always contain DON. Fusarium on Kilned wheat

Fusarium and Mycotoxins in Brewing

Las Cervezas Modelo en Zacatecas.


Key Concerns:• DON in beer

– Safety concern– Public perception

• Beer Gushing– Spontaneous over‐foaming of bottled beer upon opening (loss of a few milliliters to half the bottle)

– Caused by Fusarium, as well as species of Rhizopus, Aspergillus, Alternaria and Penicillium

• Distilling– DON is not transferred to distillate but will remain with the

spent mash• Feeding concerns with distillers grains?

Fusarium Head Blight and Beer Gushing

• Toxin does not cause gushing– Cyclic peptides– Hydophobins

• Gushing propensity increases with degree of FHB infection

• Gushing factors are very sensitive to dilution:– Gushing is less of a problem in adjunct beers– Gushing will be more of a concern in 100% malt beers– Malt from infected wheat seems to be most problematic

• Remember: many other factors in the brewery can cause gushing (rough bottle interior, some hop extracts, filter breakthroughs, etc).

Carlsberg Gushing Test(at VTT, Finland)


DON in Beer• Much of the DON present on malt will be extracted into beer

(>70%)• DON is heat stable and survives both kilning and wort boiling.• There have been many surveys of beer for DON.

– Varga et al (2013) analyzed 374 beer samples from 38 countries, and detected DON and DON3G in 77% and 93% of all beers.

– The average concentrations of DON and DON‐3‐G were 8.4 ppb and 6.9 ppb, respectively.

– Maximum levels were < 90 ppb.– The above levels in beer suggest malts with <0.1 ppm DON were

generally used. Below the LOQ for most commercial test labs.

Solutions to FHB and DON:Maltster

• Screen barley for DON– Have a “do not accept level”

• Screen all malt for DON when FHB is suspected• Have a “do not sell (or use) level”

• Barley DON is not an accurate predictor of malt DON


Grain Cleaning • Sizing and gravity separation.• DON cannot be cleaned out

of barley.– Small reductions might be

possible.• Removal of FDK (Fusarium

damaged kernels) in wheat may be more effective in reducing DON?– DON contaminated wheat does

not always show FDK.


• Storage– Fusarium losses viability and is less likely to grow when malted several months after harvest.

– Segregate and store contaminated grain for longer time periods. How long?

– Larger maltsters have diversity in supply and the capability to blend.

• Malting Practice– Empirical evidence suggests that increased overflow in steep reduces DON.

– Other?


• Chemical Control (e.g. bleach, ozone, organic acids, etc)– Nothing economical or safe has

proven effective.– Fine line between killing the Fusarium

and killing the grain.• Bio‐Control (e.g. starter cultures)

– Applied in steep or germination.– Microbes in the culture are intended

to out‐compete, or inhibit Fusarium growth.

– Several preparations have been patented.

– Nothing has proven highly effective in commercial malting practice.

Experimental bio-controlagent impacts rootlet growth.

Solutions‐FHB and DON:Grower

Grower: Integrated Management and Crop Rotation• Crop rotation

– Do not plant barley after corn, or rotate barley with wheat.

– Sugar beets, soybeans, canola, etc are better rotational crops as they are not hosts for Fusarium.

• Selection of less susceptible varieties.• Use of fungicides has proven effective.

DON: Beer and Food Safety


• Tricothecene toxins like DON cause “Alimentary Toxic Aleukia”

• Historical evidence of DON toxicoses in Russia, India, China– Associated with moldy

grain• Vomiting, diarrhea,

hemorrhage, shock as common symptoms

http://www.int.laborundmore.com/archive/853726/How-can-we-safeguard-the-quality-of-our-beer%3F.h


• FDA has provided advisory limits for DON on food and feed grains: http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/ChemicalContaminantsMetalsNaturalToxinsPesticides/ucm120184.html

– Nothing specific to malt or beer

– 1 ppm DON on finished wheat products (e.g. flour, bran, and germ), that may potentially be consumed by humans.


• Large malting and brewing companies have acceptance levels for DON on barley and malt– Barley: <1‐3 ppm– Malt: generally non‐detectable

(<0.2 to 0.5 ppm)– They have diversity in supply

and the ability to blend

• Craft maltsters must also establish limits– Supply chain issues are more

difficult

http://articles.aberdeennews.com/


Worst Case Scenario: Transfer of DON from malt to BeerAssumptions:– 68.7lbs malt/barrel– Only the contaminated malt was used– 100% extraction of DON from malt to beer

DON on Malt (ppm, mg/kg) DON in beer (ppm, mg/L)

0.1 0.027

0.5 0.133

1.0 0.266

1.5 0.399

2.0 0.532

3.0 0.798


• Example: Research suggests that consumption of 1 liter of beer with 800 ppb DON (3 ppm on malt) would not induce vomiting in a healthy 110 lb female (dose = 16 µg DON /kg BW)

– What about 2 or 3 liters?

• Thoughts:– There is likely no such thing as “DON‐free” just below the limit of detection

• DON was detected in 80% of all beers surveyed from 38 counties, but at very low levels (ave=8 ppb)

– Low levels of DON (< 1 ppm) on the malt are probably safe, and are in accordance with FDA guidelines on wheat products.

– What will your customer tolerate?– How to handle supply chain issues in years when FHB is a problem?

DON Testing and Error

Mycotoxin TestingTesting for DON is generally performed by one of two techniques:•Immunoassay: biochemical test that measures the presence and concentration of DON (or other toxin) in an extract by use of an antibody.

– Basis of “test kits” that are widely used by the grain industry.– Works well for umalted grain, but evidence suggests some kits do not

work for malt

Mycotoxin TestingTesting for DON is generally performed by one of two techniques:• Chromatographic Separation and Quantitation: DON (or

other toxin) is separated from other compounds in an extract and is then quantitated based upon chemical or physical properties.– Techniques include Gas Chromatography (GC) and Liquid

Chromatography (HPLC or UPLC).– Used in research labs and some of the larger malting and brewing

companies.• Works will for grain• Industry standard for malt

Information on Mycotoxin Testing

Mycotoxins in Crops: A Threat to Human and Domestic Animal Health

(David G. Schmale and Gary P. Munkvold)•http://www.apsnet.org/edcenter/intropp/topics/Mycotoxins/Pages/Detection.aspx

Mycotoxin Testing: Sources of Error

• Variation in DON results are always a concern for farmers, maltsters and brewers– “I sent two samples to two different labs and the results were

not the the same!”

• For any type of analysis there is error:– In the testing lab:

• sample preparation: grinding, weighing and extraction (error is small).• analytical instrument error (error is extremely small).• data analysis (error is extremely small).

– At the farm, malthouse or brewery:• Sampling error can be huge!

Mycotoxin Testing: Analytical Variation

Mean DON (ppm) Standard Deviation Range of Results (95% CI)

0.50 0.05 0.44‐0.56

1.00 0.10 0.89‐1.11

5.00 0.50 4.43‐5.57

10.00 1.00 8.87‐11.13

• The coefficient of variation (CV) for DON testing is usually 5 to • The above example is with 10% CV

Mycotoxin Testing: Why is Sampling Important?

FHB infection is not uniform!• Not all parts of the field are

infected.• Not all grains in a spike are

infected.• Not all grains will contain

DON.• DON may not be uniformly

distributed in a bin or truck• The lab only tests the sample

you send, so send a representative sample.

Information on Sampling

• US Wheat and Barley Scab Initiative– http://scabusa.org//pdfs/ptt/grain‐sampling_don‐analysis.pdf

• USDA‐GIPSA– http://www.gipsa.usda.gov/default.aspx– http://www.gipsa.usda.gov/fgis/handbook/MycotoxinHB/Mycotoxin_Handbook_2015‐09‐22.pdf

– http://www.gipsa.usda.gov/fgis/publication/ref/practical_sampling.pdf